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Overexpression of NF-E2 related factor 2 via viral-mediated gene transfer in vivo. Imbeault, Sophie 2005

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OVEREXPRESSION OF NF-E2 RELATED FACTOR 2 VIA VIRAL-MEDIATED GENE TRANSFER IN VIVO by SOPHIE IMBEAULT B.Sc, The University of Ottawa, 2002 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Neuroscience) THE UNIVERSITY OF BRITISH COLUMBIA April 2005 © Sophie Imbeault, 2005 Abstract The transcription factor NF-E2 related factor 2 (Nrf2) is responsible for the upregulation of the cellular anti-oxidant defence system. Upon stimulation by free radicals or reactive oxygen species (ROS), the cytoplasmic-bound Nrf2 translocates to the nucleus and initiates transcription of genes containing anti-oxidant response elements (AREs) within their promoter region. Many ARE-containing genes contribute to the maintenance of redox homeostasis within the cell thus promoting cell survival. To date, limited work has been done looking at Nrf2-mediated neuroprotection in vivo. Here, we use viral-mediated gene delivery via stereotaxic injection into the adult Wistar rat brain in order to overexpress Nrf2. We exploit the capacity of 3-nitropropionic acid (3-NP) to produce ROS in order to provide an oxidative challenge to our animals. Histological analyses show reduced lesion size (lesions are - 5 5 % smaller than controls, p<0.05) and increased cell number (125% and 155% for ipsi- and contralateral hemispheres, respectively, p<0.001) in Nrf2 infected animals versus green fluorescent protein infected controls. Using the same viral-gene transfer technique, we also show a significant accumulation of the Nrf2 target xCT, a cystine/glutamate antiporter, on the apical membrane of ependymal cells (200% mean pixel intensity of the basolateral membrane, p<0.001). This supports the hypothesis xCT is important in maintaining cerebrospinal fluid and extracellular fluid redox state. The inability to properly process free radicals is present in many neuropathological processes (i.e. stroke). Targeting the Nrf2 pathway or its downstream gene targets may help treat and provide prophylaxis for some of these conditions. ii Table of Contents page Abstract ii Table of Contents iii List of Figures v List of Abbreviations vi Acknowledgements v i i C H A P T E R 1: INTRODUCTION 1 C H A P T E R 2: MATERIALS AND M E T H O D S 8 2.1 Animals 8 2.2 Stereotaxic Injections 8 2.2.1 For viral dosage determination 9 2.2.2 For time of expression studies 9 2.2.3 ForxCT-GFP experiments 10 2.3 3-Nitropropionic Acid 10 2.4 Immunohistochemistry & Histology 11 2.4.1 Cresyl violet staining 11 2.4.2 GFP fluorescence and calculations of infected area 12 2.4.3 Cell-type specific marker labelling 13 2.4.4 Metallothionein-1 15 2.4.5 xCT-GFP labelling 16 2.5 Cell culture 16 2.6 Transfection 17 2.6.1 Viral transfection 17 2.7 Immunocytochemistry 17 2.8 3-NP administration to glial cells 18 2.9 Western Blotting 19 2.10 Data Analysis 20 C H A P T E R 3: R E S U L T S 21 3.1 Viral Dosage Determination - Striatum 21 3.2 Area of infection over time 22 3.3 rAd-GFP preferentially infects astrocytes 23 3.4 Nrf2 overexpression 23 3.5 rAd-Nrf2 protects against an oxidative insult 24 3.6 3-nitropropionic acid induces Nrf2 in vitro 27 3.7 Viral dosage determination - Lateral Ventricles 29 3.8 Ectopic expression of the cystine/glutamate antiporter xCT 29 iii Chapters page CHAPTER 4: DISCUSSION & CONCLUSION 31 4.1 Discussion 31 4.2 Future Directions 36 4.3 Conclusion 39 BIBLIOGRAPHY 40 APPENDIX 1: FIGURES 49 iv List of Figures Figure page 1. Viral dosage determination in the striatum 72h following rAd-GFP administration 51 2. Viral dosage determination in the striatum 7d following rAd-GFP administration 53 3. Characterization of the area and timecourse of infection by the rAd-GFP vector 55 4. rAd-GFP preferentially infects astrocytes 56 5. GFP reporter expression in rAd-Nrf2 infected animals 57 6. Expression the Nrf2 target metallothionein-1 is increased in rAd-Nrf2 versus rAd-GFP infected animals 58 7. Behavioural scores of rAd-GFP and rAd-Nrf2 infected animals treated with 3-nitropropionic acid or saline over 4 days 60 8. Lesion size is decreased in rAd-Nrf2 infected animals following 3-nitropropionic acid treatment 61 9. Determination of NeuN+ cells in 3-nitropropionic acid treated animals following rAd-GFP or rAd-Nrf2 administration 63 10. Gliosis in rAd-GFP and rAd-Nrf2 infected animals treated with 3-nitropropionic acid 6 5 11. 3-nitropropionic acid induces expression of the Nrf2 target gene Heme-Oxygenase-1 67 12. Proposed mechanisms of Nrf2 induction and neuroprotective action following 3-nitropropionic acid administration 69 13. Viral dosage determination in the right lateral ventricle 3d following administration of rAd-GFP vector 70 14. Viral dosage determination in the right lateral ventricle 7d after rAd-GFP vector delivery to this area 71 15. Ectopic expression of the cystine/glutamate antiporter xCT 72 16. xCT-GFP is enriched on the luminal side of infected ependymal cells 73 v List of Abbreviations ANOVA Analysis of Variance bHLH Basic Helix-Loop-Helix CCD Charge-coupled device CSF Cerebrospinal fluid d day(s) DIV Days in vitro DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid DOC Deoxycholine dsDNA double stranded DNA ECF Extracellular fluid ECL Enhanced chemiluminescence EtOH Ethanol F Fluoride GFP Green Fluorescent Protein GFAP Glial Fibrillary Acidic Protein h hour(s) HCI Hydrochloric acid HRP Horseradish Peroxidase IL Interleukin MK-801 (5S,10ft)-(+)-5-Methyl-10,11 -dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine, a noncompetitive NMDA receptor antagonist mins minutes MOI Motility of Infection NaOH Sodium Hydroxide NeuN Neuronal Nuclei NMDA N-methyl-D-aspartate Nrf2DN NF-E2 related factor 2 Dominant Negative » LSM Laser Scanning Microscope Na Sodium PMSF Phenylmethylsulfonylfluoride PVDF Polyvinylidene fluoride RNA Ribonucleic Acid SDS Sodium Dodecyl Sulphate SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide gel electrophoresis S.E.M. Standard error of the mean tBHQ tert-butylhydroquinone V Volts vi Acknowledgements First and foremost, Dr. Alaa El-Husseini for the use of the LSM 510 Meta Confocal Microscope without which most of this work would have been near impossible. Second, the members of my committee (Drs. Katerina Dorovini-Zis, Jane Roskams, and Steve Vincent), whose advice & recommendations were most useful, and who provided me with direction when others just simply weren't capable. Third, Arthur Vanderhorst, Sam Gopaul & everyone from the Small Wild Mammal Unit, Department of Zoology, for taking care of my rats. Andy Shih for doing behaviour, Heidi Erb for cell cultures and Ping Li for histology protocols. Many thanks and some hugs to all those who encouraged me to stick with this for the last 3 years: Dr. Stef Bennett, Dr. Mel Derry, Dr. Mike Page, Simon, Charles, and all the CBR/Biochem/Microbi crew (you will be missed). And finally my funding sources: the National Science and Engineering Research Council and Michael Smith Foundation for Health Research without which life would have been a lot less pleasant. vii C H A P T E R 1 INTRODUCTION NF-E2 related factor two (Nrf2) is part of the Cap'N'Collar family of bHLH transcription factors1 responsible for the coordinated upregulation of cellular anti-oxidant defence mechanisms, which favour cell survival in times of oxidative crisis2. Under basal conditions, Nrf2 is bound in the cytoplasm to the actin-binding protein Keapl 3 ' 4 . Keapl has recently been suggested to be an E3-ubiquitin ligase responsible for the normal targeting of Nrf2 for degradation via the 26S proteasome5. It is thought to act as a 'molecular switch' able to 'sense' the redox status of the cell because of its many free cysteine residues6'7. Binding of free radicals, reactive oxygen and/or nitrogen species (ROS and RNS, respectively)8, electrophiles, or small molecule inducers (e.g. tert-butyl hydroquinone (tBHQ), sulforaphane, diethyl maleate)9"13 to these cysteine residues induces a conformational change in Keapl that liberates Nrf2 and allows its translocation to the nucleus via actin rearrangement. Like all bHLH family members, Nrf2 comprises a basic leucine zipper domain (coded by exon 5) and a transactivation domain (exons 1-3), allowing it to dimerize to other bHLH family members. The dominant negative construct of Nrf2 typically consists of exon 5 only14. Once in the nucleus, Nrf2 has been shown to bind multiple partners, including members of the Maf family (MafG, MafK, to name a few)1 5'1 6. The resulting dimer initiates transcription of genes containing anti-oxidant response elements (AREs or EpRE, electrophilic response element) within their promoter region. Most of these genes are phase II detoxification enzymes whose main functions are to remove drugs and other 1 xenobiotics from the cell and to maintain redox homeostasis within the cell1 7. Microarray analysis of eGFP- versus Nrf2-transfected primary glia reveal upregulation of many phase II genes including glutathione-S-transferase, NAD(P)H:quinone oxidoreductase-1 (NQ01), heme oxygenase-1 (HO-1), cysteine/glutamate antiporter (xCT), metallothionein-1, and src related tyrosine kinase, to name a few 1 8 , 1 9. Electrophile-induced conformational change of Keapl is not the only way in which ARE-mediated transcription via Nrf2 can occur, various signalling pathways have also been implicated. For instance, Kang etal. (2002) have reported Nrf2-mediated induction of glutathione-S-transferase through activation of the phosphatidylinositol 3-kinase (PI3K) pathway in response to peroxynitrite20. Phosphorylation of ser40 on Nrf2 by protein kinase C has also been shown to promote dissociation of Nrf2 from Keapl 2 1 . Previous work in our laboratory has shown overexpression of Nrf2 in mixed primary cultures is protective against an oxidative glutamate insult. Furthermore, using the same toxicity paradigm, we have shown overexpression of Nrf2 in a few glia can confer protection to many untransfected primary neurons (a ratio of roughly 1:100)18. To date however, little work has been done exploring the role of Nrf2 in the in vivo brain. Null mice have been generated by others22, but they have mainly been the target of peripheral studies. These animals appear to function normally and the consequences of Nrf2 removal only become apparent once the animals are challenged. Not surprisingly, Nrf2 -/- mice are more sensitive than their wild-type counterparts to a variety of insults such as acetaminophen-induced hepatotoxicity, 2 hyperoxic lung injury, benzo[a]pyrene-induced tumourigenesis in the forestomach, butylated hydroxytoluene-induced acute pulmonary injury, and, cigarette smoke-induced emphysema23"28. Studies using these animals are the subject of other work being carried out in our laboratory. Looking at the absence of a gene and its effect on the organism is only one part of the story in order to show the function and necessity of that gene. We were interested in overexpressing Nrf2 and observing how animals challenged with a brain oxidative challenge would fare. Although many techniques exist to overexpress genes of interest (GOIs) in cells, accomplishing this in the adult rodent brain poses certain challenges, mainly because most cells of the brain are post-mitotic. Viral vectors have become, in recent years, an efficient way to insert exogenous DNA or RNA into cells 2 9 , 3 0. Some of these vectors are especially well suited for our purpose. In all cases, essential replicatory elements are removed from the viral genome in order to provide space for the GOI and also to abolish viral replication, which would be detrimental to infected cells and to the host30. We have chosen a recombinant adenovirus (rAd) vector to express our GOI because it is readily accessible to us and we have previously established its functionality in vitro18. Adenovirus was one of the first viruses used in viral gene therapy and is still widely used today31"35. Viral entry into cells is dependent on receptor-mediated endocytosis. Therefore, the presence of the Coxsackie-Adenovirus Receptor (CAR) 3 0 ' 3 6 on the cell surface will determine the type of cell that is infected and will differ accordingly in a heterogeneous cellular environment. This makes identification of infected cell type crucial to subsequent 3 experiments. Neurons, oligodendrocytes, astrocytes, microglia, and ependymal cells are a few of the cell types rAd has been shown to infect30 ,37. Following endocytosis, acidification of the endosome causes degradation of the viral capsid followed by its release into the cytoplasm. Viral DNA is then released from the capsid and enters the nucleus via nuclear pores38. Inside the nucleus, the dsDNA remains as an episomal element and is not integrated into the host genome3 0 , 3 6. Proteins of interest will only be transiently expressed making finding an appropriate time of expression very important. Although it is replication deficient, the particular type of adenovirus used in this study - human serotype 5 adenovirus with deleted E1 and E3 regions - is still capable of producing viral proteins because of intact E2 and E4 regions. Proper dosage needs to be determined prior to use in order to achieve maximum infection efficiency with minimal toxicity as host cell toxicity is observed at high titers of virus 3 9 , 4 0. It should also be noted this specific serotype undergoes retrograde transport at nerve terminals41 therefore potentially increasing the number of infected cells. Stroke currently affects approximately 50 000 Canadians per year42. During stroke, many factors contribute to cell death within the infarct region and in the surrounding penumbra. Excitotoxicity, ionic imbalance, apoptotic-like mechanisms, and oxidative and nitrosative stresses (ROS/RNS, respectively) are some of the major pathways implicated in cell death following stroke (for a review see Lo etal. (2003)/3. The mitochondrial toxin 3-nitropropionic acid (3-NP) shares some of these 4 mechanisms with stroke and we have exploited its ability to produce ROS/RNS in order to provide an oxidative challenge to our animals. The main ways in which this irreversible inhibitor of mitochondrial complex II44 leads to cell death is by a combination of the aforementioned ROS/RNS production and also by energy depletion45"47. However, the exact in vivo mechanisms of action are complicated and likely involve reversal of glutamate transporters and NMDA receptor-mediated excitotoxicity48,49. Glutamate reuptake is an ATP-dependent process that will be affected when energy levels within cells drop thus causing the 'reversal' of the transporter and accumulation of glutamate in the synaptic cleft5 0 , 5 1. Lowered energy levels will also affect other ATP-dependent transport processes leading to ionic imbalances within the cell. These imbalances can alter resting membrane potential to relieve the Mg 2 + block on NMDARs5 2. Both these situations can lead to NMDAR-mediated excitotoxicity. In fact, addition of glutamate to 3-NP treated cells53 and animals54 exacerbates 3-NP toxicity while addition of the NMDAR antagonist, MK-801 can inhibit this effect54. Removal of glutamatergic cortical inputs from organotypic slice cultures also reduces the amount of cell death assayed via lactate dehydrogenase activity in collected media53. Because Nrf2 seems to be involved in maintaining redox status within the cell, is activated by the presence of ROS/RNS, and activates many detoxification pathways, we hypothesize that overexpression of this transcription factor will be protective against an oxidative insult caused by 3-NP, in vivo. 5 Downstream protein targets of Nrf2 include xCT , the light chain of system x c": a Na+-independent cystine/glutamate antiporter56"58 that is part of the heteromeric amino acid transporter (HAT) family (for a review on the HAT family, see Chillaron etal.)59. HATs are composed of a light-chain, which confers specificity of transport, and a heavy-chain termed 4F2hc (or CD98, in the case of xCT) or rBAT (for b 0 + HAT members, although recent evidence shows xCT can form dimers with rBAT in NIH3T3 cells)60. Within the brain, especially in the striatum, the antiporter is crucial in maintaining glutamatergic tone (i.e. levels of extrasynaptic glutamate) and can be negatively regulated by mGluR2/361. Antiporter function is also important in addiction, as reduced transporter activity is responsible for the low levels of extracellular glutamate seen in the nucleus accumbens following withdrawal from cocaine treatment62. Furthermore, pharmacological rescue of antiporter function is able to inhibit cocaine-primed drug seeking62. In keeping with the role of Nrf2 and its gene products in detoxification, xCT is also responsible for the import of cystine into the cell5 7. It has been shown the import of cystine (which is oxidized to cysteine within the cell) is the rate-limiting step in glutathione biosynthesis63. Glutathione (GSH) is one of the major cellular anti-oxidants. It is conjugated to electrophiles, free radicals and/or xenobiotics by glutathione-S-transferase as part of the Phase II response of cells. Once conjugated, these compounds are less reactive and may be exported from the cell for excretion from the organism64. Export of GSH-linked substances can be achieved, notably, through the Multi-drug Resistance Protein-1 (MRP1) transporter65. xCT mRNA has 6 been localized to ependymal cells lining the ventricles and to cells of circumventricular organs66. Here, xCT is thought to maintain cerebrospinal fluid (CSF) oxidation state, which in turn, is important in maintenance of brain homeostasis66. CSF redox state has been found aberrant in certain pathological processes including schizophrenia67. Because xCT contains 12 transmembrane domains it is highly hydrophobic. This property renders it difficult to isolate and study. Furthermore, the N-, C-, and intracellular loops are quite short68 making protein-based research difficult. Here again, we have chosen the route of viral-mediated gene transfer of a GFP-tagged xCT fusion protein to explore its in vivo localization, notably in the ependymal cells of the lateral ventricles where it is known to be expressed. Further investigation of the role of xCT in CSF maintenance via determination of localization will help resolve if this transporter contributes to observed pathologies and if it is a possible therapeutic target. 7 CHAPTER 2 MATERIALS & METHODS All reagents were purchased from Sigma-Aldrich Co., St-Louis, MO, unless otherwise indicated. 2.1 Animals Adult male Wistar rats (Charles River, Canada) were housed by the Department of Zoology at the Small Wild Mammal Facility (South Campus Rd.) on a 12h light-dark cycle with food and water available ad libitum. All experimental protocols have been approved by the Animal Care Committee of the University of British Columbia. 2.2 Stereotaxic Injections Adult male Wistar rats (250-350g) were anaesthetized by intraperitoneal (IP) injection of Somnitol (MTC Pharmaceuticals, Cambridge, ON) at a dosage of 65mg/kg. Animals were placed in a stereotaxic apparatus (David Kopf Instruments (small rodent apparatus), Tujunga, CA) and prepared for surgery. A mid-line incision approximately 1cm in length was created and the skin flap held open by a loosely tied suture. The underlying connective tissue was gently rubbed with a cotton swab using circular motions until the skull was uncovered and the surface was dry. A burr hole was drilled at coordinates +1.00mm in relation to Bregma and 2.6mm lateral for injection into the right striatum OR at -0.80mm from Bregma and 1.5mm lateral for injection into the right lateral ventricle using a Dremmel drill equipped with a small diameter sterilized drill bit (Meisinger, Neuss, Germany). Injections were performed 8 to depths of 4.5 and 5.5mm dorso-ventral (starting from dura mater) for the striatum and 4.5mm (starting from the dura) for the lateral ventricle with a 10ul Hamilton gas-tight syringe with exchangeable 26g needle (type 2 tip) by manually pressing the syringe plunger three times at four minute intervals. Wounds were irrigated with 0.9% sterile saline solution and sutured with 5.0 silk sutures (Sofsilk Sutures, United States Surgical Corporation, Norwalk, CT). Animals were allowed to recover from anaesthesia under a heat lamp and then replaced in the home cage. Note: All viral doses are indicated as total plaque forming units (pfu) in 3ul of vehicle (PBS ("lOmM PBS: 0.154M NaCI, 0.0028M NaH 2P0 4, 0.0072M Na 2HP0 4, pH 7.2) sterile filtered). All viral vectors were produced by the Adenoviral Core Facility at the University of Ottawa (Ottawa, ON) using previously published methods69. 2.2.1 For viral dosage determination Animals (n=3 for each dose and timepoint, n=24 animals in total) were injected in the right striatum or lateral ventricle with 3ul of vehicle, 5x106 pfu, 5x107 pfu or 5x108 pfu rAd-eGFP and allowed to recover for a period of 3 or 7 days. 2.2.2 For time of expression studies Animals (n=3 for each timepoint, n=21 animals total) were injected in the right striatum with 5x106 pfu rAd-eGFP and allowed to recover for 24h, 48h, 72h, 5d, 7d, 14d or21d. 9 2.2.3 For xCT-GFP experiments: Animals (n=1 per group, n=2 animals total) were injected in the right lateral ventricle with 5x108 pfu rAd-eGFP or 5x108 rAd-xCT-GFP vector and allowed to recover for 72h. 2.3 3-Nitropropionic Acid For these experiments viral injections into the right striatum of rAd-eGFP or rAd-Nrf2, both at concentrations of 5x106 pfu were carried out in the manner described above (n=10 animals per viral vector group, n=20 total). 3-NP was prepared in 10mM phosphate buffer and adjusted to pH 7.4 using NaOH. Daily IP injections of 3-NP or control PBS were administered to animals (n=5 animals per treatment, per viral vector group, n=20 total) subsequent to behavioural assessment by a blind observer using a motor behaviour scale adapted from Ouary etal.70 and McBride et al.7^ Briefly, three major criteria were used: gait abnormalities, cage grasping using forepaws, and ability to remain on a small platform for 10s. Animals could score 0 (able) or 1 (unable) for the latter two criteria and up to 6 for the first (0-normal, 1 -general slowness due to hindlimb impairment, 2-incoordination and marked gait abnormality, 3-hindlimb paralysis, 4-incapacity to move from fore- and hindlimb impairment, 5-recumbancy with animals laying on one side but showing uncoordinated movements when stimulated, and 6-near death recumbancy with almost complete paralysis and rapid breathing). Animals were put on one of two dosage regimens: 40mg/kg, 40mg/kg, 20mg/kg over 3 days (n=2 animals per treatment (3-NP or PBS), per viral vector group, n=8 total) OR 40mg/kg, 20mg/kg, 20mg/kg, 20mg/kg over 4 days (n=3 animals per treatment (3-NP or PBS), per 10 vector group, n=12 total). Two regimens were tested in order to circumvent any preconditioning effects due to tissue disruptions from the stereotaxic injection. In both regimens, animals received a total of 100mk/kg 3-NP. A t-test revealed no difference in cresyl violet lesion size between the two regimens and the groups were pooled. Animals were sacrificed 24h after the last injection or upon displaying severe motor behaviour deficits such as recumbancy. 2.4 Immunohistochemistry & Histology Animals were anaesthetized using halothane (MTC Pharmaceuticals, Cambridge, ON) and perfused intracardially with 0.9% saline followed by 4% paraformaldehyde in PBS (PFA). Brains were then removed and allowed to fix for 24h in 4% PFA. Tissue was cryoprotected using 20% sucrose solution in PBS and cut serially (40u.m, 10|im, 10pm, etc.) onto Superfrost Plus slides (Fisher Scientific, Fair Lawn, NJ) using a cryostat (Reichert-Jung Frigocut 2800N, Cambridge Instruments GmbH, Nussloch, Germany). Sections were air dried for 30mins and stored at -20°C until further use. 2.4.1 Cresyl violet (CV) staining Frozen 40pm sections were thawed in PBS for 3 minutes at room temperature (RT) then defatted using the following incubations (EtOH, xylenes and acetic acid from Fisher; cresyl violet acetate powder from Kodak, Rochester, NY): H 20 x 1min, 70% EtOH x 1min, 95% EtOH x 5mins, 100% EtOH x 10mins, Xylene x 20mins. Sections were rehydrated for cresyl violet staining using: 100% EtOH x 5mins, 95% EtOH x 11 1min, 70% EtOH x 1min, 1% cresyl violet acetate x 10mins, 2 x H 20 dip, destain to effect (70% EtOH + 1% acetic acid); dehydrated (100% EtOH x 2mins, 100% EtOH x 2mins, Xylene x 10 mins) and mounted with Permount (Fisher) using 24mmx60mm No.1 glass coverslips (Corning, Big Flats, NY). CV sections were scanned at 600dpi using an Epson desktop scanner, then converted to grayscale and analyzed using Image J software (NIH). Total lesion volumes were calculated for all 3-NP-treated animals (n=10) using Excel (Microsoft) and the following formula: V t = Vi +V2 + ...+ V n, where V n is the volume of a truncated cone (based on Cavalieri's principle) and thus Vn= TTh(rn-i2 + rn-i rn + rn2)/3, where h is the distance between 2 sections and r is the radius of the lesion area72 using sections from the anatomical levels Bregma +170mm, +1.20mm, +0.70mm, +0.20mm, -0.26mm, and -0.80mm according the rat brain atlas73. 2.4.2 GFP fluorescence and calculations of infected area GFP fluorescence was observed by thawing frozen 10um sections in PBS for 3mins at RT. Counterstaining with 8uM propidium iodide (PI) in PBS was carried out at RT for 10mins in order to label nuclei, followed by washing for 1 x 10mins in PBS at RT. Slides were then mounted with Fluoromount-G (Southern Biotech, Birmingham, AL) using 24mmx60mm coverslips. Fluoromount was allowed to dry overnight. Coverslips were then secured and sealed with nailpolish. Fluorescent slides were stored at -20°C until needed. Fluorescent images were obtained using a Zeiss LSM 510 Meta confocal microscope with LSM 510 software in multi-track mode to minimize bleedthrough. In order to assess infected area, a tile section of the entire 12 ipsilateral (right) hemisphere of the rAd-GFP infected animals was collected for every brain section demonstrating GFP fluorescence. One tile section is comprised of 64 (8 x 8) individual images measuring 512 x 512 pixels captured through a 20x objective yielding a total imaged area of 4096 pixels x 4096 pixels or 3685.46u.rn x 3685.46nm. Area of GFP fluorescence was determined for each tile by thresholding images to eliminate background and calculating the total fluorescent area. For the same section, the PI counterstain was used to locate the corpus callosum and striatum. The area encompassed by these 2 anatomical regions was deemed to be total "striatal+cc" area. Volumes were calculated using the above equation using Excel (Microsoft) while sections themselves were analyzed using Image J software (NIH). 2.4.3 Cell-type specific marker labelling Frozen 10um sections were thawed in PBS for 3mins at RT. Mouse monoclonal anti-04 (Chemicon) or rabbit polyclonal anti-GFAP were diluted 1:400 and 1:200 respectively in Ab buffer (3% BSA and 0.3% Triton X-100 in 10mM PBS) and incubated overnight at 4°C in a humid chamber. Sections were rinsed 3 x 5mins in PBS and incubated with goat anti-mouse IgM Alexa546 conjugated or goat anti-rabbit Alexa546 or 633 conjugated secondary antibodies (all 1:500, Molecular Probes, Eugene, OR) in a humid chamber for 1h at RT. Sections were rinsed 3 x 10mins in PBS prior to mounting with Fluoromount-G and 24mmx60mm coverslips. Fluoromount was allowed to dry overnight and slides sealed and stored as above. Certain sections were also stained using a mouse monoclonal anti-s100p (1:1000) 13 primary and goat anti-mouse Cy3 conjugated secondary antibody (1:200, Amersham Pharmacia Biotech, Buckinghamshire, UK) as described above. All GFP fluorescence (in these sections and with all GFP "labelling") could be detected without using an anti-GFP antibody. For NeuN staining, an antigen retrieval process based on work by Jiao 7 4 was used following thawing. Briefly, sections were incubated at 80°C for 30mins in 10mM sodium citrate (Fisher) pH 8.5 and then rinsed 1 x 5mins in PBS prior to primary antibody incubation (1:100, Chemicon, Temecula, CA). Primary and secondary (goat anti-mouse Cy3, 1:200) antibody incubations were carried out as described above. In cases where triple labelling was performed (GFP, NeuN & GFAP), the antigen retrieval process was carried out prior to labelling. This process did not seem to affect GFP fluorescence. Images were again captured using a Zeiss LSM Meta 510 confocal microscope with LSM 510 software. For co-localization studies, tile sections were taken in both channels (excitation: 488nm for GFP and 546nm for Cy3/NeuN and Alexa546/GFAP or 04) using multi-track mode. Regions of interest (ROI) were selected and a z-stack of 10 images was collected at z-intervals of half the optimal slice interval calculated by the software for the particular pinhole size (approx. 1 pm). The number of GFP + cells in each field was counted as were the number of cells expressing both labels (n=10 fields per section at the 4 anatomical levels showing the most GFP + fluorescence, thus 40 fields per animal (n=3 animals) representing an average of 150 GFP + cells per animal). For NeuN cell counts (n=15 random fields of 256 pixel x 256 pixel within the striatum using the same 6 anatomical levels as for cresyl violet staining analysis, therefore NeuN+ cells within 90 fields were counted per animal, n=1 animal per viral 14 vector group) and gliosis assessment (striata were analyzed at the 6 anatomical levels previously mentioned, n=1 animal per viral vector group), tile sections were collected in multi-track mode for three channels (excitation: 488nm for GFP, 546nm for Cy3/NeuN and 633nm for Alexa633/GFAP). Gliosis was determined by thresholding the images to eliminate background and calculating mean pixel intensity for the total "striatal+cc" area (as defined above) and by determining total fluorescent area in the striatum using Adobe Photoshop (first parameter) and Image J software (second parameter). Images for s100p staining were collected using a Zeiss Axiophot epifluorescence microscope connected to a Retiga Ex/'CCD camera (Qlmaging) using Northern Eclipse software. 2.4.4 Metallothionein-1 Mouse monoclonal anti-metallothionein-1 (1:100, Zymed, San Francisco, CA) primary and goat anti-mouse Cy3-conjugated secondary antibodies were used for immunohistochemistry as described above. Images were collected and analyzed in the same manner as the co-labelling studies except the total number of MT-1 +cells per field (n=10 fields representing an average of 105 ± 5 GFP + cells and 80 ± 10 MT-1+ cells total per animal at the anatomical level showing the most GFP fluorescence, n=2 animals per viral vector group) were also counted because all MT-1 immunoreactive cells were not also GFP + in both the rAd-GFP and rAd-Nrf2 groups. 15 2.4.5 xCT-GFP labelling Immunohistochemistry was performed as above. Mouse monoclonal anti-GFP (Roche, Mississauga, ON) antibody diluted 1:1000 in Ab buffer was used as primary antibody and followed by a goat anti-mouse Alexa488 conjugated secondary antibody (1:1000, Molecular Probes). An 8uM PI counterstain in the second PBS wash prior to mounting with Fluoromount-G and glass coverslips. Fluorescence was examined using the LSM 510 Meta. All image stacks were collected using the same pinhole diameter, detector gain and scanning parameters. Stacks were analyzed individually using Image J software. Briefly, mean fluorescence intensity of the apical and basolateral membrane of infected cells was determined for each z-slice of a stack. The average intensity of the apical and basolateral membranes for a cell were then calculated and the resulting data analyzed using a paired t-test (n=26 cells for one xCTGFP infected animal). 2.5 Cell culture Primary glial cultures from P0-P2 Wistar rat pup cortices were obtained via the papain dissociation method18 followed by plating onto uncoated 100mm culture dishes (Corning) in "glial media" (Minimum Essential Media (MEM, Invitrogen, Grand Island, NY), 10% Fetal Bovine Serum (FBS, HyClone, Logan, UT), 1% 200mM L-glutamine, 1% penicillin/streptomycin (Invitrogen), and 3.6% glucose). Cultures from one brain were split into two dishes and kept at 37°C in a 95% air/ 5% C 0 2 atmosphere. Glial media was replaced at 1 DIV with fresh glial media. At 3DIV, adherent neurons were removed from culture by repeated pipetting of the media and 16 cultures were used prior to 10DIV. For immunostaining, primary glial cultures were replated at 3DIV onto poly-D-lysine coated culture dishes with 22mm x 22mm No.1 glass coverslips (Corning). 2.6 Transfection Confluent 24-well plates were transfected with 1 ug GFP or xCT-GFP using Effectene (Invitrogen) reagent according to manufacturer's protocol and allowed to express protein for 48h prior to immunostaining. 2.6.1 Viral transfection 80% confluent primary rat glial cultures were transfected with 200MOI (i.e. 200 viral particles available to infect one cell) rAd-GFP, rAd-Nrf2DN, or rAd-Nrf2 and allowed to incubate at 37°C for 48h prior to protein collection. 2.7 Immunocytochemistry Media was removed and cells rinsed once in PBS at RT. Note: PBS was used to rinse cells between every step and each step was performed at RT until primary antibody incubation. Cells were fixed using ice-cold 2% PFA for 20mins followed by permeabilization with 0.2% Triton X-100 in PBS for another 20mins. Blocking was carried out using 5% (v/v) normal goat serum (Vector Laboratories, Burlingame, CA) in PBS for 10mins. Following PBS rinse, coverslips were gently lifted from culture dishes using forceps, placed onto Superfrost slides (Fisher), and secured with a 17 drop of nailpolish in every corner. Slides were incubated in a humid chamber at 4°C overnight with mouse monoclonal anti-GFP primary antibody (Roche) diluted 1:1000 in Ab buffer. Slides were washed 3 x 5mins in PBS at RT and incubated in a humid chamber with a goat anti-mouse Alexa488 conjugated secondary antibody (1:1000, Molecular Probes) for 1 h, also at RT. Slides were then washed 3 x "lOmins in PBS at RT, and mounted using Fluoromount-G and 22mmx22mm coverslips. The Fluoromount was allowed to dry overnight at which point coverslips were secured & sealed with nailpolish. Fluorescent slides were stored at 4°C until use. Fluorescent images were captured using a Zeiss Axiophot epifluorescence microscope connected to a Retiga Ex/'CCD camera (Qlmaging) using Northern Eclipse software. 2.8 3-NP administration to glial cells Media from 60-70% confluent 100mm dishes of primary glia was replaced with "low glucose" media (MEM w/out phenol red, 5% dialyzed FBS, 1% L-glutamine and 1% penicillin/streptomycin) 24hrs prior to experiments. New "low glucose" media was put onto cells prior to addition of 0.1 mM, 0.5mM, 1mM, 3mM 3-np, 10u.M tBHQ (dissolved in DMSO), or vehicle (0.1% phosphate buffer (100mM pH 7.4) or 0.1% DMSO). All reagents are indicated as final concentrations and are 1:1000 dilutions of stock. The treatments were left on cell cultures for a period of 24h before protein collection. 18 2.9 Western Blotting Cells were rinsed once in PBS and collected in modified RIPA buffer (0.1% SDS, 0.5% Deoxycholine, 1% Triton X-100, 0.1mg/ml PMSF, 1mM NaF, 1mM Na Orthovanadate, 5u.g/ml aprotinin, 5|ig/ml leupeptin, in PBS) using a cell scraper. Lysates were passaged through a 20gauge needle in order to shear DNA followed by 30mins incubation on ice. Lysates were centrifuged at 20 000 x g for 20mins at 4°C. The supernatant (total cell lysate) was transferred to a fresh tube. Protein quantification was carried out using the BCA method according to manufacturer's protocol (Pierce, Rockford, IL). SDS-PAGE was performed using 20|jg of protein. Gels were run at 100V through the stacking gel then at 150V through the 12.5 % resolving gel until adequate separation of the appropriate molecular weight markers (Rainbow Standard, BioRad Laboratories, Hercules, CA) was achieved. Gels were transferred onto PVDF membranes (Bio-Rad) at 150V for 1 h30mins on ice. Membranes were blocked in 5% blocking solution (5% (w/v) milk protein, 0.5% Tween-20 in 1X Tris-buffered saline pH 7.2) at RT for 1h then rinsed with 0.5% blocking solution for 5 mins at RT. All antibodies were diluted in 0.5% blocking solution. Membranes were incubated with a mouse monoclonal anti-HO-1 antibody (1:2500, Stressgen, Victoria, BC) at 4°C overnight followed by incubation with a goat anti-mouse HRP-conjugated secondary antibody (1:5000, Amersham) for 1 h at RT. Antibody detection was performed using ECL reagent (Amersham) per manufacturer's protocol. The chemiluminescent reaction was captured on Biomax film (Kodak). Following HO-1 data collection, membranes were gently stripped in buffer (62.5mM Tris-HCI pH 6.7, 2% SDS and 100mM p-mercaptoethanol added 19 fresh) at 50°C for 30mins followed by 2 x 10mins PBS-T (0.3% Triton X-100 in PBS) washes at RT and 1 h incubation in 5% blocking buffer. Membranes were reprobed using the above method with a goat polyclonal anti-actin primary antibody (1:100, Santa Cruz Biotechnology) followed by a donkey anti-goat HRP-conjugated secondary antibody (1:5000, Santa Cruz). Signal was detected as above. 2.10 Data Analysis All data are expressed as mean ± S.E.M. and were analyzed using two-tailed t-tests or two-tailed paired t-tests where appropriate (xCTGFP membrane localization and ipsi- versus contralateral comparisons within animal groups) using Excel (Microsoft) except behavioural data which was analyzed using one-way ANOVA followed by a post-hoc Tukey's test with GraphPad software (Prism). * p<0.05, **p<0.01, ***p<0.001. 20 CHAPTER 3 RESULTS 3.1 Viral Dosage Determination - Striatum Based on literature findings for first generation adenovirus vectors39, three doses were tested for efficacy of infection in a PBS vehicle. Cell morphology was assessed via fluorescence of the GFP reporter gene at 72h (Figure 1) and 7d (Figure 2) following vector delivery in order to determine cell health, area of infection, and host toxicity. Administration of a high dose of vector (5 x 108 total pfu) resulted in a large area of infection (Fig.1, A3 and B3). However, many GFP+cells demonstrated apoptotic morphology (the cells appear as small balls with no processes). This can be seen in the magnified image of Figure 1 (C3). Furthermore, barely any fluorescent cells were observed at this dosage 7d post-injection (Fig 2., row 3) suggesting the "ball-like" GFP + cells seen at the 72h timepoint were being cleared from the system most likely via phagocytosis. A mid-range dose of vector (5 x107 pfu) yielded a slightly smaller area of infection (Fig 1., A2 and B2) than that obtained using 5 x 108 pfu, as assessed by empirical observation. At this titer of viral vector, more cells with normal morphology (soma surrounded by many processes) were observed but many still appeared to be "ball-shaped" (for a closer examination, see Fig. 1, C2). This observation remained true both at the 72h and 7d observation points (Fig. 2, row 2) although the GFP + cells remaining at this later timepoint did possess a healthier appearance (Fig. 2, C2) than those at the high dose for this timepoint (Fig. 2, C3) and also compared to the same mid-range dosage group at 3d post-injection (Fig. 1, C2). The lowest dose of vector (5 x 106 pfu) proved to be the 21 most effective at balancing infectivity versus host cell toxicity with many GFP + cells demonstrating normal morphology at both 72h and 7d post-infection (Fig. 1, C1 and Fig. 2, C1, respectively) and with a reasonable amount of GFP + cells in the target area (Figs. 1 and 2, A1 and B1). At all timepoints, vehicle injected animals did not show any fluorescence (Figs. 1 and 2, row 4) nor did the contralateral hemisphere of all animals (Figs. 1 and 2, column D) demonstrating spread of the viral vector was localized to the injected hemisphere. 3.2 Area of infection over time Where rAd can infect monolayer cells effectively in vitro, when placed in a 3-dimensional environment the properties of infection can vary widely as vector diffusion comes into play. In order to thoroughly assess protein expression, the lowest and most effective dose of rAd-GFP was used to look at area of infectivity and cell-type specificity of our vector. Seven timepoints were chosen for analysis: 24h, 48h, 3d, 5d, 7d, 14d, and 21 d following vector administration. GFP fluorescence was observed as early as 24h after stereotaxic injection until, at least, 21 d later (Fig. 3A). Diffusion was observed at all time points in all planes from the site of injection (Fig 3B and C). The breakdown of infected striatal + corpus callosum volume over time is: 0.83 ± 0.10% (24h, n=2), 0.17% (48h, n=1), 1.04 ± 0.68% (3d, n=3), 0.35 ± 0.12% (5d, n=3), 0.66 ± 0.35 (7d, n=2), 0.79 ± 0.04% (14d, n=2), and 0.48 ± 0.14% (21d, n=2). Overall an average of 0.66 ± 0.14% of total striatal area (including the corpus callosum and striatum) was GFP + (Fig 3D). A total of three animals were allocated to each timepoint. In some cases, no fluorescence was 22 observed anywhere in the brain (animals 24h#1, 48h#1, 7d#1,14d#3, and 21d#3) whereas others were mechanically damaged and could not be processed for GFP/PI staining (48h#2). 3.3 rAd-GFP preferentially infects astrocytes Because rAd has been reported to infect a variety of cell types in vivo (see Davidson and Breakefield for a review)30, immunohistochemistry using the cell-type specific markers glial fibrillary acidic protein (GFAP, astrocytes), neuronal nuclei (NeuN, neurons), and 04 (oligodendrocytes) was performed. A majority of GFP + cells also labelled with GFAP (62 ± 1 % , Fig. 4A,E), whereas 2.3 ± 1.9% co-labelled with 04 (Fig. 4D,E). No cells were ever observed to also label with NeuN (Fig. 4C,E). The remaining GFP + cells presented with astrocyte-like morphologies despite not labelling with GFAP. It has been previously shown that a discrete population of astrocytes within the brain does not label with GFAP, therefore certain sections were restained for the calcium binding protein s100(3, which has been shown to label a broader population of astrocytes within the brain75. A total of 91.8 ± 0.2% GFP + cells co-labelled with s100|3 (Fig. 4B,E) confirming these remaining GFAP" cells were astrocytes. 3.4 Nrf2 overexpression The GFP reporter was observed (without antibody labelling) in a few rAd-Nrf2 infected animals with a similar distribution pattern to the GFP-only controls (compare Fig 5A to 3A and C). In some of these animals immunohistochemistry for 23 metallothionein-1 (MT-1), a downstream protein target upregulated by Nrf219, was carried out. Fluorescence for MT-1 (Fig. 6B, E) was observed in both rAd-Nrf2 and rAd-GFP infected controls. When the number of GFP + and MT-1+ cells within a field are analyzed, 54 ± 5% of GFP + cells also express MT-1 and 81 ± 6% of MT-1+ cells co-label with GFP in rAd-Nrf2 infected brain (Fig. 6 bottom row and G). Comparatively, in rAd-GFP infected animals (Fig. 6 top row and G), only 32 ± 7% of GFP + cells within a field co-label with MT-1 whereas 41 ± 9% of MT-1+ cells also show GFP fluorescence. In both the GFP + and the MT-1+ cell populations, there is a significant increase in the number of co-labelled cells in the rAd-Nrf2 infected animals versus rAd-GFP infected controls (1.68 fold for GFP + co-localizing with MT-1 and 1.98 fold for MT-1+ cells also labelling with GFP) for the same number of cells counted for each viral vector group (about 105 + 5 GFP + cells and 80 ± 10 MT-1 + cells total in 10 fields per animal, n=2 animals) Taken together, these results suggest the Nrf2 transgene is being functionally expressed in rAd-Nrf2 infected animals. 3.5 rAd-Nrf2 protects against an oxidative insult As a model of neurodegenerative disease, 3-NP toxicity is associated with motor behaviour dysfunction, the severity of which usually correlates with lesion size 7 0 ' 7 6" 7 8 . We used aberrant motor behavioural function in order to assess whether our toxin treatment was having an effect in these animals. rAd-GFP animals receiving 3-NP show significant motor behaviour dysfunction on days 2, 3, and 4 of treatment (Fig. 7A, n=5 saline, n=5 3-NP, **p<0.01, ***p<0.001 one way ANOVA, post-hoc Tukey's test) relative to saline-injected controls. Similarly, rAd-Nrf2 infected animals treated 24 with 3-NP also show dysfunction when compared to saline administered controls although the difference in behaviour is only significant on days 2 and 3 of treatment (Fig. 7B, n=5 saline, n=5 3-NP, *p<0.05 one way ANOVA, post-hoc Tukey's test). However, when both rAd-GFP and rAd-Nrf2 groups that were treated with 3-NP are compared, rAd-Nrf2 infected animals show a trend toward less severe motor behavioural impairment than controls, especially on the last day of behavioural assessment (Fig. 7C). Because of the correlates between lesion size and severity of symptoms, the behavioural data suggests rAd-Nrf2 animals would have smaller lesions than the rAd-GFP controls. Following 3-NP challenge and behavioural assessment, animals were sacrificed and lesion volumes calculated by the absence of cresyl violet staining at six anatomical levels within the striatum. In the infected hemisphere (Fig. 8A, arrow), lesions were significantly smaller in animals receiving Ad-Nrf2, compared to Ad-GFP control virus (9.6 ± 2.6 mm3 versus 23.4 ± 4.2 mm3, p<0.05, two-tailed t-test, Fig. 8A and B). Interestingly, a trend toward decreased lesion volume was also observed in the hemisphere contralateral to the virus injection (12.2 ± 3.8 mm3 for rAd-Nrf2 compared to 25.9 ± 4.9 mm3for rAd-GFP controls, Fig. 8A and B), although this difference did not attain statistical significance (p=0.06, two-tailed t-test). This effect is likely 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 hemisphere79,80. 25 Reflective of the smaller lesion size, there are more NeuN+cells remaining in the ipsi- and contralateral striata of rAd-Nrf2 infected animals than rAd-GFP infected controls (40 ± 2 cells/field versus 32 ± 1 cells/field, respectively in the ipsilateral hemisphere and 43 ± 2 cells/field versus 28 ± 2 cells/field, respectively in the contralateral hemisphere, p<0.001 two-tailed t-test for both groups, Fig. 9 A and B). It is interesting to note the significant difference between cell number in the ipsilateral versus contralateral hemispheres of rAd-GFP animals (32 ± 1 cells/field versus 28± 2 cells/field, p<0.001 two-tailed paired t-test, Fig. 9B). Further investigation revealed counts at one of the anatomical levels included in the analysis were responsible for this significant difference and that differences in number of cells/field was not significant between hemispheres in rAd-GFP animals for all other anatomical levels examined (p>0.05, two-tailed paired t-test). This discrepancy could be due to a slight difference in the plane of the cut for this level resulting in inclusion of a region normally containing less NeuN+ into our counting area. To assess gliosis, mean pixel intensity was determined as an indication of GFAP immunoreactivity and upregulation. No significant difference was observed between rAd-GFP and rAd-Nrf2 groups in the ipsilateral (19 ± 3 pixel intensity versus 28 ± 8 pixel intensity, respectively, Fig. 10B) or in the contralateral hemisphere (13 ± 5 pixel intensity versus 11 ± 3 pixel intensity, respectively, Fig. 10B). The area of GFAP immunoreactivity was also calculated as a reflection of astrocyte number in the striatal area. Again, no significant difference was found in the ipsilateral striatum of rAd-Nrf2 animals in contrast to GFP controls (0.28 ± 0.03 mm2 for rAd-GFP versus 26 0.34 ± 0.06mm2 for rAd-Nrf2, Fig. 10C), nor was there a significant difference in the contralateral hemisphere (0.23 ±0.13 mm2 for rAd-GFP versus 0.13 ± 0.04mm2 for rAd-Nrf2, Fig. 10C). Of note is the significant difference in the GFAP + area between the ipsilateral and contralateral hemispheres of rAd-Nrf2 infected animals (0.34 ± 0.06 mm2 and 0.13 ± 0.04 mm2, respectively, p<0.05, two-tailed t-test, Fig. 10C). The difference in mean pixel intensity between these brain regions was almost significant (p=0.06, two-tailed t-test) and may be due to the increased area occupied by GFAP + cells. Taken together, these factors point to an increased number of glial cells in this hemisphere. 3.6 3-nitropropionic acid induces Nrf2 in vitro To verify the toxin we are using and the Nrf2 pathway are indeed interrelated, in vitro assessment of Nrf2 activation following 3-NP treatment was undertaken. Primary glial cells, the major cell type infected by our viral vector, were exposed to various amounts of 3-NP (0.1 mM, 0.5mM, 1 mM, 3mM) or to 10pm tBHQ, or to vehicle (0.1 % phosphate buffer or 0.1% DMSO, respectively) in media containing ~5mM glucose. The amount of glucose in the media was carefully chosen to best mimic the in vivo environment and also because glucose/energy substrate supplementation in media has been shown to protect against 3-NP toxicity5 3 , 5 4'8 1, most likely by compensating for the energy depletion typically caused by 3-NP. tBHQ was used as a positive control for Nrf2 induction as were glial cultures transfected with rAd-GFP, rAd-Nrf2DN and rAd-Nrf2. The concentrations of 3-NP are based on the Km of SDH for 27 succinate (0.5mM) and represent a low (0.1 mM) and two high doses (1mM and 3mM) of the irreversible inhibitor. The assay consists of measuring the amount of protein of the Nrf2 target HO-1 8 2 contained in whole cell lysates after 24h treatment with 3-NP or controls (or 48h of rAd transfection) and normalizing to the amount of actin in the same sample via densitometry. A representative blot is shown in Figure 11A with the accompanying quantifications in B. The data obtained from three individual experiments is shown in C. One should note not all doses are represented across the 3 experiments (n=1 experiment containing 0.1 mM and 3mM 3-NP, n=3 experiments using 0.1% PB and 1 mM 3-NP, n=2 experiments for all other treatments). A trend toward increasing HO-1 levels can be seen in all groups when compared to their respective controls. For the viral-transfected group, the amount of HO-1 increases 1.2 fold in rAd-Nrf2DN and 3.1 fold in rAd-Nrf2 cells versus rAd-GFP controls while the small molecule inducer tBHQ caused a 1.3 fold increase in protein levels compared to vehicle control. For 3-NP, 0.1 mM was not very different from vehicle control (0.98 fold). However, marked increases in HO-1 protein levels were observed for the remaining 3-NP doses (3.7 fold, 5.9 fold and 6.3 fold for 0.5mM, 1 mM and 3mM 3-NP, respectively). These results suggest a 24h treatment of 3-NP is able to induce Nrf2-mediated gene transcription in primary glial cells. 2 8 3.7 Viral dosage determination - Lateral Ventricles A dosage series using the same low, mid-range and high titers of viral vector was carried out in another anatomical location - the lateral ventricles via intracerebroventricular (ICV) stereotaxic injection. In contrast to the striatum, the lowest titer of vector was not able to infect any cells along the ventricular walls (Fig. 13, row 2) whereas the mid-range dose only infected a few cells (Fig. 13, row 3. However, the highest dose of vector, which proved quite toxic in the striatum, was the best at infecting the ependymal cells lining the lateral ventricles (Fig. 13, rows 4 and 5). Even cells in the ventricle contralateral to the injected hemisphere were infected (Fig. 13, C4 and D4), as were those lining the 4 t h ventricle of the brain (Fig. 13, A5 and B5). Access to this area was presumably due to vector diffusion through the cerebrospinal fluid. The above data was collected 3d following vector delivery. As with the striatum, a 7d timepoint was also investigated, however no fluorescence was observed for any of the viral titers tested (Figure 14). For subsequent studies an end-point of 3d following vector administration was chosen. 3.8 Ectopic expression of the cystine/glutamate antiporter xCT xCT is a highly hydrophobic 12TM-domain protein whose mRNA has been detected in ependymal cells lining the ventricles66. However, due to its hydrophobic nature and short cytoplasmic loops, creating an antibody targeted against xCT has proven difficult. We have used a recombinant adenovirus vector expressing a GFP-tagged version of xCT (Fig. 15A) in order to explore the localization of this protein. 29 Immunocytochemistry of primary glial cells overexpressing xCT-GFP or GFP-only reveals xCT-GFP to have a punctate distribution pattern suggesting membrane localization (Fig. 15B, right panel) compared to uniform GFP distribution in rAd-GFP transfected controls (Fig. 15B, left panel). To further explore probable membrane localization in vivo, we delivered rAd-xCTGFP or rAd-GFP via ICV injection to adult male Wistar rats and let protein expression occur for 3d as determined above. Immunohistochemistry showed a distinct difference in GFP fluorescence localization between rAd-xCTGFP infected animals and rAd-GFP controls. As can be seen in Figure 15C, GFP fluorescence in rAd-XCTGFP is limited to the area surrounding the nucleus, presumably the plasma membrane, whereas GFP controls show fluorescence throughout the cell including the nuclear area. It should be noted the GFP control images hail from the dorsal aspect of the right lateral ventricle (where cells sometimes does not appear cuboidal, depending on the plane in which the section is cut) and are included simply to enhance the striking difference in GFP localization between the groups. From these images, and given the fact many transporters, including members of the HAT family, show membrane polarization, the mean pixel intensity of rAd-xCTGFP infected cells on the apical and basolateral membrane was examined (Fig. 16 A and B). Indeed, when compared to the apical side of the membrane (mean pixel intensity = 43 ± 8), the basolateral portion shows a very significant decrease in mean pixel intensity (21 ± 3, p<0.001 two-tailed paired t-test). In other words, there is more GFP fluorescence on the apical side of the infected ependymal cells and by implication, more xCTGFP is localized to this area of the plasma membrane. 30 CHAPTER 4 DISCUSSION & CONCLUSION 4.1 Discussion Our rAd vectors are optimally used at a titer of 5 x 10 6 pfu and remain in the brain for at least 21 d, which is in accordance with the literature 3 9 , 4 1. The nature of the virulence (infection of the corpus callosum and the 'patchiness' of infection within a section) is also commonly reported with this particular serotype of vector 4 1 , 8 3" 8 6. At first glance, the total infected volume may seem low, however it is important to consider the actual spatial distribution of infected cells - a web-like "net" in the striatum which could allow secreted trophic factors (see below) to access a greater number of neurons than say, if the infected cells were all distributed in a straight line. There is also precedent, looking at the cholinergic interneurons of the striatum, for a small number of cells (these cells make up -1 -2% of striatal volume) to profoundly affect the functional output of the striatum influencing such diverse behaviours as sensorimotor function, sleep & arousal states, learning & memory, anxiety and pain sensations8 7. Furthermore, in our hands, this vector preferentially infects astrocytes. We have previously shown, in vitro, that a small number of Nrf2 overexpressing astrocytes can protect neighbouring neurons from an oxidative glutamate insult (1 astrocyte : 100 untransfected neurons)18. We believe this effect to be mediated by secretion of glutathione and/or glutathione precursors by astrocytes 6 5 , 8 8 , 8 9 and that a similar mechanism is responsible for the robust protection seen in the toxicity experiments above. In fact, S-nitrosoglutathione has been shown to provide protection in a 3-NP toxicity model 9 0. 31 One may have expected the observed motor behaviour dysfunction to differ more significantly between groups given the dramatic reduction in lesion size in the 3-NP paradigm and the behavioural correlate of lesion size. Two factors may be responsible for the lack of better significance: first, a ceiling-effect may be at play with the lesions being so large a small behavioural difference may not be observed. This leads into the second point - the motor behavioural scale we are using is perhaps not sensitive enough to detect slight differences between the groups at the earlier timepoints. Paw-placement kinematics or rotarod performance would have been good supplements to our scale. However, by the last day of treatment, there was a trend toward reduction in motor behaviour dysfunction in the rAd-Nrf2 group versus the rAd-GFP controls. In these toxicity experiments, it is worthy to note not all the animals treated with 3-NP manifested visible lesions and only those with visible cresyl violet lesions were included in the above analyses. The main factors likely contributing to the differences in responses to 3-NP are the outbred nature & normal variability of the rat stock used in the study70 and systematic errors including: unsuccessful IP injections (even seasoned experimenters miss -10-20% of IP injections) & high stress in the animals leading to altered pharmacokinetics (faster drug metabolism and clearance)64. However, the smaller lesion volumes observed in Nrf2-infected animals versus the GFP-infected controls are real and significant. This is corroborated by a higher number of NeuN+ cells in both the ipsilateral and contralateral hemispheres of rAd-Nrf2 animals compared to GFP-controls. Our 32 findings are further supported by our other studies using Nrf2 -/- mice, which are more sensitive to 3-NP toxicity than their wild-type counterparts and by the work of Calkins etal. (2005) who showed Nrf2-mediated neuroprotection against malonate-induced toxicity by competitive inhibition of SDH, in Nrf2 -/-mice transplanted with Nrf2-overexpressing astrocytes91. Results of gliosis assessment are quite interesting although a caveat attached to these (as well as the NeuN cell counts) is that only a small number (n=1 per vector group) of animals could be included in the study due to equipment failure and time constraints. Further investigation of the remaining animals would yield a clearer picture. Nonetheless, the increase in area of GFAP immunoreactivity and the mean pixel intensity of the ipsilateral hemisphere of rAd-Nrf2 animals are intriguing. Since this is the only hemisphere showing an increase in glial number/reactive astrocytes and as control rAd-GFP animals do not show this increase in glial number/reactivity, the presence of the Nrf2 transgene may be responsible for this effect. How Nrf2 could be mediating an increase in glial cell number/reactivity is speculative at best. A glance at microarrays18,19 comparing rAd-Nrf2 versus rAd-GFP transfected astrocytes yields a number of candidate genes including: numerous cell surface/adhesion molecules, chemokines (IL-7) and chemokine receptors (CXCR4 family). The ability of these genes to affect astrocyte number and reactivity should be further explored in an attempt to explain these interesting in vivo results. 33 Our in vitro experiments suggest there is a mechanistic link between 3-NP toxicity and Nrf2 activation. Other studies undertaken in our laboratory support the western blot data presented here. We have shown activation of a heat-stable placental alkaline phosphatase (hPAP) reporter gene under the control of the ARE promoter element and also accumulation of a GFP-tagged version of Nrf2 into the nucleus of COS-1 cells when these cells were exposed to 3-NP9 2. However, the concentrations of 3-NP used in these experiments is quite high (10-30mM range for activation) which is why we propose our testing method, using media containing 5mM glucose and 3-NP treatment with more relevant concentrations of the toxin, is a better model of the in vivo testing paradigms. It would be interesting to repeat the reporter and translocation experiments using the more physiological conditions shown here to verify the 3-NP-induced increases in Nrf2 target genes. One should note the variability observed in the densitometry analyses. This could be due to different developing conditions during the ECL reaction. Data however, was carefully expressed as % control to avoid any error in directly comparing the normalized optical density measurements from the 3 included experiments. As for the dosage experiments in the lateral ventricles, the optimal titer of virus (5 x 108 pfu) for this area was found to be quite toxic in the striatum, whereas this high titer was the only one to infect a fair number of cells along the ventricle walls. Presumably, this occurs because the vector is diluted into the CSF thus lowering the final concentration. Furthermore, ICV injection of the vector results in distribution throughout the CSF and this explains why GFP fluorescence is observed 34 in the contralateral and 4 ventricles even though the vector was only administered to the right lateral ventricle. Ectopic expression of xCTGFP in vivo demonstrated plasma membrane localization consistent with the punctate staining pattern observed in vitro. Interestingly, in vivo overexpression yielded an accumulation of fluorescence on the apical side of ependymal cells - an effect that would have otherwise been missed in vitro as not all glial cells are polarized in this environment. In hindsight, given the proposed function of xCT, this polar localization is not surprising. As with other members of the HAT family and with other transporters, localization to the apical or basolateral membrane is common. Our findings of apical localization for xCT supports the hypothesis of Sato et al. 6 6 who maintain xCT plays an important role in maintenance of the cysteine/cystine ratio in the CSF and therefore redox homeostasis of the CSF. The balance between cysteine and cystine could serve as a major redox buffer in the CSF in vivo as it does in cell-culture models93. Once inside the cell, cystine is converted to cysteine and either incorporated into proteins or glutathione but it can also be released via the neutral amino acid transporter back into the CSF where it can react with oxygen to form cystine, which can once again be taken up by xCT 6 6. This cycle would thus act as a buffer against oxidative stressors in the CSF. Production of the cellular anti-oxidant glutathione by ependymal cells could also contribute to the maintenance of CSF and ECF redox states. As previously mentioned, import of cystine into the cell is the rate-limiting step in glutathione biosynthesis63. Production of glutathione in cells along the CSF/ECF border could be helpful in clearing harmful xenobiotics from the system via uptake of these compounds directly into the ependymal cells for 35 conjugation and subsequent excretion into the CSF for clearance from the organism. Secretion of glutathione via MRP1, which is thought to localize to the basolateral membrane of ependymal cells, could also possibly help control redox status of the cells surrounding the ependyma, especially in times of oxidative crisis as happens in some pathological conditions. 4.2 Future Directions This study has shown the Nrf2 pathway can protect the striatum against 3-NP induced toxicity. It has also raised interesting questions about the possible mechanisms of Nrf2 protection in this paradigm. First, the accumulation of GFAP + cells in the ipsilateral hemisphere of rAd-Nrf2 infected animals was unexpected. However, a glance at microarrays comparing rAd-Nrf2 transfected glial cells to rAd-GFP transfected controls yields a few candidate molecules (IL-7, various cell surface/adhesion proteins, etc.) that could be involved in chemotaxis of untransfected glial cells toward cells overexpressing Nrf2. Boyden chamber assays using rAd-Nrf2 infected astrocytes in one compartment and untransfected astrocytes in the other would be one way of testing if rAd-Nrf2 transfected glia can cause chemotaxis of astrocytes versus rAd-GFP transfected controls. It would also be appealing to repeat the 3-NP toxicity experiments with an Nrf2DN group to verify the hypothesis animals lacking Nrf2 function would show bigger lesions and more motor behaviour dysfunction than wildtype or rAd-Nrf2 infected animals and to see how gliosis may be modulated in this group. Repeating experiments with Lewis rats, who develop lesions following 3-NP administration in a more consistent fashion than the 36 Wistar rat strain used in this study (E. Brouillet, personal communication), would also be useful. Of course, knowing 3-NP can induce Nrf2 is only one step in elucidating the actual mechanisms of activation. The most likely way in which this occurs is via production of ROS and RNS, especially peroxynitrite, by 3-NP. Although some evidence suggests ROS/RNS can be directly produced by inhibition of SDH by 3-NP 9 4 , free radicals are most likely produced as a result of secondary excitotoxicity. This secondary excitotoxicity is caused by the increased sensitivity of NMDARs to glutamate (due in part to relief of the Mg 2 + block and a decrease in the K m for agonist binding) and a subsequent increase of C a 2 + influx into the cell 5 2. 3-NP also causes mitochondrial dysfunction that impairs the ability of the mitochondrial intracellular C a 2 + buffering system to properly sequester this extra C a 2 + which can, through second messenger activities, lead to activation of phospholipases (such as phospholipase A2, implicated in arachidonic acid signalling leading to initiation of apoptotic processes) and nitric oxide synthase (leading to peroxynitrite formation).46, 9 5 By indirectly assessing the degree of lipid peroxidation 9 6 , 9 7 and also the amount of 3-nitrotyrosine98, ROS/RNS have been shown, without a doubt, to be present in excess in the brain following 3-NP administration. The 3-nitrotyrosine assay is of particular interest as it is indicative of peroxynitrite - a free radical known to activate Nrf2 through PI3K20. The presence of ROS/RNS would then induce the translocation of Nrf2 to the nucleus to activate the Phase II detoxification response. This, in turn, favours the quenching of free radicals and reactive species present in the cell and tips the balance toward cell survival instead of apoptosis. Starting in vitro, it would be interesting to monitor ONOO" levels in primary glial cells treated in our in vitro 37. paradigm via a fluorescence-based assay utilizing a dye such as 2',7'-dihydrodichlorofluorescein diacetate that becomes fluorescent upon binding to ONOO"99. Using various inhibitors or free radical spin traps at each step in the proposed pathway and looking at output (either Nrf2-induction via an ARE-reporter assay or cell survival via cell counts and immunostaining for apoptot[c markers like Annexin-V and TUNEL for more downstream manipulations (see Saraste and Pulkki (2000) and van Heerde etal. (2000) for reviews of apoptosis)1 0 0 , 1 0 1 would confirm our proposed mechanism for Nrf2-mediated neuroprotection following 3-NP administration. 3-NP can also increase Ca 2 + within astrocytes by reversal of the Na +/Ca 2 + exchanger102 which then leads to ionic imbalances, oxidative stress, second messenger signals and Nrf2 activation. The astrocytes could then secrete glutathione/glutathione precursors in order to help neurons maintain redox homeostasis. Because astrocytes express more Nrf2 than neurons103, a further look at neuronal-glial interactions in co-culture experiments is also warranted. To solidify the role of xCT in maintenance of CSF redox state as hypothesized by Sato66 and Bannai93, and confirmed by our apical localization of the xCTGFP fusion protein, immunohistochemistry verifying the in vivo localization of MRP1 to the basolateral membrane and EAAT1 (a glutamate transporter thought to be important in reuptake of glutamate effluxed by xCT into the CSF) to the apical membrane could be undertaken. A good follow up to localization of the transporter is confirming its function in pathological processes using animal models of diseases where CSF and ECF redox state are aberrant. Furthermore, the role of xCT and its 38 viability as a therapeutic target, could be expanded upon by creating xCT knockout animals to investigate what happens to CSF and ECF redox state when the antiporter is absent and whether this increases susceptibility to disease. 4.3 Conclusion We have found overexpression of the transcription factor Nrf2 reduces lesion size in a 3-nitropropionic acid toxicity paradigm in the adult male rat. This points to an important role for Nrf2 in maintaining redox homeostasis within the cell (by activation of phase II enzymes which quench ROS and other electrophiles) and in tilting the balance toward cell survival following an oxidative insult. Furthermore, in keeping with the global role of Nrf2 in redox homeostasis, a downstream gene product, xCT, has been found, when overexpressed in vivo in the ependymal cells lining the lateral ventricles, to accumulate on the apical surface of the plasma membrane. 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Murphy TH, Yu J, Ng R, Johnson DA, Shen H, Honey CR, Johnson JA. Preferential expression of antioxidant response element mediated gene expression in astrocytes. J Neurochem. 2001;76:1670-1678 48 APPENDIX 1 Figures 49 Figure 1. Viral dosage determination in the striatum 72h following rAd-GFP administration. Representative images of animals (n=3 per dose) injected with the indicated amount of viral vector (given in total plaque forming units) into the right striatum 72h post-injection. Both the corpus callosum and the striatum show GFP fluorescence in the ipsilateral hemisphere (columns A and B) and no fluorescence in the hemisphere contralateral to the vector injection site (column D). Note the apoptotic-like morphology of GFP+ cells at the highest and mid-range doses of rAd-GFP, especially upon magnification (column C). The vehicle-injected controls show no fluorescence in any area and are included, in part, as imaging controls (row 4). Scale bars - 50um. 50 IS Figure 2. Viral dosage determination in the striatum 7d following rAd-GFP administration. Representative images of animals (n=3 per dose) injected with the indicated amount of viral vector (given in total plaque forming units) into the right striatum 7d post-injection. Again, both the corpus callosum and striatum were positive for GFP fluorescence in the ipsilateral hemisphere (columns A and B, magnifications in column C) although very little GFP+ cells can be observed in the mid-range and highest doses (rows 2 and 3). In fact, there were so little GFP+ cells that an image could not be obtained for the striatal area of animals injected with 5 x 107 pfu. Contralateral hemispheres show no fluorescence (column D). Vehicle controls also do not show GFP fluorescence and have been included, in part, as imaging controls (row 4). Scale bars - 50um. 52 Figure 3. Characterization of the area and timecourse of infection by the rAd-GFP vector. A) GFP fluorescence (green, with propidium iodide counterstain in red) can be observed in the corpus callosum and striatum as early as 24h following injection (left panel) until at least 21 d later (right panel). B) Recreation of a 3-dimensional brain using sections from an animal injected with rAd-GFP and sacrificed 14d post-injection overlayed with the appropriate sections of the rat brain in order to show vector diffusion in the anterior - posterior axis, a - anterior, p - posterior. C) Serial sections demonstrating the expression of the GFP reporter (green, again with propidium iodide counterstain) over several anatomical levels (clockwise from top left: +1.00mm, +0.48mm, -0.26mm and -0.80mm from Bregma) in an animal infected with rAd-GFP and sacrificed 21 d post-injection. D) Graphical representation of % infected "striatal + corpus callosum" volume at 24h (n=2 animals), 48h (n=1), 3d (n=3), 5d (n=3), 7d (n=2), 14d (n=2), and 21d (n=2) following vector administration. All scale bars- 500pm. 54 \ • i f B c D E 100 a 80 1 70 Q. 60 u-(3 50 5 40 § 30 20 10 GFAP s100b NeuN 04 Figure 4. rAd-GFP preferentially infects astrocytes. Representative images of immunohistochemistry for the cell-type specific markers GFAP (A), s100B ( B ) , NeuN ( C ) , and 04 (D). The amount of co-localization between GFP fluorescence and these markers is quantified and graphically represented in E (for all markers: n=10 fields at each of 4 anatomical levels showing the most GFP fluorescence from 3 separate animals). Scale bars - 10pm in A , C , D and 50pm in B . . 56 i f i " Figure 5. GFP reporter expression in rAd-Nrf2 infected animals. A) Representative image of GFP fluorescence seen in an animal infected with rAd-Nrf2 confirming transgene expression. Scale bar - 500um B) Magnification of four GFP+ cells in a striatal field also stained for NeuN (red) and GFAP (blue). Scale bar - 50um. 57 A * > B 1 c **- * 1 * D • E F G ^ 1 0 0 l g 80-+ S 70-Q . BO-I L O 50 !» .? 20-O o 10-* o. • c PFP Ifl * 1 ** 1 GFP MT1/2 Co-localization Figure 6. Expression of the Nrf2 target metallothionein-1 is increased in rAd-Nrf2 versus rAd-GFP infected animals. Representative images of GFP fluorescence (A, D) and immunohistochemistry for metallothionein-1 (B,E) in rAd-GFP (A-C) and rAd-Nrf2 (D-F) infected animals. There is a marked increase in co-localization between these two markers in rAd-Nrf2 infected animals compared to controls (G). * p<0.05, ** p<0.01, two-tailed t-test (n=10 fields at the anatomical level showing the most GFP fluorescence from 2 animals per viral vector group). Scale bars - 50pm. 58 Figure 7. Behavioural scores of rAd-GFP and rAd-Nrf2 infected animals treated with 3-nitropropionic acid or saline over 4 days. A) rAd-GFP infected animals treated with 3-nitropropionic acid (n=5) demonstrate significant motor impairment on days 2, 3, and 4 of treatment compared to animals injected with saline (n=5). B) rAd-Nrf2 infected animals administered 3-nitropropionic acid (n=5) also show significant motor abnormalities on days 2 and 3 when compared to saline treated controls (n=5). C) rAd-GFP and rAd-Nrf2 infected animals administered 3-nitropropionic acid (n=5 per viral vector group) demonstrate similar motor abnormalities except on the fourth day of treatment when rAd-GFP animals perform worse than rAd-Nrf2 infected animals in motor behaviour tests. * p<0.05, ** p<0.01, ***p<0.001, one way A N O V A and post-hoc Tu key's test. 59 Ad-GFP Ad-Nrf2 Figure 8. Lesion size is decreased in rAd-Nrf2 infected animals following 3-nitropropionic acid treatment. A) Representative images of cresyl violet stained sections taken at +0.70mm from Bregma of saline (n=5 per viral vector group) or 3-nitropropionic acid (n=5 per viral vector group) treated rAd-GFP and rAd-Nrf2 infected animals. Arrow represents hemisphere in which the viral vector was delivered. Scale bar - 2mm. B) Quantification of lesion volume in the 3-nitropropionic acid treated groups using sections from +1.70mm, +1.20mm, +0.70mm, +0.20mm, -0.26mm, and -0.80mm from Bregma. * p<0.05, two-tailed t-test. 61 Figure 9. Determination of NeuN+ cells in 3-nitropropionic acid treated animals following rAd-GFP or rAd-Nrf2 administration. A) Representative immunohistochemistry images taken at -0.26mm from Bregma of NeuN staining in ipsilateral (right) and contralateral (left) hemispheres of rAd-GFP and rAd-Nrf2 infected animals. Scale bars - 500 pm. B) Graphical depiction of average NeuN+ cell counts per field (n=15 random fields at levels +1.70mm, +1.20mm, +0.70mm, +0.20mm, -0.26mm, and -0.80mm from Bregma) analyzed throughout the striatum of infected animals treated with 3-nitropropionic acid (n=1 per viral vector group). *** p<0.001 two-tailed t-test for comparison between rAd-GFP and rAd-Nrf2 infected animals. ***p<0.001 two-tailed paired t-test between the hemispheres of an animal. 62 63 Figure 10. Gliosis in rAd-GFP and rAd-Nrf2 infected animals treated with 3-nitropropionic acid. A) Representative immunohistochemistry images taken at -0.26mm from Bregma of GFAP staining in ipsilateral (right) and contralateral (left) hemispheres of animals administered rAd-GFP and rAd-Nrf2 vectors. Scale bars -500 pm. B) Graphical depiction of mean pixel intensity of GFAP immunoreactivity throughout the striatum of infected animals (n=1 animal per viral vector group) following 3-nitropropionic acid treatment at levels +1.70mm, +1.20mm, +0.70mm, +0.20mm, -0.26mm, -0.80mm from Bregma. C) Graphical representation of GFAP immunoreactive area (mm2) throughout sections of the striatum (levels same as in Figure 9) in GFP and Nrf2 overexpressing animals after receiving the 3-NP regime (n=1 per viral vector group). * p<0.05, two-tailed paired t-test between the hemispheres of an animal. 64 65 Figure 11. 3-nitropropionic acid induces expression of the Nrf2 target gene Heme-Oxygenase-1. A) Western blots for HO-1 and actin (as loading control) from rat primary glial cells treated with 1mM 3-NP (lane 1), 0.5mM 3-NP (lane 2), 0.1 mM 3-NP (lane 3), or 0.1% phosphate buffer vehicle control (lane 4) and rAd-Nrf2 infected glial cells (lane 5) as positive control of Nrf2 induction. B) Quantification of optical density (O.D.) measurements taken from the HO-1 blot and normalized to actin loading controls shown in A. C) Results of densitometry of HO-1 normalized to actin across various treatment conditions. Data is expressed as % control condition. 66 Figure 12. Proposed mechanisms of Nrf2 induction and neuroprotective action following 3-nitropropionic acid administration. Systemic administration of the succinate dehydrogenase (SDH) inhibitor 3-NP results in ATP depletion (45, 47) and production of reactive nitrogen and oxygen species (RNS and ROS, respectively).(46, 94) Secondary excitotoxicity involving NMDA receptors and glutamate transporters (49, 50, 52-54) is the most likely contributor to free radical formation following 3-NP administration although ROS formation resulting from direct inhibition of SDH is also a possibility.(94) RNS and ROS can lead to release of Nrf2 from the actin-bound cytoplasmic regulatory protein Keapl.(4, 20) The subsequent translocation of Nrf2 into the nucleus and dimerization with small Maf proteins (15) initiates transcription of genes containing anti-oxidant response elements within their promoter regions (AREs). Many of these genes are Phase 2 enzymes (18, 19) that detoxify the cell and help maintain redox homeostasis thereby combating oxidative stress and promoting cell survival. (Inset) Further [Ca2+]i increases from reverse operation of the Na+/Ca2+ exchanger (102) can also contribute to ionic imbalance, oxidative stress and Nrf2 induction in astrocytes which can then secrete glutathione (GSH), GSH precursors and other trophic/antioxidant factors to help neurons maintain redox homeostasis.(65, 88) Because astrocytes express more Nrf2 than neurons (103) this pathway may also contribute to the neuroprotection observed from oxidative stress during metabolic inhibition by 3-NP. 68 secondary excitotoxicity 69 ipsilateral contralateral Figure 13. Viral dosage determination in the right lateral ventricle 3d following administration of rAd-GFP vector. GFP fluorescence can only be observed at high titers (rows 4 and 5) in the ependymal cells lining the right (ipsilateral, columns 1 and 2) and left (contralateral, columns 3 and 4) lateral ventricles. At the highest titer used, GFP fluorescence could also be observed in the 4th ventricle (A5 and B5). Phase contrast images are included to better visualize ventricles (n=3 animals per dose). Scale bars -50pm. 70 o CD > CD o X LO o X LO o  o X LO phase contrast GFP •A1 • • ' B1 A2 B2 A3 , B3 A4 B4 Figure 14. Viral dosage determination in the right lateral ventricle 7d after rAd-GFP vector delivery to this area. At this timepoint, GFP fluorescence could no longer be observed for any of the viral titers used. Phase contrast images are included to better visualize ventricles (n=3 animals per dose). Scale bars - 200pm. 71 Overlay L Figure 15. Ectopic expression of the cystine/glutamate antiporter xCT. A) Schematic representation of the xCTGFP fusion protein. The GFP tag has been added to the C-terminal region of the protein using glycine linkers. B) Expression of GFP in GFP-transfected (left panel) and xCTGFP-transfected (right panel) primary glial cells. Arrows denote the punctate staining pattern observed in xCTGFP-transfected cells suggesting plasma membrane localization. Scale bars - 25pm. C) In vivo expression at +0.20mm from Bregma of GFP fluorescence in rAd-GFP infected (top row) and xCT-GFP infected (bottom row) animals sacrificed 3d following vector administration to the right lateral ventricle (n=1 per viral vector group). xCTGFP appears localized to the plasma membrane of cells and is excluded from the nucleus according to lack of co-localization with the propidium iodide nuclear counterstain (red), unlike cells infected with GFP-only. Scale bar - 20pm. 7 2 A 'apical' 'basolateral' Membrane localization Figure 16. xCTGFP is enriched on the apical side of infected ependymal cells. A) rAd-GFP infected controls show uniform fluorescence (inset graph) when a section (line) is taken across a particular cell. B) rAd-xCTGFP infected animals demonstrate increased fluorescence intensity on the apical side of the membrane (inset graph) when a cross-sectional line is drawn through a particular cell. Scale bar -10pm. C) Quantification of mean pixel intensity of rAd-xCTGFP infected cells (n=26 cells from 1 animal) showing a very significant decrease in mean pixel intensity on the basolateral membrane. *** p<0.001, two-tailed paired t-test. 73 


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