Rosenberg’s Molecular and Genetic Basis of Neurological and Psychiatric Disease http://dx.doi.org/10.1016/10.1016/B978-0-12-410529-4.00061-9 687 © 2015 Elsevier Inc. All rights reserved.

C H A P T E R

61

Disorders of Glutathione MetabolismKoji Aoyama and Toshio Nakaki

Teikyo University School of Medicine, Tokyo, Japan

INTRODUCTION

Glutathione (GSH) is the major endogenous low molecular-weight thiol in mammalian cells. GSH plays pivotal roles in antioxidative defense, intracellular redox homeostasis, cysteine carrier/storage, cell signaling, enzyme ac-tivity, gene expression, and cell differentiation/proliferation. In particular, GSH is one of the most important antiox-idants or antioxidant-related compounds in the brain for neuroprotection. The brain is one of the organs generating large amounts of reactive oxygen species (ROS) because of its high ratio of O2 consumption (~ 20% of total body O2 consumption) to weight of the brain (~ 2% of body weight) and also generates high levels of lipids with unsaturated fatty acids, leading to oxidative stress. However, the brain contains low antioxidant levels with low antioxidant en-zyme activities. ROS can cause lipid peroxidation, DNA damage, mitochondrial dysfunction, and protein oxidation. GSH acts directly as a potent antioxidant by itself or in collaboration with other enzymes to reduce ROS or detoxify xenobiotics.1 GSH also acts as the major redox buffer to maintain intracellular redox homeostasis. Oxidative damage alters the redox state of the cell, leading to permanent loss of the functions of proteins as enzymes, receptors, and transporters.2 GSH can preserve protein thiol groups in a reduced state (S-glutathionylation) to prevent irreversible protein oxidation.3 A variety of GSH functions are crucial to protect the cells against the oxidative damage leading to neurodegeneration.

Clinically, inborn errors in the GSH-related enzymes are very rare, while disorders in GSH metabolism are com-mon in neurodegenerative diseases, including Alzheimer disease (AD), Parkinson disease (PD), amyotrophic lat-eral sclerosis (ALS), and Huntington disease (HD). In this chapter, we focus on disorders of GSH metabolism in neurologic diseases, especially in neurodegenerative diseases related to GSH dysfunction. Additional reviews are presented in the references of this chapter.4–7

GSH AND THE γ-GLUTAMYL CYCLE

GSH is a tripeptide constituted of three amino acids: glutamate, cysteine and glycine. GSH synthesis requires two steps involving adenosine triphosphate (ATP)-dependent enzymatic reactions. The first step mediates the reaction between glutamate and cysteine by an enzyme called γ-glutamylcysteine ligase (GCL) or γ-glutamylcysteine synthe-tase. Glutamate has two carboxyl groups, one of which binds at the γ-position with the amino group of cysteine to form a dipeptide, γ-glutamylcysteine. This step is the rate-limiting reaction for GSH synthesis. GCL is a heterodimer composed of a catalytic (heavy; molecular weight of 73 kDa) subunit, GCLC, and a modulatory (light; molecular weight of 28 kDa) subunit, GCLM. GCLC, but not GCLM, has all the catalytic function and is subject to feedback inhibition by GSH,8 while the association of GCLM with GCLC increases the affinity for glutamate.9 The second step for GSH synthesis is mediated by another enzyme called GSH synthetase (GS), which binds γ-glutamylcysteine and glycine to form GSH, although the precise mechanisms for the regulation of GS activity have not been clarified yet.

GSH reacts nonenzymatically with superoxide, nitric oxide, hydroxyl radical, and peroxynitrite as an antioxidant, while GSH is utilized enzymatically by GSH peroxidase (GPx) and GSH-S-transferase (GST) as a reducing agent against oxidative damage. GPx requires GSH as an electron donor to react with H2O2 or endogenous hydroperoxides

C H A P T E R

61

688 61. DIsoRDERs of GluTATHIonE METAbolIsM

III. NEUROMETABOLIC DISORDERS

(ROOH), which are formed by the reaction of superoxide with superoxide dismutase (SOD) or lipid peroxidation, respectively. There are four types of selenium-containing GPx. The cytosolic isoform of GPx, named GPx-1, is the most abundant GPx and functions as an important antioxidative enzyme in the brain. In the processes of these GSH-associated antioxidant mechanisms, GSH is oxidized to GSH disulfide (GSSG), which is then reduced back to GSH by the reaction with GSH reductase (GR). This reaction requires nicotinamide adenine dinucleotide phosphate (NADPH), which is produced by glucose-6-phosphate dehydrogenase (G6PDH), as a substrate for supplying elec-trons to GSSG. Neuronal GR activity is sufficiently active to rapidly regenerate GSH from GSSG.10 Thus, GSSG is maintained as less than 1% of total GSH under physiological conditions and the GSSG level is doubled (~ 2%) under oxidatively insulted conditions,11 although the GSSG increase during oxidative stress is transient because of the re-duction by GR. Although catalase can also degrade intracellular H2O2 to H2O and O2

-, this enzyme can not detoxify other endogenous ROOH and therefore does not work sufficiently for the peroxide detoxification in neurons.12 GST catalyzes GSH attack on various electrophilic metabolites of xenobiotics to detoxify the compounds and release them from the cell. In mammalian species, seven classes of cytosolic GST have been reported as the isoforms alpha, mu, pi, sigma, theta, omega, and zeta.13 GSH and GSH-containing molecules, including GSSG and GSH S-conjugates, are ex-ported to the extracellular space via the multidrug resistance-associated proteins.14,15 GSH and its conjugates are then cleaved into the γ-glutamyl moiety and cysteinylglycine by the reaction with γ-glutamyl transpeptidase (γGT), which is a plasma membrane-bound enzyme with its active site on the extracellular side. Subsequently, the γ-glutamyl moiety is cleaved into the corresponding amino acid and 5-oxoproline by the reaction of γ-glutamyl cyclotransferase (GCT), while cysteinylglycine is cleaved into cysteine and glycine in a reaction mediated by membrane-bound di-peptidase. 5-Oxoproline is then converted to glutamate by the reaction of 5-oxoprolinase. These cleaved or converted amino acids are reused for GSH synthesis to form the γ-glutamyl cycle (Figure 61.1).

DISORDERS OF ENZYMES IN THE γ-GLUTAMYL CYCLE

Animal Models

GCLC-deficient mice are embryonic lethal,16 while GCLM-deficient mice are viable and fertile, although the GSH levels in their organs and plasma are low.17 Mice deficient in GPx-1, for which the total GPx activity is completely blocked in the brain, show normal growth and fertility and no histological abnormality in the brain or other organs,18 although they do show increased vulnerability to some neurotoxins.19 γGT-deficient mice develop glutathionuria, se-vere growth failure, lethargy, shortened lifespan, hypogonadism, and infertility.20,21 Mice deficient in some cytosolic GSTs have been developed to show no obvious phenotype with some increased susceptibility to toxic xenobiotics.13

Clinical Features and Molecular Genetics

GCL deficiency is a very rare hereditary (autosomal recessive) disease, which has been reported in nine patients in seven families around the world. The patients exhibit hemolytic anemia and, in some cases, neurological symp-toms, such as spinocerebellar degeneration, mental retardation, peripheral neuropathy, myopathy, and aminoacid-uria. The laboratory data show low GCL activity/levels, and low GSH levels in red blood cells and/or cultured skin

5-OP

CysGlu

GCL

CysGly

Dipeptidase

Gly

GS

GSH

GRGPx

NADP+

NADPHGSSG

GST

G6PDH

ROOHH2O2

ROHH2O + O2

AA

AA-GSH

γglutamyl-AA

γGluCys

γGT

5-OPase

AA

GCT

FIGURE 61.1 The γ-glutamyl cycle. Abbreviations: AA, amino acids; Cys, cys-teine; CysGly, cysteinylglycine; GCL, γ-glutamylcysteine ligase; GCT, γ-glutamyl cyclotransferase; γGT, γ-glutamyl transpeptidase; γGluCys, γ-glutamylcysteine; Glu, glutamate; Gly, glycine; G6PDH, glucose-6-phosphate dehydrogenase; GPx, glutathione peroxidase; GR, glutathione reductase; GS, glutathione synthetase; GSH, glutathione; GSSG, glutathione disulfide; GST, glutathione-S-transferase; H2O2, hydrogen peroxide, NADPH, nicotinamide adenine dinucleotide phosphate; 5-OP, 5-oxoproline; 5-OPase, 5-oxoprolinase; ROH, alcohol; ROOH, hydroperoxide.

ExCITAToRy AMIno ACID TRAnsPoRTERs (EAATs) 689

III. NEUROMETABOLIC DISORDERS

fibroblasts. Both human gene loci of GCLC and GCLM are described on chromosome 6p12 and chromosome 1p21, respectively. Mutation analysis of the patients has identified four different mutations in the GCLC subunit in four families. No promising treatment has been established yet. Patients with GCL deficiency should avoid drugs known to cause hemolytic anemia in patients with G6PDH deficiency—e.g., acetyl salicylic acid or sulfonamides.

GS deficiency is also a rare autosomal recessive disease reported in more than 70 patients in more than 50 families. The patients present with hemolytic anemia, metabolic acidosis, 5-oxoprolinuria, progressive neurologic symptoms, such as mental retardation, seizures, spasticity, and ataxia, and recurrent bacterial infections. The human GS gene is localized to chromosome 20q11.2, on which some different mutations or epigenetic modifications have been revealed. For this deficiency as well, the laboratory data indicate low GS activity/levels, and low GSH levels in red blood cells and/or cultured skin fibroblasts. About 25% of the patients die in the neonatal period. Early administration of vita-min C and/or vitamin E improves the long-term clinical outcome. Treatment with bicarbonate, citrate, or trometamol is useful to correct the metabolic acidosis. N-acetylcysteine (NAC) administration might be recommended to protect cells from oxidative stress, but accumulated cysteine would be rather neurotoxic in patients with GS deficiency.22–24 The patients should avoid drugs that could precipitate hemolytic anemia.

There are some other hereditary diseases with enzyme dysfunctions in the γ-glutamyl cycle although all disorders are clinically very rare.25

EXCITATORY AMINO ACID TRANSPORTERS (EAATS)

Neurons cannot assimilate extracellular GSH directly into the cell interior, but can directly assimilate cysteine. Most of the neuronal cysteine uptake is mediated by sodium-dependent systems, mainly the excitatory amino acid trans-porter (EAAT). To date, five EAATs have been reported: glutamate aspartate transporter (GLAST, also termed EAAT1), glutamate transporter-1 (GLT-1, also termed EAAT2), excitatory amino acid carrier 1 (EAAC1, also termed EAAT3), EAAT4, and EAAT5.26 GLAST and GLT-1 are localized primarily to astrocytes, and EAAC1, EAAT4 and EAAT5 to neu-rons. EAAT4 and EAAT5 are localized to cerebellar Purkinje cells and the retina, respectively, while EAAC1 is expressed widely throughout the central nervous system (CNS). In the brain, astroglial EAATs, mainly GLT-1, play a central role in removing interstitial glutamate.27,28 EAATs can transport not only extracellular glutamate but also cysteine into the cells.29 Notably, EAAC1 preferentially transports extracellular cysteine rather than glutamate into neurons (Figure 61.2). Under normal conditions, EAAC1 is mostly localized in the cytoplasm, with the expression on the plasma membrane accounting for only 20% of the total.30 Once stimulated by intracellular signaling, such as protein kinase C or phospha-tidylinositol 3-kinase activation, EAAC1 translocates to the plasma membrane for glutamate and cysteine uptake, leading to neuronal GSH synthesis.7 In contrast, the expression of EAAC1 on the plasma membrane is restricted by direct interaction with glutamate transport associated protein 3-18 (GTRAP3-18), which inhibits translocation of

PKC

Glu + Cys + Gly = GSH

PI3K

GTRAP3-18

EAAC1

ExtracellularPlasma

membrane

Glu Cys

Intracellular

FIGURE 61.2 EAAC1-mediated GSH synthesis in neurons. Abbreviations: Cys, cysteine; EAAC1, excitatory amino acid carrier 1; Glu, glutamate; Gly, glycine; GTRAP3-18, glutamate transport associated protein 3-18; GSH, glutathione; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C.

690 61. DIsoRDERs of GluTATHIonE METAbolIsM

III. NEUROMETABOLIC DISORDERS

EAAC1 to the plasma membrane.31 GTRAP3-18 is a member of the prenylated Rab acceptor family, which contains two extensive hydrophobic domains tightly attached to the endoplasmic reticulum (ER) membrane and anchors the target proteins in the ER. GTRAP3-18 negatively regulates the neuronal GSH level by controlling the EAAC1-mediated cysteine uptake.32–34

DISORDERS OF EAAC1 LEADING TO GSH DEPLETION

Animal Models

EAAC1-deficient mice develop brain atrophy, spatial learning and memory dysfunction, decreased number of dopaminergic neurons in the substantia nigra (SN), and movement disorder at advanced ages but not at adoles-cence.35,36 The total GSH levels in the brain are much lower in EAAC1-deficient mice than in the wild-type mice, while the liver GSH levels are comparable between the groups. The brains of EAAC1-deficient mice show increased oxidant levels and increased vulnerability to oxidative stress, which are alleviated by treatment with NAC to in-crease GSH synthesis.

Conversely, inhibition of GTRAP3-18 expression increases GSH levels in the brain.33,34 GTRAP3-18-deficient mice show the increased EAAC1 expression on the plasma membrane, increased cysteine and GSH contents in the brain, resistance to oxidative stress, and facilitated learning and memory functions.

Clinical Features

Several lines of putative evidence have been reported regarding EAAC1 dysfunction leading to GSH depletion in neurodegenerative diseases; however, it is still uncertain whether this finding is the primary cause of neurodegener-ative diseases (as discussed below).

NEURODEGENERATIVE DISEASES LEADING TO GSH DEPLETION

Alzheimer Disease

AD is a leading age-related neurodegenerative disease characterized by progressive dementia developed in middle or later life. Pathologically, depositions of amyloid-β plaques and neurofibrillary tangles in the brain are the hallmark of AD. Amyloid-β induces oxidative stress leading to neurofibrillary tangles formation and neuronal death.37 Changes in GSH metabolism have been reported in AD patients. Polymorphisms in the GPx-1 and GST genes have been iden-tified as possible risk factors for AD.38,39 GSH levels in erythrocytes are decreased, while plasma oxidation protein products are increased in AD patients.40 A recent clinical study using magnetic resonance spectroscopy revealed that brain GSH levels are depleted in AD patients as compared to healthy subjects.41 Postmortem brain tissue samples from AD patients also showed decreased GSH/GSSG ratios with disease progression and decreased GST activities.42,43 Mild cognitive impairment (MCI), which is considered the earliest stage of AD,44 has also been associated with decreased GSH levels in erythrocytes, increased oxidation protein products in plasma,40 and the decreased ratio of GSH/GSSG in the hippocampus.45 These findings suggest that GSH depletion would precede the onset of the disease.

The ε4 allele of the apolipoprotein E gene (APOE) is a major genetic risk factor for late-onset AD. The ApoE4 pro-tein enhances Aβ deposition in the CNS. Brain tissues from AD patients with the APOE ε4 allele also show decreased GSH levels with decreased GPx activities as compared to those of age-matched controls or AD patients homozygous for the ε3 allele.46 α-tocopherol is also a potent antioxidant in the brain. However, the analysis of α-tocopherol levels in the brain revealed no relation with the APOE polymorphism. These findings suggest a specific dysregulation of GSH biosynthesis in AD patients. Indeed, in the hippocampus of AD patients, degenerating neurons exhibit aberrant detergent-insoluble EAAC1 accumulation.47 GSH depletion via EAAC1 dysfunction would lead to neurodegenera-tion in the hippocampus of patients with AD.

Parkinson Disease

PD is the second most common neurodegenerative disease after AD, and is clinically characterized by resting tremor, rigidity, akinesia, and postural instability. PD is also a progressive, late-onset movement disorder that is

nEuRoDEGEnERATIvE DIsEAsEs lEADInG To GsH DEPlETIon 691

III. NEUROMETABOLIC DISORDERS

affected by dopaminergic neurodegeneration in the SN. Lewy bodies are eosinophilic neuronal inclusions that con-tain both α-synuclein and ubiquitin as the pathological hallmarks of PD. Oxidative stress leads to α-synuclein aggre-gation, followed by proteasome dysfunction and neuronal death.48 Postmortem analysis of normal individuals with incidental Lewy bodies, who would be considered presymptomatic PD subjects, revealed lower GSH levels in the SN than those of age-matched controls without Lewy bodies.49 As with AD patients, GSH depletion would precede the onset of the disease.50 Consequently, the brain GSH levels in PD patients have been shown to be depleted in the SN, but not in the other regions, as compared to those of age-matched controls.51 The activity of the GSH synthetic enzyme GCL was unchanged, while that of the GSH-degrading enzyme γGT was elevated in PD patients.52 The severity of GSH depletion parallels pathological and/or clinical PD severity.53 Decreased GSH may be considered not only as an early event in PD progression but even as the primary cause of neurodegeneration.50,54 Although the primary cause of GSH depletion in PD is still unclear, recent studies suggest that EAAC1 dysfunction is involved in the pathogenesis. Human dopaminergic neurons in the SN express EAAC1.36,55 Dopaminergic (DA) neurons are more vulnerable to EAAC1 dysfunction than non-DA neurons.56 Considering these findings, EAAC1 dysfunction would be a possible cause of GSH depletion in PD, although further clinical studies would be required to elucidate the involvement.

Approximately 10% of PD patients present a family history of the Mendelian form of the disease with autoso-mal dominant or recessive inheritance. To date, there are 18 specific chromosomal regions, designated PARK1-18.57 Mutations in the α-synuclein gene (SNCA), formerly termed PARK1, were found in autosomal dominant PD. Normal α-synuclein translocates into lysosomes for degradation, while mutated α-synuclein aggregates in neurons to cause neurodegeneration.58 A mutant A53T model in the α-synuclein gene decreases de novo GSH synthesis after treatment with a proteasome inhibitor.59 Loss-of-function mutations in parkin (PARK2) are found in autosomal recessive juve-nile PD patients. Parkin is an E3 ubiquitin ligase, catalyzing the addition of ubiquitin to specific substrates, including α-synuclein, which targets them for degradation by the ubiquitin–proteasome system. The active sites of parkin are cysteine-rich regions and thereby sensitive to oxidative modification, which alters the protein solubility and its func-tion.60,61 Mutations in PINK1 (PARK6) cause autosomal recessive early-onset PD. PINK1 is a mitochondrial protein ki-nase and loss of its function induces mitochondrial dysfunction. In primary fibroblasts from the PD patients, elevated GR and GST activities with increased lipid peroxidation and mitochondrial dysfunction are observed, although the GSH/GSSH ratios are unchanged, compared to controls.62 DJ-1 (PARK7) is also one of the causative genes for famil-ial PD. Mutations in DJ-1 cause an autosomal-recessive, early onset familial form of PD. DJ-1 is a redox-dependent molecular chaperone that upregulates GSH synthesis during oxidative stress.63 Oxidation of a conserved cysteine residue in DJ-1 regulates its chaperone activity against α-synuclein.64 Conversely, DJ-1 is oxidatively damaged in the brains of idiopathic PD and AD patients.65

Amyotrophic Lateral Sclerosis

ALS is a chronic progressive disease characterized by selective degeneration of motor neurons in the spinal cord and motor cortex. Although the precise etiology is still unknown, glutamate neurotoxicity induced by loss of GLT-1 has been reported in the spinal cord and motor cortex of sporadic ALS patients.66,67 Moreover, the expression of EAAC1 proteins, but not the mRNA, is also slightly downregulated in the spinal cord and motor cortex of sporadic ALS patients.67,68 GSH depletion has been shown to result in motor neuron death in vitro and in vivo.69 GSH metabo-lism seems to be altered in ALS patients. An early clinical study reported increased protein glutathionylation, which is an important adaptive cellular response to protect crucial protein functions under oxidative stress, in the spinal cords of sporadic ALS patients.70 A recent study also demonstrated decreased GSH, GR, and G6PDH levels, but in-creased lipid peroxidation in the erythrocytes of sporadic ALS patients.71 These changes correlate with the disease progression. The mRNA expression of GST pi is reduced in the spinal cord and motor/sensory cortex in the brain of ALS patients.72 These changes in GSH metabolism support the clinical evidence implicating oxidative stress in ALS pathogenesis.73 Approximately 10% of all ALS cases are familial; in turn, ~ 20% of these familial cases are inherited in an autosomal dominant pattern with mutations in the gene encoding cytosolic Cu/Zn SOD (SOD1). SOD1 mutant mice show decreased GSH levels in the spinal cord and motor neurons.69

Huntington Disease

HD is caused by the expansion of CAG trinucleotide repeats (in excess of 38 repeats) on chromosome 4 in exon 1 of the gene coding “huntingtin” with autosomal dominant inheritance. HD patients show hyperkinetic movement disor-ders based on basal ganglion dysfunction. The precise mechanisms causing HD are still elusive. However, oxidative stress is considered to be the major cause leading to the neurodegeneration. HD patients have higher plasma lipid

692 61. DIsoRDERs of GluTATHIonE METAbolIsM

III. NEUROMETABOLIC DISORDERS

peroxidation levels and lower GSH levels than their age and sex-matched controls.74 In a recent study, depleted GSH levels with elevated ROS levels were found in neurons prepared from a model of HD (HD140Q/140Q) in which a human huntingtin gene with 140 CAG repeats was inserted into the mouse genome.75 These results were attributable to EAAC1 dysfunction, which impairs cysteine uptake and thereby leads to GSH depletion in neurons. Further clinical studies are needed to clarify the mechanisms of EAAC1 dysfunction in HD patients.

Therapy

Antioxidant therapy may be a potential approach to prevent and treat neurodegenerative diseases. However, oral supplementation with ascorbate and/or α-tocopherol, which are important antioxidants in the brain, have not yet shown definitive benefit in patients with AD and PD. In addition, the levels of ascorbate and α-tocopherol in the CNS do not change in patients with AD and PD compared with controls, while GSH levels in the CNS are consistently reduced in these neurodegenerative diseases, as described above. Epidemiological studies have indicated that inci-dence of neurodegenerative diseases such as AD and PD is inversely correlated with caffeine consumption or plasma uric acid level.76,77 These purine derivatives may induce neuronal GSH synthesis by promoting cysteine uptake, leading to neuroprotection.78 Therefore, the therapeutic strategy of increasing the GSH levels in the brain in neuro-degenerative diseases is sound; however, at present there are no therapeutic drugs for increasing brain GSH levels. Cysteine is the rate-limiting substrate for GSH synthesis, but direct administration of cysteine is not recommended due to its neurotoxicity. The plasma half-life of intravenous administered GSH is 2–3 minutes. Orally administered GSH is rapidly degraded in the gut. Moreover, neither cysteine nor GSH in the blood can penetrate the blood–brain barrier (BBB) easily, and these compounds are rapidly oxidized to cystine and GSSG, respectively. NAC is a promis-ing compound as a therapeutic drug for neurodegenerative diseases. NAC acts as both a direct antioxidant and the substrate for GSH synthesis in the brain. NAC can penetrate the BBB and the plasma membrane to supply cysteine into the cells. In the mouse models of neurodegenerative diseases, NAC can increase neuronal GSH levels and reduce neuronal damages induced by oxidative stress.35,79 Based on the promising results of NAC treatment for AD models in vitro and in vivo, two clinical trials in the USA (ClinicalTrials.gov identifier: NCT01320527 and NCT01370954) have been initiated and completed to investigate whether a dietary supplement containing NAC maintains or improves cognitive performance in patients with AD or MCI, although these results are not available as of June 2014. For the same reasons as AD, NAC is also a promising agent for the treatment of PD patients. As of June 2014, two clinical studies (ClinicalTrials.gov identifier: NCT01427517 and NCT01470027) are listed in the USA. However, in a random-ized, double blind clinical trial with NAC treatments for ALS patients, no significant benefits were found in either survival or disease progression.80

An endogenous approach to increase GSH levels in neurons is an alternative strategy against neurodegeneration. Although there is no known drug for clinical use to activate neuronal GSH synthesis in the brain, the compounds facilitating EAAC1 function might be a promising treatment for neurodegenerative diseases in the future.

CONCLUSIONS

Insight into neurological disorders involving GSH metabolism has been obtained from a few rare hereditary dis-eases with enzymatic dysfunctions of GSH metabolism, as well as from some major neurodegenerative diseases such as AD, PD, ALS, and HD. GSH depletion is an early event in neurodegeneration and is related to disease progression in patients. A strategy to increase neuronal GSH levels would be a promising treatment for patients with these disor-ders, although it is still under basic and clinical investigations.

References 1. Dringen R. Metabolism and functions of glutathione in brain. Prog Neurobiol. 2000;62:649–671. 2. Klatt P, Lamas S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur J Biochem.

2000;267:4928–4944. 3. Giustarini D, Rossi R, Milzani A, et al. S-glutathionylation: from redox regulation of protein functions to human diseases. J Cell Mol Med.

2004;8:201–212. 4. Ristoff E, Larsson A. Disorders of glutathione metabolism. In: Rosenberg RN, DiMauro S, Paulson HL, et al., eds. The Molecular and Genetic

Basis of Neurologic and Psychiatric Disease. 4th ed. Lippincott Williams & Wilkins/Wolters Kluwer: Boston, MA; 2008:683–688. 5. Aoyama K, Watabe M, Nakaki T. Regulation of neuronal glutathione synthesis. J Pharmacol Sci. 2008;108:227–238.

III. NEUROMETABOLIC DISORDERS

REfEREnCEs 693

6. Aoyama K, Nakaki T. Inhibition of GTRAP3-18 may increase neuroprotective glutathione (GSH) synthesis. Int J Mol Sci. 2012;13:12017–12035.

7. Aoyama K, Nakaki T. Neuroprotective properties of the excitatory amino acid carrier 1 (EAAC1). Amino Acids. 2013;45:133–142. 8. Richman PG, Meister A. Regulation of gamma-glutamyl-cysteine synthetase by nonallosteric feedback inhibition by glutathione. J Biol Chem.

1975;250:1422–1426. 9. Dickinson DA, Forman HJ. Glutathione in defense and signaling: lessons from a small thiol. Ann N Y Acad Sci. 2002;973:488–504. 10. Dringen R, Kussmaul L, Gutterer JM, et al. The glutathione system of peroxide detoxification is less efficient in neurons than in astroglial

cells. J Neurochem. 1999;72:2523–2530. 11. Maher P. The effects of stress and aging on glutathione metabolism. Ageing Res Rev. 2005;4:288–314. 12. Cooper AJ, Kristal BS. Multiple roles of glutathione in the central nervous system. Biol Chem. 1997;378:793–802. 13. Hayes JD, Flanagan JU, Jowsey IR. Glutathione transferases. Annu Rev Pharmacol Toxicol. 2005;45:51–88. 14. Leier I, Jedlitschky G, Buchholz U, et al. ATP-dependent glutathione disulphide transport mediated by the MRP gene-encoded conjugate

export pump. Biochem J. 1996;314(Pt 2):433–437. 15. Ballatori N, Krance SM, Marchan R, et al. Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology.

Mol Aspects Med. 2009;30:13–28. 16. Dalton TP, Dieter MZ, Yang Y, et al. Knockout of the mouse glutamate cysteine ligase catalytic subunit (Gclc) gene: embryonic lethal when

homozygous, and proposed model for moderate glutathione deficiency when heterozygous. Biochem Biophys Res Commun. 2000;279:324–329. 17. Yang Y, Dieter MZ, Chen Y, et al. Initial characterization of the glutamate-cysteine ligase modifier subunit Gclm(-/-) knockout mouse. Novel

model system for a severely compromised oxidative stress response. J Biol Chem. 2002;277:49446–49452. 18. Ho YS, Magnenat JL, Bronson RT, et al. Mice deficient in cellular glutathione peroxidase develop normally and show no increased sensitivity

to hyperoxia. J Biol Chem. 1997;272:16644–16651. 19. Klivenyi P, Andreassen OA, Ferrante RJ, et al. Mice deficient in cellular glutathione peroxidase show increased vulnerability to malonate,

3-nitropropionic acid, and 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine. J Neurosci. 2000;20:1–7. 20. Harding CO, Williams P, Wagner E, et al. Mice with genetic gamma-glutamyl transpeptidase deficiency exhibit glutathionuria, severe

growth failure, reduced life spans, and infertility. J Biol Chem. 1997;272:12560–12567. 21. Kumar TR, Wiseman AL, Kala G, et al. Reproductive defects in gamma-glutamyl transpeptidase-deficient mice. Endocrinology. 2000;141:

4270–4277. 22. Puka-Sundvall M, Eriksson P, Nilsson M, et al. Neurotoxicity of cysteine: interaction with glutamate. Brain Res. 1995;705:65–70. 23. Janaky R, Varga V, Hermann A, et al. Mechanisms of L-cysteine neurotoxicity. Neurochem Res. 2000;25:1397–1405. 24. Ristoff E, Hebert C, Njalsson R, et al. Glutathione synthetase deficiency: is gamma-glutamylcysteine accumulation a way to cope with

oxidative stress in cells with insufficient levels of glutathione? J Inherit Metab Dis. 2002;25:577–584. 25. Ristoff E, Larsson A. Inborn errors in the metabolism of glutathione. Orphanet J Rare Dis. 2007;2:16. 26. Danbolt NC. Glutamate uptake. Prog Neurobiol. 2001;65:1–105. 27. Tanaka K, Watase K, Manabe T, et al. Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT-1. Science.

1997;276:1699–1702. 28. Holmseth S, Dehnes Y, Huang YH, et al. The density of EAAC1 (EAAT3) glutamate transporters expressed by neurons in the mammalian

CNS. J Neurosci. 2012;32:6000–6013. 29. Zerangue N, Kavanaugh MP. Interaction of L-cysteine with a human excitatory amino acid transporter. J Physiol. 1996;493(Pt 2):419–423. 30. Fournier KM, Gonzalez MI, Robinson MB. Rapid trafficking of the neuronal glutamate transporter, EAAC1: evidence for distinct trafficking

pathways differentially regulated by protein kinase C and platelet-derived growth factor. J Biol Chem. 2004;279:34505–34513. 31. Ruggiero AM, Liu Y, Vidensky S, et al. The endoplasmic reticulum exit of glutamate transporter is regulated by the inducible mammalian

Yip6b/GTRAP3-18 protein. J Biol Chem. 2008;283:6175–6183. 32. Watabe M, Aoyama K, Nakaki T. Regulation of glutathione synthesis via interaction between glutamate transport-associated protein 3-18

(GTRAP3-18) and excitatory amino acid carrier-1 (EAAC1) at plasma membrane. Mol Pharmacol. 2007;72:1103–1110. 33. Watabe M, Aoyama K, Nakaki T. A dominant role of GTRAP3-18 in neuronal glutathione synthesis. J Neurosci. 2008;28:9404–9413. 34. Aoyama K, Wang F, Matsumura N, et al. Increased neuronal glutathione and neuroprotection in GTRAP3-18-deficient mice. Neurobiol Dis.

2012;45:973–982. 35. Aoyama K, Suh SW, Hamby AM, et al. Neuronal glutathione deficiency and age-dependent neurodegeneration in the EAAC1 deficient

mouse. Nat Neurosci. 2006;9:119–126. 36. Berman AE, Chan WY, Brennan AM, et al. N-acetylcysteine prevents loss of dopaminergic neurons in the EAAC1-/- mouse. Ann Neurol.

2011;69:509–520. 37. Mattson MP. Pathways towards and away from Alzheimer’s disease. Nature. 2004;430:631–639. 38. Spalletta G, Bernardini S, Bellincampi L, et al. Glutathione S-transferase P1 and T1 gene polymorphisms predict longitudinal course and age

at onset of Alzheimer disease. Am J Geriatr Psychiatry. 2007;15:879–887. 39. Paz-y-Mino C, Carrera C, Lopez-Cortes A, et al. Genetic polymorphisms in apolipoprotein E and glutathione peroxidase 1 genes in the

Ecuadorian population affected with Alzheimer’s disease. Am J Med Sci. 2010;340:373–377. 40. Bermejo P, Martin-Aragon S, Benedi J, et al. Peripheral levels of glutathione and protein oxidation as markers in the development of

Alzheimer’s disease from Mild Cognitive Impairment. Free Radic Res. 2008;42:162–170. 41. Mandal PK, Tripathi M, Sugunan S. Brain oxidative stress: detection and mapping of antioxidant marker ‘Glutathione’ in different brain

regions of healthy male/female, MCI and Alzheimer patients using non-invasive magnetic resonance spectroscopy. Biochem Biophys Res Commun. 2012;417:43–48.

42. Lovell MA, Xie C, Markesbery WR. Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer’s disease. Neurology. 1998;51:1562–1566.

43. Ansari MA, Scheff SW. Oxidative stress in the progression of Alzheimer disease in the frontal cortex. J Neuropathol Exp Neurol. 2010;69:155–167. 44. Petersen RC. Mild cognitive impairment: transition between aging and Alzheimer’s disease. Neurologia. 2000;15:93–101.

694 61. DIsoRDERs of GluTATHIonE METAbolIsM

III. NEUROMETABOLIC DISORDERS

45. Sultana R, Piroddi M, Galli F, et al. Protein levels and activity of some antioxidant enzymes in hippocampus of subjects with amnestic mild cognitive impairment. Neurochem Res. 2008;33:2540–2546.

46. Ramassamy C, Averill D, Beffert U, et al. Oxidative insults are associated with apolipoprotein E genotype in Alzheimer’s disease brain. Neurobiol Dis. 2000;7:23–37.

47. Duerson K, Woltjer RL, Mookherjee P, et al. Detergent-insoluble EAAC1/EAAT3 aberrantly accumulates in hippocampal neurons of Alzheimer’s disease patients. Brain Pathol. 2009;19:267–278.

48. Dawson TM, Dawson VL. Molecular pathways of neurodegeneration in Parkinson’s disease. Science. 2003;302:819–822. 49. Dexter DT, Sian J, Rose S, et al. Indices of oxidative stress and mitochondrial function in individuals with incidental Lewy body disease. Ann

Neurol. 1994;35:38–44. 50. Jenner P. Oxidative damage in neurodegenerative disease. Lancet. 1994;344:796–798. 51. Sian J, Dexter DT, Lees AJ, et al. Alterations in glutathione levels in Parkinson’s disease and other neurodegenerative disorders affecting

basal ganglia. Ann Neurol. 1994;36:348–355. 52. Sian J, Dexter DT, Lees AJ, et al. Glutathione-related enzymes in brain in Parkinson’s disease. Ann Neurol. 1994;36:356–361. 53. Riederer P, Sofic E, Rausch WD, et al. Transition metals, ferritin, glutathione, and ascorbic acid in parkinsonian brains. J Neurochem.

1989;52:515–520. 54. Jenner P. Oxidative stress in Parkinson’s disease. Ann Neurol. 2003;53(suppl 3):S26–S36, discussion S36–S28. 55. Plaitakis A, Shashidharan P. Glutamate transport and metabolism in dopaminergic neurons of substantia nigra: implications for the

pathogenesis of Parkinson’s disease. J Neurol. 2000;247(suppl 2):II25–II35. 56. Nafia I, Re DB, Masmejean F, et al. Preferential vulnerability of mesencephalic dopamine neurons to glutamate transporter dysfunction.

J Neurochem. 2008;105:484–496. 57. Klein C, Westenberger A. Genetics of Parkinson’s disease. Cold Spring Harb Perspect Med. 2012;2:a008888. 58. Lee MK, Stirling W, Xu Y, et al. Human alpha-synuclein-harboring familial Parkinson’s disease-linked Ala-53→Thr mutation causes

neurodegenerative disease with alpha-synuclein aggregation in transgenic mice. Proc Natl Acad Sci U S A. 2002;99:8968–8973. 59. Vali S, Chinta SJ, Peng J, et al. Insights into the effects of alpha-synuclein expression and proteasome inhibition on glutathione metabolism

through a dynamic in silico model of Parkinson’s disease: validation by cell culture data. Free Radic Biol Med. 2008;45:1290–1301. 60. Wong ES, Tan JM, Wang C, et al. Relative sensitivity of parkin and other cysteine-containing enzymes to stress-induced solubility alterations.

J Biol Chem. 2007;282:12310–12318. 61. Meng F, Yao D, Shi Y, et al. Oxidation of the cysteine-rich regions of parkin perturbs its E3 ligase activity and contributes to protein

aggregation. Mol Neurodegeneration. 2011;6:34. 62. Hoepken HH, Gispert S, Morales B, et al. Mitochondrial dysfunction, peroxidation damage and changes in glutathione metabolism in

PARK6. Neurobiol Dis. 2007;25:401–411. 63. Zhou W, Freed CR. DJ-1 up-regulates glutathione synthesis during oxidative stress and inhibits A53T alpha-synuclein toxicity. J Biol Chem.

2005;280:43150–43158. 64. Zhou W, Zhu M, Wilson MA, et al. The oxidation state of DJ-1 regulates its chaperone activity toward alpha-synuclein. J Mol Biol.

2006;356:1036–1048. 65. Choi J, Sullards MC, Olzmann JA, et al. Oxidative damage of DJ-1 is linked to sporadic Parkinson and Alzheimer diseases. J Biol Chem.

2006;281:10816–10824. 66. Rothstein JD, Martin LJ, Kuncl RW. Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N Engl J

Med. 1992;326:1464–1468. 67. Rothstein JD, Van Kammen M, Levey AI, et al. Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann

Neurol. 1995;38:73–84. 68. Bristol LA, Rothstein JD. Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex. Ann Neurol. 1996;39:676–679. 69. Chi L, Ke Y, Luo C, et al. Depletion of reduced glutathione enhances motor neuron degeneration in vitro and in vivo. Neuroscience.

2007;144:991–1003. 70. Lanius RA, Krieger C, Wagey R, et al. Increased [35S]glutathione binding sites in spinal cords from patients with sporadic amyotrophic

lateral sclerosis. Neurosci Lett. 1993;163:89–92. 71. Babu GN, Kumar A, Chandra R, et al. Oxidant-antioxidant imbalance in the erythrocytes of sporadic amyotrophic lateral sclerosis patients

correlates with the progression of disease. Neurochem Int. 2008;52:1284–1289. 72. Usarek E, Gajewska B, Kazmierczak B, et al. A study of glutathione S-transferase pi expression in central nervous system of subjects with

amyotrophic lateral sclerosis using RNA extraction from formalin-fixed, paraffin-embedded material. Neurochem Res. 2005;30:1003–1007. 73. Barber SC, Mead RJ, Shaw PJ. Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target. Biochim Biophys Acta.

2006;1762:1051–1067. 74. Klepac N, Relja M, Klepac R, et al. Oxidative stress parameters in plasma of Huntington’s disease patients, asymptomatic Huntington’s

disease gene carriers and healthy subjects: a cross-sectional study. J Neurol. 2007;254:1676–1683. 75. Li X, Valencia A, Sapp E, et al. Aberrant Rab11-dependent trafficking of the neuronal glutamate transporter EAAC1 causes oxidative stress

and cell death in Huntington’s disease. J Neurosci. 2010;30:4552–4561. 76. Kutzing MK, Firestein BL. Altered uric acid levels and disease states. J Pharmacol Exp Ther. 2008;324:1–7. 77. Ribeiro JA, Sebastiao AM. Caffeine and adenosine. J Alzheimers Dis. 2010;20(suppl 1):S3–S15. 78. Aoyama K, Matsumura N, Watabe M, et al. Caffeine and uric acid mediate glutathione synthesis for neuroprotection. Neuroscience.

2011;181:206–215. 79. Aoyama K, Matsumura N, Watabe M, et al. Oxidative stress on EAAC1 is involved in MPTP-induced glutathione depletion and motor

dysfunction. Eur J Neurosci. 2008;27:20–30. 80. Louwerse ES, Weverling GJ, Bossuyt PM, et al. Randomized, double-blind, controlled trial of acetylcysteine in amyotrophic lateral sclerosis.

Arch Neurol. 1995;52:559–564.