Redox Signaling Mediated by Thioredoxin and Glutathione Systems in the Central Nervous System

Regulation of H₂O₂ metabolism via the Trx/Prx and GSH/GPx systems

Prx, GPx, and CAT are the three types of antioxidant enzymes that catalyze the decomposition of H₂O₂ with high efficiency (122). These enzymes can scavenge H₂O₂ at a reaction rate of 10⁷–10⁸ M⁻¹s⁻¹, whereas the direct reaction rate of GSH with H₂O₂ is only 0.89 M⁻¹s⁻¹ (207). Given the notable absence of CAT expression in brain mitochondria, the concentration of CAT in the brain is estimated at 1.2 μM, which is ∼50 times less than its level in the liver (154), whereas the Prx3 concentration in the brain is ∼60 μM, Prx5 is 20 μM, and GPx1 is 2 μM (44, 184).

Mitochondria are the main sites of superoxide and H₂O₂ production (138). About 0.5–2.0% of electrons leaked from mitochondrial respiration are converted into superoxide. Mitochondrial SOD2 can catalyze the conversion of superoxide to H₂O₂, a well-known signaling messenger (185). The rate of H₂O₂ production using NAD-linked substrates in the brain is 50–70 pmol/min/mg mitochondrial protein. Interestingly, mitochondria can scavenge exogenous H₂O₂ at a rate of 9–12 nmol/min/mg mitochondrial protein in a respiration- and substrate-dependent manner, which is 100 times higher than the H₂O₂ production rate. Thus, mitochondria act as a cellular source and also as a sink of H₂O₂ (6, 184).

Several studies have highlighted the essential role of the mitochondrial Trx system in counteracting ROS in the brain, with its importance potentially due to the lack of CAT expression. Inhibition of mitochondrial TrxR2 by auranofin was demonstrated to diminish the H₂O₂ scavenging capacity of mitochondria, whereas this was not affected by uncoupling of mitochondria, phosphorylation of added ADP, or the genetic ablation of GPx1 (53, 184). Mitochondrial Trx/Prx couple was suggested to reduce H₂O₂ in a respiration-dependent manner. Nicotinamide nucleotide transhydrogenase (Nnt), which uses NADH and NADP⁺ to generate NADPH, links the substrate requirement to mitochondrial Trx/Prx as the ultimate electron donor for H₂O₂ removal. In a previous study, inhibition of Nnt decreased the NADPH level and impeded the Trx system's capacity to scavenge H₂O₂ in the brain, whereas inhibition of Nnt had no effects on H₂O₂ removal in liver mitochondria since it is independent of respiration as CAT is highly expressed in mitochondria from liver cells (119).

Reversible regulation of protein thiol modification

A large part of ROS/reactive nitrogen species (RNS)-mediated signal transduction relies on modifications of cysteine thiol (-SH) within proteins. Irreversible thiol modification (sulfonic acid, -SO₃H) has been considered a hallmark for various pathological conditions and usually leads to permanent functional loss and degradation of the protein. Reversible thiol modifications, such as S-nitrosylation (-SNO), S-sulfenylation (sulfenic acid, -SOH), S-glutathionylation (-SSG), disulfide formation (-S-S-), S-sulfhydration (-S-SH), and sulfinic acid (-SO₂H), are involved in redox signaling (Fig. 3).

Several properties allow thiols to act as signal switches. First, thiols are susceptible to ROS/RNS challenge that lays a foundation for its sensitivity. Second, in different local environments, thiols exhibit different reactivity, which gives them selectivity (62). Last, most thiol modifications are reversible, which provides flexibility for sophisticated signaling regulation. Thus, great efforts have been made to identify proteins with functional cysteines and understand the regulation of reversible thiol modification in neuronal systems.

Redox regulation of receptor signaling

Activation of protein tyrosine kinases (PTKs) to phosphorylate target proteins is a common mechanism to transduce ligand–receptor-based signals, which are important for growth factor-mediated neural development. Therefore, protein tyrosine phosphatases (PTPs), which negatively regulate the activity of PTKs and downstream cascades, are important to maintain the balance of total protein phosphorylation.

Although members in the PTP superfamily share limited sequence similarity, the presence of a -CXXXXXR- (X can be any amino acid) motif in the active site is a common catalytic hallmark for PTPs (194). Due its low pKa value (4–6.5), the cysteine in the active site of PTPs exists as a thiolate (-S⁻) at physiological pH and it is sensitive to ROS or RNS modification in vivo (159). In particular, upon growth factor stimulation, ROS transiently and locally produced by NOX are required to activate PTKs and downstream cascades (187). During the last two decades, redox regulation of PTPs in signal transduction has received much attention due to its implication in various physiological and pathological settings in the CNS. Details have been discussed in recent reviews (81, 112).

Inactivation of PTPs by other oxidants, such as H₂O₂ (48), oxidized GSH (GSSG) (14), lipid peroxide (42), NO (13), and H₂S (105), results in reversible cysteine modifications, such as sulfenic acid, glutathionylation, disulfide bonds, nitrosylation, or sulfhydration. The oxidized PTPs can be reduced and restored by the Trx and GSH systems (113). Interestingly, the Trx system was reported to exhibit certain specificity toward different PTPs. A recent study showed that both Trx1 and Trp14 coupled with TrxR and NADPH can reactivate oxidized PTP1B, but not SHP2, another member of the PTP family (45). Grx was reported to reduce glutathionylated PTP1B and restore its phosphatase activity (14).

Prx (38) and GPx (42) also contribute to receptor signaling due to their high efficiency for removing peroxides and protection of PTPs from oxidation. Prx can protect PTEN from oxidation by a direct protein–protein interaction (33). Interestingly, under certain conditions, Prx acts as an oxidase of PTPs, in contrast to its typical well-characterized role as an H₂O₂ scavenger. During H₂O₂ burst, the cysteine at the active site of Prx is initially oxidized into sulfenic acid or intra-/intermolecular disulfide, then the oxidized Prx may selectively transfer the equivalent oxidation to target proteins via specific protein–protein interactions (74). However, more evidence is needed to understand this process in mammalian cells. It was also reported that growth factor stimulation inactivated Prx via phosphorylation and that dephosphorylation by PTPs may restore Prx activity (208).

Regulation of redox-sensitive apoptosis induced by ER stress

Accumulation of misfolded proteins caused by ER stress is a hallmark of neurodegenerative diseases. ER is an important organelle for protein quality control and ensures that proteins are synthesized, folded, and post-translationally modified correctly. Disturbance of ER function leads to ER stress, which may sequentially trigger undesired apoptosis (213).

Trxs are critical mediators of ER stress-induced apoptotic signaling. Trx1 has been found to be a physiological inhibitor of apoptosis-signaling kinase 1 (ASK1) via a direct protein–protein interaction. ASK1 is a mitogen-activated protein kinase kinase kinase (MAP3K) and activates MAP2K-JNK/MAPK-p38 signaling cascades, which are essential for ER stress-induced apoptosis (146). Trx1 inhibits ASK1 kinase activity via the formation of a disulfide between its active site Cys32 or Cys35 and Cys250 in the N-terminal portion of ASK1. When cytosolic Trx1 is oxidized in response to proinflammatory stimuli, ROS, and cellular stress, ASK1 is released from Trx1-ASK1 complexes and subsequent ASK1-dependent apoptosis is activated (Fig. 6). Trx2 associates with mitochondrial ASK1 via binding with the ASK1 Cys30 and activates a JNK-independent apoptosis pathway (222).

The activity of Trx is also regulated by Trx-interacting protein (TXNIP), its endogenous inhibitor (147), which is also induced during ER stress (149). The interaction between Trx and TXNIP involves disulfide formation between Cys32 in Trx and Cys247 in TXNIP, which may be regulated by an internal disulfide switching mechanism between Cys63-Cys247 and Cys63-Cys190 in TXNIP (87).

Regulation of redox-sensitive apoptosis in I/R

ROS production is involved in ischemic brain damage, thus overexpression of antioxidant enzymes, such as Trxs and Prx2, attenuates cerebral infarction following ischemia and I/R. Prx2 protects brain tissue from I/R-induced injury by suppressing the ASK1/JNK-mediated mitochondrial signaling pathway (68). Prx2 also attenuates the poly(ADP-ribose) polymerase 1 (PARP1) and p53-dependent cell death pathway (110).

The NMDA receptor is a critical mediator during ischemic brain damage. The TXNIP pathway has been shown to be involved in neuroprotection mediated by the synaptic NMDA receptor in rat neurons. Synaptic activity blocks TXNIP expression and activates the expression of sulfiredoxin and sestrin 2, thus enhancing reduction of Prx by the Trx system and facilitating intrinsic antioxidant defenses in neurons (150). Although the Trx system has a protective role in cerebral I/R, the mitochondrial Trx system has been indicated to be involved in caspase 3-mediated neuronal apoptosis in rat hippocampus (186). Cerebral I/R can activate glutamate receptor 6 (GluR6), which upregulates the expression of FasL, and thus enhances the expression of TrxR2 and Trx2. The mitochondrial Trx2 system then denitrosylates and activates procaspase 3 (186) (Fig. 5).

GSH in health and diseases of human CNS

GSH is the most abundant thiol-containing molecule and one of the most important antioxidants involved in neuroprotection (8). The GSH level in the brain is ∼2–3 mM (43); it is highest in the cortex, followed by the cerebellum, hippocampus, and striatum, and is lowest in the substantia nigra (97).

GSH reacts nonenzymatically with oxidants to inhibit oxidative stress in the cell, including superoxides, NO, hydroxyl radicals, and peroxynitrite. In addition, GSH reacts enzymatically with GPx and GSH-S-transferase (GST) against neurodegeneration (9). In neurons, GR is sufficiently active to allow the rapid reduction of accumulated GSSG (55). Under oxidative stress, GR maintains the equilibrium of the GSH/GSSG redox state in the cell. Most importantly, GSH depletion induced by oxidative stress can exacerbate oxidative injury in the brain (126).

An age-related decline in GSH level has been observed in humans, and the GSH concentration in the cerebral spinal fluid decreases with age. Such findings suggest that aging-associated decreases in GSH may underlie changes to antioxidant capacity, which occur during aging and during the onset of various aging-related diseases (193). Previous works have suggested that GSH depletion is involved in neurodegeneration (175). Indeed, the GSH levels are decreased in the brain in some neurodegenerative diseases, but it remains unclear whether the decreased GSH level is a cause or an outcome of neurodegeneration (163). In PD, although there is a 30–40% decrease in GSH concentrations, no corresponding increase in the level of GSSG has been detected, thus the reason for GSH depletion is not clear (178). In AD, the total GSH level in brain is not affected, whereas GPx and GR were found to be either elevated or unchanged in different brain regions (157). In clinically isolated syndrome and relapsing–remitting multiple sclerosis, there is evidence that GSH content and GPx activity are decreased, and the former might serve as a marker closely correlated with the neurological scoring of acute CNS inflammation (117). The low concentration of GSH during inflammation is likely to be a consequence of accelerated turnover linked to elevated oxidative stress. In a beta-N-oxalyl amino-L-alanine (L-BOAA)-induced mouse model of neurolathyrism (a motor neuron disease involving the pyramidal system), GSH loss and inhibition of mitochondrial complex I were observed in the lumbosacral cord of male mice (51). Other CNS toxicants such as ethanol also cause a decrease in GSH concentration, particularly in the cerebellum, striatum, and cortex, which may be associated with an increase in the concentration of acetaldehyde, which is normally removed from cells by GSH (11).

Mercury

Exposure to mercury (Hg), whether it is in the form of methylmercury (MeHg) in ingested fish, mercury vapor (Hg⁰) released from dental amalgams, or ethylmercury (EtHg) derived from thimerosal-containing vaccines, results in its accumulation in the CNS, which eventually leads to neurotoxicity. The neurotoxic effects are particularly serious if exposure to Hg takes place during fetal development, or in early infancy, due to the immaturity of the CNS and BBB (40).

Systemic distribution of MeHg and its accumulation in the CNS are facilitated by binding to -SH groups in cysteine due to the molecular similarity between the MeHg-Cys complex and the amino acid methionine, which enables MeHg-Cys transport across the BBB and into the brain via neutral amino acid transporters (39). On the other hand, Hg⁰ has no charge and can freely diffuse across membranes and reach the CNS. Both the organic species of Hg and Hg vapor are converted to the highly reactive Hg²⁺. In fact, Hg²⁺ is the most abundant Hg species found in the brain following chronic exposure to MeHg, EtHg, or Hg⁰ (32, 201).

One of the known mechanisms by which Hg exerts neurotoxicity is through the disruption of glutamate (Glu) transport, either by increasing its release from the presynaptic terminal or by inhibiting its reuptake from the synaptic cleft by astrocytes. Impairment of glutamate uptake is associated with increased ROS production and subsequent oxidative stress (60). The Hg electrophilic nature underlies its oxidative effects and its high binding affinity for thiols and selenols (35, 169). As a result, both the GSH and Trx systems are targets for mercurial compounds (Fig. 7).

Depletion of GSH has been shown to occur after exposure to Hg compounds in vitro and in vivo using experimental animals (47, 204). In primary cultures of neurons and astrocytes, GSH was decreased by 30–50% following a 30-min treatment with 5 μM MeHg (98). This abrupt decrease in GSH levels cannot be explained on the basis of a 1:1 complex formation. Therefore, it is likely that upon exposure to Hg, ROS generation plays an important role in GSH oxidation with the subsequent formation and excretion of GSSG. Additionally, decreased synthesis of GSH, due to impaired uptake of its precursor Glu, leads to higher ROS generation (47) (Fig. 7). Concomitantly, in the study by Kaur et al., pretreatment with diethylmaleate depleted cellular GSH and further aggravated MeHg toxicity, whereas pretreatment with N-acetylcysteine increased the intracellular GSH level and offered additional protection against toxicity (98). Most interestingly, in vivo data suggest that GR activity in the brain is increased by exposure to MeHg, which may function as a compensatory response following GSH oxidation (27).

The pivotal role of GSH in modulating Hg toxicity cannot be separated from the fact that it is an important cofactor for enzymes involved in antioxidant defense, such as GPxs and Grxs. GPx1, an important scavenger of H₂O₂, is affected by exposure to mercurials, in particular MeHg. Likewise, membrane-located GPx4 presented decreased activity and expression in the cortex and cerebellum of mice exposed to MeHg (220). Besides dependence on GSH as an electron donor, GPx activity relies on the reducing ability of Sec residue in the active site. This Sec is prone to direct interaction with MeHg (28), but not Hg²⁺, as shown in vitro by Bulato et al. (31).

Data on the interaction between Hg and Grxs are scarce. We have previously reported that both MeHg and Hg²⁺ target the thiols in purified Grx1; however, in HeLa cells, inhibition of Grx activity is only achieved at very high concentrations. To the best of our knowledge, there are no reports concerning the interaction between Hg and Grx in CNS models or in experimental animals (34).

The Trx system has been proven to be particularly sensitive to Hg. TrxR is a prime target for mercurials due to the high reactivity and location of the Sec residue at the open C-terminal (34, 35). Several in vitro and in vivo studies have shown a significant drop in TrxR activity in the brain following exposure to different Hg compounds, which is both time and concentration dependent (27, 28, 166, 220). In liver cells, inhibition of cytosolic TrxR1 by Hg²⁺ is counteracted by upregulation of TrxR1 synthesis via the Nrf2 pathway (29). This Nrf2 response is weaker during exposure to MeHg, resulting in decreased expression of TrxR1. It has been shown that a fast Nrf2 response occurs upon MeHg exposure in microglia and neurons (145); however, the relationship between Nrf2 signaling and TrxR1 in the CNS has not been fully elucidated. Contrary to its cytosolic counterpart, TrxR2 is not regulated by Nrf2 (29) and thus it might be a very important early target in the development of Hg toxicity. In fact, TrxR2 and the overall mitochondrial Trx system, via Prx3, play a vital role in the detoxification of H₂O₂ in brain mitochondria, and TrxR2 inhibition increases the sensitivity of CNS cells to other environmental stressors such as paraquat (PQ) (118).

Trx1 is also inhibited by mercurials in vitro with a 1:2.5 and 1:5 enzyme:inhibitor ratio for Hg²⁺ and MeHg, respectively (34). In SH-SY5Y cells exposed to EtHg (166) and in the brain of experimental animals exposed to MeHg and Hg²⁺, Trx activity is inhibited in a dose-dependent manner similarly to the effect on TrxR, although the reduction in activity is less significant (27, 28).

Paraquat

PQ (1,1′-dimethyl-4-4′-bipyridinium dichloride) is a typical quaternary ammonium herbicide, which kills plants nonselectively by targeting the chloroplasts in green plant tissues. In plants, PQ mainly exerts its toxic effect by acting as a redox cycler, which can constantly consume NADPH and disturb the electron transfer photosystems (25). In humans, PQ is not easily absorbed from the skin or gut. Once absorbed, PQ mainly accumulates in lungs and causes pulmonary fibrosis. Acute PQ poisoning results in respiratory failure and death within a few days or weeks. Chronic exposure to PQ leads to elevated oxidative stress in the plasma, including increased lipid peroxidation, decreased antioxidant capacity (affecting the ferric-reducing ability of plasma), and decreased blood thiol content (164). Although it is still under debate whether PQ can pass through the BBB and exert its toxic effect directly in the brain (15, 16, 30, 177), many epidemiological and etiological studies have shown a clear association between low-dose exposure (absence of acute toxicity) to PQ and neurodegenerative diseases, especially PD. Mechanistic studies also demonstrated that PQ can induce apoptotic cell death in several types of neuronal cells in association with PD, including in cerebellar granule cells and other dopaminergic neurons (71).

In mammalian cells, the mechanism of PQ-induced toxicity is very similar to the mechanism in plant cells. PQ ions (PQ²⁺) are reduced to the free radical form of PQ (PQ^•+) by NADPH. PQ^•+ can spontaneously react with oxygen, resulting in the formation of superoxide anion (O₂^•−), which then leads to the generation of other types of ROS, including H₂O₂ and hydroxyl radicals (25). The overproduction of ROS then causes damage to proteins, lipid membranes, and DNA (Fig. 8). Furthermore, PQ constantly consumes NADPH and disrupts essential NADPH-dependent biochemical processes (167) (Fig. 8). Besides acting as a redox cycler, the role of mitochondria in PQ neurotoxicity has been addressed. ROS generation following PQ exposure is dependent on respiratory chain components, especially complex III (36).

Trx and Grx systems are not only very important for the defense against PQ toxicity but are also disrupted by PQ toxicity as both systems use NADPH as the ultimate electron donor. In SH-SY5Y and SK-N-MC human neuroblastoma cell lines, PQ was found to oxidize Trx1 and activate the ASK1/JNK/caspase 3 cascade. PQ treatment also reduced the activity of TrxR and GPx (188, 198). Dopaminergic N27 cells were sensitized to PQ treatment by disruption of mitochondrial Trx2 and TrxR2 (118). Drosophila with Trx2 loss of function were sensitized to PQ toxicity (197). In Drosophila cells, Prxs also had a cytoprotective effect following PQ treatment (162). The expression of certain Trx and Grx system proteins is regulated by the transcription factor Nrf2. Although PQ treatment was found to decrease the protein level of Nrf2, it can also activate the Nrf2 pathway and increase the transcription of target genes (167, 216). TrxR appears to have a dual role in PQ toxicity. Several studies in different species have reported that TrxR has a protective role following PQ treatment (118, 142). However, in the presence of NADPH, TrxR was found to reduce PQ and generate ROS that may contribute to its toxicity (72, 189). Several known antioxidants or proteins with antioxidant properties, including vitamin C, vitamin E, Se, N-acetylcysteine, and coenzyme Q10 (CoQ10), were found to have protective roles against PQ treatment (12, 71, 132, 188, 198, 219). These findings also suggest the importance of antioxidant enzymes for protection against PQ toxicity.

Coenzyme Q

CoQ10 (ubiquinone) is an electron carrier lipid of the mitochondrial respiratory chain and an endogenously synthesized antioxidant. CoQ10 can accept electrons from complex I and complex II and transfer them to complex III, which is considered to be the rate-limiting step in ATP production (130). CoQ10 is able to protect lipids from peroxidation in cell/organelle membranes and in the circulation (168). As well as effects on lipid membranes, in vivo administration of CoQ10 can also protect membrane proteins (64) and DNA (192) from overoxidation. CoQ10 is also able to regenerate other antioxidant molecules, such as vitamin E (66). Additionally, oral administration of CoQ10 has been proven to be safe even at very high concentrations (3600 mg/day) (88).

The two major functions of CoQ10, oxidation and regeneration, both depend on changes in its redox state. In the mitochondrial matrix, CoQ10 is regenerated by the respiratory chain enzymes and the majority of CoQ10 in mitochondria is in its reduced form (1). In the other cellular compartments and plasma, several enzymes with reductase activity can regenerate CoQ10, including lipoamide dehydrogenase, TrxR, and GR. Compared with lipoamide dehydrogenase, full-length TrxR (in the presence of selenocysteine) is more effective, whereas the truncated form of TrxR does not have the ability to regenerate CoQ10 (211). This may also explain why several studies reported that the combined administration of Se and CoQ10 yields better cardiovascular protective effects than administration of CoQ10 only (3, 4).

It has been well established that mitochondrial dysfunction is associated with neurodegenerative diseases. In neuronal cells, pretreatment with CoQ10 was found to reduce ROS generation in mitochondria and protect neuronal cells from cell death induced by neurotoxins such as kainic acid (181). A decrease of CoQ10, or the reduced form of CoQ10, in SH-SY5Y neuroblastoma cells resulted in increased oxidative stress (58). In rats, oral administration of CoQ10 can significantly increase the CoQ10 level in plasma, cells, and mitochondria and decrease oxidative damage to proteins (107). Several trials in humans and in rats have validated the beneficial role of CoQ10 in neurodegenerative diseases, such as PD and motor dysfunction disorders (18, 174).

Selenium

Se is an essential micronutrient with a narrow range of optimal concentrations. Disease can be caused by both Se insufficiency (e.g., Keshan disease) and an excessive Se intake (e.g., selenosis) (165). Upon entering cells, Se-containing compounds, organic or inorganic, are reduced to selenide (Se²⁻) and then integrated in proteins in the form of selenocysteine by a fairly complex synthesis mechanism [reviewed in detailed by Papp et al. (151)]. At least 25 genes that encode selenoproteins have been identified in mammals, including genes encoding TrxR isoforms 1, 2, and 3 (TGR) and GPx isoforms 1, 2, 3, 4, and 6 (151). At physiological pH, the selenol (-SeH) in the selenocysteine residue is in the form of selenolate (-Se⁻), providing selenoenzymes with high reactivity and sensitivity to electrophilic agents (as previously discussed for Hg) (35).

The brain is a prioritized organ for Se distribution, meaning that during periods of insufficient intake, the Se stored in visceral organs (e.g., liver) is redistributed to the brain (20). In agreement, TrxR activity was unchanged in the brain of rats fed an Se-deficient diet, whereas activity was reduced in the kidney and liver (82). In fish, supplementation of water with sodium selenite did not change TrxR activity in the brain, contrary to what was observed in the liver (28). However, SH-SY5Y cells treated with 2.5 μM sodium selenite had a 40% increase in TrxR activity (166), indicating that Nrf2-mediated gene transcription was induced, as previously observed in the liver (29). The selenite amount used in these experiments is considerably higher than the reported 100 nM necessary to optimize TrxR1 synthesis physiologically (156), and thus, it is expected that redox cycling and additional ROS formation will occur. However, it should be noted that the aforementioned experiments were performed with the aim of using Se as an antagonist to the toxicity of Hg compounds and to effectively recover the TrxR activity following inhibition by mercurials (Hg²⁺; MeHg; EtHg), thus Se needs to be in a molar excess relative to the inhibitors (29, 35, 166). In fact, selenite treatment is able to remove Hg²⁺ from the active site of TrxR1 and fully restore TrxR1 activity in vitro (35). The protective effect of selenite following Hg-induced inhibition of TrxR1 has also been observed in SH-SY5Y cells (166).

Generally, GPx1 activity in the brain is not enhanced to the same extent as is TrxR1 upon Se supplementation, indicating its low ranking in the selenoprotein hierarchy (139). Nevertheless, GPx4 is essential for neuronal development and survival and has been reported to be highly expressed in 90% of the mouse brain (223). The use of the small organoselenium compound Ebselen, a mimetic of GPx activity, has been reported to offer antioxidant protection in the CNS (214). Interestingly, human subjects exposed to Ebselen exhibited reduced levels of brain GSH (128), which could be due to its GPx-like activity and GSH consumption. This contrasts with the upregulation of GSH observed in rat brain following selenite treatment (108, 183).