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Activation of the NRF2-mediated phase 2 enzyme response as a prophylactic strategy for treatment of stroke… Shih, Andy Yi-An 2006

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ACTIVATION OF THE NRF2-MEDIATED PHASE 2 E N Z Y M E RESPONSE AS A PROPHYLACTIC STRATEGY FOR T R E A T M E N T OF STROKE A N D NEURODEGENERATION by A N D Y Y I - A N SHIH B.Sc, The University of British Columbia, 2000 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH C O L U M B I A May 2006 © Andy Yi-an Shih, 2006 A B S T R A C T The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates expression of genes required for free radical scavenging, detoxification of xenobiotics, and maintenance of redox potential. We hypothesized that the multifaceted Nrf2 response was a viable neuroprotective strategy for neurodegeneration and stroke. We initially examined the role of Nrf2 in primary cortical neuron/astrocyte cultures. Nrf2 activity was robust in the astrocytes, but limited in neurons. Augmentation of astrocytic antioxidant capacity using Nrf2 over-expression conferred potent neuroprotection during in vitro toxicity paradigms involving oxidative stress. Nrf2 overexpression coordinated upregulation of many Phase 2 enzymes. In particular, Nrf2-dependent production of astrocyte GSH was both necessary and sufficient for neuroprotection. Activation of endogenous astrocyte Nrf2 by the small molecule antioxidant response element (ARE) inducer, fer/-butylhydroquinone (tBHQ), also protected against oxidative stress. Nrf2 function was critical for neuroprotection during in vivo neurodegeneration. Nrf2_/" mice exhibited hypersensitivity to the mitochondrial toxin 3-nitropropionic acid (3-NP) displaying severe motor deficits and larger striatal lesions than identically treated Nrf2 + / + and Nrf2+/" controls. Dietary supplementation with tBHQ increased brain GSH content and attenuated 3-NP toxicity in Nrf2+ /" mice, but not NrlT 7". Increased Nrf2 activity alone was sufficient to protect animals from 3-NP toxicity because intrastriatal adenovirus-mediated Nrf2 over-expression reduced lesion size compared to GFP over-expressing controls. Nrf2 activation was also essential for neuroprotection during in vivo cerebral ischemia. Acute pretreatment with tBHQ increased cortical GSH levels and reduced cortical damage and sensorimotor deficit even 1 month after ischemia-reperfusion in rats. Conversely, Nrf2"A mice exhibited larger ischemic infarcts than Nrf2 + / + controls due to decreased survival of penumbral i i tissue. Neuronal death caused by an endothelin-1 based "penumbral" model of stroke could be attenuated by t B H Q administration to N r f 2 + / + , but not N r f i 7 " mice. We conclude that increased Nrf2 activity is highly neuroprotective in both in vitro and vivo toxicity paradigms that mimic aspects of neurodegeneration and ischemic injury. Nrf2 pathways may be accessed in vivo by treatment with small molecule inducers through multiple routes of administration, providing an effective prophylactic strategy for combating neuronal death caused by neurodegenerative disease and stroke. T A B L E OF C O N T E N T S A B S T R A C T n T A B L E O F C O N T E N T S IV L I S T O F T A B L E S xii L I S T O F F I G U R E S xm L I S T O F A B B R E V I A T I O N S xvi A C K N O W L E D G E M E N T S xix C O - A U T H O R S H I P S T A T E M E N T X X C H A P T E R 1 : G E N E R A L I N T R O D U C T I O N 1 1.1. O X I D A T I V E S T R E S S A N D T H E B R A I N 1 1.2. S T R O K E : A B R A I N A T T A C K 5 1.2.1. Focal Stroke 5 1.2.2. Hemorrhagic Stroke 6 1.2.3. Global stroke 7 1.2.4. The Ischemic Cascade: Focus on ROS/RNS 7 1.2.5. Reperfusion Injury 11 1.2.6. Stroke Evolution: ATP depletion to Inflammation 12 1.2.7. Therapeutic Targets for Neuroprotection 13 1.3. T H E T R A N S C R I P T I O N F A C T O R N R F 2 18 1.3.1. Keapl Represses Nrf2-mediated Antioxidant Gene Expression 20 1.3.2. Keapl is involved in Proteosomal Nrf2 Degradation 22 1.3.3. Kinase Regulation of Nrf2 23 1.3.4. Nrf2 Knockout Mice have Increased Sensitivity to Toxicity 25 1.3.5. Phenotypes resulting from disruption of related CNC transcription factors 26 iv 1.3.6. A Link Between Nr/2 Dysfunction and Disease 28 1.4. N R F 2 A S A T H E R A P E U T I C T A R G E T F O R S T R O K E A N D N E U R O D E G E N E R A T I O N 28 1.4.1. Nr/2 A Herniates Damage Cause by Inflammation 30 1.4.2. A Role for Endogenous Nrj2 in Neuroprotection 30 1.4.3. Strategies for A ugmenting Nrf2 A ctivity 32 1.4.4. Inducers of'Nrj2 and the Phase 2 Response 33 1.5. N R F 2 R E G U L A T E S T H E P H A S E 2 R E S P O N S E 35 1.5.1. Glutathione 36 1.5.2. Glutathione S-transferase 37 1.5.3. NAD(P)H:quinone oxidoreductase 37 1.6. T H E D I S T R I B U T I O N - F U N C T I O N R E L A T I O N S H I P OF N R F 2 38 1.6.1. Increased Nrf2 Activity in Astrocytes 39 1.6.2. Antioxidant Capacity of Astrocytes and Neurons: GSH Coupling 40 1.7. M O D E L I N G S T R O K E A N D N E U R O D E G E N E R A T I O N IN VITRO 41 1.7.1. Excitotoxicity 41 1.7.2. Oxidative glutamate toxicity 42 1.7.3. Ionomycin 43 1.7.4. Oxygen glucose deprivation 44 1.7.5. Metabolic inhibitors 45 1.8. M O D E L I N G S T R O K E IN VIVO 45 1.8.1. The Cerebral Vasculature 46 1.8.2. Animal Models of Focal Ischemia 47 1.8.2.1. Intra-luminal Suture Model 47 1.8.2.2. Occlusion of Distal Middle Cerebral Artery 48 1.8.2.3. Embolism Model 50 v 1.8.2.4. Photothrombotic Model 50 1.8.2.5. Endothelin-1 Model 51 1.8.3. Animal Models of Global Ischemia 52 1.9. M O D E L I N G N E U R O D E G E N E R A T I O N IN VIVO U S I N G 3 - N I T R O P R O P I O N I C A C I D 53 1.10. P R O P E R U S E OF A N I M A L M O D E L S OF S T R O K E 55 1.11. R E S E A R C H H Y P O T H E S I S A N D O B I E C T I V E S 57 C H A P T E R 2 : C O O R D I N A T E R E G U L A T I O N O F G L U T A T H I O N E BIOSYNTHESIS AND R E L E A S E B Y NRF2 E X P R E S S I N G G L I A P O T E N T L Y P R O T E C T S N E U R O N S F R O M O X I D A T I V E STRESS 59 2.1 . I N T R O D U C T I O N 60 2.2. M A T E R I A L S A N D M E T H O D S 61 2.2.1. Materials 61 2.2.2. Plasmids and Adenoviruses 61 2.2.3. Mammalian Cell Culture 62 2.2.4. Transfections and Infections 65 2.2.5. Toxicity Treatment 65 2.2.6. Determination of Neuronal Viability 65 2.2.7. Western Blot Analysis andImmunocytochemistry 67 2.2.8. Densitometry 68 2.2.9. [3H]-glutamate uptake assay 68 2.2.10. Placental Alkaline Phosphatase and Quinone Reductase Staining 68 2.2.11. Total Intracellular GSH Assay and Effluxed GSH Assay 69 2.2.12. RT-PCR and Microarray Analysis 70 2.2.13. Statistical Analysis 72 vi 2.3. R E S U L T S 72 2.3.1. Neurons express lower levels ofNrf2 protein than astrocytes 72 2.3.2. Over-expression ofNr/2 enhances antioxidant activity of neurons and astrocytes in immature cortical cultures 74 2.3.3. Microarray analysis ofNrf2 over-expressing mixed cortical cultures and enriched glial cultures 79 2.3.4. Enhancing antioxidant potential by Nrf2 over-expression protects neurons from cell death due to oxidative stress but not staurosporine-induced apoptosis 85 2.3.5. Glutathione released from ad-Nrf2 infected glial is necessary and sufficient for protecting neurons from oxidative glutamate toxicity 88 2.3.6. Small molecule inducers ofNrj2 increase neuronal survival during oxidative glutamate toxicity 92 2.4. D I S C U S S I O N 93 2.4.1. Nrf2 over-expression boosts antioxidant potential of glia: Protection of neurons from oxidative glutamate toxicity 93 2.4.2. Characterization of the Nrf2 inducible gene set in neurons and glia using microarray analysis 99 2.4.3. Small molecule inducers of ARE gene expression in brain cells 101 CHAPTER 3 : INDUCTION OF T H E N R F 2 - D R I V E N A N T I O X I D A N T R E S P O N S E C O N F E R S N E U R O P R O T E C T I O N D U R I N G M I T O C H O N D R I A L STRESS IN VIVO .103 3.1. I N T R O D U C T I O N 104 3.2. M A T E R I A L S A N D M E T H O D S 106 3.2.1. Chemicals 106 3.2.2. In vivo 3-NP dosage regimen 106 vii 3.2.3. Semi-qualitative Behaviour Scoring of Mice 107 3.2.4. Histology andFluorojade Staining 107 3.2.5. Succinate Dehydrogenase Assay 108 3.2.6. Preparation of tBHQ-supplemented diets 109 3.2.7. Enzyme Assays for brain tissue 110 3.2.8. Stereotaxic Injections Ill 3.2.9. Immunohistochemistry Ill 3.2.10. Preparation of primary glial-enriched and COS-1 cultures 112 3.2.11. Plasmids and Adenoviruses 112 3.2.12. Transfection, infection, and treatments for astrocytes and COS-1 cells 113 3.2.13. Placental alkaline phosphatase assay: 114 3.2.14. Semi-qualitative reverse-transcriptase PCR 114 3.2.15. Glutathione assay: 115 3.2.16. Western Blot Analysis andImmunocytochemistry 115 3.2.17. Nrf2-GFP localization in COS-1 cells 116 3.2.18. Data Analysis: 116 3.3. R E S U L T S 116 3.3.1. Nrf2'/' mice are hypersensitive to systemic 3-NP treatment 116 3.3.2. Striatal SDH Inhibition by 3-NP is Not Different Among Genotypes 120 3.3.3. Pre-activation ofNrf2 by tert-butylhydroquinone attenuates 3-NP toxicity in Nrf2+/', but not Nrf2'A mice 121 3.3.4. Adenoviral overexpression ofNrf2 attenuates 3-NP induced striatal lesioning in vivo. 126 3.3.5. 3-NP activates ARE-dependent gene expression in cultured astrocytes 128 3.4. D I S C U S S I O N 134 viii 3.4.1. Nrf2~ ' mice are hypersensitive to 3-NP toxicity 134 3.4.2. The small molecule inducer, tBHQ, provided Nr/2-dependent amelioration of 3-NP toxicity in vivo 135 3.4.3. Overexpression ofNrf2 protein is sufficient to provide neuroprotection in vivo 138 3.4.4. 3-NP exposure activated ARE-mediated gene expression in vitro 139 3.4.5. Conclusion 139 C H A P T E R 4 : A S M A L L M O L E C U L E I N D U C I B L E N R F 2 - M E D I A T E D ANTIOXIDANT R E S P O N S E PROVIDES E F F E C T I V E P R O P H Y L A X I S A G A I N S T C E R E B R A L I S C H E M I A IN V I V O 140 4 . 1 . I N T R O D U C T I O N 141 4 . 2 . M A T E R I A L S A N D M E T H O D S 1 4 2 4.2.1. Reagents 142 4.2.2. Astrocyte-enrichedprimary cultures 142 4.2.3. Plasmids, Adenoviruses and Transfections 143 4.2.4. Glutamate and analogue treatments 143 4.2.5. Human placental alkaline phosphatase assay 144 4.2.6. Enzyme Assays for astrocyte cell culture and brain tissue 144 4.2.7. Glutathione assay 145 4.2.8. Oxidative glutamate toxicity and assay for neuronal viability 146 4.2.9. Animals and Experimental treatments 146 4.2.10. Stroke models 148 4.2.11. Semiqualitative Neurological Scoring for Rat Ischemia-Reperfusion 149 4.2.12. Infarct Measurement 150 4.2.13. Statistical Analyses 152 ix 4 . 3 . R E S U L T S 1 5 2 4.3.1. Induction of multiple antioxidant systems in astrocyte cultures by inducers ofNr/2-dependent transcription 152 4.3.2. Intracerebroventricular infusion oftBHQ is protective in a rat model of ischemia-reperfusion 155 4.3.3. Induction of cortical glutathione levels in vivo following tBHQ treatment 160 4.3.4. Prophylactic intraperitoneal injection oftBHQ confers protection both 24 h and 1 month after ischemia-reperfusion 163 4.3.5. Basal and Inducible Phase 2 enzyme activities are suppressed in the Nrf2'^ mouse brain 166 4.3.6. Loss of Nrf2 function exacerbates cortical infarction 7 days, but not 24 h, after permanent ischemia 171 4.3.7. Loss ofNrj2 function abrogates tBHQ-mediated neuroprotection during an endothelin-1 model of ischemia-reperfusion 174 4.3.8. Extracellular glutamate induces astrocyte Nrf2 activity 178 4.4. D I S C U S S I O N 180 4.4.1. TBHQ and the blood brain barrier 180 4.4.2. Nrf2 dependency of tBHQ-mediated neuroprotection 181 4.4.3. Nrf2 activation protects the ischemic penumbra, not the stroke core 183 4.4.4. Identifying the signals that activate endogenous Nrf2 in response to ischemia 183 4.4.5. Conclusion 184 C H A P T E R 5 : G E N E R A L DISCUSSION 186 5.1. N R F 2 - M E D I A T E D U P R E G U L A T I O N OF T H E GSH S Y S T E M IS N E U R O P R O T E C T I V E : IN VITRO A N D IN VIVO E V I D E N C E 187 X 5.2. V E R S A T I L I T Y OF T H E N R F 2 - M E D I A T E D N E U R O P R O T E C T I O N 191 5.3. T H E R O L E OF E N D O G E N O U S N R F 2 192 5.4. N R F 2 I N D U C E R S A S T H E R A P E U T I C A G E N T S A N D T H E A L T E R N A T I V E S 194 A P P E N D I X 197 U B C A N I M A L C A R E C E R T I F I C A T E ( F O R C A N A D I A N I N S T I T U T E OF H E A L T H R E S E A R C H F U N D I N G ) 197 U B C A N I M A L C A R E C E R T I F I C A T E ( F O R M I C H A E L S M I T H F O U N D A T I O N F U N D I N G ) 198 R E F E R E N C E S 199 x i LIST OF T A B L E S T A B L E 2-1 . E V A L U A T I O N O F N A + - I N D E P E N D E N T L - [ 3 H ] - G L U T A M A T E IN A D - N R F 2 - I N F E C T E D I M M A T U R E C O R T I C A L C U L T U R E S 76 T A B L E 2-2. E V A L U A T I O N OF G S H - B I M A N E A D D U C T S T A I N I N G IN I N F E C T E D M I X E D C O R T I C A L C U L T U R E S 77 T A B L E 2-3 . C O M M O N N R F 2 U P R E G U L A T E D G E N E S IN G L I A L - E N R I C H E D C U L T U R E S A N D M I X E D N E U R O N A L / G L I A L C U L T U R E S 82 T A B L E 2-4. N R F 2 U P R E G U L A T E D G E N E S SPECIFIC TO G L I A L - E N R I C H E D C U L T U R E S O R M I X E D N E U R O N A L / G L I A L C U L T U R E S 84 T A B L E 3-1. E F F E C T S OF N R F 2 G E N O T Y P E O N B A S A L A N D T B H Q - I N D U C E D L E V E L S OF A N T I O X I D A N T / D E T O X I F I C A T I O N M A R K E R S IN S T R I A T U M , C O R T E X , A N D L I V E R 123 T A B L E 4 -1 . S U M M A R Y OF P H Y S I O L O G I C A L P A R A M E T E R S F O R R A T A N D M O U S E S T R O K E S T U D I E S 158 T A B L E 4-2. P H Y S I O L O G I C A L P A R A M E T E R S F O R M O U S E P E R M A N E N T S T R O K E S T U D I E S W I T H T B H Q DIET 176 T A B L E 5-1. S U M M A R Y O F T R E A T M E N T S A N D T O X I C I T Y P A R A D I G M S U S E D IN N R F 2 - M E D I A T E D N E U R O P R O T E C T I O N STUDIES 190 xn LIST OF F I G U R E S F I G U R E 1-1. N R F 2 A N D A R E - M E D I A T E D G E N E E X P R E S S I O N 21 F I G U R E 1-2. S U M M A R Y OF I M P O R T A N T F U N C T I O N A L D O M A I N S IN N R F 2 A N D K E A P I 23 F I G U R E 1-3. S U M M A R Y OF I M P O R T A N T A N T I O X I D A N T A N D D E T O X I F I C A T I O N P A T H W A Y S U N D E R N R F 2 R E G U L A T I O N 3 0 F I G U R E 1-4. S C H E M A T I C D I A G R A M OF E X T R A C R A N I A L A R T E R I E S S U P P L Y I N G B L O O D TO T H E B R A I N A N D T H E C E R E B R A L V A S C U L A T U R E 47 F I G U R E 1-5. C A R B O N B L A C K P E R F U S I O N OF R A T A L L O W S V I S U A L I Z A T I O N O F C E R E B R A L A R C H I T E C T U R E 50 F I G U R E 2-1 . C O - C U L T U R E A N D M E M B R A N E D E L I M I T E D C O - C U L T U R E 64 F I G U R E 2-2. W E S T E R N B L O T OF H E T E R O L O G O U S L Y E X P R E S S E D N R F 2 74 F I G U R E 2-3. A D - N R F 2 - I N F E C T E D C U L T U R E S E X H I B I T E N H A N C E D A N T I O X I D A N T P O T E N T I A L 78 F I G U R E 2-4. N R F 2 O V E R E X P R E S S I O N IN A S U B P O P U L A T I O N OF C E L L S C O N F E R S W I D E S P R E A D N E U R O N A L P R O T E C T I O N F R O M O X I D A T I V E G L U T A M A T E T O X I C I T Y 87 F I G U R E 2-5. N R F 2 O V E R E X P R E S S I O N I N M I X E D I M M A T U R E C O R T I C A L C U L T U R E S P R O T E C T S N E U R O N S F R O M H 2 0 2 - M E D I A T E D T O X I C I T Y , B U T N O T S T A U R O S P O R I N E - I N D U C E D APOPTOSIS . 88 F I G U R E 2-6. A S M A L L F R A C T I O N OF I N F E C T E D G L I A L C E L L S IS S U F F I C I E N T T O P R O T E C T N E U R O N S F R O M O X I D A T I V E G L U T A M A T E T O X I C I T Y 89 F I G U R E 2-7. R E L E A S E OF G S H F R O M G L I A IS B O T H S U F F I C I E N T A N D N E C E S S A R Y F O R C O N F E R R I N G N E U R O N A L P R O T E C T I O N 91 F I G U R E 2-8. N E U R O N A L P R O T E C T I O N C A N B E A C H I E V E D B Y A C T I V A T I O N OF E N D O G E N O U S N R F 2 WITH T H E U S E OF A S M A L L M O L E C U L E I N D U C E R 93 xi i i FIGURE 2-9. SCHEMATIC D I A G R A M OF G S H BIOSYNTHESIS A N D RELEASE PATHWAYS THAT M A Y BE INVOLVED WITH N R F 2 - D E P E N D E N T COUPLING OF G S H BETWEEN ASTROCYTES A N D NEURONS. 96 FIGURE 3-1. B E H A V I O R A L SCORING, WEIGHT LOSS, A N D LESIONS OF MICE WITH VARIED N R F 2 GENOTYPE DURING 3 - N P C H A L L E N G E 119 FIGURE 3-2. 3 - N P INDUCED LESION V O L U M E IS STRONGLY CORRELATED WITH BOTH B E H A V I O U R A N D WEIGHT 119 FIGURE 3-3. INHIBITION OF B R A I N S D H is NOT DIFFERENT A M O N G N R F 2 GENOTYPES 2 H AFTER ACUTE 3 - N P INJECTION 121 FIGURE 3-4. D I E T A R Y SUPPLEMENTATION OF T B H Q ATTENUATES 3 - N P TOXICITY IN N R F 2 + / ~ MICE, BUT E X A C E R B A T E S TOXICITY IN N R F 2 _ / " MICE 126 FIGURE 3-5. A D - N R F 2 INFECTED A N I M A L S DEVELOPED S M A L L E R LESIONS T H A N A D - G F P CONTROL ANIMALS FOLLOWING 3 - N P ADMINISTRATION 127 FIGURE 3-6. 3 - N P EXPOSURE INDUCES A R E - D E P E N D E N T GENE EXPRESSION IN C U L T U R E D ASTROCYTES 130 FIGURE 3-7. R T - P C R IMAGES WERE COLLECTED WITHIN THE LINEAR R A N G E OF THE P C R REACTION 132 FIGURE 3-8. 3 - N P PROMOTES N U C L E A R TRANSLOCATION OF N R F 2 - G F P 133 FIGURE 4-1 . T I M E COURSE A N D N R F 2 DEPENDENCY OF T B H Q - M E D I A T E D PHASE 2 E N Z Y M E INDUCTION IN ASTROCYTE-ENRICHED CULTURES 154 FIGURE 4-2. T B H Q STABILITY IN MINI-OSMOTIC PUMPS 155 FIGURE 4-3 . INTRACEREBROVENTRICULAR INFUSION OF T B H Q REDUCED SENSORIMOTOR DEFICIT A N D INFARCT SIZE 2 4 H AFTER ISCHEMIA-REPERFUSION 157 FIGURE 4-4. INDUCTION OF G S H IN THE CORTEX AFTER INTRAPERITONEAL INJECTION OF T B H Q . 162 xiv FIGURE 4-5. INTRAPERITONEAL INJECTION OF T B H Q REDUCED SENSORIMOTOR DEFICIT A N D STROKE D A M A G E 24 H AFTER ISCHEMIA-REPERFUSION 164 FIGURE 4-6. INTRAPERITONEAL INJECTION OF T B H Q REDUCES SENSORIMOTOR DEFICIT A N D STROKE D A M A G E 1 MONTH AFTER STROKE ONSET 166 FIGURE 4-7. R E D U C E D B A S A L G S T A N D N Q O l ACTIVITY, BUT NOT G S H CONTENT, IN THE N R F 2 " A BRAIN 169 FIGURE 4-8. EFFECT OF DIETARY T B H Q ADMINISTRATION ON B R A I N A N D LIVER G S H CONTENT A N D PHASE 2 E N Z Y M E ACTIVITY 170 FIGURE 4-9. Loss OF N R F 2 FUNCTION IN VIVO INCREASED CORTICAL D A M A G E AFTER P E R M A N E N T ' F O C A L ISCHEMIA 173 FIGURE 4-10. EFFECT OF DIETARY T B H Q ADMINISTRATION O N P E R M A N E N T F O C A L ISCHEMIA. . .175 FIGURE 4-11 . T B H Q - M E D I A T E D PROTECTION OF CORTICAL TISSUE F R O M TRANSIENT ISCHEMIA IS LOST IN NRF2" / _ MICE 178 FIGURE 4-12. E X T R A C E L L U L A R G L U T A M A T E INDUCES GLIAL N R F 2 ACTIVITY 179 xv LIST OF A B B R E V I A T I O N S 3-NP = 3-nitropropionic acid a-GCS = a-glutamylcysteine synthetase A N O V A = analysis of variance ARE = antioxidant response element ASIC = acid sensitive ion channel ATP = adenosine triphosphate B H A = butylated hydroxyanisole BSO = L-buthionine sulfoximine C a + + = calcium cAMP = cyclic adenosine monophosphate C D K = cyclin dependent kinase cDNA = complementary deoxyribonucleic acid COS-1 = african green monkey kidney cell line CSF = cerebral spinal fluid Cu/Zn SOD = copper/zinc superoxide dismutase CREB = cyclic A M P / C a + + response element binding protein DMSO = dimethyl sulfoxide DNA = deoxyribonucleic acid DTNB = 5,5'-Dithiobis(2-nitrobenzoic acid) EDTA = ethylene diamine tetraacetic acid EGTA = ethylene glycol-bis(2-aminoethylether)-A',Ar,Ar',A7'-tetraacetic acid ET-1 = endothelin-1 FUDR = fluorodeoxyuridine/uridine xvi GFP = green fluorescent protein GSH = glutathione (reduced) GSSG = glutathione (oxidized) GST = glutathione S-transferase HBSS = Hank's/HEPES-buffered salt solution HEK293 = human embryonic kidney cell line HIF1 = hypoxia-inducible factor HO-1 = heme oxygenase-1 H 2 O 2 = hydrogen peroxide L D H = lactate dehydrogenase M A P K = mitogen activated protein kinase M C A = middle cerebral artery MCAo = middle cerebral artery occlusion mcBi - monochlorobimane M E M = minimal essential media mM = millimolar [xM = micromolar |xm = micrometer Mn SOD = manganese superoxide dismutase mRNA = messenger ribonucleic acid MRP = multidrug resistance protein N A D H = nicotinamide adenine dinucleotide (reduced) NADPH = nicotinamide adenine dinucleotide phosphate (reduced) NAIP = neuronal apoptosis inhibitor protein N M D A = N-methyl-D-aspartate xvii NO = nitric oxide NOS = nitric oxide synthase NQOl = NAD(P)H:quinone oxidoreductase O2* = superoxide anion OH' = hydroxyl radical ONOO" = peroxynitrite OGD = oxygen glucose deprivation PAP = placental alkaline phosphatase PBS = phosphate buffered saline PCR = polymerase chain reaction PDC = L-pyrrolidinedithiocarbamate PKA = protein kinase A PKC = protein kinase C rCBF = regional cerebral blood flow RNA = ribonucleic acid RT-PCR - reverse transcriptase polymerase chain reaction ROS/RNS = reactive oxygen/nitrogen species SDH = succinate dehydrogenase SDS-PAGE = Sodium dodecyl sulfate - polyacrylamide gel electrophoresis SEM = standard error of the mean tBHQ = tertiary butylhydroquinone TRP = transient receptor potential channel TTC = 2,3,5-triphenyl-tetrazolium chloride xCT = cystine-glutamate antiporter (system xc~) xviii A C K N O W L E D G E M E N T S I am grateful to have begun my career with Dr. T im Murphy. His support and guidance as a mentor has helped me realized how exciting it is to discover something for the first time, whether the finding is small or large. Dr. Murphy's positive drive, energy, curiosity, and patience wil l be traits that I w i l l strive for in the future. I owe a debt of gratitude to my committee members, including Dr. Max Cynader, Dr. Wolfram Tetzlaff, and Dr. Steve Vincent, who have taken time out of their busy lives to lead me in the right direction with their advice and insights. I was lucky to have all that brains in one room at one time. I would like to thank the all the friends that I have acquired during this degree. A l l the members of the Kinsmen Lab: Dr. Lynn Raymond, Dr. Alaa el-Husseini, Ping-a-ling, Shawn, Sabrina, Doctor Brown, The Janitor Guy (Ian), BigfeetZhi, Herbs, John, Laureny, Gautum, Sophie, Vilte, Carrie, Bippen, David, Ferman Hernandes, Austen Powers, Layvan, Jing, Cath, Mandolfini Fantastico, Christine "The Meat-asaurus" Sutton, L i l y , Esther, Tao, Sister Marie-Francis, Kunnie, Sexy Cat, J-Lo, Hamster, Kangaroo, Rochie, Rick, Rujun, Alex , Cindy, Ron-wen, Gluteus minimus (Vij), Brebs, Kitty kat, Soyon, Christina (monkey), Julie, Carine, Giada, Fred, Pascale, Joseph, and all my friends at the Brain Research Centre, and the dedicated staff of the Animal research unit and Pathology. Last but not least, I would especially like to thank those closest to me, my dear family, Jeanie (mum), Steve (pops), Evan (bro), Yuan (gramps), Jean (grandma), all the uncles and aunts, Frank, and Foxy Fiona for their unwavering support during my studies. It would not have been possible without you. xix C O - A U T H O R S H I P S T A T E M E N T For the work described in Chapter 3, "Induction of the Nrf2-driven antioxidant response confers neuroprotection during mitochondrial stress in vivo ", I performed more that 80% of the bench-work (with the aid of our technicians and a rotation graduate student Vil te Barakauskas) and 100% of data analyses for Nrf2 knockout mice studies and cell culture experiments. I prepared and revised the figures and manuscript with feedback from the senior author and co-first author. The co-first author (S. Imbeault, M.Sc . student) contributed the results for the in vivo viral infection studies in Figure 3-5. A l l other figures resulted directly from my experiments. xx Chapter 1 : GENERAL INTRODUCTION 1.1. Oxidative Stress and the Brain Free radicals are broadly defined as molecules that are capable of independent existence but are unstable and reactive because they contain one or more unpaired electrons within an atomic orbital (Halliwell and Gutteridge, 1999). Free radicals are continuously produced in biological systems as a result of normal and abnormal cell metabolism. They exert their effects either as oxidizing agents, stealing electrons from other non-radicals, or as reducing agents, donating electrons to non-radicals. In both cases, a non-radical is transformed into a free radical since it is left with an unpaired electron. With continuous electron transfer between radicals and non-radicals, a chain reaction is created that can cause damage to nearly all macromolecules within the cell, including DNA, proteins and lipids by altering their function or structure altering structure and interfering with biological reactions and functinos (Coyle and Puttfarcken, 1993; Halliwell and Gutteridge, 1999). This chain reaction can be broken when two free radicals eventually react with each other, or through reactions with electron donating molecules such as glutathione (GSH) and N A D P H , a process usually catalyzed by antioxidant enzymes. Fortunately, cells possess a complex antioxidant defense system composed of small molecules antioxidants (i.e. GSH and vitamin E) and enzymes to maintain redox homeostasis by balancing the toxic effects of free radicals (Halliwell and Gutteridge, 1988; Prestera et al., 1993; Pahl and Baeuerle, 1994). However, when the antioxidant defense of the cell is unable to neutralize free radical toxicity (for example, during disease or aging), this shift toward an oxidative environment (oxidative stress) can cause cell death (Halliwell and Gutteridge, 1984; Coyle and Puttfarcken, 1993; Jacobson, 1996; Halliwell and Gutteridge, 1999). I Free radicals generated from oxygen and nitrogen are particularly common in biological systems (reactive oxygen/nitrogen species, R O S / R N S ) . The major types and sources of ROS/RNS are briefly discussed below, but please refer to more comprehensive reviews for further information (Coyle and Puttfarcken, 1993; Hal l iwel l and Gutteridge, 1999; Love, 1999). ROS encompass many reactive molecules primarily including the superoxide anion ( O 2 ' ) , hydrogen peroxide ( H 2 O 2 ) , and hydroxyl radical (OH') (Hall iwell and Gutteridge, 1999). O 2 " is considered the "mother" of other R O S and predominantly arises from the mitochondrial electron transport chain by leakage of a small proportion of free electrons directly on to O 2 while passing electrons along the earlier components of the chain (Turrens, 1997). Thus, O2" can be a byproduct of normal cell metabolism. During excitotoxicity (discussed below), 0 2 ' production from the electron transport change can be augmented (Luetjens et al., 2000). Other sources of O2" include xanthine oxidase, auto-oxidation of some biologically important molecules (i.e. glyceraldehyde, F M N H 2 , F A D H 2 , L-dopa, dopamine, and thiol compounds such as cysteine, transition metal ions) (Coyle and Puttfarcken, 1993; Hal l iwel l and Gutteridge, 1999; Love, 1999), decomposition of oxyhemoglobin (Balagopalakrishna et al., 1996), and inflammatory cells (i.e. respiratory bursts of neurotrophils) (Henderson and Chappel, 1996). The dismutation of 0 2 *by superoxide dismutase converts O2* to H 2 O 2 (Hall iwell and Gutteridge, 1999). H 2 O 2 is not a classical free radical (it has no unpaired electrons) and is only a weak oxidizing/reducing agent. However, it is highly toxic because it can be further transformed into OH", via the Fenton reaction, a process catalyzed by F e 2 + . O H ' is the most reactive R O S and is therefore highly toxic to cells. The H 2 O 2 molecule itself also exerts its toxic effects by selectively inactivating enzymes such as glyceraldehyde-3-phosphate dehydrogenase, an enzyme of the glycolytic pathway, or by oxidizing keto-acids such as pyruvate (Brodie arid Reed, 1987). 2 The gaseous second messenger nitric oxide (NO*) has an unpaired electron and is categorized as a free radical . N O is a readily diffusible between and within cells and has several physiological roles in the nervous system, vascular system, as well as other organs of the body (Zigmond, 1999). For example, in the nervous system, N O is produced by neuronal nitric oxide synthase (NOS) in response to normal excitatory neurotransmission as well as over-excitation (Bredt and Snyder, 1990; Hope et al., 1991; Sattler et al., 1999). N O can then diffuse and act on various targets distant from its source of production (i.e. soluble guanylate cyclase) to modulate neurotransmission and synaptic plasticity (Vincent, 1994). In the vascular system, N O produced by endothelial N O S is important for regulation of blood pressure by controlling vasodilation. N O produced from the inducible N O S isoform is used by inflammatory cells to combat invading pathogens. N O can be converted into more reactive free radicals. A reaction between O2' and N O produces the R N S peroxynitrite ( O N O O ) (Bartosz, 1996; Hal l iwel l and Gutteridge, 1999). Peroxynitrite is a powerful oxidant can cause cell damage in multiple ways including lipid peroxidation, inactivation of enzymes and ion channels via protein oxidation and nitration, and inhibition of mitochondrial respiration (Heales et al., 1999; Love, 1999; Virag et al., 2003). In addition, nitric dioxide ( N 0 2 ) is formed when N O is exposed to O2, which is a R N S even more toxic than N O (Halliwell and Gutteridge, 1999). Environmental exposure to pollutants is also a major source of R O S / R N S production leading to injury or disease, and cells have evolved antioxidant defenses for protection from a wide assortment of xenobiotics (Hayes and Pulford, 1995). Exogenous sources and triggers for ROS/RNS production include: photochemical oxidants (ozone and nitrogen oxides), xenobiotics and pro-carcinogens (metabolized to electrophiles in the body), radiation, and ultraviolet light (Halliwell and Gutteridge, 1999; Talalay, 2000). 3 Of all the organs within the body, the equilibrium between oxidant and antioxidant molecules is perhaps most important in the brain. The brain undergoes rapid energy metabolism and consumes 20% of the oxygen taken into the body, yet represents only 2% of the total body weight (Zigmond, 1999). Oxidative stress is an injury mechanism central to pathological conditions of the brain such as stroke, neurodegeneration, or trauma (Coyle and Puttfarcken, 1993; Love, 1999; Lewen et al., 2000; Chan, 2001). These conditions can augment ROS/RNS production from a host of sources including dysfunction or metabolic inhibition of mitochondria, over-activity of NOS, arachadonic acid metabolism, xanthine oxidase, and monoamine oxidase (Coyle and Puttfarcken, 1993). Furthermore, neurons are highly vulnerable to damage caused by ROS/RNS. Neuronal membranes contain high proportions of polyunsaturated fatty acids, which are easily oxidized, resulting in a chain of oxidation-reduction reactions that propagate between lipid molecules (i.e. lipid peroxidation) (Halliwell and Gutteridge, 1999). As a result, the plasma membrane becomes rigid and leaky and neurons lose properties that are essential for their function (i.e. membrane ion gradients for propagation of action potentials). Neurons are also particularly vulnerable to oxidative stress because they express ionotropic glutamate receptors, which can initiate excitotoxicity and other forms of ion imbalance when over-active (Rothman and Olney, 1986; Choi, 1987; Choi et al., 1987; Rothman and Olney, 1995). The accumulation of Ca + + during excitotoxicity leads to the activation of Ca++-dependent ROS-producing enzymes, NO production to toxic levels, and mitochondrial dysfunction causing further oxidative stress and bioenergetic failure (Lipton, 1999; Sattler et al., 1999). Neurons also have a weaker antioxidant defense for handling oxidative stress, compared to glia and vascular cells (discussed below) (Raps et al., 1989; Makar et al., 1994; Lucius and Sievers, 1996). Given the inherent sensitivity of the brain to oxidative damage, a great amount of research has focused on understanding how oxidative stress is generated and controlled during pathologies such as stroke, in order to identify therapeutic interventions for neuroprotection. 4 Since our approach to this problem has focused on the use of in vitro and in vivo models that mimic aspects of human stroke, in the following sections we will briefly discuss of how various forms of stroke are thought to cause brain tissue damage. 1.2. Stroke: A Brain Attack In 2004, approximately 50,000 Canadians were hospitalized due to stroke (Heart and Stroke Foundation of Canada). Although stroke is a leading cause of death in North America, a large proportion of patients survive its devastating effects (-80%) and require further rehabilitation or long-term care to overcome disabilities (Mayo et al., 1999). Stroke is not only a great cost and burden for the economy (Moore et al., 1997), but also for the affected individual and family members. Recent advances in our understanding of why stroke occurs and the mechanisms underlying ischemic brain injury suggest that it is a preventable and treatable disease. Stroke refers to a cascade of events resulting from transient or permanent interruption of blood flow to brain tissue (i.e. ischemia) (Gilroy, 2000). Blood flow can be lost due to clot formation within the blood vessel, hemorrhage (rupture of a weak blood vessel or aneurysm), or heart failure. Each type of stroke can trigger a different pattern of tissue injury resulting in different functional'behavioural outcomes (Gilroy, 2000). The following sections briefly discuss different types of stroke based on what is known from both human clinical studies and animal models. 1.2.1. Focal Stroke Local thrombosis, the most common type of human stroke (80% of stroke cases), can be caused by atherosclerosis within the blood vessel or lodging of an embolism from peripheral circulation (Gilroy, 2000). This type of stroke generates a heterogeneous reduction of blood flow 5 in a localized brain region. The center of a focal stroke (stroke core) can exhibit less than 20% of initial blood flow and cells rapidly die through necrosis due to severe ischemia (Tamura et al., 1981; Nedergaard et al., 1986). This is an area of the stroke that is unsalvageable except by rapid reperfusion within minutes. The stroke core is surrounded by a penumbra where gradations of sub-optimal blood flow only partially impede cell metabolism (Dirnagl et al., 1999). In contrast to the stroke core, cells within the penumbra have access to limited glucose and oxygen, and die more slowly through an active apoptotic process (Ginsberg and Pulsinelli, 1994; Hossmann, 1994a; Lipton, 1999). Most neuroprotective strategies have been designed to salvage neurons in the penumbra, which are not lethally damaged, and could potentially aid in the recover of normal brain function (Dirnagl et al., 1999). Neuroprotective treatment would need to be implemented quickly, however, because the penumbra collapses with time and contributes to the expanding lesion (Garcia et al., 1993; Zhang et al., 1994; Hossmann, 1996; Dirnagl et al., 1999). 1.2.2. Hemorrhagic Stroke Hemorrhagic stroke accounts for ~ 20-30%> of stroke cases and is the most difficult to treat. Hemorrhagic transformation of ischemic cerebral injury occurs commonly in embolic strokes and most often results from vascular malformations caused by sustained hypertension. Other factors leading to intracerebral hemorrhage include traumatic brain injury, amyloidal angiopathies in eldery, coagulation disorders, bleeding from brain tumours, and drug abuse (Broderick et al., 1999). Hemorrhage can also manifest after the onset of an initial non-hemorrhagic ischemic stroke due to augmented collateral circulation into the ischemic zone, which may already have a compromised blood brain barrier (Lyden and Zivin, 1993). Intracerebral hemorrhage typically leads to injury caused by not only metabolic inhibition but also from release of blood constituents (i.e. serum glutamate, complement factors, and iron) into the parenchyma (Lyden and Zivin, 1993). Due to increased risk of hemorrhage and exacerbation 6 of injury, there is great concern for treating ischemic stroke with thrombolytic drugs (i.e. TPA, one FDA-approved stroke therapy) (Patel and Mody, 1999). If the hemorrhage can be contained, neuroprotective drugs can then be a useful therapy to treat this form of stroke. 1.2.3. Global stroke Although less common among strokes, global ischemia is a serious condition that can arise during heart failure. Short periods of global ischemia has severe consequences because to the brain uses all available oxygen within seconds (Safar, 1993). Furthermore, cellular glucose/ATP stores are exhausted within minutes, despite transition from aerobic to anaerobic metabolism (i.e. creatine recycling) (White et al., 1993; Sims and Zaidan, 1995). Prolonged global ischemia can be particularly fatal since all major areas of the brain are affected including centers necessary to control function of the autonomic nervous system, such as the hypothalamus (Lipton, 1999). However, permanent neuronal damage can still occur even if successful resuscitation is performed within minutes. Global ischemia results in a very specific pattern of cell death within the brain (Lipton, 1999). Notably the CAI region of the hippocampus (necessary for memory-related tasks), striatum and layers 2 and 5 of the cortex are particularly vulnerable to ischemia and are the first to be damaged (Cervos-Navarro and Diemer, 1991). The majority of these neurons are lost due to delayed active cell death (i.e. apoptosis). 1.2.4. The Ischemic Cascade: Focus on ROS/RNS Stroke is a complex injury that involves many mechanisms of cell death including excitotoxicity, apoptosis, necrosis and inflammation. The molecular basis of stroke begins with the metabolic and homeostatic shutdown of neurons and glia due to the inability to generate adequate levels of ATP (Dirnagl et al., 1999; Lee et al., 1999). The brain relies heavily on oxidative phosphorylation to derive energy and a sudden loss of glucose and oxygen strongly 7 influences the ATP pool. A series of events mark the eventual death of neurons in the affected area of the brain. The ensuing breakdown of ionic gradients across the plasma membrane leads to widespread depolarization of neurons and glia resulting in an uncontrolled release of synaptic glutamate (Benveniste et al., 1984; Novelli et al., 1988). The loss of membrane potential also shuts down or even reverses Na'-dependent glutamate transporters exacerbating extracellular glutamate accumulation (Rossi et al., 2000). This increase of extracellular glutamate initiates a toxic cascade called "excitotoxicity" (Olney et al., 1986; Rothman and Olney, 1986; Choi, 1987; Choi et al., 1987; Haddad and Jiang, 1993). During excitotoxicity, excessive extracellular glutamate leads to over-activation of neuronal ionotropic glutamate receptors (N-methyl-D-aspartate (NMDA)-type and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type) and a massive accumulation of cytosolic C a + + from the extracellular fluid and intracellular stores (Benveniste et al., 1988; Dingledine et al., 1999). Mitochondrial dysfunction follows intracellular C a + + overload leading to further exacerbation of ATP depletion and triggering of the intrinsic apoptotic cascade (Sciamanna et al., 1992; Zamzami et al., 1996). Further over-activation of the pro-apoptotic protein poly (ADP-ribose) polymerase (PARP), which exhaustively repairs D N A damage during apoptosis, inadvertently depletes cellular N A D H and ATP content, consequently augmenting bioenergetic failure and increasing vulnerability to oxidative damage (Eliasson et al., 1997). In the event of blood reperfusion, the reintroduction of oxygen to already dysfunctional mitochondria may cause a burst of ROS generation (reperfusion injury discussed below). In addition, the activation of Ca++-dependent cyclooxygenases and phospholipases contribute to the ROS/RNS accumulation. In the end, ROS/RNS will contribute to lipid peroxidation, protein and D N A damage (Braughler and Hall, 1989; Hall and Braughler, 1989; Siesjo et al., 1989; Hall and Braughler, 1993). N M D A receptors are an important component to neuronal excitotoxic death (Choi et al., 1987). Since N M D A receptor activation was detected in cerebral ischemia and blockers of 8 ionotropic glutamate receptor and second messenger pathways associated with excitotoxicity were highly neuroprotective in in vitro stroke models, it was argued that N M D A receptor antagonists could be of therapeutic value for stroke (Benveniste et al., 1984; Simon et al., 1984; Benveniste et al., 1988; Novelli et al., 1988). However, multiple trials to block excitotoxicity in human clinical stroke trials have been unsuccessful to date (Siesjo and Bengtsson, 1989; Benveniste, 1991; Gido et al., 1994; Hossmann, 1994b), questioning the role of excitotoxicity in human ischemic injury (see section 1.2.7). Thus, understanding alternative pathways leading to neuronal death are now of particular interest. Recent advanced have identified a role for transient receptor potential channels (TRP channels) in anoxia-induced neuronal death. Tymianski and colleagues have shown that TRPM7 (possibly also TRPM2) channels expressed on neurons account for a slow activating, outwardly rectifying non-selective cation conductance that occurs following ionotropic glutamate receptor mediated cation influx (Aarts et al., 2003; Aarts and Tymianski, 2005). TRPM7 conductance could not be inhibited by conventional glutamate receptor blockers, but could be blocked by low concentrations of G d 3 + . Furthermore, TRPM7 conductances were upregulated by decreases in extracellular C a + + concentration and increases in intracellular ROS (i.e. N M D A receptor-mediated NO free radical production); two conditions that are known to occur during stroke (Aarts et al., 2003). Interestingly, C a + + entry via the TRPM7 channel further augmented ROS production forming a damaging positive feedback loop. Thus, the T R P M family channels possess many characteristics suggesting a role in delayed non-excitotoxic C a + + influx that could promote neurodegeneration. Indeed, siRNA knockdown of TRPM7 expression blocked delayed C a + + uptake in a neuronal culture system and conferred resistance to cell death during oxygen glucose deprivation, suggesting that TRPM7 inhibition may be a viable therapeutic strategy for stroke (Aarts et al., 2003). Increased C a + + influx leading to neuronal death may also occur via acid-sensing ion channels (ASICs) (Xiong et al., 2004). During ischemia, low oxygen availability causes the brain 9 to switch to anaerobic glycolysis producing lactic acid as a by-product, consequently lowering pH levels in the affected areas. ASICs are uniquely activated by low pH levels in the physiological range (pH half max: 4.4 - 6.5), depending on the subunits that form the channel (Waldmann et al., 1997b; Waldmann et al., 1997a; Waldmann et al., 1999; Krishtal, 2003). ASICs are upregulated during ischemia and contribute to C a + + induced neuronal injury (Xiong et al., 2004; Gao et al., 2005). Blockade of ASIC conductance using either the non-specific ASIC inhibitor amiloride, or ASIC la-specific inhibitor psalmotoxin 1, was found to be neuroprotective in both cell culture and in vivo models of stroke. Similarly, cells with ASIC knockdown were resistant to oxygen glucose deprivation, but sensitivity could be re-established with exogenous ASIC expression (Xiong et al., 2004). Reduced pH during ischemia can also increase neurotoxicity by augmenting iron availability for the Fenton Reaction, leading to free radical production (Goldman et al., 1989). Further, decreased intracellular pH has the potential to increase N a + / H + pump activity leading to cellular depolarization (Piper et al., 1996). It is thus very surprising that a number of studies have shown, quite paradoxically, that lowered pH is neuroprotective in vitro and in vivo, rather than neurotoxic (Giffard et al., 1990; Tang et al., 1990; Traynelis and Cull-Candy, 1990). The mechanism(s) underlying this neuroprotection are not fully elucidated. However, N M D A receptor inhibition may be involved, since N M D A receptors contain a pH-sensitive site reduces Ca + + influx in response to extracellular H + . However, other possibilities still remain because MK-801 application and acidity have additive neuroprotective effects (Kaku et al., 1993). Finally, it is important to point out that in developing strategies for neuroprotection, our approach should not be too neuron-centric. In addition to neurons, other components of the neurovascular unit (astrocytes, microglia, oligodendrocytes, vascular smooth muscle) are also vulnerable to ischemia in different ways (Lo et al., 2003). For example, white matter composed of axonal cylinders, oligodendrocytes, and myelin are uniquely sensitive to excitotoxicity (Stys, 10 2004). Interestingly, studies by Stys and colleagues have shown that oligodendrocyte cell bodies are sensitive to intracellular C a H accumulation via A M P A receptors, while the myelin sheaths respond to overactivity of N M D A receptors (Micu et al., 2006). These findings highlight the need to develop treatments for white matter injury during stroke, perhaps using different strategies than those developed to protect neurons. 7.2.5. Reperfusion Injury A further dimension to stroke injury is the effect of blood reperfusion during transient ischemia (reperfusion injury). Although early reperfusion (within 1 h) reduces infarct size, delayed reperfusion (several hours after stroke onset) paradoxically exacerbates stroke injury (Astrup et al., 1977; Schaller and Graf, 2004). Many factors contribute to reperfusion injury. Initially, reperfusion can stress already damaged blood vessels in severely affected regions, leading to extravasation of blood. The resulting edema and hemorrhage can trigger necrotic cell death. Reperfusion also facilitates the infiltration of leukocytes (attracted by inflammatory mediators such as interleukin 1(3) that in interaction with erythrocytes and platelets lead to capillary clogging (Engler et al., 1983). Trapped leukocytes contribute to neuronal injury by releasing proteases and ROS/RNS (Fabian et al., 2000). Perhaps most important, the sudden influx of oxygen with reperfusion causes a burst in cellular metabolism and enzyme activity that leads to increased ROS/RNS generation. Mechanisms behind ROS/RNS production during reperfusion include: 1) over-activity of dysfunctional mitochondrial electron transport chain leading to pre-mature shunting of electrons to molecular oxygen to produce O2' (Love, 1999), 2) transformation of xanthine dehydrogenase into xanthine oxidase, a major source of O2' and H2O2 (Beckman, 1991), 3) increased activity of N A D P H oxidase in immune cells and endothelium, an enzyme that produces O2' and H 2 0 2 to be used as a defense against invading 11 microbes (Walder et al., 1997), and 4) redox recycling of iron released from extravasated red blood cells (Davalos et al., 1994). Confirming the central role of ROS/RNS-mediated damage, many studies have found that reperfusion injury is effectively reduced by treatment with antioxidant therapies (Horakova et al., 1997; Cadenas and Davies, 2000) . Interestingly, increased oxidative stress during reperfusion has also been linked to aberrant activation of redox-sensitive transcription factors such as N F - K B (Nichols, 2004) . The role of N F - K B activation remains somewhat controversial since its effects can be either neuroprotective (i.e. increased expression of antioxidant enzyme, Mn-SOD and N Q O l , as well as numerous anti-apoptotic genes) or injurious (i.e. increased expression of cytokines and cell adhesion molecules promoting recruitment of inflammatory cells) (Kumar et al., 2004) . 1.2.6. Stroke Evolution: A TP depletion to Inflammation As alluded to above, a further dimension of stroke injury to consider when developing therapeutic strategies is how the injury evolves over time (Dirnagl et al., 1999; Lee et al., 1999). In initial minutes after ischemic onset, the focal stroke core rapidly becomes anoxic leading to ATP depletion and widespread cellular depolarization. The resulting ionic imbalances lead to edema causing all cells to swell and burst (essentially necrosis) (Lipton, 1999). Within the core-penumbra interface amongst cells undergoing edematous death, some neurons will be exposed to elevated levels of glutamate leading to initiation of the excitotoxic cascade (Choi et al., 1987). These neurons will undergo apoptotic cell death, which unfolds over several hours to days. Also during this period, spreading waves of depolarization repeatedly emanate from the stroke core (1-3 events per hour post-stroke), a phenomenon referred to as spreading depression (Hossmann, 1996; Obeidat and Andrew, 1998). These waves may further propagate the flow of glutamate and other toxic molecules from the stroke core in the penumbra. With each successive wave, the viable regions within the penumbra recede until the stroke is a contiguous mass of dead tissue 12 (Lipton, 1999). Within hours after stroke, the brain and body mount an inflammatory response involving release of cytokines (interleukins and tumour necrosis factor) leading to the recruitment of inflammatory cells from circulation, as well as the local activation of microglia/astrocytes (Hallenbeck, 1996; del Zoppo et al., 2000). The inflammation process can cause further damage even weeks to days after stroke, since immune cells release toxic mediators (i.e. nitric oxide, peroxynitrite, lysosomal enzymes) as they function to remove debris and dead brain tissue, and permeability of blood vessels due to immune cell proliferation exacerbates edema. Although the inflammatory process can lead to further tissue damage after stroke, its facilitation of necrotic tissue removal is necessary for long-term remodeling of the remaining brain tissue, and is ultimately a beneficial process (del Zoppo et al., 2001; Stoll et al., 2002). Anti-inflammatory therapies for stroke are a subject of intense study and aim to ameliorate toxicity associated with earlier stages of inflammation without deterring its beneficial effects (Barone and Feuerstein, 1999). Although there is no well-defined stroke core or penumbra in global ischemia, glutamate receptor-dependent excitotoxicity, reperfusion injury, ROS/RNS production, apoptosis, and inflammation remain important factors that contribute to ischemic injury. However, injury caused by transient global ischemia may manifest over a longer time period since affected neurons die primarily by apoptosis, similar to neurons destined to die in the penumbra of a focal stroke (Lipton, 1999). 1.2.7. Therapeutic Targets for Neuroprotection Many strategies have been designed to salvage neurons balancing between apoptotic and survival pathways (i.e. in the stroke penumbra) (Barber et al., 2001; Lo et al., 2003). However, given the complexity of stroke progression it is clear that a neuroprotective strategy targeting one aspect of stroke damage will not suffice. As mentioned above, despite several excellent strategies 13 for neuroprotection, which have been successful in animal models, efficacy in human clinical trials have proven disappointing (De Keyser et al., 1999; Gladstone et al., 2002; Hoyte et al., 2004). Promising drug candidates included blockers specific for either N M D A or AMPA-type glutamate receptor, which should impede early stages of excitotoxic damage and thus control the progression of stroke damage (Simon et al., 1984; Prass and Dirnagl, 1998; Turski et al., 1998). For example, MK801 was thought to be consistently neuroprotective during pre-clinical testing in animal model, but its protective effects were later ascribed to hypothermia caused by the drug, rather than N M D A receptor antagonism itself (Buchan and Pulsinelli, 1990; Corbett et al., 1990). However, when core temperatures were maintained in subsequent studies, no neuroprotection was observed (Buchan et al., 1991; Valtysson et al., 1994). The failure of N M D A receptor antagonism has been linked to a number of other possibilities as well. Competitive N M D A R antagonists must be used at high concentrations in vivo due to the receptor's high affinity for glutamate. Although side effects are tolerable in many cases, these high concentrations have led to serious side effects including hallucinations and psychosis leading to termination of the research study. In fact, some health concerns were observed in preclinical testing (i.e. changes in blood pressure) that likely contributed to problems in clinical trials (Xue et al., 1992). Further, some N M D A R antagonists (i.e. selfotel and aptiganel hydrochloride) have led to increased mortality rates in human trials suggesting that extended N M D A R blockade can be neurotoxic (Davis et al., 2000; Albers et al., 2001). Indeed, synaptic N M D A R activity is important for neuronal survival, neurogenesis and repair, and antagonists may exacerbate injury by inhibiting beneficial N M D A R function in later stages of stroke (Bernabeu and Sharp, 2000; Arvidsson et al., 2001; Hardingham et al., 2002; Hardingham and Bading, 2003). The lessons learned from the failure of N M D A receptor antagonists led to other strategies to block the excitotoxic cascade without directly disrupting N M D A R function. For example, nimodipine, a voltage gated calcium channel blocker, has been tested with the hope of reducing 14 another mode of C a 4 + entry (American-Nimodipine-Study-Group, 1992). In addition, agonists of inhibitory receptors such as serotonin and G A B A - A receptors with the goal of suppressing excitation have also been tested (Hoyte et al., 2004). Notably, Aarts et al. used novel peptide-based drugs to disrupt interactions between the N M D A R and its postsynaptic scaffolding partner PSD-95, which uncouples NMDAR-mediated C a + + influx from nNOS activation leading to a reduction in peroxynitrite formation and increased neuronal survival in vitro and in vivo (Aarts et al., 2002). Multiple antioxidant-based drugs have also proven successful in animal models, but these efforts have not yet proven useful in clinical trials (Zhao et al., 1994; Schmid-Elsaesser et al., 1999; Green and Ashwood, 2005). Spin trapping agents such as a-phenyl-tert-butylnitrone (PBN) function by reacting with free radicals to form stable non-reactive adducts that can then be detoxified from the cell. In sufficient concentrations, spin trapping drugs are neuroprotective and have been found to reduce infarct size following both transient and permanent ischemia (Cao and Phillis, 1994; Folbergrova et al., 1995; Fevig et al., 1996; Floyd, 1997). Other spin trapping agents are currently promising therapeutic agents, such as the novel synthesized nitrone N X Y -059 (Zhao et al., 2001; Lapchak et al., 2004). As described above, inflammation is an important mediator of tissue damage in the early stages of some forms of stroke. Accordingly, drugs that possess some anti-inflammatory action, such as minocycline (Wang et al., 2002) and aspirin (Vartiainen et al., 2003), also reduce stroke damage in animal models (Barone and Parsons, 2000). However, anti-inflammatory drugs remain to be rigorously tested for human stroke. Strategies have also been designed to block the activity of proteins that are critical to the apoptotic process. Cyclin-dependent kinases are a large group of serine/threonine kinases that play important roles in regulating cell cycle progression, as well as other functions in neurons (i.e. transcription) under normal brain conditions (Gold and Rice, 1998; Ekholm and Reed, 15 2000). During stroke, the activity of some Cdk isoforms paradoxically contribute to neuronal death (Copani et al., 2001). To further examine the role of Cdks in in vivo ischemia, Park and colleagues have used the broad-spectrum Cdk inhibitor flavopiridol, and dominant-negative proteins to specifically inhibit the activity of Cdk isoforms (Osuga et al., 2000; Rashidian et al., 2005). Their findings suggest that Cdk4 and its activator cyclinDl are involved in delayed component of cell death (as seen in the CAI region during global ischemia), while Cdk5 and its activator p53 are involved in the excitotoxic component (as seen in the stroke core during focal ischemia). Thus, inhibition of Cdk activity may be a versatile strategy for achieving neuroprotection in multiple types of stroke damage- a promising approach considering the heterogeniety of strokes seen in the human population. In another method to inhibit the apoptotic process, Robertson and colleagues examined the neuronal apoptosis inhibitor protein (NAIP). NAIP acts as a potent inhibitor of effector caspases 3 and 7, cysteine-rich proteases that cleave numerous cellular targets that propagate the apoptotic process and compromise cellular integrity (Xu et al., 1997; Robertson et al., 2000). For example, caspase targets include enzymes involved in genome function (such as PARP, DNA-dependent protein kinase, U l small ribonucleoprotein and the 140 kDa component of D N A -replication complex C), regulators of the cell cycle progression (Rb, PKCd, mdm2), structural proteins (lamins, actin, fodrin, gelsonin), and a DNA-fragmentation factor (DFF) which mediates internucleosomal cleavage of D N A (Mattson, 2000). Loss of NAIP function due to mutation leads to abnormal apoptosis and has been linked to some hereditary neurodegenerative disorders. Conversely, over-expression of NAIP offers neuroprotection during in vivo stroke. Prolonged mild hypothermia (~32°C for 24 h) is highly neuroprotective when applied during or after focal or global ischemia (Busto et al., 1987; Corbett et al., 1990; Minamisawa et al., 1990; Karibe et al., 1994a; Meden et al., 1994). In animal models of stroke, hypothermic treatment is unsurpassed in terms of reproducibility, neuroprotection potential, versatility, and 16 preservation of functional outcome. The nature of this protection has been difficult to determine since many processes are likely to be affected by cooling (Colbourne et al., 1997). For example, one can speculate that hypothermia decreases synaptic glutamate release by limiting neuronal excitability in early stages of ischemia (Busto et al., 1989). With reduced neuronal activity, metabolic stores are preserved (Astrup et al., 1981), lactic acid production is lowered (Sutherland et al., 1992), and free radical generation is reduced (Karibe et al., 1994b). In later stages of stroke, hypothermia has also been shown to mitigate inflammatory responses (Goss et al., 1995). In line with the multi-faceted approach offered by hypothermia, recent research focuses have shifted to therapies involving combinations of multiple drugs targeting different stages of the ischemic cascade (Lo et al., 2003; Ovbiagele et al., 2003; Janardhan and Qureshi, 2004). A particularly attractive strategy is to target global genetic responses, which can induce diverse changes in heterogeneic cellular types to facilitate adaptation and survival to a new environment (for example, HIF-1 and hypoxia) (Bergeron et al., 1999; Sharp and Bernaudin, 2004). Individual cells throughout the body are able to respond to noxious stimuli by enhancing endogenous defenses to attenuate damage from recurring or prolonged injury. For example, a toxic environment arising perhaps from poor drug metabolism in the liver, abnormally high or low levels of oxygen, or high blood pressure, can induce an adaptive response within the affected cells involving activation of transcription factors, which coordinate expression of multiple genes required for cell survival. One striking example is the well-studied effect of the transcription factor HIF-1 in response to hypoxia. HIF-1 orchestrates the expression of vascular endothelium growth factor to enhance angiogenesis, erythropoietin to increase red blood cell growth, and numerous glycolytic enzymes to promote energy production from glucose without oxygen (Sharp and Bernaudin, 2004). Many global defensive responses are essential for survival and are thus highly conserved through evolution. For example, the redox sensitive transcription factors 17 OxyR is used by bacteria to reduce the damage cause by oxidative stress similar to defense mechanisms in higher mammals (Demple and Amabile-Cuevas, 1991; Zheng et al., 1998). Our studies have focused on the transcription factor, NF-E2 related factor (Nrf2), which is capable of modulating redox homeostasis within the brain and many peripheral organs by regulating the global expression of numerous antioxidant/detoxification genes. We hypothesize that increased Nrf2 activity will provide neuroprotection during stroke by limiting oxidative load during stroke. 1.3. The Transcription Factor Nrf2 Over 30 years ago, researchers had begun to view the detoxification of harmful agents from the body as a two-step process (Williams, 1967). Xenobiotic molecules entering the body would first be processed by Phase 1 enzymes altering their function through oxidation-reduction reactions. These reactions often gave rise to reactive electrophilic molecules that could lead to carcinogenesis, for example, by damaging DNA. However, a second wave of reactions catalyzed by Phase 2 enzymes served to conjugate Phase 1 reaction products to endogenous ligands for excretion from the body. Early studies suggested that drugs capable of selectively activating the beneficial Phase 2 response, but not the harmful Phase 1 response, could be valuable anti-cancer agents (Benson et al., 1978; Benson et al., 1979; Benson et al., 1980; Prochaska and Talalay, 1988). Ongoing work in this area has now broadened our understanding of how cells process and detoxify harmful agents. Phase 1 reactions are now known to by catalyzed largely by a family of cytochrome p450 enzymes, while Phase 2 enzymes are inducible through the binding of transcription factors (notably Nrf2) at the promotors of multiple antioxidant/detoxification enzymes (Rushmore and Kong, 2002; Holtzclaw et al., 2004). We will now discuss the role of Nrf2 and the Phase 2 response in more detail. 18 Nrf2 was first isolated a decade ago, and was well-known for its important but non-essential role in transcription during blood cell development (Andrews et al., 1993; Moi et al., 1994; Chan et al., 1996a). Nrf2 belongs to the CNC (cap 'n ' collar) family of transcription factors along with p45 NF-E2, Nr f l , Nrf3 (Andrews et al., 1993; Chan et al., 1993; Ney et al., 1993; Moi et al., 1994), and two distantly related factors, Bachl and Bach2 (Oyake et al., 1996). These transcription factor require heterodimerization with one of many small Maf proteins (MafF, MafG, MafK) (Itoh et al., 1997a), Some studies have shown that heterodimerization with other auxiliary proteins are sometimes required for modulation of transcription, such as CREB binding protein (Katoh et al., 2001), c-Jun, Jun-B, Jun-D, activating transcription factor (He et al., 2001), polyamine modulated factor 1 (Wang et al., 2001c), and peroxisome proliferator-activated receptor y (Ikeda et al., 2000). Many studies have shown that Nrf2 has primary control over a multi-faceted inducible antioxidant response, where enzymes involved in many protective mechanism are up-regulated in unison: ROS scavenging, glutathione biosynthesis, detoxification and export of xenobiotics, and NADPH generation (Fig. 1-1, 1-3) (Alam et al., 1999; Chan and Kwong, 2000; Hayes et al., 2000b; Ishii et al., 2000; L i et al., 2002; Sasaki et al., 2002; Thimmulappa et al., 2002). The family of protective genes that are regulated by Nrf2 are collectively known as Phase 2 detoxification enzymes (Talalay, 1989; Egner et al., 1994; Prestera et al., 1995). Nrf2 gene targets share a common 51 base pair D N A element, the cis-acting antioxidant response element (ARE), also known as a electrophile response element, located in the promotor region that is specifically recognized by the Nrf2 transcription complex (Itoh et al., 1997b; Venugopal and Jaiswal, 1998a; Jeyapaul and Jaiswal, 2000; Nguyen et al., 2003a). The core sequence of the ARE can now be defined as 5'-TGACnnnGC-3', where n = any nucleotide (Rushmore et al., 1991). Mutation of this sequence (GC box mutation) abolishes ARE-dependent gene expression (Nguyen et al., 1994). Structurally, Nrf2 consists of a number of Neh (Nrf2-erythroid cell-19 derived protein homology units) domains (Fig. 1-2). Neh4 and Neh5 are necessary for activation of transcription and Nehl is the D N A binding domain. Importantly, Neh2 is required for negative regulation by Keapl, a negative regulator of Nrf2 (discussed below), since deletion of this domain increases transcriptional activity (Itoh et al., 1999b). 1.3.1. Keapl Represses Nrf2-mediated Antioxidant Gene Expression Nrf2 is normally localized to the cytoplasm, tethered to an actin-bound regulatory protein, Keapl. Increased oxidative stress directly modifies key sulfhydryl group interactions in the Nrf2-Keapl complex that allows Nrf2 dissociation (Fig. 1-1, 1-2) (Itoh et al., 1999a; Itoh et al., 1999b; Dinkova-Kostova et al., 2002). Nrf2 then translocates to the nucleus and participates in the formation of a transcription complex (Itoh et al., 1997b; Marini et al., 1997; Venugopal and Jaiswal, 1998a). Therefore, Keapl has been described as an oxidative stress sensor protein uniquely positioned to control antioxidant gene expression. Keapl can be divided into five domains, NTR, BTB/POZ, IVR, DGR, and CTR (Fig. 1-2). Of particular importance is the DGR domain (double glycine repeat, also known as Kelch domain), which is necessary for interaction with Nrf2 and actin binding. In addition, functional Keapl exists as a dimer and the BTB/POZ domain is necessary for dimerization. Indeed, mutation of Serl40 in the BTB/POZ domain is sufficient to block dimerization (Zipper and Mulcahy, 2002). 20 P r o t e o s o m a l D e g r a d a t i o n t Oxidative Stress Electrophilic Nrf2 inducer PKC PERK MAPK? PI3K? N r f 2 - V Cytop Maf/X Nr t2 ARE ! Pha»e 2 Genes Heme Oxygenase-1 NAD{P)H:quinone oxidoreductase xCT (Cysfme Transporter) Glutathione Synthesis Enzymes Glutathione S-transferase 1 ' Detoxification of Xenobiotics Free Radical Scavenging Cel l Surv i va l Figure 1-1. Nrf2 and ARE-mediated gene expression. Schematic D i a g r a m o f N r f 2 d i s soc i a t i on f rom K e a p l and ac t iva t ion o f A R E media ted gene express ion i n the nucleus. A proper stoichiometric ratio of Nrf2 and Keap l protein is necessary for the inducible nature of the complex, since over-expressed Nrf2 accumulates in the nucleus, while Keapl over-expression retains Nrf2 outside the nucleus even upon exposure to inducers (Zipper and Mulcahy, 2002). The antagonizing function of Keap l in vivo has been consolidated by an important study showing that Nrf2 is primarily localized in the nucleus and Phase 2 genes are constitutively over-expressed in Keap l knockout mice (Wakabayashi et al., 2003). Interestingly, these mice do not survive past weaning due to excessive proliferation of keratinocytes in the esophagus and forestomach causing death by malnutrition. This suggests that in addition to 21 enhancing antioxidant status, Nrf2 may also play a role in cell proliferation. However, the authors showed that Nrf2 could directly activate ARE-mediated gene expression of specific genes involved in keratinocyte proliferation. Thus, Nrf2 over-activation was not indirectly affecting ROS signally that may have been involved in cell proliferation. Indeed, subsequent studies further confirmed these results showing that that Nrf2 could be activated by growth factors and, in conjunction with small Maf proteins, augment differentiation and hyperproliferation of keratinocytes in vitro (Braun et al., 2002; Motohashi et al., 2004). 1.3.2. Keapl is involved in Proteosomal Nrf2 Degradation Recent studies have further identified a role for Keapl in the degradation of Nrf2. Initial studies found that the expression of some Nrf2-regulated enzymes were augmented by proteosome inhibitors (Sekhar et al., 2002). It is now known that under basal conditions, Keapl actively targets Nrf2 for ubiquitination and proteosomal degradation (Zhang and Hannink, 2003). This process is dependent upon two cysteine residues within the IVR domain of Keapl (C273 and C288) which appear to act as an adaptor bridging Nrf2 to the Cullin3-based ubiquitin ligase (Fig. 1-2) (Kobayashi et al., 2004). The basal turnover rate in the cytoplasm is quite fast (half-life 10-30 min) (Nguyen et al., 2003b). Thus, Keapl controls Nrf2 activity by both enhancing its rate of degradation and its subcellular localization. However, Keapl independent Nrf2 degradation still exists since a component of Nrf2 degradation occurs in Keapl knockout mice. 22 Keapl Binding Transactivation Domains (i.e. CBP Binding) A Neh2 Neh4 Neh5 Maf Interaction and leucine zipper DNA binding domain / Nuclear Localization Signal Neh6 Neh1 Neh3 NRF2 NTR BTB/POZ IVR DGR (Kelch repeat) CTR 1 2 3 4 ] 5 6 Keapl Keapl Dimerization Cys273, Cys288 Actin Binding and Redox Sensor, Nrf2 Interaction Involved in Nrf2 Ubiquitination Figure 1-2. Summary of impor tan t funct ional domains in Nrf2 and K e a p l . Diag ram adapted f rom ( K w a k et a l . , 2004) . 1.3.3. Kinase Regulation of Nrf2 Nrf2 activity is also regulated by the direct or indirect actions of a number of protein kinase pathways, including the mitogen activated protein kinase (MAPK)(ERK, INK, and p38 kinase cascades), PI3 kinase (PDK), and PKC pathway (Fig 1-1) (Nguyen et al., 2003a; Chen and Kong, 2004). The involvement of the M A P K pathway in Nrf2 activation is supported by two lines of evidence: 1.) Classic Phase 2 enzyme inducers (BHA, tBHQ, and sulforaphane, described below) activate ERK2 and M E K (Yu et al., 1997; Chen and Kong, 2004). 2.) The M E K inhibitor, PD98059 and a dominant-negative version of ERK2, both abolished ERK2 activation and reduced induction of Phase 2 gene expression by inducers. Interestingly, activation of JNK, a stress activated protein kinase (responds to U V , free radicals, inflammatory cytokines) led to increased ARE-dependent gene expression (Shen et al., 2004). Similarily, the 23 p38 pathway, which is also stress activated has an inhibitory role on Nrf2 activity (Alam et al., 2000; Zipper and Mulcahy, 2000). However, Nrf2 activation by the M A P K pathway may be indirect because mutation of putative M A P K phosphorylation sites do not alter Nrf2 modulation (Zipper and Mulcahy, 2003). PI3K is an important lipid kinase controlling cell growth, differentiation, and apoptosis. A number of molecules that induce Nrf2 are capable of activating PI3K, including tBHQ, peroxynitrite, and hemin (Kang et al., 2002a; Kang et al., 2002b; Nakaso et al., 2003). PI3K has recently been linked to ARE-mediated gene expression based on two lines of evidence: 1.) PI3K blockers (wortmannin and LY294002) inhibit expression of Phase 2 genes (Lee et al., 2001b; Nakaso et al., 2003; Martin et al., 2004). 2.) Overexpression of constitutively active PI3K is sufficient to induce ARE-mediated gene expression (Lee et al., 2001b). However, it remains to be determined if PI3K modulates Nrf2 by direct phosphorylation. Although the molecular mechanism by which M A P K and PI3K pathways activate Nrf2 are complex and may be indirect, there is evidence to suggest that P K C directly phosphorylates Nrf2 and facilitates its translocation to the nucleus (Huang et al., 2000, 2002). Huang et al. found that ARE-directed transcription was activated by phorbol 12-myristate 13-acetate, but was blocked by selective inhibitors of PKC, staurosporine and Ro-32-0432 (Huang et al., 2000). Importantly, Nrf2 was directly phosphorylated by the catalytic subunit of P K C or immunoprecipitated PKC in an in vitro cell-free system (Huang et al., 2000). Phosphorylation occurred on Ser-40 of Nrf2 since the effect was abolished by substitution of serine to alanine (Huang et al., 2002). Recent studies have also found that Nrf2 is phosphorylated and activated by PRK-like endoplasmic reticulum kinase (PERK). PERK activation is critical for cell survival during endoplasmic reticulum (ER) stress (Cullinan et a l , 2003; Cullinan and Diehl, 2004), suggesting that Nrf2 activation by PERK may be a response necessary to combat ER stress. Accordingly, 24 fibroblast derived from Nrf2 knockout mice are more susceptible to ER-stress induced by glucose deprivation and tunicamycin, an antibiotic that blocks glycoprotein synthesis and disrupts protein trafficking. Interestingly, PERK activation (phosphorylation of PERK) has been observed in the mouse forebrain following ischemia-reperfusion by bilateral occlusion of the common carotid arteries (Kumar et al., 2001). It remains to be determined whether PERK activation leads to increased Nrf2 function during in vivo stroke. 1.3.4. Nrf2 Knockout Mice have Increased Sensitivity to Toxicity The importance of endogenous Nrf2 in cellular protection is clear in Nrf2 knockout mice studies. Nrf2 knockout mice are more susceptible to acetaminophen liver toxicity (Chan et al., 2001; Enomoto et al., 2001), acute lung injury caused by butylated hydroxytoluene, hyperoxia, and diesel exhaust fumes (Chan and Kan, 1999; Aoki et al., 2001; Cho et al., 2002b), benzopyrene induced gastrointestinal tumourigenesis (Ramos-Gomez et al., 2001), and cigarette smoke-induce emphysema (Rangasamy et al., 2004). Numerous studies have found that induction of the Nrf2 pathway by administration of Nrf2 inducers (see below) not only induces the Phase 2 response in vivo, but also attenuates toxicity from carcinogens in a number of peripheral organs (Kwak et al., 2001a; Ramos-Gomez et al., 2001). Importantly, Phase 2 enzyme induction was observed in Nrf2 wild-type (Nrf2 + / +) and heterozygous (Nrf2+/"), but was substantially reduced in knockout (Nrf2"A) mice, confirming the requirement of Nrf2 for the function of some electrophilic inducers (McMahon et al., 2001; Ramos-Gomez et al., 2001; Chanas et al., 2002; Thimmulappa et al., 2002). In addition, the basal levels of many Phase 2 enzymes (i.e. N Q O l , GST, Mn-SOD, catalase) are suppressed in peripheral tissues (i.e. liver, lung, gastrointestinal tract) of knockouts suggesting Nrf2 contributes to the constitutive expression of some Phase 2 genes (Itoh et al., 1997a; Chan and Kan, 1999; Chan and Kwong, 2000; Kwak et al., 2001a; McMahon et al., 2001). 25 1.3.5. Phenotypes resulting from disruption of related CNC transcription factors Nrf2 knockout mice have no obvious phenotype suggesting that Nrf2 is not essential for development and growth, but is instead critical for mounting a defensive response when the animal is challenged by injury or a toxic environment (Chan et al., 1996a). Although Nrf2 has a predominant role in induction of the Phase 2 response, other C N C transcription factors are also capable of inducing gene expression through the antioxidant response element suggesting that some redundancy in function may exist between C N C transcription factors. Studies have shown that Nr f l and Nrf2 function and expression patterns overlap significantly during development (Chan et al., 1993; Leung et al., 2003). N r f l is also capable of binding and inducing expression through the A R E (Venugopal and Jaiswal, 1996). However, N r f l plays an additional critical role during embryonic development because genetic disruption of N r f l leads to embryonic lethality (Fanner et al., 1997; Chan et al., 1998). N r f l knockout mice are non-viable due to decreased numbers of enucleated red blood cells and severe anemia. Extensive studies on cells derived from Nr f l knockout embryos show reduced basal expression of y G C S , H O - 1 , G S H peroxidase, and metallothionein 1/2, and increased sensitivity to oxidants in vitro suggesting that embryo death could be related to a compromised antioxidant defense (Kwong et al., 1999; Chen et al., 2003a). In accordance with this, N r f l and Nrf2 double mutants express lower levels of Phase 2 genes and exhibit more cell death than either single knockout alone (Leung et al., 2003). The closely related p45 N F - E 2 transcription factor is highly expressed in erythroid cells and exerts most of its function in this cell type (Andrews et al., 1993). Knockout of this protein led to increased oxidative stress in erythrocytes and abnormal megakaryocyte maturation and platelet production (Shivdasani et al., 1995). As a result, p45 N F - E 2 knockouts die from hemorrhage due to lack of platelets in circulation. 26 NrD shares strong homology with Nrf21 and Nrf2, and is enriched in the placenta, and B cell and monocyte lineage (Kobayashi et al., 1999). However, the contribution of NrD in mounting the Phase 2 response is unknown as mice with targeted NrD disruption have no obvious phenotype (Derjuga et al., 2004). Furthermore, double knockout of Nrf3 with either Nrf2 or p45 NF-E2 did not cause lethality. Nevertheless, NrO has been shown to be an activator of ARE-mediated gene expression and may have a partial compensatory role for Phase 2 gene induction during sub-optimal function of other CNC transcription factors (Kobayashi et al., 1999). In fact, recent studies by Braun et al. suggested that Nrfi could facilitate the skin wound healing process by inducing antioxidant genes in the chronic absence of Nrf2 (Braun et al., 2002). Bachl and Bach2 are also CNC transcription factors, but distantly related to Nrf2. In combination with Maf proteins both proteins are known to act as repressors of ARE-mediated gene expression (best studied with the HO-1 gene) (Oyake et al., 1996; Dhakshinamoorthy et al., 2005). Bachl is ubiquitously expressed, while Bach2 is restricted to monocytes and neuronal cells. Bachl knockout mice exhibited high constitutive levels of HO-1 expression under normal physiological conditions, strongly supporting the inhibitory role of Bachl on ARE-mediated gene expression (Sun et al., 2002). Further, increased HO-1 expression in Bachl knockouts could be suppressed by compound knockout of Nrf2, suggesting that a balance between Nrf2 and Bachl activities modulated HO-1 expression (Sun et al., 2002). Although this balance was clear in some tissues (i.e. lung, liver, heart), Nrf2 knockout had no effect on HO-1 over-expression in the thymus, suggesting that other factors can also contribute HO-1 expression (i.e. c-Fos, Fra-1) (Venugopal and Jaiswal, 1996). Interestingly, recent studies have shown that Bachl inhibition by nuclear export is equally important in Phase 2 gene induction as Nrf2 activation (Suzuki et al., 2004). 27 1.3.6. A Link Between Nrf2 Dysfunction and Disease Highlighting the role of Nrf2 in maintaining health, a number of studies have traced back specific disease states to altered Nrf2 function or gene expression. 1.) Cho et al. used genome-wide linkage analysis to identify susceptibility genes for sensitivity to hyperoxic lung injury in C57B1/6 mice (Cho et al., 2002a). In comparison with relatively resistant C3H/HeJ mice, C57B1/6 mice that were more sensitive to hyperoxic lung injury exhibited polymorphisms in lung Nrf2 mRNA. 2.) Bae et al. found that a breast cancer tumour suppressor gene BRCA1, was an activator of Nrf2-dependent Phase 2 gene expression. The authors propose that mutation of the BRCA1 gene and the resulting inhibition of Nrf2 could be a factor in development of breast cancer (Bae et al., 2004). 3.) Kirby et al. have found that mutant SOD1 acts as a transcriptional repressor of Nrf2 (Kirby et al., 2005), suggesting that motor neuron degeneration in mutant SOD1 mice could be related to decreased Nrf2 activity. These results suggest that identification of Nrf2 polymorphisms and abnormal protein interactions that affect Nrf2 function during human genetic disease will be an important area of future research (Yamamoto et al., 2004). 1.4. Nrf2 as a Therapeutic Target for Stroke and Neurodegeneration Why would Nrf2 succeed as a neuroprotective stroke therapy when other antioxidant treatments have failed (Lee et al., 1999)? As described above, oxidative stress leading to ischemic cell death involves the action of many different forms of ROS/RNS (Coyle and Puttfarcken, 1993). Over-expressing or enhancing the activity of a single antioxidant enzyme cannot neutralize a complex assault of many ROS/RNS (Duffy et al., 1998). For example, SOD transgenic mice are efficient at halting the accumulation of O 2 ' leading to protection in various stroke models, but the resulting formation of H 2 O 2 requires neutralization by parallel enhancement of glutathione peroxidase or catalase (Fig. 1-3) (Kinouchi et al., 1991; Murakami et al., 1997; Saito et al., 2003). Some studies in immature animals reported an exacerbation of 28 toxicity in these mice likely owing to the generation of OH' from H 2 O 2 fueled by the Fenton reaction (Ditelberg et al., 1996; Fullerton et al., 1998; Levy et al., 2002). Nrf2 can augment a powerful set of antioxidant enzymes that work in synergy to completely neutralize ROS to harmless products through sequential reactions (i.e. Cu/Zn SOD, glutathione peroxidase and catalase are up-regulated in parallel). Taken together, unlike administration of small molecule free radical scavenging compounds (i.e. Tirilazad, Vitamin C), the protective cascade initiated by Nrf2 has prolonged action (dependent upon the half-life of the Phase 2 enzymes) and is versatile against a wide range of electrophilic attacks. This suggests that enhancing Nrf2 may be a useful as a preventative strategy for patients with high probability for stroke. An increased basal antioxidant capacity may attenuate oxidative damage early in the ischemic cascade (mitochondrial inhibition or Ca + + overload), preserving tissue integrity until reperfusion can be established. Scavenging of Reactive Oxygen Species Fjerritlfv "T'~ o r OH"-«- H,0, B I ' E l l SliBtf ] H.,0 + 0, Glutathione Biosynthesis and Conjugation IJlIl Cys !GclcY;Gd(r| Glu-Cys I GSHsynmaso; Glu-Cys-Gly (GSH) ( X ) -Xenobiotic or ROS kGSTsi GSH-X i i GSH-X or GSH NADPH Production \MalicCnzyme; IG6RDH! Quinone Detoxification P450s „ Quinone >• Semiquinone 1e~ (reactive) Hydroquinone (non-reacllve) Glucuronidation UDP-Giucose LiDP-Glucuronic nctd • (X)- ffiGg fi-Glucuronide Heme Catabolism Heme j HO-1 Biliverdm Bilirubin (antioxidant) 29 Figure 1-3. Summary of important antioxidant and detoxification pathways under Nrf2 regulation. Shaded boxes depict enzymes that have been shown to be Nrf2 regulated by either biochemical or microarray approaches (Li et al., 2002; Thimmulappa et al., 2002; Lee et al., 2003c; Shih et al., 2003). Abbreviations: BRD = biliverdin reductase, G6PDH = glucose-6-phosphate dehydrogenase, Gclc/Gclm = Y-glutamyl cysteine synthetase (light and heavy chains), GRD = glutathione reductase, G P X = glutathione peroxidase, HO-1 = heme oxygenase 1, PRX = peroxiredoxin, TRX = thioredoxin, T R X R D = thioredoxin reductase, UDPGDH = UDP-glucose dehydrogenase, UGT = UDP-glucuronosyltransferase. 1.4.1. Nrf2 Attenuates Damage Cause by Inflammation In addition to reducing oxidative stress during acute stages of injury, maintained Nrf2 activation may also contribute to attenuation of oxidative stress during stroke-induced inflammation, which continues weeks to months after a stroke. A growing body of evidence suggests that Nrf2-regulated enzymes are induced by inflammatory factors (i.e. prostaglandins) and can in turn modulate the inflammatory response by changing the oxidative status of the cell/organ (Chen and Kunsch, 2004; Itoh et al., 2004; Zhang et al., 2004). Consistent with this idea, previous studies found that loss of Nrf2 function exacerbates carageenan-induced inflammation of the pleura (serous membrane encasing the lungs), and increases incidence of autoimmune disease (Yoh et al., 2001; Itoh et al., 2004; Lee et al., 2004). Nrf2 activation also occurs during vascular inflammation caused by non-laminar blood flow which may prevent the formation of atherosclerotic lesions (Chen et al., 2003b). As an effective dietary strategy, Juurlink and colleagues found that feeding broccoli sprouts laden with the Nrf2 inducer sulforaphane improved cardiovascular health in spontaneously hypertensive rats (Wu et al., 2004). In dissociated neuronal cultures, Nrf2 over-expression could protect from toxicity mediated by the potent inflammatory agent, platelet activating factor-1 (Lee et al., 2003a). 1.4.2. A Role for Endogenous Nrf2 in Neuroprotection Previous studies have shown that endogenous ARE-mediated gene expression is increased after brain ischemia, suggesting that Phase 2 gene induction may be a physiological 30 response for mitigating further damage caused by secondary ROS/RNS formation and inflammation. In particular, Campagne et al. have demonstrated using gel shift assays that antioxidant response element binding was increased in the ischemic cortical area within 6 h after stroke onset (Campagne et al., 2000). Furthermore, oligemia (blood flow reduction without acute tissue damage that occurs in shock, migraine, and the stroke penumbra) also activates Nrf2 (Liverman et al., 2004). Robust induction of Phase 2 enzymes such as N Q O l and metallothionein-1 (MT-1) within astrocytes, vascular endothelial cells, and pia-mater cells of the infarct border can be observed within hours-days after stroke onset (van Lookeren Campagne et al., 1999; Campagne et al., 2000; Laxton et al., 2001). The timing and location of Phase 2 gene induction suggests that activation of endogenous Nrf2 may be necessary for limiting oxidative stress in the ischemic penumbra. Recent studies have also found that Nrf2 can be activated by and promote neuroprotection from agents that model neurodegeneration in vitro and in vivo. Treatment of neurons with 6-hydroxydopamine (6-OHDA), a toxin used to produce lesions in models of Parkinson's disease, led to increased binding of the A R E in cultured neurons and in the striatum in vivo, shown using transgenic A R E reporter mice (Jakel et al., 2005). ARE-mediated gene expression was also induced in the striatum after in vivo 3-nitropropionic acid administration (Calkins et al., 2005). Interestingly, A R E activation in vivo was detected primarily in cells, perhaps activated astrocytes, immediately surrounding the lesioned brain tissue. Both tBHQ treatment and stable Nrf2 overexpression was able to attenuate cell death caused by 6-OHDA in vitro. Furthermore, a different group found that tBHQ treatment was able to protect against in vivo l-methyl-4-(2'-methylphenyl)-l,2,3,6-tetrahydropyridine toxicity which causes dopaminergic neuronal death in the striatum (Abdel-Wahab, 2005). These data suggest that Nrf2 activation may be a therapeutic strategy for neurodegenerative diseases that affect the striatum 31 such as Parkinson's and Huntington's disease (Hara et al., 2003; Cao et al., 2005; Jakel et al., 2005). 1.4.3. Strategies for A ugmenting Nrf2 A ctivity A number of strategies for augmenting Nrf2 activity have emerged in recent years, allowing the therapeutic potential of Nrf2-mediated gene expression to be tested in vivo. Our lab was the first to use adenovirus to over-express Nrf2 in dissociated brain cultures and in the striatum in vivo (see Chapters 2 and 3) (Shih et al., 2003; Shih et al., 2005b). From our studies, it was clear that viral Nrf2 over-expression was able to maximally augment Phase 2 gene expression and promote neuroprotection both in vitro and in vivo. However, further studies will be necessary to find the optimal viral system to ectopically express Nrf2 in vivo, as there are disadvantages to using adenovirus (notably, short duration of expression, increased toxicity, and inflammatory reactions) (Hermens et al., 1997). A recent study found that suppression of the negative regulator Keapl using siRNA could be used as a method to augment Nrf2 activity (Devling et al., 2005). SiRNAs may be used to knockdown sequence-specific gene expression in the in vivo brain, however, this is a relatively new technique that remains to be explored (Thakker et al., 2004). Viral and non-viral methods of delivering therapeutic siRNA molecules to the brain are currently an active area of research (Xia et al., 2002; Fountaine et al., 2005). Perhaps the most practical and proven approach to increase Nrf2 activity in vivo is to deliver low toxicity Nrf2 inducers (Talalay, 2000). Dietary intake of Nrf2 inducers, for example, is well tolerated by rodents and is able to promote Phase 2 gene expression throughout the body (i.e. liver, gastrointestinal tract, and lungs) (Hayes et al., 2000b; Kensler et al., 2000; McMahon et al., 2001; Mc Walter et al., 2004). Treatment with inducers can attenuate injury caused by various carcinogens and toxic environments (McMahon et al., 2001; Ramos-Gomez et al., 2001; 32 McWalter et al., 2004). The therapeutic potential of targeting Nrf2 as an anti-cancer strategy has led to the extensive study of relatively non-toxic Nrf2 inducing small molecules (Kensler et al., 2000; Talalay, 2000; Talalay and Fahey, 2001; Chen and Kong, 2004). Nrf2 inducers will now be discussed in more detail. 1.4.4. Inducers of Nrf2 and the Phase 2 Response Electrophilic inducers of Nrf2 fall into two categories: monofunctional and bifunctional (Talalay, 2000). Bifunctional inducers (i.e. polycyclic hydrocarbons, dioxins, azodyes, and flavones), are not considered beneficial since they induce Phase 1 enzymes (i.e. cytochrome p450s, CYP1A1 and 1A2), in addition to inducing protective Phase 2 enzymes. As mentioned above, Phase 1 enzymes can metabolize normally innocuous chemicals into highly reactive electrophilic intermediates which lead to cell damage and carcinogenesis. Phase 2 enzymes are required to detoxify the products of Phase 1 enzyme reactions through reactions involving sulfation, acetylation, glucuronidation, and GSH conjugation (Talalay, 2000). Bifunctional inducers co-regulate Phase 1 and Phase 2 genes by increasing binding of the A R E (Phase 2 genes), and also by activating the Aryl hydrocarbon receptor, which facilitates gene expression via the xenobiotic response element (Phase 1 genes)(Fisher et al., 1990; Whitlock, 1999). Monofunctional inducers can induce Phase 2 enzymes without influencing Phase 1 enzymes, and are thus desirable therapeutic agents since they can enhance protective gene expression but minimizing detrimental Phase 1 enzyme reaction products (Talalay, 2000). A number of monofunctional inducers are specific activators of Nrf2 (herein referred to as Nrf2 inducers) because they are not able to activate the Phase 2 response when Nrf2 function is blocked through a dominant-negative approach or in Nrf2 knockout mice. Nrf2 inducers enhance Phase 2 gene expression by preferentially targeting Nrf2 to bind the A R E . Nrf2 inducers come from diverse backgrounds, natural and synthetic, including flavonoids and phenolic 33 antioxidants (Rushmore et al., 1991), thiol-containing compounds such as isothiocyanates (Talalay and Zhang, 1996), l,2-dithiole-3-thiones (Kwak et al., 2001b), heavy metals (25), and heme complexes (Inamdar et al., 1996). Of particular interest are compounds proven to be of relatively low toxicity and safe for dietary consumption such as: butylated hydroxy anisole (BHA), tertiary butylhydroquinone (tBHQ), ethoxyquin, sulforaphane, resveratrol, curcumin, and oltipraz (Fahey et al., 1997; Kang et al., 2002b; Rushmore and Kong, 2002; Nguyen et al., 2003a; Petzer et al., 2003; Chen et al., 2005). The phenolic antioxidant B H A and its metabolite tBHQ are both Food and Drug Administration (FDA) approved antioxidants commonly used as food preservatives. Both B H A and tBHQ were observed to augment Phase 2 enzyme in various tissues and prevent tumour growth long before the discovery of Nrf2 (Rahimtula et al., 1982; Wattenberg, 1983, 1985). Since then, the Nrf2-dependency of tBHQ has been well-established in vitro, and it is widely used as a classical Nrf2 inducer (Liu et al., 1996). Exothyquin is currently used as a pet food preservative, but was previously used as a pesticide. Naturally occurring inducers such as sulforaphane (cruciferous vegetables), resveratrol (grape skin), curcumin (from turmeric, coloring agent in curry), organosulfur compounds (garlic), diterpenoids (green coffee bean) may be useful as a practical dietary strategy (Dinkova-Kostova and Talalay, 1999; Fahey and Talalay, 1999; Kensler et al., 2000; Chen and Kong, 2004; Chen et al., 2005). The dithiolethione, Oltipraz, an FDA-approved anti-shistosomal drug, is currently undergoing clinical trials in China to test its potential as an anti-cancer drug (Wang et al., 1999). Recently, gold-based anti-rheumatoid drugs that have been used for many years to treat arthritis have been shown to work, in part, through an Nrf2-dependent mechanism (Kataoka et al., 2001). Although gold-based anti-rheumatoid drugs can be locally injected into muscle, some versions can be taken orally to affect multiple organs. Although Nrf2 inducers are very diverse, Talalay and colleagues have found a common signal among them. In general, Nrf2 inducers are all Michael reaction acceptors making them 34 highly reactive with sulfhydryl groups (Talalay et al., 1988). They are able to induce Nrf2 either by increasing the oxidative state of the cell leading to accumulation of ROS, or by directly dissociating the Nrf2-Keapl complex independent of oxidative stress by oxidizing or alkylating key sulfhydryl residues on Keapl (Lee et al., 2001a; Johnson et al., 2002). Of the former group, the potency of molecule as a Michael reaction acceptor is related to the electrophilicity of the molecule (Talalay et al., 1988). Thus, the most potent Nrf2 inducers are also the most reactive and may be the most damaging to the cell. If Nrf2 inducers are to be used as therapeutic agents, it is of utmost importance to identify Nrf2 inducers that are of low toxicity yet still have reactivity to stimulate the Nrf2 pathway. 1.5. N r O Regulates the Phase 2 Response The expanding list of Phase 2 enzymes can be broadly categorized into functional networks: glutathione biosynthesis and utilization, free radical scavenging, xenobiotic detoxification, N A D P H production (Fig. 1-3). The elucidation of Nrf2-regulated gene networks is owed largely to microarray studies. Our lab has used Nrf2 over-expression to maximally upregulated Nrf2 targets in neuronal and glial cultures for detection by microarray (Shih et al., 2003). Johnson and colleagues have extensively mapped the Nrf2 gene set in neurons and glia, with and without tBHQ treatment, as well as in cultures derived from Nrf2 knockout mice (Lee et al., 2003c; Kraft et al., 2004). Thimmulappa et al. performed a comprehensive in vivo microarray study on the effects of sulforaphane on the small intestine of Nrf2 wild-type and knockout mice (Thimmulappa et al., 2002). For practicality, throughout our studies, Phase 2 response status was evaluated using individual enzyme assays for prototypic markers of Nrf2, including total GSH content, and the activities of glutathione S-transferase and NAD(P)H:quinone oxidoreductase, two enzymes 35 involved in xenobiotic detoxification. These markers will be referred to repeatedly in this thesis, and are thus described in detail below. 1.5.1. Glutathione GSH, a peptide composed of glycine, cysteine, and glutamate, is the major cellular antioxidant (Meister and Anderson, 1983; Dringen et al., 2000). GSH plays diverse roles ranging from cellular antioxidant defense (Meister and Anderson, 1983; Cooper and Kristal, 1997) to modulation of redox-sensitive signaling cascades (Sen, 1998; Janaky et al., 1999). In antioxidant defense, GSH can directly neutralize ROS/RNS by donating electrons, or can act as a cofactor to antioxidant enzymes such as glutathione peroxidase (uses electron donating capacity of GSH to decompose H 2 O 2 ) and glutathione S-transferases (described below). GSH also contributes to the regeneration of other intracellular antioxidants such as ascorbic acid (vitamin C)(Meister, 1992) and a-tocopherol (Liebler, 1993). GSH chelates free copper ions reducing their ability to generate ROS (Hanna and Mason, 1992). A l l enzymes involved in GSH biosynthesis have been shown to be Nrf2-regulated including, xCT cystine-glutamate exchanger, y-glutamyl-cysteine synthetase (y-GCS), and glutathione synthase (Fig. l-3)(Chan and Kwong, 2000; Wild and Mulcahy, 2000; McMahon et al., 2001; Shih et al., 2003). X C T imports cystine into the cell, which is rapidly reduced to cysteine, the rate-limiting precursor for GSH synthesis. The rate-limiting enzyme for GSH synthesis, y-glutamyl-cysteine synthetase, catalyzes the linkage of cysteine with glutamate. Finally, glutathione synthase links glycine with cysteinyglutamate to form the final tripeptide. Of note, other enzymes integral to the GSH utilization pathway are also upregulated by Nrf2, including glutathione reductase (catalyzes the reduction of oxidized glutathione) and multidrug 36 resistance protein-1 (exports reduced GSH and GSH-conjugates from the cell) (Hirrlinger et al., 2002; Thimmulappa et al., 2002; Hayashi et al., 2003). 1.5.2. GlutathioneS-transferase Glutathione S-transferases (GST) comprise a family of enzymes that neutralize reactive electrophiles by conjugating them with GSH (Fig. 1-3). GSTs target electrophiles arising from both biological oxidations (i.e. unsaturated aldehydes, quinones, epoxides, hydroperoxides arising as a consequence of intracellular oxidative stress) as well as xenobiotics (i.e. chemical carcinogens and environmental pollutants) (Hayes et al., 2005). The A R E was originally identified in the 5' flanking region of the GST Ya subunit (Friling et al., 1990a; Rushmore and Pickett, 1990). Since then many GST isoforms have been found to be under regulation by the ARE (Hayes et al., 2000a; Chanas et al., 2002; Ikeda et al., 2002). In the CNS, GSTs also reduce metabolites of catecholamine-based neurotransmitters including, dopamine, epinephrine, and norepinephrin, preventing their oxidative cycling and generation of ROS (Segura-Aguilar et al., 1997). In Nrf2 knockout mice, basal and inducible levels of GSTs are reduced in lever and intestines (Hayes et al., 2000b; McMahon et al., 2001). Knockout of the GST-Pi isoform leads to increased sensitivity to carcinogenesis (Henderson et al., 1998). 1.5.3. NAD(P)H:quinone oxidoreductase Quinones are highly reactive molecules that can arise endogenously (i.e. estrogen and dopamine-quinone)(Cavalieri et al., 2004) and exogenously (burnt organic materials such as cigarette smoke and automobile exhaust) and can cause cancer and neurodegeneration (Monks et al., 1992). Phase 1 enzymes can catalyze one-electron reduction of quinones to unstable semiquinones leading to recycling of ROS production (Halliwell and Gutteridge, 1999). NAD(P)H:quinone oxidoreductase (NQOl) is a Phase 2 enzyme that catalyzes the two-electron 37 reduction of quinones and their derivatives to hydroquinones, preventing their oxidative cycling (Figure l-3)(Talalay, 2000). N Q O l also maintains the reduced and active form of endogenous antioxidants including ubiquinone and a-tocopherylquinone (Beyer et al., 1996; Siegel et al., 1997). An A R E sequence was found within the promotor of the human N Q O l gene and is necessary for basal expression and induction in response to electrophiles (Li and Jaiswal, 1992). The action of N Q O l is an important component of Nrf2-mediated protection of neuroblastoma cells during oxidative glutamate toxicity since inhibition of N Q O l activity using the specific blocker dicumarol led to augmentation of toxicity (Murphy et al., 1991). Importantly, N Q O l knockout mice exhibit increased susceptibility to systemic menadione toxicity and benzopyrene-induced skin carcinogenesis, confirming the protective role of N Q O l in vivo (Radjendirane et al., 1998; Long et al., 2000). 1.6. The Distribution-Function Relationship of Nrf2 The distribution of Nrf2 expression in the mouse was revealed using in situ hybridization and Northern blot by Kan and colleagues (Chan et al., 1996b). Nrf2 mRNA was detected throughout development stages from embryo to adult in many tissue types. Nrf2 mRNA was expressed at particularly high levels in liver, intestines and kidney (organs involved in systemic detoxification), as well as heart and lung. On closer inspection, Nrf2 expression was enriched in distinct cell populations within these organs, presumably in cells that require higher Phase 2 enzymes expression to remove toxic metabolites and xenobiotics (for example, luminal cells of the stomach and intestine, the lining of the bronchi and alveoli and the renal tubules) (Chan et al., 1996b). In a follow-up study, McMahon et al. found a very similar distribution for Nrf2 mRNA using Northern blot on adult mouse tissues (McMahon et al., 2001). In both studies, Nrf2 mRNA was detected in relatively low amounts within the brain parenchyma. However, consistent with the detoxification role of Nrf2, brain Nrf2 mRNA expression was enriched in the meninges, 38 ventricular lining (ependymal cells) and choroid plexus, sites involved in secretion of CSF and fdtering of toxic molecules at the CSF-blood barrier (Strazielle et al., 2004). Accordingly, higher levels of known Nrf2 targets, N Q O l and xCT, are also expressed at the CSF-blood brain barrier (Murphy et al., 1998; Sato et al., 2002). McMahon et al. also found that Nrfl mRNA expression was not coordinately expressed with Nrf2 and that the levels of each varied greatly between tissue types (McMahon et al., 2001). Nrfl expression was high within the brain suggesting that both isoforms could contribute to ARE-mediated gene expression in this organ. 1.6.1. Increased Nrf2 Activity in Astrocytes To further define the role of ARE-mediated gene expression in the brain, Murphy et al. delivered a reporter construct bearing an ARE-containing promotor to dissociated cultures from cortex (Murphy et al., 2001). Interestingly, basal and inducible A R E binding could be detected in astrocytes, but not co-cultured neurons, suggesting transcription factors such as Nrf2 and Nrf l were more active in astrocytes. In contrast, constitutive cytomegalovirus promotor activity (not regulated through Nrf2) could be detected with the reporter in both cell types. The finding that ARE-mediated gene expression was restricted to the astrocyte population was confirmed upon examination of dissociated brain cultures derived from transgenic mice carrying the A R E -reporter in all cells (Johnson et al., 2002). In astrocytes, Nrf2 activity was partially responsible for basal Phase 2 gene expression (Lee et al., 2003c), but primarily important for electrophilic induction of Phase 2 genes, since induction was completely abolished in astrocytes derived from Nrf2 knockout mice (Lee et al., 2003c). Both astrocytes and neurons cultured from Nrf2 knockout mice were more susceptible to metabolic inhibition, C a + + overload, GSH depletion, and H 2 0 2 exposure (Lee et al., 2003c; Kraft et al., 2004; Calkins et al., 2005). 39 1.6.2. Antioxidant Capacity of Astrocytes and Neurons: GSH Coupling Glial cells (i.e. astrocytes) have a substantially higher antioxidant capacity than neurons (Slivka et al., 1987; Raps et al., 1989; Makar et al., 1994; Eftekharpour et al., 2000). This may be, in part, be due to a higher expression of total Nrf2 protein and ARE-associated genes in comparison to neurons (Sagara et al., 1993b; Ahlgren-Beckendorf et al., 1999; Eftekharpour et al., 2000; Murphy et al., 2001; Shih et al., 2003) (also see Chapter 2). Astrocytes are closely associated with neuronal bodies and synaptic junctions (Travis, 1994), and are thus ideally suited to support neuronal function and survival during ischemia by supplying energy substrates and scavenging ROS and waste products (Desagher et al., 1996; Kirchhoff et al., 2001). Astrocytes may promote neuroprotection by acting as "sinks" for extracellular ROS (Drukarch et al., 1998), and also by releasing antioxidant factors for neuronal consumption (Sagara et al., 1996; Dringen et al., 2000; Wang and Cynader, 2001). GSH is central to astrocyte-dependent neuroprotection and is constantly produced and released by astrocytes (Sagara et al., 1996). Specific blockade of GSH synthesis in astrocytes using the irreversible inhibitor L-buthionine sulfoximine (BSO) increases neuronal susceptibility to ROS exposure in vitro (Drukarch et al., 1997). Furthermore, depletion of GSH in the brain exacerbates ischemic injury in vivo, underscoring the importance of GSH in neuroprotection (Mizui et al., 1992). The mechanism of astrocyte to neuron thiol delivery has been extensively studied in recent years. Neurons and astrocytes uptake different precursors for GSH synthesis (Kranich et al., 1996b). Astrocytes in culture utilize cystine (disulfide dimer of cysteine) for replenishment of GSH stores. Wang et al. have shown that cystine is the predominant thiol species extracted from the blood by the brain (Wang and Cynader, 2000). Astrocytes are tightly associated with brain vasculature through endfeet processes, which may be able to uptake cystine from the blood. There is evidence to suggest that cystine and GSH can be transported across the B B B (Wade and Brady, 1981; Kannan et al., 1990). Also the finding that xCT is highly expressed at the blood-40 brain and blood-CSF barriers is also in support of role for cystine uptake from the blood (Sato et al., 2002). High uptake of cystine through xCT has also been detected in endothelial cells originating from the blood brain barrier (Hosoya et al., 2002). The functional role of xCT-mediated cystine uptake remains to be evaluated in vivo. In contrast to astrocytes, neurons preferentially uptake cysteine for GSH synthesis (Sagara et al., 1993b; Wang and Cynader, 2000; Aoyama et al., 2005). To generate reduced cysteine in an oxidizing extracellular space, Dringen et al. proposed the GSH released by astrocytes is cleaved by the ectoenzyme y-glutamyl transpeptidase producing the dipeptide cysteine-glycine (CysGly)(Dringen et al., 1999). CysGly may be further cleaved by a second ectoenzyme aminopeptidase N to release cysteine for rapid neuronal uptake (Dringen et al., 2001). GSH can then be resynthesized within the neuron. The Na+-dependent glutamate transporter EAAT3 (EAAC1) has been implicated in neuronal cysteine uptake leading to enhanced GSH synthesis both in vitro and in vivo (Himi et al., 2003; Aoyama et al., 2005). 1.7. Modeling Stroke and Neurodegeneration in vitro A complex injury such as stroke is often best studied as simplified models in vitro. A number of different cell culture models can be used to mimic components of stroke damage. This section briefly introduces the most commonly used in vitro models, some of which were employed in our studies. 1.7.1. Excitotoxicity Excitotoxicity results from an increased extracellular glutamate accumulation. During ischemia, excessive synaptic glutamate release is a direct consequence of neuronal depolarization due to ATP depletion. The classical method to model excitotoxic cascade uses mature neuron-enriched or mixed neuron/glial cortical cultures, which are transiently exposed (10 min) to a 41 range of glutamate concentrations (0 - 300 uM) in a C a + + containing buffered salt solution (Choi, 1985; Olney et al., 1986; Rothman and Olney, 1986; Choi, 1987). Early studies by Choi et al. showed that the process of excitotoxic neuronal death can be broadly separated into acute and delayed phases (Choi, 1987; Choi et al., 1987). The acute phase involves rapid Na + influx, osmotic imbalance, cell swelling and necrotic death. As described above, the delayed phase involves over-activation of N M D A and AMPA-type glutamate receptors allowing the accumulation of Na + and C a + + within the cell. The excessive influx of C a + + is further propagated by activation of voltage-gated C a + + channels as well as by intracellular stores via G-protein linked metabotropic glutamate receptors. C a + + overload initiates the intrinsic apoptotic cascade by causing mitochondrial damage and also initiates other intracellular cascades that contribute to neuronal cell death including arachidonic acid production by phospholipase A2, nitric oxide synthesis, calpain-dependent cytoskeletal breakdown, and endonuclease-dependent D N A degradation (Lipton, 1999). As described above, the cytotoxic mechanisms underlying neuronal excitotoxicity have recently broadened. Activation of TRPM7 channels is now thought to mediated the prolonged C a + + influx that is necessary to complete the apoptotic cascade during anoxic cell death (Aarts et al., 2003). TRPM7 channel conductance is augmented by reduction of the extracellular C a + + concentration and increased intracellular ROS. Thus, excitotoxicity in the classic sense (NMDA receptor over-activation), may represent early stages of the death process that generates signals necessary for TRPM7 activation and further C a + + influx leading to neuronal death. /. 7.2. Oxidative glutamate toxicity A non-excitotoxic component of glutamate toxicity leads to neuronal apoptosis by increasing oxidative stress. High levels of extracellular glutamate (mM range) compete with cystine uptake through the xCT cystine-glutamate exchanger (Bannai, 1986; Miura et al., 1992). 42 Since availability of cyst(e)ine is the rate-limiting precursor for generating GSH, several hours of glutamate exposure leads to depletion of intracellular GSH and oxidative stress ensues (Murphy et al., 1989; Murphy et al., 1990; Sagara et al., 1993a). This type of glutamate toxicity was most clearly demonstrated using immature neurons that have not yet expressed a full complement of glutamate receptors (Murphy et al., 1989; Murphy and Baraban, 1990; Murphy et al., 1990), where death of immature neurons could be blocked by antioxidants but not glutamate receptor antagonists (Bannai and Kitamura, 1980; Bannai, 1984, 1986). In mature neurons, it has been proposed that oxidative glutamate toxicity may be a component of excitotoxic cascade contributing to apoptotic neuronal death (Schubert and Piasecki, 2001). Non-neuronal cells relying on xCT for cystine uptake are also vulnerable to oxidative glutamate toxicity including oligodendrocytes (Back et al., 1998), N18-RE-105 neuro-retina hybridoma cells (Murphy et al., 1989), and PC 12 cells (Pereira and Oliveira, 1997). Interestingly, certain cancerous cells are more highly dependent upon xCT for glutathione synthesis than normal cells, and blockers of xCT-mediated cystine uptake are candidate drugs for reducing cancer growth (i.e. sulfasalazine) (Gout et al., 2001; Chung et al., 2005). Considering that oxidative glutamate toxicity is dependent upon the extracellular concentration of cystine, glutamate concentration need not be as high in vivo to produce neurodegeneration through this pathway, since cystine availability is substantially lower in vivo, with CSF levels of - 0.2 p;M (Lakke and Teelken, 1976; Murphy et al., 1990; Coyle and Puttfarcken, 1993). 1.7.3. Ionomycin The C a + + ionophore ionomycin leads to apoptotic neuronal cell death by allowing C a + + to directly permeate the plasma membrane leading to intracellular C a + + overload and apoptosis (Takei and Endo, 1994). Previous studies have found that ionomycin toxicity can mimic the 43 Ca++-dependent phase of glutamate toxicity involving delayed degeneration by apoptosis (Choi, 1985, 1987; Castillo and Babson, 1998). 1.7.4. Oxygen glucose deprivation Oxygen glucose deprivation (OGD) is a widely used in vitro model for stroke (Gwag et al., 1995; Newell et al., 1995; Plesnila et al., 2001). For this paradigm, mature cortical cultures are placed in an anoxic incubator in oxygen depleted (N 2 purged), glucose-free medium (Gwag et al., 1995). Reperfusion can be simulated by replacing glucose-containing medium and restoring cultures to normoxic culturing conditions. Oxygen glucose deprivation typically produces both acute necrotic neuronal cell death and slowly developing apoptosis, similar to excitotoxicity (Gwag et al., 1995). This biphasic cell death mechanism more accurately reflect the in vivo situation during stroke, in contrast to the predominantly apoptotic cell death induced by ionomycin (Takei and Endo, 1994). OGD can also be performed on organotypic and acute brain slices, where specific patterns of cell death (i.e. C A I hippocampal lesions) can be created in a highly reproducible manner, similar to the in vivo situation (Newell et al., 1995). OGD on brain slices is also advantages in many circumstances since it permits the study of relatively intact brain structure in vitro, while allowing full control of temperature, ionic composition of the bath, and simple application of drugs. Complete deprivation of oxygen and glucose causes more severe neuronal damage (i.e. stroke core) than partial deprivation. In contrast to OGD, hypoxia/hypoglycemia leads to cell damage over a longer period of time and generally initiates apoptotic cell death, perhaps providing a more accurate in vitro model of the stroke penumbra (Lipton, 1999). This toxicity paradigm involves the reduction of oxygen content to 1% from 21% and glucose to 0.2 g/L from 5.5 g/L (Nath et al., 1998). Since some oxygen is present, ROS production remains an important element in delayed neuronal death throughout the treatment (Dubinsky et al., 1995). 44 Furthermore, in addition to a glutamate receptor-based excitotoxic component, OGD and hypoxia/hyperglycemia models will also include cell death due to C a + + entry through acid sensing cation (ASIC) channels since lactic acid accumulates during metabolic inhibition (Xiong et al., 2004). 1.7.5. Metabolic inhibitors During stroke, the deprivation of oxygen and glucose leads to metabolic shutdown and the inability to generate ATP (Coyle and Puttfarcken, 1993; Dirnagl et al., 1999; Lee et al., 1999). Chemicals that inhibit enzymes in the citric acid cycle or electron transport chain have been used to mimic this aspect of stroke, producing a simple in vitro model. Sodium azide (inhibitor of electron transport chain complex I, NaCN), rotenone (complex I inhibitor), malonate (reversible complex I inhibitor), oligomycin (inhibitor of H + -ATP synthase), 3-nitropropionic acid (3-NP, an irreversible inhibitor of succinate dehydrogenase / complex II), iodoacetate (inhibitor of glyceraldehyde-3-phosphate dehydrogenase) are among the most commonly used metabolic inhibitors (Uto et al., 1995; Smith and Bennett, 1997; Massieu et al., 2000; Mattson et al., 2000; Donohoe et al., 2001; Lee et al., 2002). 1.8. Modeling Stroke in vivo In vivo stroke has been modeled in many types of mammals including rodents, cats, and non-human primates. The discussion presented here will primarily compare and contrast focal and global stroke models between rats and mice. Focal ischemia is generally achieved by occluding one major intracranial artery, usually the middle cerebral artery, leading to a localized reduction of blood flow. Global ischemia is achieved by occluding a combination of the major extracranial arteries supplying blood to the brain. To better understand the various in vivo stroke models, we will first discuss the vascular structure of the brain and arteries supplying the brain. 45 1.8.1. The Cerebral Vasculature The brain is highly dependent upon its blood supply because it is the major source for glucose and oxygen, which are indispensable for brain metabolism. Blood supply is also essential from removal of waste products such as carbon dioxide, lactic acid, and detoxification products that can lead to brain damage if accumulated (Samuels and Mayer Media., 1996; Gilroy, 2000; Kandel et al., 2000). Four major arteries provide blood to the brain (Fig. 1-4). On the ventral side, blood from the aortic arch splits into two common carotid arteries (CCA). Each CCA then gives rise to an internal carotid artery (ICA), which passes through the skull to provide blood to each cerebral hemisphere. Within the skull, the two ICAs converge at the Circle of Willis found directly beneath the brain. The Circle of Willis allows sharing of blood flow between the left and right hemispheres. For each hemisphere, three major arteries sprout from the Circle of Willis, the anterior cerebral artery (ACA), middle cerebral artery (MCA), and posterior cerebral artery (PCA). The M C A supplies blood to the largest portion of the forebrain (Fig. 1-4). The lenticulostriate arteries, the first branches that arise from the M C A , supply blood to many of the subcortical structures, while the remaining blood flow from the M C A supplies the cerebral cortex including the parietal and temporal lobes. On the dorsal side, the vertebral arteries account for the two other vessels supplying blood to the brain. The vertebral arteries merge to form the basilar artery before connecting to the Circle of Willis. The vertebral-basilar system normally supplies blood to the medulla, pons, mesencephalon, and cerebellum. The basic vascular anatomy described here is conserved between humans and rodents. 46 / 'W \ -^ An:i» Dt it>«i HOIH intwnal capsule, lower corona / .1 \ j radiata, body ol caudate) Figure 1-4. Schematic diagram of extracranial arteries supplying blood to the brain and the cerebral vasculature. During the intraluminal suture model of ischemia-reperfusion in rats/mice, a suture would be inserted in to the ICA to the origin of the M C A as depicted. Cross-section of a cerebral hemisphere showing territory supplied by middle cerebral artery (shaded area)(right panel). Figure shows human vasculature and brain structure, but vessels and brain regions involved in rodent focal models are very similar. Figure adapted from (Kandel et al., 2000) . 1.8.2. Animal Models of Focal Ischemia 1.8.2.1. Intra-luminal Suture Model Focal ischemia-reperfusion is most commonly modeled by passing a blunt-tipped nylon suture through the lumen of the I C A until the tip occludes the origin of the M C A (Fig. 1-4 and 1-5A,B,C) . This model is arguably the most relevant to human stroke since the origin of the M C A is a very common site of occlusion in human stroke cases. A n advantage of this model is that the suture can be retracted from the M C A after a period of ischemia (up to 3 h) allowing full control over reperfusion. Alternatively, the suture can be permanently left in place to model permanent M C A occlusion. Since the M C A is occluded at its origin, lenticulostriate circulation is also lost in this model producing a large infarction including both cortical and subcortical regions (Fig. 1-4 and 1-5D). The disadvantage of this model is its technical difficulty and the need to carefully 47 identify improper suture placement. Interanimal variability can be minimized by measuring cerebral blood flow (i.e. MRI or laser Doppler) or by using a series of behavioural tests to identify contralateral sensorimotor deficits during ischemia. The intraluminal suture method has been used for both rats and mice. The Sprague Dawley rat strain is most commonly used as it gives consistent results and the animals tolerate the stroke well. However, other rat strains (i.e. Long Evans and Wistar) are also used. For mice, the C57B1/6 strain has been reported to give the most reproducible results as they are a highly inbred strain with very little genetic differences in terms of stroke susceptibility and vasculature (Connolly et al., 1996). It has been shown that the cerebral cortices of C57B1/6 mice have a larger MCA-supplied territory and have larger strokes, when compared to another commonly used strain SV129 (Maeda et al., 1998). Recent advances in transgenic and gene disruption techniques in mice have allowed elegant studies evaluating the role of specific genes in stroke (Kinouchi et al., 1991; Bruce et al., 1996; Eliasson et al., 1997; Iadecola et al., 1997; Asahi et al., 2001). However, the mixed background strains of some transgenic/knockout mice can often give variable results when using the intraluminal suture method. This can be attributed to variable patency of the posterior communicating artery, which allows collateral blood flow from vertebral arteries to sufficiently supply blood to the brain even when carotid circulation is occluded (Kitagawa et al., 1998). Thus extra care should be taken to ensure ischemia was achieved when using mixed strain backgrounds. 1.8.2.2. Occlusion of Distal Middle Cerebral Artery As a more direct means to occlude the M C A , an alternative model involves exposing the distal portion of the M C A by craniotomy and directly electro-coagulating or clamping the artery (Bederson et al., 1986b; Furuya et al., 2004). Occlusion of the distal M C A does not affect 48 lenticulostriatal circulation and thus only causes cortical infarction. The advantages of this model are its simplicity and reproducibility. The disadvantage of this technique is the invasiveness of the procedure and exposure of the brain to air, which causes additional non-ischemia related injury. This method has been used as a model of transient and permanent focal ischemia in both rats and mice. However, it is most commonly used as a model of permanent ischemia in mice because it is rather difficult to temporarily clamp the M C A (with a micro-aneurysm clip) without damaging it. As with temporary focal ischemia, mouse strain also plays a significant role in stroke outcome when using permanent focal ischemia (Majid et al., 2000) . 49 F i g u r e 1-5. C a r b o n b l a c k p e r f u s i o n o f r a t a l l o w s v i s u a l i z a t i o n o f c e r e b r a l a r c h i t e c t u r e . A.) Ventral view shows origin of M C A (arrow) and expected source of blood flow from ICAs, not seen with carbon black (arrows in upward direction). B.) The sagittal view shows the M C A as it traverses the parietal lobe on the surface of the brain. C.) Dorsal view shows fan-like distribution of arteries stemming from the M C A . The shaded demarcated territory would be supplied by the M C A . D.) After M C A occlusion using the intraluminal suture method (24 h post stroke), both subcortical and cortical structures are affected. T T C staining reveals live (dark) and dead (white) tissue. 1.8.2.3. Embolism Model Injection of pre-formed clots into the vessel leading to the brain, mimicking the lodging of an embolism, has been developed as a model of focal ischemia (Wang et al., 2001a). Initial studies injected fragmented blood clots into the C C A or I C A , which usually led to M C A occlusion, but sometimes also caused extracranial or contralateral brain ischemia due to inadvertent lodging of the embolism in other vessels (Kaneko et al., 1985; Overgaard et al., 1992). Later studies increased reproducibility and accuracy by directly injecting blood clots into the M C A (Wang et al., 2001b). These clots were found to dissolve within 1-3 h after injection allowing reperfusion. The advantage of this model is that it employs a real blood clot allowing the study of thrombolytic drugs. However, as attested by earlier studies, the uncontrolled circulation of emboli even after dissolution within the M C A could lead to occlusion elsewhere in the brain with unknown effects. 1.8.2.4. Photothrombotic Model Focal ischemia has also been produced by irradiating the photosensitive dye, Rose Bengal, after it is injected either intravenously or intraperitoneally to access the vasculature and the brain (Watson et al., 1985; Dietrich et al., 1986; Dietrich et al., 1987). The photochemical reaction of the dye (irradiated with green laser light) causes a local aggregation of platelets leading to occlusion of blood flow. Irradiation of the cortical surface leads to a well-defined 50 lesion, which may penetrate into subcortical structures with higher laser powers (Boquillon et al., 1992). Alternatively, the laser light may be focused directly on the M C A to produce ischemia in downstream territories (Markgraf et al., 1993; Sugimori et al., 2004). Photothrombosis is a very attractive model because of the versatility over where ischemia can be induced and its high reproducibility. However, some have argued that the Rose Bengal reaction produced neuronal injury through production of free radicals (singlet oxygen) in addition to the clot formation, complicating the true injury mechanism (Gajkowska et al., 1997; Trieu et al., 1999). The breakup of Rose Bengal induced clots can allow reperfusion in some cases, but this event is spontaneous and reversible, making it difficult to follow (Zhang et al., 2005). Nevertheless, studies have shown that the photothrombotic model exhibits many of the same characteristics as classic suture-based and electrocautery models. For example, N M D A and A M P A receptor are involved in the ischemic cascade since antagonists can attenuate damage (Yao et al., 1994; Umemura et al., 1997). Further, spreading depression has also been documented in the photothrombotic model (Dietrich et al., 1994). L8.2.5. Endothelin-1 Model Focal ischemia has also been modeled using the potent vasoconstrictive peptide, endothelin-1 (ET-1) (Fuxe et al., 1997). In the brain, ET-1 produced in the vascular endothelium induces vasoconstriction by binding to ET-A (or ET-B in some cases) receptors located on underlying smooth muscle cells (Yanagisawa et al., 1988; Salom et al., 1995). To induce stroke, ET-1 can be injected into the parenchyma (cortex or striatum) to act on local blood vessels, or can be applied topically to the M C A to affect downstream brain regions (Macrae et al., 1993; Ueki et al., 1993; Fuxe et al., 1997; Biernaskie et al., 2001). Vasoconstriction has been shown to persist for up to 16 h at a flow rate of approximately 50% of baseline followed by return of 51 normal blood flow (Biernaskie et al., 2001). This long-lasting suboptimal blood flow leads to delayed neuronal death that may be reminiscent of the ischemic penumbra found in the intraluminal suture ischemia-reperfusion model (Biernaskie et al., 2001). This is in contrast to the severe ischemia caused in the stroke core of focal ischemia or during photoactivation of Rose Bengal where ischemia is typically more severe, causing necrotic cell death (Biernaskie et al., 2001). Unfortunately there is no control over blood flow recovery when using ET-1. However, one group has used ET-3, which also induced vasoconstriction but has a known receptor antagonist (Henshall et al., 1999). Post-injection of the antagonist was found to completely inhibit the effect of ET-3 and promote reperfusion. 1.8.3. Animal Models of Global Ischemia Global ischemia models brain damage that occurs during heart failure where there is a loss of blood flow to the whole brain (or large proportion) is lost. In rats, all four major blood vessels supplying the brain must be occluded (both CCAs and both vertebral arteries) to achieve global ischemia (Lipton, 1999). The vertebral arteries are permanently Iigated in a presurgery. On the second day, both CCAs are transiently compressed for 10-20 min. Loss of the righting reflex occurs within 15 seconds showing that blood flow is substantially reduced in the hippocampus, striatum, and neocortex. Anesthetia is commonly not used during ischemia with 4-vessel occlusion. Global ischemia can also be achieved by transiently occluding both CCAs in combination with reduction in blood pressure, by withdrawing blood from the body. The outcome of 2-vessel occlusion with hypotension is typically more severe than with 4-vessel occlusion (Lipton, 1999). A simplified version of global forebrain ischemia can performed on gerbils, where only bilateral ligation of the CCAs is necessary. Since gerbils lack posterior communicating arteries, vertebral circulation does not contribute to the forebrain blood supply. Interestingly, C57B1/6 52 mice often have poorly developed pCommAs, and global forebrain ischemia can also be produced in the same manner. However, care must be taken to ensure ischemia is achieved since there is some heterogeneity in the patency of pCommA within this strain. Similarly, this model is often unreliable with mixed backgrounds, as with transgenic/knockout mice, due to increased variability in the pCommA (Kitagawa et al., 1998). Since blood flow is very close to zero using global models, the damage caused is quite severe, compared to focal ischemia, and thus occlusion is applied only for short periods of time. Transient global ischemia primarily induces cell death in a delayed manner (apoptosis is seen between 12 h and several days) and in a very specific pattern (Lipton, 1999). Hippocampal neurons in the C A I region are particular susceptible to ischemia and are the most well studied. Neuronal death is also seen specifically in parts of the CA4 region of the hippocampus, striatum, and layers 2 and 5 of the cerebral cortex (Lipton, 1999). Although focal models typically lead to unilateral motor deficits that can be measured with motor tasks (i.e. skilled forelimb reaching test and cylinder), global stroke models primarily affect memory-related tasks (i.e. Morris water maze and T-maze) (Whishaw and Kolb, 2005). 1.9. M o d e l i n g N e u r o d e g e n e r a t i o n In Vivo U s i n g 3 - n i t r o p r o p i o n i c A c i d The brain is highly dependent upon adequate blood supply for glucose and oxygen to maintain a high level of brain metabolism. Interestingly, when injected into the brain or administered through diet, a number of metabolic inhibitor commonly used to model in vivo neurodegeneration (in vitro use described above) produce lesions that are in some ways similar to stroke-induced damage. For example, the cell death mechanisms and vulnerable cell populations are affected similarly between in vivo ischemia and chemical metabolic inhibition, although important distinction also exist (i.e. timecourse of lesion evolution and behaviour outcomes). In this study we use systemic injection of the metabolic inhibitor, 3-nitropropionic 53 acid (3-NP) to inhibit brain metabolism and model neurodegeneration. We will now discuss 3-NP administration as a model of neurodegeneration. 3-NP was originally discovered as a toxin found in fungus-contaminated sugarcane, that when accidentally ingested caused symptoms similar to that seen in Huntington's disease (HD) . 3-NP has been widely used as a model of in vivo neurodegeneration (Beal et al., 1993a). 3-NP specifically and irreversibly inhibits complex II of the mitochondrial electron transport chain (also known as succinate dehydrogenase (SDH)) thereby blocking ATP synthesis (Alston et al., 1977)(Liu et a l , 1992; Fu et al., 1995). Intraperitoneal injections of 3-NP induces bilateral striatal-specific neurodegeneration in many animal species, but has been best studied in rodents and non-human primates. In general, chronic treatment regimens of 3-NP (low doses over weeks-months) are most commonly used in rat because they adequately reproduce neurodegeneration and motor abnormalities seen in HD (Gould and Gustine, 1982; Brouillet et al., 1998). Interestingly, the dosages required to produce the same extent of lesioning and motor deficits in mice is substantially higher than with rats, which may be related to the efficiency of 3-NP removal from the body or intrinsic sensitivities of neurons (Alexi et al., 1998). In both rats and mice, however, 3-NP produces lesions that are a consequence of secondary excitotoxic mechanisms (Beal et al., 1993a; Brouillet et al., 1993). Partial reduction of neuronal toxicity can be obtained by treatment with N M D A receptor antagonists, as assessed in dissociated culture (Zeevalk et al., 1995; Fink et al., 1996; Pang and Geddes, 1997). There is also evidence to suggest that oxidative stress plays a significant role in 3-NP induced pathology (Beal et al., 1995; Schulz et al., 1996). Notably, ATP depletion causes membrane depolarization and increased neuronal/glial glutamate release. Excessive extracellular glutamate over-activates N M D A and A M P A glutamate receptors leading to accumulation of intracellular C a + + and excessive mitochondrial ROS production (Reynolds and Hastings, 1995; Schulz et al., 1995). In addition, secondary activation of Ca++-dependent enzymes such as phospholipase A 2 leads to arachidonic 54 acid metabolism and recruitment of free radical generating inflammatory cells. Ca -dependent nitric oxide synthase activation can also augment the formation of peroxynitrite (Beal, 1992, 1995). Finally, excessive dopamine release can generate H2O2 when metabolized (Reynolds et a l , 1998). With chronic 3-NP treatment, death is restricted to neurons expressing G A B A , substance P, somatostatin and neuropeptide Y , but spares N A D P H diaphorase staining NOS neurons (Beal et al., 1993a). This pattern of cell death draws similarities with HD associated neurodegeneration in humans. In contrast, acute treatment with 3-NP (large doses over 1-2 days) causes rapid motor incoordination or death. Furthermore, acute treatment does not have the same neuronal specificity as chronic treatment and also affects extra-striatal brain structures. Therefore, acute 3-NP treatment does not replicate symptoms of HD (Hamilton and Gould, 1987). Recent studies have developed a highly reproducible subacute 3-NP regimen for mice, in which successively increasing doses of 3-NP are given over a period of 4-5 days (Feraagut et al., 2002). Subacute 3-NP toxicity manifests many of the same features as chronic 3-NP treatment and is used in our studies (see Chapter 3). 1.10. Proper Use of Animal Models of Stroke Animal stroke models are the final stage of testing for a potential neuroprotectant before human clinical trials. It is therefore of highest importance that animal stroke models be designed and used to best reflect human stroke. Before performing an experiment using existing stroke models there are some caveats that must be considered (De Keyser et al., 1999; Hoyte et al., 2004). First, rodents and human brains have obvious anatomical differences. Of note, rodent brains have proportionally less white matter than human brains. The use of rodent models may overlook the importance of white matter injury, which is a significant contributor to motor 55 impairment after human stroke. It will be is an important to develop strategies to adequately test neuroprotectants of white matter injury in vivo (Stys, 2004). Other interspecies differences between rodent and human brains may also complicate the evaluation of neuroprotective therapies. Second, in human stroke patients, it is difficult to achieve the same concentration of neuroprotective drugs that are beneficial in animal models. As mentioned above, side effects associated with drugs such as N M D A receptor antagonists are difficult to assess in animals and are likely manifested at lower doses than expected in humans (Buchan, 1990). Third, hypothermia (reduction of core and brain temperature to 34°C) is highly neuroprotective even when the manipulation is applied several hours after stroke. It is therefore very important to regulate core, and if possible, brain temperature to evaluate the contributions of the drug itself and not indirect drug-induced fluctuations in body temperature. For example, earlier reports using N M D A receptor antagonist did not regulate body temperature during stroke, and there are reports to suggest that observed neuroprotection was due to drug-induced hypothermia (Buchan and Pulsinelli, 1990; Buchan et al., 1991). Fourth, the evolution of stroke damage occurs over weeks to months. The majority of research studies to date have emphasized neuroprotection seen within days after the onset of ischemia. Considering that the efficacy of neuroprotectants in humans is evaluated months after stroke, animals stroke studies should also evaluate neuroprotective efficacy on longer time-points post-stroke. Furthermore, the functional endpoint of human stroke therapy is the key measurement of its success. It is also necessary to obtain information on the long-term recovery of motor function when using animal models (Colbourne et al., 1997). Fifth, drug administration to rodents has usually been restricted to within 1-3 hours after stroke. However, human stroke patients are often not fortunate enough to receive treatment within such a short time period (usually up to 6 hours have elapsed before human patients are 56 treated) (Gladstone et al., 2002). Thus, animal studies should be designed to identify drugs with longer therapeutic windows. Finally, human strokes come in all shapes and sizes, and patients are usually aged individuals with other health complications such as high blood pressure and diabetes. However, the detailed evaluation of neuroprotective drugs requires a model with very little inter-animal variability. Thus, most stroke studies use healthy young male rats, which do not necessarily model patients that most susceptible to stroke. Despite these important caveats, with careful design and proper use, animal stroke models hold significant predictive value for the identification of neuroprotectants for human stroke. In our studies we have taken these caveats in mind when testing the neuroprotective potential of Nrf2 function. In particular, we have rigorously tested our hypothesis in multiple models of stroke using both rats and mice. Core body temperatures during and immediately after stroke were tightly regulated. Finally, favourable outcomes were observed for both histological and behavioural correlates collected after relatively long survival periods post-stroke (1 month). 1.11. Research Hypothesis and Objectives Hypothesis: By controlling a global antioxidant response. Nrf2 plays a central role in management of oxidative stress in the brain. Activation of Nrf2 can be an effective prophylactic strategy to attenuate tissue damage cause by stroke and neurodegenerative disease. To test our hypothesis, our research was divided into the following aims: Aim 1: Determine whether Nrf2 activation reduces neuronal death in in vitro models of stroke damage, and examine the mechanisms that account for Nrf2-mediated neuroprotection. 57 A i m 2: Investigate whether Nrf2 knockout mice are more susceptible to oxidative stress and neurodegeneration induced by 3-NP in vivo. Conversely, determine whether Nrf2 activation by electrophilic inducer, tBHQ, can attenuate 3-NP toxicity. A i m 3 : Investigate the utility oftBHQ administration in protection from transient and permanent focal ischemia. Examine whether Nrf2 knockout mice are more susceptible to focal ischemic injury. 58 Chapter 2 : Coordinate regulation of glutathione biosynthesis and release by Nrf2 expressing glia potently protects neurons from oxidative stress1 A version of this chapter has been published. Shih A.Y., Johnson D.A., Wong G., Kraft A.D., Jiang L., Erb H . , Johnson J.A., and, Murphy T . H . (2003) Coordinate regulation of glutathione biosynthesis and release by Nrf2 expressing glia potently protects neurons from oxidative stress. The Journal of Neuroscience. 2003, April 15, Vol. 23, Iss. 8, pgs. 3394-3406. Neuroscience Online: http://jneurosci.org/. 59 2.1. Introduction Glial cells (astrocytes) are known to interact with surrounding neurons by nourishing, protecting, and modulating growth and excitability (Travis, 1994). Although it has been repeatedly shown that astrocytes can improve neuronal survival, the mechanisms of protection remain uncertain. Astrocytes have stronger anti-oxidative potential than neurons (Raps et al., 1989; Makar et al., 1994; Lucius and Sievers, 1996) and can protect neurons from oxidative stress induced by various compounds such as dopamine, H2O2, and 6-hydroxydopamine, and nitric oxide (Desagher et al., 1996; Mena et al., 1996; Chen et al., 2001). One potential defense against the toxicity of reactive oxygen species (ROS) is the induction of a family of Phase 2 detoxification enzymes (Fahey et al., 1997; Kensler, 1997). Data from our lab and others suggest that this response is preferentially expressed in astrocytes, with considerably lower levels in neurons (Ahlgren-Beckendorf et al., 1999; Eftekharpour et al., 2000; Murphy et al., 2001; Johnson et al., 2002). Originally thought to be restricted to promoting xenobiotic conjugation with endogenous ligands, such as glutathione (GSH) (Hayes and Pulford, 1995; Primiano et al., 1997), the observed functions of Phase 2 enzymes have recently broadened. Recent microarray studies, as described in this chapter, have elucidated approximately 50-100 possible Nrf2-regulated Phase 2 enzyme genes (Li et al., 2002; Thimmulappa et al., 2002; Lee et al., 2003c; Kraft et al., 2004). Sharing common regulatory pathways, these enzymes possess chemically versatile antioxidant properties and are inducible by various agents, including those found in a normal diet (Fahey et al., 1997; Gao et al., 2001). Treatment of mammalian cells with electrophilic agents such as fer?-butylhydroquinone (synthetic food antioxidant) and sulforaphane (abundant in cruciferous vegetables) provokes a cellular response leading to the coordinated transcription of Phase 2 genes (Prestera et al., 1993). A unique cis-acting regulatory sequence, termed the antioxidant response element (ARE) is essential for the constitutive and 60 induced expression of many antioxidant genes involved in the Phase 2 pathway (Friling et al., 1990b; Rushmore et al., 1991; Nguyen et al., 2000). Several lines of evidence suggest that Nrf2 is an important transcription factor responsible for upregulating ARE-mediated gene expression (Itoh et al., 1997a; Alam et al., 1999; Itoh et al., 1999a; Hayes et al., 2000b; Ishii et al., 2000). Studies using knockout mice have shown that Nrf2 was part of a transcription factor complex required for regulation of the mouse glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase (NQOl) genes (Itoh et al., 1997a; Hayes et al., 2000b). In addition, Nrf2/small Maf heterodimers bind to the A R E sequence with high affinity during regulation of the GST and NQOl genes (Venugopal and Jaiswal, 1996). From these observations, Nrf2 appears to be the major transcription factor necessary for A R E activation, and thus essential for the induction of Phase 2 detoxification enzymes. In this study, we use replication-deficient adenoviruses to over-express Nrf2 protein in both neurons and glia to determine whether augmentation of the A R E -mediated antioxidant response might reduce neuronal vulnerability to oxidative stress. 2.2. Materials and Methods 2.2.1. Materials A l l chemicals were purchased from Sigma Canada unless otherwise stated. 2.2.2. Plasmids and Adenoviruses PEF mammalian expression plasmids carrying cDNA encoding mouse Nrf2 and Nrf2DN were a generous gift from Dr. Jawed Alam (Alton Ochsner Medical Foundation, New Orleans, LA) (Alam et al., 1999). Rat A R E sequences were obtained from the N Q O l promotor. To make the human placental alkaline phosphatase (hPAP) reporter construct (rQR51), a 51 bp ARE/EpRE fragment was excised using restriction sites (Xho I and Hind III) flanking the A R E 61 sequence from a parent luciferase expression vector (Moehlenkamp and Johnson, 1999) and subcloned into the pGEM-7zf vector (ATCC) upstream of the hPAP reporter gene. A mutant rQR51 (rQR51mut) was made by replacing the original core A R E sequence with a cassette encoding a mutant A R E core sequence. The Bgl II/Nhe I excisable cassette was constructed using the following oligonucleotides (Sigma Genosys). The 10 bp mutation is underlined and the region corresponding to the A R E core sequence is shown in bold: 5 '-CTAGCTCG AG ATCCTCAG AG ATTTC AGTCTAG AGTC AC ACGCAAACAGG A A A A T C A - 3 ' 3 '-GAGCTCTAGGAGTCTCTAAAGTCAGATCTCAGTGTGCGTTTGTCCTTTTAGTCTAG-5' Recombinant adenoviral vectors were constructed using the Cre-lox system (Canadian Stroke Network core facility, University of Ottawa) (Hardy et al., 1997). The Nrf2 and Nrf2DN cDNAs were excised from the PEF vector using restriction enzymes Not I, and Not I and Hind III, respectively. A l l viruses were titred on HEK293 cells. 2.2.3. Mammalian Cell Culture Mixed neuronal/glial cultures were prepared from the cerebral cortex of Wistar rat fetuses (El7-18) using the papain dissociation method (Murphy et al., 1990). Viable cells were plated at 1 x 106 cells/mL on poly-D-lysine coated plastic culture plates (Costar) in B27-supplemented neurobasal medium (Gibco). After 1 DIV, the medium was changed to minimum essential medium (MEM) (Gibco) supplemented with 5.5 g/L D-glucose, 2 m M glutamine, 10% fetal bovine serum (FBS) (HyClone), 1 mM Na+-pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin (MEM-pyr). This medium change was required to reduce excessive antioxidant levels from the B27 medium. Enriched neuronal cultures used for Western Blot analysis were prepared by culturing cortical cultures in the presence of 10 u.M 5-fluoro-2'-deoxyuridine/uridine that was replaced every 4 days until the cultures were harvested at 14 DIV. Enriched glial cultures were prepared from 1-2 d post-natal rat pups. Cortices were dissected, minced and used 62 in the papain dissociation method. Cells were plated in M E M supplemented with 10% FBS, 5% heat-inactivated horse serum (HyClone), 2 mM glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin in non-coated 10 cm plates (2 plates per brain). After 1 DIV, the medium was replaced and the glia were allowed to grow for 3 days. Any adherent neurons were removed from the glial culture by repeated pipetting of medium. A l l glial cultures were used within 10 DIV since older, quiescent cultures appeared to lack the antioxidant response. These conditions for isolating glial cells largely results in a population of Type I and II astrocytes. Anti-glial fibrillary acid protein (GFAP) staining suggests that the glial cultures are mainly of the astrocyte phenotype. For a simple neuron-glial co-culture setup, virus-infected glia were trypsinized and transplanted directly into naive (no contact with virus) cortical culture after 24 h of transgene expression (co-culture) (Fig. 2-1 A). Glia were transplanted with concentration ranging from 0.1 x 104 - 2 x 104 cells/mL, a plating density of 0.1 - 2% of the total number of cells per well. Co-cultures with glia physically separated from neurons (membrane delimited co-culture) consisted of naive cortical cultures prepared in 24-well plates (neuronal compartment) and infected glia separated in culture plate inserts with 0.4 u.m pore size (glial compartment) (Millipore) (Fig. 2-12?). Infected glia were trypsinized 6 h after infection and plated at 2 x 104 cells per insert, a plating density of 2% of the total cell number, into the collagen-coated inserts. For depletion of glial glutathione, the glial compartment was pre-treated with 200 \xM BSO for 24 h, then pre-incubated for 10 min in fresh medium and washed twice before transfer into the neuronal compartment. For both setups, transplanted glia were washed 3 times with PBS to prevent transfer of virus to the neuronal compartment. Human embryonic kidney 293 cells (HEK293, ATCC) were plated at a density of l x l 0 5 cells/mL on 10 cm plates (Costar). Culture medium was prepared from M E M supplemented with 10% FBS, 1 mM Na+-pyruvate, 2 mM L-glutamine, and 100 U/mL penicillin, 0.1 mg/mL 63 streptomycin. After plating, the cells were allowed to grow for approximately 16 h before commencement of transfection. A l l cultures were maintained at 37°C in a humidified 9 5 % O2, 5 % C 0 2 incubator. A B Co-culture Membrane delimited co-culture N^fc = infected glia = naive glia a>— = naive neuron Figure 2-1. Co-culture and membrane delimited co-culture A , F o r a s imple neuron-g l i a l cocul ture setup the vi rus- infected g l i a were t ransplanted d i rec t ly into na ive (no contact w i t h v i rus) m i x e d neurona l -g l i a l cultures after 24 hr o f transgene express ion . B , S o m e experiments required a setup b y w h i c h g l i a were separated p h y s i c a l l y f rom neurons (membrane -de l imi t ed coculture) . T h i s system consis ted o f naive cultures prepared i n 2 4 - w e l l plates; infec ted g l i a were separated by a culture plate insert. B o t h cocul tures were ma in ta ined for 24 hr and then exposed to 3 m M glutamate ( G l u ) for a further 24 hr, f o l l o w e d by quanti tat ion o f neuronal v i a b i l i t y . 64 2.2.4. Transfections and Infections HEK293 cells were transiently transfected using the calcium-phosphate method (10 u.g/10 cm plate) (Chen and Okayama, 1987) or with Polyfect reagent (Qiagen) according to the manufacturer's protocol. The transfection efficiency was typically 60-70% as assessed by |3 gal staining. Mature (14 DIV) primary cultures of mixed cortical neurons and glia were transiently transfected with the ballistic gene transfer method using a Helios gene gun (Bio Rad) firing 0.6 pm gold particles coated with D N A (1 pg DNA/mg gold loading ratio). For adenovirus infection, immature cortical cultures were infected at 1 DIV using virus diluted to a multiplicity of infection (MOI, plaque forming units per cell) of 200 in MEM-pyr. When calculating MOI, we used the number of cells per well at time of plating. Virus stocks were usually on the order of 1 x 1011 plaque forming units per mL. The cultures were allowed to express transgenes for 48 h before usage. A l l infected cultures were examined for adequate infection efficiency (25% of neurons, 80% of glia) assessed by GFP fluorescence. 2.2.5. Toxicity Treatment For all toxicity studies, cortical cultures were used in their immature state (1-4 DIV). MEM-pyr was replaced with M E M supplemented with 5.5 g/L D-glucose, 2 mM glutamine, 5% FBS, 100 U/mL penicillin, 0.1 mg/mL streptomycin (MEM-5% FBS) containing the indicated concentrations of L-glutamate, H2O2 or staurosporine. Cells were exposed to all toxins for 24 h. No inhibitors of GSH oxidation were added to the medium during the toxicity experiments. 2.2.6. Determination of Neuronal Viability Neuronal viability was evaluated using two methods: 1.) Manual counting of cells labeled with fluorescent antibodies to neuron specific enolase (NSE) or green fluorescent protein (GFP), 65 and 2.) [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2//-tetrazolium bromide] (MTT) cell viability assay. For cell counting after immunocytochemistry, 10 pictures of each experimental group were taken with a 20 X objective (Zeiss) using wide-field fluorescence microscopy (Zeiss Axiophot) with color CCD camera (Sony powerHAD, model DXC-950). The imaged areas were chosen randomly from at least 3 different wells per experimental group. A l l counting was performed with the rater blinded to the experimental conditions used. To confirm cell counts, 96 well plates used for counting were scanned with a Fluoroskan fluorescence plate reader (ex: 530, em: 620) (Labsystems) to measure red fluorescence from NSE-positive cells, although not all experiments could be processed this way since absolute levels of NSE fluorescence could vary due to background staining or cell density. A l l measurements made using the Fluoroskan were background subtracted where background was the fluorescence measured from ad-GFP infected wells treated with 3mM glutamate (Fig. 2-65, Q , 30 H 2 0 2 (Fig. 2-5A), or 10 pJVI staurosporine (Fig. 5B) for 24 h. For the MTT assay, cultures were incubated with 0.45 mg/mL MTT diluted in M E M - 5 % FBS with no phenol red for 2 h at 37°C, after the toxicity studies. The MTT staining solution was then removed and replaced with PBS and an equal volume of MTT lysis buffer consisting of 20% SDS in 50%> dimethylformamide, pH 4.7. The formazan crystals were allowed to dissolve overnight before the plate was read on a Multiskan plate reader (abs: 560) (Labsystems). In mixed neuron/glia cortical cultures, glial-mediated MTT turnover accounted for approximately one half of the total MTT reduction. To eliminate the glial component of MTT reduction, measurements from control wells treated with 3 mM glutamate for 24 h, where nearly all neurons had died, were used as a background that was subtracted from all other groups (Fig. 2-7C, 2-M). 66 2.2.7. Western Blot Analysis and Immunocytochemistry Cells were washed in PBS and harvested in PBS containing 2 j.ig/mL aprotinin, 1 mM phenyl-methylsulfonyl fluoride, 1 mM EGTA, and 1 m M EDTA, and sonicated for 10 s to make crude lysate. Protein concentration was measured using the bicinchronic acid method (Pierce). Samples prepared in loading buffer (7 mg/mL DTT, 6% SDS, 30% glycerol, and 0.38 M Tris pH 6.8, and pyronin Y) were denatured by boiling for 2 min prior to loading. For SDS-PAGE, 30% acrylimide gels were used to run HEK293 protein lysates (10 \xg loaded) and enriched neuron or glial lysates (30 \xg loaded), respectively. Antibody reactivity was detected using enhanced chemiluminescence substrate (Amersham). For immunocytochemistry following toxicity experiments, cultures were washed twice with 37°C PBS to remove dead cells and cellular debris and were then fixed with 2% paraformaldehyde (PFA) for 10 min and then incubated with primary and secondary antibodies. Immunostained cells were mounted in Fluoromount-G (Southern Biotechnology Associates). Antibodies used in this study include: anti-Nrf2 from rabbit (Santa Cruz, 1:200 dilution), anti-green fluorescent protein (GFP) from mouse (Boeringher Mannheim, 1:1000), anti-NSE from rabbit (Polysciences Inc., 1:2000), anti-GFAP from rabbit (Sigma, 1:100), anti-mouse Alexa Fluor 488 from goat (Molecular Probes, 1:2000), anti-rabbit Texas Red from goat (Molecular Probes, 1:2000), anti-actin from goat (Santa Cruz, 1:1000), anti-heme oxygenase 1 from rabbit (Stressgen Inc., 1:500), anti-rabbit horse radish peroxidase (HRP) from sheep (Amersham Pharmacia, 1:5000), anti-goat HRP from donkey (Santa Cruz, 1:5000). 67 2.2.8. Densitometry Densitometric analysis of Western Blots was performed using the Scion Imaging program (Version Beta 4.0.2, Scion Corporation). Bands intensities were measured by taking the mean pixel intensity. A l l band measurements were background subtracted. 2.2.9. [3H]-glutamate uptake assay L-[3H]-glutamate uptake was measured as described previously (Shih and Murphy, 2001). Briefly, infected immature cortical cultures were preincubated in Na+-free HBSS for 10 min at 37°C before being incubated'in with Na+-free HBSS containing 38 nM L-[3H]-glutamate (Amersham) and the indicated unlabeled compounds (1 mM) for a further 20 min at 37°C. The cells were then washed three times with ice-cold Na+-free HBSS and lysed with 0.5% Triton X -100 in 0.1 M potassium phosphate buffer. Radioactivity was determined using liquid scintillation counting and normalized to protein concentration for each sample. 2.2.10. Placental Alkaline Phosphatase and Quinone Reductase Staining For hPAP staining (Murphy et al., 2001), cultures were rinsed with PBS and fixed with 4% PFA and 0.2%> glutaraldehyde for 10 min. Preparations were rinsed again with PBS and incubated at 65°C for 30 min to inactivate any endogenous heat-labile alkaline phosphatase activity. The preparations were stained with 1 mg/mL nitro blue tetrazolium (NBT) and 1 mg/mL 5-bromo-4-chloro-3-indoyl phosphate dissolved in 0.1 M Tris buffer at pH 10. The staining reaction was performed at 37°C for ~ 30 - 40 min and was terminated by washing with PBS. Pictures were taken with a 60 X objective (Zeiss) using conventional light microscopy with color CCD camera. For N Q O l staining (Murphy et al., 1998), cells were fixed with 2%> PFA for 10 min, washed with PBS and preincubated in buffer A (25 m M Tris, 0.08% Triton X-100, 2 mg/mL BSA, pH 7.4) for 30 min. The preincubation solution was replaced with buffer 68 containing 100 uM NBT and 100 uM L Y 83583 (Alexis Corp.) and staining was initiated upon addition of N A D P H (1 mM final). The reaction was incubated at 37°C for 20-30 min and terminated by washing in buffer A. Differential interference contrast (DIC) and phase contrast pictures were taken before and after staining, respectively, using a 63 X oil immersion objective (Zeiss) on an Axiovert 200M microscope (Zeiss) equipped with an AxioCam HRm digital camera (Zeiss). DIC and phase contrasts images were overlayed for final presentation. 2.2.11. Total Intracellular GSH Assay and Effluxed GSH Assay Total GSH was quantified by the method of Tietze (Tietze, 1969). Briefly, cells were collected in PBS and sonicated for 10 s on ice. The acid-soluble fraction was obtained by adding perchloric acid to a final concentration of 3% followed by eentrifugation at 14,000 g for 10 min. The acid-soluble fraction was neutralized to pH 7 with 0.5 M KOH/50 m M Tris. Following the removal of precipitate (potassium perchlorate) by a second eentrifugation, 50 uL aliquots of sample were combined with 100 uL of reaction mixture consisting of 2.5 mL 1 m M DTNB, 2.5 mL 5mM N A D P H , 2.65 mL phosphate buffer solution (100 m M NaP0 4 , pH 7.5, 1 m M EDTA), glutathione reductase (5U/mL final). The increase in A 4 1 2 from GSH-mediated reduction of DTNB was measured at 30 s intervals over 30 min. GSH content among treatment groups was normalized to protein. For GSH efflux assays, M E M - 5 % FBS with no phenol red was incubated with cultures for 24 h and used in the same procedure minus the removal of proteins using perchloric acid. In some experiments, proteins were removed by filtration through a 3 kDa molecular weight cutoff membrane (Nanosep3K Omega, Pall Gelman Corp.). Monochlorobimane (Molecular Probes) staining was performed by incubating adenovirus infected cultures with 60 \xM mCBi for 20 min (Tauskela et al., 2000). Stained cultures were washed twice with PBS to remove excess mCBi and fixed with 4% PFA with 0.2% glutaraldehyde for 20 min. At this stage, cells were either viewed directly for fluorescence (ex: 69 360, em: 410) or further stained for GFP and GFAP using immunocytochemistry. Quantitative measurements of mCBi:GSH adduct fluorescence were made with Photoshop (Adobe Systems Inc.) by taking mean intensities from individual cells. A l l values were background subtracted. 2.2.12. RT-PCR and Microarray Analysis Total RNA was isolated from infected cultures using Trizol Reagent (Life Technologies, GIBCO) following manufacturer's instructions. One pg of R N A was reverse transcribed using Oligo (dT)15 primer in accordance with the RT System (Promega). The resulting cDNA was then amplified by PCR using primer sets for the genes: GFP, 5'-GAG CTG TTC A C C G G G GTG GTG-3' and 5'-GAG CTC G A G A T C TGA GTC CGG-3'; Mouse Nrf2, 5 '-TGA A G C TCA GCT CGC ATT GAT CC and 5 ' -AAG A T A C A A GGT GCT G A G C C G CC-3 ' ; Rat xCT, 5'- TTG C A A GCT C A C A G C A A T TC-3' and 5'- CGT C A G A G G A T G C A A A A C A A - 3 ' ; Actin, 5'- CCC A G A G C A A G A G A G GTA TC-3' and 5'- A G A G C A T A G CCC TCG T A G AT-3' . PCR conditions were as follows: initial denaturing step using 1 cycle at 95°C for 3 min, followed by 35 cycles at 95°C for 30 sec, the appropriate primer annealing temperature (ranging from 53.4 - 56.6°C) for 1 min, 72°C for 1.5 min, and a final cycle at 72°C for 5 min. The PCR products were separated on a 1.4% agarose gel containing ethidium bromide. Stained cDNA was then visualized using an ultraviolet light source. Microarray analysis was performed as previously described (Li and Johnson, 2002; L i et al., 2002; Stein and Johnson, 2002), using total RNA extracted from primary cultures using Trizol Reagent (Life Technologies, Gibco). Briefly, cDNA was synthesized from total RNA by reverse transcription using T7-(dT)24 primer incorporating a T7 R N A polymerase promotor, followed by a D N A polymerase reaction (MessageAmp Kit, Ambion). Biotin-labeled cRNA was prepared by an in vitro transcription reaction using the cDNA from above (MessageAmp Kit, Ambion, Biotin-labeled NTPs, Enzo Biochem, Inc.). Labeled cRNA was fragmented by incubation at 94°C for 35 min in the 70 presence of 40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, and 30 m M magnesium acetate. Fifteen pg of fragmented cRNA was hybridized for 16 h at 45°C to a Rat Genome U34A array (Affymetrix, Santa Clara, CA). After hybridization, the gene chips were automatically washed and stained with streptavidin-phycoerythrin by using a fluidics station. Probe arrays were then scanned at 3 pm resolution using the Genechip System confocal scanner made for Affymetrix by Agilent. Affymetrix Microarray Suite 5.0 was used to scan and analyze the relative abundance of each gene from the average difference of hybridization intensities. Analysis parameters used by the software were set to values corresponding to a moderate stringency (alphal = 0.04, alpha2 = 0.06, tau = 0.015; Gamma IL and 1H = 0.0025, Gamma 2L and 2H = 0.003). Output from the microarray analysis was stored as an Excel data spreadsheet. The definition of statistically significant increase or decreased for individual genes was based on the following three criteria in order as previously described (Li and Johnson, 2002; L i et al., 2002; Stein and Johnson, 2002): 1.) Rank analysis of the Difference Call (a measure of the direction and magnitude of change) from three inter-group comparisons (3 x 3, matrix analysis of 3 replicate samples) for neuronal cultures and two inter-group comparisons (2 x 2) for astrocyte cultures. Namely, No Change was given a value of zero, Marginal Increase/Decrease a value of 1/-1, and Increase/Decrease a value of 2/-2. The final rank referred to summing up the 9 values for the neuronal cultures and 4 values for the astrocyte cultures corresponding to the Difference Calls. These values varied from 18 to -18 and 9 to -9 for the neuronal and astrocyte cultures, respectively. The cutoff value for the determination of Increase/Decrease for neuronal cultures was set as 9/-9 and for astrocyte cultures set as 4/-4; 2.) Coefficient of Variation (cut-offs were set at 1.20); 3.) Fold Change (cut-offs were set at 1.3 or greater for increased genes or -1.3 or lower for decreased genes). 71 2.2.13. Statistical Analysis A l l experiments were repeated at least three times unless otherwise stated. Results are presented as mean + S.E. Statistical analysis of raw data was performed using GraphPad Prism 2.0. Experimental groups were compared by one-way A N O V A , two-way A N O V A followed by Bonferroni's post test, Kruskall-Wallis test, or / test as appropriate. A statistical probability of p < 0.05 was considered significant. 2.3. Results 2.3.1. Neurons express lower levels of Nrf2protein than astrocytes We previously demonstrated that cortical astrocytes have a higher basal and stimulated level of ARE-mediated gene expression than neurons (Murphy et al., 2001; Johnson et al., 2002). One explanation for this observation would be that astrocytes express higher levels of the transcription factor Nrf2 than neurons. To test this hypothesis, enriched neuronal and astrocyte primary cortical cultures were prepared and their extracts probed with an anti-Nrf2 antibody by Western Blot. A 105 kDa Nrf2-specific band was identified in enriched glial cultures that co-migrated with recombinant Nrf2 over-expressed in HEK293 cells (Fig. 2-2A, B). The same band was detected at very low levels in neuron-enriched cultures. Densitometry analysis revealed that glial-enriched cultures express approximately 12-fold more Nrf2 protein than neuron-enriched cultures implying that astrocytes may have more pronounced ARE-mediated gene expression because they express higher levels of Nrf2 than neurons (Fig. 2-2B). The 105 kDa band was not detectable in HEK293 cells over-expressing a (3-galactosidase control. The Nrf2 antibody, which was raised against the Nrf2 C-terminal region, also recognized the dominant negative N-terminal deleted form of Nrf2 migrating at 29 kDa (Nrf2DN). Although the Nrf2 antibody was used for Western blots, its non-specific binding activity made it unsuitable for immunocytochemistry. 72 If a lower basal level of Nrf2 protein is the reason for the lack of ARE-mediated gene expression in neurons, then over-expression of Nrf2 in neurons may be a strategy to boost their antioxidant gene expression. To test this idea, we used a reporter construct carrying a heat stable human placental alkaline phosphatase (hPAP) gene driven by a minimal A R E bearing promoter (rQR51). Previous studies in our lab show that in rQR51-transfected cortical cultures and cultures derived from transgenic ARE-hPAP reporter mice, hPAP staining is largely restricted to the astrocyte population (Murphy et al., 2001; Johnson et al., 2002). Consistent with our hypothesis, when rQR51 was co-transfected with an expression vector for Nrf2, the number of neurons staining for hPAP increased dramatically (Fig. 2-2C-G). Co-expression of rQR51 with the empty expression vector PEF did not increase neuronal staining, and co-expression with Nrf2DN not only prevented neuronal staining but also suppressed basal astrocyte staining. Thus, neurons are able to undergo ARE-mediated gene expression but appear to be constrained due to insufficient levels of Nrf2 protein. Induction of hPAP expression from rQR51 was dependent on the presence of a wild-type consensus A R E sequence and was abolished by mutation of this sequence (rQR51mut) (Fig. 2-2C). 7 3 Nrf2 actin PEF/rQR51wt D Nrf2/rQR51wt F G • 40um Figure 2-2. Western blot of heterologously expressed N r f 2 A, Western blot of heterologously expressed Nrf2 (105 kDa) and Nrf2DN (28 kDa) in HEK293 cells. B, A comigrating 105 kDa band corresponding to Nrf2 is observed in enriched cortical glial, but not neuronal, cultures. Densitometric analysis reveals an ~12-fold difference in Nrf2 protein (« = 3); *p < 0.05. C, Coexpression of Nrf2 cDNA with a hPAP-encoding reporter of ARE-mediated gene expression (rQR51) greatly increases neuronal hPAP expression. Reporter constructs carrying a mutation within the core A R E consensus sequence (rQR51Mut) were not inducible. Coexpression with Nrf2DN cDNA suppresses both neuronal and glial hPAP expression. *p < 0.05, neuron comparison to pEF (empty vector) control; #p < 0.05, glial comparison to pEF control. D, E, Representative hPAP-stained astrocyte-like cells with coexpression of pEF vector only. F, G, With Nrf2 overexpression cells of both neuronal and glial morphology show ARE-driven hPAP expression. Data from hPAP experiments represent the mean ± SEM number of cells counted from triplicate coverslips over four independent experiments. 2.3.2. Over-expression of Nrf2 enhances antioxidant activity of neurons and astrocytes in immature cortical cultures. We next evaluated whether Nrf2 over-expression could increase the antioxidant properties of cortical neurons and glia. For these experiments, replication-deficient adenoviruses (Crocker et al., 2001) were used to efficiently infect a large fraction of neurons. Three different adenovirus constructs were made with C M V promoters driving the expression of enhanced green 7 4 fluorescent protein (eGFP) alone (ad-GFP), Nrf2 and eGFP (ad-Nrf2), or Nrf2DN and eGFP (Ad-Nrf2DN). For the latter two viruses, each cDNA was driven by a separate C M V promoter. When used to infect immature rat cortical cultures (1 DIV), all three viruses were able to infect ~ 25% of the neurons and ~ 80%> of the glia, when used at multiplicity of infection of 200. To characterize the antioxidant activity of ad-Nrf2 infected cultures, the protein products of three known Nrf2 target genes were evaluated: HO-1 (Alam et al., 1999; Gong et al., 2002), xCT cystine transporter (Ishii et a l , 2000; Sasaki et al., 2002), and N Q O l (Bloom et al., 2002). Western Blot analysis showed that HO-1 protein levels increased in parallel to Nrf2 over-expression in mixed cortical cultures (Fig. 2-3A). Similarly, when infected with ad-Nrf2, both neurons (Fig. 2-3B) and glia (data not shown) exhibited robust increases in N Q O l staining. Glial staining increased over and above normal basal levels (compared with ad-GFP control). Staining was also observed selectively in ad-Nrf2 infected neurons but not in ad-GFP infected neurons, further supporting the idea that neurons are capable of having an enhanced antioxidant response but are limited by low basal Nrf2 expression. Un-infected neurons in the ad-Nrf2 infected culture do not stain for N Q O l . In addition, when infected with ad-Nrf2, xCT-mediated L-[ H]-glutamate uptake in mixed cortical cultures increased to 372 + 82 % of the ad-GFP infected group (Table 2-1). Conversely, activity was suppressed to 75.7 ± 7.1 % of the ad-GFP control in ad-Nrf2DN infected cultures. The Nrf2 induced increase in L-[3H]-glutamate uptake fit the pharmacological profile of system xc" since it was blocked by competitive inhibitors of cystine uptake such as quisqualate and homocysteic acid (Table 2-1). Nrf2 dependent upregulation of xCT mRNA was also verified using RT-PCR with two independent primer sets (Fig. 2-3C). Comparison of basal xCT mRNA levels between mixed cultures and glial-enriched cultures using RT-PCR showed an approximately 3-fold (n = 1) higher signal in glial-enriched cultures (data not shown). However, this observation should be interpreted as a qualitative difference due to potential non-linearity in the RT-PCR method. RT-PCR for GFP confirmed than an approximately equal amount of viral 75 gene expression occurred in ad-GFP and ad-Nrf2 infected cultures, as well as between the mixed and glia-enriched culture types. Table 2-1. Evaluation of Na+-independent L-[3H]- glutamate in ad-Nrf2-infected immature cortical cultures. Fold induction over ad-GFP control, and pharmacology of induced uptake Ad-GFP Ad-Nrf2 Ad-Nrf2DN 100% 372 + 8 2 % * 76.7 + 7 .1%* Compound % Ad- Nrf2 Uptake None 100 L-Cystine 26.6 ± 5.8 # Quisqualic acid 22.7 + 5.3 # Homocysteic acid 27.5 + 11 # Data represent the mean + S E M of five independent experiments Performed in quadruplicate. * p < 0.05, compared with ad-GFP control. # p < 0.05, compared with ad-Nrf2 control with no compounds. Uptake assay performed on mixed neuron/glial immature cultures. The basal rate of L-[ 3H]-glutamate uptake by ad-GFP-expressing cultures is 59.1 ± 5.7 fmol/mg protein per min. Nrf2 over-expression in mixed cultures also generated a ~ 4-5 fold increase in total intracellular glutathione (GSH + GSSG) compared to GFP control (Fig. 2-3D). Treating the cultures with 3 m M Glu for 6 h prior to harvesting for GSH measurement reduces the GSH levels in all groups without causing neuronal death, but the ad-Nrf2 infected group still contained more GSH in comparison to controls. Using monochlorobimane (mCBi) to stain for GSH in ad-Nrf2 infected cultures, we observed that the GSH:mCBi adduct fluorescence was enriched in GFAP-positive glia cells (Fig. 2-3E). This staining pattern was not observed in ad-GFP infected 76 cultures. A detailed quantitation of mCBi fluorescence revealed that both infected and uninfected neurons were also more heavily stained in the ad-Nrf2 infected mixed cultures in comparison to neurons in ad-GFP infected cultures (Table 2-2). It should be noted, however, that increased mCBi staining can be indicative of both increased GSH levels as well as enhanced GST activity (Tauskela et al., 2000) Table 2 - 2 . Evaluation of GSH-bimane adduct staining in infected mixed cortical cultures. Cell Type Ad-GFP Ad-Nrf2 Ad-Nrf2DN Infected neuron Uninfected neuron Infected glia 21.1+4.9 (60) 19.0 ±4.9 (60) 61.6 ± 11 (25) 40.6 ± 10(60) * 34.1 ± 10(60) * 119 ± 17(59)* 25.0 ±5 .2 (60) 19.9 ±4 .5 (60) 35.1 ±5 .2 (40) * # Data represent the mean + SEM, in arbitrary units of fluorescence intensity. Cell type was identified based on morphology (glial cells larger with web-like processes). The total number of cells evaluated for each group is listed in parentheses; with data collected from cells over three independent experiments performed in duplicate. * p < 0.001, compared with ad-GFP of same cell type. # p < 0.05, compared with ad-GFP of same cell type. 77 A t(h) Anti-Nrf2 Anti-HO-1 Anti-fi actin 0 8 24 48 B Infected Neurons Ad-GFP Ad-Nrf2 Quinone Reductase Ad-GFP Ad-Nrf2 Ad~Nrf2DN Phase GFAP Infected m C B i a. LL o I -o < < . • 1 m <r .« i * < * • * * *• V 1 • * HI si, * V Figure 2-3. Ad-Nrf2-infected cultures exhibit enhanced antioxidant potential. A, T i m e course eva lua t ion o f N r f 2 prote in overexpress ion w i t h pa ra l l e l i n d u c t i o n o f H O - 1 express ion . B, H i s t o c h e m i c a l s ta in ing revealed increased N Q O l ac t iv i ty i n ad -Nr f2 - in fec t ed neurons, but not ne ighbor ing uninfected neurons, v i s i b l e under D I C opt ics (bo t tom panels) . N e u r o n a l N Q O l s ta in ing was not observed i n the a d - G F P group (top panels) . Scale bar, 20 u m . C, x C T m R N A levels increase w i t h Nr f2 overexpress ion as s h o w n b y R T - P C R . N r f 2 m R N A de r ived f rom in fec t ion was detected b y us ing selective mouse pr imers . D, N r f 2 overexpress ion increases total in t race l lu la r G S H / G S S G leve l s . Suble tha l glutamate exposure (6 hr) leads to part ial deple t ion o f in t race l lu la r G S H i n a l l groups (open bars). C o n t r o l groups represent a separate group o f cultures w i t h no glutamate exposure but that were vehicle- t reated (f i l led bars). Cu l tu re were g i v e n a total o f 48 hr for express ion before b e i n g harvested for G S H analys is . E, Increase i n m C B i s ta in ing is p r i m a r i l y enr iched i n g l i a o f ad -Nr f2- in fec ted m i x e d cultures. Cu l tu res are depicted i n phase-contrast (Phase), f luorescence i m m u n o s t a i n i n g for a n t i - G F A P ( G F A P + ) and a n t i - G F P (Infected), and 60 u M m C B i s ta ining ( m C B i ) . m C B i s ta in ing images s h o w selected areas w i t h h igh numbers o f g l i a l clusters and are not representative o f actual m i x e d cul ture c o m p o s i t i o n . H O - 1 and N Q O l images are representative o f at least three separate exper iments . G S H data represent mean ± S E M o f four separate exper iments pe r fo rmed i n dupl icate; *p < 0 .05, c o m p a r e d w i t h G F P con t ro l . 78 2.3.3. Microarray analysis of Nrf2 over-expressing mixed cortical cultures and enriched glial cultures. To thoroughly define the gene set targeted by Nrf2, microarray analysis was performed on mRNA derived from mixed neuron/glia cultures (3 DIV) and glial-enriched cultures (5-10 DIV) 48 h after infection with ad-GFP or ad-Nrf2. Affymetrix Rat Genome U34A arrays were used to monitor the expression of - 7000 full length mRNAs and ~ 1000 expressed sequence tag clusters. To better define our culture systems, we initially assessed the basal expression of glial markers in GFP over-expressing cultures. As expected, the basal GFAP and vimentin signals were ~ 7-fold higher than in glial-enriched cultures in comparison to mixed cultures. The GFAP and vimentin signals from the two different culture types were normalized to various housekeeping genes, including lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and (3-actin, and similar results were obtained for each. The basal levels of the housekeeping genes varied by only ~ 10 % between the glial-enriched and mixed cultures. Conversely, the basal signal for the neuronal markers, neurofilament M and tau microtubule associated protein in mixed cultures were ~ 34 and ~ 62-fold higher, respectively, in comparison to glial-enriched cultures. The relative GFAP signal was further used to estimate that - 13 % of the mRNA from mixed cultures was of glial origin. Basal signals also indicated that glial-enriched cultures typically contain ~ 31-fold more endogenous Nrf2 mRNA than the mixed cultures. Interestingly, not all transcription factors were enriched in glia as basal signals for CREB, Maf 1, Maf 2, and c-Jun varied only slightly between the two culture types. However, the basal signal for NF-kappa B was also highly enriched in glial cultures (~ 15-fold). As expected from previous work, examination of basal mRNA levels indicated preferential glial expression of antioxidant factors such as N Q O l , GST A3, GST P2, HO-1, catalase, thioredoxin reductase, metallothionein 1 or 2, and peroxiredoxin 1 and 5 (Dwyer et al., 1995; Murphy et al., 1998; Ahlgren-Beckendorf et al., 1999; Eftekharpour et al., 2000; Murphy et al., 2001). 79 Analysis of three separate comparisons between mRNA from GFP and Nrf2 over-expressing cultures, consistently indicated the selective upregulation of a number of known and previously unknown phase 2 detoxification genes in response to acute Nrf2 over-expression (48 h) (Table 2-3, 2-4). Given the large number of Nrf2 upregulated genes, only some genes were listed and categorized into four groups based on function: Detoxification, Antioxidant/Reducing Potential/Metabolism, Signal Transduction, and Inflammation. A full gene list including increased and decreased genes is available online: http://www.pharmacy.wisc.edu/SOPDir/PersonDetails.cfm?Type=F&ID=38. Of the increased genes, 92 were common to both glial-enriched and mixed cultures, where genes of interest are presented in Table 3 (fold induction cut-off = 1.3, repression cut-off = -1.3). In addition, a subset of genes were selectively induced in either the mixed cultures or glial-enriched cultures. Genes of interest from this list are presented in Table 2-4. The increased expression of various GST isoforms, N Q O l , HO-1, y-GCS (only modifier light chain upregulation observed), thioredoxin reductase, and malic enzyme by Nrf2 over-expression in both cultures types corresponds to the gene induction profile previously observed when using electrophilic agents such as /er/-butylhydroquinone and sulforaphane (Ahlgren-Beckendorf et al., 1999; Eftekharpour et al., 2000; L i et al., 2002; Thimmulappa et al., 2002). Induction of these well-characterized Nrf2 gene targets are, in part, supported by our own immunoblot data for HO-1, histochemical stains for N Q O l , and measurements of GSH content (Fig. 2-3,4, B, D). Of particular interest was the induction of many genes associated with the biosynthesis, utilization, and export of GSH including: 1.) Uptake of cystine at the cell surface via the xCT cystine/glutamate antiporter, shown by RT-PCR (Fig. 2-3, 2-9 and Table 2-1) (Bannai, 1986; Sato et al., 1999). 2.) Synthesis of gGluCys by the rate-limiting enzyme for GSH synthesis y-GCS. 3.) Incorporation of Gly to gGluCys to make the complete GSH tripeptide by 80 GSH synthetase. 4.) Utilization of GSH by various GSTs and glutathione reductase. 5.) Possible efflux of GSH through the multidrug resistance protein ( M P v P l ) , a mechanism previously described for astrocyte GSH release (Hirrlinger et al., 2001; Hirrlinger et al., 2002). 6.) Possible extracellular cleavage of GSH by y-glutamyl transpeptidase (yGT). In support of our findings, recent studies have shown that MRP1 expression is Nrf2-dependent since its induction is abolished in cells derived from Nrf2 knockout mice (Hayashi et al., 2003). 81 Table 2-3. Common Nrf2 upregulated genes in glial-enriched cultures and mixed neuronal/glial cultures Glial-Enriched Neuron/Glial Mixed Genbank Accession Gene R Fold C V BS R Fold C V BS Detoxification M58495 NAD(P)H:quinone oxidoreductase 8 4.3 0.24 2621 18 5.4 0.56 141 K00136 Glutathione S transferase A l or 2 6 17.6 0.41 24 12 8.8 0.80 100 KOI932 Glutathione S transferase A3 8 12.3 0.77 4657 18 19.8 0.56 223 X62660 Glutathione S transferase A4 8 21-.7 0.56 443 18 4.1 0.42 769 X02904 Glutathione S transferase P2 8 3.4 0.38 22952 18 2.4 0.25 7230 J02722 Heme oxygenase 1 8 2.8 0.18 24137 18 21.3 1.2 358 M l 1670 Catalase 8 4.1 0.34 1381 16 1.7 0.22 593 M l 1794 Metallothionein 1 or 2 4 3.7 0.56 15750 16 2.83 0.35 1274 AI010083 Peroxiredoxin 1 8 2.9 0.06 32993 18 2.5 0.25 6609 AFO14009 Peroxiredoxin 6 (1-Cys peroxiredoxin) 8 3.3 0.12 6153 14 1.8 0.20 1772 M21060 Cu/Zn Superoxide dismutase 8 1.5 0.09 28627 16 1.9 0.27 14495 AF106563 p-glycoprotein/multi-drug resistance protein (MDR2) 6 1.9 0.23 1459 10 5.9 0.71 2671 X90642 Multi-drug resistance protein (MRP1) Signal Transduction 8 2.3 0.33 3432 14 1.7 0.13 1317 U09583 Src related tyrosine kinase 8 22.6 0.90 89 18 25.6 0.16 56 AI231354 Stress activated protein kinase, alpha 8 3.1 0.10 1097 14 1.65 0.26 880 D78610 Protein tyrosine phosphatase receptor E 7 6.7 0.27 83 14 3.5 0.80 358 M l 5427 c-raf 8 3.6 0.25 3558 18 1.6 0.22 7427 U31668 E2F-5 transcription factor 8 2.8 0.06 2242 18 1.7 0.16 933 U04835 cAMP responsive element modulator 8 2.0 0.20 2242 10 2.7 1.1 756 82 Antioxidant/Reducing Potential/Metabolism A1233261 Glutamate-cysteine ligase, modifier subunit (yGCS) 8 13.0 0.13 945 18 5.1 0.21 208 U73174 Glutathione reductase 1 8 3.3 0.15 1667 14 2.0 0.32 1324 U63923 Thioredoxin reductase 1 8 2.6 0.16 10344 16 3.4 0.56 3426 M26594 Malic enzyme 1 8 8.3 0.23 1264 18 10.1 0.35 227 A l l 69802 Ferritin H subunit H 7 1.8 0.23 50557 10 1.3 0.13 18452 J02791 Acyl-Coenzyme A dehydrogenase, medium chain 8 3.2 0.22 925 12 3.2 0.87 42 M60322 Aldose reductase 1 8 5.1 0.15 24812 18 2.4 0.18 9958 AA799452 Transaldolase 8 3.6 0.25 21356 18 1.8 0.23 19236 M96633 Mitochondrial intermediate peptidase 8 2.8 0.30 699 9 1.5 0.24 452 AA945054 Cytochrome b5 8 2.6 0.40 1902 14 1.6 0.27 2179 S53527 S-100 calcium binding protein |3 8 78.7 1.15 1167 10 2.3 0.68 67 U76714 Solute carrier family 39 (iron regulated transporter) Inflammation 6 21.3 0.27 402 18 5.9 0.27 182 AI176170 FK506 binding protein 6 1.5 0.20 19602 18 1.3 0.06 18354 AA818025 CD59 antigen 8 25.9 0.04 1128 18 1.4 0.08 10966 U03388 Cyclooxygenase 1 House Keeping 8 3.9 0.23 2083 18 5.6 0.50 312 V01217 (3 actin 186225 164621 X02231 Glyceraldehyde-3-phosphate-dehydrogenase 405 348 U07181 Lactate dehydrogenase B 13108 11253 Genes with Fold Change over 1.3 are listed. R = relative rank. C V = coefficient of variation . BS = Basal signal intensity from GFP-infected cultures. Data shown is from n = 2 glial-enriched cultures and n = 3 mixed cultures. 83 T a b l e 2-4. Nrf2 u p r e g u l a t e d g e n e s s p e c i f i c t o g l i a l - e n r i c h e d c u l t u r e s o r m i x e d n e u r o n a l / g l i a l c u l t u r e s Genbank G e n e R F o l d Q y B § Acce^sjon^ _ N e u r o n Increased Genes Antioxidant/Reducing Potential AF090867 Guanosine monophophate reductase 12 2.5 0.41 330 Signal Transduction M54987 Corticotropin releasing hormone 12 6.8 1.20 120 M19651 Fos-related antigen (Fra-1) 16 7.4 1.10 70 D15069 Adrenomedullin 12 12.7 0.74 106 Miscellaneous M84488 Vascular cell adhesion molecule-1 18 16.5 0.85 74 Astrocyte Increased Genes Detoxification/Metabolism S83436 Glutathione S-transferase, mitochondrial 4 3.9 0.27 341 AF041105 Organic anion transporter protein 4 11.3 0.47 21 S56936 UDP-glucuronosyltranferase, bilirubin specific 6 3.0 0.12 382 X56228 Rhodanese, thiosulfate sulphurtransferase 8 7.7 0.65 1686 J02852 Cytochrome P450 2A3 6 3.2 0.45 23 Antioxidant/Reducing Potential X03518 Y-ghnarnyl transpeptidase L38615 Glutathione synthetase 6.5 3.9 0.15 149 0.04 1328 Signal Transduction/Apoptotis AI228669 G A B A transporter protein 8 25.2 0.40 26 L00981 Tumor necrosis factor 4 6.7 0.13 182 M91595 Insulin-like growth factor binding protein 2 8 3.5 0.28 4228 U63740 Protein kinase C-binding protein Zeta 1 4 6.2 0.81 86 AI072770 Proteolipid protein 6 10.6 1.10 137 AI 145444 Neurabin, actin-binding protein 4 3.3 0.13 23 AF061726 Calpain 3 Inflammation 8 3.3 0.24 176 AFO10464 Interleukin 7 4 7.8 0.26 32 U66322 Dithiolethione-inducible gene (D1G-1) 8 3.0 0.52 1515 M16410 Tachykinin 2 Miscellaneous 4 4.5 0.65 244 AF098301 NFB42-neural F box 8 42.6 0.86 841 X99773 Neuroserpin, serine proteinase inhibitor 8 6.3 0.58 187 U09022 15 kDa Perforational protein (PERF) 8 97.0 0.62 29 AF029690 8-oxoguanine-DNA-glycosylase 8 4.7 0.13 1248 Genes with Fold Change over 1.3 are listed. R = relative rank. CV = coefficient of variation. BS = basal signal intensity from GFP-infected cultures. Data shown is from n = 2 glial-enriched cultures and n = 3 mixed cultures. 2.3.4. Enhancing antioxidant potential by Nrf2 over-expression protects neurons from cell death due to oxidative stress but not staurosporine-induced apoptosis. Can the increased antioxidant potential from Nrf2 over-expression protect neurons in in vitro models of oxidative stress? For this experiment, we used a well-established N M D A receptor-independent oxidative glutamate toxicity paradigm where neurons die from GSH depletion (Murphy et al., 1989; Murphy et al., 1990). When ad-GFP infected cultures were treated with 3 mM glutamate, only 12.5 + 5.3% of the infected neurons were viable after 24 h 85 (Fig. 2-4/4, B). With the same treatment, 103.7 ± 4.6% of the ad-Nrf2 infected neurons survived the toxic exposure compared to the ad-GFP control. Surprisingly, 88.0 ± 14.0% of the un-infected neurons in the same culture were also protected (Fig. 2-4/4, D). The extent of neuronal death in the ad-Nrf2DN infected group was very similar to the ad-GFP infected group. Doubling the glutamate concentration to 6 mM was not able to overcome the protective effect of Nrf2 (data not shown), suggesting that upregulated glutamate removal mechanisms such as: enhanced glutamate metabolism, glutamine synthesis from glutamate, or sodium-dependent glial glutamate uptake were not the main mechanisms for protection. Furthermore, after a 24 h incubation, glutamate levels in the medium were not significantly different among the ad-GFP and ad-Nrf2 groups as assessed by a fluorescence-based glutamate dehydrogenase cycling assay (data not shown) (Nicholls et al., 1987). To ensure that the observed toxicity was indeed a result of oxidative stress, lOOpM a-tocopherol was added in the presence of 3 m M glutamate and a nearly complete block of the neuronal death (80.3 ± 11.5% reduction, n = 4) was observed. We further ruled out the possibility of N M D A receptor mediated excitotoxicity in our paradigm by treating 3-4 DIV cultures with 200 u M N M D A overnight or with a short 500 p,M N M D A or glutamate exposure for 10 min in Hank's balanced salt solution followed by washout. Neither treatment caused appreciable neuronal death in these cultures (data not shown). The viability of glial cells was not affected by oxidative glutamate toxicity as observed in previous studies (Murphy et al., 1990) (Fig. 2-4/4, Q . 86 u_ CD z z Q Control 3mM Glu \ w , " A . . . A * i 80 nm """" B Infected Neurons 4, 3mM 3mM N = 6 VE N = 4 O i-Q. LL CD LU W z 10 0 Infected Glia i l l fefi i ^ i fri i f r i Con 0.3mM 1mM 3mM 3mM N = 6 N = 3 N = 3 N = 6 VE N = 4 r ~ 1 Kr!2 I 1 Nrf2DN Un-lnfected Neurons N Con 3mM 3mM 6 N = 6 VE N = 4 Figure 2-4. Nrf2 overexpression in a subpopulation of cells confers widespread neuronal protection from oxidative glutamate toxicity. A, Immunocytochemistry for eGFP (green, identifying infected cells) and NSE (red marker, a neuron-selective marker). Within a typical ad-GFP-infected culture the infected neurons (yellow, red + green), uninfected neurons (red), and infected glia (green) can be observed. (For black an white versions of this thesis, please use quantitated data graphs in panels B ,C, and D). B, Group data evaluating the vulnerability of infected neurons to oxidative glutamate toxicity. Data are expressed as a percentage of GFP + NSE + cells (presumed infected neurons) in the indicated glutamate treatment group as compared with the ad-GFP control group. VE, Vitamin E (cc-tocopherol), 100 uM. C, Viability of GFP + N S E " cells (presumed infected glia) present per image was not affected significantly with glutamate treatment. D, Uninfected neurons within cultures containing Nrf2-infected cells are more resistant to oxidative glutamate toxicity. Data represent the mean ± SEM number of cells counted over triplicate wells from at least three independent experiments; *p < 0.05, compared with the GFP control no-g!utamate group. Ad-Nrf2 infected cultures were also more resistant to direct exposure to the pro-oxidant molecule H 2 O 2 . A d - G F P or ad-Nrf2 infected cultures were exposed to 0.3 - 30 p M H 2 O 2 for 24 h. Under these conditions, the ad-GFP infected group began to show significant neuronal toxicity at 3 p M H 2 O 2 and maximum cell death at 30 p M H 2 O 2 (Fig. 2-5A). Infecting the cultures with ad-Nrf2 reduced neuronal toxicity by 53.1 + 16.9% of the ad-GFP control during exposure to 10 u M H 2 O 2 . The protective effect of Nrf2 was overcome by 30 p M H 2 O 2 . Neuronal injury from oxidative stress can exhibit features of an apoptotic pathway (Whittemore et al., 1995; Tan et al., 1998). To test whether Nrf2 upregulation could protect neurons from apoptosis induced by a different pathway than direct exposure to R O S , we treated 87 infected cultures with 0.1 - 10 u M staurosporine, a potent inhibitor of phospholipid/Ca++ dependent protein kinase (Tamaoki et al., 1986). Interestingly, over-expression of Nrf2 did not confer any protection from staurosporine-induced toxicity under these conditions (Fig. 2-5B). In both ad-GFP and ad-Nrf2 infected groups cell death was first observed with 0.3 u M while maximal toxicity was achieved with 10 uM. H 20 2 (uM) *~ Staurosporine (uM) Figure 2-5. Nrf2 overexpression in mixed immature cortical cultures protects neurons from H202-mediated toxicity, but not staurosporine-induced apoptosis. A d - G F P - and ad-Nrf2-infected cultures were allowed to express transgenes for 48 hr before exposure to 0.3-30 u M H 2 0 2 (A; n = 3) or 0.1-10 p M staurosporine (B; n = 4) for a further 24 hr. Cells were stained for N S E to evaluate neuronal viability. Data represent the mean ± S E M from the indicated number of experiments performed in quadruplicate; *p < 0.05, compared with ad-GFP-infected control. 2.3.5. Glutathione released from ad-Nrf2 infected glial is necessary and sufficient for protecting neurons from oxidative glutamate toxicity. Given that a small number of ad-Nrf2 infected glia are present in infected mixed cortical cultures (~ 2% of the total cell number according to manual cell counting data), it is possible that these enhanced glial cells contributed to the observed protection of neurons. To address this possibility, we tested whether ad-Nrf2 infected glia transplanted into naive (un-infected) mixed cultures could protect resident neurons. Ad-Nrf2 infected glia were plated directly into nai've cultures at 0.1 - 2% of the total cell number using the co-culture setup, as detailed in the 88 Materials and Methods (Fig. 2-\A). Amazingly, up to 83.3 ± 22.3% of the naive neurons were protected from 3 mM glutamate when the glia were transplanted at only 0.5%> (Fig. 2-6A, B, Q. Complete neuronal protection was achieved when glia were plated at 1.5 % of the total cell number. This protection was not observed with ad-GFP, ad-Nrf2DN infected, or un-infected glia. It should be noted, however, that the plated glial densities slightly underestimate the number of glia during exposure to toxins 24 h after plating due to glial proliferation. A No Glu 3mM Glu c OL L L CD CN • • \ j s •• • 1 » \ ^ | 80 nm' CO e3 o o o B — • — GFP -•- Nrf2 —A— Nrf2DN 200' 180' 160' 140' 120' 100 -80 60' 40 20 0 — No Glu o 200 c 180 5 O 160 140 o 120 z 100 Q_ LL 80 O 60 40 LU 20 OT Z 0 0 0.1 0.5 1.5 2.0 T r a n s p l a n t e d Gl ia (% of tota l cel ls) 3 m M Glu 0 0.1 0.5 1.5 2.0 T r a n s p l a n t e d Gl ia (% of total cel ls) Figure 2-6. A small fraction of infected glial cells is sufficient to protect neurons from oxidative glutamate toxicity. A, Representative images from glial-neuron coculture setup (see Fig. \A). Within a typical coculture infected glia (green, irregular shaped bright cells) and uninfected neurons (red, shaded small cells often in clusters) can be observed. Uninf, Uninfected glia transplanted. (For black an white versions of this thesis, please use quantitated data graphs in panels B and C). B, Group data obtained from plate scanning for NSE (red) fluorescence. C, Decreased neuronal viability is demonstrated by a loss of red fluorescence. The addition of Nrf2-overexpressing glia restores NSE expression to levels found in an ad-GFP-infected group that was not exposed to glutamate. Data represent the mean ± S E M from three separate experiments performed in quadruplicate; *p < 0.05. Scale bar, 80 um. 89 Glia are known to release a number of protective factors that enhance neuronal survival through a variety of mechanisms. Of particular importance is the release of sulfhydryl species such as GSH, cysteine-glycine (CysGly), or cysteine, which can contribute to neuronal GSH synthesis (Sagara et al., 1993a; Dringen et al., 1999; Wang and Cynader, 2000). When enriched glial cultures are infected with ad-Nrf2, both intracellular and released GSH are increased (Fig. 2-7'A, B). When glia are infected with ad-Nrf2 and plated at 2 x 104 cells/mL (equivalent to 2% of the total cell number), the GSH accumulated in the medium over 24 h was significantly increased to 11.5 ± 1.9 pM, compared to 4.6 ± 0.8 uM with ad-GFP, or 2.8 + 1.2 u M with ad-Nrf2DN. Filtering the medium (< 3 kDa size restriction) did not significantly reduce the GSH measured in the medium suggesting that the released GSH was not protein bound and was most likely in its free form. Indeed, neuronal toxicity was completely prevented when naive cultures were exposed to 3 m M glutamate in the presence of 10 p M exogenous GSH for 24 h (Fig. 2-1C). This suggests that the concentration of GSH released into the medium by a small ad-Nrf2 infected glial component was sufficient for neuronal protection from oxidative glutamate toxicity. To test whether GSH release from ad-Nrf2 infected glia was necessary for the observed neuronal protection, L-buthionine sulfoximine (BSO), an irreversible blocker of y-GCS, was used to inhibit glial GSH synthesis (Griffith and Meister, 1979). To avoid potential effects of BSO on neuronal GSH, infected glia were plated on culture plate inserts and treated with 200 pM BSO for 24 h prior to transfer into neuronal cultures using the membrane delimited co-culture setup (Fig. 2-15). For this set-up, glial cells were situated in an upper layer 1-2 mm above naive neurons in the bottom of a well. BSO pre-treatment of ad-Nrf2 infected glia suppressed levels of released GSH to ~ 3 p M for more than 24 h after washout (data not shown) and completely blocked the Nrf2-mediated protection of neurons (Fig. 2-1D). 90 A B £ 16 TO *° 12 o * 10 6 4 2 0 x co O T3 0> C D 0) <D CK Glutamate (mM) F i g u r e 2-7. R e l e a s e o f G S H f r o m g l i a i s b o t h s u f f i c i e n t a n d n e c e s s a r y f o r c o n f e r r i n g n e u r o n a l p r o t e c t i o n . A, B, Infection with ad-Nrf2 increases total intracellular GSH/GSSG as well as GSH released into the medium (MEM/5% FCS, no phenol red). C, Exogenous addition of reduced GSH concurrently with glutamate treatment protects neurons from oxidative glutamate toxicity. D, Glial GSH release is necessary for Nrf2-dependent neuronal protection. A membrane-delimited coculture (see Fig. 2-15) was used, allowing enriched glial cultures to be pretreated separately with the GSH synthesis inhibitor BSO and then to be washed and added to wells containing neurons. BSO pretreatment (200 pM) for 24 hr produces long-term reduction of intracellular GSH/GSSG and release of GSH from glia and abolishes glial-mediated neuronal protection. Data represent the mean ± SEM of at least three independent experiments performed in triplicate; #*p < 0.05. 91 2.3.6. Small molecule inducers of Nrf2 increase neuronal survival during oxidative glutamate toxicity. Compounds such as fer/-butylhydroquinone (tBHQ) and dimethyl fumarate, known inducers of Nrf2 activation and Phase 2 gene induction, have been shown to protect various cell lines against oxidative stress due to H 2 O 2 , oxidative glutamate toxicity, and dopamine exposure, and protect primary astrocytes from H 2 O 2 and platelet activating factor (Murphy et al., 1991; Duffy et al., 1998; L i et al., 2002; Lee et al., 2003c). In this study, we have found that direct pre-treatment of immature mixed cortical culture with 10-20 p M tBHQ for 24 h efficiently blocks oxidative glutamate toxicity (Fig. 2-8A). To compare the protective potential of tBHQ treated glia with ad-Nrf2 over-expressing glia, enriched glial cultures were treated with 5-20 p M tBHQ for 24 h to allow upregulation of an antioxidant response before being transplanted into naive cortical cultures using the co-culture setup (Fig. 2-1 A). Glia pre-treated with 20 p M tBHQ provided neuronal protection from 3 m M glutamate when transplanted at a plating density of 5% of the total cell number (Fig. 2-8B). However, protection was not seen when tBHQ-treated glia were transplanted at 2%, a density sufficient for neuronal protection when using ad-Nrf2 infected glia. Treating glial with tBHQ increased intracellular GSH content as previously described (Eftekharpour et al., 2000), and also increased released GSH in a dose-dependent fashion (data not shown). 92 A tBHQ • • None E S 1 0 uM C D 2 0 uM 0 1 3 Glutamate (mM) 10 20 tBHQ (uM) Figure 2-8. Neuronal protection can be achieved by activation of endogenous Nrf2 with the use of a small molecule inducer. A, Immature cortical cultures pretreated for 24 hr with 10 and 20 u M t B H Q are protected from 1-3 m M glutamate exposure. Partial protection is conferred by t B H Q treatment at 3 m M glutamate. B, Selective induction of endogenous Nrf2 in glia led to partial neuronal protection from oxidative glutamate toxicity. Glia pretreated with a range of t B H Q concentrations (0-20 uM) for 24 hr were transplanted into naive neuronal cultures at a plating density of 5% of the total cell number, using a coculture setup (see Fig. 2-1A). Data represent the mean ± S E M from three independent experiments performed in quadruplicate; *p < 0.05. • 2.4. Discussion 2.4.1. Nrf2 over-expression boosts antioxidant potential of glia: Protection of neurons from oxidative glutamate toxicity. Over-expression of the Cap 'n ' Collar transcription factor Nrf2 upregulates a strong antioxidant response in both neurons and glia dissociated from rat cortex. We show that protection of neurons can be achieved by upregulation of the glial antioxidant response alone. Central to this protection was the augmented synthesis and release of glial GSH, which can be used toward strengthening the neuronal GSH pool. This conclusion was based on several lines of evidence: First, ad-Nrf2 infection of glia significantly increased intracellular and released GSH. Second, microarray analyses indicated the coordinated induction of GSH biosynthesis, utilization and export enzymes. Third, exogenous GSH addition mimicked neuroprotection offered by ad-Nrf2 infected glia, and selective depletion of glial GSH using BSO abolished this 93 neuroprotection. Thus, Nrf2-dependent enhancement of glial GSH release appears to be necessary and sufficient for neuronal protection. However, it is important to note that suppression of glial GSH synthesis may have deleterious effects on glial health and could possibly alter the expression of other glial-derived protective factors. Although, GSH plays an important role for protection in the oxidative glutamate toxicity paradigm, we cannot completely rule out the involvement of other glial-derived factors. It is well documented that astrocytes can protect neurons from damage due to various ROS (Desagher et a l , 1996; Lucius and Sievers, 1996; Tanaka et al., 1999; Kirchhoff et al., 2001). Previous studies have shown that an adequate astrocyte GSH content is essential since depletion of GSH with BSO abolishes astrocyte-mediated neuroprotection (Drukarch et al., 1997; Chen et al., 2001). One mechanism underlying this protection is the continuous glial delivery of GSH and/or GSH precursors such as cysteine to neurons for GSH synthesis (Yudkoff et al., 1990; Sagara et al., 1993a; Sagara et al., 1993b; Dringen et al., 1999; Dringen et al., 2000). Interestingly, GSH efflux becomes markedly increased when the astrocytes are exposed to oxidative stress induced by cadmium chloride (Sagara et al., 1996), an agent that induces Phase 2 expression through Nrf2 in culture (Ishii et al., 2000). Although the events necessary for the neuronal uptake of GSH and its precursors remain uncertain, evidence suggests that a number of sulfhydryl species can be used by neurons including cysteine, cystine and the CysGly dipeptide (Murphy et a l , 1990; Sagara et al., 1993a; Sagara et al., 1993b; Kranich et al., 1996a; Wang and Cynader, 2000). Two majors paths can be initiated upon the release of GSH into the medium/cerebral spinal fluid (CSF) (Fig. 2-9). GSH can be metabolized into CysGly and Glu-X conjugates by the glial ectoenzyme y-GT (Dringen et al., 1999). CysGly can be taken up by neurons, or can be further hydrolyzed by the ectoenzyme aminopeptidase N to release cysteine (Dringen et al., 2001). Alternatively, a constant release of GSH can potentiate an extracellular thiol/disulfide exchange reaction with cystine to produce 94 cysteine and a GSH-cysteine conjugate (Wang and Cynader, 2000). There is currently little direct evidence to support the uptake of GSH by neurons. However, both sodium-dependent and independent GSH transport systems have been isolated from brain cells including astrocytes (Kannan et al., 2000), bovine brain capillary cells (Kannan et al., 1996), and immortalized mouse brain endothelial cells (Kannan et al., 1999). Furthermore, sodium-dependent GSH transport in rat synaptosomal membrane vesicles exhibited high affinity kinetics ( K m = 4.5 + 0.8 pM) (Iantomasi et al., 1999) and may be physiologically relevant for uptake considering the low GSH levels found in CSF (5.87 ± 0.29 pM) (Wang and Cynader, 2000). A recent study suggests that neuronal cysteine uptake through the high affinity glutamate transporter EAAC1 was a primary means of maintaining neuronal GSH levels in vivo (Aoyama et al., 2005). Knockouts for E A A C 1 exhibited decreased neuronal glutathione levels developed brain atrophy and behavioral changes with aging. Brain slices from EAAC1 knockouts showed increased oxidant levels and susceptibility to injury, an effect that could be reversed by N-acetylcysteine, a membrane permeable version of cysteine. This report further supports our finding that astrocytes maintain the neuronal GSH pool during oxidative stress. Cysteine would not exist in the highly oxidizing extracellular space unless there was a constant release of reducing agents such as GSH. G l u - X 95 F i g u r e 2-9. S c h e m a t i c d i a g r a m o f G S H b iosynthes i s a n d release p a t h w a y s t h a t m a y be i n v o l v e d wi th Nrf2-dependent c o u p l i n g o f G S H between as trocy tes a n d n e u r o n s . Astrocyte GSH synthesis and release are more robust with Nrf2 overexpression. Higher levels of xCT (system x c ) expression were detected, which promotes cystine uptake and provides a precursor for GSH synthesis. Microarray analyses indicate that all major enzymes involved in GSH biosynthesis are upregulated also. GSH release from astrocytes is increased, leading into several possible pathways of extracellular GSH metabolism, including an initial breakdown by Y-glutamylcysteine transpeptidase (Y GT) and possible further breakdown by aminopeptidase (Apep) into the glutathione precursors Cys, Gly, and CysGly suitable for neuronal uptake (Dringen et al., 1999; Dringen et al., 2001). Alternatively, extracellular GSH may be taken up by neurons directly or may contribute to the reduction of cystine into cysteine, which may be a source of sulfhydryl species for neuronal uptake (Sagara et al., 1993a; Wang and Cynader, 2000). Neurons may also uptake cystine for glutathione synthesis (Murphy and Baraban, 1990). Microarray analyses also indicate the upregulation of additional factors involved in detoxification, ROS scavenging, and N A D P H production that may work together with GSH to protect neurons. Genes that are upregulated significantly by Nrf2 overexpression are underlined. Intracellular and extracellular concentrations of glutamate are average values from a combination of previous studies in the human brain and CSF (Siegel, 1981). CSF levels of cystine are typically very low at ~0.2 uM (Lakke and Teelken, 1976; Araki et al., 1988; Wang and Cynader, 2000). The Invitrogen M E M used in this study is formulated to contain 100 uM cystine. Follow-up studies from our lab have used real-time mCBi fluorescence measurement to re-evaluate the rate of conjugation, total content, synthesis, and efflux of GSH in cultured astrocytes (Sun et al., 2005). The results of this study suggest that Nrf2 activation by tBHQ, a small molecule Nrf2 inducer, primarily enhances GSH synthesis and total content without affecting GSH conjugation and efflux. Thus, the function of enzymes such as xCT, yGCS and glutathione synthase may play a primary role in Nrf2-mediated neuroprotection by cultured astrocytes in the toxicity paradigms we have examined. Although multiple enzymes in the GSH-mediated antioxidant defense were identified in our microarray, we did not detect a significant change in the expression of glutathione peroxidase, an enzyme necessary for GSH-dependent detoxification of H 2 O 2 . However, recent studies in peripheral tissues (small intestines and liver) have shown that the glutathione peroxidase gene carries a functional A R E and can be regulated by Nrf2 (Cho et al., 2002b; Thimmulappa et al., 2002; Banning et al., 2005). Further biochemical experiments are required to directly examine the expression status and/or enzymatic activity of glutathione peroxidase 96 during Nrf2 overexpression in astrocytes. Glutathione peroxidase is expected to play an important role in the observed Nrf2-mediated neuroprotection from direct H 2 O 2 exposure. For a toxicity paradigm, we have primarily used oxidative glutamate toxicity, where neurons are exposed to millimolar levels of extracellular glutamate to completely inhibit cystine uptake through the cystine/glutamate antiporter, system xc", leading to GSH depletion and oxidative stress (Murphy et al., 1989; Murphy et al., 1990). Given the upregulation of GSH pathways we have observed, it is not surprising that Nrf2 activity provides robust neuroprotection during oxidative glutamate toxicity. In vivo, oxidative stress resulting from glutamate toxicity may potentially play a role in cell death during stroke, injury and neurodegenerative disease (Coyle and Puttfarcken, 1993). Oxidative glutamate toxicity has also been postulated to be a component of the excitotoxic cascade (Schubert and Piasecki, 2001). Under these conditions, extracellular levels of glutamate need not be in the millimolar range to inhibit cystine uptake since CSF cystine levels are quite low and can be easily inhibited by only 100 uM glutamate which is below the level of extracellular glutamate found in models of stroke and trauma (McAdoo et al., 1999; Wang and Cynader, 2000; Schubert and Piasecki, 2001). It is clear that Nrf2 is important for cellular resistance to pro-oxidant molecules ( H 2 O 2 , fert-butylhydroperoxide, and peroxynitrite), oxidant generating molecules (menadione), in vivo situations that potentiate ROS-mediated damage (hyperoxic lung injury), as well as cytotoxic molecules that are neutralized by GSH detoxification or glucuronidation (4-hydroxynonenal, acetaminophen) (Duffy et al., 1998; Chan and Kan, 1999; Gao et al., 2001; Cho et al., 2002b). However, it remains to be determined whether Nrf2 can provide resistance to toxicity paradigms that are not completely ROS-dependent (i.e. excitotoxicity). Our data suggests Nrf2-mediated protection by electrophilic inducers does not extend to staurosporine toxicity and cell death attributed caused by ionomycin (allows extracellular calcium influx) (Duffy et al., 1998), both of which can initiate the apoptotic cascade but may not necessarily rely on ROS accumulation 97 (Ratan et al., 1994). Another recent study also demonstrated the specificity of Nrf2-mediated neuroprotection by showing that increasing Nrf2 activity offered protection from 6-hydroxydopamine- and 3-morpholinosydnonimine toxicity but not l-methyl-1-4-phenylpyridinium toxicity (MPP +) (Cao et al., 2005). The authors suggest that the latter may not rely ROS generation to induce apoptotic cell death. Interestingly, neuronal cultures derived from Nrf2 knockout mice may not have the same specificity, since Lee et al. found these cultures to be sensitive to MPP + , ionomycin and 2,5-di-(/-butyl)-l,4-hydroquinone (inhibits endoplasmic reticulum Ca2+-ATPase)-induced toxicity (Lee et al., 2003a). The apparent discrepancy on neuroprotection from M P P + between groups may be due to differences in cell type. Cao et al. used a neuroblastoma cell line where as Lee et al. used mixed primary neuronal/glial cultures. Further, Lee et al. propose that Nrf2 may also play a role in C a + + homeostasis (Lee et al., 2003a). Loss of Nrf2 function led to an altered expression of a number of genes involved in C a + + homeostasis in Nrf2 knockout mice including: visinin-like 1, calbindin-28K, synaptotagmin-1, hippocalcin, synaptotagmin-5, nucleobindin-2, calmodulin III, and ryanodine receptor-3. However, it is unclear whether these genes are directly regulated by Nrf2 binding, or i f reduced expression of these genes could arise from compensatory changes due to development without Nrf2 function. The role of Nrf2 in C a + + regulation will need to be explored further. In a recent study, Morito et al. found that thymocytes and hepatocytes from Nrf2 knockout mice were more susceptible to death induced by anti-Fas antibody and TNFa , which can initiate the extrinsic apoptotic pathway (Morito et al., 2003). Interestingly, administration of cell-permeable GSH was sufficient to block this enhanced cell death in Nrf2 knockout. The authors concluded that Nrf2 could modulate death receptor-mediated apoptosis by controlling intracellular GSH levels. 98 2.4.2. Characterization of the Nrf2 inducible gene set in neurons and glia using microarray analysis. The Phase 2 enzyme gene set regulated by Nrf2 is rapidly broadening with the use of microarray technology (Li et al., 2002; Thimmulappa et al., 2002). These studies demonstrate that small molecule inducers of Nrf2 such as tBHQ and sulforaphane, coordinately upregulate enzymes involved in several important lines of cellular defense: 1.) antioxidant scavenging of ROS, 2.) neutralization of electrophiles and xenobiotics by glucuronidation and glutathione conjugation, and 3.) NAD(P)H generating metabolic pathways. Our results are in agreement with the enhancement of these lines of cellular defense, in particular, GSH biosynthesis and utilization pathways, during Nrf2 activation. The fact that Nrf2 upregulates GSH related enzymes is consistent with our observation that glial GSH release is important for neuroprotection. However, in light of the microarray data which implicates that action of additional factors such as malic enzyme, ferritin H, HO-1, and peroxiredoxins, we believe Nrf2 activation in vivo leads to the upregulation of multiple enzymes that work in conjunction with enhanced GSH to account for changes in cerebral metabolism after insults such as stroke. There are certain advantages to using Nrf2 over-expression as an approach to identify the Nrf2-regulated gene set. Over-expression leads to robust activation of the A R E and potentially induces more subtly activated genes that may have escaped detection while using tBHQ or other inducers as a transcriptional stimulus. In addition, acute viral over-expression also complements the comparison of gene expression between wild-type and Nrf2 knockout mice, without the added complication of potential gene compensation due to chronic absence of Nrf2 over the life span of the animal (Thimmulappa et al., 2002). Since basal Nrf2 levels in neurons are considerably lower than glia, the induction of these enzymes in neurons by endogenous Nrf2 may not necessarily follow the same profile exhibited by Nrf2 over-expression (Table 2-3, 2-4). Furthermore, the amount of Nrf2 expressed 99 in neurons is likely not the only factor regulating Phase 2 enzyme expression. For example, despite large fold changes in many Phase 2 enzyme mRNAs in mixed cultures (containing mostly neurons), after Nrf2 over-expression, the absolute mRNA levels for many phase 2 enzymes were still well below that found in glial cultures. An estimate of this discrepancy can be made by multiplying the fold induction by the basal signal in Table 2-3. Perhaps glial cells have higher levels of other ARE-specific transcription factors such as Nrfl that can contribute to a portion of the observed Phase 2 enzyme gene induction (Venugopal and Jaiswal, 1996, 1998b). Alternatively, glia may have reduced Nrf2 degradation through the ubiquitin-proteosome pathway (Nguyen et al., 2002; Sekhar et al., 2002; Stewart et al., 2002). However, it is worth noting that a follow-up study by Kraft et al, which used cell-sorting technology to purify neuronal and glial samples, also found that Nrf2 mRNA was ~4-fold higher in glia compared to neurons (Kraft et al., 2004). Nrf l enrichment in glia was not reported. Given that the expression of Phase 2 detoxification enzymes is generally lower in neurons (Eftekharpour et al., 2000; Murphy et al., 2001; Johnson et a l , 2002), the large number of genes that are apparently upregulated in the mixed cultures may reflect, in part, the presence of contaminating glial cells in these cultures. However, genes that appear to be present only in mixed cultures but not in glial-enriched cultures suggest that the gene lists may represent a set of Nrf2-regulated genes specific to neurons, including guanosine monophosphate reductase and vascular cell adhesion molecule 1 (Table 2-4). Similarly, genes that appear to be selectively upregulated in glia cells were also identified, including yGT and UDP-glucuronosyltransferase. Nevertheless, it is important to note that increased expression of a gene in mixed cultures but not glial-enriched cultures does not necessarily prove that the gene is exclusively upregulated in neurons. It is possible that glial gene expression could be altered by direct contact with neurons or response to diffusible neuron-derived factors. We have not verified the expression of these putative neuron or glial-specific genes using in situ hybridization or immunostaining. A follow-100 up study by Kraft et al. combined cell sorting and microarray analyses to more thoroughly examine the Nrf2-regulated gene sets specific to neurons and astrocytes (Kraft et al., 2004). Their results confirmed the enriched expression of antioxidant/detoxification Phase 2 enzymes in glia. However, in addition to previously identified antioxidant pathways, their studies also suggest that specific Nrf2-dependent gene sets in both astrocytes and neurons may modulate cell adhesion and integrin signaling (i.e. fibronectin, laminin, calpain), energy production (i.e. glucan branching enzyme, transketolase, facilitated glucose transporter), and synapse function (i.e. Rapsyn, neuroserpin, syntaxin bp3, synaptotagmin 4) (Kraft et al., 2004). However, the actual involvement of these genes in proposed glial-neuron interactions remains to be tested in functional assays. 2.4.3. Small molecule inducers of ARE gene expression in brain cells In recent years, small molecules that induce the activation of Nrf2 have been well characterized (Alam et a l , 1999; Ishii et al., 2000; Lee et al., 2001a; Kraft et al., 2004). In this study, we found that the selective activation of glial Nrf2 by the inducers partially protected neighboring neurons from oxidative stress, whereas direct treatment of mixed cultures provided more extensive neuronal protection. It is possible that when mixed cultures are treated with tBHQ, induced neurons are upregulating their own antioxidant defenses in addition to accepting any diffusible sulfhydryl contributions from induced glia. Consistent with this observation, Johnson et al. demonstrated using transgenic ARE-hPAP reporter mice that neurons display enhanced ARE-mediated gene expression with tBHQ treatment albeit at a considerably lower level than glia (Johnson et al., 2002). As mentioned above, later studies examined the Nrf2-regulated gene set expressed in neurons and found that it consisted of genes that enhance synapse function, cell adhesion, and energy production. These results suggest that Nrf2-mediated gene 101 expression in neurons may involve more diverse functions than enhancing antioxidant potential, as we have described in astrocytes (Kraft et al., 2004). From a practical standpoint, diet-based therapies that deliver Phase 2 enzyme inducers to the brain may prove to be an efficient and straightforward means for neuroprotection (Fahey et al., 1997). In the brain, activation of both glial and neuronal antioxidant responses with low doses of electrophilic agents may be sufficient for protecting neurons from oxidative stress. Furthermore, if glial delivery of sulfhydryl species to neurons occurs in vivo, efficient coupling is expected considering the close association of neurons and glia and the much higher glial to neuron ratio in the CNS (Travis, 1994). In conclusion, Nrf2 activation in glia produces a strong antioxidant response, particularly through the GSH synthesis and utilization pathway. Neuronal viability is significantly enhanced by an increased supply of GSH precursors from Nrf2 over-expressing glia. In subsequent chapters, we will determine whether similar neuroprotection can be achieved in vivo by induction of the phase 2 response using either administration of small molecule inducers or strategic adenoviral-mediated Nrf2 over-expression in certain areas of the brain. 102 Chapter 3 : Induction of the Nrf2-driven Antioxidant Response Confers Neuroprotection During Mitochondrial Stress in vivo2 2 A version of this chapter has been published. Shih A.Y., Imbeault S., Barakauskas V., Erb FL, Jiang L., Li P., and Murphy T.H. (2005) Induction of the Nrf2-driven Antioxidant Response Confers Neuroprotection During Mitochondrial Stress in vivo. Journal of Biological Chemistry. 2005 Jun 17, Vol. 280, Issue 8, pgs. 22925-22936 103 3.1. Introduction The Cap 'n ' Collar transcription factor NF-E2 related factor (Nrf2) controls the coordinated expression of important antioxidant and detoxification genes (Phase 2 genes) through a promotor sequence termed the antioxidant response element (ARE)(Rushmore et al., 1991; Itoh et al., 1997b; Alam et al., 1999). Phase 2 genes work in synergy to constitute a pleiotropic cellular defense that scavenges reactive oxygen/nitrogen species (ROS/RNS), detoxifies electrophiles and xenobiotics, and maintains intracellular reducing potential (Ishii et al., 2000; Talalay, 2000; Thimmulappa et al., 2002; Lee et al., 2003c; Shih et al., 2003). Normally, Nrf2 is sequestered in the cytoplasm by the actin-bound regulatory protein, Keapl (Itoh et al., 1999b; Kang et al., 2004). Multiple cysteine residues allow Keapl to act as a molecular 'switch' by responding to electrophiles/ROS with a conformational change, which releases Nrf2 for nuclear translocation and activation of Phase 2 gene expression (Dinkova-Kostova et al., 2002; Itoh et al., 2003; Wakabayashi et al., 2004). Thus, Nrf2 provides an important mechanistic link between oxidative stress leading to cell death and antioxidant gene expression supporting cell survival. Nrf2_/" mice are particularly maladapted when challenged with toxicity paradigms such as hyperoxic lung injury, butylated hydroxytoluene-induced acute pulmonary injury, acetaminophen-induced liver toxicity, benzo[a]pyrene-induced tumourigenesis in the forestomach, or cigarette smoke-induced emphysema (Ramos-Gomez, ; Chan and Kan, 1999; Chan et al., 2001; Enomoto et al., 2001; Cho et al., 2002b; Fahey et al., 2002). Although the protective function of Nrf2 is apparent in peripheral tissues, the role of Nrf2 in the brain, where substantial ROS/RNS production can occur due to a high rate of metabolism, remains unclear. We hypothesize Nrf2 function is critical for supporting neuronal survival during neurodegenerative disease when aberrant ROS/RNS production is known to be exacerbated 104 (Coyle and Puttfarcken, 1993). To study the effect of Nrf2 activity on in vivo neurodegeneration, we systemically administered 3-nitropropionic acid (3-NP), an irreversible inhibitor of succinate dehydrogenase (SDH), which causes striatal-specific cell loss leading to impairment of motor function (Beal et al., 1993b). Metabolic inhibition by 3-NP produces oxidative stress in the brain through multiple mechanisms: 1.) ATP depletion, membrane depolarization, increased neuronal/glial glutamate release, over-activation of N-methyl-D-aspartate ionotropic glutamate receptors, accumulation of intracellular Ca 2 + , and excessive mitochondrial ROS production (Reynolds and Hastings, 1995; Schulz et al., 1995). 2.) Secondary activation of Ca2+-dependent enzymes such as phospholipase A2 (production of inflammatory mediators) and nitric oxide synthase (peroxynitrite formation) (Beal, 1992, 1995). 3.) Excessive dopamine release, which can generate H2O2 when metabolized (Reynolds et al., 1998). Upregulation of Nrf2 activity in the brain is an attractive strategy for mitigating ROS/RNS overproduction during neurodegenerative disease. To test the hypothesis that augmentation of Nrf2 could protect neurons in vivo, we increased Nrf2 activity in the brain using dietary administration of the small molecule inducer, /ert-butylhydroquinone (tBHQ) or adenoviral-mediated overexpression of Nrf2 (Shih et al., 2003). In previous studies, these treatments protected cultured neurons from toxicity paradigms that mimic aspects of in vivo neurodegeneration, such as glutamate-induced glutathione depletion, dopamine toxicity, direct exposure to H2O2, metabolic inhibition with mitochondrial toxins (rotenone), platelet activating factor-induced inflammatory responses, and increased intracellular C a 2 + (Murphy et al., 1991; Duffy et al., 1998; Lee et al., 2003b; Shih et al., 2003; Kraft et al., 2004). Importantly, neuronal cultures derived from NrO"'" mice showed increased susceptibility to neurotoxicity paradigms and were not protected by Nrf2 inducers, but could be rescued by overexpression of Nrf2 (Lee et al., 2003a; Kraft et al., 2004). 105 Our in vitro and in vivo data suggest that Nrf2 activation is a protective response to counter the toxic effects of metabolic inhibition and ROS/RNS production in the brain. Loss of Nrf2 function in Nrf2"A mice exacerbated motor deficits and striatal lesions caused by 3-NP administration. Conversely, pre-activation of the Nrf2 response (using dietary administration of a Nrf2 inducer or viral gene transfer) attenuated 3-NP toxicity. Nrf2 activation may be a viable strategy to prime the antioxidant capacity of the brain, thereby decreasing injury caused by progressive neurodegeneration. 3.2. Materials and Methods 3.2.1. Chemicals A l l chemicals were obtained from Sigma-Aldrich unless otherwise stated. 3.2.2. In vivo 3-NP dosage regimen A l l experiments were approved by the U.B.C. Animal Care Committee and were conducted in strict accordance with guidelines set by the Canadian Council on Animal Care. Adult male Wistar rats (Charles River, Canada) (250-350 g) and Nrf2"A mice (C57B1/6 / SV129 background), originating from the laboratory of Dr. Yuet Wai Kan (Chan et al., 1996b), were maintained in a 12 h light-dark cycle with food and water ad libitum. 3-NP was prepared in 0.1 M phosphate buffered saline (PBS) and adjusted to pH 7.4 with NaOH and maintained at 4°C for up to 1 week. For Nrf2 mice, a similar number of males and females (aged 10-16 weeks) were used. The 3-NP treatment regimen consisted of 9 total intraperitoneal (i.p.) injections with one injection given every 12 h at successively increasing doses: 20 mg/kg x 4, 40 mg/kg x 4, 60 mg/kg x 1 (Fernagut et al., 2002). No significant difference was observed between males and females with respect to behaviour, weight loss, or lesion size in this study. For rats, daily i.p. injections of 3-NP (n = 10) or PBS control (n = 10) were administered by one of two dosage 106 regimens: (40 mg/kg, 40 mg/kg, 20 mg/kg) or (40 mg/kg, 20 mg/kg, 20 mg/kg, 20 mg/kg), each regimen resulting in the animal receiving a total of 100 mg/kg of 3-NP over 3 or 4 days. Both dosage regimens were tested in order to overcome possible preconditioning effects due to adenovirus injection. However, the average lesion volumes produced by the two dosage regimens were not different after statistical analyses (two-tailed t-test), and the groups were pooled. Since virus was injected unilaterally we conservatively only included in the study animals with measurable 3-NP lesions by histology. Animals were sacrificed 24 h after the last injection or upon displaying severe motor behaviour deficits such as recumbence (complete loss of motor control). 3.2.3. Semi-qualitative Behaviour Scoring of Mice A detailed description for the assessment of 3-NP induced motor deficits was described previously (Fernagut et al., 2002). Briefly, the scoring system involved the evaluation of 5 major symptoms each with a 3-tiered scoring scale (0, 1, or 2), with 0 = normal behaviour and 2 = severe deficit. Symptoms included: a.) hind limb clasping, b.) reduced general locomotor activity, c.) hind limb dystonia (increased muscle tone leading to abnormal jerky movements), d.) truncal dystonia, and e.) postural instability. Animals received a cumulative score out of 10 for each trial. Behaviour was assessed by an experimenter blinded to the animal genotype and treatment before each scheduled injection. 3.2.4. Histology and Fluorojade Staining Mice were deeply anesthetized with euthanyl (Bimeda-MTC) and trans-cardially perfused with room-temperature PBS followed by ice-cold 4% paraformaldehyde in PBS (PFA). The brains were post-fixed overnight in PFA and then cryoprotected with 30% sucrose for 2 days before cryostat sectioning. Cresyl violet (CV) staining was performed with standard protocols on 107 40 pm sections mounted on SuperFrost Plus slides (Fisher). Lesion volume was calculated according to the principle of Cavalieri (volume = sidi + S2d2 + S 3 d 3 + S4CI4) where s = lesion surface area and d = distance between 2 sections. Adjacent 20 pm slide-mounted sections were examined for Fluorojade (Histochem Inc.) staining as previously described (Schmued et al., 1997). CV sections were scanned at 600 DPI on an Epson desktop scanner and lesion area was quantified by an experimenter blinded to the animal genotypes using ImageJ software (National Institute of Health). Four anatomical levels of the striatum were examined (Bregma (in mm) + 1.10, +0.40, -0.26, -0.92) according to the mouse brain atlas (Paxinos and Franklin, 2001). Fluorojade-positive neurons were only assessed in the core of the striatal lesion. Fluorojade images were collected using Northern Eclipse software (ver. 6.0). The wide-field fluorescence microscopy (Zeiss Axiophot) setup was equipped with a Retiga Exz CCD camera (Qlmaging). For rats, perfusions cryosectioning and CV staining were performed as described for mice. Total lesion volumes were calculated using the following formula V t = V i +V 2 + ...+ V„, where V n = jrh(rn.i2 + r„_irn + r n 2)/3, where h is the distance between 2 sections and r is the radius of the lesion area using sections from Bregma (in mm) 1.70, 1.20, 0.70, 0.20, -0.26, and -0.80 according to the rat brain atlas (Paxinos and Watson, 1986). 3.2.5. Succinate Dehydrogenase Assay Male or female mice from each genotype were acutely injected with 60 mg/kg 3-NP and were always paired with PBS-injected littermates of the same sex and genotype. After 2 h, the mice were euthanized with halothane and then decapitated. Brains were washed with ice-cold PBS and striata and cortices were dissected from a 2 mm brain-matrix slice at approximately Bregma +1.0 mm. Crude mitochondrial samples were prepared by dounce homogenization in 0.25 M sucrose, 1 mg/ml bovine serum albumin (BSA), 1 mM EDTA, pH 7.4. To collect the 108 mitochondria, the homogenate was centrifuged at 4°C, 600 x g for 5 min. The supernatant was then re-centrifuged at 7,200 x g at 4°C for 10 min. The supernatant was aspirated, and the mitochondrial pellet resuspended in the same buffer. SDH activity was assayed immediately using a protocol based on studies by Pennington et al. (Pennington, 1961). Briefly, the reaction consisted of 0.1 M potassium phosphate buffer (pH 7.4), 0.1 M succinate, 0.05 M sucrose, 2 mg/mL p-Iodonitrotetrazolium chloride (prepared fresh), and 125 pg/mL crude mitochondria (final concentrations in 400 pL reaction volume) and incubated for 20 min at 37°C. The formazan mixture was vortexed before measurement on a microplate reader at 490 nm. Protein concentration was measured using the bicinchronic acid method (Pierce). Brain homogenates were serially diluted for the assay and data analyses were performed on values within the linear range of the SDH reaction. We also used 4 Nrf2+ /" mice to examine the dose-response relationship between acute SDH inhibition and various injected 3-NP concentrations (20 mg/kg, 40 mg/kg or 60 mg/kg). Both 20 mg/kg and 40 mg/kg doses produced little or no change 2 h after injection, compared to PBS injection (data not shown), whereas 60 mg/kg produced an observable decrease in activity. The 60 mg/kg dose was used for further SDH bioassays. 3.2.6. Preparation of tBHQ-supplemented diets A l l rats and mice were normally fed Lab Diet 5001, which contains no tBHQ, but trace amounts of B H A for preserving animal fats (2 ppm). For tBHQ feeding, food pellets were powdered in a coffee grinder and dry mixed with tBHQ (1% w/w). Distilled water was added to the powder (equal v/w), and the mixture was reshaped into food pellets. The pellets were then baked at 60°C for 3 h. Control food was processed in the same fashion without the addition of tBHQ. Initially, we determined 5% tBHQ in diet was not consumed by Nrf2+ /" mice (n = 3), and 0.5% tBHQ, although consumed, only slightly preserved motor function during 3-NP treatment 109 (n = 3). For these reasons, a concentration of 1% tBHQ was used in all further feeding experiments. 3.2.7. Enzyme Assays for brain tissue Brain and liver tissue was dissected in ice-cold PBS and homogenized with 10 strokes of a dounce homogenizer in tissue buffer containing 25 mM Tris (pH 7.4) and 250 mM sucrose. Crude homogenates were centrifuged at 15,000 g for 10 min at 4°C. Tissue homogenate supernatants were collected and promptly assayed for enzyme activity. N Q O l enzyme activity was determined by calculating the dicumarol-sensitive fraction of DCPIP reduction (Benson et al., 1980). Reactions consisting of 25 mM Tris-HCl buffer (pH 7.4) with 0.7 mg/mL BSA, 5 u M FAD, 200 p M N A D H , 30 pg/mL protein, with and without 20 p M dicumarol were pre-incubated for 10 min at 25°C (final concentrations in 200 pL reaction volume). DCPIP was then added to a final concentration of 36 p M (20 pL volume), and the reaction was monitored at 540 nm. The extinction coefficient for DCPIP was 2.1 x 104 M" 1 cm" . The GST assay consisted of 1 m M 1-chloro-2,4-dinitrobenzene (CDNB), 1 mM glutathione, and 100 pg/mL protein at 37°C in 100 mM potassium phosphate buffer (pH 6.5) (final concentration in 150 pL reaction volume) (Kelly et al., 2000). The GST reaction was monitored at 340 nm and the spontaneous non-enzymatic slope was subtracted from the total observed slope. The extinction coefficient for CDNB was 9600 M'crn" 1 . The L D H assay consisted of 3.3 m M pyruvate, 0.34 mM N A D H , and 100 pg/mL protein in PBS at 37°C (final concentrations in 150 pL reaction volume) (Everse et al., 1970). The L D H reaction was monitored at 340 nm and the spontaneous non-enzymatic slope was subtracted from the total observed slope. Purified L D H enzyme standards were used to ensure that values were obtained within the linear range of the assay. The extinction coefficient for 110 N A D H was 6219 iVT'cm"1. Protein concentration was determined using the bicinchronic acid method according to the manufacturer's protocol (Pierce). 3.2.8. Stereotaxic Injections Rats were anaesthetized by i.p. injection of Somnitol (MTC Pharmaceuticals, Cambridge, ON) and placed in a stereotaxic frame (David Kopf Instruments). A burr hole was drilled and injection was performed into the right striatum at coordinates: 1.00 mm anterior-posterior from Bregma, 2.60 mm medial-lateral, and both -4.50 & -5.50 mm dorsal-ventral, using a 10 pi Hamilton gas-tight syringe with 26 gauge needle (type 2 tip). The virus was delivered by manually pressing the syringe plunger 3 times at 4 min intervals. Ad-GFP or Ad-Nrf2 was delivered at a concentration of 5 x 106 total plaque forming units in 3 pi of PBS. Wounds were irrigated with 0.9% sterile saline solution and sutured. Animals were allowed to recover for 3 days before 3-NP treatment was initiated. 3.2.9. Immunohistochemistry For rats, frozen 10 pm brain sections were thawed in PBS for 3 min at room temperature (RT). Mouse monoclonal anti-04 (Chemicon) or rabbit polyclonal anti-GFAP antibodies were diluted 1:400 and 1:200 respectively in Ab buffer (3% BSA and 0.3% Triton X-100 in PBS) and incubated overnight at 4°C in a humid chamber. Sections were rinsed 3 x 5 min in PBS and incubated with goat anti-mouse IgM Alexa546 conjugated or goat anti-rabbit Alexa546 conjugated secondary antibodies (both Molecular Probes, 1:500) in a humid chamber for 1 h at RT. Sections were rinsed 3 x 1 0 min in PBS prior to mounting with Fluoromount-G (Southern Biotechnology Associates Inc.) and No. l glass coverslips (Corning). A l l GFP fluorescence from viral infection could be detected without using an anti-GFP antibody. To assess total striatal 111 volume, a propidium iodide (PI) counterstain was used to label all cell nuclei. Sections were incubated in 8 p M PI dissolved in PBS for 10 min at RT followed by a 10 min PBS rinse. To determine infectivity, GFP fluorescence was calculated by thresholding images to subtract background and calculating the area of fluorescence (ImageJ). In the same section, PI staining was used to locate the corpus callosum and caudate putamen. The area encompassed by these two regions was defined as the striatal area. Data was expressed as % GFP + volume within the total striatal volume. For NeuN staining, an antigen retrieval process was used following thawing. Sections were incubated at 80°C for 30 min in 10 mM sodium citrate pH 8.5 and then rinsed for 5 min in PBS prior to anti-NeuN antibody incubation overnight at 4°C (Chemicon, 1:100) and goat anti-mouse Cy3 secondary antibodies for 1 h at RT (Sigma, 1:200). Fluorescent images were captured using a Zeiss L S M Meta 510 confocal microscope and analyzed using NIH Image (for Mac) or ImageJ (for PC). 3.2.10. Preparation of primary glial-enriched and COS-1 cultures Enriched glial cultures were prepared from 0-2 d post-natal Wistar rat pups as described previously (Shih et al., 2003). The conditions used largely results in a population of Type I and II astrocytes as assessed by anti-glial fibrillary acid protein (GFAP) staining. COS-1 cells (ATCC) were maintained in D M E M supplemented with 10% fetal bovine serum, 1 m M sodium pyruvate, 2 mM glutamine, 100 U/mL penicillin, and 0.1 mg/mL streptomycin. 3.2.11. Plasmids and Adenoviruses PEF mammalian expression plasmids carrying cDNA encoding mouse Nrf2 and Nrf2DN were a generous gift from Dr. Jawed Alam (Alton Ochsner Medical Foundation, New Orleans, LA) (Alam et a l , 1999). Rat A R E sequences were obtained from the nqol promotor. The human placental alkaline phosphatase (hPAP) reporter plasmid (rQR51) was constructed as described 112 previously (Murphy et al., 2001; Shih et al., 2003). Recombinant adenoviral vectors were generated using the Cre-lox system (Canadian Stroke Network core facility, University of Ottawa), as described previously (Hardy et al., 1997; Shih et a l , 2003). The N-terminus-tagged Nrf2-GFP vector was made by PCR-amplifying the Nrf2 coding sequence from the PEF vector with specific primer sequences: forward, 5'-A C T C A G A T C T C G A G A A G A T T T G A T T G A C A T C C T T - 3 ' and reverse, 5'-CCCGGGGGTACCCTAGTTTTTCTTTGTATC-3 ' . The resulting PCR product was restriction digested with Xho I and Kpn I for ligation into the pEGFP-Nl vector (Invitrogen). The Keapl vector was a generous gift from Dr. Masayuki Yamamoto (University of Tsukuba), later modified to Keapl -FLAG by Laurie Zipper (University of Wisconsin) (Itoh et al., 1999b; Zipper and Mulcahy, 2002). 3.2.12. Transfection, infection, and treatments for astrocytes and COS-1 cells Astrocytes and COS-1 cells seeded (1 x 105 cells/mL) in 24-well plates were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol with some modifications. In particular, for each well of a 24-well plate, 1 pg of D N A and 1 pL of lipofectamine were used. The transfection efficiency was typically ~ 20% for astrocytes and ~ 50% for COS-1 cells as assessed by (3 galactosidase staining. For some experiments, adenovirus infection of astrocytes as described previously (Shih et al., 2003), was initiated immediately after lipofectamine transfection. A l l infected cultures were examined for adequate infection efficiency (-90 % of glia) as assessed by GFP fluorescence. 3-NP or tBHQ treatments were initiated 24 h after transfection. 3-NP was prepared in basic salt solution (BSS) consisting of (in mM) 3.1 KC1, 134 NaCl, 2.5 CaCl 2 , 1 MgS0 4 , 0.25 K H 2 P 0 4 , 15.7 N a H C 0 3 (pH 7.2), and filtered before use. The 3-NP stock was further diluted in BSS to the indicated concentration for experiments. Cells 113 were washed with BSS twice and then exposed to 3-NP or BSS (vehicle) for 2 h. The cells were washed again 3 times with BSS and the original culture media was replaced. The Nrf2 inducer, tBHQ, was dissolved as a lOOOx concentrated stock in pure DMSO (vehicle), sterile fdtered, and then diluted to a final concentration of 10 or 20 p M in the glial culture media. TBHQ was present in the media for the duration of the experiment until cells were harvested. 3.2.13. Placental alkaline phosphatase assay: Human placental alkaline phosphatase (hPAP) activity was measured as described previously (Henthorn et al., 1988). Briefly, cultures were collected in lysis buffer consisting of 10 mM Tris-HCl (pH 8), 1 mM MgCl 2 , and 0.1% Triton X-100. Half of the sample was saved for determination of protein concentration and (3-galactosidase activity (for normalization of transfection efficiency). The remaining sample was heated to 65°C for 30 min to inactivate endogenous phosphatase activity. The assay was initiated by mixing ~15 pg protein with diethanolamine buffer (0.73 M diethanolamine with 0.36 m M M g C ^ , pH 9.8), and 13.6 m M p-nitrophenyl phosphate (final concentrations in 150pL reaction volume). The reaction was monitored every 2 min over 30 min, at 405 nm. For all assays, hPAP activity from rQR51mut transfected astrocytes was subtracted as ARE-independent background. 3.2.14. Semi-qualitative reverse-transcriptase PCR Total RNA was isolated from infected cultures using Trizol Reagent (Life Technologies, GIBCO) following manufacturer's instructions. One pg of R N A was reverse transcribed using random hexamer primers and Thermoscript reverse transcriptase enzyme (Invitrogen). The resulting cDNA was then amplified by PCR using various primer sets (Lee et al., 2003a): Mouse Nrf2 exon 5, 5-TCTCCTCGCTGGAAAAAGAA-3'and 5-AATGTGCTGGCTGTGCTTTA-3'; 114 nqol, 5 ' - C A T T C T G A A A G G C T G G T T T G A - 3 ' and 5 ' - C T A G C T T T G A T C T G G T T G T C A G - 3 ' ; Xct, 5'- T T G C A A G C T C A C A G C A A T T C - 3 ' and 5'- C G T C A G A G G A T G C A A A A C A A - 3 ' ; gclc, 5 ' -ACAAGCACCCCCGCTTCGGT -3 ' a nd 5 ' - C T C C A G G C C T C T C T C C T C C C - 3 ' ; B-actin, 5'- C C C A G A G C A A G A G A G G T A TC-3' and 5'- A G A G C A T A G C C C T C G T A G AT-3' . PCR conditions were as follows: initial denaturing step using 1 cycle at 95°C for 3 min, followed by 26 cycles at 95°C for 30 s, the appropriate primer annealing temperature (~55°C) for 40 s, 72°C for 1 min, and a final cycle at 72°C for 5 min. For Xct and gclc, 36 and 31 cycles were used, respectively. The PCR products were separated on a 1.4% agarose gel containing ethidium bromide. cDNA was then visualized using an ultraviolet light source. cDNA templates for each sample were serially diluted and band densities were plotted. These plots were used to ensure that data represented PCR products within the linear range of the reaction (Suppl. Fig. 2). 3.2.15. Glutathione assay: Total glutathione was quantified in glial cell lysates and tissue homogenates (0.3 mg/mL) by the method of Tietze (Tietze, 1969), as described previously (Shih et al., 2003). 3.2.16. Western Blot Analysis and Immunocytochemistry Western blot and immunocytochemistry v/as performed as described previously (Shih et al., 2003). Antibodies used include: rabbit anti-heme oxygenase 1 (Stressgen Inc., 1:500), goat anti-actin (Santa Cruz, 1:1000), sheep anti-rabbit horse radish peroxidase (HRP) (Amersham Pharmacia, 1:5000), donkey anti-goat HRP (Santa Cruz, 1:5000), mouse anti-FLAG M5 (Sigma, 1:1000), goat anti-rabbit Texas Red (Molecular Probes, 1:2000). 115 3.2.17. Nrf2-GFP localization in COS-1 cells After 2 h of 3-NP treatment or 4 h of tBHQ treatment, COS-1 cells were fixed and stained. Cell counting (and nucleus/cytoplasm delineation) was performed by an experimenter blinded to the treatment groups and directed to assess whether Nrf2-GFP fluorescence was excluded from or enriched in the nucleus. We did confirmatory experiments using a nuclear counterstain (Hoechst) and found in all other experiments that it was relatively straightforward to assess Nrf2 localization even without the counterstain since COS-1 cells have a well-defined nuclear-cytoplasmic border. 3.2.18. Data A nalysis: A l l data are shown as means + SEM. Statistical analyses were performed using Student's t-test and one-way or two-way A N O V A in GraphPad Prism (ver 2.01). 3.3. Results 3.3.1. Nrf2'/' mice are hypersensitive to systemic 3-NP treatment Nrf2_/" mice were challenged with 3-NP to examine how loss of Nrf2 function affects both motor system impairment and toxicity attributed to metabolic inhibition in vivo. Our hypothesis was that Nrf2"A mice would be more sensitive to the effects of 3-NP-induced metabolic inhibition. Thus, in initial experiments we determined in vivo 3-NP dosing regimes that would provide significant impairment in Nrf2_/" mice and then assessed what impact these had on wild type animals. The extent of 3-NP toxicity was monitored through both manifestation of overt motor symptoms (i.e. reduced general locomotor activity, hind limb and truncal dystonia, and postural instability) and development of bilateral lesions restricted to the dorso-lateral striata (Fernagut et al., 2002). An advantage of using behavioral assessment as an adjunct 116 to histology is that it can be performed repeatedly on the same animals providing a measure of 3-NP toxicity progression. In assays of motor behavior Nrf2"A mice were indistinguishable from Nrf2 + / + and Nrf2 + A controls before 3-NP treatment. However, with progressively increasing doses of 3-NP (given 12 h apart), Nrf2_/" mice exhibited motor deficits more quickly than controls (Fig. 3-1 A). This increased sensitivity of Nrf27" mice became obvious after the 9 t h scheduled injection (60 mg/kg dose). 3-NP administration was ceased at this point due to increased morbidity of Nrf2"/_ mice. Preceding motor deficits, Nrf2"A mice also began to lose significant amounts of weight compared to control groups (Fig. 3-IB). By the end of the experiment, NrfT7" mice lost nearly 20% of their body weight. Consistent with behavioural scores, the doses given were insufficient to produce striatal lesions in Nrf2 + / + mice. However, the majority of 3-NP challenged Nrf2_/" mice exhibited extensive striatal lesioning (Fig. 3-1C, 3-1D left column, and 3-1E). Nrf2+ /" mice also developed striatal lesions that, on average, were ~7-fold smaller than in Nrf2"A mice (lesion volumes, +/+ = 0.00 ± 0.00 mm 3, +/- = 0.74 ± 0.41 mm , -/- = 5.24 ±1.50 mm , uncorrected for edema). Both motor deficits and weight loss were highly correlated with lesion size during 3-NP treatment (Fig. 3-2A,B) (Guyot et al., 1997). In addition, edema was likely contributing to the pathology of these mice since the striatal volume of 3-NP treated Nrf2"A mice was ~ 27% larger than controls (total striatal volume, +/+ = 7.95 ± 0.54 mm 3, +/- = 7.98 ± 0.29 mm 3, -/-= 10.14 ± 1.19 mm 3, * p < 0.05). 117 A —•— +/+ -•- +/~ - A - -i-* •k I A * 0 12 24 36 48 60 72 84 96 108 132 • 20 mg/kg 40 mg/kg • • 6 0 mg/kg • 3-NP injection t ime (h) and d o s e ^ Bregma (mm) +/+ 1.10 +/-0 . 4 0 -0 .26 -0.92 • f t / I T B +/+ -•-- A — 110-, .c O) <u 100-t <u 9 0 -to 8 0 -£ O) 7 0 -mal 6 0 -c < 5 0 -D 0 12 24 36 48 60 72 84 96 108 132 m 20 mg/kg mm 40 mg/kg mm 60 'ng/kg» 3 -NP injection t ime (h) and dose Cresyl violet Fluorojade +/+ +/-_ 100 I 90 io 70 | 60 40 30 20 10 0 CTS CD C o CD +/+ +/--/-unnnl Bregma 1.10 0.40 -0.26 -0.92 CD O T3 & CD -O TO CD X3 'o 1— o LL. 160] 140-120; 100-80 60 40 20 0 Bregma 1 o * i [f.jl 10 0.40 -0.26 -0.92 Figure 3-1. Behavioral scoring, weight loss, and lesions of mice with varied Nrf2 genotype during 3-NP challenge. Behavior (A) and weight (B) were evaluated before each scheduled injection. The 3-NP treatment regimen consisted of 9 total intraperitoneal injections with one injection given every 12 h at successively increasing doses: 20 mg/kg x 4, 40 mg/kg x 4, and 60 mg/kg x 1 (doses are depicted with black bars below the x axis). At 108 h, behavior was measured but no injections were made. All animals were sacrificed 1.5 days after the last dose of 3-NP (arrow). C, representative cresyl violet-stained slices from all genotypes after 3-NP treatment. Pallor in staining because of cell death and cell shrinkage is outlined by dotted lines. D, representative images from cresyl violet-stained slices (left column, scale bar = 1 mm), magnified image from cresyl violet striatal inset (middle column, scale bar = 100 pm), and magnified image of Fluorojade staining (marker of neuronal degeneration) from striatal inset (right column, scale bar =100 i^m). Arrow shows typical hemorrhaging seen in some cases with the Nrf2_/~ group. E, lesion area from 4 different levels relative to Bregma within the striatum were measured and expressed as percentage of total ipsilateral striatum. Left and right bars for each genotype correspond to left and right hemispheres. No significant differences were seen between hemispheres within the animals. F, Fluorojade-positive cells were counted in a region of interest within the core of the lesion. Counts from both hemispheres were averaged for each slice. Data represent the mean ± S.E. from n = 5 (Nrf2+/+), n = 7 (Nrf2+/), n = 5 (Nrf2"/_). *,p < 0.05; **,p < 0.01; ***, p < 0.001, compared with wild-type control group, two-tailed t test. A 1 2 111 101 9 8 7 6" 5-4 3 w o B -1 0 1 2 3 4 5 6 7 8 9 10 XL 03 * in ro I c < 110 105 100 95 90-85-80-75-70 65 60^ 55-l 50 © o a +/+ I 0 + / -A ./. -1 0 1 2 3 4 5 6 7 8 9 10 Lesion Volume (mm ) Lesion Volume (mm ) Figure 3-2. 3-NP induced lesion volume is strongly correlated with both behaviour and weight. Lesion volume (x-axis) was plotted against either behavioural scores (y-axis) obtained 1.5 days after the last 3-NP injection (A) or percentage start weight 1.5 days after the last 3-NP injection (B). With both comparisons, strong correlations were found suggesting that both behaviour and weight were excellent predictors of lesion size. Correlation statistics were done with Spearman test, *** p < 0.0001, r = 0.867 (A), ** p < 0.001, r = -0.693 (B). Data represent the mean ± SEM. 119 Lesions of Nrf2_/" mice contained mainly pyknotic cells which stained positive for fluorojade, a marker of neuronal degeneration (Fig. 3-1D center and right column, and 3-1F) (Schmued et al., 1997). Only a sub-population of the remaining neurons in the Nrf2+ /" lesions was fluorojade-positive, and little or no positive cells were observed in the striata of Nrf2 + / + mice. No significant extra-striatal (i.e. hippocampal or cortical) fluorojade-positive staining was observed in 3-NP treated Nrf2_/" mice, consistent with the striatal-specific action of systemic 3-NP administration. Importantly, Nrf2_/" mice were specifically sensitive to 3-NP since injection of PBS control solution caused negligible motor abnormalities and no lesions/fluorojade-positive neurodegeneration (data not shown). Although the treatment regimen we used did not cause toxicity in Nrf2 + / + mice, prolonged 3-NP administration (1-2 doses of 60 mg/kg 3-NP beyond current regimen) was able to provoke the development of motor deficits and lesions in this genotype (data not shown). 3.3.2. Striatal SDH Inhibition by 3-NP is Not Different Among Genotypes Since we used systemic administration of 3-NP, it was possible that increased toxicity in Nrf2_/" mice was due to compromised peripheral detoxification systems (i.e. liver, kidney, and intestines), leading to a higher circulating concentration of 3-NP in the brain. To address this possibility, we directly quantified SDH activity in the brain after an acute systemic injection of 3-NP (60 mg/kg). The cortex and striatum were collected after 2 h when peak brain SDH inhibition was shown to occur (Brouillet et al., 1998). SDH activity was reduced by 40-50% in total tissue from cortex and striatum of all genotypes, consistent with previous studies (Fig. 3-3)(Brouillet et al., 1998). Importantly, we observed no difference in SDH activity between genotypes, suggesting that increased damage caused by 3-NP in Nrf2"A mice is most likely due to an intrinsic sensitivity in their brains. 120 I 70 ^ 50 140 < r 30 Cortex Striatum c CD Q i _ o o O 20-10-0 JL -V+ M- -I- +H- •+/- -/- -V+ Hf - /- +/+ -/-P B S - i n j e c t e d 3 - N P - i n j e c t e d Figure 3-3. Inhibition of brain SDH is not different among Nrf2 genotypes 2 h after acute 3-NP injection. Four mice from each genotype were injected with a single dose of 60 mg/kg 3-NP or PBS. 3-NP-injected mice were compared with PBS-injected littermates of the same genotype for each experiment. Animals were sacrificed 2 h after 3-NP injection when the peak of S D H inhibition has been shown to occur (47). Mitochondria samples were prepared from striatum and cortex and used in the S D H assay. A^Q was normalized to mitochondrial protein content. Data represent the mean ± S.E. from n = 4 mice for each genotype and treatment group. *,p < 0.05, compared with PBS injected controls from same genotype, one-way A N O V A with Bonferroni post-hoc test. 3.3.3. Pre-activation of Nrf2 by tert-butylhydroquinone attenuates 3-NP toxicity in Nrf2+/~, but not Nrf2'A mice. Nrf2 induction by small electrophilic molecules represents a practical method to increase the Phase 2 response in vivo (Talalay et al., 1995; Talalay and Zhang, 1996; Talalay and Fahey, 2001). Dietary administration of Nrf2 inducers, such as sulforaphane (isolated from broccoli sprouts) have been shown to induce Phase 2 genes in peripheral tissues and reduce toxicity associated with a number of pathologies, including stomach tumour formation and hypertension (Fahey et al., 2002; Wu et al., 2004). To determine whether in vivo induction of Nrf2 could 121 provide resistance to 3-NP toxicity, mouse diets were supplemented with the potent Nrf2 inducer, tBHQ (Alam et al., 1999; Nguyen et al., 2000). The in vivo phase 2 response was assessed by measuring the activities of two prototypic phase 2 enzymes, glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase (NQOl), as well as total glutathione (GSH) content. Nrf2_/" mice exhibited lower basal striatal and cortical activity for GST and N Q O l , when compared to Nrf2 + / + littermate controls (Table 3-1). Interestingly, basal brain GSH levels were not altered, suggesting constitutive synthesis of brain GSH does not rely on Nrf2 function, at least in the young adult mice examined in here. As observed in previous studies, GST, N Q O l activities, and total GSH levels were markedly reduced in the livers of Nrf2"A mice (Chan and Kwong, 2000; Hayes et al., 2000b; Kwak et al., 2001a; Chanas et al., 2002). The housekeeping gene, lactate dehydrogenase (Ldh), is not regulated by Nrf2 and accordingly, no significant differences in activity were observed between genotypes. 122 Table 3-1. Effects of Nrf2 genotype on basal and tBHQ-induced levels of antioxidant/detoxification markers in striatum, cortex, and liver Basal Phase 2 Enzyme Levels Striatum __ Cortex Liver ~~Ti T ~ " - ' / " - " ' + /"+ - / - "+/ +" -7 GST 72.9 ± 6.9 57.4 ± 4 . 7 * * 65.9 ± 6.2 49.0 ± 4.9 *** 290.4 ± 35.8 195.0 ± 2 1 . 2 NQOl 46.7 ± 1.3 37.3 ± 1 . 6 * * 29.7 ± 2.6 24.0 ± 1 . 2 * 18.0 ± 4 . 0 5.2 ± 1.4 ' ** GSH 28.5 ± 3.4 30.2 ± 4.8 23.1 ± 4 . 1 26.5 ± 3.9 110.5 ± 15.9 70.3 ± 9.3 * LDH 590.7 ± 4 9 . 6 601.5 ± 29.4 628.6 ± 27.9 686.8 ± 53.2 1094.6 ± 51.0 1234.7 ± 7 9 . 5 tBHQ-induced Phase 2 Enzyme Levels (% control) Striatum Cortex Liver + / + and + / - - / - + / + and + / - -/- + / + and + / - - / -GST 102.3 ± 3.5 107.9 ± 0 . 8 114.3 ± 9 . 1 126.4 ± 2 7 . 1 180.2 ± 13.4 # 81.3 ± 21.7 NQOl 111.5 ± 9 . 3 109.1 ± 8.9 140.8 ± 2 2 . 6 117.8 ± 6.6 143.8 ± 8.2 # 101.3 ± 12.0 GSH 126.8 ± 9.4 # 107.1 ± 6.8 160 ± 19.8 # 109.5 ± 14 117.0 ± 12.8 151.2 ± 13.9 # LDH 102.4 ± 2.5 95.9 ± 4 . 3 99.4 ± 4.7 106.4 ± 8.4 98.2 ± 3 . 4 100.0 ± 2 . 0 Basal GST, NQOl, and LDH activities from tissue extracts are expressed as nmol/min/mg protein, and total glutathione content is expressed as pg GSH + GSSG / mg protein. Data are represented as mean + SEM. N = 6 animals, for both Nrf2+ / + and Nrf2_/" groups, were used to determine basal phase 2 enzyme levels. Enzyme activities and glutathione content were measured after 7 days of tBHQ feeding (1% w/w in mouse chow), and expressed as a percentage of values obtained from littermates receiving control diet. N = 2, 4, and 4 littermate pairs for Nrf2+ / +, Nrf2+/", and Nrf2_/" groups were used, respectively, to determine tBHQ-induced phase 2 enzyme levels. * p < 0.05, ** p < 0.01, *** p < 0.001, compared to wild-type littermate. # p < 0.05, compared littermates with same genotype recieving control diet. Two-tailed paried t-test. Primarily Nrf2 + A and Nrf2_/" mice were used in tBHQ feeding studies since we were interested in observing reduced motor deficit and lesion size during 3-NP treatment, and Nrf2 + / + mice did not develop these phenotypes. The tBHQ-supplemented diet (1% w/w) was well tolerated by Nrf2+ /" mice since no significant weight loss was observed over 1 week of feeding, except a transient loss of weight 2 d after initiation of feeding, and food consumption was similar with mice receiving control diet (Fig. 3-4A,C). However, Nrf2"A mice were less tolerant of dietary tBHQ with overall body weight ~ 10-20% lower than mice with control diet during 1 123 week of feeding (Fig. 3-4B, *** p < 0.0001 two-way A N O V A ) , and less food consumption than tBHQ-fed Nrf2+ /" mice (Fig. 3B,D). In total, Nrf2+/", and Nrf2~A mice consumed 12.9 ± 0.9, and 7.6 + 0.7 mg tBHQ/g body weight over 1 week, respectively. The 1% tBHQ diet increased striatal and cortical GSH levels in Nrf2-expressing mice (Nrf2 + / + and Nrf2+/") (Table 3-1). Importantly, the induction of brain GSH was not observed in Nrf2"/_ mice, providing evidence that tBHQ-induced changes in GSH are Nrf2 dependent. In addition, tBHQ-induced increase in liver GST and N Q O l activities was observed in Nrf2 expressing mice, but not Nrf2"/_ mice, consistent with the effects of other Nrf2 inducers used in vivo (Kwak et al., 2001a; McWalter et al., 2004). However, no induction of GST or N Q O l was detected in brain. Levels of the negative control enzyme, L D H , was unaffected by tBHQ in all tissues examined. Interestingly, we observed increased liver GSH levels in tBHQ-fed Nrf2"/_ mice. We believe that this Nrf2-independent change may reflect an inherent hepatic toxicity associated with tBHQ feeding, perhaps due to inefficient detoxification from the body by the same pathways regulated by Nrf2 (Phase 2 genes). Together, these results highlight the sensitivity of the Nrf2 - /" phenotype to tBHQ toxicity and suggest normal Phase 2 induction in response to tBHQ is abrogated in Nrf2"A mice. The tBHQ diet was given 1 week prior to the 3-NP regimen and maintained until sacrifice. TBHQ-fed Nrf2+ /" mice had significantly attenuated 3-NP induced motor deficits compared to mice receiving the control diet (Fig. 3-4E, ** p < 0.001 two-way A N O V A ) . In contrast, tBHQ-fed Nrf2"/_ mice exhibited an overall exacerbation of motor deficit (Fig. 3-4F, *** p = 0.0002 two-way A N O V A ) . The average lesion volume of Nrf2+ /" given control diet was similar to that measure in our initial experiments (0.65 + 0.29 mm 3, uncorrected for edema) (Fig. 3G,H). However, consistent with reduced behavioural scores, no tBHQ-fed Nrf2+ /" mice exhibited detectable lesions indicating protection of neurons. TBHQ feeding did not reduce lesion size in Nrf2"A mice, confirming the protective effect of tBHQ consumption was Nrf2-dependent (Fig. 3-4G,H). 124 Figure 3-4. Dietary supplementation of tBHQ attenuates 3-NP toxicity in N r f 2 + / mice, but exacerbates toxicity in Nrf2 _ / " mice. Mice received a tBHQ-supplemented diet for 1 week prior to and during 3-NP administration. Only Nrf2+ /~ and Nrf2" / _ mice were studied because we were interested in observing reduced motor deficit and lesion size with t B H Q treatment, and N r f 2 + / + mice did not develop these phenotypes. A, no significant differences were observed in body weight of Nrf2 + /~ mice between t B H Q and control diet. B, Nrf2 _ /~ mice never fully adjusted to the tBHQ-supplemented diet where body weight of tBHQ-fed Nrf2 _ /~ mice dropped by 10-20% during 1 week of feeding. C, control diet and tBHQ-fed Nrf2 + / " mice consumed similar amounts of food throughout the experiment. D, Nrf2 _ /~ mice consumed less food than Nrf2 _ /~ mice with control diet. E, tBHQ-fed Nrf2 + /~ mice exhibited reduced motor deficit at the end of the 3-NP treatment regimen. F, an overall exacerbation of 3-NP-induced motor deficits was associated with t B H Q feeding of Nrf2 _ /~ mice. G and H, t B H Q feeding prevented lesion development in Nrf2 + /~ mice, but had no effect on NrfZ"7" mice. Lesions are outlined by dotted lines. For the Nrf2 + /~ group, n = 8 and 6 animals were used for control and tBHQ-supplemented diets, respectively. For the Nrf2 _ /~ group, n = 6 and 5 animals were used for control and tBHQ-supplemented diets, respectively. Data represents mean ± S.E. *, p < 0.05; ** , p < 0.01, compared with Nrf2 + /~ mice receiving control diet, one-way A N O V A with Bonferroni post-hoc test. 3.3.4. Adenoviral overexpression of Nrf2 attenuates 3-NP induced striatal lesioning in vivo. To further address whether activation of Nrf2-dependent pathways is sufficient to protect the striatum during 3-NP toxicity, in parallel studies, we tested whether direct Nrf2 overexpression using adenovirus vectors (Ad-Nrf2 or Ad-GFP control) conferred neuroprotection to rats treated with 3-NP. Overexpression of Nrf2 causes its accumulation in the nucleus leading to even more robust ARE-dependent transcription than that observed with small molecule inducer treatment (Zipper and Mulcahy, 2002). In vivo intrastriatal adenovirus injection primarily led to infection of astrocytes (Fig, 3-5A top panel, and 3-5B), and almost no neurons or oligodendrocytes (Fig. 3-4A middle and bottom panel, and 3-4B). Vector diffusion was observed throughout the striatum (Fig. 3-5C), infecting 0.48 ± 0.08% of the total striatal volume, as assessed by the presence of the GFP marker (Fig. 3-5D). After infection, protein expression was allowed to occur for 3 days before 3-NP treatment was initiated. Following 3-NP challenge, lesion volumes in the infected hemisphere were significantly smaller in animals receiving Ad-Nrf2, compared to Ad-GFP control virus (9.6 ± 2.6 mm 3 versus 23.4 ± 4.2 mm 3, p = 0.03, two-tailed t-test, Fig. 4E,F). Interestingly, a trend toward decreased lesion volume was also observed in the hemisphere contralateral to the virus injection (12.2 ± 3.8 126 mm 3 compared to 25.9 ± 4.9 mm 3 , p = 0.06), although this difference did not attain statistical significance. This effect may be due to diffusion of secreted glial factors, possibly glutathione or glutathione precursors, by volume transmission along fiber bundles of the corpus callosum, or through the cerebral spinal fluid from the infected Nrf2 overexpressing astrocytes in the ipsilateral hemisphere (Bjelke et al., 1995; Zol i et al., 1999). F i g u r e 3-5. Ad-Nrf2 i n f e c t e d a n i m a l s deve loped s m a l l e r les ions t h a n A d - G F P c o n t r o l a n i m a l s f o l l o w i n g 3-NP a d m i n i s t r a t i o n . A, immunos ta in ing o f G F P (bright cel ls) w i t h the ce l l type-spec i f ic markers for astrocytes ( G F A P , top panel), neurons ( N e u N , middle panel), and ol igodendrocytes ( 0 4 , bottom panel) was first pe r fo rmed to characterize the spec i f i c i ty o f v i r a l infec t ion i n rats in vivo. Scale bars = 10 p m . ( F o r b l a c k a n w h i t e versions o f th i s thesis, p lease use q u a n t i t a t e d d a t a g r a p h s i n p a n e l B). B, i nc idence o f c o - l a b e l i n g between G F P and the different markers . JV represents the total n u m b e r o f G F P + c e l l s counted for two animals. C, representative images o f G F P express ion i n the s t r ia tum o f A d - G F P - i n f e c t e d an ima l s at Bregma (clockwise f r om top left) 1.00 m m , 0.48 m m , - 0 . 8 0 m m , and - 0 . 2 6 m m . Scale bars, 500 p m . ( F o r b l a c k an w h i t e v e r s i o n s o f th i s thesis, please use q u a n t i t a t e d d a t a g r a p h s i n p a n e l D). D, analysis o f G F P f luorescence as a percentage o f total striatal v o l u m e (as de te rmined b y the area ou t l i ned by a p r o p i d i u m iod ide counters ta in , n = 11 animals) . E, quan t i f i ca t ion o f 3 - N P - i n d u c e d l e s ion v o l u m e s i n the ipsilateral (right) and contralateral (left) hemispheres. Da ta represent mean ± S . E . o f n = 5 an ima l s per group. *,p < 0 .05, c o m p a r e d w i t h A d - G F P infected cont ro ls , two- ta i l ed t test. F, representative c r e s y l violet-stained sections at B r e g m a 0.70 m m for a l l the ind ica ted treatment groups . Arrow indicates hemisphere r e c e i v i n g the adenov i ra l vector. Scale bar = 2.5 m m . 127 3.3.5. 3-NP activates ARE-dependent gene expression in cultured astrocytes. Our data show that both the Nrf2 inducer tBHQ and Nrf2 overexpression can protect neurons by pre-activating the Phase 2 response. Since increased susceptibility of Nrf2_/~ mice may result from inability to activate expression of protective genes necessary for reducing toxicity associated with 3-NP, we hypothesized that metabolic inhibition by 3-NP was acting as a signal for Nrf2 activation. To test this hypothesis, we examined the effect of 3-NP on Nrf2 activity in cultured astrocytes, the primary site of ARE-mediated gene expression in brain cultures (Eftekharpour et al., 2000; Murphy et al., 2001; Shih et al., 2003). To mimic the effects of bolus in vivo injection, cultured astrocytes were treated with a wide range of 3-NP concentrations (1-30 mM) for 2 h and then ARE-mediated gene expression was assessed 24 h later using a hPAP reporter assay regulated by an ARE-bearing nqol promoter (Fig. 3-6A) (Murphy et al., 2001). Astrocytes transiently treated (2 h) with 10-30 m M 3-NP exhibited a doubling of ARE-mediated gene expression. Over these time periods and with these 3-NP concentrations no apparent astrocyte toxicity was observed. Expression of ARE-driven hPAP, with and without 3-NP exposure, was reduced to sub-basal levels when astrocytes were infected with adenovirus overexpressing a dominant-negative form of Nrf2 (Ad-Nrf2DN), but not with overexpression of GFP control (Ad-GFP) (Fig. 3-6B) (Shih et al., 2003). Exposure to tBHQ (20 p M for 24 h) induced hPAP expression to a similar extent as 3-NP treatment. Maximal hPAP activity was achieved with Nrf2 overexpression (Ad-Nrf2). Although the concentrations of 3-NP we used to activate Nrf2 mediated gene expression were high, the ability of dominant negative Nrf2 to block the induction indicates that it is nonetheless mediated by the A R E . Perhaps these high concentrations reflect the activated and toxin-resistant nature of astrocytes in vitro (Olsen et al., 1999). 128 Semi-quantitative RT-PCR confirmed that astrocytes treated with 30 m M 3-NP for 2 h expressed higher levels of prototypic Nrf2 target gene mRNAs including nqol, y-glutamylcysteine synthetase (gclc), and the cystine/glutamate exchanger (Xct) (Fig 3-6C). The expression of these mRNAs was increased by Ad-Nrf2 and suppressed by Ad-Nrf2DN infection, verifying the specificity of these target genes. For all RT-PCR experiments, D N A templates were serially diluted to ensure that band intensities were observed within the linear range of the PCR reaction (Fig. 3-7A-E). Since a number of Nrf2 target genes are involved in GSH synthesis (i.e. Xct, gclc, and glutathione synthase), treatment with 30 mM 3-NP also increased intracellular GSH levels in cultured astrocytes (Fig. 3-6D) (Chan and Kwong, 2000; Ishii et al., 2000; Chanas et al., 2002; Shih et al., 2003). Consistent with ARE-driven hPAP expression, 3-NP-mediated GSH induction was blocked with Ad-Nrf2DN infection, but not Ad-GFP control (Fig. 3-8D). Protein levels of heme oxygenase-1 (HO-1), a well characterized Nrf2 target, were also induced by 3-NP treatment (Fig. 3-6E,F)(Alam et al., 1999). Both basal and induced expression of HO-1 was suppressed with Ad-Nrf2DN infection. Although this analysis does not encompass the full scope of Nrf2 gene targets, it nevertheless indicates the Phase 2 response is activated as a dose-dependent effect of 3-NP. 129 A * B 3-NP (mM) 0 3 30 0 3 30 Ad-GFP Ad-Nrf2DN 3-NP (mM) 0 3 30 0 3 30 Ad-GFP Ad-Nrf2DN F i g u r e 3-6. 3 - N P e x p o s u r e i n d u c e s A R E - d e p e n d e n t gene e x p r e s s i o n i n c u l t u r e d as trocytes . A, cul tured cor t ica l astrocytes were transiently transfected w i t h a h P A P reporter construct regulated by an A R E - b e a r i n g nqol p romote r (41). The astrocytes were then treated w i t h v a r y i n g concentra t ions o f 3 - N P (1 -30 m M ) for 2 h i n a glucose-free bas ic salt so lu t ion (BSS vehicle), f o l l o w e d b y washout . A d o u b l i n g o f h P A P ac t iv i ty was observed 24 h after 10 or 30 m M 3 - N P treatment. B, exp res s ion o f A R E - d r i v e n h P A P , wi th veh ic le or 3 - N P exposure, was reduced to sub-basal leve ls w h e n astrocytes overexpressed a dominant-negat ive fo rm o f N r f 2 ( A d - N r f 2 D N ) , but not w i t h ove rexp re s s ion o f a G F P con t ro l ( A d - G F P ) (6). A R E - d r i v e n h P A P was i n d u c e d b y 24 h o f t B H Q exposure and m a x i m a l l y increased w i t h A d - N r f 2 infect ion. Da ta represent the mean ± S . E . f rom three independent exper iments . C , semi-quant i ta t ive R T -P C R con f i rmed that astrocytes treated w i t h 30 m M 3 - N P expressed more m R N A s f rom pro to typ ic N r f 2 130 target genes including nqol, gclc, and Xct. DNA templates were serially diluted to ensure that band intensities were observed within the linear range of the PCR (Fig. 3-7). As controls, Ad-Nrf2- and Ad-NrODN-infected cultures were also evaluated with RT-PCR. In all cases, the mRNA levels were induced with Nrf2 overexpression and suppressed with Nrf2DN overexpression. RT-PCR experiments were reproduced three times with similar results. D, cultures treated with 30 mM 3-NP produced more total intracellular GSH than vehicle treated controls. GSH induction was blocked with Ad-Nrf2DN infection, but not Ad-GFP control. E, Western blot analysis shows that protein expression of HO-1 was induced with 30 mM 3-NP treatment in Ad-GFP-infected cultures. Both basal and induced expression of HO-1 was largely blocked with Ad-Nrf2DN infection. F, densitometric analyses of HO-1 protein levels. GSH and HO-1 data represent the mean ± S.E. from three independent experiments. *,p < 0.05; **,p < 0.01; ***,/? < 0.001, two-tailed paired / test. A EB cDNA Template Dilution cDNA Template Dilution 131 Figure 3-7. RT-PCR images were collected within the linear range of the PCR reaction. R N A extracted f rom veh ic l e , 3 - N P treated g l i a (3 and 30 m M ) was used for standard semi-quant i ta t ive R T - P C R . Approp r i a t e pr imers were used to determine re la t ive quanti t ies o f N r f 2 , N Q O l , G C L C , x C T transcripts, and these values were n o r m a l i z e d wi th ac t in densi ty ( A - D ) . E . ) 3 - N P treatment d i d not affect actin m R N A levels . c D N A templates were ser ia l ly d i lu ted to ensure that band intensit ies were observed wi th in the l inear range o f the P C R react ion. Dens i tomet r i c analyses were per fo rmed w i t h ImageJ Software ( N I H ) . Da ta s h o w n is representative o f 3 independent exper iments w i t h s i m i l a r results. We further evaluated nuclear translocation of GFP-tagged Nrf2 (Nrf2-GFP) in response to 3-NP exposure. In initial experiments using astrocytes, Nrf2-GFP fluorescence was almost undetectable when co-expressed with its cytoplasmic regulator Keapl, possibly due to a faster rate of Nrf2 turnover by proteasome dependent pathways (McMahon et al., 2003). As an alternative, COS-1 cells were used because of their high level of trans-gene expression and well-defined morphology for evaluating nuclear versus cytoplasmic localization. Consistent with previous studies, overexpressed Nrf2-GFP was almost exclusively localized to the nucleus unless co-expressed with Keapl (Keapl-FLAG) (Fig. 3-8A, top row) (Zipper and Mulcahy, 2002; Itoh et al., 2003). COS-1 cells co-expressing Nrf2-GFP and Keap l -FLAG were evaluated after treatment with 3-NP or BSS vehicle control for 2 h. As expected, the proportion of cells exhibiting nucleus-enriched Nrf2-GFP fluorescence was significantly higher in 3-NP-treated cultures, when compared to vehicle control (Fig. 3-8A,B). Interestingly, 3-NP treatment also caused some aggregation of Keapl and Nrf2-GFP in the cytoplasm, which we speculate is due to 3-NP-induced endoplasmic reticulum stress causing accumulation of unfolded proteins (Cullinan et al., 2003). This effect was transient since the aggregations were not observed 24 h after 3-NP washout (data not shown). Treatment with 20 p M tBHQ for 4 h, as a positive control, also robustly increased nuclear localization of Nrf2-GFP (Fig. 3-8A,B). Both Nrf2-GFP and Keapl-F L A G constructs were functional, as assessed by their ability to augment and suppress A R E -driven hPAP expression, respectively (Fig. 3-8C). 132 Collectively, this in vitro data confirms Nrf2 translocates to the nucleus and initiates Phase 2 genes induction in response to 3-NP mediated metabolic inhibition. This effect can be mimicked using the Nrf2 inducer, tBHQ, and by adenoviral Nrf2 overexpression. F i g u r e 3-8. 3 - N P p r o m o t e s n u c l e a r t r a n s l o c a t i o n o f Nrf2-GFP. A, C O S - 1 ce l l s were t ransient ly transfected w i t h N r f 2 - G F P ( N - t e r m i n a l fus ion to N r f 2 ) w i t h and wi thou t K e a p l - F L A G . Cons is ten t w i t h p rev ious studies, overexpressed N r f 2 - G F P was a lmos t e x c l u s i v e l y loca l ized to the nucleus unless co-expressed w i t h its cy top l a smic regulator K e a p l (top row) (13 , 43 ) . C O S - 1 cel ls co-express ing N r f 2 - G F P and K e a p l - F L A G were evaluated after treatment w i t h 3 - N P or B S S vehicle cont ro l . The p ropor t i on o f cel ls e x h i b i t i n g nuclear-enr iched N r f 2 - G F P was s ign i f i can t ly h igher i n 3-NP-treated cultures, w h e n compared w i t h veh ic l e con t ro l . 3 - N P also appeared to cause a transient aggregation o f some K e a p l - F L A G and N r f 2 - G F P in the c y t o p l a s m (bottom row). Treatment w i t h 20 p M t B H Q for 4 h , as a pos i t i ve con t ro l , also robust ly increased nuc lear l o c a l i z a t i o n o f N r f 2 - G F P (scale bar -10 pm) . ( F o r b l a c k a n w h i t e v e r s i o n s o f this thesis , p lease use q u a n t i t a t e d d a t a g r a p h s i n p a n e l s B). B, summary o f N r f 2 - G F P l o c a l i z a t i o n in response to t B H Q and 3 - N P . C , bo th N r f 2 - G F P and K e a p l -F L A G constructs were func t iona l , as assessed b y their ab i l i t y to augment and suppress A R E - d r i v e n h P A P expression, respect ive ly . D a t a represents three separate exper iments scored b y a researcher b l i n d e d to the 133 treatment conditions (total cells evaluated: n = 119 and n = 158, BSS and 3-NP-treated cells, respectively). *,p < 0.05, two-tailed paired t test. 3.4. Discussion This study provides evidence to support the neuroprotective function of Nrf2 during metabolic compromise in the in vivo brain. First, loss of Nrf2 function led to an increased susceptibility to 3-NP since Nrf2"/_ mice developed motor deficits and striatal lesions more rapidly than Nrf2 expressing controls. Second, pre-activation of endogenous Nrf2 using the small molecule inducer, tBHQ, attenuated 3-NP toxicity in Nrf2+ /" mice, but not Nrf2"/_, confirming both the neuroprotective action and Nrf2-dependence of tBHQ in vivo. Third, direct overexpression of Nrf2 in the striatum is sufficient to reduce lesion size caused by 3-NP. Fourth, ARE-dependent gene expression in cultured astrocytes was activated by 3-NP, and could be completely suppressed by overexpression of a dominant-negative form of Nrf2. 3.4.1. Nrf2~A mice are hypersensitive to 3-NP toxicity The increased sensitivity of Nrf2~A mice may result from an inability to induce Phase 2 genes in response to 3-NP toxicity. Indeed, a recent study by Calkins et al. (published while this work was submitted) showed using transgenic reporter mice, that ARE-dependent gene expression occurred in the immediate border of 3-NP induced striatal lesions, perhaps in reactive astrocytes commonly found within this region (Calkins et al., 2005). Since Nrf2 is generally thought to respond to oxidative stress or signal transduction pathways associated with it (Owuor and Kong, 2002; Nguyen et al., 2003a), we speculate that 3-NP activates this protective pathway by indirectly generating ROS/RNS. 3-NP may increase brain oxidative stress through multiple mechanisms that may be implicated in our in vitro and in vivo experiments (Murphy et al., 1991; Duffy et al., 1998; Lee et al., 2003a; Shih et a l , 2003). Also supported by our data, an alternative 134 possibility for the increased sensitivity of Nrf2_/" mice may be the lower basal levels of phase 2 enzymes expressed in this genotype (Table 1, basal phase 2 enzyme levels) (83). Given that systemic 3-NP undergoes first pass metabolism before it reaches the striatum, it was possible that increased striatal damage in Nrf2"/_ mice was in part due to impaired clearance of 3-NP from the body by peripheral detoxification. Indeed previous studies of Nrf2"A mice have shown decreased Phase 2 enzyme activity in the liver and intestines (Hayes et al., 2000b; McMahon et al., 2001), organs which may affect the removal of systemically injected 3-NP. However, we have shown that after a single high in vivo dose of 3-NP, striatal SDH was inhibited to an equal extent between Nrf2 + / + and Nrf2"A genotypes, suggesting that 3-NP produces similar metabolic inhibition on a dose-wise basis regardless of genotype. Thus, it appears that the brains of Nrf2"A mice are intrinsically more sensitive to direct damage. This conclusion is also supported by a number of other findings. First, neurons and glia isolated from Nrf2~A mice are more sensitive to in vitro toxicity paradigms involving oxidative stress or metabolic compromise (Lee et al., 2003c; Lee et al., 2003a; Kraft et al., 2004). Second, direct injection of malonate (reversible SDH inhibitor) into the striatum produced larger lesions in Nrf2"/_ mice, compared to Nrf2 expressing controls (Calkins et al., 2005). Third, Nrf2_/~ mice develop larger cortical infarcts than Nrf2 + / + mice after experimental stroke (permanent distal middle cerebral artery occlusion), a model which also involves local brain metabolic inhibition (See Chapter 4) (84). 3.4.2. The small molecule inducer, tBHQ, provided Nrf2-dependent amelioration of 3-NP toxicity in vivo Although studies indicate that loss of Nrf2 potentiates neurotoxicity (Calkins et al., 2005), whether the in vivo neuroprotective effects of small molecule inducers are lost in Nrf2 knockout mice has not been previously addressed. Recent in vitro studies have shown that tBHQ 135 treatment augments ARE-dependent gene expression in cultured astrocytes and protects neurons from toxicity paradigms involving oxidative stress (Ahlgren-Beckendorf et al., 1999; Johnson et al., 2002; Shih et al., 2003; Kraft et al., 2004). We extend these findings to the in vivo situation since dietary consumption of tBHQ significantly reduced motor deficits and lesion developments in Nrf2+/" mice. Importantly, the protective effect of tBHQ was abrogated in Nrf2"/_ mice. Furthermore, dietary tBHQ administration increased brain GSH levels in Nrf2 + / + and Nrf2 + A mice, but not Nrf2_/" mice (Table 1, tBHQ-induced phase 2 enzyme levels). As expected from previous studies (Rahimtula et al., 1982), dietary tBHQ treatment also increased Phase 2 enzyme activity (GST and NQOl) in the liver. Thus, it remains possible that attenuation of 3-NP toxicity in tBHQ-fed mice could be in part due to enhanced removal of 3-NP from the body by increased liver detoxification or conceivably liver derived antioxidants such as GSH released into the blood. However, our observed increase in brain GSH would be expected to contribute to local neutralization of ROS/RNS, leading to tissue preservation. In preliminary studies, we have found that even local infusion of tBHQ in brain ventricles using osmotic pumps can lead to increases in liver phase 2 enzyme levels suggesting that it is difficult to completely dissociate brain and peripheral organ detoxification. Future studies could more directly address the role of brain Nrf2 by developing tissue and organ specific Nrf2 knockouts. However, in support of the brain being the site of tBHQ action, overexpression of Nrf2 within the striatum using adenovirus was sufficient to reduce the effect of systemically administered 3-NP. Although some uncertainty remains as to the site of Nrf2 induction mediated by tBHQ in vivo, our study nonetheless indicates that striatal-specific damage due to the systemic administration of a metabolic stressor can be strongly attenuated by this dietary strategy. Given that many neurodegenerative diseases may be triggered by exposure to diet or lung-derived toxins (Calne et a l , 1986; Jenner, 2001; Di Monte et al., 2002), it is conceivable that a dietary strategy to increase antioxidant function in multiple organs may be an ideal prophylactic strategy. To our knowledge, 136 this is the first study to show that a protective effect of a Phase 2 inducer on brain function in vivo is dependent on Nrf2. Although tBHQ is widely used as a food antioxidant, and is well-tolerated by the body (FAO/WHO, 1975; 1997), it is also important to consider the detrimental action of tBHQ and other Nrf2 inducers. As observed in this study, Nrf2"A mice do not tolerate tBHQ administration as well as controls (as evidenced by weight loss and reduced food consumption) perhaps due to inefficient detoxification of tBHQ through Nrf2-regulated pathways (Fig. 3A,B). Other groups have also found that NrO"7" mice are less tolerant to dietary administration of known Nrf2 inducers (McMahon et al., 2001). Recently, increased susceptibility to hypoxia has been linked to polymorphisms in the Nrf2 gene (Cho et al., 2002a). The question arises as to whether such mutations affect Nrf2 function in humans, and could underlie sensitivity to human disease. Since many Nrf2 inducers are essentially reactive electrophiles, the inability to properly detoxify Nrf2 inducers from the body could be harmful to individuals with such a genetic predisposition. With regard to practical application, recent studies have elucidated a variety of structurally related small molecules which are able to activate Nrf2 by reacting with thiol/disulfide groups on the cytoplasmic regulatory protein Keapl (Kensler et al., 1993; Talalay and Zhang, 1996; Fahey et al., 1997; Talalay, 2000; Fahey et al., 2001; Talalay and Fahey, 2001). Some of these inducers are found within our diet. For example, the isothiocyanate sulforaphane, a potent Nrf2 inducer abundant in cruciferous plants (i.e. broccoli sprouts), was found to inhibit gastric tumour formation induced benzo[a]pyrene (Fahey et al., 1997). Juurlink and colleagues have recently demonstrated a significant improvement of cardiovascular fitness in spontaneously hypertensive rats through chronic upregulation of Phase 2 enzymes by supplementing diets with broccoli sprouts (Wu and Juurlink, 2001; Wu et al., 2004). Such studies support the potential for mitigating oxidative tissue damage in chronic disorders involving 137 oxidative stress, by applying a change in diet -a practical and realistic therapeutic approach from a clinical standpoint. 3.4.3. Overexpression of Nrf2 protein is sufficient to provide neuroprotection in vivo We have also conducted a proof of principle experiment showing that adenoviral overexpression of Nrf2 in the striatum is sufficient to reduce 3-NP toxicity in vivo. Adenovirus was found to mainly infect astrocytes throughout the striatum as well as in the corpus callosum. In our immunohistochemical analysis, we observed that some GFP-positive cells with astrocyte morphology (-35%) were not labeled by any cell-type marker tested However, these cells were labeled by a different astrocyte marker, SlOOp (total GFP and S 100(3 co-localization = 91.8 ± 0.2%), n = 2), confirming their identity (Walz, 2000). With regard to the extent of neuroprotection achieved with Nrf2 overexpression in vivo, we have previously demonstrated a small number of Ad-Nrf2 infected astrocytes can confer protection to many neighbouring neurons during an oxidative glutamate insult in vitro (1 astrocyte can protect 100 neurons) (Shih et al., 2003). These Nrf2 overexpressing astrocytes secrete high levels of glutathione, providing a means for a small number of astrocytes to affect the survival of a much larger number of neurons (Shih et al., 2003). In support of our findings, Calkins et al. observed significant neuroprotection in vivo by transplanting a relatively small number of Nrf2 overexpressing astrocytes (from culture) directly into the striatum prior to injection of the competitive complex II inhibitor, malonate (Calkins et al., 2005). In addition, many factors contributing to oxidative stress (i.e. ROS/RNS) are cell permeable allowing them to be metabolized in a subpopulation of cells (Drukarch et al., 1998). Another factor to consider is the actual spatial localization of the astrocytes infected in situ. The patchy nature of the infection (Fig. 4C) suggests a wider area can be protected within the volume of the brain. Furthermore, there is precedent for a small number of cells playing a major role in striatal function. One such example are the cholinergic interneurons of the striatum which only 138 make up 1-2% of all striatal neurons yet have profound impact on the functional output of the striatum influencing such diverse behaviours as sensorimotor function, sleep & arousal states, learning & memory, anxiety and pain sensations (Zhou et al., 2002). 3.4.4. 3-NP exposure activated ARE-mediated gene expression in vitro Our in vitro experiments show that 3-NP exposure upregulated Phase 2 gene expression in cultured astrocytes, providing a mechanistic link between 3-NP and Nrf2 activation. The effect of 3-NP was attributed to Nrf2 since a dominant negative version of Nrf2 was able to block the effect of 3-NP. However, further experiments are necessary to determine which component of 3-NP toxicity acts as a signal for Nrf2 activation. For example, Nrf2 activation may result from 3-NP induced ROS/RNS generation, loss of ATP, or even as a direct response to SDH inhibition. In preliminary studies using cultured astrocytes, we examined the level of SDH inhibition caused by the range of 3-NP concentrations used in vitro. After 2 h of exposure to non-Nrf2 inducing 3-NP concentrations (0.3-1 mM), SDH activity was reduced by ~ 90%>. However, at Nrf2 inducing concentrations (10-30 mM), SDH activity was almost completely inhibited (~ 98%). Given this difference, it is possible that a non-linear relationship exists between SDH activity and activation of ARE-mediated gene expression. Conceivably, the requirement for high concentrations of 3-NP to induce Nrf2 mediated gene expression may in part be due the high intrinsic resistance of cultured astrocytes which show an activated phenotype to agents that induce metabolic inhibition and oxidative stress (Olsen et al., 1999). 3.4.5. Conclusion Nrf2 plays an important role in regulating neuronal survival during metabolic compromise in vivo. The absence of Nrf2 function in Nrf2"A mice exacerbates neurodegeneration caused by 3-NP administration. Augmentation of ARE-mediated gene expression in Nrf2-139 expressing animals using the small molecule inducer, tBHQ, attenuated tissue damage and preserved motor function. Dietary administration of Nrf2 inducers may have profound effects for neuronal viability in neurodegenerative disease, stroke and related forms of energy deprivation. Chapter 4 : A small molecule inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in v ivo . A version of this chapter has been published. Shih A.Y., Li P., Murphy T.H. (2005) A small molecule inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo. The Journal of Neuroscience. 2005, Nov 2, Vol. 25, Iss. 44, pgs. 10321-10335. Neuroscience Online: http://jneurosci.org/. 140 4.1. Introduction Oxidative stress leading to ischemic cell death involves the formation of reactive oxygen/nitrogen species (ROS/RNS) through multiple injury mechanisms, such as mitochondrial inhibition, Ca 2 +overload, reperfusion injury and inflammation (Coyle and Puttfarcken, 1993; Lipton, 1999; Love, 1999; Lewen et al., 2000). Although, many treatment strategies have implemented antioxidants to promote neuroprotection during ischemia, their clinical efficacy has proven disappointing (De Keyser et al., 1999; Lo et al., 2003). The Cap 'n ' Collar transcription factor NF-E2 related factor (Nrf2) regulates an expansive set of antioxidant/detoxification genes that act in synergy to remove ROS/RNS through sequential enzymatic reactions (Li et al., 2002; Thimmulappa et al., 2002; Shih et al., 2003). As an improvement upon previous neuroprotective approaches, we have examined whether a coordinated cellular defense activated by Nrf2 is an effective prophylactic treatment for stroke. Nrf2 gene targets, collectively known as Phase 2 genes, are involved glutathione (GSH) production and use, detoxification of ROS/RNS and xenobiotics, and N A D P H production (Chan and Kwong, 2000; Ishii et al., 2000; Talalay, 2000; Thimmulappa et al., 2002). Nrf2 is normally localized to the cytoplasm, tethered to the regulatory protein, Keapl (Itoh et al., 1999b). Oxidative stress, or electrophilic agents that mimic oxidative stress (Nrf2 inducers), can modify key sulfhydryl group interactions in the Keapl-Nrf2 complex allowing dissociation and nuclear translocation of Nrf2 (Itoh et al., 1999b; Dinkova-Kostova et al., 2002). When activated, Nrf2 specifically targets genes bearing an antioxidant response element (ARE) within their promoters (Venugopal and Jaiswal, 1996; Itoh et al., 1997a). A wide range of natural and synthetic small molecules are potent inducers of Nrf2 activity (Fahey et al., 1997; Fahey et al., 2001; Rushmore and Kong, 2002). These molecules have been identified from diverse chemical backgrounds including: isothiocyanates (abundant in cruciferous vegetables), l,2-dithiole-3-thiones, heavy metals, and hydroperoxides (Talalay et al., 1995; Talalay, 2000). One well-characterized inducer, 141 ter/-butylhydroquinone (tBHQ), is a metabolite of the widely used food antioxidant butylated hydroxyanisole (National Toxicology Program, 1997; FAO/WHO, 1999). TBHQ possesses an oxidizable 1,4 diphenolic structure that confers its potent ability to dissociate the Keapl-Nrf2 complex (De Long et al., 1987; Talalay, 1989; van Ommen et al., 1992). Previous studies have shown that increasing Nrf2 activity in mixed neuronal/glial cultures is highly neuroprotective during in vitro models of stroke damage, such as oxidative glutamate toxicity (a model involving GSH depletion), H2O2 exposure, metabolic inhibition by rotenone, and C a 2 + overload (Lee et al., 2003a; Shih et al., 2003; Kraft et al., 2004). However, the protective role of Nrf2 activity during in vivo stroke has not been examined. Here, we report that administration of tBHQ prior to stroke onset significantly improves behavioural and histological outcome following ischemia-reperfusion. Conversely, mice lacking Nrf2 have increased sensitivity to permanent focal ischemia and fail to respond to Nrf2 inducers. Our data suggest that Nrf2 function is important for controlling stroke damage, and application of Nrf2 inducers may be a viable prophylactic treatment for those at risk for stroke. 4.2. Materials and Methods 4.2.1. Reagents A l l reagents were obtained from Sigma unless otherwise stated. 4.2.2. Astrocyte-enrichedprimary cultures Enriched cortical astrocyte cultures were prepared from 0-2 d post-natal rat pups as described previously (Shih et al., 2003). The conditions used largely results in a population of Type I and II astrocytes as assessed by anti-glial fibrillary acid protein (GFAP) staining. 142 4.2.3. Plasmids, Adenoviruses and Transfections Mammalian expression plasmids carrying cDNA encoding mouse Nrf2 was a generous gift from Dr. Jawed Alam (Alton Ochsner Medical Foundation, New Orleans, LA) (Alam et al., 1999). The antioxidant response element sequence was obtained from the rat NAD(P)H:quinone oxidoreductase gene promotor and used to make the human placental alkaline phosphatase (hPAP) reporter constructs (rQR51wt) and mutant (rQR51mut), as described previously (Murphy et al., 2001; Shih et al., 2003). Recombinant adenoviral vectors were constructed using the Cre-lox system (Canadian Stroke Network core facility, University of Ottawa) (Hardy et al., 1997), and used as described previously (Shih et al., 2003). Astrocytes seeded (2 x 105 cell/mL) in 24-well plates were transfected using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol, except 1 pg of D N A and 1 pL of lipofectamine was used for each well and transfections were terminated after 6 h. The transfection efficiency of astrocytes was typically -20% as assessed by (3 galactosidase staining. 4.2.4. Glutamate and analogue treatments Astrocyte cultures were given various treatments 24 h after transfection of the A R E reporter and p-galactosidase vector. Glutamate and analogues (D-glutamate, L-aspartate, D-aspartate) were prepared as 100 mM stocks in H2O and dissolved by adjusting to pH 7.4 using NaOH. Stocks were diluted to indicated concentrations in HEPES-buffered salt solution (HBSS, in mM, 136 NaCl, 5 KC1, 1 M g C l 2 , 2.5 CaCl 2 , 10 HEPES, 1 N a H C 0 3 and 20 glucose; pH 7.4) for exposure to cells. After a 20 min exposure, the cells were washed 3x with HBSS and the original astrocyte conditioned media was replaced. For blocking ionotropic glutamate receptors or glutamate transporters, cultures were pre-treated with kynurenic acid or L-PDC, respectively, for 20 min and then co-applied with glutamate. HPAP activity was assessed 24 h after treatment. 143 4.2.5. Human placental alkaline phosphatase assay To assess ARE-mediated gene expression, hPAP activity was measured as described by previous studies (Henthorn et al., 1988). Briefly, astrocyte cultures were collected in lysis buffer consisting of 10 m M Tris-HCl (pH 8), 1 mM MgCl 2 , and 0.1% Triton X-100. Half of the sample was saved for determination of protein concentration and |3-galactosidase activity (for normalization of transfection efficiency). The remaining sample was heated at 65°C for 30 min to inactivate endogenous phosphatase activity. The assay was initiated by mixing ~15 pg protein with diethanolamine buffer (0.73 M diethanolamine with 0.36 mM MgCl 2 , pH 9.8, final concentration) and 13.6 m M p-nitrophenyl phosphate (final volume 150pL) and monitored at 405 nm. HPAP activity from rQR51mut transfected astrocytes was subtracted as A R E -independent background. 4.2.6. Enzyme Assays for astrocyte cell culture and brain tissue Astrocyte cells were rinsed with 10 mM phosphate buffered saline (PBS) pH 7.4, scraped into ice-cold PBS and sonicated for 10 s. These lysates were centrifuged at 15,000 g for 10 min at 4°C. Supernatants were collected and promptly assayed for enzyme activity. Brain and liver tissue was dissected in ice-cold PBS and homogenized with 10 strokes of a dounce homogenizer in tissue buffer containing 25 m M Tris (pH 7.4) and 250 mM sucrose. Crude homogenates were centrifuged at 15,000 g for 10 min at 4°C. Tissue homogenate supernatants were collected and promptly assayed for enzyme activity. N Q O l enzyme activity was determined by calculating the dicumarol-sensitive fraction of DCPIP reduction (Benson et al., 1980). Reactions consisting of 25 mM Tris-HCl buffer (pH 7.4) with 0.7 mg/mL BSA, 5 p M F A D , 200 p M N A D H , 30 pg/mL protein, with and without 20 p M dicumarol were pre-incubated for 10 min at 25°C (final 144 concentrations in 200 pL reaction volume). DCPIP was then added to a final concentration of 36 uM (20 pL volume), and the reaction was monitored at 540 nm. The extinction coefficient for DCPIP was 2.1 x 104 M" 1 cm" . The GST assay consisted of 1 mM l-chloro-2,4-dinitrobenzene (CDNB), 1 mM GSH, and 100 pg/mL protein at 37°C in 100 m M potassium phosphate buffer (pH 6.5) (final concentration in 150 pL reaction volume) (Kelly et al., 2000). The GST reaction was monitored at 340 nm and the spontaneous non-enzymatic slope was subtracted from the total observed slope. The extinction coefficient for CDNB was 9600 M ' c m 1 . The L D H assay consisted of 3.3 mM pyruvate, 0.34 mM N A D H , and 100 pg/mL protein in PBS at 37°C (final concentrations in 150 pL reaction volume) (Everse et al., 1970). The L D H reaction was monitored at 340 nm and the spontaneous non-enzymatic slope was subtracted from the total observed slope. Purified L D H enzyme standards were used to ensure that values were obtained within the linear of the assay. The extinction coefficient for N A D H was 6219 M" lcm" 1. Protein concentration was determined using the bicinchronic acid method according to the manufacturer's protocol (Pierce). 4.2.7. Glutathione assay Total GSH was quantified by the method of Tietze (Tietze, 1969). Briefly, the acid-soluble fraction was obtained from astrocyte cell lysates and tissue homogenates by adding perchloric acid to a final concentration of 3% followed by centrifugation at 14,000 g for 10 min. The acid-soluble fraction was neutralized to pH 7 with 0.5 M KOH/50 m M Tris. Following the removal of precipitate (potassium perchlorate) by a second centrifugation, 50 pL aliquots of sample were combined with 100 pL of reaction mixture consisting of 2.5 mL 1 m M DTNB, 2.5 mL 5 mM N A D P H , 2.5 mL phosphate buffer solution (100 m M NaP0 4 , pH 7.5, 1 m M EDTA), 145 and glutathione reductase (5 U/mL final). GSH-mediated reduction of DTNB was measured at 412 nm at 30 s intervals over 30 min. GSH content was normalized to protein. 4.2.8. Oxidative glutamate toxicity and assay for neuronal viability The oxidative glutamate toxicity paradigm was used as described previously (Shih et al., 2003). Briefly, immature cortical neurons (1-4 DIV) were pre-treated with tBHQ or DMSO vehicle for 24 h, followed by washout and exposure to 1 mM glutamate for a further 24 h. Neuronal viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2//-tetrazolium bromide (MTT) cell viability assay. 4.2.9. Animals and Experimental treatments Our experiments were approved by the Canadian Council for Animal Care and the University of British Columbia Animal Care Committee. A l l experiments were conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Wistar (UBC animal facility) and Sprague-Dawley rats (Charles River) weighing 250-350 g at the time of stroke were housed in pairs maintained in a 12-hour light/dark cycle with food and water ad libitum. A l l rats and mice were normally fed the Lab Diet 5001 formulation (Lab Diet), which contains no tBHQ, but trace amounts of B H A for preserving animal fats (2 parts per million). Osmotic pump studies were performed using Wistars, but due to high incidence of sub-arachnoid hemorrhage during stroke and higher post-stroke morbidity, later experiments (intraperitoneal injections) were performed on Sprague-Dawley rats. For intracerebroventricular (i.c.v.) osmotic mini-pump delivery, tBHQ was prepared at a final concentration of 1 mM in 1% DMSO in PBS (vehicle). TBHQ was first dissolved in pure DMSO and PBS was added to final volume. The mixture was rapidly and vigorously vortexed to re-dissolve any precipitates. We found that tBHQ prepared in this manner was stable for over a 146 week when maintained at 37°C, retaining its ability to induce Phase 2 enzymes and protect immature cortical neurons from oxidative glutamate toxicity. A rust color developed with time, although this transition did not affect its neuroprotective ability. Fresh tBHQ solution was sterile fdtered and loaded into pumps according to the manufacturer's protocol (Alzet, Model 1003D, designed to deliver 1 pL/h for 4 days). Each pump was then attached via a 2 cm length of tubing to a cannula (Plastics One) placed at bregma +1.5 mm M L , -0.9 mm AP, 4.5 mm DV, targeting the right lateral ventricle. TBHQ was infused continuously for 72 h at which time focal ischemia was initiated. Proper cannula placement was verified by eye during brain sectioning at the time of sacrifice. For intraperitoneal (i.p.) delivery, a more concentrated tBHQ solution was prepared at 5 mg/mL in 1% DMSO in PBS with the same method described above. The tBHQ solution was injected 3 times at 3.33 mg/kg or 16.7 mg/kg separated by 8 h intervals. The volume of each injection was set at 3.33 mL/kg. The concentrated tBHQ stock solution was prepared fresh for each injection. Nrf2_/" mice (C57B/SV129 background) originating from the laboratory of Dr. Yuet Wai Kan were maintained in a 12 h dark-light cycle with food and water ad-libitum (Chan et al., 1996b). Male mice were used between the ages of 10-16 weeks. A l l animals in this study were derived from breeding Nrf2+ /" heterzygotes since it was conceivable that some selection for mice with reduced sensitivity to oxidative stress may occur if the knockout animals were allowed to inbreed over several generations. For tBHQ feeding of mice, food pellets (Lab Diet 5001) were powdered in a coffee grinder and dry mixed with 1% tBHQ (w/w). Distilled water was added to the powder (equal v/w), thoroughly mixed, and reshaped into food pellets. The pellets were then baked at 60°C for 3 h. Control food was processed in the same fashion without the addition of tBHQ. 147 4.2.10. Stroke models Anesthesia was induced with 4% isofluorane (Baxter International Inc.) and maintained with 2% isofluorane in 30% 0 2 and 70% N 2 0 . Body temperature was maintained at 37°C with a feed-back controlled rectal probe-heat blanket system (FHC) and an overhead incandescent lamp. Ischemia-reperfusion was induced in rats using the intra-luminal suture method as previously described (Longa et al., 1989; Kawamura et al., 1991). Briefly, a ventral neck incision was made and neck muscles were pulled aside with ligatures and the right common carotid artery (CCA) was carefully dissected away from the vagus nerve. The external carotid artery (ECA) was ligated and cauterized. A 3 cm 3-0 nylon suture (Ethilon) (tip-blunted with a soldering iron) was inserted through the E C A and pushed into the internal carotid artery (ICA) until a gentle resistance was felt, leaving approximately 7-10 mm of the suture remaining outside the ECA/ICA bifurcation. The suture was clamped in place with a micro-aneurysm clip (Harvard Apparatus) and the animal was then sutured and awoken from anesthesia. After 1.5 h of ischemia, the animal was then re-anesthetized and the suture was retracted. Although the E C A was permanently ligated, blood flowing from the CCA could still pass through the ICA allowing full reperfusion. Animals were tested for stroke-induced sensorimotor deficits (described below) just before stroke was terminated to ensure proper placement of the suture. Blood samples were collected from a PE-10 femoral artery catheter (Intramedic, Becton Dickenson) and blood-gas measurements (Bayer Rapidlab 348) were made before, during, and 15 min after the ischemic period. Animals were included in the ischemia-reperfusion study based on a number of criteria: A.) Must show all signs of stroke behaviour during ischemia (behavioural score of 10 out of 12, see scoring scheme below). B.) No subarachnoid hemorrhage (assessed during removal of the brain from the skull). C.) Must have a visible infarct, regardless of size (lack of infarct is likely due to poor suture placement). D.) Must not be hypothermic at any point during or 30 min after the stroke surgery (i.e. less than 36°C). 148 For Nrf2"" mice, cortical ischemia was induced by permanently occluding the right distal middle cerebral artery (MCA) using a procedure modified from previous studies (Cechetto et al., 1989; Majid et al., 2000). Anesthesia and body temperature regulation were same as focal ischemia with rats. The mice were placed in a stereotaxic frame (Kopf instruments) for surgery. An incision was made in the skin between the right eye and ear. The temporal muscle was cut and pulled aside, and the M C A was accessed by drilling a small hole in the skull using an air-powered dental drill. The artery was carefully cauterized using either a f i n e tip hand-held heat cautery (Fine Science Tools) or a bipolar electro-coagulator (Martin, M E 102). Any animals that developed sub-arachnoid hemorrhage from this procedure were immediately sacrificed. The successful occlusion of the M C A was guided with a Laser Doppler (Perimed PF3). The Doppler probe was placed above the temporal ridge but not over an artery to ensure measurement of cerebral microcirculation. Heat cauterization itself produced negligible tissue damage in sham surgeries, where no change in regional cerebral blood flow was observed. Intracortical endothelin-1 microinjections were performed as described previously (Zhang et al., 2005), with some modifications. Briefly, a hole was drilled in the skull at +1.5 mm M L , -1.0 mm AP, and -0.8 mm DV. Glass pipettes (60 - 90 pm tip diameter) linked by PE-10 tubing to a 10 pL Hamilton syringe were used to inject 0.5 pL of endothelin-1 (1 pg/pL) at a rate of 6 pL/h using a syringe pump (World Precision Instruments). Mice were sacrificed 7 days after endothelin-1 injection. 4.2.11. Semiqualitative Neurological Scoring for Rat Ischemia-Reperfusion Sensorimotor performance was scored based on a previous description with some modifications (Reglodi et al., 2003). Briefly, the scoring system involved the evaluation of 6 tests each with a 3-tiered scoring scale (0, 1, or 2), with 0 being normal behaviour and 2 as 149 severe deficit of contralateral side. Tests included: a.) contralateral thorax twisting when elevated by tail, b.) contralateral forelimb flexion, c.) resistance to lateral push, d.) forelimb bracing against table surface, e.) forelimb placement on table edge, f.) forelimb and hindlimb table edge sensation. Each animal received a cumulative score out of 12 for each trial. Behaviour was assessed just before termination of ischemia (1.5 h) and again after 22.5 h of reperfusion. In long-term survival studies, neurological scoring was performed weekly. Only animals that received at least a score of 10 out of 12 during neurological testing at the time of occlusion were included in the study. From our experience, a score below 10 (at the time of occlusion) is most likely due to improper placement of the suture and incomplete ischemia. An experimenter blinded to the animal treatment groups performed behavioural assessments and surgeries. 4.2.12. Infarct Measurement The extent of infarction was measured with 2,3,5-triphenyl-tetrazolium chloride (TTC) for 24 h survival times (Bederson et al., 1986a), or cresyl violet (CV), which is more appropriate for assessing infarct area at longer time points since TTC vital staining will not differentiate between gliosis and viable neuronal tissue. For TTC staining, animals were deeply anaesthetized with euthanyl (Bimeda) and the brains removed, washed in room temperature (RT) PBS, and sliced into 2 mm sections for rats and 1 mm sections for mice with the aid of brain matrices (Plastics One Inc.). The sections were carefully placed into 3% TTC in PBS and incubated at RT for 10 min. The TTC solution was then removed and replaced with 4% PFA for overnight fixation at 4°C. For C V staining, animals were anaesthetized with euthanyl and transcardially perfused with RT PBS followed with ice cold 4% PFA (for each solution, 100 mL for rats and 15 mL for mice). The brains were removed and post-fixed for 24 h, then cryoprotected in 30% sucrose in PBS with 0.02%> sodium azide for a further 48 h. Coronal sections were collected with a cryostat and mounted on Superfrost slides (Fisher). C V staining was performed on 40 pm 150 sections using standard protocols. A l l stained sections were scanned at 600 dpi on a desktop scanner (Epson 1660). Infarct area was measured using ImageJ version 1.30 (NIH). Given that hemispheric volume could change as a result of the ischemia, we normalized the infarct area of each section to ipsilateral hemisphere area (or cortex area for permanent M C A occlusion in mice) and reported data as % infarct area of ischemic hemisphere or cortex. Similar results were obtained when infarct area was expressed as percentage of contralateral hemisphere/cortex. Infarct volume was calculated according to the principle of Cavalieri (volume = sidi + s2d2 + S3d3 + S4d4) where s = infarct surface area and d = distance between 2 sections. Infarct quantification was performed by a researcher blinded to the experimental conditions. Fluorojade staining used to quantify endothelin-1 induced lesions was performed on 20 pm mouse brain sections as described previously (Schmued et al., 1997). Fluorojade images were collected using Northern Eclipse software (ver. 6.0) and a wide-field fluorescence microscope (Zeiss Axiophot) equipped with a Retiga Exi CCD camera (Qlmaging). Infarct area was measured using ImageJ. Since ET-1 injection does not always produce a contiguous volume of tissue damage, infarct size was expressed as summed Fluorojade-positive area measured over 6 brain sections taken at 600 pm intervals (bregma 2.0 mm to -2.0 mm) rather than as a calculated brain volume. Since a 7 day time point was used to assess Fluorojade staining it was also not always possible to view all individually degenerating neurons and count them. Therefore, we assessed Fluorojade staining by outlining an infarcted region and measuring its area. 151 4.2.13. Statistical Analyses A l l data are shown as means + SEM. Statistical analyses were performed using the two-tailed Student's t-test, one-way A N O V A with Bonferroni's post-hoc test, or two-way A N O V A in GraphPad Prism (version 2.01). * p<0.05, ** pO.Ol , *** pO.OOl. 4.3. Results 4.3.1. Induction of multiple antioxidant systems in astrocyte cultures by inducers of Nrf2-dependent transcription. In cultured astrocytes, Nrf2-mediated induction of antioxidant enzymes can be achieved by treatment with certain low molecular weight electrophilic compounds such as tBHQ (Ahlgren-Beckendorf et al., 1999; Eftekharpour et al., 2000; Murphy et al., 2001; Lee et al., 2003a; Shih et al., 2003). Based on previous studies, three established markers of Nrf2 activation were evaluated throughout the study: total glutathione (GSH) content, glutathione S-transferase (GST), and NAD(P)H:quinone oxidoreductase (NQOl) activity (Chan and Kwong, 2000; Hayes et al., 2000b; Ishii et al., 2000; McMahon et al., 2001; Ramos-Gomez et al., 2001; Chanas et al., 2002; Shih et al., 2003). Consistent with other reports, all three markers were induced within 10-24 h in response to tBHQ treatment (Fig. 4-1A) (Ahlgren-Beckendorf et al., 1999; Lee et al., 2003c; Kraft et al., 2004). As a positive control, adenoviral-mediated over-expression of Nrf2 (Ad-Nrf2) increased all three markers, compared to GFP control (Ad-GFP), while a dominant negative version of Nrf2 (Ad-Nrf2DN) produced no change (Fig. 4-IB) (Shih et al., 2003). Importantly, these effects were specific, since no increase in lactate dehydrogenase (LDH) activity (not regulated by Nrf2) was observed with either tBHQ treatment or overexpression of Nrf2 (Fig. 4- lA,B) . 152 Previous studies have shown that tBHQ does not activate the Phase 2 response in Nrf2"" cultures, confirming its specificity for Nrf2 (Lee et al., 2003c). Consistent with this report we find that astrocytes prepared from Nrf2"A mice have reduced basal (23.8 + 4.3% of Nrf2 + / + control, n = 3) and inducible ARE-mediated gene expression as assessed by reporter assay (Fig. 4-1C). As a result, basal and inducible GSH, GST and N Q O l levels were also suppressed in Nrf2_/" astrocytes (Fig. 4-lD,E). Importantly, induction of these markers could be rescued by Nrf2 over-expression (Fig. 4-IE). Previous in vitro experiments by Kraft et al. indicate that the neuroprotective effects of tBHQ are lost when using Nrf2_/" astrocytes (Kraft et al., 2004). These results suggest that the observed tBHQ-stimulated changes in ARE-mediated gene expression, not induction of another pathway, play a causal role in neuroprotection. The three markers tested (GSH, GST, and NQOl) do not represent the full scope of Nrf2 gene targets but nonetheless indicate that the Phase 2 response is activated. For a more comprehensive description of Nrf2-regulated genes in vitro, see recent microarray studies (Li et al., 2002; Thimmulappa et al., 2002; Lee et al., 2003c; Shih et al., 2003; Kraft et al., 2004). In this in vitro paradigm, the effect of tBHQ requires 10-24 h to be manifested since new transcription and protein translation must precede its neuroprotective function. Accordingly, in our in vivo studies we first examined the effects of tBHQ pre-treatment on transient forebrain ischemia in rats. 153 Figure 4-1. Time course and Nrf2 dependency of tBHQ-mediated phase 2 enzyme induction in astrocyte-enriched cultures. A, Astrocyte cultures (7-10DIV) prepared from postnatal rats (postnatal day 0-2) were treated with 20 p M tBHQ or DMSO vehicle for various times before harvesting for GSH, GST, N Q O l , and L D H assays. Induction of GSH, GST, and N Q O l was observed within 10-24 h, whereas L D H activity (negative control) was unchanged. B, As a positive control, Ad-Nrf2 was sufficient to increase GSH content, as well as N Q O l and GST activity, compared with GFP control (Ad-GFP), where as Ad-Nrf2DN produced nochange (Shih et al., 2003). C, Nrf2 + / + and Nrf2"/' cultures were transiently transfected with an hPAP reporter construct regulated by an ARE-bearing promotor and treated with 20 u M tBHQ for 24 h. Increased hPAP activity was detected in Nrf2 + / + but not Nrf2"y" cultures. D, Basal activities of GST and NQOl , as well as GSH content, were suppressed in astrocyte cultures derived from Nrf2 / _ mice, compared with Nrf2 + / + control cultures. E, tBHQ-mediated induction of GSH, GST, and N Q O l by tBHQ was abrogated in Nrf2"/_ cultures but could be rescued with adenoviral Nrf2 overexpression (Lee et al., 2003b; Shih et al., 2003). Data represent the mean ± S E M collected from at least three independent experiments. p<0.05, p< 0.01, and p< 0.001; two-tailed t test. 154 4.3.2. Intracerebroventricular infusion of tBHQ is protective in a rat model of ischemia-reperfusion. As one strategy to induce Phase 2 enzymes in the brain in vivo, we implanted osmotic mini-pumps, which continuously delivered a small volume (1 pL/h) of 1 m M t B H Q solution (prepared in 1% D M S O vehicle) for 4 days into the right lateral ventricle (i.e.v.), bypassing the blood brain barrier. The t B H Q solution was active for the entire duration of in vivo administration since it was capable of protecting cultured neurons from oxidative glutamate toxicity even when extracted from the pump after 4 days at 37°C (Fig. 4-2). ES998SS3B9B K I —. /~"N I . . wmm No (J\U ] 3mM Glu F i g u r e 4-2. T B H Q s t a b i l i t y i n m i n i - o s m o t i c p u m p s . The 1 mM tBHQ solution (in PBS) was loaded into an Alzet mini-osmotic pump and maintained at 37°C within a PBS fdled eppendorf tube for 4 days. The remaining solution was extracted from the pump after this time and used in a bioassay of neuronal viability during oxidative glutamate toxicity as described previously (Shih et al., 2003). The tBHQ solution was diluted to a final concentration of 20 u M in culture media. The tBHQ solution remained active within the pump for at least 4 days, since the extracted solution protected neurons as effectively as freshly prepared tBHQ. Data represent the mean + S E M collected from 3 independent experiments. 155 After 3 days of continuous tBHQ delivery, rats were subjected to focal ischemia-reperfusion using the intraluminal suture model (Fig. 4-3E) (Longa et al., 1989; Kawamura et al., 1991). In animals treated with vehicle, we observed a subcortical (i.e. striatum) and cortical infarct 24 h after ischemia using vital staining with TTC (Fig. 4-3A,B,C). However, in animals treated with tBHQ we observed a significant reduction of infarction in the cortex but not of the stroke core within the subcortex (volume of cortical infarct expressed as % of total ischemic hemisphere: DMSO = 9.2 ± 2.9, tBHQ = 1.1 ± 0.7, ** p = 0.008, two-tailed t-test) (Fig. 2A, B, C). For both DMSO and tBHQ treated animals, we observed no significant hypothermia or apparent differences in temperature between the groups (Table 4-1). Both vehicle and tBHQ-treated animals had a similar level of contralateral sensorimotor deficits during occlusion, suggesting that both groups were subjected to similar levels of ischemia (Fig. 4-3D). However, 24 h after ischemia we found a significant reduction in the behavioural scores in the tBHQ-treated group when compared to vehicle-treated controls, consistent with infarct volume measurements (Fig. 4-3D). 156 A a x CD CD a5 14 § 12 cc v> 2 E 10 CC CD x: o ro c o E 0 o (/) o 9 K S u b c o r t e x i 1 2 3 4 5 6 Section (2mm) B Vehicle 1mM tBHQ 0 i« 0 a. ro CD It •2 E £ CD o D 20 18 16 14 12 10 8 6 4 2 0 C o r t e x * 1 i * V 1 2 3 4 5 6 Section (2mm) 12 CD o10 o w o 13 CD C ii 1.5 24 Time (h) Pump Implant] 72 h-Ischemia (1.5 h) 24 h Infarct Assessment Figure 4-3. Intracerebroventricular infusion of tBHQ reduced sensorimotor deficit and infarct size 24 h after ischemia-reperfusion. Intracerebroventricular delivery of 1 mM tBHQ (1 pl/h) for 72 h before focal ischemia reduced cortical infarction compared with vehicle-treated controls. A, Animals were killed 24 h after the initiation of transient M C A O (1.5 h), and infarcted brain regions were visualized using TTC staining (lighter colored tissue is infarcted). Representative examples are shown from each treatment group. B, Cortical infarction was attenuated with tBHQ treatment. Infarct areas are shown between sections 1 (anterior) and 6 (posterior). Section 2 approximately corresponds to bregma = 0.0. C, Subcortical infarction was not attenuated. D, Acute motor deficits assessed during the stroke (1.5 h) were similar, suggesting that animals from both groups received the same degree of ischemia. However, sensorimotor deficit was significantly lower in the tBHQ-treated group when evaluated 24 h after reperfusion. E, A schematic diagram summarizing the pump implant and stroke timeline. Intracerebroventricular infusion was continuous from pump implant to killing. Data represent mean ± S E M collected from n = 9 animals for tBHQ and n = 1 fox vehicle treatment groups, p < 0.05, two-tailed t test or one-way A N O V A with Bonferroni's post hoc test. 1 5 7 Table 4-1. Summary of Physiological Parameters for Rat and Mouse Stroke Studies I.C.V. O s m o t i c P u m p S t u d y V e h i c l e t B H Q Number of animals 9 7 Core temp, before occlusion (°C) 37.4 ±0.3 36.7 ±0.3 Core temp, during occlusion (°C) 38.0 ±0.2 37.8 ±0.5 Core temp. 30 min after occlusion (°C) 36.9 ±0.5 36.9 ±0.5 Weight before pump implant (g) 267 ± 15 274 ± 17 Weight before stroke (g) 272 ± 13 275 ± 13 Weight 24 h after stroke (g) 243 ± 15 253 ±20 I.P. D e l i v e r y : 24 h S u r v i v a l Number of animals 11 11 Core temp, before occlusion (°C) 36.6 ±0.2 36.9 ±0.2 Core temp, during occlusion (°C) 39.1 ±0.1 38.9 ±0.1 Core temp. 30 min after occlusion (°C) 37.6 ±0.3 37.6 ±0.2 Weight before i.p. injection (g) 309 ±7 315 ± 7 Weight before stroke (g) 292 ±7 294 ±7 Weight 24 h after stroke (g) 259 ± 6 73 ± 7 I.P. D e l i v e r y : 1 M o n t h S u r v i v a l Number of animals 5 6 Core temp, before occlusion (°C) 37.1 ±0.1 36.9 ±0.1 Core temp, during occlusion (°C) 38.7 ±0.3 38.7 ±0.3 Core temp. 30 min after occlusion (°C) 37.5 ±0.2 37.2 ±0.2 Weight before i.p. injection (g) 330 ± 12 316 ± 15 Weight before stroke (g) 299 ± 10 291 ± 14 158 Weight 24 after stroke (g) Weight 1 month after stroke (g) Blood Gas Analysis (before) pH p C O 2 (mm Hg) p O 2 (mm Hg) Cl"(mmol/L) Hematocrit (%) Blood Gas Analysis (during) pH p C 0 2 (mm Hg) p 0 2 (mm Hg) Cl (mmol /L) Hematocrit (%) Blood Gas Analysis (15 min after) pH p C 0 2 (mm Hg) p 0 2 (mm Hg) Cl"(mmol/L) Hematocrit (%) Adult Mouse Focal (24 h. survival) Number of animals Core temp. 30 min after cautery (°C) Weight before stroke (g) Weight at sacrifice (g) Adult Mouse Focal (7 d . survival) 263 ± 8 429 ± 10 7.31 ± 0 . 0 5 39.1 ± 2 . 7 111.7 ± 7.1 102.5 ± 0 . 6 41.2 ± 1.2 7.32 ± 0 . 0 7 30.4 ± 7 . 8 124.9 ± 5 . 9 101.4 ± 1.8 37.3 ± 0 . 3 7.50 ± 0 . 0 1 37.7 ± 1.4 109.5 ± 2 . 4 101.7 ± 1.2 37.3 ± 0 . 3 Nrf2 + / + 8 36.6 ± 0 . 2 26.4 ± 1.1 24.8+1.0 Nrf2 258 ± 13 420 ± 18 7.32 +0.05 40.5 ± 3 . 1 103 ± 4 . 5 103.6 ± 1.1 40.6 ± 1.0 7.39 ± 0 . 0 9 38.5 ±_1.7 116.0 ± 6 . 8 101.5 ± 1.0 37.0 ± 2 . 0 7.47 ± 0.05 36.8 ± 1.4 111.0 + 7.8 102 ± 0 . 6 3 8.5+2.1 Nrf2 -'-5 36.1 ± 0 . 7 25.9 ± 0 . 7 25.2 + 0.9 159 Number of animals 9 3 8 Core temp, before cautery (°C) 37.2 ±0.2 37.4 ± 0.1 37.2 ±0.2 Core temp. 30 min after cautery (°C) 37.1 ±0.2 37.7 ± 0.2 36.7 ±0.2 Weight before stroke (g) 27.0 ±0.8 32.0 ± 0.3 29.2 ±0.6 Weight at 24h post-stroke (g) 27.0 ± 1.0 32.1 ± 0.9 28.2 ± 1.0 Weight at sacrifice (g) 27.0 ±0.9 29.5 ± 0.2 28.8 ±0.7 Adult Mouse ET-1 (7 d. survival) Nrf2 +/+ Nrf2 -'-Diet type Control 1% tBHQ Control 1% tBHQ Number of animals 6 6 5 6 Core temp, after microinjection (°C) 37.2 ±0.1 37.4 H :0.3 37. 1 ±0.2 36.9 ±0.3 Weight before tBHQ feeding/sham (g) 31.4 ± 1.7 31.8 H : 1.2 25. 8 ± 1.0 27.9 ±0.6 Weight before stroke (g) 30.7 ± 1.4 30.0 H : 1-2 25. 7+1.0 21.3 ±0.3 Weight at sacrifice (g) 30.3 ±2.0 29.4 H : 1.4 26. 2 ± 1.0 19.5 ±0.5 All values are the mean + standard error. PC0 2, arterial carbon dioxide partial pressure; P0 2, arterial oxygen partial pressure. For rat studies, there were no significant differences among groups for physiological variables, Student's t-test or one-way ANOVA. For mouse studies, only a significant reduction of body weight in tBHQ-fed Nrf2_/" mice was observed; compared to Nrf2_/" mice fed control diet. *p < 0.05 versus control diet for same genotype. 4.3.3. Induction of cortical glutathione levels in vivo following tBHQ treatment. Given that i.c.v. delivery of tBHQ is not necessarily a viable strategy for clinical prophylactic treatment, we also tested whether intraperitoneal (i.p.) injection of tBHQ would protect against focal ischemia-reperfusion in rats. We first optimized i.p. injection of tBHQ by monitoring cortical GSH content, GST and N Q O l activities. Using a protocol consisting of three 16.7 mg/kg injections, each spaced 8 h apart, we could robustly and consistently increase GSH levels at 24 h after the first injection (Fig. 4-4). The increase in GSH was apparently due to local brain synthesis and likely not delivered to the brain after synthesis in peripheral organs since GSH levels in the serum and cerebrospinal fluid (CSF) were unaltered with tBHQ treatment 160 (serum GSH + GSSG levels 24 h after i.p. injection, expressed in pM: vehicle = 14.5 ± 2.9, tBHQ = 14.7 ± 2.8; Total CSF GSH + GSSG levels: vehicle = 3.1 ± 0.3, tBHQ = 3.2 ± 0.4). Using this treatment we were not able to resolve significant elevation of GST and N Q O l enzyme activity in the cortex perhaps because longer time-points were needed for the induction of these enzymes. It is also possible that the site of induction for these enzymes is restricted to a subpopulation of cells in the total tissue sample (i.e. specific astrocyte types or the meninges) leading to dilution of the enzyme activity. The 16.7 mg/kg dose (total 50 mg/kg injected) was used for subsequent stroke experiments since this treatment produced a significant increase in cortical GSH, whereas a dose of 3.3 mg/kg was unable to produce this induction (Fig. 4-4). With higher doses (33.3 mg/kg), animals displayed adverse behaviour with ataxia of the hind-limbs and trunk, as observed in previous studies (FAO/WHO, 1975). The 16.7 mg/kg dose sometimes produced a mild and transient ataxia observed within the first 5 min of injection and lasting for less than 10 min. However, this effect is unlikely to be related to Nrf2 due to its rapid and transient nature, and may be due to discomfort from the injected solution. 161 B o CD £ O co 00 O c/) CD = 1 50 40 30 20 10 0 Cortex P = 0.007 * * — *b 4 MX ± <b Start Sacrif ice 24 h Oh 8 h 16 h F i g u r e 4-4. I n d u c t i o n o f G S H i n t h e c o r t e x a f t e r i n t r a p e r i t o n e a l i n j e c t i o n o f t B H Q . Sprague Dawley rats were injected with tBHQ intraperitoneally (vehicle, 3.3 mg/kg, or 16.7 mg/kg) three times daily (8 h apart), and GSH content was evaluated 24 h after the first injection. Homogenized cortical tissue was protein precipitated, and the acid-soluble GSH was immediately assayed using the glutathione reductase method (Tietze, 1969). The 16.7 mg/kg dose, but not the 3.3 mg/kg dose, significantly increased cortical GSH levels. Symbols represent paired animals in each experiment. Data represent means ± SEM from n = 6 animals per treatment group, p = 0.007, two-tailed / test. 162 4.3.4. Prophylactic intraperitoneal injection of tBHQ confers protection both 24 h and 1 month after ischemia-reperfusion. Comparable to i.c.v. delivery, i.p. administration in rats also reduced ischemic damage in the anterior cortex after 24 h of reperfusion (Fig. 4-5A) (volume of cortical infarct expressed as % of total ischemic hemisphere: vehicle = 14.76 ± 3.83, tBHQ = 7.50 ± 3.04, *p < 0.05, two-tailed t-test), while no significant protection was observed in the subcortex (Fig. 4-5B). In this case, ischemia was induced 24 h after the first of the three tBHQ injections (Fig. 4-5E). We did not observe differences in core temperature between vehicle and tBHQ treated groups before, during or 30 min after the ischemic period (Table 4-1). Again, a similar level of behavioural impairment was detected between vehicle and tBHQ groups during ischemia, but a significant improvement in the tBHQ-treated animals was observed 24 h post-ischemia (Fig. 4-5C). Importantly, tBHQ injection did not significantly reduce body weight compared to controls, suggesting that the protective effect was not due to reduced caloric intake (Fig. 4-5D and Table 4-1) (Yu and Mattson, 1999). 163 A o ro <D s*_ 03 o is CD CD CL 0 CD JZ o B CD it-CD JO Q. E CD JC o E CD JO o If) E 35-30-25-20-15-10-5-0-30-20-15-10-5-0-Oh Cortex -fjL 2 3 4 5 6 Section (2 mm) Subcortex 1 1 2 3 4 5 6 7 Section (2 mm) Ischemia (1.5 h) C 0) o o 01 o Z3 CD z > < 12 10-8-6 4 2 0 D 100 TO."!5 5 co .E CA 95 90 85 80 ] V e h i c l e I t B H Q 1.5 24 T i m e a f t e r o n s e t o f i s c h e m i a ( h ) X P o s t i .p . I n j e c t i o n P o s t S t r o k e — 24 h -8 h 16 h 24 h _ ^ I n f a r c t - I A s s e s s m e n t Figure 4-5. Intraperitoneal injection of tBHQ reduced sensorimotor deficit and stroke damage 24 h after ischemia-reperfusion. Sprague Dawley rats were injected with tBHQ intraperitoneally (16.7 mg/kg; 3 times) or vehicle, as described in Figure 3, followed by focal ischemia-reperfusion. The animals were killed 24 h after initiation of transient MCAO (1.5 h), and brain slices were stained with TTC. A, tBHQ treatment significantly reduced stroke damage in anterior cortical regions of the affected hemisphere. B, Similar to intracerebroventricular delivery of tBHQ, no sparing of subcortical tissue was observed. C, Acute sensorimotor deficits assessed during the stroke (1.5 h) were similar, suggesting that animals from both groups received the same degree of ischemia. However, sensorimotor deficit was significantly lower in the tBHQ-treated group when evaluated 24 h after stroke. Avg. Neurol. Score, Average neurological score. D, Average animal weight in the two treatment groups was not different when examined both 24 h after intraperitoneal injection of tBHQ and 24 h after stroke. E, A schematic diagram summarizing the injection and stroke timeline. The short arrows represent time points of tBHQ injection. Data represent mean ± SEM collected from n = 11 animals per treatment group, p < 0.05 and p < 0.01, two-tailed / test or one-way ANOVA with Bonferroni's post hoc test. 164 To assess the long-term benefit of acute tBHQ injection, we examined a second group of rats for 1 month after ischemia. These animals were injected with tBHQ as with 24 h survival groups and no additional tBHQ supplementation was given post-stroke. For this group of animals, arterial blood-gas levels were examined before, during and 15 minutes after ischemia-reperfusion. We found no differences in blood pH, p C 0 2 , pCh, CI" or hematocrit between tBHQ-treated and control animals (Table 4-1). Neurological scoring was performed during ischemia to verify successful occlusion, and on a weekly basis thereafter. TBHQ-treated animals had on average lower neurological scores throughout the entire survival period (** p = 0.002, two-way ANOVA) (Fig. 4-6B), but recovered sensorimotor function more quickly within the first 4 days of reperfusion, compared to vehicle controls. Histological examination showed that ischemic damage in anterior cortical regions was significantly reduced with tBHQ treatment even 1 month after the ischemic event (volume of cortical infarct expressed as % of total ischemic hemisphere: vehicle = 24.9 ± 3.9, tBHQ = 10.1 ± 4.5, *p < 0.05, two-tailed t-test) (Fig. 4-6A,C). No attenuation of damage was observed in the subcortex (Fig. 4-6D). Together, these data suggest that the effects of systemic tBHQ administration extend beyond the blood brain barrier (possibly even more so in an ischemia-compromised state), providing neuroprotection and preservation of sensorimotor function for up to 1 month after stroke. 165 A 0) o 3 Q C 0) u_ SZ <n E x : o I CD O w 45 i 40-35-30-25-20-15-10-5-oJ c^ C o r t e x Vehicle tBHQ 1 2 3 4 5 6 7 Section Q D 12i o 10-o to 8-o -3 6-0 z 4-d) Av 2-0-D o CD W (ti u CO —^ c 0) a CO E CD -£= O £ 4) sz a w 20 18-16-14-12 10H 8 6 4 2 0 0 5 10 15 20 25 30 Post ischemia survival time (d) S u b c o r t e x fl r ir 1 it 3 4 5 6 7 Section Figure 4-6. Intraperitoneal injection of tBHQ reduces sensorimotor deficit and stroke damage 1 month after stroke onset. A s w i t h 24 h su rv iva l an ima l s , long- te rm su rv iva l experiments were in i t i a ted w i t h the same intraperitoneal t B H Q in jec t ion r eg imen (16.7 m g / k g ; 3 t imes) or v e h i c l e before i schemia- reper fus ion . N o addi t ional injections were g i v e n after stroke. Sensor imotor func t ion was evaluated on a w e e k l y basis . Af te r 1 month , the an imals were perfused, and brains were c ryosec t ioned for c r e sy l v io l e t s ta in ing . A, Representative examples are s h o w n f rom each treatment group ( infarct border is demarcated b y b l ack l ine) . B, t B H Q treatment s ign i f i can t ly i m p r o v e d the average n e u r o l o g i c a l score ( A v g . N e u r o l . Score) throughout the entire s u r v i v a l pe r iod ( p = 0 .002; t w o - w a y A N O V A ) . C, Infarc t ion o f the anterior cor t ical regions was attenuated i n the t B H Q - t r e a t e d an imals . D, N o s ign i f ican t subcor t i ca l p ro tec t ion was observed. Da ta represent m e a n ± S E M co l lec ted f rom n = 6 for t B H Q and n = 5 an ima l s for veh ic l e control groups, p < 0 .05, two- ta i l ed t test or one-way A N O V A w i t h Bonfe r ron i ' s post hoc test. 4.3.5. Basal and Inducible Phase 2 enzyme activities are suppressed in the Nrf2" mouse brain. Induction of Nrf2-dependent gene expression normally occurs in the ischemic penumbra following stroke raising the possibility that Nrf2 activation may be an endogenous response to 166 limit stroke damage (van Lookeren Campagne et al., 1999; Campagne et al., 2000; Laxton et al., 2001; Liverman et al., 2004). Data using gel shift assays indicated that binding to the A R E sequence found in Nrf2 target genes such as metalliothionein-1 and -2 was upregulated after transient forebrain ischemia in rat (Campagne et al., 2000). Before testing the hypothesis that Nrf2 can modify sensitivity to ischemia in vivo, we first verified the Nrf2_/" phenotype and the effect of tBHQ administration in our mouse colony by examining basal and inducible Phase 2 enzyme levels in the liver and multiple brain regions, again using GSH, GST, and N Q O l as markers. As observed in previous studies, all three markers were suppressed in the livers of Nrf2" mice when compared to Nrf2 + / + controls (Fig. 4-7B,D,F) (Chan and Kwong, 2000; Hayes et al., 2000b; Kwak et al., 2001a; Chanas et al., 2002). In brain, basal GST and N Q O l activities were modestly but significantly reduced in multiple brain regions of Nrf2"/_ mice, compared to Nrf2 + / + controls (Fig. 4-7A,C). Interestingly, basal brain GSH levels were not altered, suggesting that the constitutive synthesis of brain GSH does not rely on Nrf2 function, at least in the young adult mice examined in this study (Fig. 4-7E). The house-keeping gene, lactate dehydrogenase (LDH), is not regulated by Nrf2 and accordingly, no difference in activity was observed between genotypes (Fig. 4-7G, H). 167 LDH activity / - v (nmol / min / mg protein) ^ ^  o o o o o o o o o o o o o o o o o r~ D ug GSH + GSSG / mg protein O O l O O i O O l O U l O O l O m NQ01 activity Q (nmol / min / mg protein) -iMUJitllCFlNO) OOOOOOOOO GST activity J^> (nmol / min / mg protein) o o o o o o o o o o LDH activity (nmol / min / mg protein) N) & O) 05 O tO ^ o o o o o o o o o o o o o o o jig GSH + GSSG / mg protein M t. U> fl) O M * o o o o o o o o NQ01 activity O (nmol / min / mg protein) -J. -» K> w o oo ro cn o GST activity (nmol / min / mg protein) -»• K) ro w w ui o yi o 01 o cn o o o o o o o o 00 Figure 4-7. Reduced basal GST and NQOl activity, but not GSH content, in the Nrf2 brain. Nrf2+/+ and Nrf2"A littermate pairs were examined for markers of Nrf2 activity. Brains were dissected in cold PBS, homogenized, and immediately assayed for GSH, GST, NQOl, and LDH. A, B, GST activity in Nrf2"A mice was lower in liver and all brain regions examined, compared with Nrf2+ / + mice. C, D, NQOl activity was significantly reduced in the Nrf2_/" liver, cortex, and striatum. E, F, Total GSH content, although reduced in the Nrf2_/" liver, was not reduced in any brain regions examined. G, H, Activity levels of LDH (not an Nrf2 target) were not reduced in liver or brains of Nrf2mice. Data represent the mean ± SEM of six mice per genotype. *p < 0.05, **p < 0.01, and *"p < 0.001; paired two-tailed /test. Hippo, Hippocampus; Cereb, cerebellum. We next examined tBHQ-dependent induction of these markers in mice with different Nrf2 genotypes. We initially found that all genotypes responded poorly to intraperitoneal injections of tBHQ administered at the same dose used for rats (3 injections of 16.7 mg/kg), exhibiting hyperactivity, increased sensitivity to tactile stimulus, and vertical jumping. As an alternative we delivered tBHQ by dietary supplementation as described previously for other Nrf2 inducers such as oltipraz and sulforaphane (McMahon et al., 2001; Ramos-Gomez et al., 2001; Fahey et al., 2002; Wu et al., 2004). In previous studies, we found that 5% tBHQ in diet was not consumed by Nrf2 expressing mice, whereas 1 % tBHQ was consumed and highly protective (in Nrf2 expressing mice, but not knockouts) during 3-nitropropionic acid toxicity in vivo, a model involving striatal-specific oxidative stress (Shih et al., 2005b). A diet of 0.5% tBHQ was only partially neuroprotective against 3-nitropropionic acid (Shih et al., 2005b). For these reasons, a concentration of 1%> tBHQ was used in all further feeding experiments for the current study. Nrf2"/_ mice were less tolerant of dietary tBHQ administration losing ~ 20 % body weight during 1 week of tBHQ feeding, and consuming approximately half as much food as tBHQ-fed Nrf2 + / + and Nrf2+ /" mice (Fig. 4-8A,B). In total, Nrf2 + / + , Nrf2 + / \ and Nrf2"A mice consumed 12.5 ± 1.4, 12.7 ± 1.3, and 7.3 ± 0.5 mg tBHQ/g body weight over 1 week, respectively. The 1%> tBHQ diet increased cortical GSH levels in Nrf2 + / + and Nrf2+ /" mice, consistent with i.p. delivery of tBHQ in rats (Fig. 4-8C). Importantly, the induction of cortical GSH was not observed in 169 Nrf2"" mice, providing evidence that tBHQ-induced changes in cortical GSH are Nrf2 dependent (Fig. 4-8C). A cn.5> — c 220 f ^ 200 o « 180 T3 120 110 100' 90 80 70 60 50 87 5 CO 160 140 <3 8 120 100f 80 60 1 2 3 4 5 6 7 8 9 Days of feeding GSH Cx Str NQ01 Liv B o 20 18 16 14 12 10 8 6 4 2 0 — n — +/+ control (/) - • - +/+ tBHQ (7) —A— +/- control (12) - A - +/- tBHQ (10) - 9 - -/- control (5) - O - -/- tBHQ (8) 1-2 3-4 5-6 Days of feeding Cx Str Liv 180 Str Liv Cx Str Liv Figure 4-8. Effect of dietary tBHQ administration on brain and liver GSH content and phase 2 enzyme activity. Nrf2-/" mice and Nrf2-expressing controls were fed 1 % tBHQ for 1 week and were subsequently examined for markers of Nrf2 activity. .4, B, Summary of animal weight and food consumption from tBHQ feeding experiments. On average, Nrf2"A mice lost ~20% of their starting body weight and consumed approximately one-half as much food as Nrf2"A mice fed control diet. tBHQ feeding did not cause loss of body weight in Nrf2-expressing mice. Brains from tBHQ-fed mice were dissected in cold PBS, homogenized, and immediately assayed for GSH, GST, NQOl, and LDH. C, Consistent with intraperitoneal injection of tBHQ in rats, dietary tBHQ also produced a significant increase in GSH levels within the cortex and striatum of Nrf2-expressing mice. No increase in GSH was observed in the brains of Nrf2"/_ mice, consistent with the Nrf2-specific action of tBHQ. However, an increase of GSH was detected in the livers of Nrf2_/' mice, which may be associated with tBHQ toxicity observed in this genotype. D, E, Increases in both GST and NQOl activity were only detected in the livers of Nrf2-expressing mice but notNrfZ"'" mice. F, LDH levels were not altered by tBHQ feeding in any genotype. Cx, Cortex; Str, 170 striatum; Liv , liver. Data represent the mean ± S E M from the number of mice indicated in figure legends (in parentheses), p < 0.05, two-tailed / test. In addition, a tBHQ-induced increase in liver GST and N Q O l activities was observed in Nrf2 expressing mice, but not Nrf2"A mice, consistent with the effects of other Nrf2 inducers used in vivo (Fig. 4-8D,E) (Kwak et al., 2001a; Mc Walter et al., 2004). As with rat i.p. injection, tBHQ feeding did not induce a detectable increase in GST or N Q O l activity measured by homogenate assay from brain tissue. No changes were observed with the negative control enzyme, L D H , in any of the tissues examined (Fig. 4-8F). Interestingly, we observed an increase in liver GSH levels in tBHQ-fed Nrf2v" mice. We believe that this Nrf2-independent change may reflect an inherent hepatic toxicity associated with tBHQ feeding, perhaps due to inefficient detoxification from the body by the same pathways regulated by Nrf2 (Phase 2 genes). Together, these results highlight the sensitivity of the Nrf2_/" phenotype to tBHQ toxicity and suggest that normal Phase 2 induction in response to tBHQ is abrogated in Nrf2"A mice. 4.3.6. Loss of Nrf2 function exacerbates cortical infarction 7 days, but not 24 h, after permanent ischemia. Given the apparent changes in basal markers of A R E mediated gene expression we next examined whether loss of Nrf2 function inNrf2"/" mice increased susceptibility to ischemia. In our hands the intraluminal suture model (as used with rats) did not produce consistent strokes with Nrf2 colony mice due to variation in cerebral vasculature characteristic of the strain used (Kitagawa et al., 1998; Maeda et al., 1998). Thus, we found it necessary to use a focal ischemia model involving permanent occlusion of the distal middle cerebral artery (MCA) by cauterization as previously described (Cechetto et al., 1989). Using this model, we could consistently reduce regional cerebral blood flow (rCBF) by ~ 80% in mice expressing Nrf2 as well as knockouts (measured by Laser Doppler; Fig. 4-9B) resulting in a cortical infarct that 171 affected -10 - 15% of the ipsilateral cortical volume (Fig. 4-9A). Twenty-four h after M C A occlusion we observed no significant difference in infarct size between Nrf2_/" and Nrf2 + / + mice by TTC vital staining (Fig. 4-9C,E). However, it is conceivable that 24 h (in contrast to 7 days) is too early to observe cell death specific to the Nrf2_/" genotype within the permanently occluded lesion core because the penumbra is still evolving as collateral blood flow develops and late reperfusion may actually promote secondary generation of oxidative stress (Chan, 2001). Therefore, we also evaluated a different group of mice at 7 days after occlusion. At this time-point, we now observed a large increase (~ 2-fold) in infarct volume with Nrf2"A mice, but not with Nrf2 + / + controls (Fig. 4-9A,D,E). No significant difference in body core temperature (Table 4-1) or reduction of rCBF was observed between genotypes during the induction of ischemia (Fig. 4-9B). We propose that this continued expansion of infarct size, between the 24 h and 7 day time points, reflects cell death that is sensitive to the Nrf2"A genotype. These results suggest a model where at short time points (24 h) Nrf2-mediated gene induction is not complete or impaired in permanent ischemia models, while over time (7 days) Nrf2 activity in perhaps surviving glial cells reduces infarct evolution in Nrf2 expressing mice, but not in knockouts. 172 Section ( imm) Section Figure 4-9. Loss of Nrf2 function in vivo increased cortical damage after permanent focal ischemia. M a l e mice (10-16 weeks o f age) o f var ious N r f 2 genotypes were subjected to permanent focal i s c h e m i a by cauterizat ion o f the dis ta l m i d d l e cerebral artery, p r o d u c i n g an infarct restr icted to the cortex. T w e n t y -four hours or 7 d after stroke onset, m ice were k i l l e d for eva lua t ion o f infarct s ize u s ing c resy l v io l e t s taining. A, Representat ive examples for each genotype are s h o w n f r o m the 7 d s u r v i v a l group (infarct borders are demarcated b y b l a c k l ine) . B, T o ensure that a l l genotypes r ece ived comple te o c c l u s i o n o f regional cerebral b l o o d f l o w , surgeries were gu ided by laser D o p p l e r con t inuous measurements o f b l o o d f low through the s k u l l above the tempora l r idge . N o difference i n b l o o d f l o w reduc t ion was observed between the genotypes. C, E, A separate group o f an imals , k i l l e d 24 h after stroke, showed no s igni f icant differences i n cor t i ca l damage between genotypes (v i sua l i z ed w i t h T T C ; data not s h o w n ) . D, E, H o w e v e r , 7 d after stroke, Nr f2"" m i c e sustained cons iderab ly more damage, w h i c h u sua l l y extended to m e d i a l and dorsal cor t ica l regions, whereas N r f 2 + / + and N r f 2 + / " controls exh ib i t ed no increase i n infarct s ize b e y o n d the 24 h t ime point . D a t a represent the mean ± S E M co l lec ted f r o m n = 9, 3, and 8 m i c e for N r f 2 + / + , N r f 2 + / \ and Nrf2" / " groups , respec t ive ly (7 d su rv iva l ) , n = 8 and 5 m i c e for N r f 2 + / + and Nrf2"' ' groups , respectively (24 h su rv iva l ) , p < 0.05, p< 0 .01 , and p < 0 .001; pa i r ed two- t a i l ed t test or one -way A N O V A w i t h Bonfer ron i ' s post hoc test. It is possible that the potentially salvageable penumbral region associated with this permanent ischemia model was small or largely survived even 1-week post-ischemia due in part to the action of Nrf2-dependent factors (Hata et al., 2000a, b). To examine this possibility, we fed mice with 1% tBHQ for 1 week prior to permanent focal ischemia and evaluated infarct size 7 d post-ischemia. Consistent with Nrf2-dependent events already limiting lesion size, tBHQ feeding did not offer further reduction of infarct size in Nrf2 + / + and Nrf2+ /" mice (Fig. 4-10). In 173 contrast, the continuing growth of infarct size in Nrf2"" mice suggests that loss of Nrf2 function promotes peri-infarct (penumbral) death of neurons well after ischemia. InNrf2_/~ mice this delayed neuronal death should be insensitive to the Nrf2-dependent effects of tBHQ. Consistent with this idea, tBHQ feeding also offered no further protection to Nrf2_/" mice (Fig 4-10) Our data showing an inability of tBHQ to protect against the core ischemic lesion in Nrf2 + / + and Nrf2+/" mice is consistent with tBHQ effects being restricted to a salvageable penumbra (that is not well developed in a permanent occlusion model) (Memezawa et al., 1992b; Memezawa et al., 1992a; Hata et al., 2000a). However, this negative result also fails to directly link Nrf2 and tBHQ in protection against stroke damage in mice. 4.3.7. Loss of Nrfl function abrogates tBHQ-mediated neuroprotection during an endothelin-1 model of ischemia-reperfusion. To better test the effect of tBHQ in penumbral brain regions (with partial reduction in blood flow) we employed an endothelin-induced ischemia model and asked whether the effect of tBHQ was abrogated in Nrf2 v" mice. Endothelin-1 (ET-1), a potent vasoconstrictive peptide, was directly micro-injected into the cortical parenchyma (Yanagisawa et al., 1988; Sharkey et al., 1993), producing a long-lasting but moderate reduction in blood flow (-50%) followed by complete reperfusion several hours later (Bieraaskie et al., 2001; Zhang et al., 2005). ET-1 induced ischemia produces neurodegeneration, which can be assessed by Fluorojade staining. Infarct sizes were similar to those observed in previous studies (Fig. 4-11A) (Zhang et al., 2005). 174 35 30H 25-20 15 ^ 10 X CD tr o o " r o jo U> Q . CD E is c 0 I Control |1% tBHQ Nrf2 +/+ Nrf2 +/-Nrf2 Figure 4-10. Effect of dietary tBHQ administration on permanent focal ischemia. N r f 2 colony mice were fed 1 % t B H Q or control diet for 1 week and subjected to permanent focal ischemia. T B H Q feeding was continued for 1 additional week after stroke and the mice were then sacrificed for evaluation of infarct size. No significant difference was observed between control and tBHQ diet with any genotype. Data represent the mean + S E M from at least 4 mice for each genotype and treatment group. Body weight and core temperatures are reported in the Table 4 -2 . 175 Table 4-2. Physiological Parameters for Mouse Permanent Stroke Studies with tBHQ diet Genotype Nrf2 T7+ Nrf2 +T- Nrf2 "7" Diet Control 1% tBHQ Control 1% tBHQ Control 1% tBHQ Number of animals 5 4 6 5 5 5 Core temp. 30 min after cautery (°C) 38.0 ± 0 . 5 38.0 ± 0 . 5 37.8 ± 0 . 5 38.1 ± 0 . 5 37.2 ± 0 . 2 38.3 ± 0 . 4 Weight before stroke (g) 26.9 ± 1.2 24.0 ± 1 . 1 27.2 ± 1.3 27.6 ± 1.4 29.0 ± 0 . 8 20.8 ± 1.7 * Weight at sacrifice (7 d)(g) 26.9 ± 1.2 24.8 ± 1.1 2 6 . 6 ± 1.1 26.9 ± 1.0 28.6 ± 1.0 18.9 ± 1 . 1 * All values are the mean ± standard error. Significant reduction of body weight in tBHQ-fed Nrf2" /_ mice group, compared to Nrf2_ /" mice fed control diet. *p < 0.05 versus control group for same genotype. Feeding Nrf2 mice with tBHQ reduced ET-1 induced infarction by 66.1 + 15.5 %, when compared to Nrf2 + / + mice on control diet (Fig. 4-11 A,B,F)(* p < 0.05, unpaired t-test, n = 6 for both groups). Consistent with the protective effects of tBHQ being Nrf2-dependent, in vivo tBHQ-feeding did not reduce infarct size in Nrf27" mice (Fig. 4-11C,D,F). In fact, tBHQ appeared to exacerbate injury with infarcts on average 280.8 ± 132.2 % larger than in Nrf2"/_ mice receiving control diet (n = 5 for control diet, n = 6 for tBHQ diet); although the apparent increase in sensitivity was not significantly different from controls due to high lesion variability between Nrf2"A mice on tBHQ diets. Again, tBHQ feeding reduced body weight of Nrf2"A mice by ~ 20%, highlighting the increased sensitivity of the Nrf2"/_ genotype (Fig. 4-1 IE) and as observed previously with 3-NP toxicity in vivo (Shih et al., 2005b). Nrf2 + / + mice did not lose significant body weight during tBHQ feeding suggesting that caloric restriction did not account for neuroprotection (Fig. 4-1 IE). Interestingly, when using ET-1 induced ischemia, the infarct size of Nrf2_/" mice fed the control diet was unexpectedly smaller than those of Nrf2 + / + mice on 176 control diet (Fig. 4-11A,C and data not shown; * p < 0.05, unpaired t-test). This result could be explained by differential sensitivity of the Nrf2genotype to ET-1 induced vasoconstriction. Supporting this proposal we re-examined our microarray data obtained using adenovirus to overexpress Nrf2 in astrocytes and found that the endothelin B receptor was up-regulated ~ 30 fold by Nrf2 (Shih et al., 2003) (http://www.pharmacy.wisc.edu/facstaff/sciences/JohnsonGroup/microdata.cfm), possibly altering ET-1 induced signalling (see Discussion). In summary, using the ET-1 model we show that dietary administration of tBHQ ameliorates damage caused by ET-1 induced ischemia-reperfusion in Nrf2 + / + mice. Importantly, tBHQ offers no protection to Nrf2_/" mice from ET-1 injection, confirming the Nrf2-specific action of tBHQ in vivo. 177 Figure 4-11. tBHQ-mediated protection of cortical tissue from transient ischemia is lost in Nrf2"~ mice. Male Nrf2 + / + and Nrf2~/_ mice were fed 1 % t B H Q or control diet for 1 week before intracortical ET-1 microinjection. Mice were maintained for 1 additional week after ET-1 injection (during which respective diets were continued) and were then killed for evaluation of infarct size. A-D, Representative FluoroJade-stained images of ischemia-induced neurodegeneration after ET-1 injection (bregma = -0.9 mm). Scale bar, 500 um. E, Summary of animal weight during t B H Q prefeeding (1-7 d) and after ET-1 injection (8-15 d). F, Average infarct size, as assessed by the FluoroJade-positive area, was significantly reduced with tBHQ-fed N r f 2 + / + mice, compared with its control diet group. In contrast, the average infarct size of tBHQ-fed Nrf2_/" mice showed a trend toward exacerbation when compared with its control diet group. Data represent the mean ± S E M from the number of mice indicated in figure legends (in parentheses), p < 0.05, unpaired two-tailed t test. 4.3.8. Extracellular glutamate induces astrocyte Nrf2 activity. The increased susceptibility of the Nrf2"A phenotype to ischemia likely reflects its inability to mount a protective response to multiple toxic stimuli. Indeed, Nrf2 activation in response to toxic conditions related to ischemic injury has previously been observed (i.e. oxidative stress, glucose deprivation, and exposure to some inflammatory mediators) (Ishii et al., 2000; Cullinan and Diehl, 2004; Itoh et al., 2004; Zhang et al., 2004). However, other data indicates that oxidative stress may not necessarily be the trigger for Nrf2 activation (Lee et al., 2001a). One critical toxic event in the early ischemic cascade is an accumulation of extracellular glutamate at concentrations sufficient to cause excitotoxic neuronal death. Accordingly, we examined the possibility that extracellular glutamate acts as a signal for Nrf2 activation, by returning to an in vitro astrocyte culture system. The application of L-glutamate (100 pM) for 24 h led to a ~ 40% induction in Nrf2 as assessed by the ARE-driven hPAP reporter, that was stastically significant from control (Fig. 4-12A). This response was smaller but comparable to induction mediated by tBHQ. A response was not produced by the stereoisomer D-glutamate, nor the glutamate analogue D-aspartate indicating specificity and perhaps involvement of enzyme dependent metabolism of glutamate. Nrf2 activity was modestly induced, however, by L-aspartate. Importantly, ARE-mediated gene expression induced by L-glutamate was dependent upon Nrf2 function, since the response was 178 lost in NrtT" glial cultures (Fig. 4-12B). Co-application of 2 mM kynurenic acid, a broad-spectrum ionotropic glutamate receptor antagonist, did not significantly alter glutamate-induced Nrf2 activity (Fig. 4-12C). However, Nrf2 activity was robustly blocked by 100 p M L-trans-pyrrolidine-2,4-dicarboxylic acid (L-PDC), suggesting the involvement of a Na+-dependent glutamate transporter (Fig. 4-12C). Glutamate diffusion could provide a means for the ischemic core, which would have reduced protein synthesis (Kokubo et al., 2003), to affect the A R E -mediated gene expression in the surrounding penumbra. 220' ? 2001 S 1801 cu o 160 cu > 140 1201 100-' 80" 60 H * * B v <0 v <D 150i o c 140-o 130-O CD o 120-CU 110-> 100->. 90-' > 80-T> CO 70-Q_ 60-x: 50-* ^ / x v V Figure 4-12. Extracellular glutamate induces glial Nrf2 activity. Glial cultures were transiently transfected with a human placental alkaline phosphatase reporter construct regulated by an ARE-bearing promotor (Murphy et al., 2001). A.) Transfected cultures were treated with 100 uM L-glutamate or equal concentrations of various glutamate analogues for 20 min and hPAP and (3-galactosidase activity (for normalization of transfection efficiency) was assayed 24 h after exposure. ARE-mediated gene expression was increased by L-glutamate and L-aspartate, but not D-glutamate or D-aspartate. B.) Glial cultures derived from Nrf2+ / + and Nrf2~/_ cultures were similarly transfected and treated with glutamate. Increased hPAP activity was detected in Nrf2+ / +, but not Nrf2"A cultures confirming the Nrf2-specific action of glutamate. C.) To determine a mechanism of action, a broad spectrum inhibitor of ionotropic glutamate receptors, kynurenic acid (Kyn, 2 mM) or a competitive blocker of glutamate transporters, L-rra«5-pyrrolidine-2,4-dicarboxylic acid (L-PDC, 100 pM), was pre-applied to cultures for 20 min and then co-applied with glutamate for an additional 20 min. L-PDC completely inhibited the effect of glutamate suggesting the involvement of glutamate transporters. Data represent the mean + SEM collected from at least 3 independent experiments for each treatment. *p < 0.05, **p < 0.01, two-tailed t-test. 179 4.4. Discussion. Nrf2 plays an important role in limiting ischemic injury. We have observed the neuroprotective effects of tBHQ, a well-characterized Nrf2 inducer, with two different models of ischemia-reperfusion (MCAO and ET-1 vasoconstriction) in rats and mice, using three different routes of administration (intracerebroventricular, intraperitoneal, and dietary). In rats, prophylactic tBHQ treatment improved functional recovery up to 1 month after focal ischemia-reperfusion. Conversely, loss of Nrf2 function in vivo exacerbates ischemic damage. Interestingly, this is an effect that becomes evident not 24 h, but 7'days after stroke suggesting that Nrf2 activity affects the evolving ischemic penumbra. Consistent with a specific role for Nrf2, we found tBHQ-dependent elevation of GSH levels in rat cortex and also detected this increase in the brains of Nrf2 + / + and Nrf2+ /" mice, but not Nrf2"" mice. Further, the neuroprotective effect of tBHQ after ET-1 induced ischemia-reperfusion was evident with Nrf2 + / + mice, but not Nrf2_/" mice, confirming that in vivo neuroprotection by tBHQ is Nrf2-dependent. Together, these results highlight the potential of Nrf2 activation as a new prophylactic strategy for stroke. 4.4.1. TBHQ and the blood brain barrier During the evaluation of tBHQ (as a food preservative) on human health, the World Health Organization conducted studies of C1 4-labelled tBHQ metabolism in rats (National Toxicology Program, 1997; FAO/WHO, 1999). A single ingested dose of tBHQ (92 mg/kg) was excreted from the animal within 3-4 days and led to trace amounts of radioactivity within brain tissue (FAO/WHO, 1975). Interestingly, tBHQ reached higher levels in brain (0.09-0.38 mg tBHQ / g wet brain tissue) when supplied through diet for prolonged periods of time (5.7 mg/kg, equivalent to 0.029% in diet for 17 days) (FAO/WHO, 1975). Thus, it is reasonable to believe 180 that in our study tBHQ enters the brain to activate local Nrf2 given the doses and times we have used. Previous studies by Juurlink and colleagues have shown that chronic dietary supplementation with Nrf2 inducers can attenuate damage to the central nervous system, since feeding butylhydroxyanisole (tBHQ precursor) had a beneficial effect in an animal model of multiple sclerosis (Mohamed et al., 2002). Furthermore, dietary Nrf2 inducers can promote cardiovascular health during chronic hypertension (Wu and Juurlink, 2001; Wu et al., 2004). Also, we have recently shown that dietary administration of tBHQ attenuates striatal neurodegeneration and motor deficits caused by systemic 3-nitropropionic acid (Shih et al., 2005b). Our finding that systemic (intraperitoneal and dietary) delivery of tBHQ locally increases brain GSH and protects from ischemic brain injury, supports the idea that tBHQ, and possibly other small electrophilic Nrf2 inducers, are able to pass the blood brain barrier. However, as evidenced by the robust induction of GST and N Q O l activity in liver, systemic tBHQ delivery affects peripheral antioxidant systems as well. Although it would be difficult to dissect out the contributions of brain and peripheral systems without tissue specific disruption of Nrf2 expression, the final outcome of systemic tBHQ delivery has a clear beneficial effect during ischemic injury. 4.4.2. Nrf2 dependency of tBHQ-mediated neuroprotection Previous studies have confirmed that the neuroprotective effect of tBHQ in vitro is Nrf2 genotype dependent (Fig. l)(Kraft et al., 2004). Furthermore, our recent in vivo studies of mitochondrial stress (3-nitropropionic acid) shows tBHQ is Nrf2-dependent since its protective effect was lost in Nrf2_/" mice (Shih et al., 2005b). Whether tBHQ accesses similar pathways to attenuate brain damage during ischemia in vivo was previously unknown. To address this question, we fed mice with tBHQ prior to ET-1 induced ischemia-reperfusion. Nrf2 + / + mice 181 exhibited increased Phase 2 enzyme activity in both brain and liver, and were protected from ET-1 induced ischemic damage, whereas Nrf2"A mice have lower Phase 2 enzyme levels in brain and liver, and the neuroprotective effects of tBHQ were lost (Fig. 4-7, 4-8, 4-9). Although these results are in agreement with our hypothesis that tBHQ function in vivo is dependent on Nrf2, there are some important caveats that must be considered. First, Nrf2_/" mice were found to be less tolerant to tBHQ feeding than Nrf2 expressing controls. Overall, Nrf2"-~ mice lost more weight and consumed less food at tBHQ concentrations necessary to provide neuroprotection (Fig. 4-8A,B); also see (Shih et al., 2005b). We speculate that the reduced food consumption of the knockout phenotype is due to systemic toxicity associated with poor tBHQ metabolism and detoxification, which may sensitize these mice to further insults. Second, Nrf2"A mice may have reduced sensitivity to ET-1 induced ischemia. Indeed, the ET-B subtype of endothelin receptor (Genbank accession: S65355) was found to be highly upregulated (~30-fold) by Nrf2 over-expression in astrocyte cultures (Shih et al., 2003)(also see http://www.pharmacy.wisc.edu/facstaff/sciences/JohnsonGroup/microdata.cfm). This suggests that loss of Nrf2 function may reduce basal expression of ET-B, or perhaps prevent its induction post-ischemia, although this remains to be examined (Stenman et al., 2002). ET-B has been shown to modulate cerebral blood flow by inducing both vasodilation or vasoconstriction in different circumstances (Clozel et al., 1992; Moreland et al., 1992; Touzani et al., 1997). To control for differences in ET-1 sensitivity between genotypes, lesion volumes with tBHQ diets were normalized to mice receiving control diet for each genotype. To clearly dissociate potential systemic toxicity of tBHQ from its effects on CNS glia, future experiments would benefit from inducible and tissue-specific Nrf2 knockout strategies. 182 4.4.3. Nrf2 activation protects the ischemic penumbra, not the stroke core Nrf2 activity attenuates damage caused by transient ischemia. In contrast to the cortex, tBHQ administration was unable to salvage the striatal stroke core resulting from transient MCAO in rat. With this model, death of striatal tissue results from more severe ischemia than within the surrounding cortex (Nedergaard et al., 1986; Kita et al., 1995; Liu et al., 2003). Similarly, we also found that tBHQ was unable to protect mice against permanent focal ischemia using electrocoagulation of the distal M C A (Fig. 4-10). This was not surprising since permanent ischemia tends to result in a more severe infarction on a faster timescale (Memezawa et al., 1992b; Memezawa et al., 1992a; Lipton, 1999; Mao et al., 1999). Permanent ischemia may also produce a smaller and more short-lived penumbra than ischemia-reperfusion (Hata et al., 2000a, b). Although tBHQ did not reduce striatal damage caused by transient M C A O , it significantly attenuated striatal neurodegeneration during 3-nitropropionic acid toxicity (Shih et al., 2005b). Since 3-nitropropionic acid produces gradual and partial metabolic inhibition (Beal et al., 1993a; Sato et al., 1997; Brouillet et al., 1998; Kim et al., 2000), it is in some ways similar to the salvageable cortical stroke penumbra. 4.4.4. Identifying the signals that activate endogenous Nrf2 in response to ischemia Previous studies have shown that endogenous ARE-mediated gene expression is increased after brain ischemia, suggesting that Phase 2 gene induction may be a physiological response for mitigating further damage caused by secondary ROS/RNS formation and inflammation. In particular, Campagne et al. have demonstrated using gel shift assays that antioxidant response element binding was increased in the ischemic cortical area within 6 h after stroke onset (Campagne et al., 2000). Furthermore, oligemia (blood flow reduction without acute tissue damage that occurs in shock, migraine, and stroke penumbra) also activates Nrf2 (Liverman et al., 2004). Robust induction of Phase 2 enzymes such as N Q O l and 183 metallothionein-1 (MT-1) within astrocytes, vascular endothelial cells, and pia-mater cells of the infarct border can be observed within hours-days after stroke onset (van Lookeren Campagne et al., 1999; Campagne et al., 2000; Laxton et al., 2001). The timing and location of Phase 2 gene induction suggests that activation of endogenous Nrf2 may be necessary for limiting oxidative stress in the ischemic penumbra. Many components of ischemic injury may lead to Phase 2 gene expression. For example, mitochondrial inhibition, endoplasmic reticulum stress induced by glucose deprivation, inflammatory mediators, and increased oxidative stress in general, all promote Nrf2 activity and are events relevant to ischemic injury (Ishii et al., 2000; Kang et al., 2002a; Cullinan and Diehl, 2004; Itoh et al., 2004; Zhang et al., 2004; Calkins et al., 2005). Adding to this data, we identify extracellular glutamate as a novel mediator of Nrf2 activation. This finding may be highly relevant to ischemia since glutamate released due to anoxic depolarization is thought to cause excitotoxicity (Dirnagl et al., 1999; Lee et al., 1999). Peri-infarct depolarizations could further propagate anoxic glutamate release into penumbral zones (Hossmann, 1996) and activate Nrf2 within surviving astrocytes. Previous studies have shown that astrocytes are tightly coupled to glutamate release from active neurons and respond by increasing lactate release to support neuronal metabolism (Pellerin and Magistretti, 1994; Voutsinos-Porche et al., 2003). It is conceivable that a "glutamate-Nrf2 signal" could exist to limit oxidative stress created during excitotoxicity (Reynolds and Hastings, 1995). Thus Nrf2-targeted genes may function to strengthen neuron-glia metabolic coupling and re-establish neuronal function after ischemia (Shih et al., 2003; Kraft et al., 2004). 4.4.5. Conclusion Nrf2 inducers represent a new generation of drugs that can single-handedly activate multiple cellular defence effectors. As an improvement upon previous approaches, future 184 therapies could target these 'molecular switches' that control the coordinated expression of protective gene families. 185 Chapter 5 : General Discussion Cellular injury is induced through a combination of complex pathways during both stroke and chronic neurodegenerative disease, including excitotoxicity, metabolic inhibition, oxidative stress, and inflammation (Coyle and Puttfarcken, 1993; Dirnagl et al., 1999; Lee et al., 1999; Lo et al., 2003). It is now becoming clear that an effective neuroprotective strategy for these pathologies must attenuate multiple injury mechanisms. As a step toward this goal, we have examined the potential of Nrf2 activation as a neuroprotective strategy in animal models of stroke and neurodegeneration. Together with complementary findings from other laboratories (Lee et al., 2003c; Lee et al., 2003a; Kraft et al., 2004; Wu et al., 2004; Calkins et al., 2005; Jakel et al., 2005), our studies provide several lines of evidence to support the hypothesis that Nrf2-mediated induction of a global antioxidant response can be an effective prophylactic strategy to attenuate brain tissue damage: 1.) Nrf2 activates Phase 2 gene expression in cultured brain cells, primarily astrocytes (Shih et al., 2003). 2.) Nrf2 activation protects cultured neurons and astrocytes in a variety of toxicity paradigms (Lee et al., 2003a; Shih et al., 2003). 3.) Loss of Nrf2 function in vivo exacerbates cell death due to 3-NP toxicity and cerebral ischemia (Shih et al., 2005b; Shih et al., 2005a). 4.) Administration of small molecule Nrf2 inducers to wild-type and heterozygous mice increases brain GSH synthesis and offer neuroprotection in vivo. However, this protective effect is lost in Nrf2 knockout mice (Shih et al., 2005b; Shih et al., 2005a). These lines of evidence will now be discussed in more detail. 186 5.1. Nrf2-mediated upregulation of the G S H system is neuroprotective: in vitro and in vivo evidence Our initial in vitro studies showed that Nrf2 activation in mixed neuron/glial cultures promoted the coordinated expression of multiple antioxidant/detoxification Phase 2 enzymes (Shih et al., 2003). Using Western blots and a reporter assay system for ARE-mediated gene expression, we found astrocytes to be the primary site of Nrf2 expression and activity within these mixed cultures (Fig. 2-2). Accordingly, Nrf2 activation in astrocytes produced more robust Phase 2 gene expression than their neuronal counterparts (Ahlgren-Beckendorf et al., 1999; Eftekharpour et al., 2000; Murphy et al., 2001; Johnson et al., 2002; Shih et al., 2003). A surprising finding from our studies was that astrocytes over-expressing Nrf2 were highly neuroprotective (a single astrocyte could protect -100 neighboring neurons in culture) (Fig. 2-6). Among the wide range of Phase 2 genes involved in this astrocytic protective response, we highlighted GSH synthesis and release as an important component for neuroprotection. This was confirmed by Kraft et al. using cultures derived from Nrf2 wild-type and knockout mice (Kraft et al., 2004). Nearly all enzymes involved in the biosynthesis, use, and export of GSH were enhanced by Nrf2 over-expression. Intracellular and extracellular GSH content within the culture was doubled by Nrf2 over-expression (Table 2-3 and Fig. 2-7). A similar increase in GSH synthesis was observed with tBHQ-mediated Nrf2 activation in cultured astrocytes (Sun et al., 2005). This GSH increase was necessary for Nrf2-mediated neuroprotection since inhibition of GSH synthesis using the irreversible GSH synthesis inhibitor BSO blocked neuroprotection. Considering that GSH is rapidly diluted when released into the media of our culture system, in a compact in vivo system it is likely that the neuroprotective effect of continuously released GSH would be even more profound. Further studies by another group used a similar approach to show that Nrf2 over-expressing astrocytes conferred wide-spread protection to co-cultured motor 187 neurons during p75 neurotrophin receptor dependent apoptosis, although it was not determined whether GSH production was important for this neuroprotection (Vargas et al., 2005). Together, these results suggest that Nrf2 activation is a strong regulator of GSH synthesis and utilization, and GSH release is central to Nrf2-mediated neuroprotection in certain in vitro toxicity paradigms. Interestingly, cultured Nrf2 over-expressing astrocytes were recently shown to protect neurons in vivo after transplantation into the striatum of mice receiving intrastriatal injection of malonic acid, a competitive inhibitor of the mitochondrial chain complex II (Calkins et al., 2005). This study demonstrates that Nrf2 expressing astrocytes, even in the complex and compartmentalized milieu of living striatal tissue, could protect distant neurons perhaps through the production and release of diffusible factors such as GSH. In a separate study from our group, a similar result was found by injecting Ad-Nrf2 directly into the rat striatum leading to the specific infection of astrocytes dispersed throughout the striatum (Fig. 3-5) (Shih et al., 2005b). When the rats were subsequently challenged with chronic systemic 3-NP treatment, the Ad-Nrf2 treatment significantly reduced striatal lesion size, compared to control rats infected with the Ad-GFP control virus. In this case as well, the wide-spread protection offered by a limited number of Nrf2 over-expression astrocytes suggests the involvement of a diffusible factor such as GSH, which was clearly implicated in our in vitro studies. In support of this notion, administration of the Nrf2 inducer, tBHQ, through various delivery routes (intraperitoneal and dietary) produced robust changes in brain GSH content, but surprisingly not other co-regulated Phase 2 enzymes GST and N Q O l (Table 3-1 and Fig 4-8) (Shih et al., 2005b; Shih et al., 2005a). This dynamic increase in GSH was Nrf2-specific since it was not observed in Nrf2 knockout mice. Although these findings allude to an important role for GSH in Nrf2-mediated neuroprotection in vivo, further studies will be required to determine whether increased GSH 188 synthesis is necessary for neuroprotection. As described in Chapter 2, the xCT cystine-glutamate antiporter is the primary uptake system for the rate-limiting precursor for GSH synthesis, cyst(e)ine. Since xCT expression is strongly regulated by Nrf2 activity, future studies can examine the neuroprotective role of Nrf2-mediated GSH induction in vivo using subtle gray (sut) mice, which are known to express mutant non-functional xCT protein (Chintala et al., 2005). In principle, the over-expression of Nrf2 or administration of tBHQ should induce Phase 2 enzymes in sut mice, but the lack of xCT function will limit the induction of GSH synthesis by blocking the availability of GSH precursor cyst(e)ine. If Nrf2-mediated neuroprotection is suppressed in these mice, it would suggest that GSH is an important component in Nrf2-mediated neuroprotection in vivo. We could further confirm this idea by using xCT over-expressing adenoviruses (available in our lab) to rescue GSH synthesis in sut mice and increase neuroprotection. However, a potential caveat of this experiment is that other compensatory transport systems for cyst(e)ine may exist in these mice. For example, some members of the X A G transport system (comprising many high affinity glial transporters) mediate Na+-dependent cystine uptake (Danbolt, 2001; McBean, 2002). One member of this system, EAAT3 (also known as EAAC1) transports cysteine across neuronal membranes and is critical for maintenance of neuronal GSH in vivo (Zerangue and Kavanaugh, 1996; Aoyama et al., 2005). Finally, recent studies from our group provide a new technique to measure GSH levels at the cellular level in the in vivo brain using the GSH-specific probe monochlorobimane (Sun et al., 2006). This strategy will be useful for identifying sites of increased brain GSH synthesis during Nrf2 activation. 189 Table 5-1. Summary of treatments and toxicity paradigms used in Nrf2-mediated neuroprotection studies. Cell Type Protected Nrf2 Activation or Inhibition Strategy Toxicity Paradigm Ref. Rat immature cortical neurons Adenoviral Nrf2 overexpression - Oxidative glutamate toxicity - H 2 0 2 (Shih et al., 2003) Mouse immature cortical neuron Nrf2 inducers (tBHQ and sulforaphane) - Oxidative glutamate toxicity - H 2 0 2 (Kraft et al., 2004) Mouse immature cortical neurons Nrf2 inducer (tBHQ) - 6-OHDA (Jakel et al., 2005) Mouse mature cortical neurons - Adenoviral Nrf2 overexpression - Nrf2 wild-type vs. Nrf2 knockout -MPP + - rotenone - Ionomycin - Di-tertbutylhydroquinone (Lee et al., 2003a) Mouse mature cortical neurons Nrf2 wild-type vs. Nrf2 knockout - 3-nitropropionic acid (Calkins et al., 2005) Mouse cortical astrocytes Nrf2 wild-type vs. Nrf2 knockout ' - H 2 0 2 - Platelet activating factor (Lee et al., 2003c) Rat cortical astrocytes Acetyl-L-carnitine (nrf2 inducer?) - lipopolysaccharide - interferon y (Calabrese et al., 2005) Rat spinal cord motorneurons - Nrf2 overexpression in astrocytes - Nrf2 inducers (fibroblast growth factor and tBHQ) - p75NTR-dependent apoptosis (Vargas et al., 2005) Human neural stem cells Nrf2 inducer (tBHQ) - H 2 0 2 (Li et al., 2005) Neuroblastoma cell line (N18-RE105) Nrf2 inducer (tBHQ) - Oxidative glutamate toxicity (Murphy et al., 1991) Neuroblastoma cell line (N18-RE105) Nrf2 inducers (tBHQ and DMF) - H 2 0 2 - Dopamine (Duffy et al., 1998; Su et al., 1999) Human neuro-blastoma cell line ( I M R - 3 2 ) Nrf2 inducer (tBHQ) - H 2 0 2 (Li et al.,2002) Human neuro-blastoma cell line (SH-SY5Y) Stable Nrf2 overexpression - 6-OHDA - 3-morpholinosydnonimine (NO and 02" production) (Cao et al., 2005) PC 12 cell line Nrf2 inducer (resveratrol) - H 2 0 2 (Chen et al., 2005) Human retinal epithelial cell (ARPE-19) Nrf2 inducer (sulforaphane and DMF) - menadione - tert-butylhydroperoxide - 4-hydroxynonenal - peroxynitrite - UV light-induced photooxidation (Nelson et al., 1999; Gao et al.,2001; Gao and Talalay, 2004) 190 5.2. Versatility of the Nrf2-mediated neuroprotection Activation of Nrf2, either by Nrf2 over-expression or application of small molecule Nrf2 inducers, provides neuroprotection from a wide variety of in vitro toxicity paradigms with relevance to human neurodegeneration and stroke, attesting to the versatility of the Nrf2-mediated antioxidant response (Table 5-1). Notably, Nrf2 was particularly effective for protection against direct exposure to H2O2, GSH depletion, menadione, dopamine, metabolic inhibition, and C a + + overload (Duffy et al., 1998; Gao et al., 2001; Lee et al., 2003a; Shih et al., 2003; Kraft et al., 2004). Oxidative stress is central to toxicity paradigms involving GSH depletion and ROS exposure, but also plays an important role in cell death mediated by metabolic inhibition, excitotoxicity and C a + + overload (Murphy et al., 1989; Murphy et al., 1990; Sagara et al., 1993a; Reynolds and Hastings, 1995; Sattler et al., 1999; Schubert and Piasecki, 2001). Thus, it is perhaps not surprising that Nrf2 is neuroprotective given that prototypic Nrf2-regulated Phase 2 enzymes cooperate to reduce oxidative load and detoxify electrophilic molecules. However, this antioxidant-based view of Nrf2-mediated neuroprotection has recently been broadened. Kraft et al. have used FACS sorting to enrich neuronal and glial cell types before their microarray analyses (Kraft et al., 2004). In this way, they were able to study how astrocytes influence Nrf2-regulated gene expression in co-cultured neurons and vice versa. Some of the genes identified using this method suggested that Nrf2 performed a broader set of functions in the brain: 1.) neuron-glial metabolic coupling of energy substrates such as glucose and lactate. 2.) cell-cell adhesion, and 3.) modulation of neuronal neurotransmitter signaling and Ca + + homeostasis. This study raises an interesting possibility that Nrf2 also confers neuroprotection by influencing the expression of genes that regulate other aspects of neuronal-glia function in addition to redox homeostasis. This is a relatively new concept will need to be reinforced with further functional biochemical studies. For example, it is possible that the genes 191 identified in this study were indirectly affected by Nrf2 activity and are not ARE-regulated. Future work should examine whether Nrf2 knockout mice have some impairment of neurotransmitter signaling (i.e. changes in field potential) and energy metabolism (i.e. reduced ATP content), particularly after an acute brain injury. 5.3. The role of endogenous Nrf2 Neurons and glia cultured from Nrf2 knockout mice are more susceptible than wild-type cultures during various in vitro toxicity paradigms (Lee et al., 2003c; Lee et al., 2003a; Kraft et al., 2004). Cao et al. have found similar results by acutely knocking down expression of Nrf2 using siPvNA (Cao et al., 2005). The increased susceptibility of Nrf2 knockout mice has also been demonstrated in vivo, strongly suggesting that endogenous Nrf2 function is critical for reducing brain oxidative load under stress-inducing conditions (Calkins et al., 2005; Shih et al., 2005b; Shih et al., 2005a). Although previous work on Nrf2 knockout mice was largely conducted on peripheral tissues such as the liver, lung and small intestines (where Nrf2 expression is known to be high) (Chan et al., 1996b; Chan et al., 2001; McMahon et al., 2001; Cho et al., 2002b), our studies were the first to demonstrate that Nrf2 plays a critical role in reducing brain injury as well. In one study, we found that Nrf2 knockout mice sustained more striatal injury during the progressive toxicity caused by systemic 3-NP treatment (5 day treatment regimen), a model that mimics certain aspects (cell death specificity and motor deficits) of chronic human neurodegenerative diseases such as Huntington's disease (Beal et al., 1993a; Shih et al., 2005b). However, a potential caveat of this study was that 3-NP detoxification from the body (i.e. liver detoxification) may have been compromised in Nrf2 knockout mice, resulting in increased circulation of 3-NP and increased toxicity. It remained unclear whether Nrf2 function played an important role in local brain neuroprotection in vivo. This issue was resolved by the findings of two other studies. First, Calkins et al. circumvented complications of 192 differential peripheral toxin removal by directly injecting malonate into the striatum (Calkins et al., 2005). They found that malonate-induced lesions were larger in Nrf2 knockout mice than wild-type controls, suggesting an inherent sensitivity of neurons within the brains of Nrf2 knockout mice. Second, we showed that Nrf2 knockout mice were more sensitive to ischemic brain injury caused by permanent M C A O , further suggesting that Nrf2 function is important for neuroprotection during local brain injury (Fig. 4-9) (Shih et al., 2005a). Future studies can use brain specific knockouts to verify our conclusions. Such animals may be available in the near future, since a recent study has already generated liver-specific knockouts of Keapl to demonstrate that systemic acetaminophen toxicity can be attenuated by increased Nrf2-dependent liver detoxification (Okawa et al., 2006). To gain a better understanding of how endogenous Nrf2 might be activated during a stroke, we examined astrocyte responses to glutamate in culture. Glutamate is rapidly released from both neurons and astrocytes during anoxic depolarization (Rothman, 1984; Simon et al., 1984; Rossi et al., 2000). It is believed that glutamate release from the ischemic core could influence excitotoxic cell death in the stroke penumbra (Hossmann, 1996). We hypothesized that astrocytes within the penumbra might respond to glutamate and activate Nrf2 to promote neuroprotection. Our results show that exposure to glutamate at excitotoxic levels (100 pM) generates a modest activation of Nrf2 (Fig. 4-12A). Importantly, this response to glutamate was specific since it was not detected in Nrf2"A glial cultures and appeared to function through the activity of Na+-dependent high affinity glutamate transporters since it was blocked by L-PDC (Fig. 4-12C). These preliminary studies suggest a possible link between glutamate release and glial Nrf2 activation. However, more work is required to determine how glutamate transport activity can be linked to Nrf2 activation, and whether this link exist in vivo. Two possibilities deserve further examination: 1.) Nrf2 responds to ionic imbalances imposed by glutamate transport over-activity, 2.) Nrf2 responds to reduced ATP levels resulting from N a + - K + ATPase 193 activity necessary to re-establish the plasma membrane ionic gradients. The latter is thought to be important for induction of astrocyte lactate synthesis and release in response to neuronal glutamatergic activity (Pellerin and Magistretti, 1994; Voutsinos-Porche et al., 2003). 5.4. Nrf2 inducers as therapeutic agents and the alternatives Nrf2 can be pre-activated in vivo using the well-known small molecule inducer tBHQ to potentiate Nrf2-mediated gene expression and prime the brain for subsequent damage cause by a neurological insult. Prophylactic tBHQ delivery was able to attenuate both 3-NP induced striatal degeneration and ischemic cortical injury and was quite robust since it was observed using several routes of administration (intracerebroventricular, intraperitoneal, and dietary delivery) (Shih et al., 2005b; Shih et al., 2005a). Importantly, tBHQ treatment also preserved motor function in these injury models, which is the ultimate endpoint to any neuroprotective therapeutic strategy. Our findings show for the first time that in vivo treatment with Nrf2 inducers provides effective neuroprotection in complex diseases such as stroke by activating multiple cellular defense effectors. One important caveat arising from our in vivo studies is the toxicity associated with tBHQ administration to Nrf2 knockouts (Fig 3-4). It can be argued that tBHQ toxicity may be sensitizing these mice to injury, and thus neuroprotection cannot be observed. We believe that tBHQ toxicity is associated with inefficient excretion from knockout mice, perhaps due to compromised liver detoxification. Ironically, tBHQ may be poorly removed from Nrf2 knockout mice because it is normally detoxified through the very same pathways it activates. Indeed, we observed an Nrf2-independent increased in hepatic GSH in Nrf2 knockout mice, which may be stress response to liver injury (Fig. 4-8). However, we noted that no tBHQ toxicity was observed in the brain based on Fluorojade staining and measurements of brain enzymatic activity (lactate dehydrogenase housekeeping gene). Specific brain and liver effects of tBHQ will be difficult to 194 dissociate with the mice at hand, and tissue specific knockouts of Nrf2 will be able to address these issues more clearly. More importantly, Nrf2 wild-type mice were not affected by tBHQ toxicity at the same dose suggesting a larger therapeutic window between the protective and toxic doses of tBHQ in normal animals. Higher doses of tBHQ (5% in diet) were toxic even to wild-type mice, suggesting that drug dose must be carefully tested in humans. It is expected that human patients will possess the detoxification systems necessary to reap the benefits of electrophilic Nrf2 inducers without toxic side effects. Some groups have raised the possibility that polymorphisms in the Nrf2 gene could underlie human disease (Cho et al., 2002a; Yamamoto et al., 2004). If so, individuals with this genetic predisposition would be poor candidates for Nrf2 inducing drugs because they may not be able to detoxify the drug from their bodies. Suh et al. have shown that Nrf2 expression declines with increased age in rats (Suh et al., 2004). Given that neurodegenerative disease and stroke primarily occurs with aged individuals, it may be important to examine the status of Nrf2 expression prior to the administration of Nrf2 inducing drugs. In future studies, it will be important to identify Nrf2 inducing drugs more efficacious and less toxic than tBHQ for use in humans. To date, a number of groups have already identified many natural Nrf2 inducing molecules from fruits and vegetables commonly found in the human diet (sulforaphane, garlic, wasabi, green tea), and many safe Nrf2 inducers may remain to be discovered from natural sources and within the collection of existing FDA-approved drugs (Egner et al., 1994; Fahey et al., 1997; Kensler et al., 2000; Morimitsu et al., 2002; Rushmore and Kong, 2002). Although many of these natural and pre-tested molecules are still electrophilic agents, they may be better tolerated in vivo. Before Nrf2 inducing drugs will become a reality for treating neurodegeneration and stroke, it will also be important to achieve a greater understanding of their in vivo potency, therapeutic dose ranges, and blood brain barrier penetration. Our findings suggest that the most potent (most electrophilic) inducers of Nrf2 may 195 not necessarily be the most innocuous drugs in vivo. In fact, Nrf2 inducers with high electrophilicity are likely to be the most toxic in vivo, whereas inducers of relatively low electrophilicity may be less efficacious but sufficient for neuroprotection and better tolerated. As an improvement to the use of electrophilic molecules, some groups have set out to understand the molecular interactions between Nrf2 and its regulatory protein Keapl (Zhang and Hannink, 2003). In this promising area of research, X-crystallography studies of Keapl structure have begun to identify regions of that bind Nrf2, and this information may eventually lead to the design of non-electrophilic chemicals or peptides that can specifically disrupt Nrf2-Keapl binding without toxic side effects (Li et al., 2004b, a). With a similar goal of activating Nrf2 with minimal oxidative stress, other groups have examined the potential of siRNA-mediated Keapl knockdown to shift the Nrf2-Keapl (Devling et al., 2005). Finally, promising therapeutic targets may also exist in the signal transduction pathways leading to Nrf2 activation or Keapl inhibition (Nguyen et al., 2003a). These areas of research are still in their infancy, but also hold great promise as therapeutic strategies for stroke and neurodegeneration. In conclusion, my research gives proof of principle evidence that Nrf2 activation can be achieved in the in vivo brain. Administration of the electrophilic Nrf2 inducer, tBHQ, provides effective prophylaxis from acute focal ischemic injury and gradual chemical-induced striatal neurodegeneration. Furthermore, neuroprotection by tBHQ was observed through a number of delivery routes, including dietary administration. Since tBHQ is a Food and Drug Administration approved food antioxidant, dietary treatment with tBHQ or other low toxicity Nrf2 inducers may be a practical, safe, and effective treatment for stroke and neurodegenerative disease. 196 References (1997) NTP Toxicology and Carcinogenesis Studies of t-Butylhydroquinone (CAS No. 1948-33-0) in F344/N Rats and B6C3F(1) Mice (Feed Studies). Natl Toxicol Program Tech Rep Ser 459:1-326. Aarts M , Iihara K, Wei WL, Xiong ZG, Arundine M , Cerwinski W, MacDonald JF, Tymianski M (2003) A key role for TRPM7 channels in anoxic neuronal death. Cell 1 15:863-877. Aarts M , Liu Y , Liu L, Besshoh S, Arundine M , Gurd JW, Wang YT, Salter M W , Tymianski M (2002) Treatment of ischemic brain damage by perturbing N M D A receptor- PSD-95 protein interactions. Science 298:846-850. Aarts M M , Tymianski M (2005) TRPMs and neuronal cell death. Pflugers Arch 451:243-249. Abdel-Wahab M H (2005) Potential neuroprotective effect of t-butylhydroquinone against neurotoxicity-Induced by l-methyl-4-(2'-methylphenyl)-l,2,3,6-tetrahydropyridine (2-methyl-MPTP) in mice. J Biochem Mol Toxicol 19:32-41. Ahlgren-Beckendorf JA, Reising A M , Schander M A , Herdler JW, Johnson JA (1999) Coordinate regulation of NAD(P)H:quinone oxidoreductase and glutathione- S-transferases in primary cultures of rat neurons and glia: role of the antioxidant/electrophile responsive element. Glia 25:131-142. Alam J, Stewart D, Touchard C, Boinapally S, Choi A M , Cook JL (1999) Nrf2, a Cap'n'Collar transcription factor, regulates induction of the heme oxygenase-1 gene. J Biol Chem 274:26071-26078. Alam J, Wicks C, Stewart D, Gong P, Touchard C, Otterbein S, Choi A M , Burow M E , Tou J (2000) Mechanism of heme oxygenase-1 gene activation by cadmium in MCF-7 mammary epithelial cells. Role of p38 kinase and Nrf2 transcription factor. J Biol Chem 275:27694-27702. 199 Albers GW, Goldstein L B , Hall D, Lesko L M (2001) Aptiganel hydrochloride in acute ischemic stroke: a randomized controlled trial. Jama 286:2673-2682. Alexi T, Hughes PE, Knusel B, Tobin AJ (1998) Metabolic compromise with systemic 3-nitropropionic acid produces striatal apoptosis in Sprague-Dawley rats but not in B A L B / c ByJ mice. Exp Neurol 153:74-93. Alston TA, Mela L , Bright HJ (1977) 3-Nitropropionate, the toxic substance of Indigofera, is a suicide inactivator of succinate dehydrogenase. Proc Natl Acad Sci U S A 74:3767-3771. American-Nimodipine-Study-Group (1992) Clinical trial of nimodipine in acute ischemic stroke. The American Nimodipine Study Group. Stroke 23:3-8. Andrews NC, Erdjument-Bromage H, Davidson M B , Tempst P, Orkin SH (1993) Erythroid transcription factor NF-E2 is a haematopoietic-specific basic- leucine zipper protein. Nature 362:722-728. Aoki Y , Sato H, Nishimura N , Takahashi S, Itoh K, Yamamoto M (2001) Accelerated D N A adduct formation in the lung of the Nrf2 knockout mouse exposed to diesel exhaust. Toxicol Appl Pharmacol 173:154-160. Aoyama K, Suh SW, Hamby A M , Liu J, Chan W Y , Chen Y , Swanson R A (2005) Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient mouse. Nat Neurosci. Araki K, Harada M , Ueda Y , Takino T, Kuriyama K (1988) Alteration of amino acid content of cerebrospinal fluid from patients with epilepsy. Acta Neurol Scand 78:473-479. Arvidsson A, Kokaia Z, Lindvall O (2001) N-methyl-D-aspartate receptor-mediated increase of neurogenesis in adult rat dentate gyrus following stroke. Eur J Neurosci 14:10-18. Asahi M , Wang X , Mori T, Sumii T, Jung JC, Moskowitz M A , Fini M E , Lo E H (2001) Effects of matrix metalloproteinase-9 gene knock-out on the proteolysis of blood-brain barrier and white matter components after cerebral ischemia. J Neurosci 21:7724-7732. 200 Astrup J, Siesjo BK, Symon L (1981) Thresholds in cerebral ischemia - the ischemic penumbra. Stroke 12:723-725. Astrup J, Symon L, Branston N M , Lassen N A (1977) Cortical evoked potential and extracellular K+ and H+ at critical levels of brain ischemia. Stroke 8:51-57. Back SA, Gan X , L i Y , Rosenberg PA, Volpe JJ (1998) Maturation-dependent vulnerability of oligodendrocytes to oxidative stress-induced death caused by glutathione depletion. J Neurosci 18:6241-6253. Bae I, Fan S, Meng Q, Rih JK, Kim HJ, Kang HJ, X u J, Goldberg ID, Jaiswal A K , Rosen E M (2004) BRCA1 induces antioxidant gene expression and resistance to oxidative stress. Cancer Res 64:7893-7909. Balagopalakrishna C, Manoharan PT, Abugo OO, Rifkind JM (1996) Production of superoxide from hemoglobin-bound oxygen under hypoxic conditions. Biochemistry 35:6393-6398. Bannai S (1984) Transport of cystine and cysteine in mammalian cells. Biochim Biophys Acta 779:289-306. Bannai S (1986) Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem 261:2256-2263. Bannai S, Kitamura E (1980) Transport interaction of L-cystine and L-glutamate in human diploid fibroblasts in culture. J Biol Chem 255:2372-2376. Banning A, Deubel S, Kluth D, Zhou Z, Brigelius-Flohe R (2005) The GI-GPx gene is a target for Nrf2. Mol Cell Biol 25:4914-4923. Barber PA, Auer RN, Buchan A M , Sutherland GR (2001) Understanding and managing ischemic stroke. Can J Physiol Pharmacol 79:283-296. Barone FC, Feuerstein GZ (1999) Inflammatory mediators and stroke: new opportunities for novel therapeutics. J Cereb Blood Flow Metab 19:819-834. 201 Barone FC, Parsons A A (2000) Therapeutic potential of anti-inflammatory drugs in focal stroke. Expert Opin Investig Drugs 9:2281-2306. Bartosz G (1996) Peroxynitrite: mediator of the toxic action of nitric oxide. Acta Biochim Pol 43:645-659. Beal MF (1992) Does impairment of energy metabolism result in excitotoxic neuronal death in neurodegenerative illnesses? Ann Neurol 31:119-130. Beal MF (1995) Aging, energy, and oxidative stress in neurodegenerative diseases. Ann Neurol 38:357-366. Beal MF, Ferrante RJ, Henshaw R, Matthews RT, Chan PH, Kowall NW, Epstein CJ, Schulz JB (1995) 3-Nitropropionic acid neurotoxicity is attenuated in copper/zinc superoxide dismutase transgenic mice. J Neurochem 65:919-922. Beal MF, Brouillet E, Jenkins BG, Ferrante RJ, Kowall NW, Miller JM, Storey E, Srivastava R, Rosen BR, Hyman BT (1993a) Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. Journal of Neuroscience 13:4181-4192. Beal MF, Brouillet E, Jenkins BG, Ferrante RJ, Kowall NW, Miller JM, Storey E, Srivastava R, Rosen BR, Hyman BT (1993b) Neurochemical and histologic characterization of striatal excitotoxic lesions produced by the mitochondrial toxin 3-nitropropionic acid. J Neurosci 13:4181-4192. Beckman JS (1991) The double-edged role of nitric oxide in brain function and superoxide-mediated injury. J Dev Physiol 15:53-59. Bederson JB, Pitts L H , Germano SM, Nishimura M C , Davis RL, Bartkowski H M (1986a) Evaluation of 2,3,5-triphenyltetrazolium chloride as a stain for detection and quantification of experimental cerebral infarction in rats. Stroke 17:1304-1308. 202 Bederson JB, Pitts L H , Tsuji M , Nishimura M C , Davis RL, Bartkowski H (1986b) Rat middle cerebral artery occlusion: evaluation of the model and development of a neurologic examination. Stroke 17:472-476. Benson A M , Hunkeler MJ, Talalay P (1980) Increase of NAD(P)H:quinone reductase by dietary antioxidants: possible role in protection against carcinogenesis and toxicity. Proc Natl Acad Sci U S A 77:5216-5220. Benson A M , Cha Y N , Bueding E, Heine HS, Talalay P (1979) Elevation of extrahepatic glutathione S-transferase and epoxide hydratase activities by 2(3)-tert-butyl-4-hydroxyanisole. Cancer Res 39:2971-2977. Benson A M , Batzinger RP, Ou SY, Bueding E, Cha Y N , Talalay P (1978) Elevation of hepatic glutathione S-transferase activities and protection against mutagenic metabolites of benzo(a)pyrene by dietary antioxidants. Cancer Res 38:4486-4495. Benveniste H (1991) The excitotoxin hypothesis in relation to cerebral ischemia. Cerebrovasc Brain Metab Rev 3:213-245. Benveniste H, Drejer J, Schousboe A , Diemer N H (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 43:1369-1374. Benveniste H, Jorgensen M B , Diemer N H , Hansen A J (1988) Calcium accumulation by glutamate receptor activation is involved in hippocampal cell damage after ischemia. Acta Neurol Scand 78:529-536. Bergeron M , Y u A Y , Solway ICE, Semenza GL, Sharp FR (1999) Induction of hypoxia-inducible factor-1 (HIF-1) and its target genes following focal ischaemia in rat brain. Eur J Neurosci 11:4159-4170. 203 Bernabeu R, Sharp FR (2000) N M D A and AMPA/kainate glutamate receptors modulate dentate neurogenesis and CA3 synapsin-I in normal and ischemic hippocampus. J Cereb Blood Flow Metab 20:1669-1680. Beyer RE, Segura-Aguilar J, Di Bernardo S, Cavazzoni M , Fato R, Fiorentini D, Galli M C , Setti M , Landi L , Lenaz G (1996) The role of DT-diaphorase in the maintenance of the reduced antioxidant form of coenzyme Q in membrane systems. Proc Natl Acad Sci U S A 93:2528-2532. Biernaskie J, Corbett D, Peeling J, Wells J, Lei H (2001) A serial M R study of cerebral blood flow changes and lesion development following endothelin-1-induced ischemia in rats. Magn Reson Med 46:827-830. Bjelke B, England R, Nicholson C, Rice M E , Lindberg J, Zoli M , Agnati LF , Fuxe K (1995) Long distance pathways of diffusion for dextran along fibre bundles in brain. Relevance for volume transmission. Neuroreport 6:1005-1009. Bloom D, Dhakshinamoorthy S, Jaiswal A K (2002) Site-directed mutagenesis of cysteine to serine in the D N A binding region of Nrf2 decreases its capacity to upregulate antioxidant response element-mediated expression and antioxidant induction of NAD(P)H:quinone oxidoreductase 1 gene. Oncogene 21:2191-2200. Boquillon M , Boquillon JP, Bralet J (1992) Photochemically induced, graded cerebral infarction in the mouse by laser irradiation evolution of brain edema. J Pharmacol Toxicol Methods 27:1-6. Braughler JM, Hall ED (1989) Central nervous system trauma and stroke. I. Biochemical considerations for oxygen radical formation and lipid peroxidation. Free Radic Biol Med 6:289-301. Braun S, Hanselmann C, Gassmann M G , auf dem Keller U , Born-Berclaz C, Chan K, Kan Y W , Werner S (2002) Nrf2 transcription factor, a novel target of keratinocyte growth factor 204 action which regulates gene expression and inflammation in the healing skin wound. Mol Cell Biol 22:5492-5505. Bredt DS, Snyder SH (1990) Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc Natl Acad Sci U S A 87:682-685. Broderick JP, Adams HP, Jr., Barsan W, Feinberg W, Feldmann E, Grotta J, Kase C, Krieger D, Mayberg M , Tilley B, Zabramski JM, Zuccarello M (1999) Guidelines for the management of spontaneous intracerebral hemorrhage: A statement for healthcare professionals from a special writing group of the Stroke Council, American Heart Association. Stroke 30:905-915. Brodie AE, Reed DJ (1987) Reversible oxidation of glyceraldehyde 3-phosphate dehydrogenase thiols in human lung carcinoma cells by hydrogen peroxide. Biochem Biophys Res Commun 148:120-125. Brouillet E, Guyot M C , Mittoux V, Altairac S, Conde F, Palfi S, Hantraye P (1998) Partial inhibition of brain succinate dehydrogenase by 3-nitropropionic acid is sufficient to initiate striatal degeneration in rat. Journal of Neurochemistry 70:794-805. Brouillet E, Jenkins BG, Hyman BT, Ferrante RJ, Kowall NW, Srivastava R, Roy DS, Rosen BR, Beal MF (1993) Age-dependent vulnerability of the striatum to the mitochondrial toxin 3-nitropropionic acid. J Neurochem 60:356-359. Bruce AJ, Boling W, Kindy MS, Peschon J, Kraemer PJ, Carpenter M K , Holtsberg FW, Mattson MP (1996) Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors. Nat Med 2:788-794. Buchan A, Pulsinelli W A (1990) Hypothermia but not the N-methyl-D-aspartate antagonist, MK-801, attenuates neuronal damage in gerbils subjected to transient global ischemia. J Neurosci 10:311-316. 205 Buchan A, L i H, Pulsinelli W A (1991) The N-methyl-D-aspartate antagonist, MK-801, fails to protect against neuronal damage caused by transient, severe forebrain ischemia in adult rats. J Neurosci 11:1049-1056. Buchan A M (1990) Do N M D A antagonists protect against cerebral ischemia: are clinical trials warranted? Cerebrovasc Brain Metab Rev 2:1-26. Busto R, Dietrich WD, Globus M Y , Valdes I, Scheinberg P, Ginsberg M D (1987) Small differences in intraischemic brain temperature critically determine the extent of ischemic neuronal injury. J Cereb Blood Flow Metab 7:729-738. Busto R, Globus M Y , Dietrich WD, Martinez E, Valdes I, Ginsberg M D (1989) Effect of mild hypothermia on ischemia-induced release of neurotransmitters and free fatty acids in rat brain. Stroke 20:904-910. Cadenas E, Davies K J (2000) Mitochondrial free radical generation, oxidative stress, and aging. Free Radic Biol Med 29:222-230. Calabrese V , Ravagna A , Colombrita C, Scapagnini G, Guagliano E, Calvani M , Butterfield DA, Giuffrida Stella A M (2005) Acetylcarnitine induces heme oxygenase in rat astrocytes and protects against oxidative stress: involvement of the transcription factor Nrf2. J Neurosci Res 79:509-521. Calkins MJ, Jakel RJ, Johnson DA, Chan K, Kan Y W , Johnson JA (2005) Protection from mitochondrial complex II inhibition in vitro and in vivo by Nrf2-mediated transcription. Proc Natl Acad Sci U S A 102:244-249. Calne DB, Eisen A, McGeer E, Spencer P (1986) Alzheimer's disease, Parkinson's disease, and motoneurone disease: abiotrophic interaction between ageing and environment? Lancet 2:1067-1070. Campagne M V , Thibodeaux H, van Bruggen N , Cairns B, Lowe D G (2000) Increased binding activity at an antioxidant-responsive element in the metallothionein-1 promoter and rapid 206 induction of metallothionein-1 and -2 in response to cerebral ischemia and reperfusion. J Neurosci 20:5200-5207. Cao TT, Ma L, Kandpal G, Warren L, Hess JF, Seabrook GR (2005) Increased nuclear factor-erythroid 2 p45-related factor 2 activity protects SH-SY5Y cells against oxidative damage. J Neurochem 95:406-417. Cao X, Phillis JW (1994) alpha-Phenyl-tert-butyl-nitrone reduces cortical infarct and edema in rats subjected to focal ischemia. Brain Res 644:267-272. Castillo MR, Babson JR (1998) Ca(2+)-dependent mechanisms of cell injury in cultured cortical neurons. Neuroscience 86:1133-1144. Cavalieri E, Rogan E, Chakravarti D (2004) The role of endogenous catechol quinones in the initiation of cancer and neurodegenerative diseases. Methods Enzymol 382:293-319. Cechetto DF, Wilson JX, Smith K E , Wolski D, Silver M D , Hachinski V C (1989) Autonomic and myocardial changes in middle cerebral artery occlusion: stroke models in the rat. Brain Res 502:296-305. Cervos-Navarro J, Diemer N H (1991) Selective vulnerability in brain hypoxia. Crit Rev Neurobiol 6:149-182. Chan JY, Kwong M (2000) Impaired expression of glutathione synthetic enzyme genes in mice with targeted deletion of the Nrf2 basic-leucine zipper protein: Biochim Biophys Acta 1517:19-26. Chan JY, Han X L , Kan Y W (1993) Isolation of cDNA encoding the human NF-E2 protein. Proc Natl Acad Sci U S A 90:11366-11370. Chan JY, Kwong M , Lu R, Chang J, Wang B, Yen TS, Kan Y W (1998) Targeted disruption of the ubiquitous CNC-bZIP transcription factor, Nrf-1, results in anemia and embryonic lethality in mice. Embo J 17:1779-1787. 207 Chan K, Kan Y W (1999) Nrf2 is essential for protection against acute pulmonary injury in mice. Proc Natl Acad Sci U S A 96:12731-12736. Chan K, Han X D , Kan Y W (2001) An important function of Nrf2 in combating oxidative stress: detoxification of acetaminophen. Proceedings of the National Academy of Sciences of the United States of America 98:4611-4616. Chan K, Lu R, Chang JC, Kan Y W (1996a) NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proc Natl Acad Sci U S A 93:13943-13948. Chan K, Lu R, Chang JC, Kan Y W (1996b) NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development. Proceedings of the National Academy of Sciences of the United States of America 93:13943-13948. Chan PH (2001) Reactive oxygen radicals in signaling and damage in the ischemic brain. J Cereb Blood Flow Metab 21:2-14. Chanas SA, Jiang Q, McMahon M , Mc Walter GK, McLellan LI, Elcombe CR, Henderson CJ, Wolf CR, Moffat GJ, Itoh K, Yamamoto M , Hayes JD (2002) Loss of the Nrf2 transcription factor causes a marked reduction in constitutive and inducible expression of the glutathione S-transferase Gstal, Gsta2, Gstml, Gstm2, Gstm3 and Gstm4 genes in the livers of male and female mice. Biochem J 365:405-416. Chen C, Okayama H (1987) High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745-2752. Chen C, Kong A N (2004) Dietary chemopreventive compounds and ARE/EpRE signaling. Free Radic Biol Med 36:1505-1516. 208 Chen CY, Jang JH, L i M H , Surh Y J (2005) Resveratrol upregulates heme oxygenase-1 expression via activation of NF-E2-related factor 2 in PC 12 cells. Biochem Biophys Res Commun 331:993-1000. Chen L, Kwong M , Lu R, Ginzinger D, Lee C, Leung L , Chan JY (2003a) Nrfl is critical for redox balance and survival of liver cells during development. Mol Cell Biol 23:4673-4686. Chen X L , Kunsch C (2004) Induction of cytoprotective genes through Nrf2/antioxidant response element pathway: a new therapeutic approach for the treatment of inflammatory diseases. CurrPharmDes 10:879-891. Chen X L , Varner SE, Rao AS, Grey JY, Thomas S, Cook CK, Wasserman M A , Medford R M , Jaiswal A K , Kunsch C (2003b) Laminar flow induction of antioxidant response element-mediated genes in endothelial cells. A novel anti-inflammatory mechanism. J Biol Chem 278:703-711. Chen Y, Vartiainen NE, Ying W, Chan PH, Koistinaho J, Swanson R A (2001) Astrocytes protect neurons from nitric oxide toxicity by a glutathione- dependent mechanism. J Neurochem 77:1601-1610. Chintala S, L i W, Lamoreux M L , Ito S, Wakamatsu K, Sviderskaya EV, Bennett DC, Park Y M , Gahl W A , Huizing M , Spritz RA, Ben S, Novak EK, Tan J, Swank RT (2005) Slc7al 1 gene controls production of pheomelanin pigment and proliferation of cultured cells. Proc Natl Acad Sci U S A 102:10964-10969. Cho HY, Jedlicka A E , Reddy SP, Zhang L Y , Kensler TW, Kleeberger SR (2002a) Linkage analysis of susceptibility to hyperoxia. Nrf2 is a candidate gene. Am J Respir Cell Mol Biol 26:42-51. 209 Cho HY, Jedlicka A E , Reddy SP, Kensler TW, Yamamoto M , Zhang L Y , Kleeberger SR (2002b) Role of NRF2 in protection against hyperoxic lung injury in mice. Am J Respir Cell Mol Biol 26:175-182. Choi DW (1985) Glutamate neurotoxicity in cortical cell culture is calcium dependent. Neurosci Lett 58:293-297. Choi DW (1987) Ionic dependence of glutamate neurotoxicity. J Neurosci 7:369-379. Choi DW, Maulucci-Gedde M , Kriegstein A R (1987) Glutamate neurotoxicity in cortical cell culture. J Neurosci 7:357-368. Chung WJ, Lyons SA, Nelson G M , Hamza H, Gladson CL, Gillespie GY, Sontheimer H (2005) Inhibition of cystine uptake disrupts the growth of primary brain tumors. J Neurosci 25:7101-7110. Clozel M , Gray GA, Breu V, Loffler B M , Osterwalder R (1992) The endothelin ETB receptor mediates both vasodilation and vasoconstriction in vivo. Biochem Biophys Res Commun 186:867-873. Colbourne F, Sutherland G, Corbett D (1997) Postischemic hypothermia. A critical appraisal with implications for clinical treatment. Mol Neurobiol 14:171-201. Connolly ES, Jr., Winfree CJ, Stern D M , Solomon RA, Pinsky DJ (1996) Procedural and strain-related variables significantly affect outcome in a murine model of focal cerebral ischemia. Neurosurgery 38:523-531; discussion 532. Cooper AJ , Kristal BS (1997) Multiple roles of glutathione in the central nervous system. Biol Chem 378:793-802. Copani A , Uberti D, Sortino M A , Bruno V, Nicoletti F, Memo M (2001) Activation of cell-cycle-associated proteins in neuronal death: a mandatory or dispensable path? Trends Neurosci 24:25-31. 210 Corbett D, Evans S, Thomas C, Wang D, Jonas RA (1990) MK-801 reduced cerebral ischemic injury by inducing hypothermia. Brain Res 514:300-304. Coyle JT, Puttfarcken P (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science 262:689-695. Crocker SJ, Lamba WR, Smith PD, Callaghan SM, Slack RS, Anisman H, Park DS (2001) c-Jun mediates axotomy-induced dopamine neuron death in vivo. Proc Natl Acad Sci U S A 98:13385-13390. Cullinan SB, Diehl JA (2004) PERK-dependent activation of Nrf2 contributes to redox homeostasis and cell survival following endoplasmic reticulum stress. J Biol Chem 279:20108-20117. Cullinan SB, Zhang D, Hannink M , Arvisais E, Kaufman RJ, Diehl JA (2003) Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival. Mol Cell Biol 23:7198-7209. Danbolt N C (2001) Glutamate uptake. Prog Neurobiol 65:1-105. Davalos A, Fernandez-Real JM, Ricart W, Soler S, Molins A , Planas E, Genis D (1994) Iron-related damage in acute ischemic stroke. Stroke 25:1543-1546. Davis SM, Lees KR, Albers GW, Diener HC, Markabi S, Karlsson G, Norris J (2000) Selfotel in acute ischemic stroke : possible neurotoxic effects of an N M D A antagonist. Stroke 31:347-354. De Keyser J, Suiter G, Luiten PG (1999) Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing? Trends Neurosci 22:535-540. De Long MJ, Santamaria A B , Talalay P (1987) Role of cytochrome PI-450 in the induction of NAD(P)H:quinone reductase in a murine hepatoma cell line and its mutants. Carcinogenesis 8:1549-1553. 211 del Zoppo G, Ginis I, Hallenbeck JM, Iadecola C, Wang X , Feuerstein GZ (2000) Inflammation and stroke: putative role for cytokines, adhesion molecules and iNOS in brain response to ischemia. Brain Pathol 10:95-112. del Zoppo GJ, Becker KJ , Hallenbeck JM (2001) Inflammation after stroke: is it harmful? Arch Neurol 58:669-672. Demple B, Amabile-Cuevas CF (1991) Redox redux: the control of oxidative stress responses. Cell 67:837-839. Derjuga A, Gourley TS, Holm T M , Heng HH, Shivdasani RA, Ahmed R, Andrews NC, Blank V (2004) Complexity of CNC transcription factors as revealed by gene targeting of the Nrf3 locus. Mol Cell Biol 24:3286-3294. Desagher S, Glowinski J, Premont J (1996) Astrocytes protect neurons from hydrogen peroxide toxicity. J Neurosci 16:2553-2562. Devling TW, Lindsay CD, McLellan LI, McMahon M , Hayes JD (2005) Utility of siRNA against Keapl as a strategy to stimulate a cancer chemopreventive phenotype. Proc Natl Acad Sci U S A 102:7280-7285A. Dhakshinamoorthy S, Jain A K , Bloom DA, Jaiswal A K (2005) Bachl competes with Nrf2 leading to negative regulation of the antioxidant response element (ARE)-mediated NAD(P)H:quinone oxidoreductase 1 gene expression and induction in response to antioxidants. J Biol Chem 280:16891-16900. Di Monte DA, Lavasani M , Manning-Bog A B (2002) Environmental factors in Parkinson's disease. Neurotoxicology 23:487-502. Dietrich WD, Ginsberg M D , Busto R, Watson BD (1986) Photochemically induced cortical infarction in the rat. 1. Time course of hemodynamic consequences. J Cereb Blood Flow Metab 6:184-194. 212 Dietrich WD, Watson BD, Busto R, Ginsberg M D , Bethea JR (1987) Photochemically induced cerebral infarction. 1. Early microvascular alterations. Acta Neuropatho! (Berl) 72:315-325. Dietrich WD, Feng ZC, Leistra H, Watson BD, Rosenthal M (1994) Photothrombotic infarction triggers multiple episodes of cortical spreading depression in distant brain regions. J Cereb Blood Flow Metab 14:20-28. Dingledine R, Borges K, Bowie D, Traynelis SF (1999) The glutamate receptor ion channels. Pharmacol Rev 51:7-61. Dinkova-Kostova AT, Talalay P (1999) Relation of structure of curcumin analogs to their potencies as inducers of Phase 2 detoxification enzymes. Carcinogenesis 20:911-914. Dinkova-Kostova AT, Holtzclaw WD, Cole RN, Itoh K, Wakabayashi N , Katoh Y , Yamamoto M , Talalay P (2002) Direct evidence that sulfhydryl groups of Keap 1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants. Proc Natl Acad Sci U S A 99:11908-11913. Dirnagl U , Iadecola C, Moskowitz M A (1999) Pathobiology of ischaemic stroke: an integrated view. Trends Neurosci 22:391-397. Ditelberg JS, Sheldon RA, Epstein CJ, Ferriero D M (1996) Brain injury after perinatal hypoxia-ischemia is exacerbated in copper/zinc superoxide dismutase transgenic mice. Pediatr Res 39:204-208. Donohoe PH, Fahlman CS, Bickler PE, Vexler ZS, Gregory G A (2001) Neuroprotection and intracellular Ca2+ modulation with fructose-1,6-bisphosphate during in vitro hypoxia-ischemia involves phospholipase C-dependent signaling. Brain Res 917:158-166. Dringen R, Pfeiffer B, Hamprecht B (1999) Synthesis of the antioxidant glutathione in neurons: supply by astrocytes of CysGly as precursor for neuronal glutathione. J Neurosci 19:562-569. 213 Dringen R, Gutterer JM, Hirrlinger J (2000) Glutathione metabolism in brain metabolic interaction between astrocytes and neurons in the defense against reactive oxygen species. Eur J Biochem 267:4912-4916. Dringen R, Gutterer JM, Gros C, Hirrlinger J (2001) Aminopeptidase N mediates the utilization of the GSH precursor CysGly by cultured neurons. J Neurosci Res 66:1003-1008. Drukarch B, Schepens E, Jongenelen CA, Stoof JC, Langeveld C H (1997) Astrocyte-mediated enhancement of neuronal survival is abolished by glutathione deficiency. Brain Res 770:123-130. Drukarch B, Schepens E, Stoof JC, Langeveld CH, Van Muiswinkel F L (1998) Astrocyte-enhanced neuronal survival is mediated by scavenging of extracellular reactive oxygen species. Free Radic Biol Med 25:217-220. Dubinsky JM, Kristal BS, Elizondo-Fournier M (1995) An obligate role for oxygen in the early stages of glutamate-induced, delayed neuronal death. J Neurosci 15:7071-7078. Duffy S, So A , Murphy T H (1998) Activation of endogenous antioxidant defenses in neuronal cells prevents free radical-mediated damage. J Neurochem 71:69-77. Dwyer BE, Nishimura RN, Lu SY (1995) Differential expression of heme oxygenase-1 in cultured cortical neurons and astrocytes determined by the aid of a new heme oxygenase antibody. Response to oxidative stress. Brain Res Mol Brain Res 30:37-47. Eftekharpour E, Holmgren A, Juurlink B H (2000) Thioredoxin reductase and glutathione synthesis is upregulated by t- butylhydroquinone in cortical astrocytes but not in cortical neurons. Glia 31:241-248. Egner PA, Kensler TW, Prestera T, Talalay P, Libby A H , Joyner H H , Curphey TJ (1994) Regulation of phase 2 enzyme induction by oltipraz and other dithiolethiones. Carcinogenesis 15:177-181. 214 Ekholm SV, Reed SI (2000) Regulation of G(l) cyclin-dependent kinases in the mammalian cell cycle. Curr Opin Cell Biol 12:676-684. Eliasson MJ, Sampei K, Mandir AS, Hum PD, Traystman RJ, Bao J, Pieper A, Wang ZQ, Dawson T M , Snyder SH, Dawson V L (1997) Poly(ADP-ribose) polymerase gene disruption renders mice resistant to cerebral ischemia. Nat Med 3:1089-1095. Engler RL, Schmid-Schonbein GW, Pavelec RS (1983) Leukocyte capillary plugging in myocardial ischemia and reperfusion in the dog. Am J Pathol 111:98-l 11. Enomoto A, Itoh K, Nagayoshi E, Haruta J, Kimura T, O'Connor T, Harada T, Yamamoto M (2001) High sensitivity of Nrf2 knockout mice to acetaminophen hepatotoxicity associated with decreased expression of ARE-regulated drug metabolizing enzymes and antioxidant genes. Toxicological Sciences 59:169-177. Everse J, Berger RL, Kaplan NO (1970) Physiological concentrations of lactate dehydrogenases and substrate inhibition. Science 168:1236-1238. Fabian RH, Perez-Polo JR, Kent T A (2000) Electrochemical monitoring of superoxide anion production and cerebral blood flow: effect of interleukin-1 beta pretreatment in a model of focal ischemia and reperfusion. J Neurosci Res 60:795-803. Fahey JW, Talalay P (1999) Antioxidant functions of sulforaphane: a potent inducer of Phase II detoxication enzymes. Food Chem Toxicol 37:973-979. Fahey JW, Zhang Y , Talalay P (1997) Broccoli sprouts: an exceptionally rich source of inducers of enzymes that protect against chemical carcinogens. Proc Natl Acad Sci U S A 94:10367-10372. Fahey JW, Zalcmann AT, Talalay P (2001) The chemical diversity and distribution of glucosinolates and isothiocyanates among plants. Phytochemistry 56:5-51. Fahey JW, Haristoy X , Dolan P M , Kensler TW, Scholtus I, Stephenson K K , Talalay P, Lozniewski A (2002) Sulforaphane inhibits extracellular, intracellular, and antibiotic-215 resistant strains of Helicobacter pylori and prevents benzo[a]pyrene-induced stomach tumors Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. Proceedings of the National Academy of Sciences of the United States of America 99:7610-7615. FAO/WHO (1975) Toxicological evaluation of some food additives, including food colours, thickening agents and others. Joint FAO/WHO Expert Committee on Food Additives. Geneva, 14-23 April 1975. FAO Nutr Meet Rep Ser: 1-204. FAO/WHO (1999) Evaluation of certain food additives and contaminants. Forty-ninth report of the Joint FAO/WHO Expert Committee on Food Additives. World Health Organ Tech Rep Ser 884:i-viii, 1-96. Farmer SC, Sun CW, Winnier GE, Hogan BL, Townes T M (1997) The bZIP transcription factor LCR-F1 is essential for mesoderm formation in mouse development. Genes Dev 11:786-798. Fernagut PO, Hervier L, Labattu B, Bioulac B, Tison F (2002) Subacute systemic 3-nitropropionic acid intoxication induces a distinct motor disorder in adult C57B1/6 mice: behavioural and histopathological characterisation. Neuroscience 115:533-546. Fevig TL, Bowen SM, Janowick DA, Jones BK, Munson HR, Ohlweiler DF, Thomas CE (1996) Design, synthesis, and in vitro evaluation of cyclic nitrones as free radical traps for the treatment of stroke. J Med Chem 39:4988-4996. Fink SL, Ho D Y , Sapolsky R M (1996) Energy and glutamate dependency of 3-Nitropropionic acid neurotoxicity in culture. Exp Neurol 138:298-304. Fisher JM, Wu L, Denison MS, Whitlock JP, Jr. (1990) Organization and function of a dioxin-responsive enhancer. J Biol Chem 265:9676-9681. 216 Floyd RA (1997) Protective action of nitrone-based free radical traps against oxidative damage to the central nervous system. Adv Pharmacol 38:361-378. Folbergrova J, Zhao Q, Katsura K, Siesjo B K (1995) N-tert-butyl-alpha-phenylnitrone improves recovery of brain energy state in rats following transient focal ischemia. Proc Natl Acad Sci U S A 92:5057-5061. Fountaine T M , Wood MJ, Wade-Martins R (2005) Delivering RNA interference to the mammalian brain. Curr Gene Ther 5:399-410. Friling RS, Bensimon A, Tichauer Y , Daniel V (1990a) Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. Proc Natl Acad Sci U S A 87:6258-6262. Friling RS, Bensimon A, Tichauer Y , Daniel V (1990b) Xenobiotic-inducible expression of murine glutathione S-transferase Ya subunit gene is controlled by an electrophile-responsive element. Proc Natl Acad Sci U S A 87:6258-6262. Fu Y, He F, Zhang S, Jiao X (1995) Consistent striatal damage in rats induced by 3-nitropropionic acid and cultures of arthrinium fungus. Neurotoxicol Teratol 17:413-418. Fullerton HJ, Ditelberg JS, Chen SF, Sarco DP, Chan PH, Epstein CJ, Ferriero D M (1998) Copper/zinc superoxide dismutase transgenic brain accumulates hydrogen peroxide after perinatal hypoxia ischemia. Ann Neurol 44:357-364. Furuya K, Kawahara N , Kawai K , Toyoda T, Maeda K , Kirino T (2004) Proximal occlusion of the middle cerebral artery in C57Black6 mice: relationship of patency of the posterior communicating artery, infarct evolution, and animal survival. J Neurosurg 100:97-105. Fuxe K, Bjelke B, Andbjer B, Grahn H, Rimondini R, Agnati LF (1997) Endothelin-1 induced lesions of the frontoparietal cortex of the rat. A possible model of focal cortical ischemia. Neuroreport 8:2623-2629. 217 Gajkowska B, Frontczak-Baniewicz M , Gadamski R, Barskov I (1997) Photochemically-induced vascular damage in brain cortex. Transmission and scanning electron microscopy study. Acta Neurobiol Exp (Wars) 57:203-208. Gao J, Duan B, Wang DG, Deng X H , Zhang GY, Xu L , Xu TL (2005) Coupling between N M D A receptor and acid-sensing ion channel contributes to ischemic neuronal death. Neuron 48:635-646. Gao X , Talalay P (2004) Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage. Proc Natl Acad Sci U S A 101:10446-10451. Gao X , Dinkova-Kostova AT, Talalay P (2001) Powerful and prolonged protection of human retinal pigment epithelial cells, keratinocytes, and mouse leukemia cells against oxidative damage: the indirect antioxidant effects of sulforaphane. Proc Natl Acad Sci U S A 98:15221-15226. Garcia JH, Yoshida Y , Chen H, L i Y , Zhang ZG, Lian J, Chen S, Chopp M (1993) Progression from ischemic injury to infarct following middle cerebral artery occlusion in the rat. A m J Pathol 142:623-635. Gido G, Kristian T, Katsura K, Siesjo B K (1994) The influence of repeated spreading depression-induced calcium transients on neuronal viability in moderately hypoglycemic rats. Exp Brain Res 97:397-403. Giffard RG, Monyer H, Christine CW, Choi DW (1990) Acidosis reduces N M D A receptor activation, glutamate neurotoxicity, and oxygen-glucose deprivation neuronal injury in cortical cultures. Brain Res 506:339-342. Gilroy J (2000) Basic neurology, 3rd Edition. New York: McGraw-Hill, Health Professions Division. 218 Ginsberg M D , Pulsinelli W A (1994) The ischemic penumbra, injury thresholds, and the therapeutic window for acute stroke. Ann Neurol 36:553-554. Gladstone DJ, Black SE, Hakim A M (2002) Toward wisdom from failure: lessons from neuroprotective stroke trials and new therapeutic directions. Stroke 33:2123-2136. Gold MO, Rice AP (1998) Targeting of CDK8 to a promoter-proximal RNA element demonstrates catalysis-dependent activation of gene expression. Nucleic Acids Res 26:3784-3788. Goldman SA, Pulsinelli WA, Clarke W Y , Kraig RP, Plum F (1989) The effects of extracellular acidosis on neurons and glia in vitro. J Cereb Blood Flow Metab 9:471-477. Gong P, Stewart D, Hu B, L i N , Cook J, Nel A, Alam J (2002) Activation of the mouse heme oxygenase-1 gene by 15-deoxy-deltal2,14- prostaglandin j2 is mediated by the stress response elements and transcription factor nrf2. Antioxid Redox Signal 4:249-257. Goss JR, Styren SD, Miller PD, Kochanek P M , Palmer A M , Marion DW, DeKosky ST (1995) Hypothermia attenuates the normal increase in interleukin 1 beta R N A and nerve growth factor following traumatic brain injury in the rat. J Neurotrauma 12:159-167. Gould DH, Gustine DL (1982) Basal ganglia degeneration, myelin alterations, and enzyme inhibition induced in mice by the plant toxin 3-nitropropanoic acid. Neuropathol Appl Neurobiol 8:377-393. Gout PW, Buckley AR, Simms CR, Bruchovsky N (2001) Sulfasalazine, a potent suppressor of lymphoma growth by inhibition of the x(c)- cystine transporter: a new action for an old drug. Leukemia 15:1633-1640. Green AR, Ashwood T (2005) Free radical trapping as a therapeutic approach to neuroprotection in stroke: experimental and clinical studies with NXY-059 and free radical scavengers. Curr Drug Targets CNS Neurol Disord 4:109-118. 219 Griffith OW, Meister A (1979) Potent and specific inhibition of glutathione synthesis by buthionine sulfoximine (S-n-butyl homocysteine sulfoximine). J Biol Chem 254:7558-7560. Guyot MC, Hantraye P, Dolan R, Palfi S, Maziere M , Brouillet E (1997) Quantifiable bradykinesia, gait abnormalities and Huntington's disease-like striatal lesions in rats chronically treated with 3-nitropropionic acid. Neuroscience 79:45-56. Gwag BJ, Lobner D, Koh JY, Wie M B , Choi DW (1995) Blockade of glutamate receptors unmasks neuronal apoptosis after oxygen-glucose deprivation in vitro. Neuroscience 68:615-619. Haddad GG, Jiang C (1993) 02 deprivation in the central nervous system: on mechanisms of neuronal response, differential sensitivity and injury. Prog Neurobiol 40:277-318. Hall ED, Braughler J M (1989) Central nervous system trauma and stroke. II. Physiological and pharmacological evidence for involvement of oxygen radicals and lipid peroxidation. Free Radic Biol Med 6:303-313. Hall ED, Braughler JM (1993) Free radicals in CNS injury. Res Publ Assoc Res Nerv Ment Dis 71:81-105. Hallenbeck JM (1996) Significance of the inflammatory response in brain ischemia. Acta Neurochir Suppl 66:27-31. Halliwell B, Gutteridge JM (1984) Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219:1-14. Halliwell B, Gutteridge JM (1988) Free radicals and antioxidant protection: mechanisms and significance in toxicology and disease. Hum Toxicol 7:7-13. Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine, 3rd Edition. Oxford, England: Oxford University Press. 220 Hamilton BF, Gould DH (1987) Nature and distribution of brain lesions in rats intoxicated with 3-nitropropionic acid: a type of hypoxic (energy deficient) brain damage. Acta Neuropathol (Bed) 72:286-297. Hanna P M , Mason RP (1992) Direct evidence for inhibition of free radical formation from Cu(I) and hydrogen peroxide by glutathione and other potential ligands using the EPR spin-trapping technique. Arch Biochem Biophys 295:205-213. Hara H, Ohta M , Ohta K, Kuno S, Adachi T (2003) Increase of antioxidative potential by tert-butylhydroquinone protects against cell death associated with 6-hydroxydopamine-induced oxidative stress in neuroblastoma SH-SY5Y cells. Brain Res Mol Brain Res 119:125-131. Hardingham GE, Bading H (2003) The Yin and Yang of N M D A receptor signalling. Trends Neurosci 26:81-89. Hardingham GE, Fukunaga Y , Bading H (2002) Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat Neurosci 5:405-414. Hardy S, Kitamura M , Harris-Stansil T, Dai Y , Phipps M L (1997) Construction of adenovirus vectors through Cre-lox recombination. J Virol 71:1842-1849. Hata R, Maeda K, Hermann D, Mies G, Hossmann K A (2000a) Dynamics of regional brain metabolism and gene expression after middle cerebral artery occlusion in mice. J Cereb Blood Flow Metab 20:306-315. Hata R, Maeda K, Hermann D, Mies G, Hossmann K A (2000b) Evolution of brain infarction after transient focal cerebral ischemia in mice. J Cereb Blood Flow Metab 20:937-946. Hayashi A , Suzuki H, Itoh K , Yamamoto M , Sugiyama Y (2003) Transcription factor Nrf2 is required for the constitutive and inducible expression of multidrug resistance-associated protein 1 in mouse embryo fibroblasts. Biochem Biophys Res Commun 310:824-829. 221 Hayes JD, Pulford DJ (1995) The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev Biochem Mol Biol 30:445-600. Hayes JD, Flanagan JU, Jowsey IR (2005) Glutathione transferases. Annu Rev Pharmacol Toxicol 45:51-88. Hayes JD, Chanas SA, Henderson CJ, McMahon M , Sun C, Moffat GJ, Wolf CR, Yamamoto M (2000a) The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. Biochem Soc Trans 28:33-41. Hayes JD, Chanas SA, Henderson CJ, McMahon M , Sun C, Moffat GJ, Wolf CR, Yamamoto M (2000b) The Nrf2 transcription factor contributes both to the basal expression of glutathione S-transferases in mouse liver and to their induction by the chemopreventive synthetic antioxidants, butylated hydroxyanisole and ethoxyquin. Biochem Soc Trans 28:33-41. He CH, Gong P, Hu B, Stewart D, Choi M E , Choi A M , Alam J (2001) Identification of activating transcription factor 4 (ATF4) as an Nrf2-interacting protein. Implication for heme oxygenase-1 gene regulation. J Biol Chem 276:20858-20865. Heales SJ, Bolanos JP, Stewart V C , Brookes PS, Land JM, Clark JB (1999) Nitric oxide, mitochondria and neurological disease. Biochim Biophys Acta 1410:215-228. Henderson CJ, Smith A G , Ure J, Brown K, Bacon EJ, Wolf CR (1998) Increased skin tumorigenesis in mice lacking pi class glutathione S-transferases. Proc Natl Acad Sci U S A 95:5275-5280. Henderson L M , Chappel JB (1996) N A D P H oxidase of neutrophils. Biochim Biophys Acta 1273:87-107. 222 Henshall DC, Butcher SP, Sharkey J (1999) A rat model of endothelin-3-induced middle cerebral artery occlusion with controlled reperfusion. Brain Res 843:105-111. Henthorn P, Zervos P, Raducha M , Harris H, Kadesch T (1988) Expression of a human placental alkaline phosphatase gene in transfected cells: use as a reporter for studies of gene expression. Proc Natl Acad Sci U S A 85:6342-6346. Hermens WT, Giger RJ, Holtmaat AJ, Dijkhuizen PA, Houweling DA, Verhaagen J (1997) Transient gene transfer to neurons and glia: analysis of adenoviral vector performance in the CNS and PNS. J Neurosci Methods 71:85-98. Himi T, Ikeda M , Yasuhara T, Nishida M , Morita I (2003) Role of neuronal glutamate transporter in the cysteine uptake and intracellular glutathione levels in cultured cortical neurons. J Neural Transm 110:1337-1348. Hirrlinger J, Schulz JB, Dringen R (2002) Glutathione release from cultured brain cells: multidrug resistance protein 1 mediates the release of GSH from rat astroglial cells. J Neurosci Res 69:318-326. Hirrlinger J, Konig J, Keppler D, Lindenau J, Schulz JB, Dringen R (2001) The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress. JNeurochem 76:627-636. Holtzclaw WD, Dinkova-Kostova AT, Talalay P (2004) Protection against electrophile and oxidative stress by induction of phase 2 genes: the quest for the elusive sensor that responds to inducers. Adv Enzyme Regul 44:335-367. Hope BT, Michael GJ, Knigge K M , Vincent SR (1991) Neuronal N A D P H diaphorase is a nitric oxide synthase. Proc Natl Acad Sci U S A 88:2811-2814. Horakova L, Stole S, Chromikova Z, Pekarova A, Derkova L (1997) Mechanisms of hippocampal reoxygenation injury. Treatment with antioxidants. Neuropharmacology 36:177-184. 223 Hosoya K, Tomi M , Ohtsuki S, Takanaga H, Saeki S, Kanai Y , Endou H, Naito M , Tsuruo T, Terasaki T (2002) Enhancement of L-cystine transport activity and its relation to xCT gene induction at the blood-brain barrier by diethyl maleate treatment. J Pharmacol Exp Ther 302:225-231. Hossmann ICA (1994a) Viability thresholds and the penumbra of focal ischemia. Ann Neurol 36:557-565. Hossmann K A (1994b) Glutamate-mediated injury in focal cerebral ischemia: the excitotoxin hypothesis revised. Brain Pathol 4:23-36. Hossmann K A (1996) Periinfarct depolarizations. Cerebrovasc Brain Metab Rev 8:195-208. Hoyte L, Barber PA, Buchan A M , Hi l l M D (2004) The rise and fall of N M D A antagonists for ischemic stroke. Curr Mol Med 4:131-136. Huang HC, Nguyen T, Pickett CB (2000) Regulation of the antioxidant response element by protein kinase C- mediated phosphorylation of NF-E2-related factor 2. Proc Natl Acad Sci U S A97:12475-12480. Huang HC, Nguyen T, Pickett CB (2002) Phosphorylation of Nrf2 at Ser-40 by protein kinase C regulates antioxidant response element-mediated transcription. J Biol Chem 277:42769-42774. Iadecola C, Zhang F, Casey R, Nagayama M , Ross M E (1997) Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. J Neurosci 17:9157-9164. Iantomasi T, Favilli F, Vincenzini M T (1999) Evidence of glutathione transporter in rat brain synaptosomal membrane vesicles. Neurochem Int 34:509-516. Ikeda H, Serria MS, Kakizaki I, Hatayama I, Satoh K, Tsuchida S, Muramatsu M , Nishi S, Sakai M (2002) Activation of mouse Pi-class glutathione S-transferase gene by Nrf2(NF-E2-related factor 2) and androgen. Biochem J 364:563-570. 224 Ikeda Y, Sugawara A, Taniyama Y , Uruno A, Igarashi K, Arima S, Ito S, Takeuchi K (2000) Suppression of rat thromboxane synthase gene transcription by peroxisome proliferator-activated receptor gamma in macrophages via an interaction with NRF2. J Biol Chem 275:33142-33150. Inamdar N M , Ahn YI , Alam J (1996) The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for M A F and NF-E2 transcription factors. Biochem Biophys Res Commun 221:570-576. Ishii T, Itoh K, Takahashi S, Sato H, Yanagawa T, Katoh Y , Bannai S, Yamamoto M (2000) Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 275:16023-16029. Itoh K, Ishii T, Wakabayashi N , Yamamoto M (1999a) Regulatory mechanisms of cellular response to oxidative stress. Free Radic Res 31:319-324. Itoh K, Wakabayashi N , Katoh Y , Ishii T, O'Connor T, Yamamoto M (2003) Keapl regulates both cytoplasmic-nuclear shuttling and degradation of Nrf2 in response to electrophiles. Genes Cells 8:379-391. Itoh K, Wakabayashi N , Katoh Y , Ishii T, Igarashi K, Engel JD, Yamamoto M (1999b) Keapl represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 13:76-86. Itoh K, Mochizuki M , Ishii Y , Ishii T, Shibata T, Kawamoto Y , Kelly V, Sekizawa K, Uchida K, Yamamoto M (2004) Transcription factor Nrf2 regulates inflammation by mediating the effect of 15-deoxy-Delta(12,14)-prostaglandin j(2). Mol Cell Biol 24:36-45. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y , Oyake T, Hayashi N , Satoh K, Hatayama I, Yamamoto M , Nabeshima Y (1997a) An Nrf2/small Maf heterodimer 225 mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 236:313-322. Itoh K, Chiba T, Takahashi S, Ishii T, Igarashi K, Katoh Y , Oyake T, Hayashi N , Satoh K, Hatayama I, Yamamoto M , Nabeshima Y (1997b) An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 236:313-322. Jacobson M D (1996) Reactive oxygen species and programmed cell death. Trends Biochem Sci 21:83-86. Jakel RJ, Kern JT, Johnson DA, Johnson JA (2005) Induction of the Protective Antioxidant Response Element Pathway by 6-Hydroxydopamine In Vivo and In Vitro. Toxicol Sci 87:176-186. Janaky R, Ogita K , Pasqualotto B A , Bains JS, Oja SS, Yoneda Y , Shaw C A (1999) Glutathione and signal transduction in the mammalian CNS. J Neurochem 73:889-902. Janardhan V, Qureshi AI (2004) Mechanisms of ischemic brain injury. Curr Cardiol Rep 6:117-123. Jenner P (2001) Parkinson's disease, pesticides and mitochondrial dysfunction. Trends Neurosci 24:245-247. Jeyapaul J, Jaiswal A K (2000) Nrf2 and c-Jun regulation of antioxidant response element (ARE)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene. Biochem Pharmacol 59:1433-1439. Johnson DA, Andrews GK, Xu W, Johnson JA (2002) Activation of the antioxidant response element in primary cortical neuronal cultures derived from transgenic reporter mice. J Neurochem 81:1233-1241. Kaku DA, Giffard RG, Choi DW (1993) Neuroprotective effects of glutamate antagonists and extracellular acidity. Science 260:1516-1518. 226 Kandel ER, Schwartz JH, Jessell T M (2000) Principles of neural science, 4th Edition. New York: McGraw-Hill Health Professions Division. Kaneko D, Nakamura N , Ogawa T (1985) Cerebral infarction in rats using homologous blood emboli: development of a new experimental model. Stroke 16:76-84. Kang KW, Choi SH, Kim SG (2002a) Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction. Nitric Oxide 7:244-253. Kang KW, Lee SJ, Park JW, Kim SG (2002b) Phosphatidylinositol 3-kinase regulates nuclear translocation of NF-E2-related factor 2 through actin rearrangement in response to oxidative stress. Mol Pharmacol 62:1001-1010. Kang MI, Kobayashi A, Wakabayashi N , Kim SG, Yamamoto M (2004) Scaffolding of Keapl to the actin cytoskeleton controls the function of Nrf2 as key regulator of cytoprotective phase 2 genes. Proc Natl Acad Sci U S A 101:2046-2051. Kannan R, Mittur A , Bao Y, Tsuruo T, Kaplowitz N (1999) GSH transport in immortalized mouse brain endothelial cells: evidence for apical localization of a sodium-dependent GSH transporter. J Neurochem 73:390-399. Kannan R, Chakrabarti R, Tang D, Kim KJ , Kaplowitz N (2000) GSH transport in human cerebrovascular endothelial cells and human astrocytes: evidence for luminal localization of Na+-dependent GSH transport in HCEC. Brain Res 852:374-382. Kannan R, Kuhlenkamp JF, Jeandidier E, Trinh H , Ookhtens M , Kaplowitz N (1990) Evidence for carrier-mediated transport of glutathione across the blood-brain barrier in the rat. J Clin Invest 85:2009-2013. Kannan R, Y i JR, Tang D, L i Y , Zlokovic B V , Kaplowitz N (1996) Evidence for the existence of a sodium-dependent glutathione (GSH) transporter. Expression of bovine brain capillary 227 mRNA and size fractions in Xenopus laevis oocytes and dissociation from gamma-glutamyltranspeptidase and facilitative GSH transporters. J Biol Chem 271:9754-9758. Karibe H, Chen J, Zarow GJ, Graham SH, Weinstein PR (1994a) Delayed induction of mild hypothermia to reduce infarct volume after temporary middle cerebral artery occlusion in rats. J Neurosurg 80:112-119. Karibe H, Chen SF, Zarow GJ, Gafni J, Graham SH, Chan PH, Weinstein PR (1994b) Mild intraischemic hypothermia suppresses consumption of endogenous antioxidants after temporary focal ischemia in rats. Brain Res 649:12-18. Kataoka K, Handa H, Nishizawa M (2001) Induction of cellular antioxidative stress genes through heterodimeric transcription factor Nrf2/small Maf by antirheumatic gold(I) compounds. J Biol Chem 276:34074-34081. Katoh Y, Itoh K, Yoshida E, Miyagishi M , Fukamizu A, Yamamoto M (2001) Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription. Genes Cells 6:857-868. Kawamura S, Yasui N , Shirasawa M , Fukasawa H (1991) Rat middle cerebral artery occlusion using an intraluminal thread technique. Acta Neurochir (Wien) 109:126-132. Kelly VP, Ellis E M , Manson M M , Chanas SA, Moffat GJ, McLeod R, Judah DJ, Neal GE, Hayes JD (2000) Chemoprevention of aflatoxin B1 hepatocarcinogenesis by coumarin, a natural benzopyrone that is a potent inducer of aflatoxin BI-aldehyde reductase, the glutathione S-transferase A5 and PI subunits, and NAD(P)H:quinone oxidoreductase in rat liver. Cancer Res 60:957-969. Kensler TW (1997) Chemoprevention by inducers of carcinogen detoxication enzymes. Environ Health Perspect 105 Suppl 4:965-970. 228 Kensler TW, Curphey TJ, Maxiutenko Y , Roebuck BD (2000) Chemoprotection by organosulfur inducers of phase 2 enzymes: dithiolethiones and dithiins. Drug Metabol Drug Interact 17:3-22. Kensler TW, Davidson NE, Groopman JD, Roebuck BD, Prochaska HJ, Talalay P (1993) Chemoprotection by inducers of electrophile detoxication enzymes. Basic Life Sci 61:127-136. Kim GW, Copin JC, Kawase M , Chen SF, Sato S, Gobbel GT, Chan PH (2000) Excitotoxicity is required for induction of oxidative stress and apoptosis in mouse striatum by the mitochondrial toxin, 3-nitropropionic acid. J Cereb Blood Flow Metab 20:119-129. Kinouchi H, Epstein CJ, Mizui T, Carlson E, Chen SF, Chan PH (1991) Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc Natl Acad Sci U S A 88:11158-11162. Kirby J, Halligan E, Baptista MJ , Allen S, Heath PR, Holden H, Barber SC, Loynes CA, Wood-Allum CA, Lunec J, Shaw PJ (2005) Mutant SOD1 alters the motor neuronal transcriptome: implications for familial A L S . Brain 128:1686-1706. Kirchhoff F, Dringen R, Giaume C (2001) Pathways of neuron-astrocyte interactions and their possible role in neuroprotection. Eur Arch Psychiatry Clin Neurosci 251:159-169. Kita H, Shima K , Tatsumi M , Chigasaki H (1995) Cerebral blood flow and glucose metabolism of the ischemic rim in spontaneously hypertensive stroke-prone rats with occlusion of the middle cerebral artery. J Cereb Blood Flow Metab 15:235-241. Kitagawa K, Matsumoto M , Yang G, Mabuchi T, Yagita Y , Hori M , Yanagihara T (1998) Cerebral ischemia after bilateral carotid artery occlusion and intraluminal suture occlusion in mice: evaluation of the patency of the posterior communicating artery. J Cereb Blood Flow Metab 18:570-579. 229 Kobayashi A, Ito E, Toki T, Kogame K, Takahashi S, Igarashi K, Hayashi N , Yamamoto M (1999) Molecular cloning and functional characterization of a new Cap'n' collar family transcription factor Nrf3. J Biol Chem 274:6443-6452. Kobayashi A, Kang MI, Okawa H, Ohtsuji M , Zenke Y , Chiba T, Igarashi K, Yamamoto M (2004) Oxidative stress sensor Keapl functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 24:7130-7139. Kokubo Y , Liu J, Rajdev S, Kayama T, Sharp FR, Weinstein PR (2003) Differential cerebral protein synthesis and heat shock protein 70 expression in the core and penumbra of rat brain after transient focal ischemia. Neurosurgery 53:186-190; discussion 190-181. Kraft AD, Johnson DA, Johnson JA (2004) Nuclear factor E2-related factor 2-dependent antioxidant response element activation by tert-butylhydroquinone and sulforaphane occurring preferentially in astrocytes conditions neurons against oxidative insult. Journal of Neuroscience 24:1101-1112. Kranich O, Hamprecht B, Dringen R (1996a) Different preferences in the utilization of amino acids for glutathione synthesis in cultured neurons and astroglial cells derived from rat brain. Neurosci Lett 219:211-214. Kranich O, Hamprecht B, Dringen R (1996b) Different preferences in the utilization of amino acids for glutathione synthesis in cultured neurons and astroglial cells derived from rat brain. Neurosci Lett 219:211-214. Krishtal O (2003) The ASICs: signaling molecules? Modulators? Trends Neurosci 26:477-483. Kumar A, Takada Y , Boriek A M , Aggarwal BB (2004) Nuclear factor-kappaB: its role in health and disease. J Mol Med 82:434-448. Kumar R, Azam S, Sullivan JM, Owen C, Cavener DR, Zhang P, Ron D, Harding HP, Chen JJ, Han A, White BC, Krause GS, DeGracia DJ (2001) Brain ischemia and reperfusion 230 activates the eukaryotic initiation factor 2alpha kinase, PERK. J Neurochem 77:1418-1421. Kwak MK, Wakabayashi N , Kensler TW (2004) Chemoprevention through the Keapl-Nrf2 signaling pathway by phase 2 enzyme inducers. Mutat Res 555:133-148. Kwak MK, Itoh K, Yamamoto M , Sutter TR, Kensler TW (2001a) Role of transcription factor Nrf2 in the induction of hepatic phase 2 and antioxidative enzymes in vivo by the cancer chemoprotective agent, 3H-1, 2-dimethiole-3-thione. Mol Med 7:135-145. Kwak M K , Egner PA, Dolan P M , Ramos-Gomez M , Groopman JD, Itoh K, Yamamoto M , Kensler TW (2001b) Role of phase 2 enzyme induction in chemoprotection by dithiolethiones. Mutat Res 480-481:305-315. Kwong M , Kan Y W , Chan JY (1999) The CNC basic leucine zipper factor, Nr f l , is essential for cell survival in response to oxidative stress-inducing agents. Role for Nrfl in gamma-gcs(l) and gss expression in mouse fibroblasts. J Biol Chem 274:37491-37498. Lakke JP, Teelken A W (1976) Amino acid abnormalities in cerebrospinal fluid of patients with parkinsonism and extrapyramidal disorders. Neurology 26:489-493. Lapchak PA, Song D, Wei J, Zivin JA (2004) Coadministration of NXY-059 and tenecteplase six hours following embolic strokes in rabbits improves clinical rating scores. Exp Neurol 188:279-285. Laxton AW, Sun M C , Shen H, Murphy TH, Honey CR (2001) The antioxidant enzyme quinone reductase is up-regulated in vivo following cerebral ischemia. Neuroreport 12:1045-1048. Lee JM, Zipfel GJ, Choi DW (1999) The changing landscape of ischaemic brain injury mechanisms. Nature 399:A7-14. Lee JM, Moehlenkamp JD, Hanson JM, Johnson JA (2001a) Nrf2-dependent activation of the antioxidant responsive element by tert- butylhydroquinone is independent of oxidative 231 stress in IMR-32 human neuroblastoma cells. Biochem Biophys Res Commun 280:286-292. Lee JM, Hanson JM, Chu WA, Johnson JA (2001b) Phosphatidylinositol 3-kinase, not extracellular signal-regulated kinase, regulates activation of the antioxidant-responsive element in IMR-32 human neuroblastoma cells. J Biol Chem 276:20011-20016. Lee JM, Shih A Y , Murphy TH, Johnson JA (2003a) NF-E2-related factor-2 mediates neuroprotection against mitochondrial complex I inhibitors and increased concentrations of intracellular calcium in primary cortical neurons. Journal of Biological Chemistry 278:37948-37956. Lee JM, Shih A Y , Murphy TH, Johnson JA (2003b) NF-E2-related factor-2 mediates neuroprotection against mitochondrial complex I inhibitors and increased concentrations of intracellular calcium in primary cortical neurons. J Biol Chem 278:37948-37956. Lee JM, Chan K , Kan Y W , Johnson JA (2004) Targeted disruption of Nrf2 causes regenerative immune-mediated hemolytic anemia. Proc Natl Acad Sci U S A 101:9751-9756. Lee JM, Calkins MJ, Chan K, Kan Y W , Johnson JA (2003c) Identification of the NF-E2-related factor-2-dependent genes conferring protection against oxidative stress in primary cortical astrocytes using oligonucleotide microarray analysis. J Biol Chem 278:12029-12038. Lee WT, Yin HS, Shen Y Z (2002) The mechanisms of neuronal death produced by mitochondrial toxin 3-nitropropionic acid: the roles of N-methyl-D-aspartate glutamate receptors and mitochondrial calcium overload. Neuroscience 112:707-716. Leung L, Kwong M , Hou S, Lee C, Chan JY (2003) Deficiency of the Nrfl and Nrf2 transcription factors results in early embryonic lethality and severe oxidative stress. J Biol Chem 278:48021-48029. 232 Levy R, Glozman S, Milman D, Seruty C, Hagay Z, Yavin E, Groner Y (2002) Ischemic reperfusion brain injury in fetal transgenic mice with elevated levels of copper-zinc superoxide dismutase. J Perinat Med 30:158-165. Lewen A, Matz P, Chan PH (2000) Free radical pathways in CNS injury. J Neurotrauma 17:871-890. L i J, Johnson JA (2002) Time-dependent changes in ARE-driven gene expression by use of a noise- filtering process for microarray data. Physiol Genomics 9:137-144. L i J, Lee JM, Johnson JA (2002) Microarray analysis reveals an antioxidant responsive element-driven gene set involved in conferring protection from an oxidative stress- induced apoptosis in IMR-32 cells. J Biol Chem 277:388-394. L i J, Johnson D, Calkins M , Wright L , Svendsen C, Johnson J (2005) Stabilization of Nrf2 by tBHQ confers protection against oxidative stress-induced cell death in human neural stem cells. Toxicol Sci 83:313-328. L i X , Zhang D, Hannink M , Beamer L J (2004a) Crystal structure of the Kelch domain of human Keapl. J Biol Chem 279:54750-54758. L i X , Zhang D, Hannink M , Beamer LJ (2004b) Crystallization and initial crystallographic analysis of the Kelch domain from human Keapl. Acta Crystallogr D Biol Crystallogr 60:2346-2348. L i Y, Jaiswal A K (1992) Regulation of human NAD(P)H:quinone oxidoreductase gene. Role of API binding site contained within human antioxidant response element. J Biol Chem 267:15097-15104. Liebler DC (1993) The role of metabolism in the antioxidant function of vitamin E. Crit Rev Toxicol 23:147-169. Lipton P (1999) Ischemic cell death in brain neurons. Physiol Rev 79:1431-1568. 233 Liu P v M , Hu H, Robison TW, Forman HJ (1996) Increased gamma-glutamylcysteine synthetase and gamma-glutamyl transpeptidase activities enhance resistance of rat lung epithelial L2 cells to quinone toxicity. Am J Respir Cell Mol Biol 14:192-197. Liu S, Liu M , Peterson S, Miyake M , Vallyathan V, Liu K J (2003) Hydroxyl radical formation is greater in striatal core than in penumbra in a rat model of ischemic stroke. J Neurosci Res 71:882-888. Liu X , Luo X , Hu W (1992) Studies on the epidemiology and etiology of moldy sugarcane poisoning in China. Biomed Environ Sci 5:161-177. Liverman CS, Cui L , Yong C, Choudhuri R, Klein R M , Welch K M , Berman N E (2004) Response of the brain to oligemia: gene expression, c-Fos, and Nrf2 localization. Brain Res Mol Brain Res 126:57-66. Lo EH, Dalkara T, Moskowitz M A (2003) Mechanisms, challenges and opportunities in stroke. Nat Rev Neurosci 4:399-415. Long DJ, 2nd, Waikel RL, Wang XJ , Perlaky L , Roop DR, Jaiswal A K (2000) NAD(P)H:quinone oxidoreductase 1 deficiency increases susceptibility to benzo(a)pyrene-induced mouse skin carcinogenesis. Cancer Res 60:5913-5915. Longa EZ, Weinstein PR, Carlson S, Cummins R (1989) Reversible middle cerebral artery occlusion without craniectomy in rats. Stroke 20:84-91. Love S (1999) Oxidative stress in brain ischemia. Brain Pathol 9:119-131. Lucius R, Sievers J (1996) Postnatal retinal ganglion cells in vitro: protection against reactive oxygen species (ROS)-induced axonal degeneration by cocultured astrocytes. Brain Res 743:56-62. Luetjens C M , Bui NT, Sengpiel B, Munstermann G, Poppe M , Krohn AJ , Bauerbach E, Krieglstein J, Prehn JH (2000) Delayed mitochondrial dysfunction in excitotoxic neuron 234 death: cytochrome c release and a secondary increase in superoxide production. J Neurosci 20:5715-5723. Lyden PD, Zivin JA (1993) Hemorrhagic transformation after cerebral ischemia: mechanisms and incidence. Cerebrovasc Brain Metab Rev 5:1-16. Macrae IM, Robinson MJ, Graham DI, Reid JL, McCulloch J (1993) Endothelin-1-induced reductions in cerebral blood flow: dose dependency, time course, and neuropathological consequences. J Cereb Blood Flow Metab 13:276-284. Maeda K, Hata R, Hossmann K A (1998) Differences in the cerebrovascular anatomy of C57black/6 and SV129 mice. Neuroreport 9:1317-1319. Majid A, He Y Y , Gidday JM, Kaplan SS, Gonzales ER, Park TS, Fenstermacher JD, Wei L , Choi DW, Hsu C Y (2000) Differences in vulnerability to permanent focal cerebral ischemia among 3 common mouse strains. Stroke 31:2707-2714. Makar TK, Nedergaard M , Preuss A , Gelbard AS, Perumal AS, Cooper A J (1994) Vitamin E, ascorbate, glutathione, glutathione disulfide, and enzymes of glutathione metabolism in cultures of chick astrocytes and neurons: evidence that astrocytes play an important role in antioxidative processes in the brain. J Neurochem 62:45-53. Mao Y , Yang G Y , Zhou LF, Stern JD, Betz A L (1999) Focal cerebral ischemia in the mouse: description of a model and effects of permanent and temporary occlusion. Brain Res Mol Brain Res 63:366-370. Marini M G , Chan K , Casula L , Kan Y W , Cao A , Moi P (1997) hMAF, a small human transcription factor that heterodimerizes specifically with Nrfl and Nrf2. J Biol Chem 272:16490-16497. Markgraf CG, Kraydieh S, Prado R, Watson BD, Dietrich WD, Ginsberg M D (1993) Comparative histopathologic consequences of photothrombotic occlusion of the distal 235 middle cerebral artery in Sprague-Dawley and Wistar rats. Stroke 24:286-292; discussion 292-283. Martin D, Rojo AI, Salinas M , Diaz R, Gallardo G, Alam J, De Galarreta C M , Cuadrado A (2004) Regulation of heme oxygenase-1 expression through the phosphatidylinositol 3-kinase/Akt pathway and the Nrf2 transcription factor in response to the antioxidant phytochemical carnosol. J Biol Chem 279:8919-8929. Massieu L, Gomez-Roman N , Montiel T (2000) In vivo potentiation of glutamate-mediated neuronal damage after chronic administration of the glycolysis inhibitor iodoacetate. Exp Neurol 165:257-267. Mattson MP (2000) Apoptosis in neurodegenerative disorders. Nat Rev Mol Cell Biol 1:120-129. Mattson MP, Zhu H, Y u J, Kindy MS (2000) Presenilin-1 mutation increases neuronal vulnerability to focal ischemia in vivo and to hypoxia and glucose deprivation in cell culture: involvement of perturbed calcium homeostasis. J Neurosci 20:1358-1364. Mayo NE, Wood-Dauphinee S, Ahmed S, Gordon C, Higgins J, McEwen S, Salbach N (1999) Disablement following stroke. Disabil Rehabil 21:258-268. McAdoo DJ, Xu GY, Robak G, Hughes M G (1999) Changes in amino acid concentrations over time and space around an impact injury and their diffusion through the rat spinal cord. Exp Neurol 159:538-544. McBean GJ (2002) Cerebral cystine uptake: a tale of two transporters. Trends Pharmacol Sci 23:299-302. McMahon M , Itoh K, Yamamoto M , Hayes JD (2003) Keapl-dependent proteasomal degradation of transcription factor Nrf2 contributes to the negative regulation of antioxidant response element-driven gene expression. J Biol Chem 278:21592-21600. McMahon M , Itoh K, Yamamoto M , Chanas SA, Henderson CJ, McLellan LI, Wolf CR, Cavin C, Hayes JD (2001) The Cap'n'Collar basic leucine zipper transcription factor Nrf2 (NF-236 E2 p45-related factor 2) controls both constitutive and inducible expression of intestinal detoxification and glutathione biosynthetic enzymes. Cancer Res 61.3299-3307. Mc Walter GK, Higgins L G , McLellan LI, Henderson CJ, Song L , Thornalley PJ, Itoh K, Yamamoto M , Hayes JD (2004) Transcription factor Nrf2 is essential for induction of NAD(P)H:quinone oxidoreductase 1, glutathione S-transferases, and glutamate cysteine ligase by broccoli seeds and isothiocyanates. J Nutr 134:3499S-3506S. Meden P, Overgaard K, Pedersen H, Boysen G (1994) Effect of hypothermia and delayed thrombolysis in a rat embolic stroke model. Acta Neurol Scand 90:91-98. Meister A (1992) On the antioxidant effects of ascorbic acid and glutathione. Biochem Pharmacol 44:1905-1915. Meister A, Anderson M E (1983) Glutathione. Annu Rev Biochem 52:711-760. Memezawa H, Smith M L , Siesjo B K (1992a) Penumbral tissues salvaged by reperfusion following middle cerebral artery occlusion in rats. Stroke 23:552-559. Memezawa H, Minamisawa H, Smith M L , Siesjo B K (1992b) Ischemic penumbra in a model of reversible middle cerebral artery occlusion in the rat. Exp Brain Res 89:67-78. Mena M A , Casarejos MJ , Carazo A, Paino CL, Garcia de Yebenes J (1996) Glia conditioned medium protects fetal rat midbrain neurones in culture from L-DOPA toxicity. Neuroreport 7:441-445. Micu I, Jiang Q, Coderre E, Ridsdale A , Zhang L , Woulfe J, Y in X , Trapp BD, McRory JE, Rehak R, Zamponi GW, Wang W, Stys PK (2006) N M D A receptors mediate calcium accumulation in myelin during chemical ischaemia. Nature 439:988-992. Minamisawa H, Nordstrom C H , Smith M L , Siesjo B K (1990) The influence of mild body and brain hypothermia on ischemic brain damage. J Cereb Blood Flow Metab 10:365-374. Miura K, Ishii T, Sugita Y , Bannai S (1992) Cystine uptake and glutathione level in endothelial cells exposed to oxidative stress. Am J Physiol 262:C50-58. 237 Mizui T, Kinouchi H , Chan PH (1992) Depletion of brain glutathione by buthionine sulfoximine enhances cerebral ischemic injury in rats. Am J Physiol 262:H313-317. Moehlenkamp JD, Johnson JA (1999) Activation of antioxidant/electrophile-responsive elements in IMR-32 human neuroblastoma cells. Arch Biochem Biophys 363:98-106. Mohamed A A , Avila JG, Schultke E, Kamencic H, Skihar V , Obayan A, Juurlink B H (2002) Amelioration of experimental allergic encephalitis (EAE) through phase 2 enzyme induction. Biomed Sci Instrum 38:9-13. Moi P, Chan K, Asunis I, Cao A, Kan Y W (1994) Isolation of NF-E2-related factor 2 (Nrf2), a NF-E2-like basic leucine zipper transcriptional activator that binds to the tandem NF-E2/AP1 repeat of the beta-globin locus control region. Proc Natl Acad Sci U S A 91:9926-9930. Monks TJ, Hanzlik RP, Cohen G M , Ross D, Graham D G (1992) Quinone chemistry and toxicity. Toxicol Appl Pharmacol 112:2-16. Moore R, Laboratory Centre for Disease Control (Canada). Environmental Risk Assessment and Case Surveillance Division., Canadian Public Health Association. (1997) Economic burden of illness in Canada, 1993. [Ottawa]: Environmental Risk Assessment and Case Surveillance Division, Laboratory Centre for Disease Control. Moreland S, McMullen D M , Delaney CL, Lee V G , Hunt JT (1992) Venous smooth muscle contains vasoconstrictor ETB-like receptors. Biochem Biophys Res Commun 184:100-106. Morimitsu Y , Nakagawa Y, Hayashi K, Fujii H, Kumagai T, Nakamura Y , Osawa T, Horio F, Itoh K, Iida K, Yamamoto M , Uchida K (2002) A sulforaphane analogue that potently activates the Nrf2-dependent detoxification pathway. J Biol Chem 277:3456-3463. 238 Morito N , Yoh K, Itoh K, Hirayama A, Koyama A, Yamamoto M , Takahashi S (2003) Nrf2 regulates the sensitivity of death receptor signals by affecting intracellular glutathione levels. Oncogene 22:9275-9281. Motohashi H, Katsuoka F, Engel JD, Yamamoto M (2004) Small Maf proteins serve as transcriptional cofactors for keratinocyte differentiation in the Keap 1 -Nrf2 regulatory pathway. Proc Natl Acad Sci U S A 101:6379-6384. Murakami K, Kondo T, Epstein CJ, Chan PH (1997) Overexpression of CuZn-superoxide dismutase reduces hippocampal injury after global ischemia in transgenic mice. Stroke 28:1797-1804. Murphy TH, Baraban JM (1990) Glutamate toxicity in immature cortical neurons precedes development of glutamate receptor currents. Brain Res Dev Brain Res 57:146-150. Murphy TH, Schnaar RL, Coyle JT (1990) Immature cortical neurons are uniquely sensitive to glutamate toxicity by inhibition of cystine uptake. Faseb J 4:1624-1633. Murphy TH, De Long MJ, Coyle JT (1991) Enhanced NAD(P)H: quinone reductase activity prevents glutamate toxicity produced by oxidative stress. J Neurochem 56:990-995. Murphy TH, So AP, Vincent SR (1998) Histochemical detection of quinone reductase activity in situ using L Y 83583 reduction and oxidation. J Neurochem 70:2156-2164. Murphy TH, Miyamoto M , Sastre A, Schnaar RL, Coyle JT (1989) Glutamate toxicity in a neuronal cell line involves inhibition of cystine transport leading to oxidative stress. Neuron 2:1547-1558. Murphy TH, Y u J, Ng R, Johnson DA, Shen H, Honey CR, Johnson JA (2001) Preferential expression of antioxidant response element mediated gene expression in astrocytes. J Neurochem 76:1670-1678. 239 Nakaso K, Yano H, Fukuhara Y , Takeshima T, Wada-Isoe K, Nakashima K (2003) PI3K is a key-molecule in the Nrf2-mediated regulation of antioxidative proteins by hemin in human neuroblastoma cells. FEBS Lett 546:181-184. Nath R, Probert A, Jr., McGinnis K M , Wang K K (1998) Evidence for activation ofcaspase-3-like protease in excitotoxin- and hypoxia/hypoglycemia-injured neurons. J Neurochem 71:186-195. National Toxicology Program (1997) NTP Toxicology and Carcinogenesis Studies of t-Butylhydroquinone (CAS No. 1948-33-0) in F344/N Rats and B6C3F(1) Mice (Feed Studies). Natl Toxicol Program Tech Rep Ser 459:1-326. Nedergaard M , Gjedde A, Diemer N H (1986) Focal ischemia of the rat brain: autoradiographic determination of cerebral glucose utilization, glucose content, and blood flow. J Cereb Blood Flow Metab 6:414-424. Nelson K C , Carlson JL, Newman M L , Sternberg P, Jr., Jones DP, Kavanagh TJ, Diaz D, Cai J, Wu M (1999) Effect of dietary inducer dimethylfumarate on glutathione in cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 40:1927-1935. Newell DW, Barth A, Papermaster V , Malouf A T (1995) Glutamate and non-glutamate receptor mediated toxicity caused by oxygen and glucose deprivation in organotypic hippocampal cultures. J Neurosci 15:7702-7711. Ney PA, Andrews NC, Jane SM, Safer B, Purucker M E , Weremowicz S, Morton CC, Goff SC, Orkin SH, Nienhuis A W (1993) Purification of the human NF-E2 complex: cDNA cloning of the hematopoietic cell-specific subunit and evidence for an associated partner. Mol Cell Biol 13:5604-5612. Nguyen T, Rushmore TH, Pickett CB (1994) Transcriptional regulation of a rat liver glutathione S-transferase Ya subunit gene. Analysis of the antioxidant response element and its 240 activation by the phorbol ester 12-0-tetradecanoylphorbol-13-acetate. J Biol Chem 269:13656-13662. Nguyen T, Huang HC, Pickett CB (2000) Transcriptional regulation of the antioxidant response element. Activation by Nrf2 and repression by MafK. J Biol Chem 275:15466-15473. Nguyen T, Sherratt PJ, Pickett CB (2003a) Regulatory mechanisms controlling gene expression mediated by the antioxidant response element. Annu Rev Pharmacol Toxicol 43:233-260. Nguyen T, Sherratt PJ, Huang HC, Yang CS, Pickett CB (2002) Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element: degradation of Nrf2 by the 26S proteasome. J Biol Chem 22:22. Nguyen T, Sherratt PJ, Huang HC, Yang CS, Pickett CB (2003b) Increased protein stability as a mechanism that enhances Nrf2-mediated transcriptional activation of the antioxidant response element. Degradation of Nrf2 by the 26 S proteasome. J Biol Chem 278:4536-4541. Nicholls DG, Sihra TS, Sanchez-Prieto J (1987) Calcium-dependent and -independent release of glutamate from synaptosomes monitored by continuous fluorometry. J Neurochem 49:50-57. Nichols TC (2004) NF-kappaB and reperfusion injury. Drug News Perspect 17:99-104. Novelli A , Reilly JA, Lysko PG, Henneberry RC (1988) Glutamate becomes neurotoxic via the N-methyl-D-aspartate receptor when intracellular energy levels are reduced. Brain Res 451:205-212. Obeidat AS, Andrew RD (1998) Spreading depression determines acute cellular damage in the hippocampal slice during oxygen/glucose deprivation. Eur J Neurosci 10:3451-3461. Okawa H, Motohashi H , Kobayashi A , Aburatani H, Kensler TW, Yamamoto M (2006) Hepatocyte-specific deletion of the keapl gene activates Nrf2 and confers potent resistance against acute drug toxicity. Biochem Biophys Res Commun 339:79-88. 241 Olney JW, Price MT, Samson L, Labruyere J (1986) The role of specific ions in glutamate neurotoxicity. Neurosci Lett 65:65-71. Olsen C, Rustad A, Fonnum F, Paulsen RE, Hassel B (1999) 3-Nitropropionic acid: an astrocyte-sparing neurotoxin in vitro. Brain Res 850:144-149. Osuga H, Osuga S, Wang F, Fetni R, Hogan MJ, Slack RS, Hakim A M , Ikeda JE, Park DS (2000) Cyclin-dependent kinases as a therapeutic target for stroke. Proc Natl Acad Sci U S A 97:10254-10259. Ovbiagele B, Kidwell CS, Starkman S, Saver JL (2003) Neuroprotective agents for the treatment of acute ischemic stroke. Curr Neurol Neurosci Rep 3:9-20. Overgaard K , Sereghy T, Boysen G, Pedersen H, Hoyer S, Diemer N H (1992) A rat model of reproducible cerebral infarction using thrombotic blood clot emboli. J Cereb Blood Flow Metab 12:484-490. Owuor ED, Kong A N (2002) Antioxidants and oxidants regulated signal transduction pathways. Biochem Pharmacol 64:765-770. Oyake T, Itoh K, Motohashi H, Hayashi N , Hoshino H , Nishizawa M , Yamamoto M , Igarashi K (1996) Bach proteins belong to a novel family of BTB-basic leucine zipper transcription factors that interact with MafK and regulate transcription through the NF-E2 site. Mol Cell Biol 16:6083-6095. Pahl HL, Baeuerle PA (1994) Oxygen and the control of gene expression. Bioessays 16:497-502. Pang Z, Geddes JW (1997) Mechanisms of cell death induced by the mitochondrial toxin 3-nitropropionic acid: acute excitotoxic necrosis and delayed apoptosis. J Neurosci 17:3064-3073. Patel SC, Mody A (1999) Cerebral hemorrhagic complications of thrombolytic therapy. Prog Cardiovasc Dis 42:217-233. 242 Paxinos G, Watson C (1986) The rat brain in stereotaxic coordinates, 2nd Edition. Sydney ; Orlando: Academic Press. Paxinos G, Franklin K B J (2001) The mouse brain in stereotaxic coordinates, 2nd Edition. San Diego, Calif. London: Academic. Pellerin L, Magistretti PJ (1994) Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc Natl Acad Sci U S A 91:10625-10629. Pennington RJ (1961) Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem J 80:649-654. Pereira C M , Oliveira CR (1997) Glutamate toxicity on a PC 12 cell line involves glutathione (GSH) depletion and oxidative stress. Free Radic Biol Med 23:637-647. Petzer JP, Navamal M , Johnson JK, Kwak M K , Kensler TW, Fishbein JC (2003) Phase 2 enzyme induction by the major metabolite of oltipraz. Chem Res Toxicol 16:1463-1469. Piper H M , Balser C, Ladilov Y V , Schafer M , Siegmund B, Ruiz-Meana M , Garcia-Dorado D (1996) The role of Na+/H+ exchange in ischemia-reperfusion. Basic Res Cardiol 91:191-202. Plesnila N , Zinkel S, Le DA, Amin-Hanjani S, Wu Y , Qiu J, Chiarugi A , Thomas SS, Kohane DS, Korsmeyer SJ, Moskowitz M A (2001) BID mediates neuronal cell death after oxygen/ glucose deprivation and focal cerebral ischemia. Proc Natl Acad Sci U S A 98:15318-15323. Prass K, Dirnagl U (1998) Glutamate antagonists in therapy of stroke. Restor Neurol Neurosci 13:3-10. Prestera T, Zhang Y , Spencer SR, Wilczak CA, Talalay P (1993) The electrophile counterattack response: protection against neoplasia and toxicity. Adv Enzyme Regul 33:281-296. 243 Prestera T, Talalay P, Alam J, Ahn YI, Lee PJ, Choi A M (1995) Parallel induction of heme oxygenase-1 and chemoprotective phase 2 enzymes by electrophiles and antioxidants: regulation by upstream antioxidant-responsive elements (ARE). Mol Med 1:827-837. Primiano T, Sutter TR, Kensler TW (1997) Antioxidant-inducible genes. Adv Pharmacol 38:293-328. Prochaska HJ, Talalay P (1988) Regulatory mechanisms of mono functional and bifunctional anticarcinogenic enzyme inducers in murine liver. Cancer Res 48:4776-4782. Radjendirane V , Joseph P, Lee Y H , Kimura S, Klein-Szanto AJ , Gonzalez FJ, Jaiswal A K (1998) Disruption of the DT diaphorase (NQOl) gene in mice leads to increased menadione toxicity. J Biol Chem 273:7382-7389. Rahimtula A D , Jernstrom B, Dock L, Moldeus P (1982) Effects of dietary and in vitro 2(3)-t-butyl-4-hydroxy-anisole and other phenols on hepatic enzyme activities in mice. Br J Cancer 45:935-944. Ramos-Gomez M Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice.[see comment]. Ramos-Gomez M , Kwak M K , Dolan P M , Itoh K, Yamamoto M , Talalay P, Kensler TW (2001) Sensitivity to carcinogenesis is increased and chemoprotective efficacy of enzyme inducers is lost in nrf2 transcription factor-deficient mice. Proc Natl Acad Sci U S A 98:3410-3415. Rangasamy T, Cho CY, Thimmulappa RK, Zhen L, Srisuma SS, Kensler TW, Yamamoto M , Petrache I, Tuder R M , Biswal S (2004) Genetic ablation of Nrf2 enhances susceptibility to cigarette smoke-induced emphysema in mice. J Clin Invest 114:1248-1259. Raps SP, Lai JC, Hertz L , Cooper A J (1989) Glutathione is present in high concentrations in cultured astrocytes but not in cultured neurons. Brain Res 493:398-401. 244 Rashidian J, lyirhiaro G, Aleyasin H, Rios M , Vincent I, Callaghan S, Bland RJ, Slack RS, During MJ, Park DS (2005) Multiple cyclin-dependent kinases signals are critical mediators of ischemia/hypoxic neuronal death in vitro and in vivo. Proc Natl Acad Sci U S A 102:14080-14085. Ratan RR, Murphy TH, Baraban JM (1994) Oxidative stress induces apoptosis in embryonic cortical neurons. J Neurochem 62:376-379. Reglodi D, Tamas A , Lengvari I (2003) Examination of sensorimotor performance following middle cerebral artery occlusion in rats. Brain Res Bull 59:459-466. Reynolds DS, Carter RJ, Morton A J (1998) Dopamine modulates the susceptibility of striatal neurons to 3-nitropropionic acid in the rat model of Huntington's disease. J Neurosci 18:10116-10127. Reynolds IJ, Hastings TG (1995) Glutamate induces the production of reactive oxygen species in cultured forebrain neurons following N M D A receptor activation. J Neurosci 15:3318-3327. Robertson GS, Crocker SJ, Nicholson DW, Schulz JB (2000) Neuroprotection by the inhibition of apoptosis. Brain Pathol 10:283-292. Rossi DJ, Oshima T, Attwell D (2000) Glutamate release in severe brain ischaemia is mainly by reversed uptake. Nature 403:316-321. Rothman S (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death. J Neurosci 4:1884-1891. Rothman SM, Olney JW (1986) Glutamate and the pathophysiology of hypoxic—ischemic brain damage. Ann Neurol 19:105-111. Rothman SM, Olney JW (1995) Excitotoxicity and the N M D A receptor-still lethal after eight years. Trends Neurosci 18:57-58. 245 Rushmore TH, Pickett CB (1990) Transcriptional regulation of the rat glutathione S-transferase Ya subunit gene. Characterization of a xenobiotic-responsive element controlling inducible expression by phenolic antioxidants. J Biol Chem 265:14648-14653. Rushmore TH, Kong A N (2002) Pharmacogenomics, regulation and signaling pathways of phase I and II drug metabolizing enzymes. Curr Drug Metab 3:481-490. Rushmore TH, Morton MR, Pickett CB (1991) The antioxidant responsive element. Activation by oxidative stress and identification of the D N A consensus sequence required for functional activity. J Biol Chem 266:11632-11639. Safar P (1993) Cerebral resuscitation after cardiac arrest: research initiatives and future directions. Ann Emerg Med 22:324-349. Sagara J, Miura K, Bannai S (1993a) Cystine uptake and glutathione level in fetal brain cells in primary culture and in suspension. J Neurochem 61:1667-1671. Sagara J, Makino N , Bannai S (1996) Glutathione efflux from cultured astrocytes. J Neurochem 66:1876-1881. Sagara Jl , Miura K , Bannai S (1993b) Maintenance of neuronal glutathione by glial cells. J Neurochem 61:1672-1676. Saito A, Hayashi T, Okuno S, Ferrand-Drake M , Chan PH (2003) Overexpression of copper/zinc superoxide dismutase in transgenic mice protects against neuronal cell death after transient focal ischemia by blocking activation of the Bad cell death signaling pathway. J Neurosci 23:1710-1718. Salom JB, Torregrosa G, Alborch E (1995) Endothelins and the cerebral circulation. Cerebrovasc Brain Metab Rev 7:131-152. Samuels M A , Mayer Media. (1996) Functional neuroanatomy. In. Wobura, M A : Butterworth-Heinemann,. 246 Sasaki H, Sato H, Kuriyama-Matsumura K, Sato K, Maebara K, Wang H, Tamba M , Itoh K, Yamamoto M , Bannai S (2002) Electrophile response element-mediated induction of the cystine/glutamate exchange transporter gene expression. J Biol Chem 13:13, Sato H, Tamba M , Ishii T, Bannai S (1999) Cloning and expression of a plasma membrane cystine/glutamate exchange transporter composed of two distinct proteins. J Biol Chem 274:11455-11458. Sato H, Tamba M , Okuno S, Sato K, Keino-Masu K, Masu M , Bannai S (2002) Distribution of Cystine/Glutamate Exchange Transporter, System xc-, in the Mouse Brain. J Neurosci 22:8028-8033. Sato S, Gobbel GT, Honkaniemi J, L i Y , Kondo T, Murakami K, Sato M , Copin JC, Chan PH (1997) Apoptosis in the striatum of rats following intraperitoneal injection of 3-nitropropionic acid. Brain Res 745:343-347. Sattler R, Xiong Z, Lu W Y , Hafner M , MacDonald JF, Tymianski M (1999) Specific coupling of N M D A receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science 284:1845-1848. Schaller B, Graf R (2004) Cerebral ischemia and reperfusion: the pathophysiologic concept as a basis for clinical therapy. J Cereb Blood Flow Metab 24:351-371. Schmid-Elsaesser R, Zausinger S, Hungerhuber E, Baethmann A , Reulen HJ (1999) Neuroprotective effects of combination therapy with tirilazad and magnesium in rats subjected to reversible focal cerebral ischemia. Neurosurgery 44:163-171; discussion 171-162. Schmued L C , Albertson C, Slikker W, Jr. (1997) Fluoro-Jade: a novel fluorochrome for the sensitive and reliable histochemical localization of neuronal degeneration. Brain Research 751:37-46. 247 Schubert D, Piasecki D (2001) Oxidative glutamate toxicity can be a component of the excitotoxicity cascade. J Neurosci 21:7455-7462. Schulz JB, Henshaw DR, MacGarvey U , Beal MF (1996) Involvement of oxidative stress in 3-nitropropionic acid neurotoxicity. Neurochem Int 29:167-171. Schulz JB, Matthews RT, Jenkins BG, Ferrante RJ, Siwek D, Henshaw DR, Cipolloni PB, Mecocci P, Kowall NW, Rosen BR, et al. (1995) Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo. J Neurosci 15:8419-8429. Sciamanna M A , Zinkel J, Fabi A Y , Lee CP (1992) Ischemic injury to rat forebrain mitochondria and cellular calcium homeostasis. Biochim Biophys Acta 1134:223-232. Segura-Aguilar J, Baez S, Widersten M , Welch CJ, Mannervik B (1997) Human class Mu glutathione transferases, in particular isoenzyme M2-2, catalyze detoxication of the dopamine metabolite aminochrome. J Biol Chem 272:5727-5731. Sekhar KR, Yan X X , Freeman M L (2002) Nrf2 degradation by the ubiquitin proteasome pathway is inhibited by KIAA0132, the human homolog to INrf2. Oncogene 21:6829-6834. Sen CK (1998) Redox signaling and the emerging therapeutic potential of thiol antioxidants. Biochem Pharmacol 55:1747-1758. Sharkey J, Ritchie IM, Kelly PA (1993) Perivascular microapplication of endothelin-1: a new model of focal cerebral ischaemia in the rat. J Cereb Blood Flow Metab 13:865-871. Sharp FR, Bernaudin M (2004) FLTFl and oxygen sensing in the brain. Nat Rev Neurosci 5:437-448. Shen G, Hebbar V , Nair S, Xu C, L i W, Lin W, Keum YS, Han J, Gallo M A , Kong A N (2004) Regulation of Nrf2 transactivation domain activity. The differential effects of mitogen-activated protein kinase cascades and synergistic stimulatory effect of Raf and CREB-binding protein. J Biol Chem 279:23052-23060. Shih A Y , Murphy T H (2001) xCt cystine transporter expression in HEK293 cells: pharmacology and localization. Biochem Biophys Res Commun 282:1132-1137. Shih A Y , L i P, Murphy TH (2005a) A small-molecule-inducible Nrf2-mediated antioxidant response provides effective prophylaxis against cerebral ischemia in vivo. J Neurosci 25:10321-10335. Shih A Y , Imbeault S, Barakauskas V , Erb H, Jiang L, L i P, Murphy TH (2005b) Induction of the Nrf2-driven Antioxidant Response Confers Neuroprotection during Mitochondrial Stress in Vivo. J Biol Chem 280:22925-22936. Shih A Y , Johnson DA, Wong G, Kraft A D , Jiang L, Erb H, Johnson JA, Murphy TH (2003) Coordinate regulation of glutathione biosynthesis and release by Nrf2-expressing glia potently protects neurons from oxidative stress. Journal of Neuroscience 23:3394-3406. Shivdasani RA, Rosenblatt MF, Zucker-Franklin D, Jackson CW, Hunt P, Saris CJ, Orkin SH (1995) Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development. Cell 81:695-704. Siegel D, Bolton E M , Burr JA, Liebler DC, Ross D (1997) The reduction of alpha-tocopherolquinone by human NAD(P)H: quinone oxidoreductase: the role of alpha-tocopherolhydroquinone as a cellular antioxidant. Mol Pharmacol 52:300-305. Siegel GJ (1981) Basic neurochemistry, 3rd — Edition. Boston: Little Brown. Siesjo BK, Bengtsson F (1989) Calcium fluxes, calcium antagonists, and calcium-related pathology in brain ischemia, hypoglycemia, and spreading depression: a unifying hypothesis. J Cereb Blood Flow Metab 9:127-140. Siesjo BK, Agardh CD, Bengtsson F (1989) Free radicals and brain damage. Cerebrovasc Brain Metab Rev 1:165-211. Simon RP, Swan JH, Griffiths T, Meldrum BS (1984) Blockade of N-mefhyl-D-aspartate receptors may protect against ischemic damage in the brain. Science 226:850-852. 249 Sims NR, Zaidart E (1995) Biochemical changes associated with selective neuronal death following short-term cerebral ischaemia. Int J Biochem Cell Biol 27:531-550. Slivka A, Mytilineou C, Cohen G (1987) Histochemical evaluation of glutathione in brain. Brain Res 409:275-284. Smith TS, Bennett JP, Jr. (1997) Mitochondrial toxins in models of neurodegenerative diseases. I: In vivo brain hydroxyl radical production during systemic MPTP treatment or following microdialysis infusion of methylpyridinium or azide ions. Brain Res 765:183-188. Stein TD, Johnson JA (2002) Lack of neurodegeneration in transgenic mice overexpressing mutant amyloid precursor protein is associated with increased levels of transthyretin and the activation of cell survival pathways. J Neurosci 22:7380-7388. Stenman E, Malmsjo M , Uddman E, Gido G, Wieloch T, Edvinsson L (2002) Cerebral ischemia upregulates vascular endothelin ET(B) receptors in rat. Stroke 33:2311-2316. Stewart D, Killeen E, Naquin R, Alam S, Alam J (2002) Degradation of transcription factor Nrf2 via the ubiquitin-proteosome pathway and stabilization by cadmium. J Biol Chem 18:18. Stoll G, Jander S, Schroeter M (2002) Detrimental and beneficial effects of injury-induced inflammation and cytokine expression in the nervous system. Adv Exp Med Biol 513:87-113. Strazielle N , Khuth ST, Ghersi-Egea JF (2004) Detoxification systems, passive and specific transport for drugs at the blood-CSF barrier in normal and pathological situations. Adv Drug Deliv Rev 56:1717-1740. Stys PK (2004) White matter injury mechanisms. Curr Mol Med 4:113-130. Su JY, Duffy S, Murphy T H (1999) Reduction of H202-evoked, intracellular calcium increases in the rat N18-RE-105 neuronal cell line by pretreatment with an electrophilic antioxidant inducer. Neurosci Lett 273:109-112. 250 Sugimori H , Yao H, Ooboshi H , Ibayashi S, lida M (2004) Krypton laser-induced photothrombotic distal middle cerebral artery occlusion without craniectomy in mice. Brain Res Brain Res Protoc 13:189-196. Suh JH, Shenvi SV, Dixon B M , Liu H, Jaiswal A K , Liu R M , Hagen T M (2004) Decline in transcriptional activity of Nrf2 causes age-related loss of glutathione synthesis, which is reversible with lipoic acid. Proc Natl Acad Sci U S A 101:3381-3386. Sun J, Hoshino H, Takaku K, Nakajima O, Muto A, Suzuki H, Tashiro S, Takahashi S, Shibahara S, Alam J, Taketo M M , Yamamoto M , Igarashi K (2002) Hemoprotein Bachl regulates enhancer availability of heme oxygenase-1 gene. Embo J 21:5216-5224. Sun X , Erb H, Murphy TH (2005) Coordinate regulation of glutathione metabolism in astrocytes by Nrf2. Biochem Biophys Res Commun 326:371-377. Sun X, Shih A Y , Johannssen HC, Erb H, L i P, Murphy T H (2006) Two-photon imaging of glutathione levels in intact brain indicates enhanced redox buffering in developing neurons and cells at the cerebrospinal fluid and blood-brain interface. J Biol Chem. Sutherland GR, Lesiuk H, Hazendonk P, Peeling J, Buist R, Kozlowski P, Jazinski A , Saunders JK (1992) Magnetic resonance imaging and 3IP magnetic resonance spectroscopy study of the effect of temperature on ischemic brain injury. Can J Neurol Sci 19:317-325. Suzuki H, Tashiro S, Hira S, Sun J, Yamazaki C, Zenke Y , Ikeda-Saito M , Yoshida M , Igarashi K (2004) Heme regulates gene expression by triggering Crml-dependent nuclear export of Bachl. Embo J 23:2544-2553. Takei N , Endo Y (1994) Ca2+ ionophore-induced apoptosis on cultured embryonic rat cortical neurons. Brain Res 652:65-70. Talalay P (1989) Mechanisms of induction of enzymes that protect against chemical carcinogenesis. Adv Enzyme Regul 28:237-250. 251 Talalay P (2000) Chemoprotection against cancer by induction of phase 2 enzymes. Biofactors 12:5-11. Talalay P, Zhang Y (1996) Chemoprotection against cancer by isothiocyanates and glucosinolates. Biochem Soc Trans 24:806-810. Talalay P, Fahey JW (2001) Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism. JNutr 131:3027S-3033S. Talalay P, De Long MJ, Prochaska HJ (1988) Identification of a common chemical signal regulating the induction of enzymes that protect against chemical carcinogenesis. Proc Natl Acad Sci U S A 85:8261-8265. Talalay P, Fahey JW, Holtzclaw WD, Prestera T, Zhang Y (1995) Chemoprotection against cancer by phase 2 enzyme induction. Toxicol Lett 82-83:173-179. Tamaoki T, Nomoto H, Takahashi I, Kato Y , Morimoto M , Tomita F (1986) Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun 135:397-402. Tamura A, Graham DI, McCulloch J, Teasdale G M (1981) Focal cerebral ischaemia in the rat: 2. Regional cerebral blood flow determined by [14C]iodoantipyrine autoradiography following middle cerebral artery occlusion. J Cereb Blood Flow Metab 1:61-69. Tan S, Sagara Y , Liu Y , Maher P, Schubert D (1998) The regulation of reactive oxygen species production during programmed cell death. J Cell Biol 141:1423-1432. Tanaka J, Toku K, Zhang B, Ishihara K, Sakanaka M , Maeda N (1999) Astrocytes prevent neuronal death induced by reactive oxygen and nitrogen species. Glia 28:85-96. Tang C M , Dichter M , Morad M (1990) Modulation of the N-methyl-D-aspartate channel by extracellular H+. Proc Natl Acad Sci U S A 87:6445-6449. 252 Tauskela JS, Hewitt K, Kang LP, Comas T, Gendron T, Hakim A, Hogan M , Durkin J, Morley P (2000) Evaluation of glutathione-sensitive fluorescent dyes in cortical culture. Glia 30:329-341. Thakker DR, Natt F, Husken D, Maier R, Muller M , van der Putten H, Hoyer D, Cryan JF (2004) Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference. Proc Natl Acad Sci U S A 101:17270-17275. Thimmulappa RK, Mai K H , Srisuma S, Kensler TW, Yamamoto M , Biswal S (2002) Identification of Nrf2-regulated genes induced by the chemopreventive agent sulforaphane by oligonucleotide microarray. Cancer Res 62:5196-5203. Tietze F (1969) Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione: applications to mammalian blood and other tissues. Anal Biochem 27:502-522. Touzani O, Galbraith S, Siegl P, McCulloch J (1997) Endothelin-B receptors in cerebral resistance arterioles and their functional significance after focal cerebral ischemia in cats. J Cereb Blood Flow Metab 17:1157-1165. Travis J (1994) Glia: the brain's other cells. Science 266:970-972. Traynelis SF, Cull-Candy SG (1990) Proton inhibition of N-methyl-D-aspartate receptors in cerebellar neurons. Nature 345:347-350. Trieu V N , Dong Y, Zheng Y, Uckun F M (1999) In vivo antioxidant activity of genistein in a murine model of singlet oxygen-induced cerebral stroke. Radiat Res 152:508-516. Turrens JF (1997) Superoxide production by the mitochondrial respiratory chain. Biosci Rep 17:3-8. Turski L, Huth A , Sheardown M , McDonald F, Neuhaus R, Schneider HH, Dirnagl U , Wiegand F, Jacobsen P, Ottow E (1998) ZK200775: a phosphonate quinoxalinedione A M P A 253 antagonist for neuroprotection in stroke and trauma. Proc Natl Acad Sci U S A 95:10960-10965. Ueki A, Rosen L, Andbjer B, Agnati LF, Hallstrom A, Goiny M , Tanganelli S, Ungerstedt U , Fuxe K (1993) Evidence for a preventive action of the vigilance-promoting drug modafinil against striatal ischemic injury induced by endothelin-1 in the rat. Exp Brain Res 96:89-99. Umemura K, Shimakura A, Nakashima M (1997) Neuroprotective effect of a novel A M P A receptor antagonist, YM90K, in rat focal cerebral ischaemia. Brain Res 773:61-65. Uto A , Dux E, Kusumoto M , Hossmann K A (1995) Delayed neuronal death after brief histotoxic hypoxia in vitro. J Neurochem 64:2185-2192. Valtysson J, Hillered L , Andine P, Hagberg H, Persson L (1994) Neuropathological endpoints in experimental stroke pharmacotherapy: the importance of both early and late evaluation. Acta Neurochir (Wien) 129:58-63. van Lookeren Campagne M , Thibodeaux H, van Bruggen N , Cairns B, Gerlai R, Palmer JT, Williams SP, Lowe D G (1999) Evidence for a protective role of metallothionein-1 in focal cerebral ischemia. Proc Natl Acad Sci U S A 96:12870-12875. van Ommen B, Koster A , Verhagen H, van Bladeren PJ (1992) The glutathione conjugates of tert-butyl hydroquinone as potent redox cycling agents and possible reactive agents underlying the toxicity of butylated hydroxyanisole. Biochem Biophys Res Commun 189:309-314. Vargas MR, Pehar M , Cassina P, Martinez-Palma L, Thompson JA, Beckman JS, Barbeito L (2005) Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival. J Biol Chem 280:25571-25579. 254 Vartiainen N , Goldsteins G, Keksa-Goldsteine V , Chan PH, Koistinaho J (2003) Aspirin inhibits p44/42 mitogen-activated protein kinase and is protective against hypoxia/reoxygenation neuronal damage. Stroke 34:752-757. Venugopal R, Jaiswal A K (1996) Nrfl and Nrf2 positively and c-Fos and Fral negatively regulate the human antioxidant response element-mediated expression of NAD(P)H:quinone oxidoreductasel gene. Proc Natl Acad Sci U S A 93:14960-14965. Venugopal R, Jaiswal A K (1998a) Nrf2 and Nrf l in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17:3145-3156. Venugopal R, Jaiswal A K (1998b) Nrf2 and Nrfl in association with Jun proteins regulate antioxidant response element-mediated expression and coordinated induction of genes encoding detoxifying enzymes. Oncogene 17:3145-3156. Vincent SR (1994) Nitric oxide: a radical neurotransmitter in the central nervous system. Prog Neurobiol 42:129-160. Virag L, Szabo E, Gergely P, Szabo C (2003) Peroxynitrite-induced cytotoxicity: mechanism and opportunities