Gasotransmitter Hydrogen Sulfide Signaling in Neuronal Health and Disease

Abstract

Hydrogen sulfide is a gaseous signaling molecule or gasotransmitter which plays important roles in a wide spectrum of physiologic processes in the brain and peripheral tissues. Unlike nitric oxide and carbon monoxide, the other major gasotransmitters, research on hydrogen sulfide is still in its infancy. One of the modes by which hydrogen sulfide signals is via a posttranslational modification termed sulfhydration/persulfidation, which occurs on reactive cysteine residues on target proteins, where the reactive –SH group is converted to an –SSH group. Sulfhydration is a substantially prevalent modification, which modulates the structure or function of proteins being modified. Thus, precise control of endogenous hydrogen sulfide production and metabolism is critical for maintenance of optimal cellular function, with excess generation and paucity, both contributing to pathology. Dysregulation of the reverse transsulfuration pathway which generates hydrogen sulfide occurs in several neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease and Alzheimer’s disease. Accordingly, treatment with donors of hydrogen sulfide or stimulation of the reverse transsulfuration have proved beneficial in several neurodegenerative states. In this review we focus on hydrogen sulfide mediated neuronal signaling processes that contribute to neuroprotection.

Graphical Abstract

1. Introduction

Ever since the discovery of hydrogen sulfide (H₂S) three centuries, ago in 1713, the conceptions of its functions have evolved in diverse directions [1]. H₂S was considered to be an environmental toxin before it was discovered to be produced endogenously. Associated with the smell of rotten eggs and sewers, H₂S was variously labeled as “swamp gas, marsh gas, sewer gas and stink damp”. In nature, H₂S and its derivatives occur in volcanic gases, hot springs, cold springs, natural gas and are also produced by bacteria and higher organisms. The history of the earth is intimately linked to H₂S, with the early atmosphere being a predominantly reducing one with oxygen existing as a trace element. H₂S was utilized by several bacteria as a source of energy. As the atmosphere became oxygen rich, various oxidation products of sulfur emerged which contribute to a rich array of signaling metabolites. H₂S is now recognized as the third major gaseous signaling molecule or gasotransmitter akin to nitric oxide (NO) and carbon monoxide (CO) [2].

H₂S is synthesized by three enzymes in mammals: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) which synthesize the gas using pyridoxal 5-phosphate (PLP) as a cofactor and cysteine as a substrate (Figure 1). CSE acts on cysteine to produce H₂S, pyruvate and ammonia [3–7] and is present mostly in the peripheral tissues. CBS condenses cysteine and homocysteine to produce H₂S in addition to cystathionine. 3-MST generates H₂S in conjunction with cysteine amino transferase (CAT). CAT metabolizes cysteine and α-ketoglutarate to form 3-mercaptopyruvate (3-MP). 3-MST utilizes the 3-MP formed to generate H₂S with pyruvate as a by-product. 3-MST also generates H₂S using D-cysteine as a substrate in conjunction with D-amino acid oxidase. CBS and 3-MST occur mostly in the central nervous system [8], although these enzymes are also present in peripheral tissues. In addition to these pathways, H₂S can be generated from the acid labile pool, consisting of iron-sulfur protein clusters and the sulfane sulfur pool, which functions in the presence of endogenous reductants [9]. The other important yet underappreciated source of H₂S is the microbiota that colonize the gut of mammals. Half of fecal H₂S is derived from commensal bacteria [10]. Sulfate-reducing bacteria (SRB), which include the genus Desulfovibrio predominantly produce H₂S in mammals [11, 12]. H₂S produced by these bacteria may influence colon health and function. A sulfate-rich diet causes increased growth of Desulfovibrio piger and high rates of H₂S production in the colon of humans and mice. In addition, several anaerobic bacterial such as Escherichia coli, Salmonella enterica, Clostridia and Enterobacter aerogenes generate H₂S from cysteine by the enzymatic activity of cysteine desulfhydrase [13, 14]. In addition to these pathways, H₂S may be generated by the action of sulfite reductase of the bacteria, E. coli, Rhodococcus Salmonella, Enterobacter, Klebsiella, Bacillus, Staphylococcus and Corynebacterium [15].

Figure 1. Endogenous biosynthesis of hydrogen sulfide (H₂S) in mammals.

H₂S is generated by three enzymes, cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3- mercaptopyruvate sulfurtransferase (3-MST). Dietary methionine is converted to homocysteine, via the intermediates S-adenosylmethionine and S-adenosylhomocysteine, which is then condensed with serine by CBS to generate cystathionine, which is acted on by CSE to generate cysteine. Cysteine can either enter the glutathione biosynthetic pathway or be utilized as a substrate for H₂S biosynthesis. CBS generates H₂S efficiently from a combination of cysteine and homocysteine, whereas CSE can utilize either cysteine or homocysteine by itself to generate the gasotransmitter. The third enzyme 3-MST utilizes the 3-mercaptopyruvate generated by cysteine aminotransferase (CAT) by forming a persulfide on its active site (R-SH to R-SSH). The persufide releases H₂S in the presence of a reductant (R’-SH).

Study of H₂S in the central nervous system were triggered by the discovery of sulfides in the brain. In 1989, while studying H₂S poisoning, Warenycia and associates discovered that inhalation of H₂S resulted in an elevation of brain sulfide that was directly proportional to the dose of the gas inhaled and mortality in rats. More interestingly, untreated brain also revealed the presence of sulfide [16]. In two cases of fatal H₂S inhalation in humans, elevated levels of sulfide were detected [17], prompting detailed studies on H₂S biosynthesis in the brain. Expression of CBS was found to be high in the hippocampus and cerebellum and the production of H₂S was found to be dependent on the presence of cysteine and PLP. While the production of H₂S was significantly inhibited by the use of the CBS inhibitors, hydroxylamine and amino-oxyacetate, S- adenosyl methionine (SAM/AdoMet) stimulated CBS activity. Early studies reported higher concentrations of H₂S (50–160 μM) in the brains of mammals. These estimations employed, the methylene blue method, which uses harsh acidic conditions, thus including the H₂S released from acid labile sulfur and iron-sulfur clusters [18] leading to overestimates. Endogenous concentrations measured by other methods revealed much lower 10 nM to 3 μM levels [18, 19].

2. Cellular functions of H₂S

H₂S participates in a plethora of cellular processes and it is becoming increasingly evident that almost all aspects of physiology are impacted by this gasotransmitter. The processes regulated are as diverse as response to inflammation to control of cellular senescence (Figure 2).

Figure 2. Physiological processes modulated by H₂S.

H₂S modulates several aspects of cellular physiology. The gasotransmitter regulates antioxidant defense, oxygen sensing, vascular functions, response to inflammation, mitochondrial function, second messenger signaling, aging, memory and long term potentiation and proteostasis. With increasing studies on this newly identified gaseous signaling molecule, additional functions are being discovered.

2.1. Antioxidant defense

One of the earliest studies demonstrating a cytoprotectant function of H₂S involved its role in antioxidant defense in neurons. Although H₂S by itself is not a strong reducing agent like glutathione, the major cellular antioxidant, it can exert potent antioxidant effects indirectly. H₂S stimulates the activity of the cysteine transporter and the cystine/glutamate antiporter to increase the uptake of cystine into neurons, where the reducing environment converts it to cysteine [20, 21]. In addition, H₂S enhances the activity of gamma-glutamylcysteine synthetase to increase glutathione, thereby protecting cells from glutamate induced oxidative stress or oxytosis. H₂S has also been shown to be scavenger of the highly reactive peroxynitrite radical ONOO⁻ [22].

2.2. H₂S in vascular function

H₂S protects against ischemia-reperfusion injury and heart failure [23–27]. Like NO and CO, H₂S mediates vascular functions and has been recognized as an endothelial derived relaxation factor (EDRF) that maintains vascular tone, endothelial cell function [28, 29], regulation of blood pressure [30] and angiogenesis [31, 32]. H₂S, like NO and CO modulates cGMP signaling [33, 34], which plays important roles not only in the peripheral vasculature but also that of the cerebral vascular system. Thus H₂S seems to regulate almost all aspects of vascular functions. In addition to these effects, H₂S protects against a variety of stress stimuli, including endoplasmic reticulum (ER) stress in a variety of cell types ranging from cardiac myocytes to neurons [35–39]. Induction of ER stress causes endothelial cell apoptosis, which has been linked to age-related endothelial dysfunction [40, 41]. Thus administration of H₂S donors could be beneficial under these conditions.

2.3. H₂S and bioenergetics

One of the first reported effects of H₂S was the inhibition of cytochrome c oxidase, a component of the mitochondrial electron transport chain, inducing a state of suspended animation [42]. In contrast, lower concentrations of H₂S stimulate mitochondrial function by stimulating the electron transport process during oxidative phosphorylation [43–45]. Thus, the effects of H₂S follow a bell-shaped dose response curve. The effects of H₂S on mitochondria are not surprising given the fact that the earth was once a sulfide-rich environment [46] and several life forms including those living in hydrothermal vents utilized sulfide as an energy source. One of the components of the electron transport chain is Coenzyme Q (CoQ), an electron carrier for sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the H₂S oxidation pathway. CoQ deficiency therefore impairs mitochondrial sulfite oxidation, leading to elevated H₂S levels [47].

2.4. H₂S and aging

Accumulating evidence shows that the stimulation of the reverse transsulfuration pathway responsible for the production of H₂S increases lifespan. The discovery that H₂S prolongs survival in Caenorhabditis elegans triggered studies on aging in different species [48]. It was found earlier that increased flux of methionine to the reverse transsulfuration pathway occurs in the long lived Ames dwarf mice [48]. Ames dwarf mice harbor a homozygous recessive mutation at the Prophet of Pit-1 (Prop-1) locus, which encodes a paired-like homeodomain transcription factor, leading to a lack of differentiation of somatotrophic, lactotrophic, and thyrotrophic pituitary cells causing a dwarf phenotype. As a result, these mice lack growth hormone (GH), prolactin, and thyroid stimulating hormone [49]. These mice have increased levels and/or activities of the transmethylation and transsulfuration enzymes in the liver, brain and kidney compared to wild type mice. One of the consequences of increased flux via the transsulfuration pathway is increased glutathione production. The Ames dwarf mice have higher glutathione production compared to wild type mice. Availability of cysteine is the rate-limiting step for glutathione biosynthesis and these mice displayed elevated CSE expression and activity [50, 51]. The contribution of the transsulfuration pathway to lifespan was also observed in the fruitfly Drosophila, where it was shown that the beneficial lifespan extending effects of dietary restriction required an active reverse transsulfuration pathway [52, 53]. Restriction of methionine and cysteine, the amino acids of the transmethylation and reverse transsulfuration pathways respectively, induces several benefits including extension of longevity across several species. Methionine deprivation extends lifespan in yeast [54]. In Drosophila, deprivation of essential amino acids, in particular, methionine, promotes longevity [55]. More recently, it was definitively shown that endogenously produced H₂S mediates increased longevity in yeast, C. elegans, Drosophila and mice [56]. The reverse transsulfuration pathway is affected in Werner’s syndrome, an autosomal recessive genetic disorder characterized by premature aging due to a mutation in the Werner protein, WRN, a DNA helicase, which results in its truncation. The disease is characterized by defects in DNA replication, recombination and maintenance of telomere ends at the molecular level and genomic instability [57, 58]. The enzymes CSE and CBS are downregulated in the disease, which could explain the elevated oxidative stress associated with the disease. H₂S is beneficial in fibroblasts derived from patients with Werner’s syndrome, attenuating oxidative stress and modulating the mTOR pathway [59]. Thus H₂S influences aging in multiple ways. Several of the cytoprotective actions of H₂S involve the very processes whose disruption leads to aging and senescence. These include mitochondrial metabolism, antioxidant defense, sulfhydration of reactive cysteines to prevent irreversible oxidation of cysteine residues, protein degradation and turnover, response to stress, glucose homeostasis, second messenger signaling, DNA repair pathways and several more.

2.5. H₂S and autophagy

Autophagy is an evolutionarily conserved quality control process which is involved in degradation and recycling of cellular components [60]. H₂S has been reported to have both stimulatory as well as inhibitory effects on autophagy. In cardiomyocytes subjected to hypoxia/reoxygenation, excessive autophagy caused apoptosis, however treatment with NaHS decreased autophagy and promoted cell survival [61]. A vast majority of studies reported stimulation of autophagy by H₂S. H₂S stimulates autophagy in hepatocellular carcinoma and colon cancer cell lines by inhibiting the mTOR pathway [62, 63]. The mechanisms underlying these diverse effects are not clear and may reflect differences arising due to the concentration of H₂S involved and/or tissue or cell-specific effects. Autophagy is utilized by tissues under conditions of starvation to maintain adequate nutrient supply. During nutrient deprivation, the elevated H₂S levels have been observed, although the substrate for H₂S production is limiting. The source of H₂S produced has been attributed to cysteine derived from protein degradation by autophagy, as knockdown of the autophagy protein 5 (ATG5) or 7 (ATG7), key players in the process resulted in lowered H₂S production [64]. In the long living Snell dwarf mice, where the flux through the reverse transsulfuration pathway responsible for cysteine and H₂S production, autophagy is upregulated [64, 65]. Thus modulation of H₂S production may promote longevity via increased autophagy.

3. Modes of signaling by H₂S in the nervous system

3.1. Protein sulfhydration

H₂S signals in several distinct modes, one of which is sulfhydration or persulfidation, wherein a reactive –SH group of a cysteine residue on proteins is converted to an –SSH group [66] in a fashion analgous to nitrosylation which results in formation of S-NO groups [67]. Sulfhydration is a substantially prevalent modification with about 25% of proteins being basally modified in the liver. One of the best characterized sulfhydrated protein is glyceraldehyde 3-phosphate dehydrogenase (GAPDH), wherein sulfhydration of C150 of GAPDH increases its catalytic activity sevenfold [66]. Nitrosylation of this residue abolishes its catalytic activity [68]. Sulfhydration participates in a plethora of physiologic pathways ranging from response to inflammation to neuroprotection [4–7]. In addition to modulating the function of target proteins, we predicted that sulfhydration would protect cysteine residues from irreversible oxidation [4, 7]. Sulfhydration usually occurs at cysteine residues with a low pKa (acid dissociation constant), which are more susceptible to sulfhydration, because they exist as thiolate anions (S⁻) at physiological conditions. However, these residues are also more prone to attack by oxidants such as H₂O₂ that generate sulfenic acid (SOH), sulfinic acid (SO₂H), or sulfonic acid (SO₃H) derivatives [69–71]. Formation of SOH moieties is usually reversible, but higher oxidation states of the thiol group of cysteine are irreversible. Thus, sulfhydration could protect reactive cysteine residues on proteins from irreversible oxidation by generating a persulfide group. The CySSO₂H (perthiosulfinic) and CySSO₃H (perthiosulfonic) oxidation products of persulfides can be recycled back by the reduction of their S–S moieties, which does not occur in the case of the CySO₃H oxidation product of unmodified (unsulfhydrated) cysteine residues on proteins. The protection of cysteine residues by sulfhydration was subsequently demonstrated for phosphatase and tensin homolog deleted on chromosome 10, PTEN [72, 73]. Sulfhydration results in increased reactivity of the modified cysteine by virtue of enhanced nucleophilicity. Nitrosylation, on the other hand, decreases the reactivity of the modified cysteine residue. Similar to nitrosylation, sulfhydration can be reduced by endogenous enzymes [74–76]. The enzymes, thioredoxin reductase-1 (TrxR1), Trx1, and the alternative TrxR1-dependent redox active thioredoxin-related protein of 14 kDa (TRP14) reduce polysulfides and protein persulfide [76]. Several of the protective functions of H₂S can be ascribed to sulfhydration. For instance, the antioxidant defenses bolstered by H₂S is mediated in part by sulfhydration of Kelch-like ECH-associated protein 1 (Keap1), a negative regulator of NRF2, the master regulator antioxidant stress response [77, 78]. Sulfhydration of Keap1 causes its dissociation from NRF2, facilitating its nuclear translocation and transcriptional activation of tits target genes. In addition to its widespread roles in the periphery, H₂S and sulfhydration play significant roles in the optimal functioning of the nervous system. Dysregulation of H₂S metabolism and sulfhydration has been frequently observed in neurodegenerative diseases such as Parkinson’s disease, Alzheimer’s disease and during traumatic brain injury.

3.1.1. Sulfhydration and LTP

H₂S participates in various aspects of brain physiology, which includes its influence on long term potentiation (LTP) in the brain following activation of N-methyl-D-aspartate receptor (NMDA) receptors [79]. The NMDA receptor is an ion channel which is activated by glutamate and its coagonists (glycine, D-serine or D-cysteine), which is involved in regulation of memory and synaptic plasticity [80]. CO and NO also regulate LTP but in an NMDA-independent manner [81]. H₂S by itself does not augment LTP but does so in the presence of weak electrical stimulation, suggesting that it operates on active synapses [79]. The NR2B subunit of the NMDA receptor was found to be regulated by the H₂S donor sodium hydrogen sulfide (NaHS). Cognitive dysfunction induced by hepatic ischemia/reperfusion injury, which was associated with decreased NR2B expression, was mitigated by NaHS treatment along with restoration of NR2B levels [82].

It was recently shown that the facilitation of LTP by H₂S, generated by CBS, occurs by sulfhydration of serine racemase (SR), the biosynthetic enzyme for D-serine and a coagonist of the NMDA receptor [83]. LTP declines during old age along with levels of sulfhydration and activity of SR in rats. The study also showed that H₂S and sulfide levels increased after high frequency stimulation (HFS) stimulation of hippocampal neurons. Supplementation of exogenous H₂S restored sulfhydration and LTP with improvement in memory and cognition [83]. H₂S formation results in the generation of polysulfides which can mediate the physiologic functions of H₂S. Treatment with sodium tetrasulfide (Na₂S₄), a polysulfide donor, was found to increase hippocampal LTP just as with H₂S administration. H₂S was also found to promote surface expression the AMPA Receptor subunit, GluR1/GluA1, which is also involved in regulation of synaptic plasticity [84] via phosphorylation of GluR1 through a sulfhydration-dependent mechanism [85].

3.1.2. Sulfhydration in Parkinson’s disease

Parkinson’s disease (PD), the second most prevalent neurodegenerative disease, affects the substantia nigra of the brain leading to motor dysfunction. Misfolding and aggregation of the protein α-synuclein is a characteristic of the disease. We have shown that dysregulated H₂S signaling occurs in the disease, wherein sulfhydration levels of the E3-ubiquitin ligase parkin responsible for clearance of toxic, misfolded proteins occurs [86]. The main target of sulfhydration on parkin is Cys95, with Cys59 and Cys182 also being modified. Analysis of postmortem patient striata reveals diminished sulfhydration of parkin with reduced enzymatic activity; whereas nitrosylation of parkin, which inactivates it, is elevated [86, 87]. H₂S donors are protective in animal models of PD and offer therapeutic benefits (Figure 3; [88, 89]. Similarly, overexpression of CBS or use of H₂S donors in the 6 hydroxydopamine (6-OHDA)- induced PD model affords neuroprotection [90, 91].

Figure 3. Neuroprotective mechanisms mediated by H₂S in Parkinson’s disease (PD).

PD is characterized by elevated oxidative stress and accumulation of misfolded proteins such as α-synuclein. H₂S mediates sulfhydration of the E3-ubiquitin ligase, parkin, which increases its catalytic activity and results in clearance of toxic proteins. In PD, this pathway is impaired by suboptimal sulfhydration of parkin and accumulation of parkin substrates. Neuroprotection mediated by H₂S donors in PD therapy act in multiple ways. H₂S scavenges reactive nitrogen species (RNS) and reactive oxygen species (ROS) either directly or indirectly by the activation of antioxidant defense pathways such as those regulated by Nrf2. Sulfhydration of Kelch-like ECH-associated protein 1 (Keap1), the inhibitor of Nrf2, causes their dissociation to facilitate nuclear translocation of Nrf2 and transcription of its target genes involved in maintenance of redox homeostasis. Another mode by which H₂S protects cells is by maintenance of mitochondrial function, which is affected in PD.

3.2. Altered H₂S production and signaling in neurodegenerative diseases

3.2.1. H₂S production in Huntington’s disease

Huntington’s disease (HD) is a devastating neurodegenerative disorder that affects the corpus striatum of the brain leading to involuntary movements and motor dysfunction along with psychiatric disturbances. The disease is caused by expansion of polyglutamine repeats in the protein huntingtin, which aggregates and affects several cellular processes ranging from transcriptional dysfunction to motor disabilities [92, 93]. We have shown recently that cysteine metabolism is compromised in HD [93]. The biosynthetic enzyme for cysteine, CSE is depleted in cell culture, mouse models of HD as well as human HD brain due to sequestration of its transcription factor SP1 by mutant huntingtin. Decreased CSE expression in neurons causes decreased cysteine and H₂S synthesis which contribute to redox imbalance and altered stress responses (Figure 4). In addition to suboptimal cysteine biosynthesis, impaired function of cysteine as well as cystine transporters have been reported in HD, all of which lead to a cysteine deficit accompanied by elevated ROS levels in cells [94, 95]. CSE is regulated by activating transcription factor 4 (ATF4) under conditions of stress such as amino acid limitation and ER stress. This pathway was also found to be affected in HD which contributed to neurotoxicity [96]. Mitigating oxidative stress by treating mice with the antioxidant and cysteine precursor, N-acetyl cysteine (NAC) reverses symptoms in HD mice. Treating the striatal cells with sodium ascorbate relieved the oxidative burden on cells and corrects defective stress responses. Another triplet repeat disorder, where CSE depletion was observed is spinocerebellar ataxia type 3, where overexpression of CSE was beneficial in a Drosophila model of the disease [97]. Thus, optimal activity of the reverse transsulfuration pathway is vital for the maintenance of redox homeostasis in cells.

Figure 4. Disrupted cysteine and H₂S homeostasis in Huntington’s disease (HD).

HD is characterized by elevated oxidative stress and impaired cysteine metabolism, which stems from deficiency of its biosynthetic enzyme, cystathionine γ-lyase (CSE). CSE is the sole biosynthetic enzyme for cysteine, whose basal transcription is controlled by specificity protein 1 (SP1) under basal conditions. In addition to cysteine production by CSE, cysteine is also taken up from the diet via the cystine (xCT) and cysteine (EAAC1) transporters. During conditions of stress, such as amino acid deprivation, in normal cells, the activating transcription factor 4 (ATF4) upregulates CSE expression to cope with cysteine deficit. ATF4 is also the transcription factor for xCT, which imports cystine under these conditions. In HD, all these pathways are compromised. SP1 is sequestered by mutant huntingtin leading to decreased CSE expression. This causes elevated oxidative stress such that ATF4, which would normally act to produce more CSE fails to do so, further contributing to the redox burden of cells in a vicious cycle, which leads to neurotoxicity.

3.2.2. H₂S and Alzheimer’s disease

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease that affects the cognitive functions and memory of affected individuals [98, 99]. The disease predominantly affects the cerebral cortex and the hippocampus. The cause of the disease is multifactorial with both familial and sporadic causes, unlike HD, which is a monogenic disorder. Among the familial cases of AD, which account for a majority of early onset AD, mutations in the amyloid precursor protein (APP), presenilin-1 and 2 (PS1 and 2) have been frequently encountered. APP undergoes sequential proteolysis which is catalyzed by proteases, termed secretases and include α,β and γ secretases. Aggregation of β-amyloid (Aβ) and Tau proteins are hallmarks of the disease, which cause deposition of amyloid plaques and neurofibrillary tangles respectively. Aβ is a peptide which is generated by the proteolytic processing the amyloid precursor protein (APP). Aberrant processing of APP and accumulation of Aβ (1–42) is observed in AD. Several mouse models of AD harboring APP mutations either alone or in combination with mutations in other genes have been developed for the study of AD [100]. Soluble Aβ can reduce cysteine and glutathione levels in cells by inhibiting the excitatory amino acid transporter 3 (EAAT3/EAAC1), which is the neuronal cysteine transporter [101], which could affect H₂S signaling and elevated oxidative stress. The depletion of EAAC1 results in elevated oxidative stress and age-dependent neurodegeneration [102]. H₂S levels in plasma of AD patients was decreased as compared to normal individuals and the there was a negative correlation of H₂S levels with the severity of the disease [103]. In parallel, elevated homocysteine levels were observed in patients. An independent study revealed that S-adenosylmethionine (SAM) levels were diminished in AD [104]. SAM is an allosteric activator of CBS, the homocysteine metabolizing enzyme so that reduction in its activation would be expected to decrease H₂S production. Supplementation with H₂S donors in rats afforded protection against homocysteine- induced cognitive dysfunction [105]. H₂S caused an increase in expression and activity of the antioxidant enzyme, aldehyde dehydrogenase 2 and reduced homocysteine-induced generation of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), markers of oxidative stress in the hippocampus of treated rats. In the APP/PS1 mouse model of AD [106], H₂S donors were found to reduce cognitive impairment. H₂S has pleiotropic effects and influences several pathways which are affected in AD. One of them includes mitigation of oxidative stress. Administration of the H₂S donor, NaHS increased the expression of Nrf2, which plays central roles in maintenance of redox balance. As described earlier, Keap1, the inhibitor of Nrf2, is sulfhydrated, which could also contribute to increased Nrf2 nuclear localization.

Another therapeutic approach in AD involves inhibition of the BACE1, the β secretase, which is responsible for production of β-amyloid peptides. In a study utilizing the APP/PS1 mice, intraperitoneal injection of NaHS, downregulated BACE1 and PS1 via a PI3/Akt pathway, decreased Aβ and improved spatial memory [107]. Thus, H₂S mediates neuroprotection via numerous pathways (Figure 5).

Figure 5. Neuroprotective mechanisms mediated by H₂S in Alzheimer’s disease (AD).

H₂S therapy is beneficial in mouse models of AD. Treatment with H₂S donors such as NaHS or GYY4137 improved spatial memory, cognitive functions, reduced oxidative stress and inflammation. At the molecular level, H₂S prevents Tau phosphorylation and decreased expression of amyloid precursor protein (APP), β-secretase (BACE1) and γ-secretase. In addition decreased accumulation of amyloid beta peptides and neurofibrillary tangles were observed upon treatment with H₂S donors.

3.2.3. Elevated H₂S production as a cause of neurological deficits: Amyotrophic lateral sclerosis (ALS) and Down syndrome

Low concentrations of H₂S are generally cytoprotective while higher doses may be detrimental. ALS has been associated with high levels of H₂S [108]. ALS is a fatal neurodegenerative disease leading to selective degeneration of upper and lower motor neurons [109]. The causes of ALS may be sporadic or familial. Among the genetic causes of ALS, about 20% are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Of these, the most commonly studies is the G93A mutation, a gain of function mutation that aggregates and causes death of motor neurons [110, 111]. In ALS, H₂S production by CBS is elevated, with the G93A mouse model of familial ALS (fALS) exhibiting increased H₂S production in tissues and spinal cord cultures where increased intracellular Ca²⁺ levels are observed. Elevated H₂S is also detected in cerebrospinal fluid of ALS patients, indicative of aberrant gasotransmitter signaling in ALS.

Another disease where elevated H₂S production is observed is Down syndrome, which manifests trisomy of chromosome 21, which is the chromosome on which CBS is located [112]. The urine of Down syndrome patients has high levels of thiosulfate, a catabolic product of H₂S. Interestingly, the overexpression of CBS is greater than expected from the trisomy of chromosome 21. Down syndrome patients develop Alzheimer’s disease-like pathology as they age with cognitive deficits. Immunohistochemical analysis localized CBS in astrocytes adjacent to the senile plaques, indicating a role for H₂S in the process.

4. Future Perspectives

It is becoming increasingly clear that H₂S signaling modulates diverse aspects of physiological processes. An area that merits further investigation are the conditions or stimuli that modulate the production of H₂S in the brain and the levels of the gasotransmitter that promote beneficial effects. The conditions and concentrations at which the effects of H₂S switch from protective to deleterious also requires further investigation. The dose-response curve for H₂S is bell shaped, with lower concentrations stimulating several physiological processes such as mitochondrial function or antioxidant effects, and higher concentrations leading to cell death or other adverse effects. The varying effects of H₂S reported in literature could be due to the dual effects of H₂S. Accordingly, there is a need to develop more specific and sensitive reagents to detect the presence and dynamics of this gasotransmitter in vivo. Especially confounding in the field is the lack of methods to accurately estimate endogenous concentrations of H₂S. In addition to development of H₂S specific probes, there exist several areas that would benefit from further study. One of these involves the switch from cysteine production to H₂S production in various tissues. Cysteine is the precursor of metabolites such as biotin, taurine, glutathione, and coenzyme A. Thus, distinguishing effects modulated by H₂S as opposed to cysteine and its metabolites may be challenging. In addition to these aspects, the development of tissue-specific knockout models of CSE, CBS and 3-MST would be valuable tools to dissect the roles of the three enzymes in normal signaling pathways. At the molecular level, we need proteomic approaches to arrive at sites of sulfhydration and its interplay with nitrosylation both in normal and diseased states. Understanding the interplay of H₂S with the other gasotransmitters NO and CO may lead to a better understanding of their role in homeostatic regulation of cellular processes as well as novel precision therapeutics.

Abbreviations used

3-MST

3-mercaptopyruvate sulfurtransferase

4-HNE

4-hydroxynonenal

6-OHDA

6–hydroxydopamine

Aβ

β-Amyloid

AD

Alzheimer’s disease

ALS

Amyotrophic lateral sclerosis

AMPA

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

APP

Amyloid precursor protein

ATF4

Activating transcription factor 4

BACE1

β-secretase 1

CAT

Cysteine aminotransferase

CBS

Cystathionine β-synthase

CO

Carbon monoxide

CoQ

Coenzyme Q

CSE

Cystathionine γ-lyase

EAAT3

Excitatory amino acid transporter 3

EDRF

Endothelial derived relaxation factor

fALS

Familial Amyotrophic lateral sclerosis

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

GH

Growth hormone

GluA1

Glutamate receptor subunit A1

H₂S

Hydrogen sulfide

HD

Huntington’s disease

HFS

High frequency stimulation

Keap1

Kelch-like ECH associated protein 1

LTP

Long-term potentiation

MDA

Malondialdehyde

mTOR

mechanistic target of rapamycin

NaHS

Sodium hydrogen sulfide

NMDA

N-methyl-D-aspartate

NO

Nitric Oxide

NR2B

N-methyl D-aspartate receptor subtype 2B

Nrf2

Nuclear factor erythroid 2-related factor 2

PLP

Pyridoxal 5-phosphate

PD

Parkinson’s disease

pKa

Acid dissociation constant

PLP

Prop-1, Prophet of Pit-1

PS1

Presenilin 1

PTEN

phosphatase and tensin homolog deleted on chromosome 10

ROS

Reactive oxygen species

RNS

Reactive nitrogen species

SAM

S-adenosyl methionine

SP1

Specificity Protein 1

SQR

Sulfide:quinone oxidoreductase

SR

Serine racemase

SRB

Sulfate reducing bacteria

TRP14

Thioredoxin-related protein of 14 kDa

TrxR1

Thioredoxin reductase-1

TSH

Thyroid stimulating hormone

WRN

Werner protein.