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research-article2014
MSJ0010.1177/1352458514533400Multiple Sclerosis JournalA Carvalho, J Lim
MULTIPLE SCLEROSIS MSJ JOURNAL
Topical Review
Glutathione in multiple sclerosis: More than just an antioxidant? Andreia N Carvalho, Jamie L Lim, Philip G Nijland, Maarten E Witte and Jack Van Horssen
Multiple Sclerosis Journal 2014, Vol. 20(11) 1425–1431 DOI: 10.1177/ 1352458514533400 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Abstract: Oxidative stress has been strongly implicated in both the inflammatory and neurodegenerative pathological mechanisms in multiple sclerosis (MS). In response to oxidative stress, cells increase and activate their cellular antioxidant mechanisms. Glutathione (GSH) is the major antioxidant in the brain, and as such plays a pivotal role in the detoxification of reactive oxidants. Previous research has shown that GSH homeostasis is altered in MS. In this review, we provide a comprehensive overview on GSH metabolism in brain cells, with a focus on its involvement in MS. The potential of GSH as an in vivo biomarker in MS is discussed, along with a short overview of improvements in imaging methods that allow non-invasive quantification of GSH in the brain. These methods might be instrumental in providing realtime measures of GSH, allowing the assessment of the oxidative state in MS patients and the monitoring of disease progression. Finally, the therapeutic potential of GSH in MS is discussed. Keywords: Antioxidant, biomarker, diagnostics, glutathione, imaging, metabolism, multiple sclerosis, oxidative stress, therapeutics Date received: 21 March 2014; accepted: 5 April 2014
Introduction Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting both the white matter (WM) and gray matter (GM) of the central nervous system (CNS) and has an immune as well as a neurodegenerative component.1 Although its etiology remains unknown, it is suggested that both genetic susceptibility and environmental factors are involved in the disease. The pathophysiological mechanisms involved in the onset and progression of MS include inflammation, demyelination, axonal and neuronal damage, oxidative stress and excitotoxicity.1 Relapsing–remitting MS (RRMS) is the most common form of MS at disease onset and is characterized by the episodic occurrence of clinical symptoms, which in most cases later on develops to secondary progressive MS (SPMS). Clinical symptoms in RRMS are caused by immune-mediated processes involving infiltrated leukocytes and activated microglia, whereas disease progression in SPMS is mainly characterized by neurodegeneration and extensive cortical demyelination, leading to brain atrophy.1 Importantly, reactive oxygen species (ROS) play a crucial role in several pathological processes in both
early and late-stage MS, making them an interesting therapeutic target.2 Oxidative stress and antioxidant mechanisms in MS ROS are highly reactive molecules that play important roles in various physiological cellular processes, such as cell signaling, gene expression and host defense; however, increased ROS levels induce oxidative stress, which is a common pathological feature in several neurological disorders, including MS.2,3 To protect themselves from ROS-induced damage and cell death, cells are equipped with an elaborate antioxidant machinery and under physiological conditions, the levels of cellular ROS are in equilibrium with this endogenous antioxidant system; however, when this balance is altered by either an increase in ROS production or reduced antioxidant protection, oxidative stress and subsequent damage to proteins, lipids and deoxyribonucleic acid (DNA) occurs.2,3 The CNS is particularly sensitive to oxidative stress, due to its high cellular metabolic activity and enrichment in polyunsaturated fatty acids.4 Furthermore, the activities of antioxidant enzymes, such as superoxide
Correspondence to: Andreia N Carvalho Vrije Universiteit (VU) University Medical Center Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. [email protected] Jamie Lim Philip G Nijland Maarten E Witte Jack Van Horssen Vrije Universiteit (VU) University Medical Center, Amsterdam, The Netherlands Authors Witte and Van Horssen contributed equally.
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Multiple Sclerosis Journal 20(11) dismutase (SOD), catalase and glutathione peroxidase (GPx) are reported to be lower in the brain, compared to other tissues.4 Altogether, this suggests that detoxification and antioxidant protection against ROS are essential processes within the brain, and many reports underscore the importance of oxidative stress in MS pathogenesis.2, 5–7 Sources of ROS in MS: Activated macrophages and microglia, and mitochondrial dysfunction Two different sources are mainly responsible for ROS production within the cell: ROS-producing enzymes and mitochondria. ROS-producing enzymes are highly expressed in macrophages, neutrophils and microglia, in order to kill invading pathogens in a process called oxidative burst. In the active stage of MS lesion formation, the expression of several ROSgenerating enzymes, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs),5 myeloperoxidase (MPO)8 and inducible nitric oxide (NO) synthase (iNOS)9 have been found to increase, particularly in microglia and macrophages. Mitochondria produce ROS as a byproduct of cellular respiration and impaired mitochondrial function is commonly associated with enhanced production of ROS.10 Dysfunctional mitochondria were recently shown to be implicated in both early and chronic stages of MS pathology, actively contributing to both axonal and neuronal injury.11–13 The cause of mitochondrial dysfunction in patients with MS has been debated, but evidence is emerging that ROS production by macrophages/microglia might induce mitochondrial dysfunction in patients with MS,10,13 possibly via oxidative damage to mitochondrial DNA.12 Oxidative damage contribution to MS lesion formation In the initial phase of MS lesion formation, locally produced ROS can induce blood-brain barrier (BBB) disruption and promote leukocyte migration. In vitro studies demonstrate that ROS promote BBB permeability, by causing cytoskeletal rearrangements in endothelial cells, and redistribution and loss of tight junctions.14 These monocyte-induced barrier changes and the subsequent monocyte migration are preventable by ROS scavengers,14,15 providing evidence that ROS play a key role in transendothelial leukocyte migration. In turn, ROS produced by infiltrated leukocytes and resident microglia are thought to contribute to MS pathology, by promoting myelin phagocytosis16 and causing oxidative damage-induced
oligodendrocyte cell death, axonal injury and mitochondrial dysfunction.2 Concurrently, several markers of oxidative stress were found to be increased in the serum and brain of MS patients.6,7,17 Together, this suggests that oxidative stress plays an important role in the early, active phase of MS. Although inflammation is less prominent in progressive MS, microglial activation is still widespread and is strongly linked to local oxidative damage, suggesting ongoing ROS production by activated microglia. Moreover, mitochondrial dysfunction in the cortical neurons of progressive MS patients is extensive and likely to contribute to the ongoing oxidative stress in advanced stages of the disease.10,13 Given the susceptibility of the CNS to ROS, it is conceivable that oxidative stress significantly contributes to neurodegeneration in progressive MS. Antioxidant mechanisms in MS To counter-balance the potential detrimental effects of ROS, the CNS is endowed with antioxidant defense mechanisms consisting of antioxidant enzymes, such as SOD, GPx, glutathione reductase (GR) and catalase.18 The expression of the vast majority of antioxidant enzymes is controlled by binding of the transcription factor called nuclear factor-erythroid 2-related factor 2 (Nrf2) to the antioxidant response element in the promoters of target genes, such as heme oxygenase-1, NADPH:quinone oxidoreductase-1, peroxiredoxins and several proteins involved in glutathione (GSH) metabolism.19,20 In inflammatory MS lesions, the expression of Nrf2-regulated antioxidant proteins increases.7 This suggests that in response to oxidative and inflammatory damage, the main protective cellular antioxidant mechanisms, namely those regulated by Nrf2, are activated early in lesion formation; however, enhanced levels of endogenous antioxidants are detected concomitantly with several oxidative stress markers, indicating that the endogenous antioxidant response is not sufficient to restore the delicate redox-balance and prevent oxidative damage.6,7 Glutathione metabolism in the CNS Glutathione metabolism and transport in brain cells Nrf2, the main regulator of the orchestrated cellular antioxidant response, modulates the cellular levels of a vast array of antioxidants, including the most abundant mammalian intracellular thiol-containing antioxidant, GSH (designating the tripeptide
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A Carvalho, J Lim et al. intracellular ratio of > 100:1 (GSH:GSSG) and serves as an important buffer to maintain the cellular reduction-oxidation (redox) status. Glutathione has a diverse array of functions in the cell: It functions as a co-factor in the detoxification reactions catalyzed by glutathione S-transferases (GSTs) and is involved in both direct (non-enzymatic) and indirect (enzymatic, GPx-mediated) inactivation of free radicals21 (Figure1). GSH is also important in the storage and transport of cysteine and the maintenance of protein sulfhydryl groups in a reduced form.3,18,21
Figure 1. Glutathione synthesis and metabolism. GSH is synthesized intracellularly from its three precursor amino acids, Glu, Cys and Gly; in two sequential ATPconsuming reactions, catalyzed by γ-GCS and GS, respectively. GSH synthesis is limited by Cys availability and the rate-limiting step is the reaction catalyzed by γGCS. GSH can be exported to the extracellular space and metabolized into its constituent amino acids, which can then be re-used in GSH synthesis. GSH can react nonenzymatically with free radicals or be an electron donor in the detoxification of peroxides (e.g. hydrogen peroxide), by GPx. In this reaction, GSH is oxidized to GSSG, which can subsequently be regenerated to GSH by GR at the expense of NADPH. ATP: Adenosine triphosphate; Cys: cysteine; γ-GCS: gamma glutamylcysteine synthetase; Glu: glutamate; Gly: glycine; GPx: glutathione peroxidase; GR: glutathione reductase; GS: glutathione synthetase; GSH: reduced form of glutathione; GSSG: oxidized form of glutathione; NADPH: nicotinamide adenine dinucleotide phosphate.
γ-glutamyl-cysteinyl-glycine). A crucial component of the non-enzymatic branch of the cellular detoxification of ROS, GSH is ubiquitously distributed in cells throughout the body,3,18 and impairment of GSH metabolism is involved in the pathogenesis of several neurodegenerative diseases.3 GSH is synthesized in two sequential ATP-dependent reactions: first, γ-glutamylcysteine is formed from glutamate and cysteine through the action of γ-glutamylcysteine synthetase (γ-GCS); and second, GSH is synthesized by γ-glutamylcysteine binding to glycine, in a reaction catalyzed by GSH-synthetase (GS)18,21 (Figure1). The synthesis of glutathione is limited by cysteine bioavailability and the γ-GCS-catalyzed reaction is the rate-limiting step.18,21 Glutathione is present in the cell in both the reduced (GSH) and oxidized (GSSG) forms, in a typical
In CNS cells, the GSH concentration is typically around 1–2 mM, several hundred times greater than seen in the cerebrospinal fluid (CSF), which is ~4µM, and in the blood, which is ~2µM. To maintain this rather disparate concentration ratio, active intracellular GSH synthesis is required.3 It has been suggested that GSH import from the blood into the brain occurs through a transporter in brain capillaries and endothelial cells; however, the concept of transport of the intact GSH molecule through the BBB is controversial and the characterization of such a transporter is limited.18 Therefore, it is debatable whether direct uptake can significantly contribute to GSH levels in brain cells; and probably, the major source of GSH within the brain is re-synthesis, through recycling of its constituents. In the brain, GSH is synthesized by both astrocytes and neurons, and it is indispensable for several important antioxidant-related processes in both cell types.18 Antioxidant coupling between neurons and astrocytes Brain cells are differentially vulnerable to oxidative stress, with neurons and oligodendrocytes being more susceptible to oxidative stress than astrocytes, which in general have the highest GSH levels. Moreover, astrocytes have a more efficient GSH metabolism, as they are able to utilize a wider variety of precursor substrates for GSH synthesis.20,21 Astrocytes take up cystine through the cystine/glutamate exchanger (Xc-) and cystine is immediately reduced to cysteine and secreted or used for the synthesis of GSH.21 Astrocytes also display higher basal and stress-induced activity of Nrf2, leading to enhanced expression of Phase II detoxification enzymes and antioxidant enzymes, such as γ-GCS, which contribute to increased GSH concentration.20 On the other hand, neurons, although capable of synthesizing GSH, depend on neighboring astrocytes for the supply of GSH precursors. Neuronal GSH content is not complemented by direct GSH uptake, because extracellular GSH concentration is very low and so
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Multiple Sclerosis Journal 20(11) would cost a disproportionate amount of energy. Therefore, neurons take up cysteine, which has been metabolized from astrocyte-derived GSH, from the extracellular space, through the excitatory amino acid transporter 3 (EAAT3/EAAC1). Interestingly, mice deficient for EAAT3 manifest increased neurodegeneration with age, in association with reduced neuronal GSH levels and increased vulnerability to oxidative stress.22 Moreover, astrocytes efficiently take up glutamate, which can then be converted to glutamine. Neurons can, in turn, take up astrocyte-secreted glutamine and use it for GSH synthesis.20,21 Although the de novo synthesis of GSH is essential to replenish its levels, a considerable amount can also be recycled by GR, which reduces GSSG and regenerates GSH at the expense of NADPH (Figure 1). NADPH is produced in the pentose phosphate pathway (PPP). Both astrocytes and neurons possess PPP capacity; however, neurons are more dependent on high PPP activity to maintain adequate GSH pools.20 Thus, sufficient neuronal NADPH production and astrocytic release of glutamine and cysteine are essential to maintain adequate neuronal antioxidant capacity. Glutathione metabolism impairment in MS Glutathione metabolism dysfunction in MS The glutathione oxidation status (GSH/GSSG) is considered one of the best markers of cellular redox equilibrium. In the CSF of MS patients, the GSH levels were shown to be significantly lower when compared to controls, likely reflecting lower GSH in the CNS.23 Moreover, the excitatory neurotransmitter glutamate was shown to be elevated in peripheral blood, CSF and the CNS, in both acute MS lesions and normalappearing white matter (NAWM) of MS patients.24 Aside from inducing excitotoxicity, excess glutamate can also compete with cystine for the use of the Xc-, blocking the synthesis of GSH and inducing oxidative stress.25 Active MS lesions display high expression levels of glutaminase, which converts glutamine to glutamate in both macrophages and microglia in the close vicinity of dystrophic axons, indicating that glutamate production by macrophages might underlie axonal degeneration and oligodendrocyte death in MS.24 Concordant with this, several studies show that oligodendrocytes have particularly low levels of GSH, rendering them more susceptible to oxidative stress damage.2,18, 23 Oxidative stress has been strongly suggested to play an important role in the early, active phase of MS and also to contribute to neurodegeneration in the later
stages.2,10 Interestingly, a genetically determined ability to remove toxic products of oxidative stress, namely through GSTs-catalyzed reactions, has been shown to influence the long-term prognosis in MS.26 The authors reported that polymorphisms in the GSTs of the class mu (GSTM) and class pi (GSTP), such as the GSTM3 AA genotype and homozygosity for both the GSTM1*0 and GSTP1*Ile105-containing allele are associated with more severe disability in patients with a disease duration of more than 10 years.26 In fact, GSTP polymorphisms can affect substrate selectivity and stability, thereby increasing individual susceptibility; and individuals with the GSTP1*B allele have lower GSTP enzyme activity, thus displaying less efficient detoxification capacity. In line with this, the genetic markers of GSTs were differentially expressed in both acute and chronic MS plaque tissue, when compared to NAWM.27 Moreover, in a recent study, peripheral blood levels of several oxidative stress markers were shown to be increased in RRMS patients, whereas the levels of GPx, GST and total antioxidant capacity were decreased.17 Curiously, GSH levels in the blood of MS patients increased, which might in part be explained by the reported increase in GR activity that regenerates GSH from GSSG.17,23 GPx activity in blood cells (erythrocytes and leukocytes) of MS patients is reported to be lower than in controls.28 In accordance, Calabrese and co-workers29 reported decreased GPx activity and increased GR activity in the CSF of MS patients; however, Sorensen and coworkers30 found no changes in the activity of GPx in the lymphocytes of MS patients during acute relapses, and although having reported decreased GPx activity in erythrocytes of MS patients, Szeinberg and colleagues31 reported normal GPx activity in lymphocytes. Nevertheless, a significant increase in GPx gene expression is found in inflammatory MS lesions.27 In a chronic experimental autoimmune encephalomyelitis (EAE) model, daily intraperitoneal injection of GPx or catalase reduces the loss of BBB integrity,32 suggesting that GPx might have a protective role against oxidative stress and neuroinflammation. Recently, a decrease in GSH levels and GPx activity in the erythrocytes of MS patients was reported: The decrease in GSH correlated with neurological and radiological scoring of acute CNS inflammation.33 Taken together, due to its central role in the antioxidant process and high sensitivity to cellular redox changes, GSH is an important indicator of oxidative status in the human brain and constitutes a potentially interesting candidate as a biomarker in MS. Moreover,
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A Carvalho, J Lim et al. given the variations in the measurements of GSH and GSH-related enzymes in blood and CSF between diverse studies, imaging methods that allow the noninvasive quantification of GSH in the brain might be useful in providing accurate real-time measures of GSH. Imaging methods for measuring brain GSH Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) provide an ideal method for assessing GSH levels and fluctuations.34,35 GSH is consumed in detoxification and antioxidant processes; therefore, its levels need to be replenished. The dynamic nature of this process makes GSH measurement one of the best tools to assess current oxidative stress status in the brain.18 In line with this, Choi et al.34 show that GSH levels in the frontal-parietal brain regions of SPMS patients are lower, when compared to controls, as measured with magnetic resonance chemical shift imaging (CSI) at a field strength of 3T, indicating the presence of oxidative stress in SPMS. Furthermore, in another study using a MRS editing scheme, Srinivasan et al.35 were able to generate an unobstructed in vivo detection and quantification of GSH at 7T, eliminating the technical challenges associated with overlapping resonances and low GSH concentrations in the human brain. As a result, they obtained normative differences in white and gray matter GSH concentrations over a 2-dimensional region in the human brain. The authors demonstrate that GSH levels are significantly higher in GM, relative to WM, in normal subjects; and they report a significant reduction of GSH in the GM of MS patients, when compared to controls.35 No significant differences in GSH levels were found between controls and MS patients in NAWM, but in WM lesions the GSH levels decreased. This preliminary investigation demonstrates the potential of this marker to probe the oxidative state in MS; however, further research is needed to better elucidate the processes underlying GSH variations during disease progression. The method reported in this study allows non-invasive, in vivo measurement of GSH and it might be used to explore both its sensitivity to detect changes in the oxidative state in MS patients and to monitor the progression of the disease. Glutathione as a therapeutic Current MS therapeutics are primarily immunomodulatory and are relatively successful in reducing the number of inflammatory lesions in RRMS; however, there is a complete lack of therapeutics capable of slowing down disease progression in the later stages
of the disease. As ROS are crucial players in both the inflammatory and degenerative disease processes, they provide an interesting therapeutic target in both RRMS and progressive MS.2,10 GSH plays a central role in the maintenance of cellular redox homeostasis and protection against ROS. Given the involvement of decreased GSH levels and alterations in glutathione-related enzyme activities in MS, maintaining or restoring GSH levels represents a promising therapeutic strategy.3 The apparently most straightforward approach for increasing GSH levels, through its direct administration, failed due to the solubility, absorption and stability constrains that limit its bioavailability.3,36,37 GSH has a low half-life in human plasma and, as a hydrophilic molecule, it is not able to freely diffuse through the BBB. Therefore, GSH delivery to the CNS is particularly challenging. Extremely high doses of GSH would be required in order to achieve a therapeutic effect. The direct delivery of the precursor amino acid cysteine was also a failure, due to its toxic effects.37 Hence, research efforts turned to the identification and synthesis of GSH precursors and analogues that could mimic GSH’s cellular redox protective effects. A promising therapeutic approach is the supplementation of endogenous GSH levels, by using prodrugs. For instance, GSH ester prodrugs are able to cross cell membranes, due to their lipophilic characteristics, and are able to increase cellular GSH content in several tissues and restore the GSH pool in mitochondria.36 Another candidate would be cysteine prodrugs that are able to bypass the toxic effects that direct administration of cysteine causes. In the search for GSH precursors and analogues that could improve its pharmacological properties and bioavailability, the antioxidant N-acetyl-cysteine (NAC) was found to have promising protective effects for several neurodegenerative diseases, by potentiating intracellular GSH synthesis, promoting detoxification and acting as a ROS scavenger.3 Administration of NAC reduced inflammation and induced the replenishment of GSH levels in EAE animals38,39; however, no clinical trials have been conducted using NAC in MS. Although direct oral GSH intake does not seem to have an impact on brain GSH levels, several compounds can indirectly induce GSH production. Therefore, another effective approach involves the use of compounds that activate the Nrf2 pathway, as most enzymes involved in glutathione synthesis are induced by Nrf2 activation. Monomethylfumarate is a
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Multiple Sclerosis Journal 20(11) well-known Nrf2 activator and was demonstrated to enhance glutathione levels, and increased both cellular antioxidant and anti-inflammatory potential, in a Nrf2dependent manner.40–42 Recent clinical trials with oral dimethylfumarate (Tecfidera® on the pharmaceutical market) clearly highlight the therapeutic potential of Nrf2 activation in MS (review, Fox et al.43). In conclusion, due to its central role in antioxidant metabolism and its involvement in the several phases of MS progression, glutathione constitutes a privileged tool. As a biomarker, it can be safely used to predict disease progression by non-invasive imaging methods, constituting a potentially helpful tool in diagnosis. In addition, glutathione represents an interesting therapeutic target to combat oxidative stress, not only in the initial inflammatory period of the disease, but also in preventing the neurodegenerative processes associated with progressive MS. Conflict of interest The authors declare that there are no conflicts of interest. Funding This work was supported by the ECTRIMS Postdoctoral Research Fellowship Programme (Post doctoral Fellowship to ANC).
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33. Ljubisavljevic S, Stojanovic I, Cvetkovic T, et al. Glutathione homeostasis disruption of erythrocytes, but not glutathione peroxidase activity change, is closely accompanied with neurological and radiological scoring of acute CNS inflammation. Neuroimmunomodulation 2014; 21: 13–20. 34. Choi IY, Lee SP, Denney DR, et al. Lower levels of glutathione in the brains of secondary progressive multiple sclerosis patients measured by 1H magnetic resonance chemical shift imaging at 3 T. Mult Scler 2011; 17: 289–296. 35. Srinivasan R, Ratiney H, Hammond-Rosenbluth KE, et al. MR spectroscopic imaging of glutathione in the white and gray matter at 7 T with an application to multiple sclerosis. Magnet Res Imag 2010; 28: 163–170. 36. Cacciatore I, Baldassarre L, Fornasari E, et al. Recent advances in the treatment of neurodegenerative diseases based on GSH delivery systems. Oxidat Med Cell Longev 2012; 2012: 240146. 37. Wu JH and Batist G. Glutathione and glutathione analogues; therapeutic potentials. Biochim Biophys Acta 2013; 1830: 3350–3353. 38. Ljubisavljevic S, Stojanovic I, Pavlovic D, et al. Aminoguanidine and N-acetyl-cysteine supress oxidative and nitrosative stress in EAE rat brains. Redox Rep 2011; 16: 166–172. 39. Stanislaus R, Gilg AG, Singh AK, et al. N-acetyl-Lcysteine ameliorates the inflammatory disease process in experimental autoimmune encephalomyelitis in Lewis rats. J Autoimmune Dis 2005; 2: 4. 40. Lin SX, Lisi L, Dello Russo C, et al. The antiinflammatory effects of dimethyl fumarate in astrocytes involve glutathione and haem oxygenase-1. ASN Neuro 2011; 3. 41. Linker RA, Lee DH, Ryan S, et al. Fumaric acid esters exert neuroprotective effects in neuroinflammation via activation of the Nrf2 antioxidant pathway. Brain 2011; 134: 678–692. 42. Scannevin RH, Chollate S, Jung MY, et al. Fumarates promote cytoprotection of central nervous system cells against oxidative stress via the nuclear factor (erythroid-derived 2)-like 2 pathway. J Pharmacol Exp Ther 2012; 341: 274–284. 43. Fox RJ, Kita M, Cohan SL, et al. BG-12 (dimethyl fumarate): A review of mechanism of action, efficacy and safety. Curr Med Res Opin 2014; 30: 251–262.
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research-article2014
MSJ0010.1177/1352458514533400Multiple Sclerosis JournalA Carvalho, J Lim
MULTIPLE SCLEROSIS MSJ JOURNAL
Topical Review
Glutathione in multiple sclerosis: More than just an antioxidant? Andreia N Carvalho, Jamie L Lim, Philip G Nijland, Maarten E Witte and Jack Van Horssen
Multiple Sclerosis Journal 2014, Vol. 20(11) 1425–1431 DOI: 10.1177/ 1352458514533400 © The Author(s), 2014. Reprints and permissions: http://www.sagepub.co.uk/ journalsPermissions.nav
Abstract: Oxidative stress has been strongly implicated in both the inflammatory and neurodegenerative pathological mechanisms in multiple sclerosis (MS). In response to oxidative stress, cells increase and activate their cellular antioxidant mechanisms. Glutathione (GSH) is the major antioxidant in the brain, and as such plays a pivotal role in the detoxification of reactive oxidants. Previous research has shown that GSH homeostasis is altered in MS. In this review, we provide a comprehensive overview on GSH metabolism in brain cells, with a focus on its involvement in MS. The potential of GSH as an in vivo biomarker in MS is discussed, along with a short overview of improvements in imaging methods that allow non-invasive quantification of GSH in the brain. These methods might be instrumental in providing realtime measures of GSH, allowing the assessment of the oxidative state in MS patients and the monitoring of disease progression. Finally, the therapeutic potential of GSH in MS is discussed. Keywords: Antioxidant, biomarker, diagnostics, glutathione, imaging, metabolism, multiple sclerosis, oxidative stress, therapeutics Date received: 21 March 2014; accepted: 5 April 2014
Introduction Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease affecting both the white matter (WM) and gray matter (GM) of the central nervous system (CNS) and has an immune as well as a neurodegenerative component.1 Although its etiology remains unknown, it is suggested that both genetic susceptibility and environmental factors are involved in the disease. The pathophysiological mechanisms involved in the onset and progression of MS include inflammation, demyelination, axonal and neuronal damage, oxidative stress and excitotoxicity.1 Relapsing–remitting MS (RRMS) is the most common form of MS at disease onset and is characterized by the episodic occurrence of clinical symptoms, which in most cases later on develops to secondary progressive MS (SPMS). Clinical symptoms in RRMS are caused by immune-mediated processes involving infiltrated leukocytes and activated microglia, whereas disease progression in SPMS is mainly characterized by neurodegeneration and extensive cortical demyelination, leading to brain atrophy.1 Importantly, reactive oxygen species (ROS) play a crucial role in several pathological processes in both
early and late-stage MS, making them an interesting therapeutic target.2 Oxidative stress and antioxidant mechanisms in MS ROS are highly reactive molecules that play important roles in various physiological cellular processes, such as cell signaling, gene expression and host defense; however, increased ROS levels induce oxidative stress, which is a common pathological feature in several neurological disorders, including MS.2,3 To protect themselves from ROS-induced damage and cell death, cells are equipped with an elaborate antioxidant machinery and under physiological conditions, the levels of cellular ROS are in equilibrium with this endogenous antioxidant system; however, when this balance is altered by either an increase in ROS production or reduced antioxidant protection, oxidative stress and subsequent damage to proteins, lipids and deoxyribonucleic acid (DNA) occurs.2,3 The CNS is particularly sensitive to oxidative stress, due to its high cellular metabolic activity and enrichment in polyunsaturated fatty acids.4 Furthermore, the activities of antioxidant enzymes, such as superoxide
Correspondence to: Andreia N Carvalho Vrije Universiteit (VU) University Medical Center Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. [email protected] Jamie Lim Philip G Nijland Maarten E Witte Jack Van Horssen Vrije Universiteit (VU) University Medical Center, Amsterdam, The Netherlands Authors Witte and Van Horssen contributed equally.
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Multiple Sclerosis Journal 20(11) dismutase (SOD), catalase and glutathione peroxidase (GPx) are reported to be lower in the brain, compared to other tissues.4 Altogether, this suggests that detoxification and antioxidant protection against ROS are essential processes within the brain, and many reports underscore the importance of oxidative stress in MS pathogenesis.2, 5–7 Sources of ROS in MS: Activated macrophages and microglia, and mitochondrial dysfunction Two different sources are mainly responsible for ROS production within the cell: ROS-producing enzymes and mitochondria. ROS-producing enzymes are highly expressed in macrophages, neutrophils and microglia, in order to kill invading pathogens in a process called oxidative burst. In the active stage of MS lesion formation, the expression of several ROSgenerating enzymes, including nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs),5 myeloperoxidase (MPO)8 and inducible nitric oxide (NO) synthase (iNOS)9 have been found to increase, particularly in microglia and macrophages. Mitochondria produce ROS as a byproduct of cellular respiration and impaired mitochondrial function is commonly associated with enhanced production of ROS.10 Dysfunctional mitochondria were recently shown to be implicated in both early and chronic stages of MS pathology, actively contributing to both axonal and neuronal injury.11–13 The cause of mitochondrial dysfunction in patients with MS has been debated, but evidence is emerging that ROS production by macrophages/microglia might induce mitochondrial dysfunction in patients with MS,10,13 possibly via oxidative damage to mitochondrial DNA.12 Oxidative damage contribution to MS lesion formation In the initial phase of MS lesion formation, locally produced ROS can induce blood-brain barrier (BBB) disruption and promote leukocyte migration. In vitro studies demonstrate that ROS promote BBB permeability, by causing cytoskeletal rearrangements in endothelial cells, and redistribution and loss of tight junctions.14 These monocyte-induced barrier changes and the subsequent monocyte migration are preventable by ROS scavengers,14,15 providing evidence that ROS play a key role in transendothelial leukocyte migration. In turn, ROS produced by infiltrated leukocytes and resident microglia are thought to contribute to MS pathology, by promoting myelin phagocytosis16 and causing oxidative damage-induced
oligodendrocyte cell death, axonal injury and mitochondrial dysfunction.2 Concurrently, several markers of oxidative stress were found to be increased in the serum and brain of MS patients.6,7,17 Together, this suggests that oxidative stress plays an important role in the early, active phase of MS. Although inflammation is less prominent in progressive MS, microglial activation is still widespread and is strongly linked to local oxidative damage, suggesting ongoing ROS production by activated microglia. Moreover, mitochondrial dysfunction in the cortical neurons of progressive MS patients is extensive and likely to contribute to the ongoing oxidative stress in advanced stages of the disease.10,13 Given the susceptibility of the CNS to ROS, it is conceivable that oxidative stress significantly contributes to neurodegeneration in progressive MS. Antioxidant mechanisms in MS To counter-balance the potential detrimental effects of ROS, the CNS is endowed with antioxidant defense mechanisms consisting of antioxidant enzymes, such as SOD, GPx, glutathione reductase (GR) and catalase.18 The expression of the vast majority of antioxidant enzymes is controlled by binding of the transcription factor called nuclear factor-erythroid 2-related factor 2 (Nrf2) to the antioxidant response element in the promoters of target genes, such as heme oxygenase-1, NADPH:quinone oxidoreductase-1, peroxiredoxins and several proteins involved in glutathione (GSH) metabolism.19,20 In inflammatory MS lesions, the expression of Nrf2-regulated antioxidant proteins increases.7 This suggests that in response to oxidative and inflammatory damage, the main protective cellular antioxidant mechanisms, namely those regulated by Nrf2, are activated early in lesion formation; however, enhanced levels of endogenous antioxidants are detected concomitantly with several oxidative stress markers, indicating that the endogenous antioxidant response is not sufficient to restore the delicate redox-balance and prevent oxidative damage.6,7 Glutathione metabolism in the CNS Glutathione metabolism and transport in brain cells Nrf2, the main regulator of the orchestrated cellular antioxidant response, modulates the cellular levels of a vast array of antioxidants, including the most abundant mammalian intracellular thiol-containing antioxidant, GSH (designating the tripeptide
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A Carvalho, J Lim et al. intracellular ratio of > 100:1 (GSH:GSSG) and serves as an important buffer to maintain the cellular reduction-oxidation (redox) status. Glutathione has a diverse array of functions in the cell: It functions as a co-factor in the detoxification reactions catalyzed by glutathione S-transferases (GSTs) and is involved in both direct (non-enzymatic) and indirect (enzymatic, GPx-mediated) inactivation of free radicals21 (Figure1). GSH is also important in the storage and transport of cysteine and the maintenance of protein sulfhydryl groups in a reduced form.3,18,21
Figure 1. Glutathione synthesis and metabolism. GSH is synthesized intracellularly from its three precursor amino acids, Glu, Cys and Gly; in two sequential ATPconsuming reactions, catalyzed by γ-GCS and GS, respectively. GSH synthesis is limited by Cys availability and the rate-limiting step is the reaction catalyzed by γGCS. GSH can be exported to the extracellular space and metabolized into its constituent amino acids, which can then be re-used in GSH synthesis. GSH can react nonenzymatically with free radicals or be an electron donor in the detoxification of peroxides (e.g. hydrogen peroxide), by GPx. In this reaction, GSH is oxidized to GSSG, which can subsequently be regenerated to GSH by GR at the expense of NADPH. ATP: Adenosine triphosphate; Cys: cysteine; γ-GCS: gamma glutamylcysteine synthetase; Glu: glutamate; Gly: glycine; GPx: glutathione peroxidase; GR: glutathione reductase; GS: glutathione synthetase; GSH: reduced form of glutathione; GSSG: oxidized form of glutathione; NADPH: nicotinamide adenine dinucleotide phosphate.
γ-glutamyl-cysteinyl-glycine). A crucial component of the non-enzymatic branch of the cellular detoxification of ROS, GSH is ubiquitously distributed in cells throughout the body,3,18 and impairment of GSH metabolism is involved in the pathogenesis of several neurodegenerative diseases.3 GSH is synthesized in two sequential ATP-dependent reactions: first, γ-glutamylcysteine is formed from glutamate and cysteine through the action of γ-glutamylcysteine synthetase (γ-GCS); and second, GSH is synthesized by γ-glutamylcysteine binding to glycine, in a reaction catalyzed by GSH-synthetase (GS)18,21 (Figure1). The synthesis of glutathione is limited by cysteine bioavailability and the γ-GCS-catalyzed reaction is the rate-limiting step.18,21 Glutathione is present in the cell in both the reduced (GSH) and oxidized (GSSG) forms, in a typical
In CNS cells, the GSH concentration is typically around 1–2 mM, several hundred times greater than seen in the cerebrospinal fluid (CSF), which is ~4µM, and in the blood, which is ~2µM. To maintain this rather disparate concentration ratio, active intracellular GSH synthesis is required.3 It has been suggested that GSH import from the blood into the brain occurs through a transporter in brain capillaries and endothelial cells; however, the concept of transport of the intact GSH molecule through the BBB is controversial and the characterization of such a transporter is limited.18 Therefore, it is debatable whether direct uptake can significantly contribute to GSH levels in brain cells; and probably, the major source of GSH within the brain is re-synthesis, through recycling of its constituents. In the brain, GSH is synthesized by both astrocytes and neurons, and it is indispensable for several important antioxidant-related processes in both cell types.18 Antioxidant coupling between neurons and astrocytes Brain cells are differentially vulnerable to oxidative stress, with neurons and oligodendrocytes being more susceptible to oxidative stress than astrocytes, which in general have the highest GSH levels. Moreover, astrocytes have a more efficient GSH metabolism, as they are able to utilize a wider variety of precursor substrates for GSH synthesis.20,21 Astrocytes take up cystine through the cystine/glutamate exchanger (Xc-) and cystine is immediately reduced to cysteine and secreted or used for the synthesis of GSH.21 Astrocytes also display higher basal and stress-induced activity of Nrf2, leading to enhanced expression of Phase II detoxification enzymes and antioxidant enzymes, such as γ-GCS, which contribute to increased GSH concentration.20 On the other hand, neurons, although capable of synthesizing GSH, depend on neighboring astrocytes for the supply of GSH precursors. Neuronal GSH content is not complemented by direct GSH uptake, because extracellular GSH concentration is very low and so
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Multiple Sclerosis Journal 20(11) would cost a disproportionate amount of energy. Therefore, neurons take up cysteine, which has been metabolized from astrocyte-derived GSH, from the extracellular space, through the excitatory amino acid transporter 3 (EAAT3/EAAC1). Interestingly, mice deficient for EAAT3 manifest increased neurodegeneration with age, in association with reduced neuronal GSH levels and increased vulnerability to oxidative stress.22 Moreover, astrocytes efficiently take up glutamate, which can then be converted to glutamine. Neurons can, in turn, take up astrocyte-secreted glutamine and use it for GSH synthesis.20,21 Although the de novo synthesis of GSH is essential to replenish its levels, a considerable amount can also be recycled by GR, which reduces GSSG and regenerates GSH at the expense of NADPH (Figure 1). NADPH is produced in the pentose phosphate pathway (PPP). Both astrocytes and neurons possess PPP capacity; however, neurons are more dependent on high PPP activity to maintain adequate GSH pools.20 Thus, sufficient neuronal NADPH production and astrocytic release of glutamine and cysteine are essential to maintain adequate neuronal antioxidant capacity. Glutathione metabolism impairment in MS Glutathione metabolism dysfunction in MS The glutathione oxidation status (GSH/GSSG) is considered one of the best markers of cellular redox equilibrium. In the CSF of MS patients, the GSH levels were shown to be significantly lower when compared to controls, likely reflecting lower GSH in the CNS.23 Moreover, the excitatory neurotransmitter glutamate was shown to be elevated in peripheral blood, CSF and the CNS, in both acute MS lesions and normalappearing white matter (NAWM) of MS patients.24 Aside from inducing excitotoxicity, excess glutamate can also compete with cystine for the use of the Xc-, blocking the synthesis of GSH and inducing oxidative stress.25 Active MS lesions display high expression levels of glutaminase, which converts glutamine to glutamate in both macrophages and microglia in the close vicinity of dystrophic axons, indicating that glutamate production by macrophages might underlie axonal degeneration and oligodendrocyte death in MS.24 Concordant with this, several studies show that oligodendrocytes have particularly low levels of GSH, rendering them more susceptible to oxidative stress damage.2,18, 23 Oxidative stress has been strongly suggested to play an important role in the early, active phase of MS and also to contribute to neurodegeneration in the later
stages.2,10 Interestingly, a genetically determined ability to remove toxic products of oxidative stress, namely through GSTs-catalyzed reactions, has been shown to influence the long-term prognosis in MS.26 The authors reported that polymorphisms in the GSTs of the class mu (GSTM) and class pi (GSTP), such as the GSTM3 AA genotype and homozygosity for both the GSTM1*0 and GSTP1*Ile105-containing allele are associated with more severe disability in patients with a disease duration of more than 10 years.26 In fact, GSTP polymorphisms can affect substrate selectivity and stability, thereby increasing individual susceptibility; and individuals with the GSTP1*B allele have lower GSTP enzyme activity, thus displaying less efficient detoxification capacity. In line with this, the genetic markers of GSTs were differentially expressed in both acute and chronic MS plaque tissue, when compared to NAWM.27 Moreover, in a recent study, peripheral blood levels of several oxidative stress markers were shown to be increased in RRMS patients, whereas the levels of GPx, GST and total antioxidant capacity were decreased.17 Curiously, GSH levels in the blood of MS patients increased, which might in part be explained by the reported increase in GR activity that regenerates GSH from GSSG.17,23 GPx activity in blood cells (erythrocytes and leukocytes) of MS patients is reported to be lower than in controls.28 In accordance, Calabrese and co-workers29 reported decreased GPx activity and increased GR activity in the CSF of MS patients; however, Sorensen and coworkers30 found no changes in the activity of GPx in the lymphocytes of MS patients during acute relapses, and although having reported decreased GPx activity in erythrocytes of MS patients, Szeinberg and colleagues31 reported normal GPx activity in lymphocytes. Nevertheless, a significant increase in GPx gene expression is found in inflammatory MS lesions.27 In a chronic experimental autoimmune encephalomyelitis (EAE) model, daily intraperitoneal injection of GPx or catalase reduces the loss of BBB integrity,32 suggesting that GPx might have a protective role against oxidative stress and neuroinflammation. Recently, a decrease in GSH levels and GPx activity in the erythrocytes of MS patients was reported: The decrease in GSH correlated with neurological and radiological scoring of acute CNS inflammation.33 Taken together, due to its central role in the antioxidant process and high sensitivity to cellular redox changes, GSH is an important indicator of oxidative status in the human brain and constitutes a potentially interesting candidate as a biomarker in MS. Moreover,
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A Carvalho, J Lim et al. given the variations in the measurements of GSH and GSH-related enzymes in blood and CSF between diverse studies, imaging methods that allow the noninvasive quantification of GSH in the brain might be useful in providing accurate real-time measures of GSH. Imaging methods for measuring brain GSH Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) provide an ideal method for assessing GSH levels and fluctuations.34,35 GSH is consumed in detoxification and antioxidant processes; therefore, its levels need to be replenished. The dynamic nature of this process makes GSH measurement one of the best tools to assess current oxidative stress status in the brain.18 In line with this, Choi et al.34 show that GSH levels in the frontal-parietal brain regions of SPMS patients are lower, when compared to controls, as measured with magnetic resonance chemical shift imaging (CSI) at a field strength of 3T, indicating the presence of oxidative stress in SPMS. Furthermore, in another study using a MRS editing scheme, Srinivasan et al.35 were able to generate an unobstructed in vivo detection and quantification of GSH at 7T, eliminating the technical challenges associated with overlapping resonances and low GSH concentrations in the human brain. As a result, they obtained normative differences in white and gray matter GSH concentrations over a 2-dimensional region in the human brain. The authors demonstrate that GSH levels are significantly higher in GM, relative to WM, in normal subjects; and they report a significant reduction of GSH in the GM of MS patients, when compared to controls.35 No significant differences in GSH levels were found between controls and MS patients in NAWM, but in WM lesions the GSH levels decreased. This preliminary investigation demonstrates the potential of this marker to probe the oxidative state in MS; however, further research is needed to better elucidate the processes underlying GSH variations during disease progression. The method reported in this study allows non-invasive, in vivo measurement of GSH and it might be used to explore both its sensitivity to detect changes in the oxidative state in MS patients and to monitor the progression of the disease. Glutathione as a therapeutic Current MS therapeutics are primarily immunomodulatory and are relatively successful in reducing the number of inflammatory lesions in RRMS; however, there is a complete lack of therapeutics capable of slowing down disease progression in the later stages
of the disease. As ROS are crucial players in both the inflammatory and degenerative disease processes, they provide an interesting therapeutic target in both RRMS and progressive MS.2,10 GSH plays a central role in the maintenance of cellular redox homeostasis and protection against ROS. Given the involvement of decreased GSH levels and alterations in glutathione-related enzyme activities in MS, maintaining or restoring GSH levels represents a promising therapeutic strategy.3 The apparently most straightforward approach for increasing GSH levels, through its direct administration, failed due to the solubility, absorption and stability constrains that limit its bioavailability.3,36,37 GSH has a low half-life in human plasma and, as a hydrophilic molecule, it is not able to freely diffuse through the BBB. Therefore, GSH delivery to the CNS is particularly challenging. Extremely high doses of GSH would be required in order to achieve a therapeutic effect. The direct delivery of the precursor amino acid cysteine was also a failure, due to its toxic effects.37 Hence, research efforts turned to the identification and synthesis of GSH precursors and analogues that could mimic GSH’s cellular redox protective effects. A promising therapeutic approach is the supplementation of endogenous GSH levels, by using prodrugs. For instance, GSH ester prodrugs are able to cross cell membranes, due to their lipophilic characteristics, and are able to increase cellular GSH content in several tissues and restore the GSH pool in mitochondria.36 Another candidate would be cysteine prodrugs that are able to bypass the toxic effects that direct administration of cysteine causes. In the search for GSH precursors and analogues that could improve its pharmacological properties and bioavailability, the antioxidant N-acetyl-cysteine (NAC) was found to have promising protective effects for several neurodegenerative diseases, by potentiating intracellular GSH synthesis, promoting detoxification and acting as a ROS scavenger.3 Administration of NAC reduced inflammation and induced the replenishment of GSH levels in EAE animals38,39; however, no clinical trials have been conducted using NAC in MS. Although direct oral GSH intake does not seem to have an impact on brain GSH levels, several compounds can indirectly induce GSH production. Therefore, another effective approach involves the use of compounds that activate the Nrf2 pathway, as most enzymes involved in glutathione synthesis are induced by Nrf2 activation. Monomethylfumarate is a
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Multiple Sclerosis Journal 20(11) well-known Nrf2 activator and was demonstrated to enhance glutathione levels, and increased both cellular antioxidant and anti-inflammatory potential, in a Nrf2dependent manner.40–42 Recent clinical trials with oral dimethylfumarate (Tecfidera® on the pharmaceutical market) clearly highlight the therapeutic potential of Nrf2 activation in MS (review, Fox et al.43). In conclusion, due to its central role in antioxidant metabolism and its involvement in the several phases of MS progression, glutathione constitutes a privileged tool. As a biomarker, it can be safely used to predict disease progression by non-invasive imaging methods, constituting a potentially helpful tool in diagnosis. In addition, glutathione represents an interesting therapeutic target to combat oxidative stress, not only in the initial inflammatory period of the disease, but also in preventing the neurodegenerative processes associated with progressive MS. Conflict of interest The authors declare that there are no conflicts of interest. Funding This work was supported by the ECTRIMS Postdoctoral Research Fellowship Programme (Post doctoral Fellowship to ANC).
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