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Two-photon imaging of glutathione levels in intact brain indicates sites of enhanced redox buffering 2006

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TWO-PHOTON IMAGING OF GLUTATHIONE L E V E L S IN INTACT BRAIN INDICATES SITES OF ENHANCED REDOX BUFFERING by XIAOJIAN SUN B.Sc, Tsinghua University, Beijing, P. R. China, 2003 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science In The Faculty of Graduate Studies Neuroscience THE UNIVERSITY OF BRITISH COLUMBIA February, 2006 ©Xiaojian Sun 2006 Abstract Oxidative stress, the metabolic imbalance between oxidant creation and destruction is proposed as a final common pathway for neurodegenerative disease and injury. To map sites of antioxidant homeostasis in brain, I used two-photon imaging of monochlorobimane (MCB) fluorescence, a selective enzyme-mediated marker, for reduced glutathione (GSH) in both brain slices and in vivo preparations. I found that cells at the CSF or blood brain interface such as lateral ventricle ependymal cells, meningeal cells, and astroglia contain both high levels of MCB conjugation activity (glutathione S-transferase dependent) and GSH content. In comparison, cortical neurons in Layer II contained approximately 20% of the GSH content of their astrocyte counterparts. Regional mapping of GSH indicated that the highest levels were present in cells lining the lateral ventricles, specifically eperidymal cells and the subventricular zone. The enrichment of GSH content along the lateral ventricle suggested a possible function in oxidant homeostasis for developing neuronal progenitors. Consistent with this, I observed that developing neurons found in the subgranular zone of dentate gyrus, contained 3-fold more GSH than older neurons found in the neighbouring granular layer. Besides imaging GSH distribution with MCB, I also developed an assay to measure different kinetic parameters of GSH metabolism. With this assay, I found that meninges are more active than cortical cells in GSH metabolism. In conclusion, I have developed a powerful approach to map sites of antioxidant homeostasis in brain directly, demonstrating a unique role for GSH in developing neurons and cells at the CSF and blood-brain interface. T A B L E O F C O N T E N T S ABSTRACT ii TABLE OF CONTENTS iv LIST OF TABLES . ix LIST OF FIGURES x LIST OF ABBREVIATIONS xii ACKNOWLEDGEMENTS :xvi 1 CHAPTER 1 Introduction : 1 1.1 Overview of GSH metabolism in brain 1 1.1.1 Functions of GSH 1 1.1.2 Basic metabolism of GSH 2 1.1.3 GSH metabolism in brain 5 1.1.3.1 Synthesis and Consumption of GSH in brain 5 1.1.3.2 Release of GSH from astrocytes 6 1.1.3.3 Functions and fate of extracellular GSH in brain 7 1.1.3.4 Export ofGSSG from astrocytes during oxidative stress 11 1.1.4 GSH deficiency and neurological diseases 12 1.1.4.1 GSH in Parkinson's disease (PD) 13 1.1.4.2 GSH and stroke 14 1.1.4.3 Therapeutic approaches for neurodegenerative diseases 15 1.2 Imaging and measurement of GSH in brain 16 1.2.1 Different dyes to image GSH 17 1.2.2 Two photon imaging of GSH with MCB 18 1.3 Structure and function of meninges 19 1.3.1 Function of meninges 20 1.3.2 Meninges express high level of xCT, cystine/glutamate antiporter 21 1.4 Rationale of research 21 2 CHAPTER 2. Materials and methods 23 2.1 Chemicals 23 2.2 Astrocytes and meninges culture 23 2.3 Plate reader GSH assay , 24 2.4 Calibration of the GSH-MCB fluorescence , 25 2.5 Rat brain slices preparation 25 2.6 Animal preparation for in vivo imaging 26 2.7 Two-photon microscopy 27 2.8 Immunostaining .' 28 2.9 Glutathione S-transferase (GST) assay. 29 2.10 Total intracellular GSH assay 30 2.11 Data and image analysis 30 3 CHAPTER 3. Results 32 3.1 Specificity of MCB labelling 32 3.1.1 MCB labelling of GSH is dependent on GST 32 3.1.2 MCB could be used to label intracellular GSH with the plateau level of fluorescence intensity indicating the cellular GSH level 35 3.2 G S H metabolism assay . .39 3.2.1 Plate reader assay is a powerful system to measure intracellular G S H level in cultures 39 3.2.2 Plate reader assay can measure rate of G S H precursor uptake and synthesis 40 3.2.3 Plate reader assay can measure efflux of G S H - M C B conjugate 42 3.2.4 Plate reader assay can detect changes in G S H metabolism under oxidative stress 45 3.2.5 Conclusion 50 3.3 Feasibil i ty o f two-photon laser scanning microscopy ( T P L S M ) measurement o f cellular glutathione level 50 3.3.1 Assumption 1: Ef f lux of G S H - M C B at room temperature is i negligible 52 3.3.2 Assumption 2: M C B labelled al l G S H content in cel ls 55 3.3.3 Conclus ion 57 3.4 M C B labelling o f different brain regions in slices. 57 3.4.1 Meningeal and astrocytes in neocortex are labelled strongly by M C B in rat brain slices 59 3.4.2 M C B labelling of neurons in slices is much weaker than labelling o f astrocytes 62 3.4.3 Lateral ventricle ependymal cells have the highest G S H content in brain slices 66 vi 3.4.4 A subpopulation of developing neurons in dentate gyrus are labelled by MCB 73 3.4.5 Measurement of GSH concentration in different brain regions in acute brain slices 80 3.4.6 Conclusion 84 3.5 MCB labels a subpopulation of meningeal and neocortical cells in vivo 84 3.6 Meningeal cells have a more active GSH system than astrocytes in neocortex 89 3.6.1 Meninges contain higher level GSH and GST activity than cortical cells... 89 3.6.2 Meninges cells are more efficient in taking up GSH precursors and synthesizing GSH 90 3.6.3 Immunostaining of cortical cell cultures 94 CHAPTER 4. Discussion 96 4.1 MCB labelling with two-photon microscopy resolves cellular GSH in situ..96 4.2 Cellular GSH distribution in brain slices 98 4.2.1 GSH is highly expressed in cells at CSF interface 98 4.2.2 GSH in neurons 99 4.2.3 Summary of types of cells labelled by MCB in slices 103 4.3 Manipulation of GSH metabolism 106 4.4 Measurement of GSH within the cortex of live animals 109 4.5 Meninges may be an ideal position for protecting brain against oxidative load i 110 4.6 Conclus ion I l l R E F E R E N C E S 113 viii 4.5 Meninges may be an ideal position for protecting brain against oxidative load 110 4.6 Conclusion 112 REFERENCE 113 ( viii LIST OF TABLES Table 3-1. Comparison of all kinetics parameters of G S H metabolism between tBHQ treatment and control (DMSO) groups 49 Table 3-2. Comparison of kinetic parameters of GSH metabolism between meninges and cortical cell cultures 92 Table 4-1. Types of cells labelled by M C B 105 LIST OF FIGURES Figure 1-1. Metabolism of G S H 4 Figure 1-2. Metabolic interaction between astrocytes and neurons in the G S H metabolism of the brain 9 Figure 3-1. M C B can specifically label GSH 34 Figure 3-2. Monitoring GSH metabolism in astrocytes cultures with plate reader. 37 Figure 3-3. Fluorescence images (10X) of G S H - M C B in untreated (A) and MK571-treated (B) astrocytes cultures 44 Figure 3-4. Changes in G S H metabolism induced by activation of ARE-mediated gene expression 48 Figure 3-5. Standard curve for the relationship between fluorescence intensity and G S H - M C B concentration.... 51 Figure 3-6. Efflux of G S H - M C B in slices at room temperature can be neglected. 53 Figure 3-7. G S H - M C B efflux can be neglected in room temperature 54 Figure 3-8. Time course curve of M C B labelling in different brain regions 56 Figure 3-9. M C B and PI labelling of slices 58 Figure 3-10. M C B labels the meninges and astrocytes robustly 61 Figure 3-11. M C B labelling of mature cortical neurons 64 Figure 3-12. M C B labelling of GSH-containing ependymal cells lining the lateral ventricle 67 Figure 3-13. Many cells labelled by MCB in subventricular zone (SVZ) are labelled by doublecortin (DCX) 69 Figure 3-14. Some cells labeled by MCB along lateral ventricle and in SVZ are also labeled bySlOOp 70 Figure 3-15. Cells labeled by MCB along lateral ventricle and in SVZ are not labeled by GFAP 71 Figure 3-16. Mature neurons in SVZ are not labelled by MCB 72 Figure 3-17. MCB labelling of GSH-containing cells in dentate gyrus 74 Figure 3-18. Many cells in subgranular zone are colabelled by MCB and SlOOp. 76 Figure 3-19. MCB labelled cells in dentate gyrus are GFAP-positive 77 Figure 3-20. Many cells in subgranular zone are colabelled by MCB and DCX. 78 Figure 3-21. Mature neurons labelled by NeuN are not labelled by MCB 79 Figure 3-22. Quantification of GSH concentration in different brain regions 83 Figure 3-23. In vivo two-photon fluorescence image of the somatosensory cortex of a live anesthetized mouse after application of MCB and SR 101 87 Figure 3-24. GSH metabolism is more active in meninges than in cortical cells 93 Figure 3-25. MCB labelled cells in cortical cells culture are si00(3 positive 95 Figure 4-1. Comparison of MCB labelling in young and mature rat slices 101 Figure 4-2. DEM depletion of intracellular GSH content 108 X I LIST OF ABBREVIATIONS °C Degrees centigrade yGT y-glutamyl transpeptidase urn micrometer uM micromolar ABC ATP-binding cassette ACSF artificial cerebral spinal fluid ANOVA analysis of variance between groups ApN aminopeptidase N ATP adenosine triphosphate BSO DL-buthionine-(S,R)-sulfoximine CDNB l-chloro-2,4-dinitrobenzene Cktl cocktail CM AC t-butoxycarbony l-Leu-Met-7-am ino-4-chloromethylcoumarin CMAC-blue 7-amino-4-chloromethylcoumarin CMFDA 5-chloromethylfluorescein diacetate CSF cerebral spinal fluid DCX doublecortin DEM diethyl maleate DG dentate gyrus DTNB 5,5'-dithio-bis-2-nitrobenzoic acid FITC fluoresceine isothiocyanate GFAP glial fibrillary acidic protein GPx glutathione peroxidase GR glutathione reductase GSH glutathione GSSG glutathione disulfide GST glutathione S-transferase h hour HBSS Hank's Balanced Salt Solution HPLC high performance liquid chromatography LV lateral ventricle Mapll microtubule associated protein 2 MBB monobromobimane MCB monochlorobimane min minute mM millimolar MRP multidrug resistance protein NADPH nicotinamide adenine dinucleotide phosphate (reduced form) NDA 2,3-naphthalenedicarboxaIdehyde NMDA N-methyl-D-aspartate NSE neuronal specific enolase Nrf2 NF-E2-related factor 2 OPD o-phthaldehyde PBS phosphate buffered saline PFA para formaldehyde PI propidium iodide PMT photomultiplier tube QBB monobromotrimethylammoniobimane ROS reactive oxygen species RT-PCR real-time polymerase chain reaction sec second SEM standard error of the mean SGZ subgranular zone SOD superoxide dismutase SRIOI sulforhodamine 101 SVZ subventricular zone tBHQ tert-Butylhydroquinone TPLSM two-photon laser scanning microscopy Acknowledgements First, I would like to thank my husband for his unconditioned support all the time. I would also like to thank my parents for their kindly support during my study. I would also like to thank Dr. Timothy H. Murphy for his guidance, inspiration, supportiveness and encouragement during the course of my Master study. I would also like to thank Dr. Lynn Raymond, who gave me lots of advices in our lab meeting. I would like to thank all the members of the Murphy lab and Raymond, especially Andy Y . Shih, Shengxiang Zhang and Zhi Liu, for advice and friendship; and Ping L i , Heidi Erb, Lei Jiang, for help on my experiments, as well as other members, all of whom are wonderful colleagues and friends to work with. I would also like to thank my supervisory committee, including Dr. Brian MacVicar, Dr Yutian Wang, and Dr Lynn Raymond for helpul suggestions on my projects. xvi Chapter 1 Introduction The cells of the human brain utilize 20% of the oxygen consumed by the body but constitute only 2% of the body weight (Clarke and Sokoloff, 1999). This indicates the . potential generation of a high quantity of reactive oxygen species (ROS) during oxidative phosphorylation in the brain. ROS generation has to be counter-balanced by an appropriate antioxidative defense to enable a long human life. Glutathione (GSH), as an essential cellular antioxidant, plays a key role in the defense of brain cells against oxidative stress (Cooper, 1997; Cooper and Kristal, 1997; Dringen, 2000). Insufficient antioxidative defense or increased generation of ROS can cause oxidative stress. For the brain, oxidative stress has been connected with the loss of neurons during the progression of neurological diseases, e.g., Parkinson's disease (PD), Alzheimer's disease (AD) , Huntington's disease (HD) and stroke (Bains and Shaw, 1997; Cooper, 1997; Dringen, 2000; Schulz et al., 2000; Bharath et al., 2002). /./ Overview of GSH metabolism in brain 1.1.1 Functions of GSH The tripeptide G S H is the major cellular thiol in mammalian cells (Cooper, 1997). It has important functions as antioxidant, is a reaction partner for the detoxification of xenobiotica, is a cofactor in isomerization reactions, and is a storage and transport form of cysteine (Meister and Anderson, 1983; Cooper, 1997). In addition, G S H is essential for cell proliferation (Poot et al., 1995) and maintains the thiol redox potential in cells, keeping sulfhydryl groups of proteins in the reduced form (Cotgreave and Gerdes, 1998). Besides, G S H also plays a role in the regulation of apoptosis (van den Dobbelsteen et al., 1996; Ghibell i et al., 1998; Hal l , 1999). The G S H system is very important for the cellular defense against R O S . A high intracellular concentration of G S H protects against a variety of different R O S . G S H reacts directly with radicals in nonenzymatic reactions (Saez et al., 1990; Winterbourn and Metodiewa, 1994) and is also an electron donor in the reduction of peroxides catalyzed by glutathione peroxidases (GPx) (Chance et al., 1979). It should be noted that the G S H system is only part of the cellular defense system against R O S . Other enzymes, such as superoxide dismutase (SOD) and catalase, as well as antioxidants, such as ascorbate and a-tocopherol, are also involved in ROS detoxification (Meister, 1994; W o l f et al., 1998; Gate et al., 1999). 1.1.2 Basic metabolism of GSH G S H is synthesized in vivo by the consecutive action of two enzymes (Meister, 1974; Figure 1-1). yGluCys synthetase uses glutamate and cysteine as substrates forming the dipeptide yGluCys, which can be combined with glycine in a reaction catalyzed by glutathione synthetase to generate G S H . Adenosine triphosphate (ATP) is a cosubstrate for both enzymes. The intracellular level of G S H is regulated by a feedback inhibition of yGluCys synthetase by the endproduct G S H (Richman and Meister, 1975; Misra and Griffith, 1998). Therefore, cellular synthesis and consumption of G S H are balanced. 2 During detoxification of ROS, G S H is involved in two types of reactions: (i) G S H reacts nonenzymatically with radicals such as the superoxide anion, nitric oxide or the hydroxyl radical (Saez et al., 1990; Clancy et al., 1994; Winterbourn and Metodiewa, 1994; Singh et al., 1996) and (ii) G S H is the electron donor for the reduction of peroxides in the G S H peroxidases (GPx) reaction (Chance et al., 1979). The final product of the oxidation of G S H is glutathione disulfide (GSSG). Within cells G S H is regenerated from G S S G by the reaction catalyzed by glutathione reductase (GR). This enzyme transfers electrons from nicotinamide adenine dinucleotide phosphate (reduced form) ( N A D P H ) to G S S G , thereby regenerating G S H . During the reaction catalyzed by G P x and G R , G S H is not consumed but recycled (Figure 1-1). In contrast, during the generation of glutathione-S-conjugates by glutathione-S- transferases (GST) (Commandeur et al., 1995; Salinas and Wong, 1999) or by release of G S H from cells (Akerboom and Sies, 1990; Kaplowitz et al., 1996), the level of total intracellular G S H is lowered. Therefore, the G S H used for these processes has to be replaced by resynthesis from the constituent amino acids. Extracellular G S H and G S H conjugates are substrates for the ectoenzyme y-glutamyl transpeptidase (yGT). This enzyme catalyzed the transfer of the y-glutamyl moiety from G S H or a G S H conjugate onto an acceptor molecule, thereby generating the dipeptide CysGly or the CysGly conjugate, respectively (Meister et al., 1981; Commandeur et al., 1995). CysGly can be hydrolyzed by ectopeptidases (Tate, 1985) to cysteine and glycine, which are subsequently taken up by cells and can serve again as substrates for cellular glutathione synthesis (Figure 1-1). 3 Figure 1-1 C1SSG cell membrane T GSSG Figure 1-1. Metabolism of GSH (modified from Dringen 2000). ' X ' represents an acceptor of the y-glutamyl moiety transferred by yGT from G S H . ' Y ' , a substrate of G S T . 1, y-glutamylcysteine synthetase; 2, G S H synthetase; 3, G S H peroxidase; 4, G S H reductase; 5, G S T ; 6, y-glutamyl transpeptidase; 7, ectopeptidase. 4 1.1.3 GSH metabolism in brain 1.1.3.1 Synthesis and Consumption of GSH in brain G S H synthesis depends on the intracellular availability of the substrates, glutamate, cysteine and glycine. In the brain, these amino acids are not present extracelluarly in high concentrations, since glutamate and glycine are neurotransmitters (Moss and Smart, 2001; Nedergaard et al., 2002) and cysteine in higher concentration is neurotoxic (Janaky et al., 2000b). Therefore G S H synthesis in certain type of brain cells w i l l depend on its ability to use available extracellular G S H precursors. Neurons rely on the presence of extracellular cysteine for G S H synthesis and cannot use the cysteine-oxidation product cystine as G S H precursor (Sagara et al., 1993; Kranich et al., 1996). For neurons the best extracellular precursor for the glutamate moiety of G S H is glutamine (Kranich et al., 1996). In contrast to neurons, astrocytes prefer glutamate and cystine as extracellular G S H precursors (Kranich et al., 1996, 1998; Dringen and Hamprecht, 1998). This difference preference for extracellular G S H precursors prevents competition between neurons and astrocytes. However, since neurons cannot use extracellular cystine, these brain cells depend for their G S H synthesis on the supply of cysteine or a cysteine- precursor from neighboring astrocytes (Dringen et a l , 2000). Responsible for the cellular export of G S H , G S S G and GSH-S-conjugates are members of the family of multidrug resistance proteins (MRPs for human transporters; Mips for the transporters of other species) (Borst et al., 1999; Konig et al., 1999; Leslie et al., 2001). These transporters belong to the subgroup A B C C of the ATP-binding cassette 5. transporters and are ATP-driven export pumps of organic anion (Borst and Oude Elferink, 2002). For the human genome, nine paralogs have been reported (MRPT to M R P 9 ) (Borst et al., 1999; Konig et al., 1999). M R P l / M r p l and M R P 2 have been reported to mediate export of G S H and G S S G (Borst et al., 1999; Konig et al., 1999; Leslie et al., 2001). O f these two transporters, M R P l / M r p l is expressed in parenchymal brain cells in vitro (Decleves et al., 2000; Hirrlinger et al., 2001) and in vivo (Sisodiya et al., 2002). Therefore M R P l / M r p l is responsible for cellular export of G S H in brain. 1.1.3.2 Release of GSH from astrocytes Release of G S H from brain cells has been reported so far only for astrocytes (Yudkoff et al., 1990; Sagara et al., 1996; Hirrlinger et al., 2002). These cells release under unstressed conditions G S H and not G S S G (Sagara et al., 1996; Stone et al., 1999) and even protect the G S H exported against oxidation by a factor released into the medium (Stone et al., 1999; Stewart et al., 2002). In contrast to astrocytes, only marginal amounts o f G S H are released from cultures of neurons, microglial cells and oligodendrocytes (Hirrlinger et al., 2002c). The release of G S H from astrocytes was initially underestimated due to the consumption of extracellular G S H by the ectoenzyme yGT. When this enzyme is inhibited, the extracellular amount of G S H increases as a constant rate of about 3 nmol/(h xmg protein) (Dringen et al., 1997; Hirrlinger et al., 2002c). Astroglial cultures export within l h about 10% of their intracellular G S H (Dringen et al., 1997), which has continuously to be resynthesized from its precursors in order to maintain a constant cellular concentration. These data and the reported half life of about 5h for astroglial 6 G S H (Devesa et al., 1993) indicate that the export of G S H is quantitatively the most important process consuming astroglial G S H . M R P 1 and M R P 1 have been reported to mediate cellular export of G S H and G S S G (Borst et al., 1999; Konig et al., 1999; Leslie et al., 2001). O f these two transporters only M r p l is expressed in cultured rat astrocytes (Decleves et al., 2000; Hirrlinger et al., 2001). In the presence of the competitive Mrp-inhibitor M K 5 7 1 , at a concentration of 50 u M , the rate of G S H release from astrocytes was strongly reduced, indicating that M r p l is predominately responsible for the observed G S H export from astrocytes (Hirrlinger et al., 2001). Mrpl-mediated G S H export was only observed for cultured astrocytes, although substantial amount of m R N A of this transporter have also been found in cultured neurons, oligodendrocytes and microglial cells (Hirrlinger et al., 2002a). Currently it is unclear whether these brain cell types do not express function M r p l protein or whether the expressed protein is not functional as G S H exporter in these cells. A n astrocyte-specific cosubstrate necessary for Mrpl-mediated G S H export has been discussed to lower the high K m value of this transporter for G S H (Loe et al., 2000; Leslie et al., 2001). However, such a cosubstrate in astrocytes remains to be identified. Alternatively, Mrpl-mediated G S H export might depend on cell type-specific modifications of the transporter or on the presence of an additional regulatory protein that is expressed only in astrocytes. 1.1.3.3 Functions and fate of extracellular GSH in brain Besides the intracellular functions of G S H (Cooper, 1997; Cooper and Kristal, 1997; Dringen, 2000) this tripeptide has special extracellular functions in the brain. 7 Extracellular G S H is a key requirement for the metabolic interaction between astrocytes and neurons in G S H metabolism. The availability of cysteine determines the level of neuronal G S H (Dringen et al., 1999). Since the presence of astrocytes maintains (Sagara et al., 1993; Keelan et al., 2001) or even increases G S H in cultured neurons (Bolanos et al., 1996; Dringen et al., 1999), cysteine is provided to neurons in the presence of astrocytes. The mechanism of the supply of cysteine from astrocytes to neurons has been resolved (Figure 1-2). First step is the export of G S H from astrocytes. Using extracellular G S H as substrate the astroglial ectoenzyme yGT produces the dipeptide CysGly in equimolar concentrations to the G S H consumed (Dringen et al., 1997). Inhibition of yGT completely prevented the astroglia-induced effect on the G S H content in neurons, indicating the key role of astroglial yGT in the supply of cysteine for neuronal G S H synthesis (Dringen et al., 1999). On the other hand, inhibition of the neuronal aminopeptidase N (ApN) prevented the use of extracellular CysGly for G S H synthesis, demonstrating that intact CysGly is not taken up into neurons by a peptide transporter, but is hydrolyzed by A p N (Dringen et al., 2001). Consequently, cysteine availability for neuronal G S H synthesis depends on astroglial G S H export and extracellular processing of G S H by yGT and A p N . With the release of the glutamate precursor glutamine by astrocytes(Hertz et al., 1999) and the extracellular generation of CysGly from G S H , all three constituent amino acids of G S H are provided from astrocytes to neurons (Dringen et al., 2000). 8 Figure 1-2 astrocyte neuron Figure 1-2. Metabolic interaction between astrocytes and neurons in the G S H metabolism of the brain (modified from Dringen and Hirrlinger, 2003). The G S H released from astroglial cells via M r p l is a substrate for the astroglial ectoenzyme yGT. The CysGly generated serves as extracellular precursor of neuronal G S H . After hydrolysis of the dipeptide by aminopeptidase N (ApN), the amino acids cysteine and glycine are taken up into neurons. ' X ' represents an acceptor of the y-glutamyl moiety transferred by yGT from G S H . 9 G S H reacts non-enzymatically with a variety of different radicals. Therefore, extracellular G S H might function as a first line of defense against R O S generated in the extracellular space. Extracellular G S H could also serve as substrate for extracellular G P x (Brigelius-Flohe, 1999). However, the expression of this enzyme in brain cells and its function in the brain remains to be elucidated. In addition, the G S H released from astrocytes may, at least in part, contribute to the maintainance of the G S H level in cerebrospinal fluid (Anderson et al., 1989). Interestingly, a-reduced concentration of G S H in the cerebrospinal fluid of schizophrenic patients can be correlated with a lowered amount of G S H in the prefrontal cortex of the brain (Do et al., 2000). The cellular G S H consumed by export or by reactions of GSTs has to be replaced by synthesis of G S H from the constituent amino acids to maintain a constant intracellular steady state level. Alternatively, cellular uptake of intact G S H could replenish intracellular G S H levels. However, since extracellular concentrations of G S H in brain are at best 2 u M (Yang et al., 1994; Lada and Kennedy, 1997; Han et al., 1999), such an uptake would require high amounts of energy to accumulate G S H in cells against the steep concentration gradient established by the millimolar intracellular concentration of G S H . Elevation of cellular G S H content after incubation of cultured neurons (Sagara et al., 1996) or astrocytes (Dringen, 2000) with extracellular G S H has not been observed. Therefore, such uptake processes can most likely not substantially contribute to the replenishment of cellular G S H . > 10 1.1.3.4 Export of GSSG from astrocytes during oxidative stress For several tissues and cell types, a release of G S S G during oxidative stress has been reported and proposed as a mechanism of cellular self-defense (Sies et al., 1972; Akerboom et al., 1982; Sies and Akerboom, 1984; Ishikawa and Sies, 1989). The transporters responsible for the cellular export of G S S G have been identified as the M R P - family members M R P 1 (Leslie et al., 1996) and M R P 2 (Fernandez-Checa et al., 1992; Paulusma et al., 1999). O f these two transporters rat astrocytes express only M r p l (Hirrlinger et al., 2001, 2002a). G S S G is effectively transported by M R P 1 with a K m value of 93 u M (Leier et al., 1996). Under non-stressed conditions astrocytes do not export G S S G , since cellular G S S G is hardly present in the cells due to the function of G R (Hirrlinger et al., 2001). However, after application of peroxide, G S S G is transiently detectable in astrocytes (Dringen and Hamprecht, 1997; Dringen et al., 1998; Kussmaul et al., 1999) and a loss of cellular G S H was observed (O'Connor et al., 1995; Peuchen et al., 1996; Dringen and Hamprecht, 1997; Dringen et al., 1998; Kussmaul et al., 1999). In contrast to a bolus application of peroxides, application of a chronic hydrogen peroxide- induced oxidative stress by a peroxide generating system caused in cultured astrocytes a rapid and prolonged increase in intracellular G S S G (Hirrlinger et al., 2001). This export of G S S G from astrocytes was strongly inhibited in the presence of MK571 (Hirrlinger et al., 2001), demonstrating that M r p l mediates the export of G S S G from cultured astrocytes during oxidative stress. Consequently, besides its involvement in astroglial G S H export under unstressed conditions, M r p l mediates also G S S G export during oxidative stress. n Accumulation of extracellular G S S G can be a consequence of the oxidation of G S H released from astrocytes or of intracellular accumulation of G S S G during oxidative stress and subsequent export from cells. Consequently, extracellular G S S G can be considered as an indicator for oxidative stress. Nevertheless, G S S G may also have important extracellular functions in the brain. G S S G has been discussed as agonist and modulator of glutamate receptors in brain (Sucher and Lipton, 1991; Varga et al., 1994, 1997; Ogita et al., 1995;Janaky et al., 1999). Such functions may be especially important to protect neurons against glutamate excitoxicity, since extracellular G S S G downregulates the response to stimulation of the N M D A type of glutamate receptors on neurons (Sucher and Lipton, 1991). Whether such an antioxidative function of G S S G is important for the brain remains to be elucidated. 1.1.4 GSH deficiency and neurological diseases Oxidative stress was reported to connect with physiological processes such as aging (Finkel and Holbrook, 2000) and with pathological processes in neurological disorders (Bains and Shaw, 1997; Jenner and Olanow, 1998; Schulz et al., 2000; V i l a et al., 2001; Bharath et al., 2002). A reduction in G S H content of given areas of the human brain has been reported for P D (Sofic et al., 1992; Sian et al., 1994), schizophrenia (Do et al., 2000), Alzheimer's disease (Gu et al., 1998) and epilepsia (Mueller et al., 2001) as well as in rat models for Huntington's disease (Cruz-Aguado et al., 2000a, b). Presently it is not known whether the decline in the G S H concentration in pathological brains is due to insufficient synthesis of G S H , or to elevated consumption of G S H , which is not 12 compensated by increased G S H synthesis. Disturbance of G S H metabolism as well as G S S G export from brain cells during oxidative stress could contribute to the reduced G S H levels of diseased brain. L 1.4.1 GSH in Parkinson's disease (PD) Best evidence for a disturbed G S H metabolism in brain as an important factor contributing to the pathogenesis of a disease has been reported for P D (Schulz et al., 2000; Bharath et al., 2002). This disease is characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta. The etiology of the disease is unknown, but biochemical analysis of post mortem tissues provides evidence for oxidative stress in the substantia nigra during this disease (Jenner and Olanow, 1998; Schulz et al., 2000). The G S H content in this brain region is decreased by 40-50% compared with controls (Sofic et al., 1992; Sian et al., 1994). On the cellular level a significant loss of G S H in the surviving nigral neurons has been reported (Pearce et al., 1997). The importance of the decline of G S H levels during the progression is under scored by the lowered G S H level in the substantia nigra found for incidental Lewy body disease, a presymptomatic form of P D (Dexter et al., 1994). A loss of G S H alone appears not to be responsible for the nigrostriatal damage in P D , since reduction of brain G S H levels by chronical infusion of buthionine sulfoximine, an inhibitor of yGluCys synthetase, did not reduce the number of dopaminergic neurons (Toffa et al., 1997). The G S H depletion may rather enhance the susceptibility of brain cells against other harmful events, such as the reduction of mitochondrial energy production. A synergistic effect of 13 a lowered intracellular G S H concentration and a reduced A T P production in increasing the susceptibility of dopaminergic neurons has been described for the in vitro and in vivo situation (Zeevalk et al., 1997, 1998). 1.1.4.2 GSH and stroke Stroke is one of the main causes of adult disability and death. This disorder most commonly results from the occlusion of a major blood vessel in the brain (Ameriso and Sahai, 1997), resulting in localized reductions in blood flow. The affected tissue typically contains a severely ischemic core or focus surrounded by more moderately ischemic perifocal or penumbral regions (Jacewicz et al., 1992; Memezawa et al., 1992a). Reactive oxygen species (ROS) have been implicated in many studies as one important contributor to ischemic cell death (see for example L i u et al., 1989; Kinouchi et al., 1991; Cao and Phillis, 1994; Y u et al., 1998). G S H is a central component in the antioxidant defences of cells, acting both to directly detoxify R O S and as a substrate for several peroxidases (Dringen, 2000). Thus, alterations in the availability of this metabolite during ischemia or early reperfusion are likely to influence tissue damage. Consistent with this possibility, a treatment that depletes G S H by inhibiting synthesis has been found to increase infarct volume resulting from focal ischemia in rat brain (Mizui et al., 1992). Anderson et al. (2002) have found that total G S H was substantially decreased in mitochondria prepared from severely ischemic focal tissue in both cerebral cortex and striatum. Injecting G S H monoethyl ester, which can be converted to G S H in cells, to third ventricle prevented the G S H loss in mitochondria and significantly decreased infarct size after stroke (Anderson et al., 2004a, 2004b). 1.1.4.3 Therapeutic approaches for neurodegenerative diseases If alterations in glutathione metabolism play an important role in the pathogenesis of the neurodegenerative diseases, treatments that lead to enhanced synthesis of G S H or that inhibits its degradation may result in a slowing of disease progression. Because G S H itself penetrates the blood-brain barrier only poorly and cannot be taken up by neurons directly, treatments with G S H monoethyl ester, G S H precursors or other G S H analogs have been used in patients or animal models. The G S H analog Y M 7 3 7 provides protection against cerebral ischemia in rats by inhibiting l ipid peroxidation (Yamamoto et al., 1993). Since G S H synthesis in neurons is limited by the availability of cysteine (Dringen et al., 2000), compounds that can be metabolized to cysteine could be used as pro-drugs to increase neuronal G S H concentrations. In mice, treatment with the G S H precursor N-acetyl-L-cysteine resulted in a significant reduction of motor neuron loss and elevated G S H peroxidase levels with the cervical spinal cord (Henderson et al., 1996). Treatment with L-2-oxothiazolidine-4- carboxylate, a cysteine precursor, stimulates growth and normalizes tissue G S H concentrations in rats fed a sulphur amino acid deficient diet (Jain et al., 1995). Unfortunately, the therapeutic window for treatment with substances that increase brain cysteine may be narrow, because cysteine is potentially toxic for neurons (Olney et al., 1990). Alternatively, G S H in brain can be increased by intracerebroventricular 15 administration of the dipeptide y-glutamylcysteine (Pileblad et al., 1992). Neurons can utilize either glutamylcysteine or cysteinylglycine for the synthesis of G S H (Dringen et al., 1999). Although a disturbance of G S H homeostasis has been implicated in the pathogenesis of several neurodegenerative diseases it remains open to debate whether: (1) at least in some illnesses, this is a primary defect or only a consequence of ROS generation; (2) brain G S H can be increased safely using different treatment strategies; and (3) an increase of brain G S H wi l l result in clinical benefit and neuroprotection in animal models or in human diseases. 1.2 Imaging and measurement of GSH in brain Quantitative measurements of G S H usually involve extraction of the tissues followed by derivatization to give a chromogenic or fluorgenic compound that can be analysed by spectrophotometry or high performance liquid chromotography ( H P L C ) (Newton et al., 1981), or by absorption measurement after oxidation by 5,5'-dithio-bis-2-nitrobenzoic acid) ( D T N B ) (Anderson, 1985) or following conjugation to l-chloro-2,4-dinitrobenzene ( C D N B ) in the presence of glutathione S-transferase (GST) (Hermsen et al., 1997). Although specific and sensitive, these methods require extraction of the tissue and only provide an average estimate for all cells in the'tissue. 16 1.2.1 Different dyes to image GSH In principle, imaging G S H with GSH-sensitive fluorescent dyes would permit monitoring of the relative cellular distribution of G S H . Choice of an optimum dye for use is dependent on several factors. Adducts are formed between the dye and the sulfhydryl group of G S H , resulting in a dramatic increase in fluorescence intensity and/or altered excitation/emission spectral properties of the dye. A primary concern is that dyes may react with intracellular thiol other than G S H , particularly protein sulfydryls, resulting in high levels of background fluorescence. Adduct formation between G S H and the dyes containing a chloromethyl group is catalyzed by GST, confering a high degree of specificity of these dyes to G S H , thereby allowing staining at concentrations that impart very low background labelling of proteins (Meister et al., 2001). However, GSTs consist of several isoenzymes and, due to differing specificities for fluorophores, differ in their ability to catalyze adduct formation (Lowndes et al., 1994). Furthermore, the isoenzyme classes differ markedly with regard to cellular distribution and expression levels during development in the brain (Johnson et al., 1993; Lowndes et al., 1994; Beiswanger et al., 1995; Philbert et al., 1995). GST-independent dyes isolate the G S H - dependent component of cellular fluorescence, but may not possess the higher selectivity for G S H over other thiols. Other factors affecting the utility of GSH-sensitive dyes include feedback inhibition by the conjugate on G S T activity, new synthesis of G S H , transport of the fluorescent adduct out of the cell and decomposition to other fluorescent products (van der Ven et al., 1994; Barhoumi et al., 1995; Poot et al., 1996). 17 There are six fluorophores that can be used to evaluate G S H . Four of the dyes require G S T to form a fluorescent conjugate. These include: t-butoxycarbonyl-Leu-Met-7-amino- 4-chloromethylcoumarin ( C M A C ) , 7-amino-4-chloromethylcoumarin (CMAC-b lue ) , monochlorobimane ( M C B ) , and 5-chloromethylfluorescein diacetate ( C M F D A ) . The other two dyes don't require G S T for adduct formation with G S H . These include: 2, 3- naphthalenedicarboxaldehyde ( N D A ) and o-phthaldehyde (OPD) (Tauskela et al., 2000). In Tauskela et al. (2000) paper, they tested the specificity of these dyes for G S H in neurons and astrocytes cultures. They found that three of the four GST-sensitive dyes- C M A C , C M A C - b l u e , and MCB-displayed substantial sensitivity to G S H , as staining was lost when immature cultures were depleted of G S H by pretreatment with D E M (diethyl maleate) or B S O (DL-buthionine-(S,R)-sulfoximine). O f these three dyes, C M A C and M C B seem most appropriate for use in immature cultures, although the basis for why these dyes produce different intracellular staining patterns is unknown. C M A C - b l u e did not stain all cells in immature cultures. C M F D A was not sensitive to G S H , indicating that generation of the fluorescent 5-chloromethylfluorescein by esterase hydrolysis within the cells preceded the reaction with thiol-containing species. O f the two G S T - independent dyes, O P D was partially specific to G S H , while N D A was insensitive to G S H (Tauskela et a l , 2000). 1.2.2 Two-photon imaging of GSH with MCB In our study, we used M C B as a specific probe for G S H . M C B can be conjugated with G S H , catalyzed by GST. The fluorescent adduct can be excited at 390 nm, and emits at 18 450~575nm. It can also be excited with two-photon at 780nm (Meister et al, 2000). The ability to measure G S H at the cellular level by two-photon laser scanning microscopy ( T P L S M ) overcomes some of the limitations inherent with conventional biochemical techniques, which require extraction of the tissue and therefore give average levels in whole tissues. Several features of T P L S M combine to allow imaging deeper into highly scattering biological tissues and generate images with enhanced contrast (Meister et al, 2000). Near infrared light typically penetrates biological material significantly better than blue or U V light with less scattering and refraction (Duck, 1990). In our study, we were able to use T P L S M to image as deep as -250 urn in live animal brain, providing direct evidence on the G S H distribution in neocortex. 1.3 Structure and function of meninges Meninges include the three membranous layers of connective tissue that envelop the brain and spinal cord. The outermost layer, or dura mater, is extremely tough and is fused with the membranous lining of the skull. In the brain it forms a vertical sheet that separates the cerebral hemispheres and a horizontal sheet that lies between the cerebrum and the cerebellum. The thin arachnoid membrane lies below and in close contact with the dura mater. The innermost layer, or pia mater, is in direct contact with the brain and spinal cord and contains the blood vessels that supply them. The pia mater and arachnoid membrane are separated by the subarachnoid space containing the cerebrospinal fluid, which carries nutrients, absorbs the impact of shocks, and acts as a barrier to disease 19 organisms. Thus, the meninges provide a fluid-filled jacket for the protection of neural tissues and allow for the flexing and twisting of the vertebral column about the spinal cord (Zigmond et al., 1999). 1.3.1 Function of meninges Little attention has been paid to the meninges except for the physical role at the C S F - blood barrier (Nilsson et al., 1992; Smith and Shine, 1992; Tanno et al., 1993). So far, there are a few physiological findings suggesting their participation in the trophic support of neurons. For example, fetal brain neurons transplanted into the subarachnoid or meningeal space can survive, grow over the brain surface, and exhibit facilitated neuritic elongation (Ueda et al., 1989; Kyoshima et al., 1992; Risling et al., 1992). When fetal meningeal tissues are transplanted onto the median eminence of adult rats, regenerating fibers of vasopressin neurons innervate the grafts heavily following hypophysectomy (Ishikawa et al., 1995). Furthermore, the medium conditioned with cultured meningeal cells promotes the survival of various brain neurons in vitro. These findings may raise the possibility that the meninges secrete some biologically active substances that play an important role in the maintenance or regulation of brain function. Consistent with this, Ohe et al. (1996) examined the profile of the proteins secreted from cultured meningeal cells with sodium dodecyl sulphate-polyacryl-amide gel electrophoresis, and found that meningeal cells can secrete cerebrospinal fluid proteins that play important rols in certain biological events in the brain (Ohe et al., 1996). 20 1.3.2 Meninges express high level of xCT, cystine/glutamate antiporter Recently, Sato et al. (2002) used in situ hybridization to show that meninges express a high level of x C T . x C T is a subunit of system x c ", which transports an anionic form of cystine in exchange for glutamate (Bannai, 1986). x C T has 12 putative transmembrane domains, whereas the other subunit, 4F2hc , is predicted to have a single transmembrain domain (Sato et al., 1999). The x C T subunit is responsible for the specificity of this antiporter, and is expressed in the area postrema, subfornical organ, habenular nucleus, hypothalamic area, ependymal cells of the lateral wall of the third ventricle, and meninges (Sato et al., 2002). Since meninges faces C S F , which has plenty supply of cystine, it is possible that meninges can take up cystine efficiently from x C T . With this high level of cystine, meninges may have high level of G S H . In our study, we looked into the G S H metabolism in meninges, and found meningeal cells are very active in G S H metabolism. 1.4 Rationale of research There have been several biochemical assays to measure G S H level in tissue. However, these methods can only give an average value of G S H concentration in tissue homogenate. In my project, I used T P L S M to image G S H distribution in brain with M C B , which can give a detailed map of G S H distribution and measure G S H concentration in single cells. First I established that M C B is a specific probe for G S H . The fluorescence o f the conjugate can be imaged with T P L S M , and the intensity can reflect intracellular 21 G S H level. After that, I set out to measure G S H distribution in different brain regions in rat brain slices with T P L S M . B y converting the fluorescence intensity to G S H concentration, I can get a detailed map of G S H distribution in brain. With T P L S M , I was also able to measure G S H distribution in vivo with live animals. This is closer to biological reality since there is no blood supply in slices. With this approach, I found that in vivo, G S H is highly expressed in meninges and astrocytes. Since there is some preliminary data in our lab that meninges may be efficient in protecting neurons, I examined the G S H metabolism in meninges in more detail. I developed an assay, with M C B and plate reader, to measure different kinetic parameters of G S H metabolism in cultures. With this assay, I found that meninges are more active in G S H metabolism than cortical cells, which may be able to explain the neuroprotection function of meninges. 22 Chapter 2 Materials and Methods 2.1 Chemicals A l l reagents were purchased from Sigma-Aldrich Canada (Ontario, Canada) unless mentioned. Monochlorobimane was purchased from either Fluka or Molecular Probes and made as a 100 m M stock solution in dimethyl sulfoxide ( D M S O ) and stored at - 20°C. Sulforhodamine 101 (SR 101) was prepared as a 10 m M stock solution in D M S O and also stored at - 2 0 ° C . MK-571 was purchased from Alexis Biochemicals. Propidium iodide (PI) was purchased from Molecular Probes and made as a 10 m M solution in P B S , and stored at 4 °C. Primary antibodies used in this study included anti-laminin (rabbit), anti-S-100p (mouse), anti-NeuN (mouse, Chemicon), anti-doublecortin (goat, Santa Cruz). Secondary antibodies included FITC-anti rabbit, FITC anti-mouse, Alexa 594-anti mouse (Molecular Probes), and Alexa 488-anti goat (Molecular Probes). 2.2 Astrocytes and meninges culture Astrocyte cultures were prepared from 0 to 2 day old Wistar rats. The animals were anesthetized with cold, sacrificed by decapitation, the skulls opened and the brains removed in sterile PBS. The meninges were carefully removed from the brains. The remaining cortices were digested in papain at 37°C for lOmin. The meningeal and cortical cells were plated separately in M E M (Invitrogen Cat# 51200-038) amended to 15.6mM glucose, 2 m M glutamine, 10% fetal bovine serum, and lOOU/mL penicillin/ 23 streptomycin (Invitrogen Cat# 15140). Cells were passaged by trypsinization at 1 week at a concentration to yield 75 to 90% confluence on the day of the M C B assay. Cells were plated into 24 well plates from either Corning or Falcon. To inhibit the G S H synthesis, the cultures were incubated with 10 u M L-Buthionine-sulfoximine (BSO) overnight. To induce the phase II pathway, the cultures were incubated with 20 u M tert- Butylhydroquinone (tBHQ) overnight. 2.3 Plate reader GSH assay Fluorometric assays were performed using a plate reader (Fluoroskan Ascent F L ) ; A, e x = 355 ± 19nm, 'Kem = 527 ± 5nm. Although these excitation/emission filters are sub- optimal for M C B detection, they prevented saturation of the detector within the plate reader permitting linearity between the concentration of M C B - G S H and detected fluorescence over the range associated with the fluorescence values in our measurements. Purified 200 u.M G S H and 10 u M M C B were added together, with varying concentrations of purified G S T enzyme (0, 0.01, 0.02, 0.04 units). Fluorescence was measured using a plate reader over a 4 h reaction period. A l l cell reactions were carried out in Hank's Balanced Salt Solution (HBSS) (138mM N a C l , 5 m M K C I , 0.34mM N a 2 H P 0 4 , l O m M N a + H E P E S , I m M N a H C 0 3 , 20mM Glucose, 2 .5mM C a C l 2 , and I m M MgS04) . During the assay cells were bathed in either H B S S as control, or H B S S with the following: lOOuM G S H , lOOuM cystine, or a 100 u M cocktail of G S H precursors (glutamate, cystine, glycine). Efflux was estimated by measuring the change in cell fluorescence after changing the media. To inhibit the efflux, 2 5 u M M K 5 7 1 was added to 24 the cultures. To test temperature effect on G S H - M C B efflux, room temperature (20°C) and 37°C were present respectively during plate reader assay. Plates were not agitated between the measurements, so that cells sedimented and formed a thin layer at the bottom of the wells. 2.4 Calibration of the GSH-MCB fluorescence A 5 m M stock solution of G S H - M C B was made from 20 m M G S H and 3 m M M C B in the presence of a GST. Excess G S H and G S T were used to ensure that all M C B was conjugated. A dilution series of 5 m M , 1.5 m M , 0.5 m M , 0.15 m M was made from this stock. The standard solutions were contained within in vitrocells (glass microcuvettes, ID = 50 pm VitroCom), and imaged with two-photon microscopy. This method of G S H - M C B calibration was routinely used in our studies in brain slices. 2.5 Rat brain slices preparation Coronal brain slices were prepared from P15-21 or P1-2 Wistar rats (Charles River, Canada). Under halothane anaesthesia, animals were decapitated with a guillotine and the brain was quickly removed from the skull and placed in ice-cold cutting solution containing (in mM): 200 sucrose, 2.5 KC1, 0.5 C a C l 2 , 26.2 N a H C 0 3 , 10 M g S 0 4 7 H 2 0 , 1 N a H 2 P 0 4 , 11 glucose (pH maintained at 7.4 by saturation with 9 5 % 0 2 and 5 % C 0 2 ) . Using a scalpel blade (FisherScientific, 08-916-5A, No . 10), the brain was manually sliced into two hemispheres. One of the hemispheres w i l l be sectioned to slices. The total 25 period from decapitation to cutting brain to two halves is restrained in 5 minutes. Slices (300 urn) were obtained with a vibratome (Leica V T 1000 S) and then transferred to a static bath chamber where they were maintained at room temperature for at least 1 h before imaging. The slices were kept in artificial cerebral-spinal-fluid ( A C S F ) containing (in m M ) : 120 N a C l , 26.2 N a H C 0 3 , 24.2 glucose, 2.5KC1, 1.25 N a H 2 P 0 4 , 1 M g C l 2 6 H 2 0 , 2 C a C l 2 , saturated with 95% 0 2 and 5% C 0 2 . For imaging, individual slices were transferred to a recording chamber and perfused with A C S F saturated with 95% 0 2 and 5% C 0 2 . M C B was added to the perfusion system at a final concentration of 60 u M . To identify dead cells, we added 10 p.M propidium iodide together with M C B . 2.6 Animal preparation for in vivo imaging A l l experiments were approved by the University of British Columbia Animal Care Committee and were conducted in strict accordance with guidelines by the Canadian Council on Animal Care. C57B1/6 mice were purchased from the Jackson Laboratory (Bar Harbor, M E ) and bred at the University of British Columbia animal facilities. Cranial windows for in vivo imaging were produced as described previously in (Zhang et al., 2005). In brief, mice aged 3-5 months were deeply anesthetized with an intraperitoneal injection of urethane (0.12% w/w, supplemented with 0.02% w/w as needed) (Kleinfeld et al., 1998) and 20 m M glucose in PBS was supplemented to maintain animal hydration (0.2- 0.3 ml , intraperitoneal injection, every 1 h). Body temperature was maintained at 37°C using a feedback regulated heating pad. A n air- powered dental dril l was used to produce a 2 x 2 mm cranial window over the 26 somatosensory cortex at coordinates of 0.8 mm from bregma and 2.0 mm lateral, leaving the dura intact. A stainless-steel chamber that surrounded the craniotomy was glued to the skull with Krazy Glue (Elmer's Products, Columbus, OH). To reduce movement artifacts, the area between the chamber and the skull was filled with dental acrylic (Kleinfeld and Denk, 2000). The exposed cortical surface and chamber were filled with 2% (w/v) agarose (diluted in PBS or a HEPES-buffered A C S F ) and sealed with a cover glass. For in vivo labeling, M C B and SR 101 stocks were dissolved in P B S to a final concentration of 100 u.M and applied directly to the exposed brain surface. During the entire imaging session, mice were maintained under urethane anesthesia. 2.7 Two-photon microscopy Two-photon excitation of G S H - M C B conjugates was achieved using a Coherent (Santa Clara, C A ) M i r a 900 Ti:sapphire laser pumped by a 5 W Verdi laser tuned to 780 nm. Images were acquired using custom software routines (IgorPro; Wavemetrics, Eugene, OR) and by using an Olympus (Tokyo, Japan) I R - L U M P l a n F l water-immersion objective (40x; 0.80 numerical aperture). When M C B was used in combination with SR 101, the laser was operated at 800 nm and fluorescence was collected using photo- multiplier tube 1 (PMT1) for the G S H - M C B signal (512-562 nm), and P M T 2 for the SR 101 signal (620-645 nm). For immunostaining, F ITC, Alexa 488 and Alexa 594 were used as secondary antibody fluorophores. A l l fluorophores were excited at 800 nm. FITC and Alexa 488 were collected on P M T 1 and Alexa 594 was collected on P M T 2 . A l l images were taken at 1024x1024 pixels. For imaging in rat brain slices to measure G S H concentration, certain laser intensity was used for entire imaging and a standard curve 27 was made at this intensity with G S H - M C B O . l m M , 0 .3mM, l m M , and 3 m M . For imaging in vivo, to minimize photodamage, the excitation laser intensity was adjusted depending on the depth of the focal plane (lower intensity at shallower depths) and always kept at a minimum for a sufficient signal-to-noise ratio. 2.8 Immunostaining After M C B labeling and two-photon imaging, slices were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 1 h. Slices were then rinsed twice with P B S , and then incubated in Triton X-100 (0.5% in PBS) for 0.5 h to make the membrane permeable to antibodies. After that, slices were immunostained with primary antibody against S-100|3 (from mouse, 1:1000 in antibody buffer), NeuN (from mouse, 1:100 in antibody buffer), G F A P (from mouse, 1:200 in antibody buffer), laminin (from rabbit, 1:500 in antibody buffer), or doublecortin (from goat, 1:200 in antibody buffer) for 48 h at 4 °C. The antibody buffer contained 0.1% Triton X-100 and 2% goat serum in P B S . After washing twice with P B S , the slices were incubated with secondary antibodies (FTTC-anti rabbit, FITC-anti mouse, Alexa 488-anti goat, Alexa 594 anti-mouse; diluted 1:200 in antibody buffer) for 48 h in dark at 4 °C. After secondary antibody labelling, slices were washed with P B S twice, and imaged with two-photon microscopy. For immunostaining of meninges, astrocytes, or cortical cell cultures after plate reader assay with M C B labelling, we fixed the cells with 0.2% glutaraldehyde (in 4% P F A ) for 0.5h. Experiments showed that under these conditions, M C B - G S H conjugate was retained within the cells after treatment with Triton X-100 (0.5% in PBS) for 0.5h. 28 Cel l cultures were then treated with a blocking solution (4% normal goat serum in PBS) and incubated with mouse IgG anti-slOOp, or mouse IgG MapII, 1:1000 diluted in antibody buffer (as we described above). After three washing steps of 10 min each with 0 .1M P B S , cell cultures were incubated overnight with a fluorescent secondary antibody 1:200 (goat antimouse IgG, Alexa 594-labeled) following which these cells were washed and embedded with a drop of Immunomount. Fluorescence images were collected at a Axiophot photomicroscope (Zeiss) equipped with a C C D camera. 2.9 Glutathione S-transferase (GST) assay Mouse brain and live tissue were dissected in ice-cold P B S and homogenized with 10 strokes of a dounce homogenizer in tissue buffer containing 25 m M Tris, p H 7.4 and 250 m M sucrose. Crude homogenate supernatants were collected and promptly assayed for enzyme activity. The G S T assay consisted of I m M l-chloro-2, 4-dintrobenzene ( C D N B ) , I m M G S H , and 100 | ig/ml protein at 37°C in 100 m M potassium phosphate buffer, p H 6.5 (final concentration in 150 \il reaction volume). The G S T reaction was monitored at 340 nm, and the spontaneous nonenzymatic slope was subtracted from the total observed slope. The extinction coefficient for C D N B was 9600 M / c m . Protein concentration was measured using the bicinchronic acid method according to the protocol of the manufacturer (Pierce, Rockford, IL). 29 2.10 Total intracellular GSH assay Total G S H was quantified by the method of Tietze (1969). Briefly, the acid-soluble fraction was obtained by adding perchloric acid to a final concentration of 3%, followed by centrifugation at 14,000xg for 10 min. The acid-soluble fraction was neutralized to p H 7 with 0 .5M K O H / 5 0 m M Tris. After 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 of I m M 5, 5', dithiobis-(2-nitrobenzoic acid) ( D T N B ) , 2.5 ml of 5 m M N A D P H , and 2.65 ml of phosphate buffer (100 m M N a P 0 4 , p H 7.5, I m M E D T A ) , glutathione reductase (5U/ml final). The increase in A412 from G S H - mediated reduction of D T N B was measured at 30 sec intervals over 30 min. G S H content among treatment groups was normalized to protein. 2.11 Data and image analysis A l l plate reader experiments were done at least five different times unless otherwise stated. Results are presented as the mean±SE. Statistical analysis of raw data was performed with Excile X P and Origin 6.0. Experimental groups were compared by Student's t-test. A statistical probability of p<0.05 was considered significant, p O . O l was considered very significant. Image analysis was performed using N I H ImageJ software (http:// rsb.info.nih.gov/ij/). To reduce photon and P M T noise, a median filter (radius = 1) was applied. The 30 fluorescence intensity was converted to G S H - M C B concentration according to a standard curve. The time course curves of M C B labeling of rat brain slices were fitted with Origin 6.0. For statistical analyses, two-tail paired Mest or repeated measures one-way A N O V A was used with GraphPad Prism (version 2.01). Data were expressed as mean ± S E M . 31 Chapter 3 Results1 , 2 3.1 Specificity of MCB labelling Currently, monochlorobimane ( M C B ) is the probe of choice for measuring G S H levels in intact cells. M C B is essentially non-fluorescent in the absence of G S H . In a reaction catalyzed by GSH-S-transferase (GST), G S H is added to M C B to make a product with blue-green fluorescence (Figure 3-1A, Cook et al., 1991, Ublacker et al., 1991). M C B has advantages over other bimane derivative dyes for measuring G S H level, such as monobromobimane ( M B B ) and monobromotrimethylammoniobimane (QBB) , since it shows very low affinity for other low molecular weight or protein thiols (Meyer etal. , 2001). 3.1.1 MCB labelling of GSH is dependent on GST We further tested the specificity of M C B in in vitro experiments using purified G S T , the reduced form G S H , and M C B to produce a fluorescent G S H - M C B adduct quantified by plate reader. In the absence of GST, addition of 200 u M G S H to 10 u M M C B resulted in a modest linear increase in fluorescent product over time. Addition of purified G S T , resulted in a robust single exponential phase increase in the fluorescence intensity, 1 A version of part of this chapter has been published: Sun X, Erb H, Murphy TH (2005) Coordinate regulation of glutathione metabolism in astrocytes by Nrf2. Biochemical and Biophysical Research Communication. 326: 371-377. 2 A version of part of this chapter has been submitted to Journal of Neuroscience: Sun X, Shih AY, Johannssen HC, Erb H, Li P, Murphy TH (2005): Two photon imaging of glutathione levels in intact brain indicates sites of enhanced redox buffering. 32 indicating that this is an enzyme-catalyzed reaction following first order kinetics (Figure 3-IB). The curve can be fitted with an equation: F=Fo+Fm a x(l-exp(-(t-to)/T)) (Equation 1) F is the fluorescence intensity, F m a x is the maximum level of fluorescence intensity, and x is the time constant of the reaction. The plateau level of the reaction was only related to the amount of G S H and M C B added, regardless of G S T amount, while the initial conjugation rate was proportional to the G S T amount (Copeland, 2000). The time constants of the exponential phase were 1437, 691, 344 sec, respectively for 0.01, 0.02, 0.04 units of GST. The dependence on G S T confers the specificity of M C B for labelling G S H . 33 Figure 3-1 Figure 3-1. MCB can specifically label GSH. (A). Reaction between G S H and M C B , catalyzed by GST. The product has blue-green fluorescence. (B). In vitro experiments with the reduced form of G S H (200 mM) , M C B (10 mM) and the indicated amounts of purified G S T (0, 0.01, 0.02, 0.04 units). The reaction was carried out at 37°C. The fluorescence intensity was measured with a plate reader (A,ex = 355 ± 38 nm, A.em = 527 ± 5 nm). 34 3.1.2 MCB could be used to label intracellular GSH with the plateau level of fluorescence intensity indicating the cellular GSH level The formation of a highly fluorescent G S H - M C B conjugate is catalyzed by glutathione-S-transferase (GST), which has three isoforms, alpha (a), mu (u), and pi (TE), with the u form being the most efficient. The u G S T isoform is present in neurons and glia and in most brain regions (Beiswanger et al., 1995). Since M C B is a membrane permeable dye (Chatterjee et al., 1999) and G S T is widely expressed in brain cells, we assumed that the G S H in brain cells should be able to be labelled by M C B and the fluorescence intensity of G S H - M C B wi l l be related to the cellular G S H level. We confirmed this by showing that M C B labelling of astrocyte cultures which had been pretreated with glutathione synthesis inhibitor buthionine sulfoximine (BSO) have a significantly reduced M C B fluorescence (Sun et al., 2005a). We applied l O u M M C B to the astrocyte culture to label intracellular G S H and monitored the time course of the reaction by measuring the fluorescence intensity of G S H - M C B conjugates with a plate reader, with A, e x -355±19nm, A. e m=527±5nm. After about 15min, the reaction reached a plateau amount of fluorescence reflecting the G S H content of the cells (Figure 3-2). After the plateau had been reached exogenous G S H was added and the fluorescence intensity showed a further linear increase indicating that M C B is in large excess over G S H and that the fluorescence values observed were not saturating (data not shown). In order to confirm that the plateau level represented intracellular G S H content, we depleted the cells of G S H by addition of B S O , which is a specific inhibitor of y-glutamylcysteine synthetase, the rate-limiting enzyme for glutathione synthesis (Anderson, 1998). 35 Pretreatment with BSO is known to deplete the level of nonprotein thiols such as GSH while having a minimal effect on other cellular sulphydryls (Hedley and Chow, 1994; Thomas et al., 1995). For astrocytes BSO is reported to decrease the level of glutathione with a half-life of approximately 5 h (Devesa et al., 1993). M C B labelling of astrocytes cultures which have been pretreated with lOuM BSO for 24h showed a significant decrease in the fluorescence intensity, supporting our hypothesis that the G S H - M C B fluorescence reflects the amount of GSH within cells (Figure 3-2 A). The reduced fluorescence was not due to a direct effect of the BSO itself since addition of exogenous glutathione produced GSH-MCB fluorescence within these cells (Sun et al, 2005a). Consistent with the proposal that the intial rate of GSH conjugation with M C B reflects GST activity and not necessarily glutathione levels, we observed that treatment with BSO to lower GSH did not affect the initial GSH-MCB conjugation rate (data not shown). From the results described above we confirmed that M C B could be used to label intracellular GSH. The plateau level of fluorescence intensity indicates the G S H level, while the initial conjugation rate reflects the GST activity. 36 Figure 3-2 Figure 3-2. Monitoring GSH metabolism in astrocytes cultures with plate reader. (A). Addition of 10 mM BSO inhibits GSH synthesis, reduces the GSH content of the culture and lowers the plateau level of M C B fluorescence (in this experiment 30 m M M C B was used) (B). The exponential phase reflects M C B labelling of a limited amount of intracellular GSH. The four curves all fit well to exponential equations: F=l 2.5(1- exp(-t/521.6)), R2=0.9985(HBSS); F=13.5(l-exp(-t/507.4)), R2=0.9935(GSH); F=12.1(l- exp(-t/505.1)), R2=0.9982 (Cys); F=12.9(l-exp(-t/490.6)), R2=0.9981 (Cocktail). Linear phase is in response to the addition of lOOmM GSH, or cystine, or a cocktail of GSH precursors (Glu, Gly, Cys, all at 100 mM). In efflux stage, we washed cells to determine how much of the fluorescence was associated with the cells versus the media. (C, D). GSH-MCB can be exported out of cells through Mrp, which is temperature-dependent and MK571 sensitive. (C). Reaction at 37°C and 24°C. (D). Reaction with or without MK571, an inhibitor of MRP-1 mediated efflux (25mM). 38 3.2 GSH metabolism assay We developed an assay to monitor the metabolism of GSH in astrocytes, using a fluorescence plate reader and monitoring the kinetics of the GST catalyzed reaction between GSH and the substrate M C B (Sun et al., 2005a). 3.2.1 Plate reader assay is a powerful system to measure intracellular GSH level in cultures We applied 10uM M C B to the astrocyte culture to label intracellular G S H and monitored the time course of the reaction by measuring the fluorescence intensity of G S H - M C B conjugates with a plate reader. Since M C B is in large excess over intracellular GSH (based on the volume of cells versus the media), the reaction between GSH and M C B can be regarded as a pseudo-first order reaction. The time course of GSH- M C B production fit well to Equation 1 (Figure 3-2 B): F=F0+Fmax(l-exp(-(t-t0)/T)) F is the fluorescence intensity normalized to protein amount. Fmax is the maximum level of fluorescence intensity per mg protein, and T is the time constant of the reaction. After subtracting the background from the measured value, the curve usually starts from the origin, and Fo and to are usually zero. F m a x represents the plateau level of fluorescence, while the initial slope of the curve can represents the initial formation rate of GSH-MCB conjugate and is related to the GST activity. Together, this data suggest that our plate reader time course assay is a powerful system to study the astrocytic intracellular GSH 39 levels. Its advantage over other techniques lies in that it can not only measure the intracellular GSH level, but also provide a measurement of kinetics parameters for GST- catalysed conjugation reactions in situ. 3.2.2 Plate reader assay can measure rate of GSH precursor uptake and synthesis Astrocytes actively synthesize GSH through the consecutive reactions of two enzymes, y-glutamylcysteine synthetase and GSH synthetase (Dringen and Hirrlinger, 2003). Our assay can also be used to monitor the GSH synthesis in astrocytes. After the M C B labelling of endogenous GSH in astrocytes has reached a plateau level, we added 100 u M cystine, the rate-limiting precursor for GSH synthesis, or a cocktail of precursors, glutamate, glycine and cystine (100 u M for each component), to the cultured cells. We found a significant linear increase in the fluorescence intensity, indicating that the astrocytes are active in taking up the precursors and synthesizing GSH (Figure 3-2 B). The slopes are 0.111 ±0.0075 and 0.126±0.0079 (fluorescence unit per mg protein/min), respectively for cystine and cocktail, averaged from 5 different experiments. When exogenous GSH (lOOuM) was added, the fluorescence also showed a linear increase, with a slope of 0.067±0.0095 (fluorescence units per mg protein/min), significantly smaller than cystine (p=0.013<0.05) and the cystine, glutamate, glycine cocktail (p=0.0059O.05), indicating that astrocytes are more efficient at taking up the precursors for GSH synthesis and then synthesizing GSH rather than taking up GSH directly. 40 However, when we add HBSS, a cerebrospinal fluid (CSF)-like solution, to the culture, the fluorescence level doesnot change (Figure 3-2), indicating that astrocytes could not synthesize detectable levels of GSH. This suggests that even in the condition of complete depletion of the GSH pool, astrocytes cannot synthesize GSH unless exogenous precursors are available. Since HBSS is widely used in most in vitro electrophysiology experiments, our data suggest that oxidative stress may be apparent in these conditions. Our assay can also be used to explore astrocytes' preference for different precursors. When we replace the glutamate in cocktail to glutamine, the fluorescence intensity also shows a linear increase, with a slope of 0.118±0.0065 (fluorescence unit per mg protein/min). The slopes are similar between cocktail with glutamate or glutamine (p=0.278>0.05), indicating that astrocytes donot show significant preference for glutamate or glutamine. This is inconsistent with other groups' data that astrocytes prefer glutamate rather than glutamine for G S H synthesis (Kranich et al, 1996, 1999; Dringen and Hamprecht 1998). One possible explanation is that in their study, they examined GSH synthesis in astrocytes after a starvation in a minimal medium lacking glucose and amino acids. While in our assay, we used normal medium, which consists enough substrates for energy metabolism. Maybe the difference in energy metabolism condition will affect astrocytes' preference for different precursors. .41 3.2.3. Plate reader assay can measure efflux of GSH-MCB conjugate After labelling with M C B , the glutathione S-conjugates can be effluxed from astrocytes through M r p l , a member of a family of multi-drug resistance proteins (Mrps) (Borst et al., 1999; Leslie et al., 2001). Mrp transporters constitute a subgroup of the ATP-binding cassette transporters and are ATP-driven export pumps of organic anions (Borst and Oude Elferink, 2002). We can also use our assay to examine the efflux of G S H - M C B conjugates through the M r p l transporter. Since the fluorescence plate reader reads both intracellular fluorescence and fluorescence from medium with almost equal efficacy, we were not able to determine what proportion of the G S H was effluxed to medium versus cell associated during the exponential phase of the M C B labelling. However, i f we washed the wells with H B S S after the reaction, the fluorescence conjugate within the medium would be washed out, and the fluorescence signal left in the well represents the conjugate associated with cells (Figure 3-2 B). Comparing the fluorescence intensity before and after washing the cells, we can determine the amount of conjugate effluxed from cells. We found that the efflux process was highly temperature dependent (Figure 3-2 C) . At 37°C, greater than 75% of the fluorescence product was associated with the medium, while at room temperature (24 °C), the efflux percentage was less than 50%, which is consistent with previous results that the A B C pump is highly temperature dependent (Borst and Oude Elferink, 2002). Treatment with M K 5 7 1 (25uM), a competitive inhibitor of M r p l (Hirrlinger et al., 2002), resulted in a nearly complete retention of G S H - M C B fluorescence within cells (Figure 3-2 D), which further confirmed that the M r p l is predominately responsible for the G S H conjugate efflux from astrocytes. 42 Fluorescent microscope images showed that the MK571 treated cells contain more GSH- M C B fluorescence, confirming that more of the conjugates were kept within the cells in the presence of MK571 (Figure 3-3). 43 Figure 3-3 Figure 3-3. Fluorescence images (10X) of G S H - M C B in untreated (A) and MK571- treated (B) astrocytes cultures. Cultures were either maintained in normal HBSS, or HBSS with 25 mM MK571 added for 80 min at 37 °C. Addition of MK571 blocks the efflux of MCB-GSH and increases its content within cells. 44 3.2.4 Plate reader assay can detect changes in GSH metabolism under oxidative stress It has been well established that G S H is an important intracellular antioxidant that protects against a variety of different reactive oxygen species (ROS) (for review, see Schulz et al., 2000; Dringen et al., 2000; Anderson et al., 2003). We are particularly interested in how the metabolism of G S H wi l l be affected under oxidative stress. Data from our lab and others have found that astrocytes can induce a family of phase II detoxification enzymes that control R O S accumulation (Murphy et al., 2001; Johnson et al., 2002; Eftekharpour et al., 2000). The activation of the phase II pathway is dependent on the translocation of NF-E2-related factor 2 (Nrf2), an important transcription factor responsible for upregulating antioxidant response element (ARE)-mediated gene expression from the cytoplasm into the nucleus (Alam et al., 1999; Itoh et al., 1999). We activated the phase II response by incubating the astrocyte culture with 2 0 u M tert- butylhydroquinone ( tBHQ), a well-characterized Nrf2 activity inducer (Ishii et al . , 2000;Lee et'al., 2001; Shih et al., 2003, Eftekharpour et al., 2000), for 24h. Labelling these astrocytes with M C B and monitoring the time course of the reaction showed that the G S H level ( F m a x ) in t B H Q treated cells was 1.5 times of that of control groups ( tBHQ: 14.9+0.75 fluor unit per mg protein; D M S O : 10.1 ±0.14 fluor unit per mg protein, p=0.0057O.05), while the initial conjugation rates were similar between the two groups, indicating the bulk G S T activity was not rate limiting or affected by t B H Q significantly ( tBHQ: 0.043±0.004 fluor unit per mg protein/sec, D M S O : 0.045±0.003 fluor unit per 45 mg protein/sec, p=0.92>0.05)(Figure 3-4 A ) . We then added the cocktail of G S H precursors to the cells after plateau level had been reached and observed that t B H Q treated cells showed a larger slope indicating an increased synthesis/precursor uptake rate ( tBHQ: 0.163±0.018 fluor unit per mg protein/min; D M S O : 0.095+0.015 fluor unit per mg protein/min, p=0.0049O.05) (Figure 3-4 A ) . This difference was not due to more efficient G S H conjugation since no significant difference in fluorescence slope was observed after addition of exogenous G S H ( tBHQ: 0.073±0.016 fluor unit per mg protein/min; D M S O : 0.069±0.01 fluor unit per mg protein/min, p=0.845>0.05) When we washed the wells to assess efflux, and did not find much difference in the percentage of M C B efflux between t B H Q and D M S O groups ( tBHQ: 84.5±3.9%, D M S O : 87.7±1.5%, p=0.25) However, we only monitored efflux at a single time and not the full time course thus these conclusions should be taken with some caution. If we add cocktail of G S H precursors again to the culture after washing, we can also see a much faster linear increase in fluorescence intensity than the increase before washing, with t B H Q group having much larger slope than D M S O group (tBHQ: 0.266±0.021 fluor unit per mg protein/min; D M S O : 0.153+0.015 fluor unit per mg protein/min. p=0.000470.05) (Figure 3-4 A ) . This is consistent with our hypothesis that t B H Q can activate the antioxidant system and make cells more efficient in taking up precursors and synthesizing G S H . We conclude that the t B H Q treatment increases the synthesis of G S H in astrocytes, while apparently G S T and Mrp activity were relatively unaffected (Table 3-1). This is consistent with our previous finding that y-glutamylcysteine synthetase, the rate- limiting enzyme for G S H synthesis, and xCT, the cystine-glutamate transporter responsible for precursor uptake, are two important enzymes for G S H synthesis that are 46 upregulated by Nrf2 (Shih et al., 2003). Although G S T and Mrp m R N A s are induced by the Nrf2 pathway in astrocytes (Shih et al., 2003), they did not appear to be greatly affected by t B H Q in the M C B assay. It is possible that this is due to the preference of specific isozymes for MCB-conjugation or some other factor being rate-limiting over the time scales. 47 Figure 3-4 Figure 3-4. Changes in G S H metabolism induced by activation of ARE-mediated gene expression. Astrocyte cultures were treated with tBHQ (20 m M ) for 24 hrs before assessing G S H metabolism at 37°C using 10 m M M C B . D M S O (0.01%) was used as the vehicle for t B H Q and was added to control cultures. The M C B labeling phase was fitted with a single exponential equation. (A). After a plateau level of fluorescence was reached, a lOOuM cocktail of G S H precursors was added to the cultures to measure G S H synthesis. After washing the wells to measure efflux, the cocktail was added again. (B). H B S S was used as a control. 48 Table 3-1. Comparison of all kinetics parameters of G S H metabolism between t B H Q treatment and control (DMSO) groups. Treatment t B H O D M S O Fmax (fluor unit per mg protein) 14.9+0.75 10.1±0.14 Time constant (sec) 346.5±47.2 224.4±20.6 Initial slope (fluor unit per mg protein/sec) 0.043±0.004 0.04510.003 Slope after 1 s t time addition of Ckt l 0.163+0.018 0.095±0.015 (fluor unit per mg protein/min) Slope after 1 s t time addition of G S H 0.073±0.016 0.06910.010 (fluor unit per mg protein/min) Efflux percentage (%) 84.513.9 87.7+1.5 Slope after 2 n d time addition of Cktl 0.266±0.021 0.15310.015 (fluor unit per mg protein/min) Data represent the m e a n i S E M of five independent experiments. Fluor is an abbreviation for fluorescence. 49 3.2.5. Conclusion In conclusion, we have established a reliable and powerful assay to monitor glutathione metabolism in astrocyte culture, enabling us to measure the various kinetics parameters of glutathione metabolism, such as the rates of precursor uptake, synthesis, conjugation with xenobiotics, and efflux. The assay is sensitive to detect the changes in G S H metabolism under different conditions, for example, phase II inducers. Since there are still many unaddressed issues about glutathione's mechanism of neuroprotection (Dringen, 2000), we feel our glutathione metabolism assay wi l l provide new insight into this interesting area. 3.3 Feasibility of two-photon laser scanning microscopy (TPLSM) measurement of cellular glutathione level G S H - M C B conjugate can be excited by T P L S M at 780 nm and emits significant fluorescence at 450-575nm (Meyer et al., 2000). The in vitro production of G S H - M C B using purified enzymes allowed us to produce known amounts of G S H - M C B for a standard curve in two-photon imaging experiments. The standard curve indicated that two-photon microscopy was a viable technique to measure up to 5 m M concentrations of G S H (Figure 3-5). The G S H level measurement from T P L S M imaging is based on two assumptions: 50 Figure 3-5 GSH-MCB (mM) Figure3-5. Standard curve for the relationship between fluorescence intensity and GSH-MCB concentration. G S H - M C B solutions of known concentration were made from a G S T catalyzed reaction between the reduced form of G S H and M C B . The standard solution was then filled into Vitrocells (micro-cuvettes) and imaged with two- photon microscopy. Over a range of 0 - 5 m M , a linear relationship was observed between G S H - M C B concentration and fluorescence ( R 2 = 0.9937). 51 3.3.1 Assumption 1: Efflux of GSH-MCB at room temperature is negligible A l l of experiments with rat brain slices were carried on at room temperature. As mentioned above, cells can efflux the G S H - M C B conjugates through Mips , a family of multi-drug resistance proteins (Mrps) (Leslie et al., 2001; Borst et al., 1999). We have examined the efflux of G S H - M C B through Mrps in astrocyte cultures (Sun et al., 2005). We found that in culture, Mrps wi l l export around 75% of G S H - M C B conjugate at 37 °C within 10's of min. In comparison to 37 °C, at room temperature efflux was reduced considerably (Sun et al., 2005a). We further tested whether M r p l w i l l play a significant role.in slices by adding 5 0 u M M K 5 7 1 , a competitive inhibitor of M r p l (Hirrlinger et al., 2002), to rat brain slices and compared the fluorescence intensity after M C B labelling with control slices (Figure 3-6). We compared the fluorescence intensity of M C B - labelled cells in lateral ventricle ependymal cells (Figure 3-6 A , B) and in meninges (Figure 3-6 C, D). We found no significant difference with or without MK-571 (p>0.05 for all regions, from 4 separate slice experiments) in neither of these regions (Figure 3-7), indicating in brain slices at room temperature the efflux of G S H - M C B by M r p l can be neglected. 52 Figure 3-6. ^ 50 .urn $" 1 LV with MK B LV without MK C Meninges with MK D Meninges without MK Figure 3-6. Eff lux of G S H - M C B i n slices at room temperature can be neglected. A l l slices were imaged at room temperature and after 60 | i M M C B labelling for 30 min. In (A) and (C), M K 571 were added to the perfusion system at the same time with M C B , to get to a final concentration of 50uM. In (B) and (D), only M C B was used to label slices. 53 Figure 3-7 Figure 3-7. G S H - M C B efflux can be neglected in room temperature. In all these regions, the fluorescence with or without M K 571 did not show significant difference. Data were averaged from 4 experiments. Error bar represents standard error. L V , S V Z , men and astro are abbreviations for lateral ventricle, subventricular zone, meninges, and astrocytes, respectively. 54 3.3.2 Assumption 2: MCB labelled all GSH content in cells In each experiment, by doing a time course we demonstrate that the reaction between M C B and G S H goes to completion; fluorescence intensity usually reaches a maximum level after M C B labelling for 20 min (Figure 3-8). In Figure 3-8, we showed the time course curve of M C B labelling at different regions of brain, including meningeal cells, astrocytes and neurons in cortex (Figure 3-8 A ) , high and low fluorescent cells in dentate gyrus, and lateral ventricle ependymal cells (Figure 3-8 B) . A l l curves showed an exponential increase over time and the plateau level is usually reached after 20 min. Therefore for al l our experiments with slices, we only took two-photon images after M C B labelling for 20 minutes. We assumed that at this time, M C B has labelled all G S H content in cells. We further tested this by adding more M C B to slices after the fluorescence had reached the plateau level and we found no further increase in fluorescence intensity, indicating that M C B is in excess and presumably labels all intracellular G S H (data not shown). 5 5 Figure 3-8 Figure 3-8. T ime course curve of M C B labelling in different brain regions. (A). M C B labelling in meninges, cortical astrocytes, cortical neurons. (B) M C B labelling high fluorescence cells of dentate gyrus (subgranular layer, presumed neuronal progenitors), low fluorescence cells in dentate gyrus (lateral medial granule layer, presumed neurons), and ependymal cells along lateral ventricle. Data points were averaged from five cells within a single slice. 6 3.3.3 Conclusion Based on the above two assumptions, we expect the G S H - M C B fluorescence intensity measured using the two-photon would be proportional to G S H concentration. To ensure that differences in fluorescence path length do not contribute to the observed differences in G S H levels between cells and tissues, we optically sectioned each cell and made maximal intensity projections over regions of cells that were considerably larger than the axial resolution of the microscope (<3 um), thus being in a regime where fluorescence intensity is proportional to G S H - M C B concentration and not structure path length or volume (Meyer et al., 2000). 3 . 4 MCB labelling of different brain regions in slices After establishing that M C B can be a relatively specific label for glutathione, we examined the G S H distribution in various brain tissues in acute PI5-20 rat slices at room temperature. At surface of the slices there are many unstained cell bodies showing up as black holes which were labelled by the polar membrane impermeable marker PI (St John et al., 1986), indicating that they had ruptured membranes (Figure 3-9 A - C ) . A s we imaged deeper into slices the incidence of unstained cell bodies became lower (Figure 3- 9 D-F) , indicating that the death of cells at the slice surface was caused by vibratome sectioning. 57 Figure 3-9 4 * ' M C B A ' D e e p PI Merge Figure 3-9. M C B and PI labelling of slices. (A). M C B labelling of rat brain slice surface. Many cells are not labelled by M C B , showing as black holes. (B). PI labelling of the same region as (A). (C). Overlay of (A) and (B). Green color represents M C B signal, red color represents PI. The black holes in panel (A) are labelled by PI. (D). M C B labelling in deeper level of slices. (E). PI labelling of same region. (F). Overlay of (D) and (E). 58 3.4.1 Meningeal and astrocytes in neocortex are labelled strongly by MCB in rat brain slices Within brain slices M C B robustly labelled the meninges and a subpopulation of cells in neocortex with astroglial morphology, as well as perivascular cells (Figure 3-10 A ) . In order to verify the cell type of these GSH-positive cells we performed immunostaining with the calcium-binding protein S-lOOp, a specific marker of astrocytes (Matthias et al., 2003). We have used S-lOOp since well used astrocyte markers such as G F A P fail to label all astrocyte cell populations (Walz and Lang, 1998). Since M C B tends to leak out o f cells in paraformaldehyde fixed tissue we also stained the slices with either laminin, which is a major component of basement membranes in blood vessels, or PI as landmarks to locate the same region from which we had imaged G S H - M C B (Figure 3-10 B) . A s fixation of slices caused some distortion of cell position, we examined co-localization over a relatively small area based on cell morphology and the relative position o f cells. Most of the highly fluorescent MCB-label led neocortical cells were S-lOOp positive and thus were presumably astrocytes (75% MCB/S-100P co-staining, n = 47, from 2 slices of 2 animals, Figure 3-10 C). The perivascular cells were also S-100P positive astrocytes with their end-feet tightly ensheathing the vessel wall . A n increased level of G S H in astrocytes is consistent with their crucial role in the antioxidant defence of the brain (Cooper and Kristal, 1997; Dringen, 2000; Anderson et al., 2003). It has been demonstrated that astrocytes can support other brain cell types by defending them against R O S (Dringen, 2000). For example, astrocytes can protect neurons from oxidative stress 59 via G S H dependent mechanisms and therefore require an intracellular reservoir of G S H (Iwata-Ichikawa et al., 1999; Chen et al., 2001; Shih et al., 2003; Kraft et al., 2004). In addition to astrocytes, we also found that meningeal cells (most likely pial and arachnoid cell layers) were intensely labelled by M C B , indicating a very high G S H level. Meningeal cells have received little attention with respect to neuroprotection except for their role in the regulation of blood flow and the blood-cerebrospinal fluid (CSF) barrier (Tanno et al., 1993; Ghersi-Egea et al., 1994). Related structures such as the choroid plexus have been long known to filter and remove exogenous and endogenous toxins from the C S F (McKinnon, 1998). Recently, Sato et al. found that the high affinity cystine-glutamate antiporter system x c" (xCT) is expressed at particularly high levels in meninges (Sato et al., 2002). In many cell types, the uptake of cystine is the rate-limiting step for G S H synthesis. Meningeal cells may use xCT-dependent cystine uptake as an efficient means to supply cysteine for maintainence of a large G S H pool. This enhanced G S H production by the meninges may play an important role in buffering brain oxidative stress. Consistent with this proposal, we found meningeal cells to be more potent than cortical astrocytes in protecting neurons from an in vitro oxidative stress model (Sun et al., 2004; 2005a and Shih et al. in preparation). 60 Figure 3-10 Figure 3-10. MCB labels the meninges and astrocytes robustly. (A). Green fluorescence of rat brain slices labelled with M C B . (B). Laminin labelling pattern of same region, from green detection channel after immunostaining. The M C B signal was lost during paraformaldehyde fixation for immunostaining, therefore it did not interfere with the secondary antibody (FITC anti-rabbit) signal. For presentation a blue color was added. (C). Red fluorescence of cells stained with S-100P as primary antibody and Alexa 594 anti-mouse as secondary antibody. Panel A and C show that the M C B labelled cells with high fluorescent are astrocytes. Arrow and arrowhead point to an astrocyte and a perivascular cell labelled by both M C B and S-100p, respectively. Images were taken using two-photon microscopy of a rat brain slice using a 40x objective. 61 3.4.2 MCB labelling of neurons in slices is much weaker than labelling of astrocytes When imaging deeper layers of the neocortex in PI 5-20 rat slices, such as Layer II and III (Figure 3-11 A ) , we identified another group of cells that were MCB-label led but with lower fluorescence. These cells had the morphology of neurons with apical dendrites oriented towards the meninges (Figure 3-11 B) . Co-labelling the acute live slices with PI showed that these cells had intact membranes, since there was no overlap between the M C B and PI signal, and were thus viable (Figure 3-11 C). After fixation with paraformaldehyde (PFA), the M C B signal was completely lost (data not shown), while the PI signal was preserved within cells that had died during vibratome sectioning and also spread to all fixed cells that were originally labelled by M C B , including those with neuronal morphology (Figure 3-11 E). Immunostaining with NeuN, a neuron- specific nuclear protein (Mullen et al., 1992) confirmed that the cells with a lower level of fluorescence were indeed neurons (Figure 3-11 D, F). A s fixation of slices caused some distortion of cell position, we examined co-localization over a relatively small area based on cell morphology and the relative position of cells. In previous studies using largely developing neurons and glia in cell culture based assays, inferences regarding the cellular distribution of G S H in brain have been inconsistent. Some studies suggest that G S H is substantially lower in cortical neurons than in glia (Slivka et al., 1987; Lowndes et al., 1994; Beiswanger et al., 1995; Tauskela et al., 2000), whereas others do not (Amara et al., 1994; Hjelle et al., 1994; Rice and Russo-Menna, 1998). Our results provide direct evidence that in the cerebral cortex in 62 situ there is heterogeneity in the distribution of G S H between neurons and glia, with neurons in general exhibiting much lower levels of G S H . Figure 3-11 64 Figure 3-11. M C B labelling of mature cortical neurons. (A) . Coronal section o f the rat brain showing the location of two-photon imaging, modified from The Rat Brain in stereotaxic coordinates (Paxinos and Watson, 1986). (B). M C B labelling of cortical Layers I and II in a rat brain slice. In Layer II, there are many cells with neuronal morphology labelled by M C B , with much lower fluorescence intensity than the M C B - labelled astrocytes in Layer I. (C). PI labelling of the same region as (B) in an acute slice, showing dead cells with ruptured membranes permeable to PI (healthy cells are normally membrane impermeable). (D). Immunostaining of the same region in panel A with NeuN, a neuron specific nuclear protein (primary antibody) and F ITC anti-mouse as the secondary antibody, showed that the cells with relatively lower fluorescence in Layer II in panel A are neurons. (E). PI labelling of same region after fixation. (F). Overlay of NeuN and M C B signals (region from inset in panels B and D). Since fixation caused irregular of stretching of slices and M C B staining can be performed on live tissue only, we only overlaid the inset region where cells of interest were best aligned. Images were taken with two-photon microscopy using a 40x objective. 65 3.4.3 Lateral ventricle ependymal cells have the highest GSH content in brain slices In addition to meningeal cells and astrocytes, M C B also strongly labelled ependymal cells along the lateral ventricle (Figure 3-12 B) . These cells were significantly brighter than any other region of brain slices examined. A s with the meninges, ependymal cells also face C S F (Figure 3-12 A ) and are known to express a high level of cystine-glutamate transporter x C T , allowing the extraction of cystine for G S H synthesis (Sato et al., 2002). Ependymal cells may also contribute to maintenance of brain redox homeostasis by synthesizing and exporting G S H . 66 Figure 3-12 B v, I Ependymal -^ cells fl 1 40um SVZ Figure 3-12. MCB labelling of GSH-containing ependymal cells lining the lateral ventricle. (A). Coronal section of rat brain showing the location of two-photon imaging, modified from The Rat Brain in stereotaxic coordinates (Paxinos and Watson, 1986). (B). Ependymal cells lining the lateral ventricle show very strong fluorescence after MCB labelling. Although lower in fluorescence than the ependymal layer, cells within the subventricular zone (SVZ) were clearly labelled by MCB. Image was taken with 40x objective. 67 In close proximity to ependymal cells is the subventricular zone where neurogenesis can occur (even in mature brain) (Doetsch and Alvarez-Buylla, 1996). Cells within the subventricular zone were also strongly labelled by M C B (Figure. 3-13 A ) . Immunostaining with doublecortin ( D C X ) , a marker of neurogenesis, which is expressed in migrating and differentiating premature neurons (Francis et al., 1999; Brown et al., 2003), indicated that many of these GSH-containing subventricular zone cells were neuronal precursor cells (Figure 3-13). This suggests that a high level of G S H may be required to support the high proliferation rate and consequent increase in metabolic activity of these precursor cells. When cells are dividing, they also express SlOOp (Deloulme et al., 2004). Immunostaining with SlOOp showed that some M C B labelled S V Z cells are also labelled by S100P (Figure 3-14). SlOOp can also label ependymal cells (Figure 3-14), which are not labelled by D C X (Figure 3-13). However, G F A P can hardly any cells in this region (Figure 3-15). We also immunostained the slices with NeuN, a mature neuron marker, and we found no colocalization between NeuN signal and M C B signal (Figure 3.-16). When doing this, we used laminin to label blood vessels, which can help us to find the previous imaged regions after immunostaining. In conclusion, many M C B labelled cells in subventricular zone are D C X positive, some of them are S100P positive, but almost none of them are G F A P or NeuN positive, indicating these cells are precursor cells. 68 Figure 3-13 40Mm c • v • * D C X Figure 3-13. Many cells labelled by MCB in subventricular zone (SVZ) are labelled by doublecortin (DCX). (A). M C B labelling of lateral ventricle ependymal cells and cells in S V Z . (B). Immunostaining of the same region in panel A with D C X , a marker of neurogenesis (primary antibody) and Alexa 488 anti-goat as the secondary antibody. (C). Overlay of D C X and M C B signals (region from inset in panels A and B) . Since fixation caused irregular of stretching of slices and M C B staining can only be performed on live tissue, we only overlaid the inset region where cells of interest were best aligned. Images were taken with two-photon microscopy using a 40x objective. 69 Figure 3-14 Figure 3-14. Some cells labeled by M C B along lateral ventricle and in S V Z are also labeled by S100p\ (A). M C B labelling of lateral ventricle ependymal cells and cells in S V Z . (B). Immunostaining of the same region in panel A with S 100(3 (primary antibody) and FITC-anti-mouse as the secondary antibody. (C). Overlay of S 100(3 and M C B signals. Green and red colors are added artificially to M C B and S 100(3, respectively. 70 Figure 3-15 Figure 3-15. Cells labeled by M C B along lateral ventricle and in S V Z are not labeled by G F A P . (A). M C B labelling of lateral ventricle ependymal cells and cells in S V Z . (B). Immunostaining of the same region in panel A with G F A P (primary antibody) and FITC-anti-rabbit as the secondary antibody. (C). Overlay of G F A P and M C B signals. Green and red colors are added artificially to M C B and G F A P , respectively. 71 Figure 3-16 A 40Lim B / . f »* • • 5" • • # M C B Lamin in C D • *' * -T *+JC\~ J NeuN Figure 3-16. Mature neurons in S V Z are not labelled by M C B . (A). Green fluorescence of rat brain slices labelled with M C B . (B). Laminin labelling pattern of same region, from green detection channel after immunostaining. For presentation a blue color table was added. (C). Red fluorescence of cells stained with NeuN as primary antibody and Alexa 594 anti-mouse as secondary antibody. (D). Overlay of panel (A-C) , showed that mature neurons are not labelled by M C B . Images were taken with two- photon microscopy of a rat brain slice using a 40x objective. 72 3.4.4 A subpopulation of developing neurons in dentate gyrus are labelled by MCB. Given the high levels of G S H in the lateral ventricle ependymal cells and associated subventricular zone and their potential role in neurogenesis, we also examined G S H - M C B labelling in the dentate gyrus. Granule neurons of different developmental stages are found within medial and lateral aspects of the granule cell layers (Kempennann et al., 2003). The most strongly M C B labelled cells were located at the border between the condensed granular cell layer and the hilus (Figure 3-17 A , B , also termed subgranular layer). Cells at this location may be dividing precursor cells that have a unique immunocytochemical profile (Kempermann et al., 2004; L i and Pleasure, 2005) and are expected to be S-100P and D C X positive, but NeuN and G F A P negative . 73 Figure 3-17 B 50um t* - A • Dentate gyrus Figure 3-17. M C B labelling of GSH-containing cells in dentate gyrus. (A). Coronal section of the rat brain showing location of two-photon imaging, modified from The Rat Brain in stereotaxic coordinates (Paxinos and Watson, 1986). (B). M C B labelled two groups of cells in dentate gyrus. The cells within the_most medial portion of granular cell layer and subgranular cell layers (border between condensed granular cell layer and hilus) showed relatively higher fluorescence^ while the more laterally positioned cells_within the medial granular cell layer_showed lower fluorescence. Cells deep within the lateral portion of the granular cell layer were unlabelled by M C B (not significantly above background neuropil) and appeared as black holes. 74 Approximately 40% of these highly fluorescent cells in dentate gyrus were S-100(3 positive (Figure 3-18; n = 226 cells from 2 slices). Immunostaining with G F A P showed no co-localization (0%, 1 G F A P + / M C B + cell in 402 M C B positive cells from 3 slices) between GFAP-positive cells and GSH-containing cells (Figure 3-19). Since S-100P is present in dividing cells but not astrocytes until they are mature (Deloulme et al., 2004), the S-100P and M C B cb-labelled cells could represent developing neuronal precursors. Accordingly 55% of M C B labelled cells were also labelled by D C X (Figure 3-20, n = 225 cells in 2 slices). This suggests that a high level of G S H may be required to support the high proliferation rate and consequent increase in metabolic activity o f these precursor cells. Lateral to the highly fluorescent group of cells (within the condensed granular cell layer), we found a population of cells labelled weakly by M C B (Figure 3- 17B). The most lateral group of cells in the granular cell layer were not labelled by M C B , but strongly labelled by NeuN a marker for mature neurons (Figure 3-21). PI labelling of the live slices showed that the majority of these cells in the granular cell layer had intact membranes and were presumably viable since there was an absence of overlap between M C B and PI signals (Figure 3-21). Thus the reason why the mature neurons do not stain with M C B is not because they are dead. Since newborn granular cells are first present in the inner granular cell layer and migrate to the outer portion as they develop (Kempermann et al., 2003), and the levels of N e u N are correlated with the extent of differentiation (Mullen et al., 1992), we conclude that neurons gradually reduce their G S H content as they mature. 75 Figure 3-18 Figure 3-18. M a n y cells in subgranular zone are colabelled by M C B and SlOOp. (A). M C B labelled two groups of cells in dentate gyrus. Approximate border between medial (M) and lateral (L) granular layers is indicated with a dashed line. (B). S-100P staining of the same region as (A), showing that the highly G S H - M C B fluorescent cells are S-100P positive. (C). Overlay of M C B and S-100p signals indicated significant co-localization of M C B and S-lOOp positive labelling. The arrow shows a co-localized cell 76 Figure 3-19 Figure 3-19. M C B labelled cells in dentate gyrus are GFAP-pos i t ive . (A) M C B labelling of dentate gyrus. (B) G F A P labelling of the same region as (A). Primary antibody was derived from mouse. FITC-anti mouse was used as secondary antibody. (C) Overlay of panel (A) and (B) showed no colocalization between M C B and G F A P . 77 Figure 3-20 MCB / : ' DCX Merge Figure 3-20. Many cells in subgranular zone are colabelled by M C B and DCX. (A). M C B labelled two groups of cells in dentate gyrus. Approximate border between medial (M) and lateral (L) granular layers is indicated with a dashed line. (B). D C X staining of the same region as (A), showing that the highly GSH-MCB fluorescent cells are D C X positive. (C). Overlay of M C B and D C X signals indicated significant co-localization of M C B and D C X positive labelling. The arrows show some co-localized cells. 78 Figure 3-21 A " r_ ^™ „ m t B 50)im • • • * PI - JHV%*- \ MCB c * * * t,« • . • H NeuN * D • Merge Figure 3-21. Ma tu re neurons labelled by N e u N are not labelled by M C B . (A). MCB labelling of dentate gyrus. (B). Co-labelling the live slice in figure (A) with PI indicated the dead surface neurons in dentate gyrus. There is no overlap between the MCB and PI signals. (C). NeuN staining of the same slice showing that the black holes (no MCB label and no PI label) in outer portion of granular cell layer are viable neurons. (D). Overlay of MCB and NeuN signal showed that the cells in the lateral portion of granular cell layer that were not labelled by MCB could be labelled by NeuN. Images were all taken with 40x objective. 79 3.4.5 Measurement of GSH concentration in different brain regions in acute brain slices. Our data indicates that the fluorescence intensity of G S H - M C B is directly proportional to G S H concentration (Figure 3-5). Therefore, when the M C B reaction reaches a plateau we can estimate intracellular G S H concentration in brain slices. We measured the G S H concentration in different brain regions by converting the plateau level of fluorescence to a G S H concentration with the standard curve (Figure 3-22A). In order to standardize for effects of imaging depth on fluorescence intensity (excitation and emission efficiency are reduced at greater depths) (Oheim et al., 2001), we made all of the measurements from the cells at about 50 pm below the surface of the slice. B y surveying various brain regions we found that lateral ventricle ependymal cells show the highest level of G S H (2.73 ± 0.56 m M , *p < 0.05 when compared to all other brain regions measured), which is consistent with their potential role in regulating the redox state of the C S F system. The G S H level in meningeal cells was significantly higher than in cortical astrocytes (meninges, 1.45 ± 0.09 m M ; astrocytes, 0.91 ± 0.08 m M , ***p < 0.001). Since astrocytes have been described as a crucial part of antioxidant defence in brain cells (Dringen, 2000; Shih et al., 2003), our finding suggest that meninges which have received little attention in this context so far, may also be an important component of the brain antioxidant system. The G S H level in astrocytes is about 5 times greater than that of cortical Layer II neurons (astrocytes, 0.91 ± 0.08 m M ; neurons, 0.21 ± 0.02 m M , ***p < 0.001), suggesting that neighboring astrocytes support neuronal viability by releasing G S H during oxidative stress. In dentate gyrus, presumed neuronal progenitors 80 (based on cell position and staining with markers) express a relatively high level of G S H (1.75 ± 0.24 m M ) and differentiated dentate gyrus neurons contained a significantly lower amount in comparison (0.50 ± 0.08 m M , **p < 0.01). Our approach with T P L S M to image G S H provides a more detailed mapping of G S H distribution in brain than the widely used biochemical analyses that are unable to provide information about G S H concentration in at the level of single cells. A s mentioned before, since M C B is in excess and the conjugation between G S H and M C B can be regarded as a first-order reaction, the time course curve should fit a single exponential curve. Figure 3-8 A and B are the time course curves of M C B labelling in meninges, cortical astrocytes, cortical neurons, progenitor cells in dentate gyrus, young neurons in dentate gyrus, and lateral ventricle ependymal cells, respectively. Within the dentate gyrus the putative progenitor cells based on their subgranular position were termed high-fluorescence group, while the medial young neurons of the granule cell layer (weakly N e u N positive) were termed low fluorescence group; lateral granule layer neurons only weakly labelled by M C B and were not plotted. A l l o f these curves exhibited an exponential increase over time and they all reached a plateau level after labelling for 20 min, again indicating that G S T activity was not limiting and that M C B staining reflects G S H content. These curves can all be fitted well with Equation 1: F = Fo+Fmax(l-exp(-(t-to)/T)). The initial slope of the curve reflects G S T activity in these cells (Figure 3-22 B) . Comparing G S T activity between different cell groups, we found there is a trend but not significant difference between meninges, astrocytes, and neurons. The cells with higher G S H level also exhibit a higher G S T activity. This is not surprising since the cells with high G S H level (such as meninges, astrocytes, and lateral ventricle 81 ependymal cells) are usually more important for antioxidant defence, therefore they need high G S T activity to conjugate exogenous and endogenous toxins to G S H and export them out of cells. 82 Figure 3-22 B men astro neuronOG_Ngh06Jow LV men astro neuron DGJilghDGJow LV Figure 3-22. Quantification of G S H concentration in different brain regions. (A). G S H concentrations at different regions of P15-P21 rat brain slices. Data for different brain regions were averaged from five experiments respectively. (1): *** p < 0.001, significantly higher G S H in meninges when compared to astrocytes and neurons. (2): *** p < 0.001, when astrocytes were compared to neurons. (3): **p < 0.01, S V Z cells (high fluorescence) contain significantly higher G S H when compared to the more lateral dentate gyrus cells found in the granule,cell layer_(low fluorescence). (4): * p < 0.05, lateral ventricle ependymal cells contain more G S H than all other cell types examined. (B). The M C B conjugation activity (proportional to G S T activity) at different brain regions estimated from the exponential fitting of the time course curves. Average values from 3 separate experiments are shown. (6): There was a trend but no significant difference in conjugation activity between meninges, astrocytes and neurons. (5): *p < 0.05 lateral ventricle ependymal cells had significantly higher G S T activity than all other cell types except meninges. Data were given as mean + S E M and analyzed with repeated-measures one-way A N O V A . 83 3.4.6 Conclusion B y applying T P L S M to rat brain slices, we have found that G S H is enriched in meninges, lateral ependymal cells and subventricular cells, astrocytes, and immature neurons in dentate gyrus. Neocortical neurons in slices from mature animals can be labelled by M C B but at a much lower level of fluorescence suggesting that glial, meningeal, and ependymal cell sources may play a dominant role in buffering brain oxidative stress. 3.5 MCB labels a subpopulation of meningeal and neocortical cells in vivo. It is conceivable that G S H metabolism in slices may be different from that in vivo for a variety of reasons including: the lack of slice blood supply and exposure to G S H precursors as well as the possibility of trauma in making brain slices. With two-photon in vivo microscopy we were able to image G S H distribution in the intact brain directly. Exposure of the cortical surface of anaesthetized mice to M C B resulted in staining of a subpopulation of neocortical cells with a plateau level of fluorescence reached in 30 min (Figure 3-23 G). In these experiments, small cranial windows (2x2 mm) were made on C57B1/6 mice and 100 u M M C B was applied directly to the somatosensory cortex surface and MCB-containing agarose was layered over the. brain surface to continuously 84 supply M C B for on-going labelling. MCB-label led cells were imaged down to 250 pm below the brain surface. At the brain surface, M C B labelled many cells, apparently meningeal cells (Figure 3-23 A ) . Side view projections demonstrate that in the neocortex, all the MCB-label led cells showed astroglial morphology (Figure 3-23 E). These cells have multiple processes originating from the cell body, often forming end-feet attached to unstained blood vessels (Figure 3-23 C, arrow). In in vivo experiments we did not include M K 5 7 1 and the preparation was at 37 °C so we expect the plateau level of fluorescence reached to reflect the steady state balance between G S H synthesis and efflux. Recently, Nimmerjahn and colleagues have demonstrated that sulforhodamine 101 (SR 101) can be a relatively specific marker of astroglia in the neocortex in vivo (Nimmerjahn et al., 2004). In these experiments SR 101 was co-localized with astrocyte markers in neocortex, but failed to stain neurons, microglia, or oligodendrocytes. B y co-labeling with M C B and SR 101 in vivo and collecting the signal from the green and red channels respectively, we found that these two probes labelled the same population of astrocyte-like cells in the cortical parenchyma (Figure 3-23D). The meningeal cells labelled by M C B can also be labelled by SR 101 (Figure 3-23B). These results are consistent with our data from cortical slices where M C B robustly labels the meninges and astrocytes. A three- dimensional projection from the side view of the in vivo data stack showed that both in Layer I (approximately 0 ~ -100 pm) and Layer II (approximately -100 ~ -200 pm), MCB-label led cells are astrocytes (Figure 3-23 E , F). N o neurons in Layer II (SR 101 negative cells) were labelled by this approach using the mature mice we studied (>2 months of age). However, we have found that neurons in brain slices from young rats can be labelled by M C B , although with a very low fluorescence level (-20% o f astrocyte 85 levels). The reason why neurons in vivo are not labelled cannot be due to a lack of M C B tissue penetration, since astrocytes at the same depth level as neurons can be labelled. Perhaps in brain slices less attenuation of fluorescence signals takes place allowing the observation of Layer II neurons, which exhibit approximately 20% of the G S H found in astrocytes. It is also possible that the slices from young rats (P15~P21) contain more immature neurons that have relatively higher G S H . Consistent with this, using the Tietze assay (Tietze, 1969) we have seen higher G S H levels in tissue homogenates from young and embryonic rats when compared to adults (Shih et al., in preparation). 86 Figure 3-23 Figure 3-23. In vivo two-photon fluorescence image of the somatosensory cortex of a live anesthetized mouse after application of M C B and SR 101. (A). Image recorded at the brain surface after M C B labelling. A population of meningeal cells are labelled by M C B . Blood vessels appear as dark gaps (arrow). (B). SR 101 labelling of the same region in panel A . M C B and S R 101 labelled the same group o f cells. (C). Optical section of M C B fluorescence recorded about 50 pm below the pial surface showing that a subpopulation of cells had taken up the dye deeper within the parenchyma. These cells had astroglial morphological features and some formed end-feet surrounding unstained blood vessels (arrow). (D). Optical section of fluorescence for the astrocyte marker S R 101 (applied to the brain surface simultaneously with M C B ) at the same region as panel C. (E). Side view ( X - Z image) of the M C B labelled meninges and astrocytes in neocortex (maximal intensity projections) from surface of the brain to a depth of approximately - 250 pm, reconstructed from planar scans acquired every 1 pm after M C B addition. (F). Side view of SR 101 labelled cells in meninges and neocortex. A l l images were taken with 40x objective. (G). Time course curve of M C B in vivo labelling showing that the fluorescence intensity reaches a plateau level within 30 min. Average values from 3 different experiments are shown. 88 3.6 Meningeal cells have a more active GSH system than astrocytes ir neocortex The meninges have received little attention in antioxidant defense and have mainly been highlighted for their physical role at the CSF-blood barrier (Nilsson et al., 1992; Smith and Shine, 1992; Tanno et al., 1993). Our findings provide a new insight into the possible role of the meninges in protecting the brain against oxidative stress. In order to look further into the G S H metabolism system in meninges and astrocytes, we prepared meninges and cortical cells cultures from new-born rats and used M C B to label the cultures. A s described above, we have developed an assay to measure several parameters of G S H metabolism in glial cells, such as G S H content, G S T activity, rate of G S H biosynthesis, etc, using plate reader (Sun et al., 2005). This assay is also applicable to measure parameters of G S H metabolism in meninges and cortical cells cultures. 3.6.1 Meninges contain higher level GSH and GST activity than cortical cells In meninges and cortical cell cultures, the M C B labelling time course curve also fit well with a single exponential equation (Equation 1): F=Fo+Fm a x(l-exp(-(t-t 0)/x)) F is the fluorescence intensity normalized to protein amount. F m a x is the maximum level of fluorescence intensity per mg protein, and x is the time constant of the reaction. From Figure 3-24A, the curve from meninges showed a much faster increase and reached a 89 higher plateau than cortical cell cultures. The highest fluorescence level of meninges am cortical cultures are 1.541 ± 0 . 1 9 3 , 1.084 + 0.153 (fluorescence intensity/ug protein), respectively (p=0.016). The initial slopes of them are 0.652 + 0.082, 0.182 + 0.132 (fluorescence intensity per ug protein/min) (p=0.0022). Therefore, we confirmed that meninges have higher G S H level and higher G S H activity than cortical cells. 3.6.2 Meninges cells are more efficient in taking up GSH precursors and synthesizing GSH After the M C B labeling of endogenous G S H has reached a plateau level, we added 100 (aM cystine, the rate-limiting precursor for G S H synthesis, or a cocktail o f precursors, glutamate, glycine and cystine (100 u M for each component), to the cultured cells. In both cultures, we found a significant linear increase in fluorescence intensity, with meningeal cultures showing a much larger slope, indicating meningeal cells are much more efficient than cortical cells in taking up precursors and synthesizing G S H (Figure 3-24 B) . The slopes of increase after cystine addition are 0.096±0.018 and 0.0088±0.0018 (fluorescence intensity per ug protein/min) for meninges and cortical cells, respectively (p=0.011). The slopes after cocktail addition are 0.071±0.0054 and 0.011±0.0017 (p=0.0016), averaged from 4 experiments. H B S S was used as a control. Neither of them showed significant increase after H B S S addition, with the slope of 0.003210.0007 and 0.001010.0002 (fluorescence intensity per ug protein/min), respectively. When exogenous G S H (lOOuM) was added, the fluorescence also showed a 90 linear increase, with a slope of 0.066±0.0069 and 0.024±0.0071 (fluorescence intensity per pg protein/min), respectively for meninges and cortical culture. The slope o f meninges after G S H addition is significantly smaller than cystine (p=r0.048), indicating that meninges are more efficient at taking up the precursors for G S H synthesis and then synthesizing G S H rather than taking up G S H directly (see Table 3-2). 91 Table 3-2. Comparison of kinetic parameters of G S H metabolism between and cortical cell cultures. Meninges culture Cortical culture Fmax (fluor unit per mg protein) * 1.541 ± 0 . 1 9 3 1.084+0.153 Initial slope(fluor unit per mg protein/min) * * 0.652±0.082 0.182±0.132 Slope (Cys) (fluor unit per mg protein/min) * 0.096±0.018 0.0088±0.0018 Slope (Ckt) (fluor unit per mg protein/min)** 0.071±0.0054 0.011±0.0017 Slope (GSH) (fluor unit per mg protein/min)** 0.066±0.0069 0.024±0.0071 Slope (HBSS) (fluor unit per mg protein/min) 0.0032±0.0007 0.0010±0.0002 Figure 3-24 Figure 3-24. G S H metabolism is more active in meninges than in cortical cells. (A) Time course curve of M C B labelling of meningeal and cortical cells in culture at room temperature, showing that meningeal cells contain higher level o f G S H and are more active in G S T activity. (B). After the G S H - M C B fluorescence got to plateau level, we added Cys, G S H rate-limiting precurosr, or H B S S , as a control to the culture, and found meningeal cells are more efficient in taking up precursors and synthesizing G S H . 9 3 3.6.3 Immunostaining of cortical cell cultures In order to confirm the types of cells in the cortical cell cultures, we did immunostaining after M C B labeling. In order to fix M C B signal, we added 0.2% glutaldehyde to 4% P F A , and stained the cultures with astrocytes marker, slOOp. A l l the cells labeled by M C B are also labeled by slOOp (Figure 3-25 A - C ) . While counterimmunstaining with neuron marker, MapII, showed no staining at all in the cortical cultures (Figure 3-25 D-E) . Therefore the GSH-containing cells in the cortical cultures are all astrocytes. 94 Figure 3-25 Figure 3-25. M C B labelled cells in cortical cells culture are S 100(3 positive. M C B labelled cells in cortical cells cultures (A) are also labelled by S100 (3 (B). (C). The two signals are colocalized. (D, E). MapII cannot label any cells in the cortical cells cultures that are labelled by M C B . (F) is a picture of the cultured cells with light microscopy. 95 Chapter 4 Discussion 4.1 MCB labelling with two-photon microscopy resolves cellular GSH in situ. Accumulating evidence indicates that alteration of G S H content and oxidative stress play substantial roles in neurological disorders such as stroke, Parkinson's, Alzheimer's, and Huntington's disease (Bains and Shaw, 1997; Schulz et al., 2000; Keelan et al., 2001; Perry et al., 2001; Shih et al., 2005b; Shih et al., 2005a). In view of the pivotal role of G S H in protecting cells (Cooper and Kristal, 1997; Herzenberg et al., 1997; Shih et al., 2003), there has been a considerable interest in the development of methods to quantify G S H content using biochemical assays (Tietze, 1969; Newton et al., 1981; Anderson, 1985). However, biochemical assays using cell lysates or tissue homogenates are unsuitable for examining G S H levels in intact single cells. The ability to measure G S H at the cellular level using two-photon imaging overcomes limitations inherent with conventional biochemical techniques and reveals a marked cellular heterogeneity in labelling patterns that would be lost in a biochemical assay for G S H . However, it is important to note that light scattering by tissues limits the depth of two-photon microscopy to about 500 um (Oheim et al., 2001; Helmchen and Denk, 2005). When measuring G S H concentration in slices we standardized for the effect of depth by measuring the fluorescence intensity of all regions at the same depth level, 96 about 50 pm. A t this level, the damage caused by vibratome sectioning is negligible and the image depth w i l l have similar effects on different brain regions. Another potential caveat is that the M C B - G S H labelling technique is a means of measuring the total G S H content of a cell of interest (by converting all G S H to a fluorescent M C B conjugate) and wi l l not provide a real-time sensor of G S H level. If one wants to measure G S H content at different time points within the same preparation they would need to allow for efflux of G S H - M C B and then reload with M C B . I f reloading is done in the presence of G S H precursors the rate of G S H synthesis can also be measured (Sun et al., 2005). A further caveat is that M C B labelling depletes the intracellular reservoir of reduced G S H as the M C B - G S H reaction proceeds to completion. Thus, long-term imaging may lead to increased oxidative stress and promote cellular apoptosis. Actually some groups have used M C B to cause G S H depletion. For example, Vesce et al. (2005) have reported that by exposing neurons cultures with M C B for l h , mitochondria within neurons failed to hyperpolarize upon addition of oligomycin to inhibit their A T P synthesis. M C B progressively decreased cell respiration, but with no effect on mitochondrial proton leak or maximal respiratory capacity. However, in our experiments, we did not observed MCB-label led cells show response under oxidative stress caused by G S H depletion. The possible reason is that with slices and cultures, we only exposed our slices to M C B for about half an hour, which is much shorter than the incubating time in Vesce et al (2005). When we performed M C B labelling in vivo, the labelling time is usually longer than l h . But our in vivo labelling only showed M C B labelling of meninges and cortical astrocytes, which are much more efficient in defending against oxidative stress than neurons. 97 Therefore the M C B labelled cells in vivo seemed healthy during the total imaging procedure. Although these limitations exist and are acknowledged to aid future researchers, feel that two-photon imaging of M C B labelling in brain slices is major improvement previously employed biochemical or imaging assays for G S H . we over 4.2 Cellular GSH distribution in brain slices. 4.2.1 GSH is highly expressed in cells at CSF interface In this study we measure the G S H concentration in single cells in intact tissues directly with two-photon microscopy. O f all regions studied, lateral ventricle ependymal cells contain the highest level of G S H . Also of note, the meninges contain considerably higher G S H than neocortical astrocytes. Sato et al. have reported (Sato et al., 2002) that x C T (a high affinity cystine uptake system that is likely the rate limiting step in providing the precursor cysteine for G S H synthesis), is highly expressed both in meninges and in ependymal cells. Both o f these regions are in direct contact with C S F making them ideally positioned to take up cystine for G S H synthesis and release to protect the brain against oxidative stress. The meninges have received little attention in antioxidant defense and have mainly been highlighted for their physical role at the CSF-blood barrier (Nilsson et al., 1992; Smith and Shine, 1992; Tanno et al., 1993). Our findings provid new insight into the possible role of the meninges in protecting the brain against oxidative stress. In another article under preparation, we found that cultured meningeal e a 98 cells possess an active G S H antioxidant system and are even more efficient in protecting neurons against oxidative glutamate toxicity than astrocytes (Shih et al., in preparation; Sun et al, 2005a). However, in the intact brain, it is likely that all the GSH-enriched cell- types we have identified (meninges, ependyma, and astrocytes) contribute to redox buffering in the brain. 4.2.2 GSH in neurons It has been well established that heterogeneity in G S H levels exists between neurons and astrocytes in brain parenchyma (Dringen, 2000). However, the relationship between G S H concentration in neurons versus astrocytes has not been consistent between all studies (Beiswanger et al., 1995; Rice and Russo-Menna, 1998; Tauskela et al., 2000). Perhaps these differences in part may be related to the developmental state or culture conditions of the preparations used. In our study, we found that in P15-P20d rat slices astrocytes contain 5 times more G S H than neurons in cortex. We also examined brain slices from young (Pl-2) rats and found cells of presumably neuronal origin to have high G S H content (Figure 4-1). In these young slices, most cells are labelled by M C B . They can be separated to two groups. The high fluorescence group has G S H concentration of 1 .70±0 .03 m M , the low group has G S H of 0.74 + 0.04 m M ( * * * pO.OOl) . Both of these groups contain significantly higher G S H than cortical neurons in mature (P15-20) rat slices (neurons: 0.21 +0 .02mM. *** pO.OOl significantly lower than the two groups in young slices). We have hoped to identify the types of these cells in young slices. However, in these young animals it was difficult to costain M C B labelled neurons with 99 antibody markers since the neurons were not fully differentiated and the morphology of the slices was altered much by fixation. 100 Figure 4-1 A B 50um • P2 • * * f P18 Figure 4-1. Comparison of M C B labelling in young and mature rat slices. (A). M C B labels almost all cells in PI-2 rat slices, with a subpopulation of cells show high fluorescence ([GSH]=1.70 ±0.03 mM, others show lower fluorescence ([GSH]=0.74± 0.04 mM. (B). M C B labelling in mature (PI8) rat slices. Only meningeal cells and cortical astrocytes are labelled robustly by M C B . 101 Developing neurons in mature brain also contained higher GSH-MCB levels. In the mature brain there are two major regions where neurogenesis is known to occur postnatally: the subventricular zone and the subgranular zone of the dentate gyrus (Li and Pleasure, 2005). Our results indicate that both neurogenic regions contain relatively high levels of GSH. In dentate gyrus, young neurons in the medial portion of granular cell layer contain GSH at a level much lower than the dividing precursor cells (subgranular zone), while the mature neurons in the lateral portion of granular cell layer contain only background levels of GSH. This suggests that when neurons are proliferating they are in great need of antioxidant buffer and thus contain high GSH. During the process of maturation, neurons lose most of their GSH content and henceforth seem to depend on astrocytes to provide them with this antioxidant buffer. This is consistent with the findings of Beiswanger et al (1995) who visualized GSH distribution in the mouse nervous system by mercury orange histochemistry and found that all neuronal and glial progenitor cells contain GSH, while most neurons lose the bulk of their GSH content by P5 and become surrounded by a GSH-rich neuropil. Other groups also reported that neurons lose their GSH content as they grow in co-culture with astrocytes (Sagara et al., 1993; Keelan et al., 2001). This low level of GSH content in mature neurons may explain their unique vulnerability to oxidative stress (Murphy et al., 1990; Ratan et al., 1994; Bolanos et al., 1995; Dringen et al., 1999; Schubert and Piasecki, 2001). In addition to the cell types described here it will be interesting to apply the MCB labelling approach to study GSH in oligodendrocytes that have a unique developmentally-dependent vulnerability to oxidative stress (Oka et al., 1993; Back et al, 1998). 102 4.2.3 Summary of types of cells labelled by MCB in slices We have used immunostaining to verify the types of cells labelled by M C B . M C B - G S H signal can be fixed with 0.2% glutaraldehyde in 4% P F A . With immunostaining of meninges and cortical cell cultures, we fixed the culture with 0.2% glutaraldehyde in 4% P F A , and M C B - G S H signal was well preserved (Figure 3-25). However, glutaraldehyde wi l l make slices impossible to be stained with antibodies (data not shown). In order to perform immunostaining, we can only fix slices with 4% P F A , which wi l l cause completely lost of M C B - G S H signal. To find the previously imaged region after immunostaining, we used laminin, a marker for base membrane of blood vessels, or PI, a dead cell marker, as landmarks. Since M C B wi l l label perivascular astrocytes, making blood vessel visible when imaging acute slices. After immunostaining, laminin wi l l also label the same blood vessels, which can be used as landmarks. For PI, we usually colabeled the acute slices with M C B and PI together. PI wi l l label dead cells in acute slices, which is usually on the surface of slices. After fixation, PI signal was preserved within these cells that had died during vibratome sectioning, which can also be used as landmarks. However, the fixation always caused some distortion of cell position, therefore we usually only examined colocalization over a relatively small area, based on cell morphology and the relative position of cells. M C B can robustly label cells at the C S F interface, such as meninges and lateral ventricle ependymal cells. In cortex, M C B labelled cells can be separated to two groups. The high fluorescence group are SlOOp positive, and NeuN negative, and are mostly found in Layer I, indicating they are astrocytes. The low fluorescence group are usually 103 found in Layer II, showing neuronal morphology and having their apical dendrites orienting towards the meninges. Immunostained with NeuN confirmed that they are neurons. In subventricular zone (SVZ) under the ependymal cells, lots of cells are also strongly labelled by MCB. Many of them are DCX positive, indicating they are neuron precursors, and need high GSH reservoir for their high metabolic activity. In dentate gyrus, the highest fluorescence cells are found in subgranular zone (SGZ), many of which are also DCX positive. In medial portion of granular cell layer, cells showed low MCB and NeuN signal, indicating they are young neurons. In lateral portion of granular cell layer, cells were labelled by NeuN strongly, indicating they are mature neurons. However, they have lost almost all of their GSH content (Table 4-1). 104 Table 4-1. Types of cells labelled by M C B Cortex SVZ SGZ High fluorescence group Low fluorescence group s i o o p + + + NeuN - + - DCX - - + + 105 4 . 3 Manipulation of GSH metabolism We have developed an assay to monitor the metabolism of G S H in cultures, using a fluorescence plate reader and monitoring the kinetics of the G S T catalyzed reaction between G S H and the substrate M C B . We were able to measure various kinetics parameters of G S H metabolism, such as the rates of G S H synthesis, conjugation and efflux under basal conditions and after t B H Q induction of Nrf2 driven antioxidant response element-mediated gene expression. Our assay can also be applied to meninges and cortical cells cultures, which showed that meninges are more active in G S H metabolism. They contain high level of G S H , higher activity of GST, and are more efficient in taking up G S H precursors and synthesizing G S H . We have hoped to measure different kinetic parameters of G S H metabolism in rat brain slices, besides G S H content and G S T activity. We added G S H lOOuM, or Cystine 100 p M , to the perfusion system and used two-photon microscopy to record the fluorescence change over time. However, we did not find significant increase in fluorescence intensity as we have hoped. A possible reason is that cells in slices cannot take up these substrates as efficiently as in cultures. We also tried to examine whether we can increase intracellular G S H level by applying G S H precursor, cystine to animals. We injected Cystine ( ImM) from tail vein to mice and measured G S H concentration in tissue homogenate with G S H assay. However, in all brain regions that we have examined, including cortex, striatum, hippocampus, and cerebellum, there is no significant difference between animals injected with Cystine and 106 control (data not shown). We only found significant increase in liver homogenate and blood (Liver: Cys group: 156.1 + 17.9 m M , control group: 70.9 +10.3 m M , p<0.01; , Blood cells: Cys group: 417.9 ± 2 5 . 4 m M , control group: 5 3 . 2 ± 7 . 6 m M , p<0.01). We supposed that cells in brain might already have enough G S H , therefore they wonot take up G S H precursors. In order to test this, we first depleted G S H content with diethyl maleate ( D E M ) (Szaszi et al., 2005), which caused almost complete depletion of intracellular G S H , but did not affect G S T activity (Figure 4-2). After G S H depletion, we injected Cystine through tail vein. However, we still cannot find significant difference between control group and Cystine group (data not shown). A possible reason is that it is difficult for cystine to cross blood-brain barrier (data not shown). We also used two- photon microscopy to image fluorescence level after tail vein injection of G S H or Cystine, and we did not find significant increase either (data not shown). 107 Figure 4-2 B H control CZJ DEM hippo ce liver Figure 4-2. D E M depletion of intracellular G S H content. (A). G S H content in tissue homogenate from different brain regions and liver. D E M caused a significant decrease in G S H concentration in all regions (p<0.05, for all groups). (B). G S T activity in different regions. D E M did not affect G S T activity. Data were averaged from 3 animals. 108 4.4 Measurement of GSH within the cortex of live animals. In addition to examining M C B - G S H labelling in brain slices, we adapted the technique to an in vivo preparation. Brief exposure of the intact brain to M C B followed by two-photon imaging was proved to be a robust method for labelling GSH-containing cells in the somatosensory cortex in vivo. The specificity o f M C B labeling o f astrocytes in neocortex was verified by co-labelling with SR 101, a neocortex astrocyte marker (Nimmerjahn et al., 2004). This approach offers the opportunity to measure G S H content in live animals, which is closer to biological reality than measures performed in brain slices or cultures. The G S H antioxidant system can be activated when the brain is under oxidative stress, such as during the reperfusion period after an ischemic insult when G S H demand may be relatively higher (Rehncrona et al., 1980; Lyrer et al., 1991). It w i l l also be affected by many neurological disorders, such as Parkinson's disease (Schapira et al., 1990), and Alzheimer's disease (Abramov et al., 2003). Recent data also indicates a role for G S H and its precursors in maintaining the viability of brain tumors (Chung et al., 2005). However, in most cases, data linking the G S H system to these disorders was derived from tissue homogenates, which cannot detect cellular heterogeneity. In addition there are some contrary reports on how the G S H levels change during these neurological diseases (Uemura et al., 1991; M i z u i et al., 1992; Gotoh et al., 1994; Guegan et al., 1998). Our approach can be applied to animal models of these diseases and mouse mutants with abnormal handling of oxidative stress (Aoyama et al., 2005; Siddiq et al., 2005) to measure G S H levels in different brain regions directly. Recent studies from our lab have used electrophilic inducers of the redox-sensitive transcription factor Nrf2 to 109 4.4 Measurement of GSH within the cortex of live animals. In addition to examining M C B - G S H labelling in brain slices, we adapted the technique to an in vivo preparation. Brief exposure of the intact brain to M C B followed by two-photon imaging was proved to be a robust method for labelling GSH-containing cells in the somatosensory cortex in vivo. The specificity of M C B labeling of astrocytes in neocortex was verified by co-labelling with SR 101, a neocortex astrocyte marker (Nimmerjahn et al., 2004). This approach offers the opportunity to measure G S H content in live animals, which is closer to biological reality than measures performed in brain slices or cultures. The G S H antioxidant system can be activated when the brain is under oxidative stress, such as during the reperfusion period after an ischemic insult when G S H demand may be relatively higher (Rehncrona et al., 1980; Lyrer et al., 1991). It w i l l also be affected by many neurological disorders, such as Parkinson's disease (Schapira et al., 1990), and Alzheimer's disease (Abramov et al., 2003). Recent data also indicates a role for G S H and its precursors in maintaining the viability of brain tumors (Chung et al., 2005). However, in most cases, data linking the G S H system to these disorders was derived from tissue homogenates, which cannot detect cellular heterogeneity. In addition there are some contrary reports on how the G S H levels change during these neurological diseases (Uemura et al., 1991; M i z u i et al., 1992; Gotoh et al., 1994; Guegan et al., 1998). Our approach can be applied to animal models of these diseases and mouse mutants with abnormal handling of oxidative stress (Aoyama et al., 2005; Siddiq et al., 2005) to measure G S H levels in different brain regions directly. Recent studies from our lab have used electrophilic inducers of the redox-sensitive transcription factor Nrf2 to 109 induce multiple antioxidant systems as a therapeutic strategy for neurodegeneration and stroke (Shih et a l , 2005b; Shih et al., 2005a). Interestingly, evaluation of brain tissue homogenates showed dynamic increases in G S H content after the administration of Nrf2 inducers. Animals receiving inducers exhibited reduced lesion size and improved recovery compared to controls. In future studies, the technique we have described here could be used to image and quantify the cellular sites of G S H induction by Nrf2 inducers, as well as other drugs that influence the G S H system (Kamencic et al., 2001). 4.5 Meninges may be an ideal position for protecting brain against oxidative load Little attention has been paid to the meninges covering the brain parenchyma except for the physical role at the CSF-blood barrier (Nilsson et al., 1992; Smith and Shine, 1992; Tanno et al., 1993). In the current study, we have found that meninges contain significantly higher G S H content than astrocytes. With plate reader assay, we found that meninges are more active in G S H metabolism than cortical cells. They contain higher activity of G S T , and are more efficient in taking up G S H precursors and synthesizing G S H . This suggests that meninges may play an important role in antioxidant defence. Consistent with this, Shih A Y , a PhD student in our lab, used R T - P C R and western blot to show that meninges express higher level of x C T than cortex, striatum and liver. x C T is a subunit of xc~, an exchange agency specific for anionic forms of cystine and glutamate (Bannai, 1986). The x c" antiporter is composed of two subunits, x C T and 4F2hc. Whereas x C T confers cystine specificity, 4F2hc is present as a structural subunit no in a variety of amino acid transporters (Sato et al, 1999). With this high x C T level, meninges can take up 3 5S-cystine 20-fold greater than cortical cells (Shih A Y et al., paper in preparation). Shih A Y further tested whether meninges are more efficient in protecting neurons against oxidative stress than astrocytes with a well-established N M D A receptor- independent oxidative glutamate toxicity paradigm in which neurons die from G S H depletion (Murphy et al., 1989, 1990). He cocultured neurons with glia, meninges or fibroblast cells, which wi l l protect neurons against the oxidative stress caused by addition of 3 m M glutamate. B y comparing the percentage of viable neurons in each group, he found that meninges are the most efficient in neuroprotection. The percentage of N S E + (a neuron-selective marker) cells in meninges-neuron coculture is significantly higher than in other coculture (Shih et al., paper in preparation). Meninges are filled with C S F , whose antioxidant potential seems to be important for keeping brain healthy. The highly expressed x c system seems to have two functions. First, clearance of cystine via x c system and release of cysteine from the cells via neutral amino acid transporters may contribute to maintain the reduced state in the C S F against oxidative stress. Second, the uptake of cystine from Xc" provides meninges with plenty of precursors for G S H synthesis, which makes meninges efficient in protecting brain against oxidative stress. From all the above, we supposed meninges may be an ideal position for protecting brain against oxidative stress. i n 4.6 Conclusion I have described a new technique to measure glutathione, the major cellular antioxidant, in the intact brain using two-photon scanning laser microscopy. B y applying and imaging the glutathione specific dye, monochlorobimane, in rat brain slices and the neocortices of live anesthetized mice, I was able to resolve glutathione levels within individual cells. This is a considerable improvement on conventional biochemical assays for glutathione since brain homogenates and cell lysates only provide information about the average glutathione level in the sample. Using this technique, I revealed the heterogeneity of glutathione distrubution throughout the brain and identify ependymal and meningeal cells (CSF-brain barrier), and astrocytes as the major glutathione reservoirs within the brain. Surprisingly, I also find that developing neurons found within two neurogenic regions (subventricular zone and sugranular zone of the dentate gyrus) are particularly rich in glutathione, suggesting that adequate redox buffering is important for the metabolic demands of neuronal proliferation. 112 I have described a new technique to measure glutathione, the major cellular antioxidant, in the intact brain using two-photon scanning laser microscopy. B y applying and imaging the glutathione specific dye, monochlorobimane, in rat brain slices and the neocortices of live anesthetized mice, I was able to resolve glutathione levels within individual cells. 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