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Astrocytes release small molecules to protect neurons from oxidative stress Wang, Xue-Feng 2001

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ASTROCYTES RELEASE SMALL MOLECULES TO PROTECT N E U R O N S F R O M O X I D A T I V E STRESS By  Xue-Feng Wang M.D., Beijing Medical University, 1987 M . S c , Chinese Academy o f Medical Sciences, 1990 A THESIS SUBMITTED IN PARTIAL F U L F I L M E N T OF T H E REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY In  The Faculty of Graduate Studies Graduate Program In Neuroscience We accept this thesis as conforming to the required standard  The University o f British Columbia September 2001 ©  Xue-Feng Wang,  2001  Page 1 of 1  UBC Special Collections - Thesis Authorisation Form  In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e requirements f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d b y t h e h e a d o f my department o r by h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o rf i n a n c i a l gain s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n .  ) The U n i v e r s i t y o f B r i t i s h V a n c o u v e r , Canada  Columbia  16/09/2001  ABSTRACT  The aim of the present experiments is to explore the mechanisms by which astrocytes may protect neurons from oxidative stress with a focus on small molecules. A non-contact astrocyte-neuron co-culturing method was first developed. It was observed that neuronal survival was promoted substantially when neurons were cocultured with a confluent astrocyte feeder layer. The mechanisms by which a stable cysteine level is maintained by astrocytes have been explored in recent years. However, it is still unclear how cysteine is released. In my experiments, cysteine, glutathione, and related compounds in astrocyte conditioned medium were analyzed with HPLC methods. My data suggest that glutathione is released by astrocytes directly and that cysteine is generated from the extracellular thiol/disulfide exchange reaction of cystine and glutathione. Neuron conditioned medium, CSF, and plasma of the carotid artery and internal jugular vein were also analyzed. The results indicate that cystine, rather than cysteine, is transported from blood to the CNS and that the thiol/disulfide exchange reaction occurs in the brain in vivo. Though cysteine plays a critical role in regulating intracellular levels of glutathione, its cytotoxicity has also been noted, particularly in neurons. In the experiments of this thesis, I found that copper substantially accelerates the autoxidation rate of cysteine even at submicromolar levels, whereas iron and other transition metal ions, including manganese, chromium, and zinc, are less efficient. In tissue culture tests, it was found that cysteine toxicity depends highly on its autoxidation rate and on the total amount of cysteine being oxidized, suggesting that the toxicity can be attributed to the free radicals produced from cysteine autoxidation, but not to cysteine itself. The in vivo mechanisms that protect against cysteine toxicity were further explored. Catalase and pyruvate were each found to inhibit the production of hydroxyl radicals generated by cysteine autoxidation. In tissue culture, they both protected primary neurons against cysteine toxicity catalyzed by copper. Pyruvate, but not catalase or glutathione peroxidase, was detected in astrocyte conditioned medium and CSF. Herein, a concerted mechanism of neuroprotection by astrocytes is outlined.  TABLE OF CONTENTS  ABSTRACT  »  TABLE OF CONTENTS  »»  LIST OF TABLES  vi  LIST OF FIGURES  vii  ABBREVIATIONS  ix  ACKNOWLEDGEMENTS Chapter 1 GENERAL INTRODUCTION 1. 2. 3. 4. 5. 6. 7. 8. 9.  Oxygen and Our Life Oxidative Stress in the CNS and the Vulnerability of Neurons Free Radical Chemistry Multiple Roles of Astrocytes Antioxidative Systems of the Organisms Theories of Thiol Provision by Astrocytes Theories of Cysteine Toxicity Neurological Diseases and Oxidative Stress Experimental Objectives  Chapter 2  MATERIALS AND METHODS  Materials 1. Animals 2. Chemicals and Reagents 3. Equipment Methods 1. Preparation of Solutions and Media 2. Tissue Culture 3. Detection of Neuronal Death and Apoptosis 4. H P L C Assay of Thiols, Disulfides, and Related Compounds 5. Spectrophotometric Assay of Cysteine with Ellman's Reagent (DTNB) 6. Immunohistochemical Staining 7. Enzymatic Assays 8. Procedures of Obtaining CSF and Plasma 9. Fluorimetric Assay of Hydroxyl Radicals 10. Pyruvate Assay 11. Stati stical Analysis Chapter 3  xii 1 1 2 3 10 14 17 22 25 29 32 32 32 32 33 33 33 35 39 41 42 43 43 45 45 46 46  EFFECTS OF ASTROCYTES ON NEURONAL SURVIVAL AND ATTACHMENT SHOWN IN A CO-CULTURE SYSTEM 47  Introduction Results 1. Effects of Astrocytes on Neuronal Survival 2. Neuronal Apoptosis Induced by Deprivation of Astrocyte Feeder Layer 3. Effects of Astrocytes on Neuronal Attachment Discussion Chapter 4  47 49 49 51 52 54  PROVISION OF CYSTEINE BY ASTROCYTES TO NEURONS BY WAY OF RELEASING GLUTATHIONE 59  Introduction 59 Results 60 1. Cysteine-glutathione Disulfide (CSSG) Found in Cystine-containing Astrocyte Conditioned Medium 60 2. GSH Released by Astrocytes in Cystine-free Medium 63 3. Analysis of Cysteine and Related Compounds in CSF and in Plasma of Artery and Vein 64 4. Thiols not Found in Neuron Conditioned Medium 65 5. Contents of GSH and Related Compounds in Astrocytes and Neurons 66 6. Autoxidation of Cysteine and GSH 67 Discussion 67 Chapter 5  NEUROTOXICITY INDUCED BY CYSTEINE AUTOXIDATION AND T H E CENTRAL ROLE OF COPPER  Introduction Results... 1. Cysteine Autoxidation in the Presence of Transition Metal Ions 2. Cysteine Autoxidation in CSF 3. Neurotoxic Effects of Cysteine 4. Generation of Hydroxyl Radicals During Cysteine Autoxidation Discussion..... Chapter 6  73 73 74 77 78 80 80  NEUROPROTECTIVE EFFECTS OF PYRUVATE ON CYSTEINE TOXICITY AND T H E RELEASE OF PYRUVATE BY ASTROCYTES 85  Introduction Results 1. Effects of Catalase and Pyruvate on Cysteine Toxicity 2. Effects of Catalase and Pyruvate on Generation of Hydroxyl Radicals 3. Pyruvate Released by Astrocytes 4. Effects of Cysteine Provision on Neuronal Survival Discussion Chapter 7  73  GENERAL DISCUSSION AND CONCLUSIONS  General discussion 1. Thiol Sources in the CNS 2. Mechanisms of Cysteine Toxicity 3. Free Radical Sources in the CNS  85 86 86 86 88 89 90 94 94 94 95 96  iv  4. Antioxidative Mechanisms of Astrocytes 5. Significance of the Present Findings 6. Perspectives Conclusions REFERENCES  97 98 100 101 103  v  LIST OF TABLES  Table 1-1. Half-lives of ROS and Related Species  4  Table 1-2. Standard Redox Potentials  9  Table 1-3. Observations of Neuroprotective Effects of Astrocytes  14  Table 2-1. HPLC Retention Times of the Standard Compounds  41  Table 3-1. Effects of Coating Methods on Neurite Outgrowth and Cell Viability  54  Table 4-1. Content of Cysteine and Related Compounds in A C M Made from CystineFree Medium Table 4-2. Content of Cysteine and Related Compounds in CSF, artery and vein  63 65  Table 4-3. Content of Thiols, Disulfides and Acidic Amino Acids in Astrocytes and Neurons Table 6-1. Pyruvate Content in A C M , N C M and CSF  66 89  vi  LIST OF FIGURES  Figure 1-1. Atmospheric Oxygen and the Course of Evolution  2  Figure,l-2. Free Energy Change of Stepwise Reduction of Oxygen  10  Figure 1-3. Molecular Structures of Glutathione and Related Compounds  18  Figure 1-4. G S H Synthesis and Some of Reactions of GSH  20  Figure 2-1. Micrographs of Subcultured Astrocytes  35  Figure 2-2. A Side View of the Glial-Neuron Non-Contact Co-Culture Situation  38  Figure 2-3. Schematic Mechanism of Assay of Glutathione Peroxidase  44  Figure 3-1. Effects of Astrocyte Feeder Layers on Neuronal Viability  49  Figure 3-2. Micrographs of the Co-Cultured Neurons  50  :  Figure 3-3. Neuronal Death Induced by Glial Cell Deprivation  51  Figure 3-4. Morphological Characterization and T U N E L Staining of Apoptotic Neurons 53 Figure 3-5. Nuclear Morphology of Apoptotic Neurons  53  Figure 3-6. Micrographs of Cultured Neurons under Different Coating Conditions  55  Figure 4-1. HPLC Graphs of Cysteine and Related Compounds  61  Figure 4-2. Time Course of Cysteine and Related Compounds' Content in A C M  62  Figure 4-3. Time Course of Cysteine and CSSG Content in N C M  66  Figure 4-4. Autoxidation of Cysteine and GSH  68  Figure 4-5. A Model Showing How Astrocytes may Provide Cysteine to Neurons ....... 69 Figure 5-1. The Factors Influencing Cysteine Autoxidation  75  Figure 5-2. Comparisons of Cysteine Autoxidation in the Presence of Several Transition Metal Ions  76  Figure 5-3. Cysteine Autoxidation Rates in CSF  77  Figure 5-4. Neurotoxic Effects of Cysteine in the Presence of Copper  78  Figure 5-5. Effect of Total Amount of Cysteine Being Oxidized on Neuronal Survival 79 Figure 5-6. Generation of Hydroxyl Radical by the Autoxidation of Cysteine and Other Thiols Figure 6-1. Protective Effects of Catalase and Pyruvate on Cysteine Neurotoxicity  81 87  vii  Figure 6-2. Effects of Catalase and Pyruvate on the Generation of -OH from Cysteine Autoxidation  88  Figure 6-3. Time Course of Pyruvate Release by Astrocytes  90  Figure 6-4. Effect of Cysteine Supplement on Neuronal Survival  91  Figure 6-5. Diagram of the Proposed Mechanism of Protection by Astrocytes  93  viii  ABBREVIATIONS  •o -  superoxide radical  •OH  hydroxyl radical  2-ME  2-mercaptoethanol  7-OHCCA  7-hydroxylcoumarin-3 -carboxylic acid  AP  P-amyloid peptide  ACM  astrocyte conditioned medium  AD  Alzheimer's disease  ALS  amyotrophic lateral sclerosis  ANOVA  analysis of variance  ApoE  Apolipoprotein E  APP  amyloid precursor protein  ASC system  Alanine-Serine-Cysteine transport system  BBB  blood-brain barrier  BMAA  P-methylamino-L-alanine  BOAA  P-oxaloamino-L-alanine  CA  cysteic acid  CCA  coumarin-3-carboxylic acid  CD-MEM  chemically defined M E M  CNS  central nervous system  CSA  cysteine sulfinic acid  CSF  cerebrospinal fluid  CSH  cysteine  esse  cystine  CSSG  cysteine-glutathione disulfide  CysGly  cysteine-glycine dipeptide  DAB  3,3'-diaminobenzidine tetrahydrochloride  DIV  days in vitro  2  DTNB  5,5'-dithiobis(2-nitrobenzoic acid)  DTT  dithiothreitol  EBSS  Earle's balanced salt solution  FBS  fetal bovine serum  FDNB  l-fluoro-2,4-dinitrobenzene  GFAP  glial fibrillary acidic protein  GPx  glutathione peroxidase  GSH  glutathione  GSSG  glutathione-disulfide  H2O2  hydrogen peroxide  HBSS  Hank's balanced salt solution  HPLC  high-performance liquid chromatography  HSH  homocysteine  LDH  lactic dehydrogenase  MEM  minimum essential medium  MTT  3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide  NAC  N-acetyl-cysteine  NCM  neuron conditioned medium  NSE  neuronal specific enolase  PBS  phosphate buffered solution  PD  Parkinson's disease  PLL  poly-L-lysine  ROS  reactive oxygen species  SOD  superoxide dismutase  TBARS  thiobarbituric acid reactive species  TBI  traumatic brain injury  TBS  tris buffered solution  t-BuOOH TUNEL  ter/-butyl  hydroperoxide  terminal deoxynucleotidyl transferasemediated dUTP-biotin nick end labeling  P-NADH  P-nicotinamide adenine dinucleotide reduced form  p-NADPH  P-nicotinamide adenine dinucleotide phosphate reduced form  y-GT  y-glutamyl-transpeptidase  ACKNOWLEDGEMENTS  I wish to express my deep gratitude to my supervisor, Dr. Max Cynader, for his support, guidance, understanding and tolerance throughout. His encouragement always gives me confidence to step further ahead. I also enjoy the inspiring and pleasant discussion with him. I owe a great debt of gratitude to Joan Martin, not only for her generous provision of the HPLC equipment at the crossroads of my experiments, but also for her willingness to help me in every way that she could. My thanks extend to Dr. John Phillips for his generosity and kindness. I will never forget my first tissue culture experiment, guided by Dr. Yihong Wang. She helped me to kick off my research project with great patience and time. I wish her all the best in future. I sincerely thank my supervisory committee members, Dr. Vanessa Auld, Dr. Tim Murphy and Dr. Wolfram Tetzlaff, for their constructive criticism, helpful discussion and valuable suggestions. It is impossible for me to list all of the help which I have received from the members of my department. To merely mention their names, I am very grateful to Drs. Joanne Matsubara, Qiang Gu, William Jia, Shiv Prasad and Jing Cui. I am indebted to Virginia Booth, Howard Meadows, Zheng Chen, Bing Zhu, Lilian Luo, Tara Stewart, Christian Wong and Eugene Au. Thanks also go to Faye Pileberg, Swarni Sunner, Chris Crossfield and Barry Gibbs. I must thank Mr. Spencer Kong of the B C Cancer Agency for the use of the luminescence spectrometer. I thank Dr. Steven Vincent for being on my final doctoral exam committee and Dr. Kenneth Baimbridge for being on my comprehensive exam committee. I appreciate their knowledge and help. I thank Liz Wong for her warm-hearted secretary assistance throughout. Finally, I thank my wife Ying and my daughter Kelly for their boundless support, and for sharing my anxieties, pains and joys.  Chapter 1 Introduction  C H A P T E R 1 G E N E R A L INTRODUCTION  1. Oxygen and Our Life We cannot live without oxygen (O2), whose atmospheric levels are rather stable (21%). Life on the earth, however, was not born under aerobic conditions. Isotope labeling studies suggest the first living cells were born about 3.8 x 10 years ago (Mojzsis 9  et al., 1996). O2 was absent from the atmosphere at that time. It is not until about 3 x 10  9  years ago that O2 was released to the air by the appearance of the photosynthetic cells, cyanobacteria (or blue-green algae) (Nunn, 1997; Xiong et al., 2000). The increase in atmospheric O2 was very slow at first because 0 produced by early cyanobacteria was 2  almost completely utilized by converting Fe(II) to Fe(III), causing the precipitation of huge amounts of ferric oxides. After exhaustion of ferrous ion, 0 levels began to rise, 2  and reached their current levels about 1 x 10 years ago (Fig. 1-1). With the availability 9  of O2, some bacteria evolved the ability to respire, i.e. making use of the high redox potential O2 to oxidize completely the food they ingested for the synthesis of ATP. Mitochondria appeared when primitive eucaryotic cells endocytosed such respirationdependent bacteria and formed a symbiotic relationship about 1.5 x 10 years ago. They 9  provided the cells highly efficient utilization of chemical fuels. Oxidation of each molecule of glucose by O2 can produce 30 molecules of ATP in mitochondria, compared with only 2 molecules of ATP by anaerobic glycolysis. Today, mitochondria have become the major cell energy-converting organelles, and 0 has become indispensable 2  for life in virtually all eucaryotes (Alberts et al., 1994; Rosen et al., 1999). However, O2 also has been shown to be toxic to living cells. The toxicity arises from oxidative damage to biologically important macromolecules (Gilbert, 1981). Over the course of evolution, organisms have developed various protective mechanisms against oxidation toxicity. Life is a dynamic balance between challenge and adaptation. Organisms have evolved such a complicated and efficient antioxidative system in order to survive. However, our antioxidative systems are not perfect. Mammals, along with other organisms, are still bothered everyday by numerous diseases closely related with reactive oxidative species (ROS) damage. Oxidative stress has been found to be involved in  1  Chapter 1 Introduction  PUFAs are particularly easy targets of free radical attack, and facilitate the initiation and propagation of the free radical chain reaction (Aust, 1987; Gutteridge, 1987). This process leads to lipid peroxidation, which is a common and significant contributor to oxidative stress and tissue damage in vivo (Mattson, 1998). c). Neurons are excitable cells. Glutamate is the major excitatory neurotransmitter. Excitotoxicity of glutamate has been found to be mediated by massive C a  2+  influx through the N M D A receptors  (Choi, 1987; Michaels and Rothman, 1990; Tymianski et al., 1993). Further evidence supports the notion that elevated [Ca ]j induces sustained mitochondrial depolarization 2+  and collapses mitochondrial membrane potential (Ai|/) (Schinder et al., 1996; White and Reynolds, 1996; Brustovetsky and Dubinsky, 2000). Impairment of mitochondrial function increases the generation of free radicals, and is a primary event in glutamate neurotoxicity (Coyle and Puttfarcken, 1993; Castilho et al., 1998; Luetjens et al., 2000). d). The antioxidative capacity of neurons is weak. The activities of antioxidative enzymes, such as glutathione transferase and y-glutamylcysteine synthetase, were lower in cultured neurons than in cultured astrocytes (Makar et al., 1994). Peroxide detoxification ability is less efficient in neurons than in astrocytes (Dringen et al., 1999b). Intracellular glutathione (GSH) levels were lower in neurons than in astrocytes (Raps et al., 1989; Sagara et al., 1993). Taken together, these lines of evidence suggest that neurons of the CNS face a great oxidative stress and are vulnerable to free radical injury.  3. Free Radical Chemistry A free radical is defined as a molecule or a chemical species that has one or more unpaired electrons. Each orbital within atoms or molecules can hold two electrons, which spin in opposite directions. If an orbital contains only one electron, that electron is said to be unpaired. This broad definition of free radical encompasses hydrogen atom (H), most transition metals, oxygen molecule (O2), superoxide anion  (O2"),  hydroxyl  radical (OH), nitric oxide ONO), nitric dioxide ONO2), alkoxyl radical (RO), peroxyl radical (ROO), semiquinone radical (HQ-), cysteinyl radical (CS-), carbon radical (L-), tocopherol radical (TocO), ascorbyl radical (AscO), etc. Though this vast variety of chemical species can all be called free radicals by definition, their reactivity and stability differ greatly. Among them, the oxygen-centered partial reduced oxygen products, such  3  Chapter 1 Introduction  as O2", OH, RO-, and ROO- etc., are the most reactive species, and are implicated in various physiological and pathological processes (Halliwell and Gutteridge, 1984; Symons, 1991; Roberfroid and Calderon, 1995). Other species are either relatively stable or less involved in tissue injury. On the other hand, some important species in oxidative stress, such as hydrogen peroxide ( H 0 ) , lipid peroxide (ROOH) and peroxynitrite 2  2  (ONOO), do not belong to free radicals by definition. They do not have unpaired electrons, and generally have longer half-lives (Pryor, 1986; Borg, 1993). However, they are chemically reactive, and participate in many important reduction-oxidation reactions. The term of reactive oxidative species (ROS) encompasses these non-radical oxidants and those partial reduced oxygen radicals. Even this term is still not good enough to include all the biologically important species, such as carbon-centered radical ( L ) and thiol-centered radical (RS-). The term "ROS and related species", therefore, are often used to indicate all the species, though mainly oxygen radicals, which have the potential to cause radical reaction to occur in biological system. Here, I alternately use free radicals, ROS, or oxygen radicals, to implicate the same species without distinction in particular. The chemical characteristic of free radicals and related species is their high reactivity with other molecules. As free radicals have unpaired electrons, they tend to lose or take electrons from other molecules to become paired. Non-radical oxidants, such as  H2O2,  are electrophilic as well, and tend to abstract electrons. Reactivity of free  radicals and related species is different from species to species. The half-life of these species is one of the indexes reflecting the reactivity of free radicals (Table 1-1).  Table 1-1. Estimate of the half-lives of ROS and related species (modified from Pryor, 1986) Species  Half-life  Substrate  •OH  10" sec  1 M Lenoleate  RO-  10" sec  100 m M Lenoleate  ROO-  10 sec  1 m M Lenoleate  L-  10" sec  0.02 m M 0  9  6  8  3  2  4  Chapter 1 Introduction  H 0 2  Stable  2  •0 "  b  2  "Substrate chosen as representative of typical reactive target molecules for ROS and related species. b  Half-life varies according to different substrates and enzymes.  O2 and partial reduced oxygen intermediates play the central role in oxidative stress. The ground-state O2, designated as 2 ", has two unpaired electrons. Its electronic 3  ,  g  structure is as follows: (a Is) (o* Is) (a 2s) (a* 2s) (a 2p ) (Tt 2p ) (n 2p ) (71* 2p )' (71* 2p )' 2  2  2  2  2  2  z  x  2  y  x  y  The two unpaired electrons spin in parallel. This characteristic of O2 makes it hard to accept a pair of electrons in an orbital, which spin in opposite, from another molecule at a time (Kanfer and Turro, 1981). Instead, O2 prefers to accept one electron at a time. The stepwise reduction of O2 results in the formation of -02,  H2O2,  OH, and  H2O  in  sequence, with addition of total four electrons (Halliwell and Gutteridge, 1984; Ingraham and Meyer, 1985):  0  2  ->  0" 2  ->  H 0 2  2  -> -OH ->  H 0. 2  Peroxynitrite (ONOO") is another important ROS causing tissue injury. It is formed from the reaction of nitric oxide N O and O2": -NO + 0 " —» ONOO". N O is synthesized 2  from L-arginine under the catalysis of N O synthetase (NOS) (Bredt et al., 1991; Vincent and Hope, 1992). Unlike the weak reactivity of N O and -02", ONOO" is a potent oxidant and nitrating agent capable of modifying proteins, lipids and D N A (Bartosz, 1996; Demiryurek et al., 1998). Evidence has shown that ONOO" is involved in the initiation of neurodegenerative diseases (Smith et al., 1997; Torreilles et al., 1999). Lipid peroxidation is one of the major oxidative injuries. PUFAs (e.g., linoleic, linolenic, and arachidonic acids) are readily attacked by free radicals, eventually forming lipid peroxides. The whole process can be described as the following steps (Gutteridge, 1987; Borg, 1993): (1)  Initiation: a free radical, such as OH, can initiate the process of chain  reaction by abstracting a hydrogen atom ( H ) from a methylene carbon of a PUFA. L H + OH -> L- + H 0 2  5  Chapter 1 Introduction  (2)  Propagation: the resulting carbon radical (L-) reacts rapidly with O2 to  form a peroxy radical (LOO). L- + 0 - » LOO2  LOO- can further abstract an H- from another PUFA, leaving an L- and lipid peroxide (LOOH). L H + LOO- -> L O O H + LL- can either abstract an H- from a L H or react with O2 to give a LOO-, and reinitiate the process, producing more lipid peroxides. (3)  Termination: the chain reaction will propagate until two free radicals react  with each other to terminate the chain. L- + L- -> L L LOO- + LOO- -> L O O L + 0  2  L- + LOO- -> L O O L Another form of termination is the reaction of free radicals with antioxidants, such as Vitamin E (tocopherol). This process is also called interception. L- + TocOH -> L H + TocOLOO- + TocOH -> L O O H + TocOTocO- is a stable and low reactive radical. It can be recycled to TocOH by other antioxidants (Acworth et al., 1997). The produced lipid peroxides can break down into a great diversity of aldehydes in biological system. The most intensively studied aldehydes are malonaldehyde (MDA), and 4-hydroxyalkenal, such as 4-hydroxynonenal (HNE) and 4-hydroxyhexenal. These aldehydes, unlike free radicals, have long half-lives (minutes to hours) and can diffuse to relatively distant sites. They are the chemically active end products of lipid peroxidation, and can form adducts with proteins by binding covalently to cysteine, lysine and histidine residues via Michael addition and Schiff-base crosslink (Nair et al., 1986; Esterbauer et al., 1991; Uchida and Stadtman, 1992), altering protein functions. These aldehydes are cytotoxic (Esterbauer, 1993; Kruman et al., 1997; Mark et al., 1997). DNA damage by free radicals has long been considered as an important contributor to aging and aging-related diseases, such as cancer and neurodegeneration (Ames and  6  Chapter 1 Introduction  Shigenaga, 1992; Mecocci et al., 1993). The major damaging free radical was found to be OH, as neither  O2"  nor  H2O2  causes any modifications in D N A strand i f transition  metal ions are completely removed (Rowley and Halliwell, 1983; Mello Filho and Meneghini, 1984; Aruoma et al., 1989a; Aruoma et al., 1989b). Though most experiments used iron, copper was also found to be efficient in inducing D N A damage (Dizdaroglu et al., 1990). The role of transition metal ions in the reaction system is to catalyze the formation of -OH, which will be discussed below. -OH can attack all four D N A bases by adding hydroxyl groups onto the base residues, producing hydroxyl bases. 8-hydroxy-2'-deoxyguanosine (8-OH-2-dG) represents one of the major products of free radical attack (Fraga et al., 1990; Dizdaroglu, 1991). Besides -OH, aldehydes (Douki and Ames, 1994) and peroxynitrite (Douki et al., 1996) can attack D N A as well. Free radical attack can also cause other kinds of modifications, such as methylation of D N A bases (Augusto, 1993) and DNA-protein cross-linking (Gajewski and Dizdaroglu, 1990). The resulting D N A adducts can lead to D N A mutation (Echols and Goodman, 1991; Shibutani et al., 1991) or inhibit D N A replication (Moore and Strauss, 1979). Protein damage by free radicals mostly involves modification of amino acid residues. Like the attack on lipid and D N A , the major damaging free radical is O H , which can oxidize proteins to produce protein carbonyls (Amici et al., 1989). ONOO" can also attack proteins, forming carbonyls and oxidizing thiols to disulfides or nonreducible products (Radi et al., 1991). These modifications will generally induce increased cellular proteolysis and removal of the damaged proteins. The roles of transition metal ions in oxidative stress have been extensively studied. Transition elements belong to the d-block in the periodic table. The important transition metals are the elements that have incompletely filled 3d-subshell in oxidation states. Among these elements, iron (Fe), copper (Cu), manganese (Mn), zinc (Zn), nickel (Ni), and chromium (Cr), are biologically important. Transition metal ions have the ability to combine with O2,  O2"  or H2O2, to form complexes. The biological significance of this  characteristic is to facilitate and control the oxygen utilization in vivo. Transition metal ions are usually chelated by proteins, and exert the well-controlled role in various physiological processes, such as reduction-oxidation reaction (in oxidases, superoxide dismutase, peroxidases) and transport of O2 (in hemoglobin, myoglobin). The 7  Chapter 1 Introduction  importance of transition metal ions in oxidative stress is largely due to their involvement in the formation of free radicals, particularly -OH. Iron-dependent formation of -OH has been widely considered the major source of -OH in vivo. Iron(II)-catalyzed generation of •OH from H 0 is called the Fenton reaction (1898) (Fenton, 1898): 2  2  H 0 + Fe 2  -> -OH + OH" + F e  2+  2  3+  Copper has a similar effect (Fenton-like reaction): H 0 + C u -> O H + OH" + C u +  2  2+  2  The Haber-Weiss reaction was first considered as another important source of -OH without transition metals: •0 " + H 0 -> O H + OH" + 0 2  2  2  2  Later, it was found that this reaction is too slow to induce any toxic effect if the transition metal ions were carefully removed, and that this reaction can be greatly accelerated by iron (Halliwell, 1978; McCord and Day, 1978). Redox potential  is an important term in reduction-oxidation reaction. Electrons are  transferred from one compound to another in reduction-oxidation reaction. Reducing agent (reductant) is the electron donor, and oxidizing agent (oxidant) is the electron acceptor: Oxidant + e ±5 Reductant Each paired electron donor/acceptor is rated according to its propensity to give up or take up electrons. Such ratings are termed redox (reduction-oxidation) potential. The standard reduction potential (EQ) is a measure of the electromotive force generated at 25°C and pH 7.0 by a sample half-cell (containing paired electron donor/acceptor) with respect to a reference half-cell. The reference half-cell contains 1 M H in equilibrium +  with H at a pressure of 1 atm. The redox potential of H / H couple is defined to be 0 +  2  2  volt (V). A strong reductant has a negative redox potential, whereas a strong oxidant has a positive redox potential. The standard redox potential of some important biological compounds are listed below:  8  Chapter 1 Introduction  Table 1-2. Standard redox potentials of some oxidant/reductant couples Electron acceptor  Electron donor  n  E6 (V)  Reference  •OH  H 0  1  +2.33  (Fee and Valentine, 1977)  •o -  H 0  1  +0.87  (Fee and Valentine, 1977)  2  2  2  2  V0  H 0  2  +0.82  (Fee and Valentine, 1977)  Fe  Fe  2+  1  +0.77  (Prohaska, 1997)  •OH  1  +0.38  (Fee and Valentine, 1977)  1  +0.22  (Stryer, 1988)  1  +0.16  (Prohaska, 1997)  2  2  2  3+  H 0 2  2  Cytochrome c-Fe Cu  3+  Cytochrome c-Fe Cu  2 +  2+  +  Ubiquinone (ox)  Ubiquinone (red)  2  +0.10  (Stryer, 1988)  Dehydroascorbate  Ascorbate  2  +0.08  (Stryer, 1988) (Stryer, 1988)  Cytochrome b-Fe  Cytochrome b-Fe  1  +0.07  2H  H  2  0  +  2  Pyruvate  Lactate  2  -0.19  (Stryer, 1988)  esse  2CSH  2  -0.22  (Jocelyn, 1967)  GSSG  2 GSH  2  -0.24  (Jocelyn, 1967)  NAD  NADH  2  -0.32  (Stryer, 1988)  NADPH  2  -0.32  (Stryer, 1988)  •0 "  1  -0.33  (Fee and Valentine, 1977)  DTT(ox)  DTT(red)  2  -0.331  (Jocelyn, 1987)  Succinate  a-Ketoglutarate  2  -0.67  (Stryer, 1988)  +  NADP  o  +  2  2  The free energy change ( A G ' ) for a reduction-oxidation reaction can be calculated 0  from the standard redox potential: A G ° ' = -ftFAEo = -nF(Eo, acceptor - EQ, donor)  Where n is the number of electrons transferred and F is Faraday's constant (23 kcal/V). The utility of determining A G ° ' lies in that its magnitude can predict whether or not a given chemical reaction can take place spontaneously. If A G ° ' = 0, the system is at equilibrium; if A G ° < 0, the reaction will proceed spontaneously as written; if A G ° ' > 0,  9  Chapter 1 Introduction  the reaction will not proceed as written, but will, instead, proceed in the opposite direction. The free energy change of stepwise reduction of O2 can be calculated from the redox potential shown in Table 1-2 (Fig 1-2). The addition of the first electron to O2 is an uphill and unfavorable process, which is crucial for the stabilization of O2. It is reversible between O2 and 0 ". A l l 2  subsequent steps to H2O are downhill processes, and irreversible. Energy is released during reduction of O2. AG°' can also be calculated from the equilibrium constant, K, of the reaction: AG°'  = - RT ln K  R is the gas constant (1.987 cal/deg-mole), and T is the absolute temperature (0°C = 273°K). If the reactants and products are present in concentrations other than 1 M each, the actual redox potential (£") can be calculated from the Nernst equation: £,_£, 0  +  R T j [oxidized form] nF [reduced form]  4. Multiple Roles of Astrocytes For decades, glial cells, originally viewed as just the glue that holds the brain together, have been considered as passive structural and metabolic support elements. Accumulating evidence now indicates that these cells, and especially astrocytes, can nourish, protect, and interact with surrounding neurons. Neuroglia outnumber neurons 10- to 50-fold, and comprise about half the total volume of the brain and spinal cord (Travis, 1994). There are four major types of neuroglia in the CNS: (1) astrocytes, (2) oligodendrocytes, (3) microglia, and (4) ependymal cells. Oligodendrocytes are mainly responsible for the formation of the myelin sheath of nerve fibers in the CNS (Szuchet, 1995). Schwann cells in the peripheral nervous system (PNS) are the functional equivalents of oligodendrocytes. Microglial cells serve as the immune cells of the CNS.  10  Chapter 1 Introduction  They are inactive in the normal brain. In inflammation, they proliferate and become actively phagocytic (Streit, 1995; Kreutzberg, 1996). Ependymal cells line the cavities of the brain and the central canal of the spinal cord, assisting in the circulation of the CSF (Reichenbach and Robinson, 1995). Unlike other types of neuroglia, the functions of astrocytes are quite diverse. They can serve as scaffolding for the migration of immature neurons (Rakic, 1995), participate in the formation of B B B (Wolburg and Risau, 1995), regulate extracellular K ions +  (Newman, 1995), absorb glutamate and y-aminobutyric acid (GABA) released by the nerve terminals (Martin, 1995), release neuroactive amino acids and neurotrophic factors (Banker, 1980; Levi and Gallo, 1995), and supply substrates of energy (Coles, 1995). The microenvironment of the CNS is quite different from the external environment of the body. For example, not only the concentrations of amino acids of CSF are lower than that of plasma, but also the CSF/plasma ratios of amino acids are different from each other (Perry et al., 1975; McGale et al., 1977), indicating the selective absorption or regulation by the brain. Except for small lipophilic molecules and gaseous agents, most nutrients enter the brain via transporters, channels, or pinocytosis (Pardridge and Oldendorf, 1977; Rowland et al., 1991). The various components of CSF are maintained at stable levels while plasma components may fluctuate widely (Cutler and Spertell, 1982). Given the diverse functions of neuroglia, particularly astrocytes, and the special microenvironment of the brain, we could consider neuroglia functioning as neuronal caregivers, maintaining the optimal internal milieu of the brain. The glia-neuron co-culturing experiments provide a direct proof of neurosupportive effects of astrocytes. The data from different researchers have repeatedly confirmed the fact that astrocytes can improve neuronal survival. Banker (Banker, 1980) reported that the cultures of hippocampal neurons from rat fetuses have higher number of processbearing cells when they are co-cultured with astrocytes or cultured in astrocyte conditioned medium (ACM), than that cultured alone. Similarly, astrocytes can also support the survival of cortical neurons (Walsh et al., 1992; Sass et al., 1993) and retinal ganglion cells (McCaffery et al., 1984) in co-culture systems. Different co-culture methods have been used. The primary neurons and confluent astrocytes can be cocultured either by directly plating neurons on the surface of an already confluent glial  11  Chapter 1 Introduction  monolayer (McCaffery et al., 1984; Baughman et al., 1991), or by using the non-contact methods (Goslin and Banker, 1991; Walsh et al., 1992; Sass et al., 1993). The evidence from astrocyte conditioned medium (ACM) also supports a neurosupportive role of astrocytes. A C M can improve neuronal survival and neurite outgrowth (Vaca and Wendt, 1992; Yoshida et al., 1995). Some researchers have found that the trophic effects of astrocytes have regional specificity (Yuzaki et al., 1993; Le Roux and Reh, 1994; Yoshida et al., 1995). For example, cerebellar astrocytes specifically support the survival of Purkinje cells in culture (Yuzaki et al., 1993). As neurosupportive effects of astrocytes are well supported, it is agreed upon that astrocytes release or secrete some trophic factors to exert those functions. A large number of compounds have been found to be released by astrocytes. They include amino acid transmitters (Bowery et al., 1976; Holopainen and Kontro, 1990; Kimelberg et al., 1990), eicosanoids (Hartung and Toyka, 1987; Jeremy et al., 1987), neuropeptides (Deschepper et al., 1986; Melner et al., 1990), and neurotrophic factors. The neurotrophic factors that can be secreted by astrocytes includes nerve growth factor (NGF) (Furukawa et al., 1986), glial cell line-derived neurotrophic factor (GDNF) (Lin et al., 1993), basic fibroblast growth factor (bFGF) (Oh and Yong, 1996), epidermal growth factor (EGF) (Gomes et a l , 1999), interleukin-6 (IL-6) (Lee et al., 1993; Maeda et al., 1994), granulocyte-macrophage colony-stimulating factor (GM-CSF) (Ohno et al., 1990), and erythropoietin (Masuda et al., 1994; Sakanaka et al., 1998), all of which can improve neuronal survival with varying degrees of effects. The role of neurotrophic factors in glial-neuron communication has been studied intensively over the past 10 years (Barres and Barde, 2000). It has become increasingly clear that neurotrophic factors not only have important influence on neuronal survival (Goldberg and Barres, 2000), but also participate in a variety of nervous activities, such as regulating synaptic plasticity (Thoenen, 1995) and activating sodium channels (Kafitz et al., 1999). Though so many factors have been found, there is still no agreement as to which one or which category may play a more important role in supporting neuronal survival. Perhaps a synergic action of the many factors is essential for astrocytes to exert their neurotrophic effects. Varon et al. (1984) found that a trophic activity of glial cells resides with low molecular weight agents rather than with protein macromolecules. While some  12  Chapter 1 Introduction  researchers indicated that each neurotrophic factor has a specific supportive effect on distinct classes of neurons (for review see Lindsay, 1995; Schatzl, 1995; Davies, 1997). The effort of searching for more potent neurosupportive factors has never stopped, and the new members continue to be discovered. Astrocytes have shown the capacity to protect neurons from oxidative stress, as shown in Table 1-3. Various compounds were used in these experiments to induce oxidative stress. However, the mechanisms by which astrocytes protect neurons from oxidative stress are still uncertain. Desagher et al (1996) reported that the neurotoxic effect of H2O2 on neurons co-cultured with astrocytes was strongly attenuated compared with that observed with a pure population of neurons. These researchers found that catalase activity was enriched in astrocytes compared with neurons, and attributed neuroprotective effect of astrocytes to their strong H202-scavenging capacities. Data from many other experiments also identified the stronger antioxidative capacities of astrocytes than neurons (Makar et al., 1994; Lucius and Sievers, 1996; Iwata-Ichikawa et al., 1999). However, the evidence that astrocytes are more resistant to oxidative stress than neurons cannot automatically prove the neuroprotective functions of astrocytes. If astrocytes can only scavenge the oxidants that entered their cytoplasm, their role will be limited to attenuate, but not efficiently prevent, oxidative stress that neurons could face. For example, extracellular H2O2 can cross cell membranes without distinction. No matter how much H2O2 astrocytes may take and neutralize, there is still a certain proportion of H 2 0 that will enter neurons. H 2 0 permeating cell membrane itself is risky for the 2  2  neurons. Therefore, in order to protect neurons efficiently, astrocytes are assumed not only to defend themselves, but also to set up a defense for neurons and/or to enhance neuronal antioxidative capacities. So far, only a few reports have addressed this question. Tanaka et al. (1999) reported that astrocytes secrete laminin and fibronectin, which attenuate reactive oxygen/nitrogen species-induced neuronal damage. Neurotrophic factors, which can be secreted by astrocytes, can protect neurons from oxidative stress to a certain degree (Kirschner et al., 1996; Mena et al., 1997). Iwata-Ichikawa et al. (1999) found that glial conditioned medium enhances GSH synthesis of neurons by upregulating neuronal mRNA expression of y-glutamylcysteine synthetase. Astrocytes were also found to release GSH (Yudkoff et al., 1990) and cysteine (Sagara et al., 1993),  13  Chapter 1 Introduction  whose (cysteine) levels regulate neuronal GSH synthesis. The latter will be discussed in detail in section 6 of this chapter. Given the above evidence, it is reasonable to hypothesize that astrocytes release or secrete some factors that could either exert their antioxidative functions extracellularly or enhance antioxidative capacities of neurons.  Table 1-3. Observations of neuroprotective effects of astrocytes against various stimuliinduced oxidative stress Neurons  Toxins  References  Cortical neurons  •OH and ONOO"  (Tanaka et al., 1999)  y-radiation  (Noel and Tofilon, 1998)  Retinal ganglion cells  •OH and ONOO"  (Lucius and Sievers, 1996)  Cerebellar neurons  Dopamine  (Hochman et al., 1998)  Striatal neurons  H 0  2  (Desagher et al., 1996)  Mesencephalic neurons  H 0  2  (Langeveld et al., 1995)  2  2  (Han et al., 1996; Mena et  L-Dopa  al., 1997) 6-hydroxydopamine  (Bronstein et al., 1995; Hou et al., 1997)  5. Antioxidative Systems of the Organisms As mentioned at the beginning, ever since 0 was generated in the atmosphere, 2  organisms started developing defenses against oxidative stress. In the long odyssey of evolution, many efficient antioxidative systems appeared and have been adopted by most organisms. These systems can be largely classified as large molecule antioxidative defenses and small molecule antioxidative defenses. Antioxidative enzymes play a very important role in cellular antioxidative defenses. They include superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx). These enzymes are present in almost all cell types, indicating a universal requirement of these antioxidative enzymes. SOD catalyzes the dismutation of 0 ": 2O2" + 2 H —> H 0 +  2  2  2  + 0 . Cu/Zn SOD is highly expressed in cytoplasm and some organelles, such as 2  lysosomes and peroxisomes, in virtually all cell types, including neurons and non-  14  Chapter 1 Introduction  neuronal cells (Slot et al., 1986; Pardo et al., 1995). C u site of the protein, while Z n  2+  2+  in Cu/Zn SOD is at the active  helps stabilizing the enzyme (Fridovich, 1995). M n SOD,  catalyzing the same reaction as Cu/Zn SOD, exists largely in the mitochondria. M n  3 +  is  at the active site of the enzyme (Fridovich, 1995). Gene knockout animals have shown the importance of SOD for survival. For example, M n SOD deficient mice suffered severe neurodegeneration and perinatal death (Lebovitz et al., 1996). H2O2 is usually removed in human tissues by two enzymes: catalase and GPx. Catalase decomposes H2O2  directly: 2H2O2  2 H 2 O + O2.  Catalase is largely present in subcellular organelles  known as peroxisomes of all major tissues, being especially concentrated in liver (Chance et al., 1979). GPx removes H 0 by using tripeptide glutathione (GSH): H 0 + 2 G S H 2  2  2  2  —» GSSG + H2O. GPx requires selenium as its cofactor. Glutathione disulfide (GSSG) is converted back to GSH by glutathione reductase with N A D P H : GSSG + N A D P H + H  +  —» 2 G S H + N A D P . The GPx family is comprised of several distinct forms of Se+  dependent GPx. They are (1) classical GPx existing in the cytoplasm of almost all cell types (Levander, 1987), (2) phospholipid hydroperoxide GPx in the cell membrane (Ursini et al., 1985), and (3) plasma or extracellular GPx in plasma and lung fluid (Avissar et al., 1989; Avissar et al., 1996). Besides these enzymes that remove ROS directly, there are some other enzymes, which do not remove free radicals directly but are related to antioxidative defenses, such as glutathione reductase (GR), glutathione synthetase, glutathione S-transferase, and glucose-6-phosphate dehydrogenase ( G 6 P D ) . Proteins that bind transition metal ions are considered as very important antioxidative defenses. Transition metal ions, such as iron and copper, can mediate electron transfer, and their physiological functions are usually well controlled. Free or low-molecular-weight complexes of transition metal ions can catalyze autoxidation of reducing agents and the Fenton reaction, generating ROS (Halliwell and Gutteridge, 1984). The organism has evolved many binding proteins for these transition metal ions to minimize their availability as free forms (Murray et al., 1993). Transferrin is a glycoprotein that transports iron to sites where it is needed. One molecule of transferrin can bind two molecules of F e (Bates and Schlabach, 1973), preventing them from 3+  participating in catalyzing reactions. Intracellular iron is stored as F e in the binding 2+  form with ferritin. Heme, released from hemoglobin, can stimulate the generation of  15  Chapter 1 Introduction  ROS. Hemopexin, a plasma protein, can bind heme tightly and inhibit heme-induced lipid peroxidation (Gutteridge and Smith, 1988). Plasma copper is tightly bound to ceruloplasmin, each molecule of which binds 6 molecules of copper. Albumin can also bind, but with weak ability, to heme and copper. Metallothioneins (MTs) are a family of single chain molecules, which are rich in cysteine content and mainly bind zinc and copper (Dunn et al., 1987). There are four isoforms of MTs. MT-I and MT-II are extensively expressed in all mammalian tissues, including astrocytes in the brain and spinal cord (Blaauwgeers et al., 1993). MT-III and MT-IV are predominantly expressed in the CNS (Palmiter et al., 1992; Kobayashi et al., 1993). MTs are implicated in the storage of metal ions in a non-toxic form and regulation of cellular zinc and copper metabolism. Their expression in the CNS is up-regulated during oxidative stress and inflammation (Aschner, 1998; Hidalgo and Carrasco, 1998). Taken together, organisms have evolved many mechanisms to bind transition metal ions, preventing them from catalyzing the generation of ROS. Our body contains a complex mixture of small molecule antioxidants. These compounds can be classified into those synthesized in vivo and those obtained from diet. Glutathione is the major synthesized cellular antioxidant. It can remove H2O2 with GPx, or neutralize free radicals by itself. The metabolism of glutathione will be discussed in the next section. ct-Lipoic acid, previously classified as a vitamin, can be synthesized by humans (Carreau, 1979). Lipoic acid and its reduced form, dihydrolipoic acid, have antioxidative functions, such as scavenging free radicals, chelating transition metals, and regenerating other antioxidants (Packer et al., 1995). Uric acid is the oxidized end product of purine bases, such as adenine, guanine, hypoxanthine, and xanthine. It can react with free radicals, and its plasma levels are considerably high (-300 uM) (Ames et al., 1981). cc-Ketoacids, including pyruvate, a-ketoglutarate, a-ketobutyrate and oxaloacetate, are H 0 scavengers. Though the reactivity of a-ketoacids with H 0 was 2  2  2  2  first noticed many years ago (Holleman, 1904; Hatcher and Hill, 1928; Bunton, 1949), the protective effects of pyruvate against oxidative stress in biological system have not been reported until relatively recently (O'Donnell-Tormey et al., 1987; Desagher et al., 1997; Giandomenico et al., 1997). CSF has high levels of pyruvate (-200 uM) (Vannucci and Duffy, 1976). There are also some other small molecule antioxidants  16  Chapter 1 Introduction  synthesized in vivo, such as bilirubin, ubiquinone (coenzyme Q), melatonin, and melanin, all of which have capacities of scavenging free radicals. There are many small molecule antioxidants, which cannot be synthesized by humans but can be obtained from the diet. This class of antioxidants includes vitamins. Ascorbic acid (vitamin C) is a water-soluble vitamin. As a reducing agent, ascorbic acid can donate electrons and be oxidized to dehydroascorbic acid (DHA). It participates in many reduction-oxidation reactions, such as synthesis of epinephrine from tyrosine and collagen synthesis (Murray et al., 1993). The antioxidative functions reside in its capacities of scavenging various free radicals, including O2", OH, ROO-, ONOO" etc., and regenerating ct-tocopherol (Halliwell, 1996). It is postulated that ascorbic acid is especially important in the brain, as its concentration in CSF is about 10 times higher than that in plasma (Spector and Eells, 1984). However, it often acts as a pro-oxidant, usually by interaction with transition metal ions (Halliwell, 1996). In fact, many in vitro experiments make use of the mixtures of ascorbic acid and iron to generate OH (Barrier et al., 1998; Tanaka et al., 1999). a-Tocopherol (vitamin E) and carotenoids are lipidsoluble vitamins. Vitamin E is probably the most important inhibitor of lipid peroxidation. It scavenges lipid peroxyl radicals and breaks chain-reaction of lipid peroxidation (Burton et al., 1982). Vitamin E supplements have been reported to be beneficial for preventing cardiovascular disease, cataracts, various cancers (Landvik, 1997), as well as for the treatment of Alzheimer's disease (Grundman, 2000; Pratico and Delanty, 2000). Carotenoids, which are a group of compounds that are rich in plant tissues, can also scavenge free radicals (Everett et al., 1996). Besides these vitamins, there are many other plant antioxidants, such as flavanols, flavones, anthocyanidins, and phenylpropanoids. Although these non-vitamin antioxidants continue to be identified, the importance of their antioxidative roles in biological systems has yet to be elucidated.  6. Theories of Thiol Provision by Astrocytes Glutathione (GSH) is a tripeptide made up of glutamate, cysteine, and glycine. The structures of G S H and related compounds are shown in Fig. 1-3. It is the major cellular antioxidant. GSH is ubiquitously present in mammalian cells at intracellular concentrations ranging from 0.5 to 10 m M (Kosower and Kosower, 1983; Meister and  17  Chapter 1 Introduction  Glutatftione  Glutathione disulfide 1  H  fH N\  O  3  ^CH  —  CH2 CH^s. -  rOOC  I  X  C i  O Cysteine* Cystine •—*•  IN.  Cysteine-glutathione 1 disulfide O CH-CH CH2 2  OOC  \  |  coo  Y  y  H  ! -  -  f  k  <fH  2  y  CH  2  Figure 1-3. Molecular structures of glutathione and related compounds.  Anderson, 1983). As GSH is the major cellular thiol, it is considered as a storage form of thiols. G S H plays diverse roles in cellular antioxidative defenses and other functions (Meister and Anderson, 1983; Beutler, 1989; Cooper and Kristal, 1997). 1). GSH acts as a cofactor of GPx and decomposes  H2O2,  with the concomitant conversion of G S H to  GSSG. 2). As a reducing agent, GSH can directly react with free radicals, stopping the free radical chain-reaction. 3). GSH acts as a cofactor of GSH S-transferase to convert electrophilic xenobiotics (RX) to glutathione conjugates, which are excreted into bile. The reaction is as follows: R X + GSH -> RSG + H X . 4). GSH can regenerate ascorbic acid (Meister, 1992; Rose, 1993) and tocopherol (Heslop et al., 1996), and exert antioxidative roles synergistically. 5). GSH has been found to chelate copper ions and diminish their ability to generate free radicals in the reaction mixture of Cu (I) and  H2O2  (Hanna and Mason, 1992). 6). By regulating disulfide bonds, G S H can maintain protein - S H groups, or play roles in protein folding and the degradation of proteins (Carelli et al., 1997). 7). GSH plays the critical roles in cellular redox signaling (Sen, 1998; Janaky et al., 1999). 8). GSH is involved in uptake of amino acids into cells by the y-glutamyl cycle (Orlowski and Meister, 1970; Meister and Anderson, 1983). It is now realized that  18  Chapter 1 Introduction  though y-glutamyl cycle does operate in vivo, it may not play a major role in amino acid transport. Though GSH is solely synthesized in the cytoplasm, it also functions in some other organelles. For example, GSH is transported into the mitochondria, which are the major intracellular sources of ROS, scavenging free radicals and maintaining the normal mitochondrial membrane potential (Avy) (Beatrice et al., 1984; Garcia-Ruiz et al., 1995; Reed and Savage, 1995; Marchetti et al., 1997). G S H is synthesized from glutamate, cysteine and glycine (Fig. 1-4). The synthesis of GSH has two steps. First, y-glutamylcysteine synthetase catalyzes the formation of yglutamylcysteine. Next, y-glutamylcysteine is joined by glycine catalyzed by glutathione synthetase, forming GSH. Both reactions require the presence of ATP. y Glutamylcysteine synthetase is feedback inhibited by GSH. The availability of sulfur amino acids influences GSH content, and is considered the rate-limiting substrate for GSH synthesis (Beutler, 1989; Phelps et al., 1992). y-Glutamyltranspeptidase can break down GSH and transfer the glutamate residue onto other amino acids such as cysteine, methionine, and glutamine. The enzyme is located on the plasma membrane with its active site facing outwards (Meister, 1995). The resulting cysteinyl-glycine can further be hydrolyzed by a peptidase. GSH is oxidized to GSSG when it exerts antioxidative functions. GSSG is converted back to GSH by glutathione reductase (GR) with N A D P H as cofactor. GSH can be released from cells. Liver is the major organ releasing G S H to plasma (Fariss and Reed, 1983; A w et al., 1986). In summary, GSH metabolism is very active, mirroring its critical roles in antioxidative defenses and other functions. The roles of GSH in the CNS have been extensively studied. Much evidence indicates that GSH is critical for neuronal survival. GSH depletion results in increased generation of ROS, loss of mitochondrial function, and rapid cell death (Jain et al., 1991; Wullner et al., 1999). Supplementation of N-acetylcysteine to neuronal cultures can increase intracellular GSH, and prevent PC 12 cell death caused by withdrawal of serum and N G F (Ferrari et al., 1995). Ratan et al. (1994) reported that inhibition of cystine uptake by homocysteate decreases intracellular GSH levels and induces neuronal death concurrently. They also found that macromolecular synthesis inhibitors prevent neuronal death by a mechanism of shunting cysteine from protein synthesis to glutathione synthesis. Murphy et al. (1990) found that glutamate toxicity of immature cortical  19  Chapter 1 Introduction  GSSG NADPH  H 0 2  11 Glu Cys Gly  y-Glu-Cys  H 0 2  II  J a s  2  NADP  +  XR * y-Glu-Cys-Gly (GSH)  GSR + X H  Figure 1-4. GSH synthesis and some of reactions of GSH. 1, y-glutamylcysteine synthetase. 2, glutathione synthetase. 3, glutathione peroxidase. 4, glutathione reductase. 5, glutathione-S-transferase.  neurons involves the inhibition of cystine uptake. L-Buthionine sulfoximine (BSO), which irreversibly blocks y-glutamylcysteine synthetase, can abolish the astrocytemediated enhancement of neuronal survival (Drukarch et al., 1997). A l l these data demonstrate the importance of GSH in the CNS. It has been found that neurons and astrocytes have different preferences in the utilization of amino acids for GSH synthesis (Kranich et al., 1996). These researchers found that refeeding cultured neurons temporarily deprived of amino acids with cysteine, but not with cystine, can restore G S H content to a maximal degree. For astrocytes, it is cystine, but not cysteine, that restores a maximal GSH content. The utilization of cysteine by neurons requires a stable extracellular thiol source. As thiols are easily oxidized under aerobic conditions, the maintenance of stable thiol levels in the extracellular fluid is very important. Cysteine and GSH can be transported across the blood-brain barrier (BBB) (Wade and Brady, 1981; Kannan et a l , 1990). Plasma cysteine concentration of human venous blood was reported to be 11.2 + 0.9 u M (Chawla et al., 1984) or 11.9 u M (ranging from 4.96 to 21.1 uM) (Kleinman and Richie, 2000). {Cystine concentration of human venous plasma is 46 ± 8 jaM (Chawla et al., 1984) or 90.8 u M (Felig et al., 1973)}. GSH concentration of human venous plasma is 4.5 ± 0.4 u M (Chawla et al., 1984) or undetectable (lower than 8 nM) (Kleinman and Richie,  20  Chapter 1 Introduction  2000). Whether plasma thiols contribute to the thiol pool of CNS is uncertain, as no data have shown arterio-venous differences across the brain yet. Blood thiol levels fluctuate a lot under different nutritional conditions (Boebel and Baker, 1983; Suberville et al., 1987). Depending on blood as thiol supply would be a poor way to maintain stable levels of thiols in the CNS. The mechanisms of thiol maintenance by astrocytes have been explored in recent years. Yudkoff et al. (1990) followed the incorporation of [ N] glutamate into GSH, and found that GSH is released by cultured astrocytes, and the 15  G S H concentration in the medium can reach to ~ 3 uM. Bannai and his colleagues have contributed a lot to unraveling the mechanisms of thiol maintenance in the CNS. They reported that the cysteine concentration in glial cultured medium is 2.6 ± 0.4 uM, and neuronal cultured medium cysteine is 0.6 ± 0.2 u M (Sagara et al., 1993). They also found that the uptake rate of cysteine by neurons is much faster than cystine. They suggest that neurons maintain their GSH levels by taking up cysteine provided by glial cells (Sagara et al., 1993). Later, they observed a GSH efflux from cultured astrocytes, and studied the kinetics of the GSH efflux (Sagara et al., 1996). But they admitted that the physiological significance of the GSH efflux is not clear, as they had confirmed that neurons do not take up GSH directly to replenish their GSH pool. The studies from analyzing perfusates of brain slices are not quite consistent. Keller et al. (1989) found that brain slices release cysteine on Ca -dependent depolarization. The same research 2+  group later reported that more compounds, including cysteine, GSH, glutamate and aspartate, are released under the same conditions (Zangerle et al., 1992). L i et al. (1999) have shown more compounds are released by hippocampal slices during oxygen/glucose deprivation, which include cysteine, GSH, cysteine sulfinate, glutamate, y-GluGlu, yGluGln, and y-GluCys. Thus, there are two unsolved questions: first, what compound(s) is (are) released by astrocytes, cysteine, or GSH, or both? Second, i f astrocytes release GSH, how and what compound will it be converted into for neuronal uptake? Dringen et al. (1999a) suggest that GSH released by astrocytes is decomposed by ectoenzyme yglutamyl transpeptidase to generate dipeptide CysGly that is subsequently used by neurons as a precursor for GSH synthesis. Their experiments show that CysGly, like cysteine, can serve as a G S H precursor when it is supplemented in the culture medium. Acivicin, an inhibitor of the astrocyte ectoenzyme y-glutamyl transpeptidase, decreases  21  Chapter 1 Introduction  the GSH content of neurons in neuron-astrocyte co-cultures. However, there is no evidence for the existence of CysGly in CSF or astrocyte conditioned medium (ACM). The finding that y-glutamyl transpeptidase is mainly located in the endfeet of astrocytes (Zhang et al., 1997) also raises a question that this enzyme may be not accessible to extracellular GSH. In summary, GSH plays a critical role in cellular antioxidative defenses. Cysteine has been found to be the rate-limiting precursor of GSH synthesis. Thiol sources in the CNS must be maintained at a stable levels, which is a distinguishing feature of G S H metabolism in the CNS. What antioxidative compounds are released by astrocytes and provided to neurons are important questions and deserve to be investigated in depth.  7. Theories of Cysteine Toxicity Cysteine is naturally present in the human body and in the environment. It is critical for GSH synthesis. However, it has long been noted that cysteine can cause neurotoxicity. Though this finding was first reported more than three decades ago (Olney and Ho, 1970), the mechanism is still uncertain. There are several theories about cysteine neurotoxicity. 1. Excitotoxic metabolites of cysteine. Neuronal death was first reported in the infant mouse retina and hypothalamus following oral administration of high doses of cysteine (3 mg/g weight) (Olney and Ho, 1970), or subcutaneous injection (1.5 mg/g weight) (Olney et al., 1971). Widespread degenerative changes, including cerebral cortex, hippocampus, amygdala, caudate, thalamus, pons, medulla oblongata, spinal cord and cerebellum, were reported later in infant and fetal mice (Olney et al., 1972), and infant rats (Karlsen et al., 1981). In their studies, Olney et al. (1971; 1972) compared the pathological patterns of cysteine with other excitatory amino acids, such as glutamic acid, aspartic acid, cysteic acid (CA), cysteine sulfinic acid (CSA), N-methyl DL-aspartic acid and N-methyl DLglutamic acid. Some differences were observed. For example, cysteine toxicity destroys neurons in several major brain areas never affected by glutamic acid and other acidic amino acids, such as amygdala, some areas of hippocampus and cerebral cortex and several neuronal groups within the thalamus. On the other hand, some structures, like the midline periventricular area, are damaged by acidic amino acids, but not affected by  22  Chapter 1 Introduction  cysteine. Even though there exist discrepancies between cysteine and other excitatory amino acids, the first explanation of the mechanism of cysteine neurotoxicity was that the conversion of - S H terminal of cysteine to acidic group (SO2H or SO3H) could account for its neurotoxicity (Olney et al., 1971). Cysteine has one rather than two acidic groups and does not obey the structure-activity requirements for excitotoxins, while CSA (S 0 H ) and C A (-SO3H) are NMDAmimetic. Lehmann et al. (1993) studied whether 2  cysteine toxicity depends on its conversion to CSA or C A . They found that the distribution of brain injury after subcutaneous administration of cysteine (1 mg/g) to infant rat is quite different from that caused by CSA (3 mg/g). In addition, the concentrations of CSA and C A converted from injected cysteine are much lower than the toxic dose levels of CSA and C A (Lehmann et al., 1993). Similar findings from other researchers also showed that cysteine is about 10-fold more toxic to neuronal cell lines than C S A and C A , suggesting that it is not cysteine metabolites that mediate cysteine neurotoxicity (Pean et al., 1995). 2. Bicarbonate-dependent excitotoxicity. Olney et al. (1990) reported that excitotoxic potency of cysteine is substantially increased in the presence of physiological concentrations of bicarbonate ion, and that N M D A antagonists can block cysteine toxicity. In parallel, Weiss and Choi (1988) reported that beta-N-methylamino-L-alanine ( B M A A ) , an environmental toxin that causes Guam ALS-parkinsonism-dementia, requires bicarbonate as a cofactor to produce neurotoxic effects. It was suggested that cysteine, similar to B M A A , forms noncovalent hydrogen bond with bicarbonate, simulating the acidic Q terminal of excitatory amino acids, to act as a glutamate agonist (Olney et al., 1990). However, whether cysteine can form a hydrogen bond with bicarbonate has not been proven using analytical chemistry. 3. Redox modulation of the N M D A receptor. It has been found that the responses of the N M D A receptor are modulated by reduction and oxidation (Aizenman et al., 1989; Janaky et al., 1993; Kohr et al., 1994; Gozlan and Ben-Ari, 1995; Choi and Lipton, 2000). The extracellular part of N M D A receptor channel contains several redox sites. While the reducing agent dithiothreitol (DTT) potentiates the electrophysiological response of cultured neurons to N M D A , the thiol oxidant 5,5-dithio-bis-2-nitrobenzoic acid (DTNB) decreases the magnitude of the response (Aizenman et al., 1989). Similar  23  Chapter 1 Introduction  results were confirmed with the recombinant N M D A receptors with GSH and DTT (Kohr et al., 1994). G S H was also found to increase the glutamate- and NMDA-induced C a  2+  influx (Janaky et al., 1993), and to increase neuronal vulnerability to azide-induced chemical hypoxia and glucose deprivation (Regan and Guo, 1999). Cysteine, like DTT and GSH, is a reducing agent and can modulate redox state of proteins as well. PukaSundvall et al. (1995) reported that cysteine and its N-acetylated analog, N-acetylcysteine (NAC), can enhance glutamate toxicity in both neuronal cultures and in vivo experiments. These results show that reducing agents, such as cysteine and GSH, can sensitize the N M D A receptor and enhance glutamate excitotoxicity. However, high doses or elevated levels of glutamate are the prerequisite of cysteine toxicity in all these experiments. Evidence showed that cysteine injection (s.c.) cannot itself induce a significant glutamate release (Puka-Sundvall et al., 1995). As cysteine toxicity can be induced just by cysteine administration itself and no glutamate is required, the significance of sensitized N M D A receptor in cysteine toxicity is still in question. 4. Free radical theory. Cysteine is a very unstable amino acid. It will be oxidized under aerobic conditions, during which process free radicals are produced (Hanaki and Kamide, 1975; Gardner and Jursinic, 1981; Munday, 1988, 1989). Saez et al. (1982) detected spin adducts of hydroxyl radicals by electron paramagnetic resonance (EPR) during the spontaneous autoxidation of cysteine. Ions of iron and copper increase the yield of spin adducts. They also found that incubation of cysteine (4 mM) with cultured hepatocytes causes release of lactate dehydrogenase (LDH) and decrease of intracellular ATP and GSH concentrations. Nath and Salahudeen (1993) found that cysteine autoxidation generates H2O2, which is catalyzed by plasma, kidney extract, and copper ions. Autoxidation of cysteine (4 mM) in the presence of copper is toxic to cultured kidney cell lines. However, the cysteine concentrations used in these experiments are very high. It is still uncertain whether it can reflect the in vivo cysteine toxicity. In addition, there are no reports showing the effect of cysteine autoxidation on neuronal survival thus far. Cysteine is a double-edged sword. It is an essential and rate-limiting precursor of GSH synthesis. On the other hand, different research groups have proven its cytotoxicity repeatedly in different cell types. The existence of different theories reflects the  24  Chapter 1 Introduction  complexity of cysteine toxicity. Only if the mechanisms are fully understood, can we make use of the beneficial role of cysteine while harnessing its side effects.  8. Neurological Diseases and Oxidative Stress Though the pathogenesis of neurodegeneration is still elusive, accumulative evidence has indicated the important role of oxidative stress and free radical damage in neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease, Friedrich's disease, multiple system atrophy, and olivopontocerebellar atrophy (OPCA) (Gotz et al., 1994; Markesbery, 1997; Behl, 1999; Smith et al., 2000). Alzheimer's disease (AD) is the leading cause of senile dementia, and is characterized by progressive decline in memory, language, and other cognitive functions. The pathological findings include the accumulation of extracellular senile plaques containing (5-amyloid peptides (AP), intracellular neurofibrillary tangles containing polymerized and hyperphosphorylated tau proteins, and severe neuronal loss (Martin, 1999). The pathogenesis of A D remains unknown, though there are several hypotheses for A D , such as genetic defects, latent virus infection, energy metabolism deficits, excitotoxicity, mitochondrial defects, and oxidative stress (for review see Markesbery, 1992). There has been heightened interest in the oxidative stress hypothesis in recent years. Different lines of evidence have shown the widespread free radical damages in A D . For instance, protein carbonyl, the protein oxidation products, is increased in some regions, such as the frontal lobe and the hippocampus, in A D subjects (Smith et al., 1991; Hensley et al., 1995). The oxidized nucleoside, 8-hydroxy-2'-deoxyguanosine (8OHdG), the D N A oxidation product, is 3-fold higher in mitochondria and significantly increased in the nuclei of A D subjects compared with age-control subjects (Mecocci et al., 1994). Lipid peroxidation, as measured by thiobarbituric acid reactive substances (TBARS), is also increased in A D brain compared with controls (Hajimohammadreza and Brammer, 1990; Subbarao et al., 1990; Balazs and Leon, 1994; Lovell et al., 1995). Nitration (Smith et al., 1997; Torreilles et al., 1999) and advanced glycosylation end products (AGE) (Vitek et al., 1994; Yan et al., 1994; Smith et al., 1995) are also oxidative modifications found in A D .  25  Chapter 1 Introduction  The sources of oxidative stress in A D are still uncertain. It is generally believed that aging is associated with an increased production of free radicals and a decreased ability of antioxidative defenses. The modified proteins can further form intra- and intermolecular crosslinking, and cause subsequent accumulation of damaged proteins. Ap is a heterogeneous peptide with 42 (or 43) amino acids, derived from amyloid precursor protein (APP). Though A p neurotoxicity has been extensively investigated, there still lack of consensus regarding its toxic mechanism. Recent studies have shown that AP and APP is involved in the production of free radicals. A p can produce H2O2 in the presence of F e  3+  and C u  2 +  (Huang et al., 1999a; Huang et al., 1999b). APP was found  to have copper binding sites and to produce H2O2 and -OH in the presence of Cu (Multhaup et al., 1998; White et al., 1999). C u  2+  can also bind A p (Atwood et al., 2000).  Pathological findings show that senile plagues are rich in C u  2 +  (Lovell et al., 1998),  which contain Ap. Sayre et al. (2000) reported that neurofibrillary tangles and senile plaques are major sites for catalytic redox reactivity. These lesions catalyze the ^ i n dependent oxidation of 3,3'-diaminobenzidine (DAB), and pretreatment with chelators of iron and copper abolishes the ability of these lesions. The apolipoprotein E-4 (Apo E-4) genotype is an important risk factor in late-onset familial and sporadic A D (Corder et al., 1993; Saunders et al., 1993). ApoE has been reported to be a strong chelator of iron and copper (Miyata and Smith, 1996), and to be susceptible to crosslinking by products of lipid peroxidation (Montine et al., 1996). Oxidative theory has lead to the application of antioxidants in the treatment of A D . In fact, clinical trials have demonstrated potential benefits of the antioxidants, such as Vitamin E, co-enzyme Ojo, flavonoids, selegiline, and melatonin (Pratico and Delanty, 2000). A m y o t r o p h i c lateral sclerosis ( A L S ) is a progressive degenerative disorder affecting upper and lower motor neurons. Up to 10% of all A L S cases are familial forms, and the others are sporadic. Though the pathogenesis is still to be ascertained, there has been important progress in recent years. Rosen et al. (1993) identified the genetic linkage of mutations in the Cu/Zn superoxide dismutase (SOD-1) gene with the development of some forms of familial A L S . Since the initial report, approximately 50 mutations of the SOD-1 gene have been found in 20% of familial A L S cases (Siddique and Deng, 1996). Mechanistic studies have consistently pointed to a gain of function of mutant SOD-1,  26  Chapter 1 Introduction  which involves the generation of free radicals, as the damaging properties of these mutations (Louvel et al., 1997; Morrison and Morrison, 1999). Transgenic mice expressing the mutant SOD-1 gene have normal or elevated enzyme activities, and yet develop the symptoms and pathology resembling A L S (Borchelt et al., 1994; Gurney et al., 1994; Ripps et al., 1995; Bruijn et al., 1997). On the other hand, SOD-1 knockout mice that are completely deficient of this enzyme do not develop motor neuron degeneration and A L S symptoms (Reaume et al., 1996). Further investigation revealed that the loosely bound copper to the mutant SOD-1 plays a critical role in the acquisition of the toxicity of the mutant gene (Carri et al., 1994; Estevez et al., 1999; Gabbianelli et al., 1999). Excitotoxicity is another important theory of A L S . Lathyrism, a form of motor neuron disease, was found to be causally associated with eating chickling pea, which contains P-oxaloamino-L-alanine (BOAA), an excitatory amino acid (Spencer et al., 1986). These researchers also found that ALS-Parkinsonism-dementia complex of Guam can be linked to the digestion of cycad component p-methylamino-L-alanine ( B M A A ) (Spencer et al., 1987). B M A A does not possess the Q acid, as shared by glutamate, B O A A , and other excitotoxins. Weiss and Choi (1988) explained that B M A A requires bicarbonate as a cofactor. The two molecules form a hydrogen bond, mimicking the structure of excitotoxin. However, Duncan et al. (1990) indicated that the levels of B M A A in cycad flour are too low to cause the delayed and widespread neurodegeneration. A n important finding reported by Rothstein et al. (1995) showing that there is a selective loss of glial glutamate transporter GLT-1 in motor cortex and spinal cord of postmortem A L S cases. GLT-1 and G L A S T are selectively localized to astrocytes (Rothstein et al., 1994; Lehre et al., 1995), and are critical for the uptake of extracellular glutamate. Inhibition of these glutamate transporters using chronic antisense oligonucleotide administration produced elevated extracellular glutamate levels, neurodegeneration characteristic of excitotoxicity, and a progressive paralysis of rat (Rothstein et al., 1996). However, it is not explained why and how the selective loss of glutamate transporters occurs. There is a lack of genetic and etiological evidence to support this hypothesis.  27  Chapter 1 Introduction  In stroke and traumatic brain injury (TBI), brain tissue develops a series of pathological events that evolve in time and space. Oxidative stress has been found to play a critical role in secondary neuronal damage (Juurlink and Paterson, 1998; Dirnagl et a l , 1999; Lewen et al., 2000). In animal model studies, free radicals (Peters et al., 1998) and the D N A damage product, 8-OHdG (Nagayama et al., 2000), were detected in the peri-infarct region (or penumbra) after focal cerebral ischemia. Generation of free radicals after TBI was also detected (Awasthi et al., 1997). Excitotoxicity is an important pathogenic factor in the secondary neuronal injury of stroke and TBI. Impairment of cerebral blood flow restricts the delivery of oxygen and glucose. In the core region of the affected brain tissue, neurons undergo energy depletion and anoxic cell death. In the penumbral region, energy supply is partially impaired, which rapidly leads to modest decrease of ionic gradients and depolarization of membrane potential. Depolarized membrane potential will cause glutamate release into the extracellular space (Katsura et al., 1994; Martin et al., 1994). Over-stimulation of N M D A receptors, as discussed earlier, causes C a subsequently leads to mitochondrial C a  2+  2+  influx and overload, which  accumulation and failure of mitochondrial  membrane potential (A^F) (Schinder et al., 1996; White and Reynolds, 1996). Mitochondria are the major source of free radicals, and the dysfunction of mitochondria increases the generation of free radicals (Luetjens et al., 2000). Free radicals, therefore, are the executioners in the excitotoxicity process. Inflammation is another pathogenic factor in stroke and TBI, and is closely interconnected with oxidative stress. Free radicals are the major mediators of inflammation. In stroke and TBI, microglial cells are rapidly activated. Neutrophils and macrophages enter the injury site via the compromised B B B . During phagocytosis, 0  2  consumption of these inflammatory cells substantially increases, which is known as respiratory burst. The N A D P H oxidase complex in the plasma membrane of inflammatory cells makes use of N A D P H to reduce 0 , generating 0 ~: N A D P H + 2 0 2  2  2  -» N A D P + H + 2-0 ". 0 " will be further converted to other inflammatory oxidants, +  +  2  2  including H 0 , OO", and O H . These free radicals are responsible for the oxidative 2  2  degradation of engulfed particles, and release of the produced free radicals helps phagocytes to attack extracellular targets (Conner and Grisham, 1996). As free radicals'  28  Chapter 1 Introduction  attack is indiscriminate, they will cause tissue injury, as in the cases of stroke and TBI (Awasthi et al., 1997; Nagayama et al., 2000). On the other side, free radicals produced in inflammation may play a role in chemotaxis.  H2O2  produced by activated phagocytes  can up-regulate adhesion molecules, such as ICAM-1, to facilitate phagocyte adherence to endothelium (Lo et al., 1993). Free radicals can also act as second messengers to propagate inflammatory signal (Suzuki et al., 1997; Hensley et al., 2000). For example, the important oxidative stress-related transcription factor  NF-KB  can be activated by free  radicals (Schreck et al., 1991; Pahl and Baeuerle, 1994). These results demonstrate an important role of free radicals in inflammation-induced secondary tissue injury in stroke and TBI. Taken together, different lines of evidence suggest that oxidative stress is involved in many neurological diseases, and is a major pathogenic factor. Neuroprotective effects of antioxidants in the treatment of neurological diseases have been widely reported (Gotz et al., 1994; Louvel et al., 1997; Cuajungco et al., 2000; Pratico and Delanty, 2000). Further clarification of some basic oxidative stress-related mechanisms will be of benefit to the antioxidative intervention, and may lead to the final success of disease control.  9. Experimental Objectives The overall goal of my experiments is to explore the mechanisms by which astrocytes enhance antioxidative capacities of neurons via small molecule pathways. The overall hypothesis is that astrocytes can release small molecules to protect neurons from oxidative stress. Through experiments, I will test the following hypotheses: 1) . Astrocytes improve neuronal survival. 2) . Astrocytes provide cysteine to neurons. 3) . Free radicals are generated during cysteine autoxidation. 4) . Cysteine toxicity can be prevented by antioxidants. 5) . Astrocytes release antioxidant to protect neurons from cysteine toxicity. The experiments will be done in the following sequence: First, a tissue culture system will be set up for the assessment of neurosupportive effects of astrocytes. This will be the basis of further experiments. Neuronal survival  29  Chapter 1 Introduction  with or without co-cultured astrocyte feeder layer will be compared. Neurosupportive effects of astrocytes will also be observed from another perspective, i.e., the neuronal death induced by deprivation of astrocyte feeder layer. Apoptotic neuronal death will be assessed. Second, the provision of cysteine and its mechanisms by astrocytes will be investigated. These mechanistic studies are based on the evidence that astrocytes improve neuronal survival, and that cysteine is critical for GSH synthesis. As discussed earlier, it is still unclear how cysteine is released and what the mechanisms of cysteine maintenance by astrocytes entail. Cysteine, GSH, and related compounds in astrocyte conditioned medium (ACM), neuron conditioned medium (NCM), plasma, and cerebrospinal fluid (CSF) will be analyzed using HPLC. A hypothesis of indirect cysteine provision by astrocytes is proposed. Third, the mechanism of cysteine neurotoxicity will be addressed. Cysteine is critical for GSH synthesis, particularly for neurons. However, its toxicity has long been noted. To solve this paradox, I will study the relationship of cysteine autoxidation and the generation of hydroxyl radicals. The factors influencing cysteine autoxidation will be investigated. In these experiments, it was found that copper is the most efficient catalyst of cysteine autoxidation. In further experiments, the influence of copper on cysteine neurotoxicity will be studied in cell culture. The generation of hydroxyl radicals during cysteine autoxidation in the absence or presence of copper will be assayed with fluorimetric methods. A proposed mechanism of cysteine toxicity will be defined based on these results. Fourth, the in vivo mechanisms which defend against cysteine toxicity will be explored. It is known that cysteine is essential for neurons and naturally present in the CNS, whereas it has neurotoxic effects. Therefore, the organism must have developed some defensive mechanisms to prevent the side effects of cysteine. Pyruvate has antioxidative capacity by neutralizing H2O2, as does catalase. It was found in experiments to be reported here that both pyruvate and catalase prevent cysteine toxicity. In order to identify which mechanism may represent the in vivo situation, pyruvate concentrations and activities of antioxidative enzymes, including catalase and glutathione peroxidase, will be assayed in A C M and CSF.  30  Chapter 1 Introduction  From these experiments, a concerted mechanism of neuroprotection by astrocytes will be outlined, with a focus on small molecule antioxidants. The significance of the findings in neurological diseases will be discussed.  31  Chapter 2 Materials & Methods  CHAPTER 2  MATERIALS AND METHODS  MATERIALS  1. Animals  Both pregnant and adult Long-Evans rats were obtained from Charles River (Laval, QC). Pregnant rats were bred until the embryos were 18 day old.  2. Chemicals and Reagents  The following items were obtained from Boehringer Mannheim (Mannheim, Germany): DNase I and biotin-16-dUTP. The following items were purchased from GIBCO BRL (Grand Island, NY): fetal bovine serum (FBS), 0.25% trypsin solution, Hank's balanced salt solution (HBSS), 10 mM Dulbecco's phosphate buffered saline (PBS), terminal deoxynucleotidyl transferase, recombinant (rTdT) and 5x TdT buffer. The following items were made by Molecular Probes (Eugene, OR): Calcein AM, ethidium homodimer, Hoechst 33258, and 7-hydroxycoumarin-3-carboxylic acid (7OHCCA). The following items were obtained from Sigma (St. Louis, MO): cysteine (CSH), glutathione (GSH), glutathione disulfide (GSSG), glutathionesulfonic acid (GA), glutamic acid (Glu), aspartic acid (Asp), CysGly, cysteic acid (CA), homocysteic acid (HCA), sodium pyruvate, sodium lactate, cupric sulfate, cuprous chloride, ferric chloride, ferrous sulfate, manganese sulfate, chromium chloride, zinc chloride, hemin, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 0.4% trypan blue solution, iodoacetic acid, l-fluoro-2,4-dinitrobenzene (FDNB), 5,5'-dithiobis(2nitrobenzoic acid) (DTNB), 3,3'-diaminobenzidine tetrahydrochloride (DAB), isopropanol, dithiothreitol, 2-mercaptoethanol,tert-butylhydroperoxide (t-BuOOH), hydrogen peroxide (H2O2), catalase, glutathione peroxidase, glutathione reductase, lactic dehydrogenase (LDH), p-nicotinamide adenine dinucleotide reduced form ((3-NADH), pnicotinamide adenine dinucleotide phosphate reduced form (P-NADPH), anti-human  32  Chapter 2 Materials & Methods  glial fibrillary acidic protein (GFAP) polyclonal antibody from rabbit, Earle's balanced salts (EBSS), Eagle's Minimum Essential Medium (MEM), cystine-free Eagle's M E M (M-2289), phenol red-free Eagle's M E M (M-4144), insulin, transferrin, selenium, fibronectin, and PLL. The following items were obtained from Vector Labs (Burlingame, CA): biotinylated anti-rabbit IgG from goat, A B C kit (avidin-biotinylated horseradish peroxidase complex. Cysteine-glutathione disulfide (CSSG) was purchased from Toronto Research Chemicals (North York, ON). Coumarin-3-carboxylie acid (CCA) was obtained from Aldrich (Milwaukee, WI). Anti-rat neuronal specific enolase (NSE) polyclonal antibody from rabbit was made by Polysciences (Warrington, PA). 40% formaldehyde solution was from B D H (Toronto, ON).  3. Equipment C O 2 water-jacketed incubator was manufactured by NuAire (Plymouth, MN). The inverted microscope with phase contrast and fluorescence was made by Nikon (Tokyo, Japan). Orbit shaker was produced by Lab-Line (Melrose, IL). HPLC was performed using the 712 Gilson gradient system from Gilson Medical Electronics (Middleton, WI). The 3-amino propyl ion-exchange column, with a particle size of 5 um and dimensions of 4.6 x 200 mm, was obtained from C E L Associates (Houston, TX). Fluorescent measurement was accomplished using the luminescence spectrometer Model LS 50B from Perkin Elmer (Buckinghamshire, England). The Angiocath catheter was obtained from Becton Dickinson (Sandy, UT). Centricon-3 was purchased from Amicon (Beverly, M A ) . Tissue culture dishes were manufactured by Corning (Corning, N Y ) . Glass coverslips were produced by FGRSteinmetz (Vancouver, BC). Microfilament glass was made by Frederick Haer (Brunswick, ME).  METHODS 1. Preparation of Solutions and Media  33  Chapter 2 Materials & Methods  Tissue Culture: •  Brain dissecting buffer: HBSS was supplemented with NaHC03 (0.375 g/1), glucose (5 g/1), HEPES (3.57 g/1), and penicillin/streptomycin (100 units/ml of each). This buffer was Ca /Mg free.  •  Poly-L-lysine (PLL) solution: PLL was dissolved in d H 0 as stock solution (1 mg/ml) 2  and stored at -20°C. The working solution was prepared by 1:10 dilution of the stock PLL in 0.15M, pH 8.4 sodium tetraborate solution. •  DNase I solution: DNase I was dissolved in HBSS as stock solution (1 mg/ml) and stored at -20°C, and the final concentration was 0.1 mg/ml.  •  Basal M E M : M E M was supplemented with glucose (33 mM), glutamine (2 mM), N a H C 0 (26 mM). 3  •  Chemically defined M E M (CD-MEM): basal M E M was supplemented with insulin (10 mg/1), transferrin (5.5 mg/1), sodium selenite (5 ug/1), and pyruvate (1 mM).  •  10% fetal bovine serum-supplemented M E M (10% FBS-MEM): basal M E M was supplemented with 10% (v/v) FBS.  HPLC and Others: •  m-cresol purple solution (0.2 mM): m-cresol purple was dissolved in d H 0 , and 2  stored in a dark container. •  Iodoacetic acid solution (100 mM): it was dissolved in 0.2 mM m-cresol purple solution, and stored at 4°C for at least 7 days.  •  1 -Fluoro-2,4-dinitrobenzene (FDNB) solution (1% v/v): it was dissolved in ethanol, and stored at 4°C in a dark container.  •  DTNB stock solution (10 mM): DTNB was dissolved in methanol, and stored at 4°C for at least 2 weeks.  •  Hemin stock solution (10 mM): hemin was dissolved in 50 mM NaOH, and stored at 4°C.  •  The stock solutions of transition metal ions (100 uM to 10 mM): these transition metals, including FeS0 , FeCl , CuCl, CuS0 , M n S 0 , CrCl , and ZnCl , were 4  3  4  4  3  2  dissolved in d H 0 . They were freshly prepared before use. 2  34  Chapter 2 Materials & Methods  astrocytes. The astrocytes were identified by morphology and GFAP immunochemical staining (Fig. 2-1). The confluent astrocyte cultures in the flasks were ready for the preparations of A C M and subcultures, and were maintained in 10% F B S - M E M .  2.1.2. Preparation of A CM Two- to six-week astrocyte-enriched cultures in the flask were used to make A C M . The cultures were first rinsed with Hank's solution 3 times. Twenty milliliters of basal M E M was then added to each flask. The medium was collected 24 hr later and filtered-" through a 0.45-pm pore-size filter. The A C M was stored at 4°C for use. The attachmentimproving effect of the A C M can be observed for at least 3 months. To prepare A C M for HPLC analysis, serum-free M E M was added to the confluent astrocyte cultures at a concentration of 1.33 ml per 1 x 10 cells (10 ml per flask). The 6  density of confluent astrocytes was ~1 x 10 cells/cm . The A C M was collected 24 hr 5  2  later.  2.1.3. Subculturing Astrocytes onto Coverslips For the preparation of astrocyte feeder layer, the confluent astrocytes in the flasks were further subcultured onto coverslips. Two- to six-week-cultured astrocytes were first rinsed with Hank's solution three times, then digested with 5 ml of 0.25% trypsin for 5 min. The flask was gently shaken to help detach the cells. The dissociated cells were collected into a tube and centrifuged at 300 x g for 2 min. The supernatant was discarded. The pellet cells were rinsed with 10% F B S - M E M . Repeat this step once and suspend the cells in 10% F B S - M E M . The dissociated cells were plated onto the P L L coated coverslips at a density of 4 x 10 cells/cm in 10% F B S - M E M . After 1 week, the 4  2  subcultured astrocytes become confluent and ready to use.  2.2.  Tissue Culture of Primary Neurons  2.2.1. Coating the Coversi ips with PLL and A CM To make the surfaces of coverslips hydrophilic were required for neuronal cultures. The coverslips were immersed in concentrated nitric acid overnight. The next day, the  36  Chapter 2 Materials & Methods  coverslips were fully washed in tap water for 1 hr and distilled water for 15 min in a glass container. The coverslips were autoclaved at 121 °C for 15 min and dried in an oven. The working solution of PLL was prepared in borate solution before use. The coverslips were incubated with 0.5 ml of P L L solution for 30 min, then rinsed with dri^O three times and dried in the flow culture hood for 0.5 hr. The dried P L L coated coverslips can be used subsequently or stored at 4°C for several weeks. For astrocyte cultures, the dissociated cells in the serum-supplemented medium can be plated directly onto the PLL-treated coverslips or flasks. The procedures for coating flasks with P L L were the same as those of coverslips. For neuronal cultures, further coating the coverslips with A C M was required. Before plating the cells, each PLL-treated coverslip was incubated with 0.5 ml of A C M for 5 min. Then, the A C M was withdrawn. The coverslips were rinsed with HBSS twice and aspirated. Alternatively, serum-supplemented medium and fibronectin can replace A C M to coat PLL-treated coverslips. The procedures were similar to that of A C M . The coverslips were coated with P L L and dried. Before plating the cells, the coverslips were covered with 0.5 ml of 10% F B S - M E M each for 5 min, and then rinsed with HBSS twice and aspirated. Fibronectin is a plasma protein and is often used to promote cell attachment. The coating procedures with fibronectin (40 pg/ml) were the same as for A C M and serum-supplemented medium.  2.2.2. Preparation of Cell Suspensions E-18 Long-Evans rat embryos were used. The cerebral cortices were taken and placed in Ca /Mg -free brain dissecting buffer. The meninges were removed. The 2+  2+  tissue was cut into small pieces with a pair of scissors. The cortical tissue was transferred to a tube with 5 ml of 0.25% trypsin and 0.1 mg/ml DNase mixed solution. The tissue was digested for 15 min in a 37°C water bath. The tube was gently shaken every 2 min. After digestion, 1 ml of ice-cold FBS was added to inactivate the trypsin activity. The suspension was then centrifuged at 300 x g for 2 min. The supernatant was discarded. The pellet was resuspended in basal M E M and gently dispersed with the pipet five times. It then stood in ice for 5 min. This step allowed the undigested small tissue debris to deposit at the bottom. The cell suspension was carefully transferred to a new tube. The  37  Chapter 2 Materials & Methods  suspension was again centrifuged and the supernatant was discarded. The pellet was resuspended in C D - M E M . The suspended cells were counted in a hematocytometer and the concentration adjusted as needed.  2.2.3. Culturing Primary Cortical Neurons The dissociated cells (0.5 ml) in C D - M E M were plated onto the coverslips, previously coated with P L L and A C M . The cell density was 4 x 10 cells/cm or 1 x 10 4  2  5  2  cells/cm , according to different situations. The cultures were maintained in a humidified incubator of 5% CO2 and 95% air. For different purposes, some neuronal cultures were used directly, and some others were co-cultured with astrocyte feeder layer. 2.3.  Co-cultures of Neurons with Astrocyte Feeder Layer Two hours after plating the dissociated neurons, 2.5 ml of C D - M E M was added to  each culture dish (35 mm diameter x 10 mm high). Then a glass holder was inserted and the astrocyte feeder layer was applied facing down. As shown in Fig. 2-2, the two coverslips bearing primary neurons and confluent astrocytes respectively were separated by a V-shaped glass holder of 5 mm height. The medium was changed twice a week. The glass holder was used to support the astrocyte feeder layer in the co-culture dish. It was made from microfilament glass. The thin glass tube was heated and bent into a glass holder (Fig. 2-2). First, the middle part of glass tube was heated over a gas flame and bent to a " V " . After cooling, the top of the V-shaped tube was again heated and allowed to fall down a little bit to form a foot of the holder. Lastly, the two ends of the  Figure 2-2. A side view of the glial-neuron non-contact co-culture situation. The primary cortical neurons are plated on the bottom coverslip. The confluent astrocytes are attached to the top coverslip facing down. A V-shaped holder separates the two coverslips at a height of 0.5 cm. As shown on the right side, the holder is bent from a thin glass microfilament. It contains two arms and three feet. It is removable and can be reused.  38  Chapter 2 Materials & Methods  tube were heated and bent at a 90° angle to form the two other feet. The holder has two arms and three feet, with a height of 5 mm. It is removable, and can be used repeatedly after sterilization. When preparing neuron-enriched cultures, the dissociated cells were first plated onto the bottom coverslip. After adding 2.5 ml of medium, the glass holder was seated on the bottom coverslip, and then the coverslip that carried the confluent astrocyte feeder layer was put on top of the glass holder facing down.  3. Detection of Neuronal Death and Apoptosis  3.1.  Trypan Blue Staining  Trypan blue staining was used to count the viable cell number. While live cells do not take up trypan blue, dead cells do. Before counting the cells, 0.2 ml of 0.4% trypan blue solution was added to 1 ml serum-free medium. If medium contains serum, it requires changing the medium to serum-free medium. Trypan blue was incubated with cell cultures for 15 min. Trypan blue negative cells represent the live cells and were counted. Three random fields in each dish were counted, and the average was calculated. Neuronal viability was expressed as a percentage of the control, usually the average number at DIV 0. This method was used in the experiments of Chapter 3. Later, the M T T method was applied to measure the cell viability. 3.2.  MTT Colorimetric Assay of Neuronal Viability The M T T method was modified from Denizot and Lang (1986). The tetrazolium salt  M T T is reduced into a blue formazan by the mitochondrial enzyme succinatedehydrogenase (Slater et al., 1963), and the amount of formazan produced is proportional to the number of living cells. The cultured neurons were incubated with 0.5 mg/ml of M T T in phenol red-free M E M . After a 3 hr incubation at 37°C in a humidified atmosphere of 5% C O 2 , the medium was removed, and 1 ml of propanol was added to each dish to solubilize the formazan. The optical density was measured at 560 nm with 690 nm as a reference (OD o-69o)- The cell viability was normalized as a percentage of 56  control.  39  Chapter 2 Materials & Methods  3.3.  TUNEL Staining T h e terminal d e o x y n u c l e o t i d y l transferase ( T d T ) - m e d i a t e d d U T P - b i o t i n n i c k end  labeling ( T U N E L ) assay is an i n situ detection o f D N A fragments i n apoptotic cells ( G a v r i e l i et a l . , 1992). T h i s m e t h o d is based o n the specific b i n d i n g o f T d T to 3 ' - O H ends o f D N A .  T d T catalyzes template-independent addition o f labeled d e o x y u r i d i n e  triphosphate ( d U T P ) , w h i c h is then v i s u a l i z e d using i m m u n o h i s t o c h e m i c a l stains.  The  procedures are as f o l l o w s : cultured neurons o n c o v e r s l i p s were rinsed w i t h P B S 3 times, and fixed w i t h 4 % paraformaldehyde overnight at 4 ° C . A f t e r that, 3 % H 0 2  2  was used to  inactivate intrinsic peroxidase activity for 5 m i n . T h e cells were rinsed w i t h T r i s B u f f e r e d saline ( T B S ) 3 times. T h e n the f o l l o w i n g reaction mixture was added onto the c o v e r s l i p s : 80 units o f T d T , 54 ul o f T d T buffer, 10 ul o f 1 m M d U T P , and 200 ul d H 0 . 2  T h e m i x t u r e was incubated at 3 7 ° C for 1 hr. A f t e r r i n s i n g the cells w i t h T B S , a d d a v i d i n biotinylated horseradish peroxidase c o m p l e x ( A B c o m p l e x ) for 30 m i n at r o o m temperature.  A f t e r r i n s i n g w i t h T B S , the cells were stained w i t h D A B . T h e m i x t u r e  i n c l u d e d 2.8 m g D A B , 12 u l o f 3 0 % H 0 , and 4 m l T B S . T h e cells were then 2  2  counterstained w i t h methylgreen for 10 m i n . F i n a l l y , the cells were dehydrated i n a series o f gradient ethanol solutions, and m o u n t e d .  3.4.  Fluorescent Probes E s t i m a t i o n o f c e l l death was also assisted w i t h fluorescent dyes. C a l c e i n A M is an  esterase substrate that is h y d r o l y z e d to a green fluorescent product, c a l c e i n . It serves as a viability probe that measures both esterase activity and c e l l - m e m b r a n e  integrity.  E t h i d i u m h o m o d i m e r is a red fluorescent dye that has h i g h affinity to n u c l e i c acids.  It  c a n o n l y pass t h r o u g h the c o m p r o m i s e d m e m b r a n e s , and serves as a dead indicator. H o e c h s t 33258 is cell-permeant and D N A b i n d i n g stain that fluoresce bright blue u p o n b i n d i n g to D N A .  It stains n u c l e i o f both live cells and dead cells. B e c a u s e o f this  property, H o e c h s t 33258 c a n be used to c o m p a r e the m o r p h o l o g i c a l differences o f n o r m a l cells f r o m apoptotic c e l l s , w h i c h is featured by c h r o m a t i n condensation. C a l c e i n A M and e t h i d i u m h o m o d i m e r cannot be used to determine the differences o f cell death, i.e., apoptotic or necrotic. T h e s e probes were d i s s o l v e d i n D M S O as 1 m M stock solutions,  40  Chapter 2 Materials & Methods  and stored at -20°C. They were added to culture medium to a final concentration of 10 uM, and observed under fluorescence microscope.  4. HPLC Assay of Thiols, Disulfides, and Related Compounds Cysteine and related compounds were analyzed using the HPLC method developed by Reed and co-workers (Reed et al., 1980; Fariss and Reed, 1983) with minor modifications. In principle, thiols of the sample are first reacted with iodoacetic acid to block free thiol groups, and then with fluoro-dinitrobenzene (FDNB) to produce N dinitrophenyl (DNP) derivatives, which are analyzable by spectrophotometric detection at 365 nm. This method is capable of evaluating thiols, disulfides, and acidic amino acids, etc. The components of the sample are determined by comparing the retention time of each peak with that of standard chemicals. In our procedure, 200 ul of the sample was first reacted with 25 ul of 100 m M iodoacetic acid in 0.2 m M m-cresol purple and 25 (al of N a H C 0 (0.24 M) / NaOH (0.12 M) buffer for 30 min. Then, 225 ul of 1% (v/v) 3  FDNB in ethanol was added, and the mixture was stored at 4°C overnight. Thereafter, 25 ul of 1 M lysine was added to eliminate unreacted FDNB and the sample was then ready for analysis. In cases in which only a small amount of sample was available, such as CSF, the volumes of the above reactants were adjusted proportionately according to the sample amount. HPLC solvent A was 80% methanol in water. Solvent B was 0.8 M sodium acetate in 64% methanol (for preparation of solvent B, refer to Reed et al., 1980). 20 ul of the samples was injected into HPLC column. The mobile phase was maintained at 80% A , 20% B for 5 min followed by a gradient elution to 1% A , 99% B over 10 min, and held for 5 min. The flow rate was 1.5 ml/min. The retention values for several important compounds in our experiments are shown in Table 2-1.  Table 2-1. HPLC retention times for thiols, disulfides and acidic amino acids. Chemicals  Retention time (min)  Glutamic acid (Glu)  6.29  Cystine (CSSC)  7.78  41  Chapter 2 Materials & Methods  Homocysteic acid (HCA)  8.48  CysGly  9.32  Cysteic acid (CA)  9.47  Aspartic acid (Asp)  9.80  Cysteine (CSH)  10.75  Cysteine-glutathione disulfide (CSSG)  13.13  Glutathione (GSH)  14.23  Glutathione disulfide (GSSG)  16.02  The listed standard chemicals are reacted with iodoacetic acid and l-fluoro-2,4-dinitrobenzene to produce N-dinitrophenyl derivatives, which are spectrophotometrically detected at 365 nm.  The H P L C  mobile phase starts with 5 min of isocratic elution at 80% A , 20% B, followed by 10 min of gradient elution to 1% A , 99% B, and held at 1% A, 99% B for 5 min. The flow rate is 1.5 ml/min.  The concentrations of cysteine and related compound in the samples were calculated from the peak area on the basis of external standards. Two injection loops were used. For 20 pi injection volume, the calculating equations are: thiol concentration (uM) = Peak Area (arbitrary unit)/1054, and disulfide concentration (uM) = Peak Area (arbitrary unit)/2108. The minimal detectable concentrations were 0.50 u M for thiols and acidic amino acids, and 0.25 u M for disulfides. For 100 pi injection volume, thiol concentration (uM) = Peak Area (arbitrary unit)/5017, and disulfide concentration (pM) = Peak Area (arbitrary unit)/10034. The minimal detectable concentrations were 0.10 u M for thiols and acidic amino acids, and 0.05 p M for disulfides.  5. Spectrophotometric Assay of Cysteine with Ellman's Reagent (DTNB) This is an alternative method for quantitating cysteine. It is based on the thiol/disulfide reaction of thiol and DTNB, a disulfide, liberating the chromophore 5mercapto-2-nitrobenzoic acid (Ellman, 1959; Hu, 1994). The advantage of this reagent is that its reaction with thiols is faster (in seconds) than iodoacetic acid (in minutes), which is employed in our HPLC analysis. Though it cannot detect multiple components of thiols and disulfides, it is suitable for simple systems, such as the study of cysteine autoxidation in pure solutions. The reaction mixture included transition metal ions and  42  Chapter 2 Materials & Methods  cysteine in PBS. After the reaction, 0.1 ml of DTNB was added to 0.9 ml of the reaction mixture. It was measured against a reference of 1 mM of DTNB in PBS. An additional blank control containing all components except cysteine was evaluated to correct the absorption of transition metal ions. Cysteine concentrations were measured from absorbance at 412 nm, and calculated on the basis of cysteine standards: cysteine concentration (uM) = 80.0 x Absorbance. 6. Immunohistochemical Staining  Anti-NSE and anti-GFAP antibodies were used to stain primary neuron cultures and astrocyte cultures respectively. The procedures for NSE staining are as follows: 1). The coverslips bearing cultured neurons were washed in PBS andfixedwith 4% paraformaldehyde in PBS for 10 min at room temperature. 2). Endogenous peroxidase activity was blocked by incubating the coverslips with 1% H2O2 solution for 5 min. 3). Non-specific binding was blocked by incubating with 5% normal goat serum for 30 min. 4). The cultures were incubated with primary anti-NSE antibody (1:2000) in PBS for 2 h at room temperature. 5). After removing the primary antibody and rinsing with PBS, the cultures were incubated with biotinylated goat anti-rabbit IgG (1:800) for 1 h at room temperature. 6). The cultures were incubated with avidin and biotinylated horseradish peroxidase complex (1:1000) for 1 h at room temperature and rinsed 3 times with PBS. 7). The cells were then stained with 1 ml of nickel DAB solution, which was composed of 49.5 ml Nickel solution, 0.5ml 2% DAB and 20 (al 1% H 0 , for 10 min. Nickel 2  2  solution is prepared from 0.5 g nickel ammonium sulphate, 2.5 ml of 1 M imidazole, and 47 ml of 0.05 M Tris (pH 7.6). 8). The cultures were dehydrated in graded ethanol solutions, and transferred to xylene. Mounted the coverslips onto glass slides with a polyvinyl alcohol-based mountant. For the anti-GFAP immunostaining of astrocyte cultures, the primary anti-GFAP antibody was diluted 1:200. The procedures were the same as above. 7. Enzymatic Assays  43  Chapter 2 Materials & Methods  7.1.  Glutathione Peroxidase  Glutathione peroxidase (GPx) activity was determined using a method modified from Takahashi et al. (1987). GPx activity was assessed from the oxidation of N A D P H in the presence of glutathione reductase and oxidized glutathione formed by GPx. The reaction mixture included Tris-HCl (0.1 M , pH 8.0), N A D P H (0.2 mM), E D T A (0.5 mM), G S H (2 mM), GSH reductase (1 U/ml), sample (100 ul), and t-BuOOH (100 uM). The total volume was 1 ml, and t-BuOOH was added last. The oxidation of N A D P H was monitored against a reference mixture without sample and t-BuOOH. A negative control containing all components except the sample was used to correct the oxidation of G S H and N A D P H by t-BuOOH. The samples were adult rat CSF and A C M . The latter was 1 Ox concentrated A C M prepared by filtering A C M in a centricon with 3 K D molecularweight cutoff. Calculation of GPx activity was based on the consumption of N A D P H at A o 34  (ENADPH  = 6220 M " cm" at 340 nm) (Fig. 2-3). 1  1  Mechanism:  NADP  NADPH  GSH  GSSG  t-BuOOH  H 0 2  Reaction mixture: Tris-HCl: 0.1 M NADPH: 0.2 mM GSH: 2mM EDTA: 0.5 mM GSH reductase: 1 units t-BuOOH: 100 uM Sample: CSF or ACM. The decrease of NADPH is measured at A . 340  Figure 2-3. Schematic mechanism of assay of glutathione peroxidase (GPx).  7.2. Catalase Catalase activity was measured by monitoring the absorbance change of H2O2 (Duffy et al., 1998). The reaction mixture included PBS, E D T A (0.5 mM), H 0 (10 mM), and 2  2  100 ul of sample. The total volume was 1 ml. The reference contained all components except the sample. The samples were adult rat CSF and the concentrated A C M as same as above. Absorbance was monitored at 240 nm from 300 to 600 s. The minimal detectable enzyme activity of this test was 5 mU/ml. One unit of enzyme activity was defined as the amount of the enzyme that decomposes 1 umole of H2O2 per minute.  44  Chapter 2 Materials & Methods  8. Procedures of Obtaining CSF and Plasma 8.1.  CSF CSF was obtained from the rat cerebellomedullary cistern. Three-month-old male  Long-Evans rats were anesthetized with a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg). The rat was placed in a stereotaxic frame. The skin was incised along the midline over the occipital crest, and the muscles were separated. A puncture through occipital foramen magnum was made with an Angiocath catheter. About 100 pi of CSF was slowly drawn over 2 min. The CSF was centrifuged at 300 g for 2 min to remove a tiny amount of contaminating blood cells. To examine the cysteine autoxidation rate in CSF, 10 pi of 1 m M cysteine solution was added to 90 pi of CSF, to reach a final concentration of 100 u M . The original cysteine concentration in the CSF (-1.12 uM) can be ignored. The CSF was incubated at 37°C in a humidified atmosphere with 5% CO2 and 95% air.  8.2.  Plasma of Artery and Vein  For obtaining the blood samples, the anesthetized 3-month-old rat was laid on its back. The carotid artery or internal jugular vein was carefully exposed by separating the surrounding muscles and connective tissues. A vessel puncture was made with the Angiocath catheter. About 1 ml of blood was drawn into the heparin rinsed syringe. The blood was then centrifuged at 300 x g for 2 min. Plasma was taken and processed for H P L C assay.  9. Fluorimetric Assay of Hydroxyl Radicals Production of O H was estimated by using coumarin-3-carboxylic acid (CCA). Nonfluorescent C C A was converted by -OH to highly fluorescent 7-hydroxycoumarin-3carboxylic acid (7-OHCCA) (Collins et al., 1994a). For measuring -OH produced during autoxidation of 100 u M cysteine in the presence of 0.2 u M C u , the reaction mixture 2+  was prepared as follows in sequence: 0.885 ml of PBS, 0.1 ml of 10 m M C C A (final concentration: 1 mM), 10 pi of 10 m M cysteine, and 5 pi of 40 u M C u . The solution 2+  45  Chapter 2 Materials & Methods  was left at 37°C and 100% air to finish the reaction completely. After 4 hr incubation, the samples were measured using a fluorescence spectrometer, with excitation wavelength of 400 nm and emission of 450 nm. C C A (1 mM) in PBS was used as the reference. A standard curve was calculated by measuring the fluorescence intensities of a series of concentrations of 7-OHCCA. The produced O H from cysteine autoxidation was represented by the corresponding 7-OHCCA concentrations.  10. Pyruvate Assay Pyruvate in A C M , N C M , and the CSF was measured using an enzymatic method described by Von Korff (1969). Lactic dehydrogenase (LDH) catalyzes the conversion of pyruvate to lactate with N A D H : pyruvate + N A D H U lactate + N A D . The reaction +  mixture was prepared as follows: 198 pi of PBS, 100 pi of 1 m M N A D H , 100 pi of the sample, and 2 pi of 0.4 units/ul L D H . The decrease of N A D H was monitored at 340 nm with a spectrophotometer for 300 s until a constant value was obtained. Pyruvate concentration was calculated from the oxidized N A D H  (ENADH  = 6290 M " cm" at 340 1  1  nm).  11. Statistical Analysis The data are analyzed by using analysis of variance (ANOVA). The post hoc tests with Fisher's PLSD to determine significant differences between means of individual groups are followed when necessary.  46  Chapter 3 Neuronal Survival and Attachment  CHAPTER 3  EFFECTS OF ASTROCYTES ON NEURONAL  SURVIVAL AND ATTACHMENT SHOWN IN A CO-CULTURE SYSTEM  INTRODUCTION  It is our aim to discover the laws of nature, which are hidden behind phenomena. A solid fact may constitute a basis of further exploratory work. M y experiments started from tissue cultures of embryonic cortical neurons. The initial results were not satisfactory. Repeated experiments showed that the cultured neurons hardly survive more than several days. Increasing the plate density of the dissociated cells does improve neuronal survival. It was noticed that the cells died rapidly during the first several days even in the high-density cultures in 10% FBS supplemented M E M . After DIV 3 or 4, the flat, polygonal glial cells appear and start to proliferate, forming isolated islands. The cultured neurons aggregate around these glial islands, and the number of neurons tends to be stable. The glial cells proliferate until a confluent monolayer is formed within two weeks. The neurons grow on top of this layer, and survive for several weeks. The neurosupportive effects of astrocytes have long been recognized (Banker, 1980; Walsh et al., 1992; Sass et al., 1993). These pioneering researchers have designed different methods to make use of this property of astrocytes to culture neurons. Neurons can be directly plated on an already confluent astrocyte feeder layer (McCaffery et a l , 1984; Baughman et al., 1991). In this method, neurons are grown with astrocytes, and cannot be manipulated separately. Another disadvantage is that one cannot tell whether neurons benefit from a direct attachment to astrocytes or from the factors released by astrocytes. The non-contact co-culture method has been developed to solve these problems. The coverslip bearing the neurons is dotted with paraffin drops as supporters, and inverted against a confluent glial cell feeder layer (Goslin and Banker, 1991); Or astrocytes and neurons are co-cultured in transwells (Walsh et al., 1992); Or two coverslips bearing primary neurons and astrocytes respectively can be put together in a larger dish (Sass et al., 1993). These methods have been tried, and there existed various  47  Chapter 3 Neuronal Survival and Attachment  problems. For example, the height of paraffin dots was so low that gas exchange in the space between the two coverslips was severely compromised. It was also inconvenient to sterilize and repetitively use the coverslips. For transwell co-culture method, it was hard to subculture astrocytes into the inside plastic membrane. For the method of leaving two coverslips bearing astrocytes and neurons respectively in one big dish, the neurosupportive effects of astrocytes were not obvious, speculating that released factors by astrocytes were diluted in the relatively large volume of medium. In these co-culture methods, the media used are either serum-supplemented medium (Goslin and Banker, 1991; Sass et al., 1993) or N chemically defined medium (Walsh et al., 1992). The 2  disadvantage in the use of serum-supplemented medium is that the complicated components of serum make it difficult to analyze released factors of astrocytes. Also, addition of antimitotic agents was required to inhibit the proliferation of non-neuronal cells. Likewise, N chemically defined medium contains many additives (Bottenstein and 2  Sato, 1979), which make it hard to differentiate the neurosupportive effects of astrocytes from that of supplemented components. In this part of experiments, I optimized the astrocyte-neuron non-contact co-culture method. A n astrocyte feeder layer was used to promote neuronal survival. A specific glass holder was devised to support the coverslip bearing the astrocyte feeder layer in the co-cultures. A simple chemically defined medium was used in this co-culture method. In addition, the neurosupportive effects of astrocytes were studied from another perspective, i.e. neuronal death induced by deprivation of astrocyte feeder layer. Neuronal survival was estimated after removal of the co-cultured astrocyte feeder layer. Morphological changes of the whole neurons and their nuclei were observed during the process of cell death. T U N E L staining was used to estimate the apoptotic cells in situ. Healthy attachment of the dissociated cells onto the surfaces of glass coverslips or petri dishes is the prerequisite of neuronal cultures. Usually, the dissociated cells were suspended in serum-supplemented medium, and plated in the P L L coated coverslips. Serum is thought to contain factors to help neuronal attachment (Barnes and Sato, 1980; Romijn et al., 1984). However, serum proteins seldom penetrate the B B B . It is assumed that the true "attachment factors", which exist in the extracellular matrix of the CNS, are  48  Chapter 3 Neuronal Survival and Attachment  A s t r o c y t e f e e d e r layer Control  0  1  2  3  4  5  14  7  days in vitro  Figure 3-1. Effects of astrocyte feeder layers on neuronal viability. E-18 rat embryo cortical neurons were cultured in C D - M E M at the density of 4 x l 0 cells/cm . Results show that co-culture with the astrocyte feeder layer substantially improved neuronal survival. Control cultures were grown with the blank coverslips. The medium was changed twice a week. Neuronal viability was quantified by counting the number of trypan blue negative stained cells and expressed as a percentage of the average number at DIVO. Each point represents the mean ± S E M of five dishes. * p < 0.001 compared with control; ** p < 0.02 compared with control. 4  2  produced inside. Therefore, whether astrocytes release the factors that can promote neuronal attachment was investigated.  RESULTS 1. Effects of Astrocytes on Neuronal Survival The effects of astrocytes on neuronal survival were observed by co-culturing primary neurons with astrocyte feeder layer in chemically defined M E M (CD-MEM). The cell density is 4 x 10 cells/cm . The co-culture method has been described in Materials and 4  2  Methods 2.3. A V-shaped glass holder was made (see Fig. 2-2) to support astrocyte feeder layer. The C D - M E M was M E M supplemented with glucose (33 mM), glutamine (2 mM), insulin (10 mg/1), transferrin (5.5 mg/1), sodium selenite (5 jag/1), and pyruvate (1 mM). Blank coverslips, instead of the astrocyte feeder layers, were put on top of neurons as controls. As shown in Fig. 3-1, neuronal survival by co-culturing with astrocyte feeder layer was compared with that of blank controls. While the neurons of  49  Chapter 3 Neuronal Survival and Attachment  cell death was assessed by T U N E L staining and morphological observations. Cell body observation under amplified phase-contrast microscope showed the cell shrinking and blebbing of the plasma membrane (Fig. 3-4A, B). Apoptotic neurons were revealed in situ by T U N E L staining (Fig. 3-4C, D). Nuclear staining with Hoechst 33258 revealed chromatin condensation (Fig. 3-5). These results are consistent with the characterization of apoptotic cells.  3. Effects of Astrocytes on Neuronal Attachment Neuronal attachment was observed by examining neuron shape, neurite outgrowth, and 24 hr cell viability after plating. The cell suspensions in serum-free C D - M E M were plated onto the surfaces of coverslips treated with different methods. The neurite outgrowth after cell plating is assessed in terms of percentage of neurons, each of which has one or more extended neurites longer than 10 urn, an arbitrary length equal to about one neuronal diameter, (a) For the coverslips treated only with PLL, most of the cells at 4 hr after plating showed round bodies, the plasma became flat and transparent under the phase-contrast microscope (Fig. 3-6A). The neurons had few extended neurites, and died within 24 hr of plating (Table 3-1). (b) For PLL- and ACM-treated coverslips, the neurons showed a polygonal cell body shape with one or more neurites extended (Fig. 36B). There was a high proportion of cells with neurites of more than 10 urn long at 4 hr, and the survival was high 24 hr after plating (Table 3-1). (c, d) After P L L precoating, further alternative treatments were tried, including serum-supplemented medium (10% F B S - M E M ) and fibronectin (40 ug/ml). These coating methods also produced healthy neuronal attachment as shown by polygonal cell bodies and extended neurites (Fig. 3-6C, D). Neurite length and 24-hr survival revealed no obvious differences compared with that of ACM-coated coverslips (Table 3-1). (e) If the coverslips were coated only with A C M without precoating with PLL, most of the neurons did not adhere to the coverslips (Fig. 3-6E). Only a few neurons extended neurites and the 24-hr survival was low (Table 3-1). (f) I also prepared cell suspensions in 10% F B S - M E M and plated them onto P L L coated coverslips. The results showed healthy attachment using the assay of 4-hr neurite outgrowth and 24-hr survival rate (Fig. 3-6F and Table 3-1).  52  Chapter 3 Neuronal Survival and Attachment  These experiments provide several methods to plate dissociated neurons. Coating the PLL-pretreated coverslips with A C M , or serum-supplemented medium or fibronectin, can all produce healthy attachment and neurite outgrowth. The same effects can also be obtained by plating cell suspensions in serum-supplemented medium, which is frequently employed in many serum-free culture protocols.  Table 3-1. Effects of different coating methods on neurite outgrowth and cell viability Coating methods  Culture mediums  Neurons (%) with one  Cell viability  of cell suspensions  or more neurites (>  (%) (24 hr)  10 nm) (4hr) A. P L L only  CD-MEM  4.4 ± 0.9  B. P L L + A C M  CD-MEM  84.7 ± 2.3  84.5 ± 5 . 4  C. P L L + 1 0 % F B S - M E M  CD-MEM  82.7 ± 1.8  86.6 ± 4.3  D. PLL+Fibronectin  CD-MEM  79.5 + 3.1  83.0 ± 6 . 4  E. A C M only  CD-MEM  . 17.0 ± 4.0  F. P L L only  10%FBS-MEM  84.2 ± 2 . 5  a  b  0  C  4.7 ± 2.2  d  81.6 ± 5.8  The dissociated cells were suspended in serum-free chemically defined MEM (CD-MEM) (A-E) or 10% FBS-MEM (F). Different coating methods were used as shown above. 4 hr after plating, neurite outgrowth was measured by counting the number of neurons with one or more neurites longer than 10 urn, and expressed as a percentage of total cells. Cell viability was assayed by measuring trypan blue-negative stained live cells at 24 hr, and expressed as a percentage of D1V0 cell number. The results were means ± SEM. 'V<0.001 . g a  v s  p B (PLL+ACM); ' p<0.001 vs. group B (PLL+ACM). No obvious differences c d  r0U  were found among group B, C, D and F.  DISCUSSION  This co-culture system shares with other astrocyte-neuron co-culture methods the advantage that primary neurons can be cultured at low density and with high viability. In addition, my protocol possesses the following characteristics: (a) A removable glass holder was employed, which can be made easily from a glass microfilament, to separate the two coverslips in a culture dish. Therefore, the coverslips can be handled  54  Chapter 3 Neuronal Survival and Attachment  overflow of the culture dish, require extra medium unnecessarily, and lower neuronal viability, (c) A simplified formula of C D - M E M , which is supplemented with insulin, transferrin, selenium, and pyruvate, was developed. It is simpler than N and many other 2  chemically defined media, while it maintains a high neuronal survival. A simple formula will facilitate the analysis of astrocyte-released factors. In addition, pure neuronal populations can be obtained because glial cells do not proliferate in this serum-free medium. More than 95% of the cells in this culture are neurons, as identified by neuron specific enolase (NSE) immunostaining. No mitotic inhibitors are required with this method. This experiment has generated in vitro evidence that neuronal survival is highly astrocyte-dependent. The mechanisms by which astrocytes support neuronal survival remain unclear though extensive studies have been done in this field. It has been found that astrocytes can play diverse functions. They can optimize the composition of extracellular fluids, by regulating extracellular potassium (Newman, 1995), inactivating neurotransmitters (Martin, 1995), and secreting bioactive factors, such as amino acids, substrates of energy, neuropepetides, and neurotrophic factors (Vernadakis, 1988; Martin, 1992; Schmalenbach and Muller, 1993). In addition, astrocytes have been found to protect neurons from oxidative stress (Langeveld et al., 1995; Desagher et al., 1996; Hochman et al., 1998; Tanaka et al., 1999). The neurosupportive effects of astrocytes may reflect a synergistic action of multiple factors. Each factor may exert different influence according to particular circumstances. As noticed in my initial experiments, the cultured primary neurons die rapidly during the first several days in vitro in serumsupplemented medium. Then glial cells start to proliferate, and neurons tend to aggregate around the islands of glial cells. This phenomenon could be explained by the neurosupportive effect of astrocytes. The cultured neurons are very fragile due to the insufficient support from the small number of glial cells at the beginning of cultures. Once the glial cells, mainly astrocytes, proliferate and grow to a confluent layer, the cultured neurons obtain sufficient support for survival and live in a healthy state. Apoptosis has been known to be a form of cell death distinct from necrosis, and the term was first coined more than 20 years ago (Kerr et al., 1972). Apoptosis has been suggested to be involved in the abnormal neuronal death that occurs following axonal  56  Chapter 3 Neuronal Survival and Attachment  injury (Edstrom et al., 1996; Kogawa et al., 2000) or in neurodegenerative diseases (Nishimoto et al., 1997; Desjardins and Ledoux, 1998; Tatton and Olanow, 1999). While necrotic cell death is typified by rapid cell swelling and lysis, apoptotic cell death is characterized by cell shrinkage, membrane blebbing, and nuclear condensation (Kerr et al., 1972; Wyllie et al., 1980). A variety of strategies have been applied to induce experimental apoptosis, including treatment with oxidants (Slater et al., 1995), hypoxia (Beilharz et al., 1995), heat shock (Fairbairn et al., 1995), glutamate (Ankarcrona et al., 1995), calcium (Trump and Berezesky, 1995), glucocorticoids (Schwartzman and Cidlowski, 1994), hormone withdrawal (Gould and McEwen, 1993), and growth factor withdrawal (Raff et al., 1993; Collins et al., 1994b). In the present experiment, neuronal apoptosis was induced by withdrawal of the astrocyte feeder layer. This can be used as a model of neuronal apoptosis. The mechanisms of cell death in this model can be attributed to the loss of diverse neurosupportive effects of astrocytes. This apoptosis model induced by withdrawal of astrocytes may reflect the physiological and pathological conditions in vivo. Cautionary notes have been reported about the T U N E L staining for the detection of apoptosis, showing that T U N E L assay is non-specific for apoptosis (Charriaut-Marlangue and Ben-Ari, 1995; Grasl-Kraupp et al., 1995). In addition, more investigations have focused on the mechanistic studies of cell death, other than the differentiation between apoptosis and necrosis in recent years. In my further experiments, apoptosis is no longer emphasized. It has significant physiological meaning that A C M helps neuronal attachment. The factors in A C M may represent the true extracellular matrix proteins surrounding neurons in vivo. In many serum-free culture protocols, the dissociated cell suspensions are first plated in serum-supplemented medium, which is then replaced by serum-free medium several hours or 1 day later (Bottenstein and Sato, 1979; Romijn et al., 1984; Pardo et al., 1997). Serum contains fibronectin or other attachment factors (Barnes and Sato, 1980), which may contribute to this function of serum. However, these serum proteins cannot penetrate the blood-brain barrier, and therefore may not reflect the in vivo condition of neuronal growth. As for the unknown "attachment factor" in A C M , our initial analysis with an HPLC size exclusion column suggests that it has a high molecular weight (> 670  57  Chapter 3 Neuronal Survival and Attachment  kDa). My observations reveal that plating dissociated cells in serum-supplemented medium has a similar effect on neuronal attachment and 24 hr cell viability as simply coating the coverslip with A C M , serum-supplemented medium, or fibronectin. This result supports the hypothesis that the critical role of serum played in the first-step culture procedure is simply due to its functions in neuronal attachment and neurite outgrowth. Therefore, the procedure of plating cells in serum-supplemented medium can be replaced by the simple coating with A C M , which is closer to the in vivo situation. It is also shown that P L L is indispensable for A C M , serum, or fibronectin, etc., to exert their attachmentimproving effects, though P L L itself cannot produce a healthy attachment of neurons. This positively charged polymer is presumed to assist the firm coating of those attachment factors to the surface of the coverslip.  58  Chapter 4 Astrocytes Provide Cysteine to Neurons  CHAPTER 4  PROVISION OF CYSTEINE BY ASTROCYTES TO  NEURONS BY WAY OF RELEASING GLUTATHIONE  I N T R O D U C T I O N  In order to study the neurosupportive mechanisms of astrocytes, I have tested many chemicals and growth factors in neuronal cultures. The neurosupportive effects were observed with the supplements of cc-tocopherol or cysteine. Addition of ct-tocopherol to the culture medium improved neuronal survival, and the results were stable. The results obtained from cysteine supplements, however, were unstable. Addition of cysteine to the culture medium could be beneficial or cytotoxic, varying with cysteine concentrations. Also, cysteine was toxic in basal M E M , while non-toxic in D M E M . These contradictory data implicate that the role of cysteine is complicated. Cysteine is a very special amino acid. Unlike other amino acids, cysteine is very unstable owing to autoxidation under aerobic conditions. It is also the rate-limiting precursor for the synthesis of GSH (Beutler, 1989), which is the major cellular antioxidant. The extracellular abundance of thiol-containing compounds substantially influences intracellular GSH levels (Meister, 1989). Neurons, in particular, require cysteine, instead of cystine, to synthesize G S H (Kranich et al., 1996). In the following experiments, the mechanisms of cysteine maintenance in the CNS were investigated. The mechanisms by which a stable cysteine level is maintained by astrocytes have been explored in recent years. Release of thiols by astrocytes has been reported and is considered to play an important role to increase neuronal GSH synthesis (Yudkoff et al., 1990; Sagara et al., 1993; Sagara et al., 1996; Dringen et al., 1999a). However, it is still uncertain as to how thiols are provided. Yudkoff et al. (1990) found GSH in conditioned medium of astrocytes. Sagara et al. (1993) reported that glial cells can release cysteine to the cultured medium to supply neurons. In a later report (Sagara et al., 1996), they found GSH efflux from cultured astrocytes. Recently, Dringen et al. (1999a) suggested that the ectoenzyme y-glutamyl transpeptidase uses GSH released by astrocytes as a substrate to generate the dipeptide CysGly that is subsequently used by neurons as a precursor for  59  Chapter 4 Astrocytes Provide Cysteine to Neurons  GSH synthesis. In the present experiments, I tried to determine whether astrocytes release cysteine, or GSH, or both. What is the relationship of cysteine and GSH? What mechanism represents the in vivo situation? I have analyzed cysteine, GSH and other related compounds in A C M , neuron conditioned medium (NCM), CSF, plasma of the carotid artery and internal jugular vein of rat using H P L C methods developed by Reed et al. (1980). These methods are capable of evaluating thiols (cysteine, GSH, homocysteine, CysGly), disulfides (cystine, glutathione disulfide, cysteine-glutathione disulfide), and acidic amino acids (glutamic acid, aspartic acid, cysteic acid, homocysteic acid), etc. This advantage makes it possible to analyze the relationships among these compounds. These experimental results suggest that astrocytes release GSH directly, and that cysteine is generated by an extracellular thiol exchange reaction: Glutathione (GSH) + Cystine (CSSC) U Cysteine (CSH) + Cysteine-glutathione disulfide (CSSG). This reaction can occur non-enzymatically (Jocelyn, 1967). The results show that astrocytes can provide small molecules to enhance GSH synthesis, and therefore the antioxidative capacities of neighboring neurons.  RESULTS 1. Cysteine-glutathione Disulfide (CSSG) Found in Cystine-containing Astrocyte Conditioned Medium I first analyzed the components and concentrations of cysteine and related compounds in A C M . M E M was added to the confluent astrocyte cultures at a concentration of 1.33 m l / l x l O cells (10 ml per flask). As shown in Fig. 4-1, the control medium contains 6  mainly cystine (0 hr in culture dishes). Analysis of the control medium after it has been in the incubator for 48 hr shows no difference with that at 0 hr. Several new peaks appeared in A C M compared with control medium. Three of them were clearly identified as cysteine, CSSG and GSH. The finding of CSSG in A C M was meaningful. The direct efflux of CSSG is unlikely, because no CSSG was detected in astrocytes (see Table 4-3). On the other hand, extracellular CSSG can be produced when GSH reacts with cystine non-enzymatically: GSH + CSSC ^ C S H + CSSG.  60  Chapter 4 Astrocytes Provide Cysteine to Neurons  o'.OO  8'.OO  lo'.OO  16 '.00  2o'.00  Time (min) Figure 4-1. H P L C graphs of cysteine and related compounds in control medium, A C M and the standards. Control medium was serum-free cystine-containing M E M . The A C M was made by adding the culture medium to the confluent astrocytes for 24 hr. The peaks in the control medium and A C M were identified by referring to the standards. The graphs show that: A . Control M E M contains mainly cystine. B. Several new peaks besides cystine were found in 24 hr A C M . Three of them can be identified as CSH, CSSG and G S H . C. The standards were CSSC, C S H , CSSG, G S H and GSSG. The concentration of each chemical is 10 uM. The peak area of disulfide is about double that of thiol. A U F S , absorbance units of full scale.  61  Chapter 4 Astrocytes Provide Cysteine to Neurons  At 37°C and pH 7.4, the equilibrium constant K of the above reaction is 3.2 and velocity constant k is 610 liter/moles x min (Jocelyn, 1967). That means the reaction can proceed forward rapidly. The appearance of CSSG in A C M leads us to suggest that cysteine may not be released, but instead is produced extracellularly by the reaction of GSH, which is released by astrocytes, and the stable amino acid cystine. This suggestion is consistent with the fact that GSH, not cysteine, is maintained at a high intracellular level. CysGly was not detected in the A C M .  Time of incubation (hr)  Figure 4-2. Time course of cysteine and related compounds' content in A C M . Confluent astrocytes in flasks were rinsed with Hank's solution 3 times. The serum-free cystine-containing M E M was added at a concentration of 1.33 ml/1 x 10 cells. Samples of the A C M were taken at 0, 6, 12, 24 and 48 hr, and processed immediately for H P L C analysis. The calculated values of the equilibrium constant K for the equation, G S H + CSSC ±5 C S H + CSSG, are as follows: K, =4.66, K h=4.19, K48 =4.57. Data are shown as mean ± S.E.M. of five determinations. 6  2h  24  h  The changes in concentrations of cysteine and related compounds in A C M were measured as shown in Fig. 4-2. Cystine decreased with time of incubation, with a beginning concentration of 76.9 ± 3 . 4 uM. Cysteine and CSSG increased rapidly from zero, then decreased when cystine content diminished at 24 hr and 48 hr. G S H was barely detectable at 6 hr. This can be explained by the high concentration of cystine in the medium. The theoretical value of G S H calculated from the K value (3.2) and concentrations of cystine, cysteine and CSSG is 0.07 u M at 6 hr, well below the minimal  62  Chapter 4 Astrocytes Provide Cysteine to Neurons  detectable level, 0.5 uM. Therefore, G S H may not be detected in this situation. The values of the equilibrium constant K calculated from Fig. 4-2 are 4.66 at 12 hr, 4.19 at 24 hr, and 4.57 at 48 hr. A small amount of GSSG was found at 48 hr. GSSG may be generated by two possible mechanisms: autoxidation of GSH, and the thiol/disulfide exchange reaction. The latter can also occur non-enzymatically (Jocelyn, 1967): CSSG + GSH ±5 C S H + GSSG.  2. GSH Released by Astrocytes in Cystine-free Medium In order to obtain direct evidence for GSH release from astrocytes, I analyzed the conditioned medium from cystine-free M E M . Cystine-free M E M (M-2289) was added to the confluent astrocyte cultures at a concentration of 0.66 m l / l x l O cells (5 ml per flask). 6  In this case, the extracellular thiol/disulfide exchange reaction between cystine and G S H can be excluded. As shown in Table 4-1, cysteine and CSSG were no longer found, while G S H was the only component detected. This result indicates that there is no direct release of cysteine and CSSG by astrocytes. Astrocytes release GSH to the medium from an intracellular reservoir of GSH. Because there is no cystine as a source for GSH synthesis, the detected G S H concentration may not reflect the potential levels the astrocytes can release. The small content of GSSG is assumed to be the autoxidation product from its reduced form. The dipeptide CysGly was not detected in these experiments.  Table 4-1. Content (uM) of cysteine and related compounds in A C M made from cystinefree medium. Time  esse  CSH  CSSG  GSH  GSSG  CysGly  2h  ND  ND  ND  0.66 ±0.11  ND  ND  4h  ND  ND  ND  1.06 ±0.03  ND  ND  6h  ND  ND  ND  1.43 ±0.09  ND  ND  12h  ND  ND  ND  3.18 ±0.09  0.32 ± 0 . 1 9  ND  The confluent astrocyte cultures were rinsed with Hank's solution 3 times, and the cystine-free MEM was added at a concentration of 0.66 ml/lxlO cells. The samples were collected at 2, 4, 6 and 12 hr, and 6  63  Chapter 4 Astrocytes Provide Cysteine to Neurons  analyzed using HPLC methods. Data are shown as mean ± S.E.M (uM) of five determinations. ND, not detectable.  3 . Analysis of Cysteine and Related Compounds in CSF and in Plasma of Artery and Vein It is of course critical to determine whether the in vitro mechanisms studied above are applicable to the in vivo situation. Plasma of the carotid artery and of the internal jugular vein, which represent blood supply and return of the brain, and CSF were studied. Our results (Table 4-2) showed that the components of cysteine and related compounds in CSF and blood are surprisingly different. No cystine was detected in CSF, while it has a high concentration in plasma. Vein plasma clearly has a lower level of cystine than artery plasma. G S H showed a relatively high concentration in CSF, while it was not detected in plasma. Cysteine and CSSG were detected in both CSF and plasma, with a low level in CSF. No clear differences in the concentrations of cysteine and CSSG were detected in the artery and vein. GSSG was detected at a low concentration in CSF. No CysGly was detected in CSF, artery or vein. A supplementary experiment was done later on to analyze cysteine and related compounds in CSF and in plasma of artery and vein with enhanced sensitivity. The injection volume of the sample in H P L C analysis was changed from 20 pi to 100 pi. The detection sensitivity was correspondingly increased about 5 times. Three-month-old rats and the analytical procedures used the same as in the previous experiment. Some components, which were not detected with 20 pi injector, were found with 100 pi injector. In CSF, CSSC is 0.09 ± 0.02 (mean ± SEM) u M , ranging from 0.05 to 0.13 u M (n = 5). In plasma of carotid artery, GSH is 1.26 ± 0.24 uM, ranging from 0.16 to 1.68 u M (n = 5); GSSG is 0.19 ± 0.08 uM, ranging from 0.05 to 0.34 u M (n = 5). In plasma of internal jugular vein, GSH is 1.17 ± 0.31 u M , ranging from 0.65 to 1.71 p M (n = 5); GSSG is 0.20 ± 0.01 u M , ranging from 0.19 to 0.21 u M (n = 5). There are no significant differences of GSH and GSSG concentrations between artery and vein (p > 0.05). CysGly was not detected in CSF, artery and vein. The results of other components have no obvious differences from that listed in Table 4-2.  64  Chapter 4 Astrocytes Provide Cysteine to Neurons  Table 4-2. Content (uM) of cysteine and related compounds in CSF, and in plasma of the carotid artery and internal jugular vein CSSC  CSH  CSSG  GSH  GSSG  CysGly  ND  1.12 ± 0.14  0.50 ± 0 . 0 8  5.87 ± 0 . 2 9  0.22 ± 0 . 1 2  ND  b  0.68-1.75  0.28-0.83  4.92-7.11  0-0.80  ND  b  ND  b  ND  b  ND  b  ND  b  ND  b  CSF  Mean ± S E M  n=7  Range  Artery  Mean ± S E M  35.2 ± 1.2  4.94 ± 0.69  3.17 ± 0.21  n=7  Range  30.8-40.1  3.32-7.49  2.37-3.72  Vein  Mean ± S E M  26.3 ± 0.9"  5.42 ± 0.79  3.06 ± 0 . 3 5  n=5  Range  23.9-29.2  2.48-7.00  2.34-4.35  b  Three-month-old rats were used.  CSF was taken from the cerebellomedullary cistern.  Plasma of  artery and vein was taken from the carotid artery and internal jugular vein respectively. All the samples were centrifuged at 300 x g for 2 min, and immediately processed for HPLC analysis. Values represent mean ± S.E.M. (uM) and concentration range with 5 to 7 samples as indicated. The contents of cysteine and CSSG have no difference between artery and vein. ND, not detectable. a  b  p < 0.001, different from artery value.  , see results of supplementary experiments in text.  4 . Thiols not Found in Neuron Conditioned Medium Whether neurons can provide thiols by themselves was studied in order to determine whether astrocytes are the major releasers of thiols. Neuron-enriched cultures were prepared by using the glial-neuron co-culture method in serum-free medium as described in the methods. The final density is 1 x 10 cells/cm . At DIV 7, the astrocyte feeder 5  2  layers were removed. Neuron-enriched cultures were rinsed with Hank's solution 3 times. Cystine-supplemented or cystine-free M E M (M-2289) was added at a concentration such that the medium volume/cell number ratio was the same as that of A C M . As shown in Fig. 4-3, no cysteine or GSH was detected in cystine-supplemented N C M . Cystine levels showed no obvious change. Low levels of CSSG were found in the cystine-supplemented N C M , suggesting that there is still a tiny amount of GSH released by neurons and the extracellular thiol/disulfide exchange reaction, but the amount of thiols is too small to be detected. It cannot be excluded that residual glial contamination (<5%) of these cultures account for the GSH release observed.  65  Chapter 4 Astrocytes Provide Cysteine to Neurons  100  5  a. O  o w  </) (0  (0  o  o 12  24  36  Time of incubation (hr)  Figure 4-3. Time course of cysteine and CSSG content in N C M . Neuron-enriched cultures were prepared with the glial-neuronal co-culture method in serum-free medium. At DIV 7, the cultures were rinsed with Hank's solution 3 times. The serum-free cystinecontaining M E M was added at a concentration of 1.33 m l / l x l O cells, the same as medium volume/cell number ratio of A C M . CSH, GSH, GSSG and CysGly were not detected. Data are shown as mean ± S.E.M. of five determinations. 6  N C M prepared from cystine-free M E M was also analyzed. The medium was added at a concentration of 0.66 m l / l x l O cells. The samples were collected at 6 hr and 12 hr. 6  The thiols and disulfides, including cysteine, cystine, CSSG, GSH, GSSG, and CysGly, were not detected in five determinations.  5. Contents of GSH and Related Compounds in Astrocytes and Neurons As the cross-membrane concentration gradient influences the kinetics of membrane transport, the intracellular contents of GSH and related compounds were investigated. Confluent astrocytes (2 to 6 weeks in culture) and neuron-enriched cultures (DIV 7) were analyzed. As shown in Table 4-3, there are high levels of GSH in astrocytes and neurons, with higher concentrations in astrocytes. Low levels of cysteine and GSSG were found in astrocytes, but not in neurons. GSH/CSH ratio is 11.8:1, and GSH/GSSG ratio is 81.5:1 in astrocytes. Cystine, CSSG and the dipeptide CysGly were not detectable in either astrocytes or neurons. In contrast with the relatively lower levels of GSH, neurons contain much higher levels of glutamate and aspartate than astrocytes (Table 4-3).  Table 4-3. Content (nmol/mg of protein) of thiols, disulfides and acidic amino acids [glutamic acid (Glu) and aspartic acid (Asp)] in astrocytes and neurons 66  Chapter 4 Astrocytes Provide Cysteine to Neurons  GSH  CSH  GSSG  Astrocytes  59.1 ± 6 . 0  5.02 ±0.38  0.73 ±0.12  Neurons  50.8 ± 1.5  ND  ND  Asp  Glu  4.50 ±0.94  33.7 ± 6 . 6 194 ± 6  63.0 ± 3 . 6  The confluent astrocyte cultures and neuron-enriched cultures at DIV 7 were ruptured and lysed in dH 0 by freezing and thawing. Protein content was determined by the Bradford method. The samples 2  were immediately analyzed by HPLC. The results represent mean ± S.E.M. (nmol/mg of protein) of five determinations. Cystine, CSSG and CysGly were not detected. ND, not detectable.  6. Autoxidation of Cysteine and G S H Cysteine and G S H are unstable under aerobic conditions. We assayed the autoxidation rates of cysteine and GSH, 100 u M each, in phosphate buffered solution. As shown in Fig. 4-4, cysteine was oxidized to cystine, and GSH oxidized to GSSG under conditions of 37°C, pH 7.4 and 100% air. A very small amount of cysteic acid (~ 1-2 uM) was also found as a byproduct of cysteine autoxidation.  DISCUSSION  In this study, I demonstrate the mechanism by which astrocytes provide cysteine to neurons indirectly by releasing GSH. 1). In the absence of cystine, as the results from cystine-free A C M show (Table 4-1), it is GSH, not cysteine, that is found in the A C M . This result indicates that GSH is the direct product released by astrocytes. Astrocytes release G S H from their intracellular reservoirs without requiring further supplies of thiol in the cystine-free medium. Hence, the GSH concentrations measured here under these conditions may not reflect the potential levels of secretion that astrocytes may reach. 2). In the presence of cystine, as the results from cystine-containing A C M show (Fig. 4-1 & 4-2), cysteine, CSSG and GSH were found in the A C M . Intracellular CSSG was not detected in astrocytes, and cysteine was present at much lower intracellular levels than GSH (Table 4-3). The high levels of CSSG and cysteine in the A C M cannot be explained by direct cellular release. However, extracellular CSSG and cysteine can be produced when GSH reacts with cystine non-enzymatically: GSH + CSSC ±5 C S H + CSSG. The reaction can rapidly proceed forward with an equilibrium constant K 3.2  67  Chapter 4 Astrocytes Provide Cysteine to Neurons  S  •3-  7 5  • CSH  o W  CO  50 5 0  • esse  o  I  25  o  3-  o  M—• 0  5  12  24  48  Time (hr)  75  CO  5  X  25  • GSH • GSSG  0  CO o  0  6  12  24  48  Time(hr)  B  Figure 4-4. Autoxidation of cysteine and GSH. 100 u M of cysteine, or GSH, in phosphate buffered solution was incubated under conditions of pH 7.4, 37°C and 100% air. The samples were collected at 0, 6, 12, 24, and 48 hr, and analyzed by H P L C . Values represent mean ± S.E.M. of four determinations. A . Cysteine is oxidized to cystine. B. GSH is oxidized to GSSG.  (Jocelyn, 1967). The data suggest that astrocytes release GSH directly, which then reacts with the stable amino acid cystine extracellularly to generate cysteine and CSSG (Fig. 45). It should be noted that CSSG does not accumulate in A C M (Fig. 4-2). I found that CSSG can replace cystine to support astrocyte survival, while astrocytes cannot survive in cystine-free medium without the addition of other thiols or disulfides. It is possible that CSSG might either be taken up by astrocytes directly or further react with G S H to generate cysteine and GSSG (Jocelyn, 1967). This hypothesis is supported by the results of transporter studies, and provides a new understanding of the physiological significance of GSH efflux. G S H efflux has been found in fibroblasts (Bannai and Tsukeda, 1979), hepatocytes (Fariss and Reed, 1983; A w et al., 1986), and astrocytes (Sagara et al., 1996). Kinetic studies from astrocytes suggest that G S H efflux is carrier-mediated, and the K value is 127 nmol/mg of protein m  (Sagara et al., 1996). GSH transporters of astrocytes are ion-independent, and net transport depends on the concentration gradient of GSH. Since the intracellular  68  Chapter 4 Astrocytes Provide Cysteine to Neurons  Blood Vessel  Astrocyte  Neuron  Figure 4-5. A model showing how astrocytes may provide cysteine to neurons. M y data suggest that blood mainly supplies cystine to the brain. Cystine is taken up by astrocytes, and utilized to synthesize GSH. Astrocytes release GSH to the extracellular fluid, where GSH reacts with cystine to generate cysteine and CSSG non-enzymatically. Cysteine is then taken up by neurons for GSH synthesis. concentration of GSH is several orders of magnitude greater than the extracellular concentration, the transport of G S H operates as an efflux process. Cysteine transporters, in contrast, belong to the Na -dependent system A S C (Kilberg et al., 1981; Franchi+  Gazzola et al., 1982). Recently, two members of the A S C transporter family, ASCT1 and ASCT2, have been cloned and functionally studied (Arriza et al., 1993; Shafqat et al., 1993; Utsunomiya-Tate et al., 1996). Cysteine, as well as some other neutral amino acids, is taken up by cells with high affinities, coupling to the cotransport of N a . These +  results suggest a cross-membrane movement of GSH efflux and cysteine influx. Consistent with the transporter data, I show that it is GSH, not cysteine, that is released by astrocytes. Cysteine is generated extracellularly, and then taken up by neurons for G S H synthesis. Cooper and Kristal (1997) have previously indicated that transfer of sulfur amino acids and peptides between astrocytes and neurons occurs, and that astrocytes play an important role in sulfur homeostasis in the CNS. It was found that astrocytes and neurons have completely different capacities in the utilization of cystine and cysteine. The uptake rate of radio-labeled cystine by astrocytes is much higher than that of neurons (Sagara et al., 1993). Kranich et al. (1996) found that cultured astrocytes could make use of cystine  69  Chapter 4 Astrocytes Provide Cysteine to Neurons  to synthesize G S H to their maximal degree. Cultured neurons, instead, make use of cysteine, rather than cystine. Similar to these results, my data show that cystine was used rapidly within 48 hr in astrocyte-enriched cultures (Fig. 4-2), while cystine levels showed almost no change in neuron-enriched cultures (Fig. 4-3). (Note: the ratios of medium volume/cell number of A C M and N C M are the same). These results indicate that astrocytes have a strong reducing ability, converting cystine to cysteine in their cell bodies. This ability may account for the enhanced synthesis and release of G S H by astrocytes. These data show that astrocytes release substantial GSH in A C M , while neurons release little. Whether y-glutamyl transpeptidase (yGT) is involved in thiol generation is still unclear. Dringen et al. (1999a) found that adding the dipeptide CysGly, the product of yGT catalyzed reaction, to neuron-enriched primary cultures increases the GSH content of neurons. Acivicin, a yGT inhibitor, suppresses the astrocyte-mediated increase in neuronal GSH content. They suggested that the ectoenzyme yGT uses G S H released by astrocytes to generate CysGly that is subsequently used by neurons. However, no evidence thus far shows the existence of CysGly in astrocyte conditioned medium or CSF. The localization of yGT in the CNS is crucial for the study of its function. yGT is used extensively as a marker of brain microvessels. It is abundant in the choroid plexus (Tate et al., 1973). yGT positive cells include endothelial cells (Orte et al., 1999), pericytes (Risau et al., 1992), and the endfeet of astrocytes (Zhang et al., 1997). This type of localization is consistent with the suggested role of yGT in the transport of amino acids across the blood-brain barrier (BBB) (Meister and Anderson, 1983; Cooper and Kristal, 1997). However, it does not prove that y-GT can generate thiol in the extracellular space of the CNS. GSH released by astrocytes into the extracellular space may not reach the ectoenzyme yGT due to the restrictive distribution of this enzyme (only around brain microvessels). A recent experiment has shown the detection of several yglutamyl derivatives of amino acids in the perfusates of brain slices (Li et al., 1999). This result could be explained by the existence of a large amount of exposed brain microvessels in brain slices. GSH released by astrocytes could react with the ectoenzyme y-GT on the surface of these microvessel cells, which may not represent the situation of  70  Chapter 4 Astrocytes Provide Cysteine to Neurons  integral tissue. Further screening of y-glutamyl derivatives in conditioned medium and CSF is required to clarify the involvement of yGT in thiol generation. I am most interested in the applicability of the in vitro results to in vivo situations. By analyzing cysteine, GSH and related compounds in CSF, and in plasma of the carotid artery and internal jugular vein, the following conclusions are obtained. First, cystine is substantially transported from blood to the CNS. Both artery and vein contain high concentrations of cystine, and there is also an obvious positive arterio-venous (A-V) concentration difference, indicating an uptake by the brain. Second, blood GSH cannot be the thiol source of the brain. GSH concentrations were very low in plasma of the carotid artery (1.26 ± 0.24 uM) and internal jugular vein (1.17 ± 0.31 uM), while GSH has a relatively high level (5.87 ± 0.29 uM) in CSF. Third, the other substances considered, such as cysteine, CSSG and CysGly, are less likely to be the thiols or disulfides transported from blood to the brain. Cysteine and CSSG have no A - V differences, suggesting no net uptake across the B B B within the CNS. The dipeptide CysGly was not detectable in blood and CSF, nor was it found in A C M in our in vitro studies. Fourth, the thiol/disulfide exchange reaction occurs in the CNS. Cysteine, cystine, GSH, and CSSG were all found in CSF. Cystine in CSF was low (0.09 ± 0.02 uM), and was detected only with 100 pi injector. This result is consistent with the data reported by Perry (1975) (0.1 ± 0.2 u M of human CSF). Several discrepancies exist in the study of thiol and disulfide transport across the B B B . Wade and Brady (1981) found that little [ S]-cystine is taken up by the brain 35  using a carotid injection technique. By contrast, Hwang et al. (1980) showed [ S]35  cystine uptake by isolated brain capillaries. My finding of cystine levels in plasma is not unique, as Felig et al. (1973) observed a similar positive arterial-jugular venous concentration difference of cystine (9.0 + 4.2 pM) in humans. For cysteine and GSH, my data from plasma levels suggest no substantial uptake of cysteine and GSH by the brain, in contrast with the observation of [ S]-cysteine uptake (Wade and Brady, 1981) and 35  [ S]-GSH uptake (Kannan et al., 1990). It is postulated that cysteine and GSH are 35  permeable across the B B B , and that the movement is bidirectional. GSH is also important in the y-glutamyl cycle. However, their net transport across the B B B contributes little to the thiol/disulfide pool of the CNS. 71  CHAPTER 4 ASTROCYTES PROVIDE CYSTEINE TO NEURONS  It should be mentioned that there exists a sharp gradient of GSH in different blood vessels. It has long been known that the liver is the major organ releasing GSH into blood (Anderson et al., 1980; Fariss and Reed, 1983). The hepatic vein plasma has the highest levels of G S H observed (26 uM) (Anderson et al., 1980). I found that G S H levels are very low in the carotid artery and internal jugular vein. The explanation for this could be that GSH released from the liver rapidly proceeds to the thiol/disulfide exchange reaction in blood. Due to its critical role in intracellular GSH synthesis, cysteine must be maintained at a stable level. However, cysteine is unstable and will be oxidized to cystine under aerobic conditions. Likewise, GSH and other thiols will also be oxidized to the corresponding disulfides. My experiments show that the stable levels of thiols in the brain are maintained by astrocytes. The choroid plexus might also play a role in the formation and recycling of GSH in the CSF (Anderson et al., 1989). Extensive studies have shown that astrocytes play diverse functions in maintaining the stable internal milieu of the brain. In this study, I show that astrocytes indirectly provide cysteine to neurons for GSH synthesis. These data indicate that astrocytes play a critical role in protecting neurons from oxidative stress.  72  Chapter 5 Cysteine Neurotoxicity and Copper  CHAPTER 5  N E U R O T O X I C I T Y INDUCED B Y C Y S T E I N E  AUTOXIDATION AND T H E C E N T R A L R O L E OF COPPER  I N T R O D U C T I O N  Though cysteine plays a pivotal role in regulating intracellular levels of GSH, its toxicity has long been noted, particularly to neurons. Extensive degenerative changes in the CNS are induced following subcutaneous injection of cysteine to newborn mice (Olney et al., 1972) and rats (Karlsen et al., 1981). Cysteine is also toxic to cultured neurons (Puka-Sundvall et al., 1995), hepatocytes (Saez et al., 1982), and kidney cell lines (Nath and Salahudeen, 1993). The underlying mechanisms of cysteine toxicity have been studied, and there are several theories. 1). Olney et al. (1990) found that N M D A antagonists can prevent cysteine toxicity, and that cysteine is a bicarbonate-sensitive excitotoxin. They suggest a direct toxicity of cysteine. 2). Some researchers reported that glutathione potentiates glutamate toxicity by modulating the redox site of the N M D A receptor-channel complex (Sucher and Lipton, 1991; Janaky et al., 1993; Regan and Guo, 1999). Like GSH, cysteine can also regulate redox status by participating in the thiol/disulfide exchange reaction, thus exerting an indirect toxic effect. 3). Cysteine autoxidation can generate free radicals, which are cytotoxic (Saez et al., 1982; Nath and Salahudeen, 1993). This last mechanism has not been studied in neurons. Because neurons are very vulnerable to oxidative stress, due to the high oxygen consumption of the brain, their high proportion of membrane polyunsaturated fatty acids, and the weak activities of their antioxidative enzymes (Makar et al., 1994), free radicals may play an important role in cysteine neurotoxicity in brain. In the present experiments, I explore the free radical mechanisms of cysteine toxicity. I investigate the influence of cysteine autoxidation, the generation of free radicals from cysteine autoxidation, and its neurotoxicity to cultured neurons.  R E S U L T S  73  Chapter 5 Cysteine Neurotoxicity and Copper  1. Cysteine Autoxidation in the Presence of Transition Metal Ions To study the factors determining cysteine autoxidation, I first investigated the autoxidation rate of cysteine in the following three solutions: Earle's balanced salt solution (EBSS), cystine-free M E M (MEM-CSSC"), and MEM-CSSC" with cultured neurons. EBSS contains the basal salts of M E M . The medium of the primary cortical neurons was replaced by MEM-CSSC" before testing. Only a small amount of cell debris was present in the cell cultures. A l l these solutions were incubated at 37°C and pH 7.4 in a humidified atmosphere of 5% C O 2 . Cysteine was added to a final concentration of 100 p M . Cysteine and cystine were measured by HPLC at 0, 5, 15, 30, 60, and 120 min after cysteine was added to the three solutions. The results showed that cysteine was gradually oxidized to cystine. Cysteine concentrations in MEM-CSSC" and neuronal culture medium had no significant differences from the corresponding values in EBSS (p > 0.05) (Fig. 5-1 A). This result demonstrates that the components of culture medium and cultured cells have no obvious influence on cysteine autoxidation. Transition metal ions, such as F e  3+  or C u , are considered to catalyze the oxidation 2+  of reducing agents, including thiol-containing compounds (Halliwell and Gutteridge, 1985). The effects of F e  3+  and C u  2 +  on cysteine autoxidation were measured under the  same conditions as described above. Fe , at concentrations of 0.1, 1.0, and 10 uM, had 3+  no obvious effects on the autoxidation rate of cysteine, as assessed by comparing cysteine concentrations with the corresponding value in EBSS (p > 0.05) (Fig. 5-IB). There were also no obvious differences among the three different concentrations (p > 0.05). Our results showed that C u  2 +  substantially accelerated the autoxidation rate of cysteine at  concentrations between 0.1 to 10 u M (Fig. 5-1C). The efficacy of submicromolar levels of C u  2 +  in catalyzing cysteine autoxidation has significant physiological meaning as these  are well within the physiological range. The concentrations of loosely bound copper in human CSF have been reported to be in the range of 0.13 to 0.75 p M (Gutteridge, 1984). I compared the effects of variable oxidation valences of iron (Fe and Fe ) and 2+  3+  copper (Cu and Cu ) on cysteine autoxidation. The effects of hemin, the low+  2+  molecular-weight complex of Fe , and some other biologically important transition 3+  metal ions, including M n , C r , and Z n , on cysteine autoxidation were also studied. 2 +  3+  2+  As shown in Fig. 5-2, cysteine autoxidation with F e  2+  was faster than that with F e  3+  at  74  Chapter 5 Cysteine Neurotoxicity and Copper  concentrations of 100 and 200 u M , but was not significantly different at concentrations of 1 and 10 u M . The effects of C u and C u +  2+  on cysteine autoxidation were not  significantly different at concentrations of 0.01 to 1 u M . Hemin was more efficient in catalyzing cysteine autoxidation than F e and Fe , but still much less efficient than C u 2+  3+  +  and C u . The concentration for hemin to catalyze the half oxidation of 100 u M cysteine 2+  in 60 min was -10 u M , while the concentration for C u and C u +  0.2 uM, about a 50- to 100-fold difference. M n  2 +  2 +  was between 0.1 and  was efficient at concentrations of 100 Figure 5-2. Comparisons of cysteine autoxidation in the presence of several transition metal ions. A series of concentrations of FeSC»4,  FeCl , hemin, M n S 0 , C r C l , ZnCl , CuCl, and C u S 0 , were reacted with 100 u M cysteine respectively. The control was 100 u M cysteine without transition metal ions. The reaction mixtures were incubated in PBS at pH 7.4 and 37°C in a humidified 100% air. Cysteine concentrations were determined with Ellman's reagent after 60 min. A . Cysteine autoxidation with Fe , F e (1 to 200 uM), and hemin (1 to 100 uM). B . Cysteine autoxidation with M n , C r , and Z n (1 to 200 uM). C. Cysteine autoxidation with C u and C u (0.01 to 1 uM). Data are the mean ± S E M of three independent experiments in duplicate. *p<0.0\, significantly different from the control. p<0.0\ vs. corresponding values of Fe3+ groups. 3  4  3  2  1  100  10  1000  Fe(ll), Fe(lll), H e m i n ( M ) ( l  2+  1  100  10  Mn(ll), Cr(lll), Zn(ll) (nM) 120  1000  2 +  4  3+  3+  2+  +  2+  ab  J  0.01  0.1  Cu(l), Cu(ll) ^ M )  76  Chapter 5 Cysteine Neurotoxicity and Copper  Figure 5-3. Cysteine autoxidation rates in CSF and comparisons with those in Cu supplemented solutions. CSF was taken from 3-month-old rats as described in Methods. Cysteine was added to the CSF with a final concentration of 100 p M . The CSF was incubated under conditions of pH 7.4 and 37°C in a humidified atmosphere of 5% CO2 and 95% air. Samples were taken at several time points for H P L C assays. A . Time course of the concentrations of cysteine and related compounds in the CSF during cysteine autoxidation. B. Comparison of cysteine autoxidation in CSF and in C u supplemented solutions. C u was prepared in EBSS at concentrations of 0.1 u M , 0.2 u M and 0.3 p.M, and incubated under the same conditions as the CSF. Data are the mean ± S E M of three independent experiments in duplicate. CSSC, cystine; CSSG, cysteine-glutathione disulfide. 2+  2 +  and 200 p M . No catalyzing effects of C r  J+  and Z n  2+  were observed at concentrations  between 1 to 200 u M .  2. Cysteine Autoxidation in C S F Cysteine autoxidation in CSF was examined. As described in the methods, cysteine was added to rat CSF to a concentration of 100 uM. The samples were taken for test at 15, 30, 60, and 120 min after this addition. The results showed that cysteine was oxidized at a moderate rate in the CSF (Fig. 5-3 A). Most of cysteine was converted to cystine (-80%). The other small portion of cysteine participated in the thiol/disulfide 77  Chapter 5 Cysteine Neurotoxicity and Copper  CSH (pM) Figure 5-4. Neurotoxic effects of cysteine in the presence of copper. Primary cortical neurons were cultured in serum-free M E M . Cysteine was added at concentrations as indicated in the presence of 0, 0.2, and 1.0 u M C u . Neuronal viability was estimated 24 hr later using the M T T assay. Results are expressed as the percentage of surviving neurons compared with control cultures (without addition of cysteine and Cu ). Data represent the mean ± S E M of three independent experiments in triplicate. 2+  2+  exchange reaction and formed mixed disulfides, such as cysteine-glutathione disulfide and cysteine-protein disulfides. I compared cysteine autoxidation in CSF and in Cu supplemented solutions (Fig. 5-3B). The autoxidation rate of cysteine in CSF was roughly equal to that observed in solutions of 0.2 to 0.3 u M C u . The kinetics of 2+  cysteine autoxidation in Cu  solution and in CSF, however, was different. Cysteine  autoxidation in Cu -supplemented solution was a zero-order reaction, and that in CSF 2+  was a first-order reaction (Fig. 5-3B). In the discussion I propose that copper is likely the major catalyst in CSF for cysteine autoxidation. I chose 0.2 u M C u  2 +  to mimic the  cysteine autoxidation in CSF in the following toxicity experiments.  3. Neurotoxic Effects of Cysteine Cysteine neurotoxicity was investigated in the cell culture system. The effects of the autoxidation rate of cysteine, the amount of cysteine being oxidized, as well as cysteine concentrations on cysteine neurotoxicity were observed. In Fig. 5-4, primary neurons were cultured in the presence of a series of concentrations of cysteine (0, 10, 20, 50, 100, 200, 500, 1000, and 2000 pM) and in the absence or presence of copper (0, 0.2, and 1.0  78  Chapter 5 Cysteine Neurotoxicity and Copper  uM) in 2 ml culture medium (MEM). Neurotoxicity was estimated 24 hr later by the M T T assay. The role of copper in this experiment is to modulate the autoxidation rate of cysteine. Without addition of cysteine, copper itself had no visible effect on neuronal survival. The neuronal survivals in the presence of copper and absence of cysteine were 99.6 ± 11.3% (mean ± S E M % , C u  2 +  - 0.2 uM) and 105 ± 5% ( C u = 1.0 uM), no 2+  significant differences from the control 100 ± 1.5% ( C u = 0) (p > 0.05). Without C u , 2+  2+  cysteine was toxic only at relatively high concentrations (EC o -600 uM). With addition 5  of C u , the toxic concentrations of cysteine decreased (EC50 -30 u M at 0.2 u M C u 2+  EC50  2 +  and  -12 u M at 1.0 u M Cu ). This result demonstrates that cysteine toxicity is closely 2+  related to its autoxidation rate. The effect of the total amounts of cysteine being oxidized on its toxicity was further investigated. C u  2 +  (0.2 pM) was added for the purpose of catalyzing cysteine  autoxidation. Cysteine (10 uM) was added to the culture medium either once, 5, or 10 times at a rate of once every 5 min, respectively. 10 u M cysteine was completely 120 & ^  100 2  80  O  60  O  40  "~  20  0 1 2 3 4 5  Tim e (m in)  1 Cu * 2  CSH  Control  1x  0.2uM  0.2nM 1x10nM  1  5x  1  10x  0.2nM  0.2nM  5x10nM  10x10nM  Figure 5-5. Effect of total amount of cysteine being oxidized on neuronal survival. Primary cortical neurons were cultured in serum-free M E M . The concentration of C u was 0.2 u M . 10 u M of cysteine was added each time, and was added once or repetitively every 5 min for a total of 5 and 10 times. 10 p M of cysteine was completely oxidized within 5 min in the presence of 0.2 u M C u (upper right figure). Neuronal viability was estimated 24 hr later using the M T T assay. Results are expressed as the percentage of surviving neurons compared with control cultures (without addition of cysteine). Data represent the mean ± S E M of three independent experiments in triplicate. * p < 0.01, significantly different from the control.  2 +  2 +  79  Chapter 5 Cysteine Neurotoxicity and Copper  oxidized in 5 min (Fig. 5-5). While a single dose of cysteine showed no obvious toxicity (91 ± 5.1% of viability), multiple additions of cysteine substantially increased cysteine toxicity. Neuronal survival was 91.5 ± 5.1% (mean ± SEM%,/? > 0.05 vs. control) with addition of 10 u M cysteine once, and decreased to 28.9 ± 4.4% (p < 0.01 vs. control) with 5 additions of 10 u M cysteine, and 12.6 ± 1.5% (p < 0.01 vs. control) with 10 applications. Note the repeated addition increased the total amounts of cysteine being oxidized, while the maximal concentration of cysteine never exceeded 10 u M at any one time. These results demonstrate that cysteine toxicity is closely related with the total amount of cysteine being oxidized. Taken together, these data suggest that cysteine autoxidation, rather than cysteine itself, is responsible for cysteine toxicity.  4. Generation of Hydroxyl Radicals During Cysteine Autoxidation Using coumarin-3-carboxylic acid (CCA) as a probe, I measured the production of •OH generated from cysteine autoxidation. The hydroxyl radical reacts with C C A to generate 7-hydroxycoumarin-3-carboxylic acid (7-OHCCA). Cysteine in PBS was incubated at 37°C and pH 7.4 for 4 hr. As shown in Fig. 5-6A, little -OH was produced from cysteine autoxidation without the presence o f C u . In the presence of 0.2 u M C u , 2+  2+  hydroxyl radicals were generated in substantial amounts during cysteine autoxidation, and the amount of O H increased with cysteine concentration. I also compared the generation of O H with some other thiols. Glutathione, N-acetyl-cysteine, homocysteine, dithiothreitol, and 2-mercaptoethanol, can all generate -OH with C u  2 +  as a catalyst to  variable degrees (Fig. 5-6B). Disulfides (cystine and glutathione disulfide) and sulphurcontaining amino acid (cysteic acid) did not generate -OH.  DISCUSSION The biological role of cysteine is double-edged. It is a very important amino acid for the synthesis of glutathione, which is the major cellular antioxidant. Neurons, in particular, prefer to take up cysteine, rather than cystine, to synthesize glutathione  80  Chapter 5 Cysteine Neurotoxicity and Copper  (Kranich et al., 1996). On the other hand, cysteine has been found to be cytotoxic. Subcutaneous injection of cysteine in newborn mice (1 mg/g weight) (Olney et al., 1972) and rats (1.2 mg/g weight) (Karlsen et al., 1981) caused extensive neuronal death. Cysteine was toxic to cultured hepatocytes (4 mM) (Saez et al., 1982), kidney cell lines (4 mM) (Nath and Salahudeen, 1993), and primary neurons (1 mM) (Puka-Sundvall et al., 1995). In my neuronal cultures, 1 m M cysteine decreased neuronal survival to 30.5% (EC50  -600 uM) in the absence of copper (Fig. 5-4), similar to other findings (Puka-  Sundvall et al., 1995). Importantly, my results showed that cysteine toxicity was greatly increased in the presence of even submicromolar levels of copper, while copper itself was 0.04  Cu 0 Cu 0.2  50  100  CSH  150  11M  200  (|iM)  0.03  3  0.02  <  o o X  0.01  J  o I  r-  0  B Figure 5-6. Generation of hydroxyl radical ( O H ) by the autoxidation of cysteine and other thiols. Cysteine and related compounds were incubated in PBS under the conditions of pH 7.4 and 37°C in a humidified atmosphere of 100% air. C C A (1 mM) was added to react with the generated -OH, producing 7-OHCCA. Fluorescence was measured 4 hr after the reaction began. A . Cysteine, at concentrations of 0 to 200 u M , was incubated in the presence or absence of 0.2 u M C u . B. Generation of -OH from the autoxidation of thiols. Thiols, disulfides and sulphur-containing amino acid (100 u M of each) were incubated with 0.2 u M C u . The thiols include cysteine (CSH), glutathione (GSH), N-acetyl-cysteine (NAC), homocysteine (HSH), dithiothreitol (DTT) and 2-mercaptoethanol (2-ME). The disulfides include cystine (CSSC) and glutathione disulfide (GSSG). The sulphur-containing amino acid is cysteic acid (CA). Data represent the mean ± S E M of three independent experiments in duplicate. 2+  2+  81  Chapter 5 Cysteine Neurotoxicity and Copper  non-cytotoxic without cysteine (Fig. 5-4). Copper appears to function as a catalyst to accelerate cysteine autoxidation. Cysteine can be oxidized to form cystine, and donate an electron to an oxidant. O2 generally acts as an oxidant, accepting electrons one by one to form reactive oxygen species: superoxide anion (-02"), hydrogen peroxide  and hydroxyl radical ( O H ) .  (H2O2),  However, the electron transfer from cysteine to oxygen requires transition metal ions (Halliwell and Gutteridge, 1985), which can act as the electron carriers to catalyze cysteine oxidation. Our experiment showed that cysteine was oxidized to cystine very slowly in three kinds of solutions, including basal salt solution, culture medium and neuronal culture medium (Fig. 5-2A). Iron is generally considered the major transition metal ion in mediating the production of free radicals. My experiments show that copper is much more efficient in catalyzing cysteine autoxidation than iron and some other transition metal ions, such as manganese, chromium, and zinc. Variable valences of copper do not influence its catalyzing efficacy. For iron, the soluble low-molecular-weight F e more efficient in catalyzing cysteine autoxidation than F e  2+  3+  complex, hemin, is  and Fe . Whether the 3+  solubility causes the differences of catalyzing capacities of these iron forms is unknown. While FeCl is soluble in d H 0 (74.4 g/lOOml), it is insoluble in buffered salt solution as 2  3  dictated by the low solubility (K = 3.16 x 10" ) of Fe(OH) formed at neutral pH. M y 38  sp  3  FeCl stock solution was prepared in d H 2 0 . When FeCl is added into the final solution, 3  3  the availability of FeCl in the reaction mixture is accurate, though FeCl will form 3  deposits in the reaction solution.  3  FeS0  4  is soluble at neutral pH in the testing  concentration range. However, the iron oxidation status changes between F e  2+  and F e  3+  in iron-catalyzed cysteine autoxidation. In addition, F e will be oxidized to F e in 2+  aerobic conditions. Therefore, though  FeS0  4  3+  is dissolved in the reaction solution  completely at first, it gradually forms deposits. This could explain why the catalyzing efficacy of F e S 0 is just moderately higher than that of FeCl . The insolubility could be 3  4  the factor hampering the catalyzing effect of both  FeS0  4  and FeCl . However, even the 3  catalyzing efficacy of hemin is still much lower than copper. M y finding that copper is a more potent catalyst than iron is not unique. It has been reported that copper is more  82  Chapter 5 Cysteine Neurotoxicity and Copper  efficient than iron in mediating paraquat toxicity (Chevion, 1988), and in catalyzing dialuric acid autoxidation (Munday, 1988). My data show that cysteine was oxidized at a moderate rate in CSF. The autoxidation rate of 100 u M cysteine in CSF is equivalent to that in the presence of-0.2 u M Cu (Fig. 5-3). According to different authors, total copper concentrations in human CSF are in the range of 0.22 to 1.7 u M (14.2 to 109 ug/1) (Kapaki et al., 1997; Jimenez-Jimenez et a l , 1998; Joergstuerenburg et al., 1999; Stuerenburg, 2000). CSF also contains some other physiologically important transition metal ions, such as iron, manganese, chromium, and zinc. Their concentrations in human CSF are 1.1 to 3.8 u M for Fe (Jimenez-Jimenez et al., 1998; LeVine et al., 1998), 12 to 60 n M for M n (D'Amico and Klawans, 1976; Kapaki et al., 1997; Jimenez-Jimenez et al., 1998), 0.28 p M for Cr (Aguilar et al., 1998), and 0.16 to 2.6 u M for Zn (Palm and Hallmans, 1982; Kapaki et al., 1997; Jimenez-Jimenez et al., 1998). The binding status of copper in CSF is of particular importance with regard to its catalyzing effect, because only protein-unbound or loosely-bound copper, which can form a low-molecular-weight complex with cysteine, can catalyze cysteine autoxidation (Halliwell and Gutteridge, 1985). The concentration of loosely bound copper detected in human CSF, ranges from 0.13 to 0.75 p M (Gutteridge, 1984). By comparing the efficient catalyzing concentrations of these transition metal ions and their physiological ranges, I hypothesize that copper is the major determinant influencing cysteine autoxidation rate in CSF. The kinetics of cysteine autoxidation in CSF was a first-order curve, different from the zero-order reaction in copper-supplemented solutions. The first-order kinetics may reflect the regulation of copper chelating status. It is quite possible that protein-copper binding is modulated by the redox status of the CSF. The mechanism of copper-catalyzed cysteine autoxidation has been extensively investigated (Cavallini et al., 1969; Munday, 1989; Kachur et al., 1999). It has been suggested that the intermediate cysteine-Cu complex is initially formed in a 2:1 ratio. Cysteine, as well as other low-molecular-weight thiol compounds, can donate electrons via catalysts. 0 generally acts as an oxidant, accepting electrons one by one to generate 2  reactive oxygen species: 0 ", H 0 , and OH. 2  2  2  83  Chapter 5 Cysteine Neurotoxicity and Copper  Like cysteine, other low-molecular-weight thiol compounds, including glutathione, N-acetyl-cysteine, homocysteine, etc., can generate -OH during copper-catalyzed autoxidation (Fig. 5-6B). In CSF, cysteine (1.12 ± 0.14 pM) and glutathione (5.87 ± 0.29 uM) are the major low-molecular-weight thiols. The constant autoxidation of these thiols will place neurons in a situation of oxidative stress if no mechanisms exist to remove the generated oxygen radicals. The hydroxyl radical, which has an extremely short half-life 10" s (Pryor, 1986), is the major damaging radical. Once O H is produced, it rapidly 9  attacks poly-unsaturated fatty acids to initiate the chain reaction of lipid peroxidation, as well as D N A , proteins and carbohydrates. H2O2 itself is stable and non-toxic, and is the one-step precursor of OH. Eliminating H2O2 can therefore block the formation of -OH. In the following experiments, I will explore the in vivo mechanisms preventing the cysteine neurotoxicity.  84  Chapter 6 Neuroprotective Effects of Pyruvate  CHAPTER 6  NEUROPROTECTIVE EFFECTS OF PYRUVATE ON  CYSTEINE TOXICITY AND T H E RELEASE OF PYRUVATE BY ASTROCYTES  INTRODUCTION  The above data show that the extracellular fluids of the CNS contain a certain level of cysteine, and cysteine will constantly generate reactive oxygen intermediates via an autoxidation process. Therefore, the organism must have developed certain mechanisms to remove the free radicals produced. H2O2 itself is stable and non-toxic, and is the onestep precursor of O H . Eliminating H2O2 can therefore block the formation of -OH. Though cytoplasmic enzymes, such as GPx and catalase, can eliminate membranepermeable H2O2 intracellularly, H202-permeating the cell membrane itself is risky for cells. In addition, extracellularly generated H2O2 will be reduced to O H i f it is not scavenged immediately. Therefore, it will be more efficient and beneficial i f extracellularly derived H2O2 can be eliminated in situ in the extracellular space. In this chapter, I will explore two possible extracellular protective mechanisms: an enzymatic mechanism and a small molecule antioxidant mechanism. Of the two enzymes, which can catalyze the degradation of H2O2, catalase is known to be located in subcellular particles known as peroxisomes (Houdou et al., 1991). High activities of catalase are present in liver, kidney and erythrocytes but there is very little in brain (Halliwell and Gutteridge, 1985). Glutathione peroxidase (GPx), however, exists ubiquitously in cytoplasm of all cell types. The secreted forms of GPx have also been found in plasma (Avissar et al., 1989) and lung extracellular fluid (Avissar et al., 1996). Here, I will investigate whether astrocytes can secrete catalase and GPx. Pyruvate, as well as other a-ketoacids, can react with H2O2 nonenzymatically, being converted to carbon dioxide and the carboxylic acid with one less carbon: R - C O C O O H + H 0 -> R-COOH + H 0 + C 0 (Holleman, 1904; Bunton, 1949). Removing H 0 will 2  2  2  2  2  2  prevent the formation of OH, which is the major damaging radical. The protective effects of pyruvate against oxidative stress in biological systems have been reported 85  Chapter 6 Neuroprotective Effects of Pyruvate  (O'Donnell-Tormey et al., 1987; Desagher et al., 1997; Giandomenico et al., 1997). Pyruvate, and the enzymatic activities of glutathione peroxidase and catalase were examined in A C M and in the CSF.  RESULTS 1. Effects of Catalase and Pyruvate on Cysteine Toxicity When cysteine is oxidized, 0 accepts electrons one by one, and reactive oxygen 2  intermediates are produced. The hydroxyl radical is the major reactive oxygen species to cause tissue damage, and is generated from H2O2. It is therefore reasonable to expect that catalase, which decomposes H2O2, could prevent -OH generation from cysteine autoxidation, and thus reduce cysteine toxicity. Likewise, pyruvate would also be expected to have the same effect owing to its reactivity with H2O2. The effects of catalase and pyruvate on cysteine neurotoxicity were tested in our primary neuronal cultures. In the presence of C u  2 +  (0.2 uM), cysteine (100 uM) was toxic to neurons (16.8  ± 2.8% of viability) (Fig. 6-1 A). Addition of catalase (10 units/ml) or pyruvate (1 mM) completely prevented cysteine toxicity, with 101 ± 4 and 104 ± 5% of viability, respectively. Pyruvate and lactate are both glucose metabolites, and important energy suppliers to neurons (Selak et al., 1985; Pellerin and Magistretti, 1994; Tsacopoulos and Magistretti, 1996). To demonstrate that the preventive effect of pyruvate against cysteine toxicity is due to its specific reactivity with H20 , other than its energy supplying effect, 2  sodium lactate was used as control. The results showed that lactate could not prevent cysteine neurotoxicity (Fig. 6-1 A). The protective capacity of pyruvate was dosedependant. In the presence of 0.2 u M C u  2+  and 50 or 100 u M cysteine, increasing  concentrations of pyruvate (up to 1 mM) produced a progressive enhancement of neuronal protection (Fig. 6-IB).  2. Effects of Catalase and Pyruvate on Generation of Hydroxyl Radicals Owing to their ability to remove H2O2, catalase and pyruvate are expected to prevent the formation of -OH generated from cysteine autoxidation. In the presence of 0.2 u M Cu  2 +  and 100 u M cysteine, the generation of -OH was measured in PBS at 37°C and pH  86  Chapter 6 Neuroprotective Effects of Pyruvate  120 -,  o  .2 >  +*  co ra u c o o  3 Q> Z  100 80 60 40 20 0-  i  u  Cu  2 +  i  I  Contr  CSH  Cat  Pyr  Lac  +  +  +  +  +  +  +  +  +  -  Cat  Pyr  0.2uM  CSH100uM Other addition  -  Lac  CSH 0 •CSH 50nM • CSH 100nM  B  o  1§  ra « o ° «-  s=  a> Z 100 Pyruvate  1000  (\xM)  Figure 6-1. The protective effects of catalase and pyruvate on cysteine neurotoxicity. Primary cortical neurons were cultured in serum-free M E M . Cysteine toxicity was induced by addition of 100 u M cysteine and 0.2 u M C u . Neuronal viability was estimated 24 hr later using the M T T assay. Results are expressed as the percentage of surviving neurons compared with control cultures (without addition of cysteine). A . Catalase (10 U/ml), pyruvate (1 mM) and lactate (1 mM) were added immediately before addition of cysteine and C u . Data represent the mean ± S E M of three independent experiments in triplicate. B . Dose-response curve illustrating the neuroprotective effect of pyruvate. Cysteine concentrations were 0, 50, and 100 u M , respectively. Data represent the mean ± S E M of three independent experiments in duplicate. * p < 0.01, significantly different from the control. 2+  2+  87  Chapter 6 Neuroprotective Effects of Pyruvate  7.4. Catalase and pyruvate substantially decreased -OH production, while lactate did not (Fig. 6-2A). Lactate even increased the OH production, and the reason for this is unknown. The inhibitory effect of pyruvate on OH production from cysteine autoxidation was dose-dependent (Fig. 6-2B).  3. Pyruvate Released by Astrocytes I explored two potential extracellular antioxidative mechanisms, which may prevent the toxic effects of cysteine autoxidation: (1) enzymatic mechanisms and (2) small molecule antioxidant mechanisms. For the former, I tested the activities of glutathione peroxidase (GPx) and catalase in A C M and CSF. The concentrated A C M prepared from 0.04  CSH  Cat  Pyr  Lac  0.03  s  0.02  X  0.01  < o o  o I  0.1  B  10  1  Pyruvate (mM)  Figure 6-2. Effects of catalase and pyruvate on the generation of -OH from cysteine autoxidation. Cysteine (100 uM) and C u (0.2 uM) were incubated in PBS under the conditions of pH 7.4 and 37°C in a humidified atmosphere of 100% air. C C A (1 mM) was added to react with the generated OH, producing 7-OHCCA. The fluorescence was measured 4 hr after the reaction. Data represent the mean ± S E M of three independent experiments in duplicate. A . Catalase (10 U/ml), pyruvate (1 mM), and lactate (1 mM) were added immediately before addition of cysteine and C u . B. Pyruvate, at concentrations of 0, 0.1, 1, and 10 m M , was added immediately before addition of cysteine and C u . *,**/?< 0.01, significantly different from the control (cysteine only). 2 +  2+  2+  Chapter 6 Neuroprotective Effects of Pyruvate  primary confluent astrocyte cultures was used as described in the Methods. CSF was taken from 3-month-old rats. The results showed that the activities of the two enzymes were not detected in A C M and CSF. GPx activity of A C M was 1.6 ± 0.2 mU/ml (mean ± S E M , n = 6), not significantly different from the negative control (1.9 ± 0.4 mU/ml, n = 6, p > 0.05). GPx activity of CSF was 2.2 ± 0.2 mU/ml (n = 6), not significantly different from the negative control (1.7 ± 0.2 mU/ml, n = 6, p > 0.05). Catalase activity was not detected in A C M and CSF (< 5 mU/ml, n = 6, respectively). As pyruvate can scavenge  H2O2,  it may act as an extracellular antioxidant in vivo.  Pyruvate concentration was therefore assayed in A C M , N C M , and CSF. Relatively high levels of pyruvate were found in A C M (254 ± 15 uM) and in CSF (131 ± 9 pM). Pyruvate was also detected in N C M at relatively low concentrations (45.6 ± 4.4 uM) (Table 6-1), suggesting that pyruvate pool of the extracellular space and CSF was mainly contributed by astrocytes. The time course of pyruvate release by astrocytes was also measured (Fig. 6-3). In the A C M made from pyruvate-free medium, pyruvate concentration increased rapidly within 12 hr, reached a peak at 24 hr.  Table 6-1. Pyruvate content in A C M , N C M , and CSF Pyruvate concentrations (juM)  A C M (n = 6) N C M (n = 6) CSF (n = 6)  Mean ± S E M  Range  254 ± 15  184-297  45.6 ± 4 . 4 131 ± 9  36.9-62.3 98.9-145  ACM was prepared from confluent astrocyte cultures, and NCM was from 7 D1V neuronal cultures in serum-free MEM. The conditioned media were collected 24 hr later for pyruvate assay. CSF was obtained from 3-month-old male rats.  4. Effects of Cysteine Provision on Neuronal Survival As cysteine is critical for GSH synthesis, it is essential to inhibit the neurotoxic effect of cysteine in order to exert its beneficial effects. I observed the effect of cysteine provision on neuronal survival in pyruvate-containing C D - M E M . Cysteine concentration was 20 u.M, and was added every 24 hr from the beginning of neuronal cultures. The 89  Chapter 6 Neuroprotective Effects of Pyruvate  300  Time (hr)  Figure 6-3. Time course of pyruvate release by astrocytes. Confluent astrocytes in flasks were rinsed with Hank's solution. The serum-free M E M was added at a concentration of 1.33 ml per 1 x 10 cells. Samples of the A C M were taken at 0, 6, 12, 24, and 48 hr and used for pyruvate assays. Data represent the mean ± S E M of three independent experiments in triplicate. 6  control was C D - M E M without cysteine. As shown in Fig. 6-4, addition of cysteine clearly increased the survival of cultured neurons as compared with controls. This beneficial effect of cysteine supplement, however, is still less efficient than that of astrocyte feeder layer, suggesting that neurosupportive mechanisms of astrocytes are much more complicated.  DISCUSSION  I have explored two possible extracellular H202-eliminating mechanisms, including enzymatic mechanisms and small molecule antioxidant mechanisms. The activities of the two most plausible antioxidative enzymes, GPx and catalase, were not detected in A C M or CSF. Pyruvate is generally considered as an energy substrate for cultured neurons, though its trophic effect on neuronal survival has long been reported (Selak et al., 1985; Katoh-Semba et al., 1988; Izumi et al., 1994; Matsumoto et al., 1994). Recently, pyruvate was reported to protect neurons (Desagher et al., 1997) or cell lines (Giandomenico et al., 1997) from H 0 -induced toxicity. I found that pyruvate, as well 2  2  as catalase, can inhibit the production of -OH generated from cysteine autoxidation, by removing H 0 . The neuroprotective effect of pyruvate cannot be attributed to its role in 2  2  90  Chapter 6 Neuroprotective Effects of Pyruvate  pyruvate from astrocytes to the extracellular pool, then to neurons. In agreement with this, there are reports showing that astrocytes release pyruvate (Pellerin and Magistretti, 1994), and neurons can utilize pyruvate for their function recovery or survival (Selak et al., 1985; Matsumoto et al., 1994; Yoshioka et al., 2000). Given the evidence above, it is suggested that the extracellular pool of pyruvate is supplied mainly by astrocytes rather than neurons. It is still unknown whether other brain cells such as choroidal epithelial cells or ependymocytes contribute to the pyruvate pool. Another possible source of pyruvate is from blood. Pyruvate can be transported through the blood-brain barrier by the monocarboxylate transporter (Pardridge and Oldendorf, 1977; Miller and Oldendorf, 1986). However, the arterio-venous difference of pyruvate across the brain of adult fed rats was found to be negligible (arterial blood, 141 ± 14 u M ; sinus blood, 138 ± 14 uM), whereas the arterio-venous differences for glucose was remarkable (510 ± 50 uM) (Hawkins et al., 1971). It suggests that though pyruvate is permeable across the blood-brain barrier, the net transport from blood to brain contribute little to the pyruvate pool of the CNS under normal conditions. Instead, the brain utilizes glucose and produces pyruvate on its own. Taken together, these findings lead to the set of mechanisms outlined in Fig. 6-5 as those underlying the neurosupportive effect of astrocytes against cysteine toxicity. It should be noted that cysteine and some other reducing agents play a dual role. They generate reactive oxygen species when they are oxidized under the catalysis of transition metal ions, functioning as prooxidants. Meanwhile, they can donate electrons to electrophilic substances, such as 02~ and OH, functioning as antioxidants. Due to the extremely short half-life of OH, these reducing agents cannot completely prevent the damage induced by -OH. That may explain why cysteine itself cannot prevent the toxicity induced by its autoxidation. Pyruvate, however, is not a reducing agent. It functions only as an antioxidant, but not prooxidant, by reacting with H2O2. From this point of view, pyruvate is a special and indispensable antioxidant of the CNS. It needs to be noted that though addition of cysteine can improve neuronal survival when neurotoxicity is prevented, this neurosupportive effect of cysteine addition is still far less than that of astrocyte feeder layer. Two reasons are considered: 1). Due to the limit of our techniques, it is impossible to keep a stable low level of cysteine by adding  92  Chapter 6 Neuroprotective Effects of Pyruvate  Figure 6-5. Diagram of the proposed mechanism of protection by astrocytes in preventing cysteine toxicity catalyzed by copper. Astrocytes release glutathione and indirectly produce cysteine in the extracellular fluid of the CNS. Cysteine, as well as glutathione or other thiols, will be oxidized to disulfide under the catalysis of proteinunbound or loosely bound copper. Molecular oxygen, as the oxidant, accepts electrons step by step to produce superoxide radicals, hydrogen peroxide and hydroxyl radicals. The latter is the major damaging free radical to the cells. In parallel, astrocytes also release pyruvate, which can react with hydrogen peroxide, preventing the formation of hydroxyl radicals. cysteine to the culture dishes. Addition of a high dose of cysteine and the rapid autoxidation will produce a fluctuation of cysteine concentration. Considering the complex effects of cysteine, this difference may affect neuronal survival. 2). More likely, the neurosupportive effect of astrocytes must reflect a synergistic action of multiple factors. The antioxidative effect of astrocytes described here via small bioactive molecules may represent only one of the several mechanisms. Other factors, such as neurotrophins, hormones, energy substrates, uptake of excitatory amino acids, and regulation of pH and ions, may all contribute to the neurosupportive effect of astrocytes. Each factor may exert different influences according to special circumstances.  93  Chapter 7 General Discussion & Conclusions  C H A P T E R 7  G E N E R A L DISCUSSION A N D CONCLUSIONS  G E N E R A L DISCUSSION  I have studied the neuroprotective effect of astrocytes from the following perspectives: a) the neurosupportive effect of astrocytes in a co-culture system, b) the mechanism of thiol maintenance in the CNS, c) the effect of cysteine neurotoxicity on cultured neurons and its mechanism, d) the in vivo mechanism that protect against cysteine toxicity. The experimental evidence has shown astrocytes can release small molecules that are able to protect neurons from oxidative stress. As this area has not been extensively investigated, some important questions have to be clarified.  1. Thiol Sources in the CNS It has been found that cysteine is the rate-limiting precursor of GSH synthesis, and that extracellular cysteine regulates neuronal GSH levels (Beutler, 1989; Meister, 1989; Kranich et al., 1996). Cysteine, unlike other amino acids, is unstable and will be oxidized under aerobic conditions. The mechanisms by which the cysteine's level is maintained in the CNS have been explored by early researchers, as discussed in section 6 of Introduction. My results, consistent with most other reports, suggest that astrocytes release GSH, but not cysteine. However, there is no agreement so far on the functions of the released GSH. Sagara et al. (1996) found that neurons do not take up GSH directly, whereas they did not explain the physiological meaning of GSH efflux. As regard to the y-GT hypothesis proposed by Dringen et al. (1999a), it is not supported by several critical experiments, as discussed in Discussion of Chapter 4. My present results suggest a mechanism of thiol/disulfide exchange. Evidence from the analysis of conditioned medium and CSF supports the hypothesis that GSH released by astrocytes constantly converts cystine to cysteine and keeps cysteine at a stable level. It is conceivable that the functions of the released GSH are not limited to the maintenance of cysteine. GSH is also able to regulate the redox state of many proteins, including, for example, the N M D A receptor, and to maintain a stable redox potential of the CNS. From a broad point of  94  Chapter 7 General Discussion & Conclusions  view, GSH released by astrocytes can be considered as a source of reducing power in the CNS. My results suggest that the ultimate thiol/disulfide source of the brain is cystine, taken from blood. The present data show that plasma cystine concentration is much higher than the concentrations of cysteine and GSH (Table 4-2). There is also an A - V difference of cystine concentration, but not of cysteine and GSH. This demonstrates that the brain does not depend on blood as a thiol provider. Instead, the brain depends on astrocytes to provide thiols from disulfide sources and to regulate thiol/disulfide ratios. The brain, by this mechanism, can avoid the influence of fluctuations in the blood nutrient concentrations on its microenvironment, and maintain stable thiol levels and redox state in the CNS.  2. Mechanisms of Cysteine Toxicity Cysteine neurotoxicity has been reported many years ago (Olney and Ho, 1970). So far, there exist at least four theories about cysteine toxicity, as discussed in section 7 of Introduction: a) excitotoxic metabolites of cysteine, b) bicarbonate-dependent excitotoxicity, c) redox modulation of the N M D A receptors, d) free radical toxicity. M y experiments have investigated the last theory in cultured neurons. A l l the other three theories are related to excitotoxicity. First, CSA and C A are metabolites of cysteine autoxidation and are NMDAmimetic. However, my experiments show that the major oxidized product of cysteine is cystine (> 98%), rather than CSA and C A . The results from other researchers show that the distribution of brain injury caused by cysteine administration is quite different from that caused by C S A (Lehmann et al., 1993), and that cysteine is about 10-fold more toxic to neurons than C S A and C A (Pean et al., 1995). A l l these data do not support the hypothesis that CSA and C A mediate cysteine toxicity. Second, so far there is no evidence that proves the existence of a hydrogen bond between cysteine and bicarbonate. The bicarbonate-dependent excitotoxicity theory therefore cannot be verified. Third, it has been found that the reducing agents can sensitize N M D A receptors, i.e. increasing glutamate-induced response (Aizenman et al., 1989; Janaky et al., 1993; Kohr et al., 1994; Regan and Guo, 1999; Choi and Lipton, 2000). Cysteine, like GSH and other  95  Chapter 7 General Discussion & Conclusions  reducing agents, is assumed to have a similar effect on the N M D A receptors. A n important question that has to be answered is whether the sensitized N M D A receptors will kill neurons even with normal glutamate levels and metabolism, as cysteine administration cannot trigger a significant glutamate release (Puka-Sundvall et al., 1995). My experiments did not test this theory. However, there is no conflict if both the redox modulation mechanism and the free radical mechanism are verified. Fourth, the data presented here suggest that cysteine neurotoxicity is a result of hydroxyl radicals generated during cysteine autoxidation. This mechanism can also explain the early observation of cysteine neurotoxicity in animals. The high dose administration of cysteine in infant mice or rats will subsequently increase cysteine levels in CSF. Presumably it is easier for cysteine to penetrate to the brain areas due to the incomplete B B B in infant animals. Cysteine will undergo autoxidation under the catalysis of loosely bound copper, whose levels in CSF are 0.13 to 0.75 u M (Gutteridge, 1984). It is suggested that the brain damage is caused by free radicals generated from autoxidation of the large amount of cysteine under those conditions. Since cysteine is an endogenous naturally occurring constituent in the extracellular fluid of the CNS and in all cells, the complete understanding of cysteine toxicity is very important. Further experiments are required to elucidate the unsolved questions.  3. Free Radical Sources in the C N S Mitochondria are probably the major sources of free radicals in vivo. Considering over 90% of 0 taken up by organisms is used in mitochondria, even a small percentage 2  of electron leakage from electron transport chain will generate rather large amounts of reactive oxygen intermediates (Fisher, 1987; Barja et al., 1994). Some 0 -utilizing 2  enzymes, such as xanthine oxidase, tryptophan dioxygenase, indoleamine dioxygenase and nitric oxide synthetase, can generate free radicals directly (Halliwell and Gutteridge, 1985; Culcasi et al., 1994; Gotz et al., 1994). Autoxidation of some biologically important molecules, such as levodopa, dopamine and cysteine, can also generate free radicals (Saez et al., 1982; Nath and Salahudeen, 1993; Lai and Yu, 1997; Shen and Dryhurst, 1998; Soto-Otero et al., 2000). Iron is generally considered as the major transition metal ion that catalyzes autoxidation reactions.  96  Chapter 7 General Discussion & Conclusions  The studies presented here demonstrate that copper, but not iron or other transition metal ions, is the most important transition metal ion in catalyzing the generation of free radicals from autoxidation reactions. The toxic effects of copper in extracellular fluids or cytoplasm could be quite different. Extracellularly, copper is able to catalyze the autoxidation of cysteine, GSH, as well as other reducing agents, such as ascorbic acid in the CNS. This kind of reactions could occur under physiological conditions owing to the existence of loosely bound copper in CSF. The copper-catalyzed autoxidation is suggested to be an important extracellular source of free radicals in the CNS. Intracellular, copper is well bound to proteins and there is no free copper under normal conditions (Rae et al., 1999). Under pathological conditions, however, copper's binding status could be compromised and copper could unexpectedly catalyze the generation of free radicals from autoxidation reactions. This assumption may explain the toxic mechanism of SOD mutants in certain familial A L S cases.  4. Antioxidative Mechanisms of Astrocytes Ever since neurons were born at the perinatal stage when neuroblasts undergo their final round of division to give rise to postmitotic neurons, they started to set up cell-cell connections. We cannot afford the death of neurons, which will result in the loss of memory and many other neural functions, as in the cases of neurodegenerative diseases. As discussed earlier, neurons of the CNS face a great oxidative stress. To live or to die is always a critical question for neurons. Fortunately, astrocytes have evolved to serve as neuronal protectors. Evidence obtained from other reports and the present experiments shows that astrocytes play diverse functions to protect neurons from oxidative stress. 1). Glutamate uptake by astrocytes. Glutamate is released into the synaptic cleft to activate the next neuron. Overstimulation of glutamate receptors will lead to the generation of free radicals, as discussed earlier. Released glutamate is taken up by glutamate transporters, which are Na -dependent systems, and five of which have been cloned (Kanai, 1997). +  Selective depletion of the astrocyte glutamate transporter results in elevated extracellular glutamate levels and neuronal death (Rothstein et al., 1996). 2). Provision of cysteine by astrocytes. Release of thiols by astrocytes has been noted for a decade (Yudkoff et al.,  97  Chapter 7 General Discussion & Conclusions  1990; Sagara et al., 1993). My experiments demonstrate a mechanism by which astrocytes provide cysteine to neurons. The significance of astrocytes in maintenance of thiol levels in the CNS is explained. 3). Extracellular H2O2 scavenging mechanism. Removal of H2O2 is the efficient and last defense in preventing -OH attack. Pyruvate has been found to react with H2O2. Though catalase can remove H2O2 as well, my data show that astrocytes have selected pyruvate as the extracellular H2O2 scavenger. Pyruvate is maintained at relatively high levels by astrocytes in the extracellular fluid of the CNS. Taken together, these mechanisms have revealed the neuroprotective role of astrocytes against oxidative stress. It is speculated that this antioxidative role of astrocytes is ubiquitous in the CNS. The juxtaposition of astrocytes and neurons in the tissue allows astrocytes to exert their functions efficiently.  5. Significance of the Present Findings The present data clearly indicate the importance of copper in oxidative stress. This finding is useful for studying the pathogenic mechanisms of neurological diseases. Wilson's disease is an inherited disorder characterized by hepatic cirrhosis and neuronal degeneration due to an impairment of copper excretion. The Wilson's disease gene has been found to encode a copper transporting P-type ATPase (Bull et al., 1993; Tanzi et al., 1993; Yamaguchi et al., 1993). This ATPase is expressed predominantly in the liver and is responsible for the excretion of copper into the bile. Mutations of the gene result in copper accumulation in the hepatocytes and subsequently in extrahepatic tissues, such as the brain and the cornea (Loudianos and Gitlin, 2000). The pathological changes in the CNS include copper deposition (ten- to fifteen-fold over normal) in virtually all parts of brain with resulting extensive neuronal loss and gliosis in the gray matter (Scheinberg and Sternlieb, 1983). Plasma loosely bound copper and free radical production have been found to be markedly increased in Wilson's disease (Ogihara et al., 1995). However, the molecular mechanisms of copper-induced oxidative stress and cytotoxicity have not been explained. My studies suggest that elevated free copper in the CNS accelerates the autoxidation of cysteine and other reducing agents, resulting in increased production of free radicals and subsequent cytotoxicity in Wilson's disease.  98  Chapter 7 General Discussion & Conclusions  Recently, several research groups have intensively studied copper's role in the pathogenesis of Alzheimer's disease. It was found that copper could form a complex with A p (Huang et al., 1999b) or APP (Multhaup et al., 1998; White et al., 1999), catalyzing a site-specific reduction-oxidation reaction. Neurofibrillary tangles and senile plaques in tissue slices of Alzheimer's cases show the high redox reactivity, as compared with controls (Sayre et al., 2000). The physiological functions of A(3 and APP, however, are still unknown. One possibility is that they participate in copper transport. These results indicate that copper-induced toxicity is a critical pathogenic factor. In fact, copper chelators have been suggested for the treatment of Alzheimer's disease (Cuajungco et al., 2000) . Mutations of Cu/Zn SOD have been identified in patients with familial amyotrophic lateral sclerosis (FALS) (Rosen et al., 1993). Further studies have found that oxidative stress caused by altered copper coordination is the major pathogenic factor in this F A L S model (Estevez et al., 1999; Gabbianelli et al., 1999). Copper chelation has been used in the treatment of A L S in these transgenic mice (Nagano et al., 1999; Andreassen et al., 2001) . In studies of apoptosis, the release of cytochrome c from mitochondria is generally considered as a critical trigger of the apoptosis process (Liu et al., 1996; Green and Reed, 1998). However, the role of copper, the major cofactor of cytochrome c, has not been extensively studied. It is shown that cysteine and other reducing agents will be oxidized under physiological conditions in vivo. This may explain why cysteine is maintained at a relatively low level in the CSF, compared with other amino acids (McGale et al., 1977). The organism requires minimizing the generation of free radicals from cysteine autoxidation. However, the existence of certain levels of extracellular cysteine and its autoxidation are inevitable, as cysteine is essential for GSH synthesis within neurons. The brain utilizes pyruvate to scavenge H2O2 as an efficient defense. At present, some thiols, including GSH, N-acetyl-cysteine (NAC), have been applied or are being tested as therapeutic drugs or components of nutritional products. A caution in their application should be raised based on above considerations. Large doses of thiols could increase the generation of free radicals from their autoxidation in vivo. GSH and N A C can undergo an autoxidation reaction just as can cysteine, simply with varying degrees. Pyruvate, on  99  Chapter 7 General Discussion & Conclusions  the other hand, should be considered as a good candidate for antioxidant defense in clinical applications. It is a rare antioxidant without prooxidant properties. The beauty of great nature is beyond imagination. Over billions of years, the living world has become such a complicated, dynamic and well-arranged system. The organisms have faced countless challenges from the environment. To survive, they must evolve certain mechanisms to overcome these challenges. Adaptation is a great gift of life. The living species may adopt an existing system from another species, or develop a new system by themselves. It is unknown when copper was picked up by the living species from the environment as an enzyme cofactor, exerting its powerful role in redox reactions. Presumably, this event occurred after the appearance of 0 in the atmosphere. 2  The organisms must have found that the uncontrolled catalyzing effect of copper ions is devastating and intolerable in vivo. Then the specialized proteins appeared in the body fluids to carefully chelate copper ions, making full use of their catalyzing property in a controlled manner. It is unknown how the living species obtained the capacities to synthesize GSH, and to absorb so many antioxidants they cannot synthesize by themselves. When pyruvate was reacted with H2O2 in an organism for the first time, the organism must have sensed the benefit of pyruvate, then employed this chemical as a defense later on, and passed on this mechanism to the next generation. Along the history of evolution, living species have constructed many efficient and delicate antioxidative systems. To elucidate these mechanisms will have significant value for the health of humanity.  6.  Perspectives In the present study, copper was found to play a pivotal role in neurotoxicity.  However, the knowledge of copper metabolism in the brain is still patchy. As free copper can cause cell damage, it has to be carefully transported. Ceruloplasmin gene expression has been detected in glial cells of the brain and retina (Klomp et al., 1996; Levin and Geszvain, 1998). Ceruloplasmin levels are increased in subjects of Alzheimer's disease, Parkinson's disease, and Huntington's disease (Loeffler et al., 1996). However, it is unknown what the relationship of ceruloplasmin is with other copper-binding peptides or proteins, such as Ap, APP or apoE. The copper chaperone for  100  Chapter 7 General Discussion & Conclusions  SOD (CCS), which binds copper and delivers it to target protein, has recently been investigated (Rosenzweig and O'Halloran, 2000). Its role in A L S and other neurodegenerative diseases is unclear. A critical question about copper's role in neurodegeneration is what are the initial factors that could trigger the abnormal copper coordination. The therapeutic value of copper chelators deserves to be explored. Copper chelators in the treatment of Alzheimer's disease and A L S have been mentioned earlier. In stroke and traumatic brain injury (TBI), it is expected that copper levels at the site of damage will substantially increase owing to the breakdown of blood-brain barrier. The copper concentration in blood is much higher than in CSF, with ratio of CSF/plasma equal to 1.2% (Joergstuerenburg et al., 1999). Chelation of copper could decrease copperinduced oxidative stress in these pathological situations. It has been shown that astrocytes use diverse mechanisms to protect neurons from oxidative stress. It is not hard to imagine that once the functions of astrocytes are weakened, the survival capacity of neighbouring neurons will be severely compromised. Senescence can lead to the gradual functional decrease of many cell types, particularly mitotic cells like astrocytes. Senescent astrocytes may have lower capacities of uptaking glutamate and of releasing GSH and pyruvate. Once these changes in astrocytes reach beyond threshold, neurons will be affected. It is also known that aging is the common etiological factor of neurodegenerative diseases. Even for cases with hereditary tendency, like early onset Alzheimer's disease, most of them strike only after maturity. Based on these considerations, the functions of senescent astrocytes and their role in the pathogenesis of neurodegeneration should be examined.  CONCLUSIONS  The following conclusions can be drawn from the experiments: 1) . Astrocytes improve neuronal survival. 2) . Astrocytes promote neuronal attachment. 3) . Astrocytes provide cysteine to neurons indirectly by releasing GSH. 4) . Autoxidation of cysteine, as well as other reducing agents, generates free radicals, which are responsible for their cytotoxic effects.  101  Chapter 7 General Discussion & Conclusions  5) . Copper is the major catalyst of cysteine autoxidation in the CNS. 6) . Catalase and pyruvate can prevent cysteine toxicity by removing H2O2. 7) . Astrocytes release pyruvate. 9). Cysteine can improve neuronal survival when its neurotoxic effects are prevented.  102  References  REFERENCES  Acworth IN, McCabe DR, Maher TJ (1997) The analysis of free radicals, their reaction products, and antioxidants. In: Oxidants, antioxidants, and free radicals (Baskin SI, Salem H, eds), pp 23-78. Washington, DC: Taylor & Francis. 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