Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease

Abstract

Significance: Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive.

Recent advances: Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms.

Critical issues: GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss.

Future directions: In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

FIG. 1.

Oxidative stress and thiol-redox signaling in Parkinson's disease (PD). Oxidative stress in PD is linked primarily to mitochondrial dysfunction. Decreased activity of the mitochondrial complex I in the substantia nigra pars compacta (SNpc) of patients with PD has been reported, and mitochondrial toxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, used in experimental PD, act primarily by inhibition of complex I leading to an increased generation of reactive species (RS) (1). α-Synuclein and α-synuclein-metal complexes induce mitochondrial dysfunction and oxidative damage, and metallothioneins (MTs) have been shown to exert a protective effect (2). Mutant α-synuclein and environmental toxins impair vesicular dopamine transporter 2 (VMAT2) augmenting cytosolic dopamine levels, where dopamine is metabolized by monoamine oxidase (MAO) or auto-oxidized generating RS and dopamine-quinone species (DAQ) (3). DAQ are highly reactive products that can inactivate proteins by reacting with protein thiols (4). Environmental toxins have been shown to induce oxidative damage through a variety of mechanisms, including impairment of mitochondrial electron transport chain (ETC) (1), auto-catalytic generation of RS (5), increased intracellular dopamine catabolism, activation of plasma membrane nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases, and extracellular sources through the activation of glial cells and induction of inflammatory processes (6). An important cellular target of RS is the thiol group of protein cysteine residues. Protein sulfonic acid (PSO₃H) and protein nitrosylation (PSNO) of the PD-associated genes DJ-1 and Parkin have been shown to impair their redox sensing and ubiquitin E3 ligase activity, respectively (7). Broken arrows depict the magnification of the indicated region.

FIG. 2.

Glutathione (GSH) homeostasis and GSH-dependent antioxidant systems. GSH (l-γ-glutamyl-l-cysteinyl-glycine) synthesis occurs in the cytosol from the combination of glutamate and cysteine by the γ-glutamylcysteine synthetase (γ-GCS), also known as γ-glutamylcysteine ligase. Glycine is subsequently added to the γ-glutamylcysteine by the activity of glutathione synthetase (GS) (1). Export of GSH, GSSG, and GSH adducts is an important step in its catabolism (2), which is mediated extracellularly through the removal of the γ-glutamyl moiety from GSH and GSH-conjugated compounds, producing cysteinylglycine or cysteinylglycine conjugates by the activity of the γ-glutamyl transpeptidase (γ-GT). These products are hydrolyzed by dipeptidases (DPs) and then both cysteine and γ-glutamyl amino acids formed are taken up by the activity of specific transporters (3). γ-Glutamyl derivatives are substrates converted to glutamate by the activity of the 5-oxoprolinase (4). Cysteine availability is rate limiting for the synthesis of GSH, and cystine is the prevailing form of cysteine in the extracellular space due to cysteine auto-oxidation. Cystine is taken up by the xc⁻ system, which exchanges cystine for glutamate (5). GSH can catalytically detoxify cells from peroxides in both the cytosol and the mitochondrial compartments by the action of glutathione peroxidase (GPX) and phospholipid hydroperoxide GPXs (GPX4) leading to the formation of glutathione disulfide (GSSG). A long and a short form of GPX4 have been described, which are distinguishable by the presence or lack of a mitochondrial signal peptide at the N-terminus (6). GSSG is reduced by the action of glutathione reductase (GR), which requires reduced NADPH as the electron donor. In the cytosol, glucose-6-phosphate dehydrogenase (G6PD) is indispensable for the regeneration of NADPH from NADP⁺, while in the mitochondria the nicotinamide nucleotide transhydrogenase (NNT)-catalyzed reduction of NADP⁺ accounts for more than half of the mitochondrial NADPH pool (7). Glutathione-S-transferases (GSTs) catalyze the conjugation of GSH electrophilic centers facilitating the detoxification and excretion of different compounds (8). A significant pool of GSH is compartmentalized in the mitochondria by the dicarboxylate carrier (DIC) or 2-oxoglutarate transporter (OGC) (9). Glutaredoxins (GRXs) are oxidoreductases that under reducing conditions utilize the reducing power of GSH to catalyze protein deglutathionylation. GRX1 has been found in both cytosolic and intermembrane space compartments (10). Mitochondrial matrix GRX2 contains a Fe-S cluster that bridges two GRX2 monomers and serves as a redox sensor (11).

FIG. 3.

Thioredoxin–peroxiredoxin (TRX-PRDX) system. PRDXs catalyze the reduction of hydrogen peroxide (H₂O₂) to H₂O. H₂O₂ oxidizes the peroxidatic cysteine of PRDXs to protein sulfenic acid (PSOH), which can react with the thiol (SH) group of the resolving Cys to yield the formation of an inter- (typical) or intramolecular (atypical) disulfide bond. TRX/thioredoxin reductase (TRXR) system mediates the reduction of the PRDX disulfide bond. TRX reduced state is maintained by the flavoenzyme TRXR in the presence of NADPH. When H₂O₂ exceeds the normal levels, PRDXs are overoxidized from PSOH to protein sulfinic acids (PSO₂H). The latter can be reduced back to the native form of the enzyme by sulfiredoxin (SRX) in the presence of ATP. However, further oxidation of PRDXs to PSO₃H is irreversible.

FIG. 4.

Thiol-redox signaling and apoptotic signaling cascades. Studies on postmortem brain tissue of PD patients have reported the presence of apoptotic markers in the substantia nigra supporting a role for apoptosis (programmed cell death) in neuronal cell loss. Accordingly, activation of caspases and pro-apoptotic members of the B-cell lymphoma 2 (Bcl-2) family of proteins was reported in the substantia nigra of PD patient autopsies, suggesting a role for the mitochondrial pathway of apoptosis in neuronal cell death. Oxidative stress and alterations in GSH/GSSG balance are known to regulate the apoptotic machinery. However, very little is known about the mechanisms by which thiol-redox signaling regulates the progression of apoptosis in PD. Mitochondrial dysfunction in PD conveys the formation of RS, leading to the activation of the intrinsic pathway (1). Activation of the mitochondrial pathway mediates the release of cytochrome C (Cyt C) that is regulated by the Bcl-2 protein family. The BH3-only members derepress Bcl-2 associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak) by direct inhibition of the antiapoptotic Bcl-2 proteins to promote apoptosis. Bax and Bak induce the permeabilization of the outer mitochondrial membrane and the release of Cyt C, leading to the recruitment of apoptotic protease activating factor 1 (Apaf1) into an apoptosome and activation of caspase-9. Initiator caspases further activate/cleave executioner caspases (3, 6, and 7), which mediate cell demise (2). Proteins of the inhibitor of apoptosis family, including X-linked inhibitor of apoptosis protein (XIAP), inhibit executioner caspases through the occupation of their active site. In addition, XIAP can prevent caspase 9 dimerization that is required for its activation. XIAP-SNO is found in PD brains, and this oxidative modification impairs its ability to inhibit caspase activation (3). Apoptosis signal-regulating kinase-1 (ASK-1) is a member of the mitogen-activated protein kinase (MAPK) kinase family that activates c-Jun N-terminal kinase (JNK) and the p38 MAPK family members involved in apoptosis (4). TRX1 associates with the N-terminal portion of ASK-1 inhibiting its kinase activity. When oxidation of TRX1 cysteine residues occurs, the TRX1-ASK-1 complex dissociates leading to the phosphorylation and activation of ASK-1, which mediates the expression of pro-apoptotic genes (5).

FIG. 5.

Oxidative cysteine modifications. Redox-sensitive cysteines undergo reversible and irreversible thiol modifications in response to reactive oxygen species (ROS) or reactive nitrogen species (RNS), thereby modulating protein function, activity, or localization. Almost all physiological oxidants react with protein thiols (PSH). The two-electron oxidation (S-hydroxylation) involves the reaction between protein cysteine thiols (PSH) and ROS/RNS (H₂O₂, ONOO⁻) to generate the unstable PSOH (1), which subsequently reacts with another thiol to produce inter- or intramolecular mixed disulfides (2). ROS/RNS can further react with the PSOH to yield a hyperoxidized PSO₂H and ultimately PSO₃H (3). Likewise, PSOH can form mixed disulfides with GSH (PSSG) through reaction of PSH with GSSG (4). One-electron oxidation of PSH residues with free radicals and transition metal ions leads to the formation of protein thiyl radicals (PS^•) (5). PS^• can form inter- or intraprotein disulfide bonds (PSSP) by reaction with another PS^• or a PSH (this last one through a disulfide radical anion intermediate, broken line) (6), or can generate protein peroxyl radicals (PSOO^•) by reaction with O₂ (7). Similar reactions can lead to PSSG formation by the reaction of a PS^• with GS^• or GSH. If either PSSP or PSSG bonds are not formed, protein radicals (PS^• and PSOO^•) can lead to the amplification of radical reactions (8). Covalent adduction of a nitroso (NO) group to a PSH residue is referred to PSNO, which is mediated by nitrosylating agents, such as dinitrogen trioxide (N₂O₃), transition-metal-catalyzed addition of NO (9), or transfer of the NO group between PSNO or nitrosoglutathione (GSNO) and a PSH residue (transnitrosation) (10). Interestingly, PSNO has also been shown to act as an intermediate for PSSG formation by reaction with GSH (11).

FIG. 6.

Enzymatic regulation of glutathionylation and nitrosylation. GRXs are oxidoreductases that under reducing conditions utilize the reducing power of GSH to remove PSSG residues (1). However, GRXs can also catalyze the formation of PSSG residues in the presence of high levels of oxidized GSH (GSSG or GS^•) (2). Then, GRX1 activity depends upon the redox environment of the cell, with the potential to act as a glutathionylating enzyme under oxidative stress, and as a deglutathionylase under reducing conditions (high GSH availability). Removal of PSNO residues is catalyzed by the TRX system that involves the release of nitroxyl (HNO) and the formation of oxidized TRX that is further reduced by TRXR. This mechanism might involve the intermediate formation of either TRX-SNO or the intermolecular disulfide between TRX and the protein target (TRX-SS-P) (3). Transfer of NO groups by GSNO has been reported as one of the major mechanisms mediating PSNO. PSNO residues can also be denitrosylated by GSH leading to GSNO formation. GSNO reductase (GSNOR) prevents GSNO-mediated nitrosylation by its metabolism to N-hydroxysulphenamide (GSNHOH) (4).

FIG. 7.

Antioxidant gene regulation by nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Under resting conditions kelch-like ECH-associated protein 1 or inhibitor of Nrf2 (INrf2) (Keap1) targets Nrf2 for proteosomal degradation through ubiquitylation of Nrf2 Lys residues that are located at its N terminus (1). Oxidation/alkylation of Keap1 disrupts the association of Nrf2 with Keap1, leading to its nuclear translocation (2). Nrf2 activates the transcription of a variety of antioxidant genes through antioxidant response elements (AREs) also known as electrophile-responsive elements (EpREs) (3).

FIG. 8.

Metallothioneins regulate metal homeostasis and redox signaling in PD. MTs contain two metal-thiolate clusters located in separate protein domains that bind seven divalent metal ions (Zn²⁺). There are three bridging (broken lines) and six terminal cysteine ligands (continuous lines) in the N-terminal β-domain (Zn3 cluster) and four bridging and six terminal ligands in the C-terminal α-domain (Zn4 cluster) (1). Scavenging of RS by MTs induces the release of Zn²⁺. GSH reduces and stabilizes Zn²⁺ binding to MTs (2). MTs protect against oxidized dopamine products, such as DAQ, by direct scavenging and formation of covalent adducts (3). Cu²⁺ has the ability to displace Zn²⁺ binding to MTs and MT-III removes Cu²⁺ from α-synuclein-Cu²⁺ preventing the generation of RS (4). MT gene expression is regulated by AREs and metal-responsive elements (MREs) within their promoter region (5). Once Zn²⁺ is liberated from MTs it binds to the zinc (Zn) fingers of metal-responsive transcription factor-1 (MTF-1) formation, while oxidative stress promotes Nrf2 binding to ARE and activation of MT genes. Once MTs are synthesized they immediately bind Zn²⁺ (1). MT-I and -II are highly inducible in response to metals, oxidative stress, and inflammation, and increased MT-I/II levels have been found in reactive astrocytes in PD brains. Release of MT-I/II from astrocytes has been reported to exert neuroprotective effects associated to their ability to scavenge metals and RS (6).

FIG. 9.

Methionine sulfoxide and methionine sulfoxide reductase (MSR) redox cycle. Methionine oxidation by RS generates methionine sulfoxide (MetSO), which can be further hyperoxidized to MetSO₂. MSRs share similar catalytic mechanism involving the initial formation of PSOH or protein selenocysteine sulfenic acid (PSeOH) (MSRB1) on the catalytic cysteine or selenocysteine, with consequent methionine release (1). In MSRA and MSRB1 the catalytic PSOH or PSeOH subsequently forms an intradisulfide bond with the resolving cysteine, which is ultimately reduced by TRX. In MSRA, a third cysteine mediates a thiol-exchange reaction to transfer the disulfide bond to the protein surface prior to its reduction by TRX (2). In contrast, MSRB2 and MSRB3 have only one cysteine, which is reduced directly by TRX. Because MSRA reduces both free and protein-based Met-S-SO, its protective effects can be associated to enhanced GSH synthesis through the transsulfuration pathway (3). In the transsulfuration pathway methionine is converted to S-adenosylmethionine (SAM) in an ATP-dependent reaction catalyzed by methionine adenosyltransferase (MAT). Subsequently, S-adenosylhomocysteine (SAH) is generated via methyltransferase (MTF) activity. SAH is hydrolyzed by SAH hydrolase (SAHH) to adenosine and homocysteine. Homocysteine is conjugated with serine to provide cystathionine by the action of cystathionine beta synthase (CBS). Finally, cystathionine-γ-lyase (CTH, or γ-cystathionase) catalyses the conversion of cystathionine to cysteine.