Hydrogen sulfide signalling in the CNS ‐ Comparison with NO

Abstract

Hydrogen sulfide (H₂S) together with polysulfides (H₂S_n, n > 2) are signalling molecules like NO with various physiological roles including regulation of neuronal transmission, vascular tone, inflammation and oxygen sensing. H₂S and H₂S_n diffuse to the target proteins for S‐sulfurating their cysteine residues that induces the conformational changes to alter the activity. On the other hand, 3‐mercaptopyruvate sulfurtransferase transfers sulfur from a substrate 3‐mercaptopyruvate to the cysteine residues of acceptor proteins. A similar mechanism has also been identified in S‐nitrosylation. S‐sulfuration and S‐nitrosylation by enzymes proceed only inside the cell, while reactions induced by H₂S, H₂S_n and NO even extend to the surrounding cells. Disturbance of signalling by these molecules as well as S‐sulfuration and S‐nitrosylation causes many nervous system diseases. This review focuses on the signalling by H₂S and H₂S_n with S‐sulfuration comparing to that of NO with S‐nitrosylation and discusses on their roles in physiology and pathophysiology.

Abbreviations

3MP

3‐mercaptopyruvate

ADAM17

disintegrin and metalloproteinase domain‐containing protein 17

Aβ1–42

amyloid β‐protein

BACE

β‐secretase

CAT

cysteine aminotransferase

CBS

cystathionine β‐synthase

CFTR

cystic fibrosis transmembrane conductance receptor

CSE

cystathionine γ‐lyase

EE

ethylmalonyl encephalopathy

ETHE1

sulfur dioxygenase

FADH₂

flavin adenine dinucleotide

GCL

glutamate cysteine ligase

H₂S_n

hydrogen polysulfides

HSNO

thionitrous acid

HSSNO

nitrosopersulfide

Keap1

Kelch‐like ECH‐associated protein 1

MPST

3‐mercaptopyruvate sulfurtransferase

Nrf2

nuclear factor erythroid 2‐related factor 2

TRPA1

transient receptor potential ankyrin 1

γ‐GCS

γ‐glutamyl cysteine synthetase

1. INTRODUCTION

A diffusing factor, which is released from vascular endothelium by a stimulation with ACh to relax vascular smooth muscle, was discovered and called endothelium‐derived relaxation factor (EDRF) (Furchgott & Zawadzki, 1980). It was later identified as NO (Ignarro, Buga, Wood, Byrns, & Chaudhuri, 1987; Palmer, Ferrige, & Moncada, 1987), which activates soluble guanylate cyclase and subsequently PKG through cGMP (Arnold, Mittal, Katsuki, & Murad, 1977). In the brain, an excitatory neurotransmitter glutamate had been recognized to induce cGMP (Mao, Guidotti, & Costa, 1974) and the activation of NMDA receptors by glutamate was found to induce a release of a diffusible factor which had similar properties to EDRF in a Ca²⁺‐dependent manner (Garthwaite, Charles, & Chess Williams, 1988). NO producing activity was identified as an enzymatic reaction with arginine as a substrate in NADPH and Ca²⁺/calmodulin‐dependent manner (Bredt & Snyder, 1990). The activity is localized to neurons (neuronal NOS [nNOS]), vascular endothelial cells (endothelial NOS [eNOS]) and the inducible activity (inducible NOS [iNOS]) (Bredt et al., 1991).

Hydrogen sulfide (H₂S), which was identified in the brain (Goodwin et al., 1989; Savage & Gould, 1990; Warenycia, Goodwin, et al., 1989), has physiological roles including cognitive function (Abe & Kimura, 1996) and vascular tone regulation (Hosoki, Matsuki, & Kimura, 1997; Zhao, Zhang, Lu, & Wang, 2001). H₂S is produced by four enzymes cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE), 3‐mercaptopyruvate sulfurtransferase (MPST) and l‐cysteine:2‐oxoglutarate aminotransferase (CAT) or d‐amino acid oxidase, from d/l‐cysteine as a source of sulfur (Abe & Kimura, 1996; Chiku et al., 2009; Hosoki et al., 1997; Mikami, Shibuya, Kimura, Ogasawara, & Kimura, 2011; Nagahara, Yoshii, Abe, & Matsumura, 2007; Shibuya et al., 2009; Shibuya et al., 2013; Stipanuk & Beck, 1982). CBS is localized to astrocytes in the brain but most intensively in cerebellar Bergmann glia (Enokido et al., 2005), while the levels of CSE and its activity are significantly low in the brain (Ishii et al., 2004). MPST is localized to both neurons and astrocytes in the brain (Nagahara, Ito, Kitamura, & Nishino, 1998; Shibuya et al., 2009). DAO is expressed in the brain stem, cerebellum and spinal cord, exclusively astrocytes including Bergmann glia (Horiike, Tojo, Arai, Nozaki, & Maeda, 1994). CAT, which is identical to aspartate aminotransferase, is expressed mainly in the cerebellum (Lin & Chen, 1983).

H₂S induces Ca²⁺ influx in astrocytes by activating transient receptor potential ankyrin 1 (TRPA1) channels and we later found that hydrogen polysulfides (H₂S_n, n > 2) activate the channels more effectively than H₂S does (Kimura et al., 2013; Nagai, Tsugane, Oka, & Kimura, 2004, 2006; Oosumi et al., 2010). H₂S₂ and H₂S₃ were identified in the brain and MPST produces H₂S_n, and other persulfurated molecules such as cysteine persulfide, GSH persulfide and persulfurated proteins (Kimura et al., 2017; Kimura et al., 2015; Koike et al., 2017; Nagahara, 2018; Nagahara, Koike, Nirasawa, Kimura, & Ogasawara, 2018; see also Kimura, 2020). H₂S_n are oxidized by sulfur dioxygenase to sulfite (H₂SO₃), which is further oxidized by rhodanese to thiosulfate (H₂S₂O₃).

Two modes of action have been proposed for both H₂S and NO signalling. One is that both molecules diffuse to the haem for redox reaction (Ruetz et al., 2017; Vitvitsky, Yadav, Kurthen, & Banerjee, 2015) as well as to cysteine residues of the target proteins to S‐sulfurate, covalently react sulfur with a thiol of cysteine residues (S‐sulfhydrate, persulfidate; see Toohey, 2012) or S‐nitrosylate, covalently react NO with a critical protein thiol, them (Lancaster, 2017; Mustafa et al., 2009). The other is that sulfur and NO are transferred from respective enzymes to the target proteins for S‐sulfuration and S‐nitrosylation. An example of the first mode of action of NO is that of EDRF, which diffuses from endothelium to vascular smooth muscle (Furchgott & Zawadzki, 1980). In the nervous system, NO diffuses from postsynapse to presynapse as a retrograde transmitter to induce a release of neurotransmitter glutamate (Garthwaite, 1991; Garthwaite et al., 1988; Zhuo, Small, Kandel, & Hawkins, 1993). On the other hand, the enzyme‐mediated S‐nitrosylation proceeds by clusters of enzymes which generate NO, synthesize S‐nitrosylated proteins, and transnitrosylate it that is the reaction to transfer of an NO group from one protein to another (Nakamura & Lipton, 2013; Seth et al., 2018).

H₂S diffuses to haem of target proteins to regulate their activity or to the target cysteine disulfide to reduce it (Abe & Kimura, 1996; Aizenman, Lipton, & Loring, 1989; Matsui et al., 2018). H₂S and H₂S_n diffuse to the targets to S‐sulfurate cysteine residues to modify their activity (Kimura et al., 2015; Mustafa et al., 2009). As to enzyme‐mediated S‐sulfuration, MPST transfers sulfur from 3‐mercaptopyruvate (3MP) to cysteine residues of target proteins (Kimura et al., 2017; Nagahara, Nirasawa, Yoshii, & Niimura, 2012).

H₂S exerts regulatory, beneficial, and protective effects at physiological concentrations, while it is toxic at higher concentrations. In Down's syndrome and ethylmalonyl encephalopathy, the levels of H₂S and/or H₂S_n are increased and cause damage to the brain (Panagaki, Randi, Augsburger, & Szabo, 2019; Tiranti et al., 2009). For Down's syndrome and ethylmalonyl encephalopathy, a decrease in the levels of H₂S has been proposed to have therapeutic potential. In contrast, in Huntington's disease and Alzheimer's disease, H₂S levels are not enough to properly function (Cao et al., 2018; Paul et al., 2014; Sbodio, Snyder, & Paul, 2016, 2018; Vandini et al., 2019). For these diseases, a supplementation of H₂S may have a benefit. In schizophrenia, both beneficial and toxic effects of H₂S and H₂S_n have been reported (Ide et al., 2019; Topcuoglu et al., 2017; Ünal, Erzin, Yüksel, Alisik, & Erel, 2018; Xiong et al., 2018). The balance of H₂S, H₂S_n and NO as well as S‐sulfuration together with S‐nitrosylation plays an important role for the pathogenesis of these neuronal diseases.

2. S‐SULFURATION BY H₂S, H₂S_n AND MPST

Mustafa et al. (2009) have demonstrated that S‐sulfuration of cysteine residues of target proteins as a mode of action of H₂S. GAPDH is activated through S‐sulfuration by H₂S (Mustafa et al., 2009). It was later reported that the activity of GAPDH is suppressed by S‐sulfuration by H₂S_n rather than H₂S (Jarosz et al., 2015). S‐sulfuration depends on the redox condition of the target cysteine residues; H₂S S‐sulfurates oxidized cysteine like Cys‐SOH or Cys‐SNO, while H₂S_n S‐sulfurate cysteine (Cys‐SH) (Figure 1) (Mishanina, Libiad, & Banerjee, 2015). The activity of GAPDH depends on the redox state of its active cysteine residues.

FIGURE 1.

H₂S_n can transmit signals across cells, while S‐sulfuration by 3‐mercaptopyruvate sulfurtransferase (MPST) is restricted only inside the cells. (a) H₂S and H₂S_n can pass through plasma membrane of the cell in which they are produced and reach the nearby cells as well as react with the targets inside the produced cell. H₂S S‐sulfurates the S‐nitrosylated cysteine residues, while H₂S_n S‐sulfurate the cysteine residues. (b) MPST transfers sulfur from 3MP to targets inside the cell

H₂S_n activate TRPA1 channels which have two sensitive cysteine residues at the amino terminus, suggesting that the two residues are S‐sulfurated or one of them is S‐sulfurated to react with the other to make cysteine disulfide bridge (Hatakeyama, Takahashi, Tominaga, Kimura, & Ohta, 2015; Kimura, 2015a; Kimura et al., 2013; Nagai et al., 2004, 2006; Oosumi et al., 2010; Streng et al., 2008). A similar mechanism was reported for the regulation of tumour suppressor phosphatase and tensin homologue in which one cysteine residue is S‐sulfurated by H₂S_n and then reacts with another non‐S‐sulfurated one to produce a cysteine disulfide bond, leading to the conformational change (Greiner et al., 2013). Protein kinase G‐1α (PKG1α) is inactive at its monomer, while it is activated by forming dimer which is generated by S‐sulfuration of one cysteine residue of a monomer that reacts with a counterpart cysteine residue of another monomer to produce cysteine disulfide bridge between the two (Kimura, 2020; Stubbert et al., 2014).

MPST associated with thioredoxin produces H₂S and H₂S_n as well as other persulfurated molecules (Figure 2) (Kimura et al., 2017; Kimura et al., 2015; Mikami et al., 2011). The endogenous levels of H₂S and H₂S₂ in the brain are 0.030 ± 0.004 μmol·g⁻¹ protein (approximately 3.0 μM) and 0.026 ± 0.002 μmol·g⁻¹ protein (2.6 μM), respectively (Koike et al., 2017). Because H₂S₂ and H₂S₃ efficiently S‐sulfurate cysteine and GSH to produce cysteine persulfide and GSH persulfide (Kimura et al., 2017), once H₂S_n are produced, cysteine and GSH exist nearby can immediately be S‐sulfurated. Since no enzyme has been identified to mediate the reaction of H₂S with haem containing proteins such as Hb, neuroglobin and catalase as well as sulfur quinone oxidoreductase and cupper/zinc SOD (Olson et al., 2018; Olson et al., 2017; Ruetz et al., 2017; Searcy, 1996; Searcy, Whitehead, & Maroney, 1995; Vitvitsky et al., 2015), H₂S must reach these targets by diffusion.

FIGURE 2.

S‐sulfuration by H₂S_n diffusion and that by 3‐mercaptopyruvate sulfurtransferase (MPST). (a) MPST produces H₂S_n which reach by diffusion to S‐sulfurate H₂S, cysteine, GSH, and cysteine residues of proteins. (b) MPST transfers sulfur from 3‐mercaptopyruvate (MP) to H₂S, cysteine, GSH, and cysteine residues of proteins without being mediated by H₂S_n (trans‐sulfuration). This figure is produced by modifying Kimura et al. (2017)

How do H₂S_n, oxidized molecules of H₂S, survive in the relatively reducing atmosphere of the cell? In the intracellular milieu, all the cysteine residues are not necessarily thiol, but some of them are oxidized. Under basal conditions, 10% to 25% of proteins are S‐sulfurated (R‐SSH) with a few % S‐nitrosylated (R‐SNO), and remaining 70% to 90% are proteins with thiol (R‐SH) (Jaffrey, Erdjument‐Bromage, Ferris, Tempst, & Snyder, 2001; Mustafa et al., 2009). It is also supported by our observations that brain homogenates absorb H₂S as bound sulfane sulfur (S‐sulfurated molecules including proteins), which releases H₂S under reducing conditions such as in the presence of dithiothreitol (DTT) (Ishigami et al., 2009), suggesting that cells contain oxidized cysteine residues such as S‐nitrosylated ones, which can react with H₂S to produce S‐sulfurated cysteine residues or bound sulfane sulfur. H₂S is released from bound sulfane sulfur in brain homogenates (without the pre‐absorption of H₂S) in the presence of DTT (1,481 + 174 nmol·g⁻¹ protein of brain homogenates at pH 7.4) (Ishigami et al., 2009). These observations suggest that S‐sulfurated and S‐nitrosylated proteins exist together with proteins with thiol in cells. The ratio of S‐sulfurated, S‐nitrosylated proteins to intact cysteine residues of proteins must well be balanced with that of corresponding free molecules in cells (Figure 3).

FIGURE 3.

The ratio of S‐sulfurated, S‐nitrosylated proteins to intact cysteine residues of proteins must well be balanced with that of corresponding free molecules in cells. In the extracellular milieu, oxidized glutathione (GSH) and cystine are dominant with much less amount of cysteine. Pr, protein; R, cysteine and GSH

MPST transfers sulfur from 3MP to cysteine as observed in the following. (1) MPST can produce H₂S, H₂S_n, cysteine persulfide, and GSH persulfide by transferring sulfur to cysteine (Kimura et al., 2017; Kimura et al., 2015). (2) The levels of persulfurated molecules, which were measured as H₂S released in the presence of a reducing agent DTT called as bound sulfane sulfur (Ishigami et al., 2009; Kimura, 2015a; Ogasawara, Ishii, Togawa, & Tanabe, 1993; Ogasawara, Isoda, & Tanabe, 1994; Warenycia et al., 1990), were greater in cells expressing MPST compared to a control (Shibuya et al., 2009). (3) Administration of a substrate cysteine to mice increased the amounts of bound sulfane sulfur in tissues expressing MPST, while there was no change in those administered saline (Shibuya et al., 2013). (4) The levels of bound sulfane sulfur were less than half in the brains of MPST knockout mice than those in the wild‐type mice (Kimura et al., 2017). S‐sulfuration by MPST is specific but restricted inside cells, while that by diffusion extends the signalling to nearby cells.

3. S‐NITROSYLATION AND THE INTERACTION BETWEEN H₂S AND NO

For S‐nitrosylation, two mechanisms have been proposed. NO was initially thought only to diffuse after being produced by NOS to the target proteins to S‐nitrosylate them (Lancaster, 2017). However, it has been demonstrated that S‐nitrosylation proceeds by clusters of enzymes which generate NO, synthesize S‐nitrosylated proteins, and transnitrosylate it (Seth et al., 2018). Signalling by H₂S, H₂S_n and NO as well as S‐sulfuration and S‐nitrosylation must have both diffusion‐mediated and enzyme‐oriented mechanisms for regulating the activity of targets.

Synergistic effect of H₂S with NO on vascular relaxation led to the identification of two mechanisms (Hosoki et al., 1997). One is that the chemical interaction between H₂S and NO produces H₂S_n, nitrosothiol, thionitrous acid (HSNO), and nitrosopersulfide (HSSNO) that have the relaxation effect greater than each parental molecule (Cortese‐Krott et al., 2015; Eberhardt et al., 2014; Filipovic et al., 2012; Miyamoto et al., 2017; Moustafa & Habara, 2016; Nagai et al., 2006; Oosumi et al., 2010; Stubbert et al., 2014; Whiteman et al., 2006; see also Kimura, 2020). Another mechanism is that H₂S and NO mutually regulate their synthesizing enzymes (Kimura, 2016; King et al., 2014; Kondo et al., 2013; Minamishima et al., 2009; Zhao et al., 2001). For chemical interaction between H₂S and NO, both molecules should diffuse to interact with each other.

After the identification of EDRF as NO, there appeared a discrepancy. EDRF hyperpolarizes the membrane of vascular smooth muscle, while NO has little hyperpolarizing effect. Based on this observation, it has been suggested that EDRF contains an additional factor, endothelium‐derived hyperpolarizing factor (EDHF) (Chen, Suzuki, & Weston, 1988). Because H₂S activates K_ATP channels and hyperpolarizes the membrane potential, it has been proposed as a potential EDHF (Hosoki et al., 1997; Mustafa et al., 2011; Zhao et al., 2001). Another possibility is H₂S_n, which are produced by the chemical interaction of H₂S with NO and activate PKG1α to relax vasculature and are also potent substances to activate K_ATP channels by S‐sulfurating the active cysteine residue (Cortese‐Krott et al., 2015; Eberhardt et al., 2014; Filipovic et al., 2012; Miyamoto et al., 2017; Moustafa & Habara, 2016; Mustafa et al., 2011; Nagai et al., 2006; Oosumi et al., 2010; Stubbert et al., 2014; Whiteman et al., 2006; see also Kimura, 2020).

4. PHYSIOLOGICAL ROLES OF H₂S, H₂S_n, AND NO

4.1. Neuronal plasticity

When a neurotransmitter glutamate activates postsynaptic NMDA receptors, Ca²⁺ influx is induced and Ca²⁺/calmodulin‐dependent nNOS is subsequently activated to produce NO (Garthwaite et al., 1988). NO produced at post‐synaptically crosses synaptic cleft to pre‐synaptic terminal as a retrograde transmitter, which modifies a release of neurotransmitter glutamate, leading to the facilitation of hippocampal LTP (O'Dell, Hawkins, Kandel, & Arancio, 1991). NO also modifies LTP formation to alter the activity post‐synaptically (Taqatqeh et al., 2009).

H₂S enhances the activity of NMDA receptors at the active synapses by reducing the cysteine disulfide bond located at the hinge of a ligand binding domain of the receptors to facilitate LTP induction (Abe & Kimura, 1996; Aizenman et al., 1989). On the other hand, H₂S_n activate TRPA1 channels in astrocytes surrounding the synapse to induce Ca²⁺ influx, which triggers a release of a gliotransmitter d‐serine to the synaptic cleft to enhance the activity of NMDA receptors (Kimura et al., 2013; Nagai et al., 2004, 2006; Oosumi et al., 2010; Shigetomi, Jackson‐Weaver, Huckstepp, O'Dell, & Khakh, 2013). Genetic knockdown of CBS impairs LTP, while S‐sulfuration of serine racemase, which generates d‐serine from l‐serine, restores LTP (Li et al., 2017).

4.2. Neuroprotective role

There are two forms of glutamate toxicity; excitotoxicity is caused by the immoderate excitation of NMDA receptors which transport excessive Ca²⁺ into neurons to death (Choi, 1988). In oxidative glutamate toxicity called oxytosis, which is quite similar to ferroptosis (Lewerenz, Ates, Methner, Conrad, & Maher, 2018), high concentrations of glutamate suppress cystine/glutamate antiporter, leading to the decreased transport of cystine into cells that causes decreased production of GSH, an intracellular major antioxidant, resulting in making cells vulnerable to oxidative stress (Murphy, Miyamoto, Sastre, Schnaar, & Coyle, 1989; Tan, Schubert, & Maher, 2001). H₂S protects embryonic neurons from oxidative glutamate toxicity (Kimura & Kimura, 2004). H₂S enhances the activity of cystine/glutamate antiporter xCT to transport cystine, which is reduced to cysteine to be used for the production of GSH. H₂S also augments the activity of glutamate cysteine ligase (GCL), a rate limiting enzyme for GSH production, also known as γ‐glutamyl cysteine synthetase (γ‐GCS) (Kimura, Goto, & Kimura, 2010; Kimura & Kimura, 2004). The activity of ATP‐dependent K⁺ channels and cystic fibrosis transmembrane conductance receptor (CFTR) Cl⁻ channels is also activated by H₂S to suppress the excess excitation of the neurons (Kimura, Dargusch, Schubert, & Kimura, 2006). H₂S_n S‐sulfurate Kelch‐like ECH‐associated protein 1 (Keap1) to release nuclear factor erythroid 2‐related factor 2 (Nrf2) from Keap1/Nrf2 complex to nucleus where Nrf2 up‐regulates antioxidant genes, leading to the protection of neurons from oxidative stress.

H₂S and H₂S_n are further oxidized to thiosulfate, sulfite and sulfate. Sulfite protects embryonic neurons from oxidative stress as efficiently as H₂S and H₂S_n, while thiosulfate and sulfate do not (Kimura, Shibuya, & Kimura, 2019). It reacts with cystine to produce cysteine, which is more efficiently transported into cells than cystine, leading to the effective production of GSH (Clarke, 1932). A counterpart product of this reaction S‐cysteine sulfonate is agonist of NMDA receptors (Clarke, 1932; Kumar et al., 2018). Because matured neurons express NMDA receptors, of which the activity is enhanced by H₂S and S‐cysteinesulfonate (Abe & Kimura, 1996; Choi, 1988; Kumar et al., 2018; Murphy et al., 1989; Tan et al., 2001), the simultaneous application of inhibitors for NMDA receptors must be required for the protection of matured neurons.

Nagahara et al. have proposed that S‐sulfuration has a role to protect proteins from oxidative stress. In subsequent oxidation of cysteine–SH to –SOH, –SO₂H, and –SO₃H, only –SOH is reversible to –SH, but further oxidized forms are irreversible. In contrast, the oxidation of per‐sulfurated cysteine–SSO₂H and –SSO₃H can be converted to cysteine–SH by the reduced form of thioredoxin (Nagahara et al., 2012). These observations were reproduced and confirmed by other groups (Dóka et al., 2020; Zivanovic et al., 2019). The levels of S‐sulfuration are declined associated with aging that is a risk factor for the neurodegenerative diseases (Zivanovic et al., 2019).

NO protects neurons from excitotoxicity by decreasing the excessive influx of Ca²⁺ by suppressing the activity of NMDA receptors through S‐nitrosylation (Choi et al., 2000; Jaffrey et al., 2001). NO also exerts cytoprotective effects by suppressing caspase activity through S‐nitrosylation of its active site cysteine (Melino et al., 1997). Because the suppression of soluble guanylate cyclase showed a similar cell death effect to that by the deprivation of NO, cGMP may be involved in the cytoprotective effect of NO (Contestabile & Ciani, 2004).

4.3. Mitochondrial energy formation

H₂S is well‐known toxic gas at high concentrations and its toxicity is attributed to the inhibition of mitochondrial cytochrome c oxidase by suppressing the binding of oxygen (Hill et al., 1984), though its effect is not so potent compared to that of azide (Umemura & Kimura, 2007). There has been the hypothesis that mitochondria originated from sulfide‐oxidizing symbionts. Yong and Searcy (2001) demonstrated that chicken liver mitochondria consumed oxygen at an accelerated rate when supplied with low concentrations of H₂S, and H₂S oxidation is coupled to ATP generation. ATP synthesis requires less than 5‐μM H₂S and maximum respiration is induced at 10 μM and less efficient up to 60 μM (Yong & Searcy, 2001).

The balance between H₂S as the electron donor and the inhibitor of cytochrome c oxidase is likely controlled by H₂S and oxygen availability. Low concentrations of H₂S preserve the respiratory rate, while high concentrations inhibit it (Abou‐Hamdan et al., 2016). In contrast to NO and carbon monoxide, H₂S binds to sulfur quinone oxidoreductase that transfers two electrons and two protons from H₂S to coenzyme Q. A pair of electrons ultimately reduces a single oxygen atom at cytochrome c oxidase (Figure 4) (Goubern, Andriamihaja, Nübel, Blachier, & Bouillaud, 2007). Because sulfide oxidation requires three times more oxygen than that of NADH or flavin adenine dinucleotide (FADH₂), sulfide may be a poor energy substrate (Lagoutte et al., 2010). However, Szabo et al. suggested that this low energy yield is balanced by unique properties of H₂S. (1) H₂S freely diffuses across membrane without the need of transporters and (2) the affinity of H₂S to sulfide oxidation unit including sulfur quinone oxidoreductase is high and 100% is oxidized (Szabo et al., 2014). In the brain, the expression of sulfur quinone oxidoreductase is very low (Linden et al., 2012); it is predicted that a haemoprotein neuroglobin, which is primarily expressed in neurons, plays a role in H₂S oxidation (Ruetz et al., 2017).

FIGURE 4.

H₂S is used for ATP production at physiological concentrations, while it inhibits cytochrome c oxidase at higher concentrations in mitochondria. H₂S is metabolized by sulfur quinone oxidoreductase (SQR), sulfur dioxygenase (ETHE1, and rhodanese (TST) to thiosulfate through sulfite. Electrons are sent to coenzyme Q to complex IV through III and used for pumping out H⁺ from matrix to intermembrane space. ATP synthase produces ATP using the gradient of H⁺. In contrast, high concentrations of H₂S suppress cytochrome c oxidase and the energy formation

5. PATHOPHYSIOLOGICAL ROLE OF H₂S, H₂S_n AND NO

The overproduction of H₂S suppresses cytochrome c oxidase that is likely to be involved in the pathogenesis of Down's syndrome and ethylmalonic encephalopathy (Fernandez Cardoso et al., 2017; Marechal et al., 2019; Panagaki et al., 2019; Tiranti et al., 2009). In contrast, the lack of H₂S production may cause the pathology of Parkinson's disease, Huntington's disease and Alzheimer's disease (Cao et al., 2018; Chung et al., 2004; He et al., 2016; Paul et al., 2014; Sbodio et al., 2016, 2018; Vandiver et al., 2013; Wright et al., 2016; Xie et al., 2013). The detrimental effects caused by the excess production of H₂S and H₂S_n (Ide et al., 2019) as well as those by lacking in the neuroprotective effect of these molecules (Topcuoglu et al., 2017; Ünal et al., 2018; Xiong et al., 2018) have been reported for the pathogenesis of schizophrenia.

6. TOXICITY OF H₂S AND H₂S_n

6.1. Down's syndrome

Down's syndrome is characterized by impaired brain growth and maturation, which causes mental retardation, and has a trisomy of chromosome 21. CBS is encoded on chromosome 21 (21q22.3), and the expression of CBS mRNA is 12 times greater in myeloblasts of Down's syndrome children than those of normal individuals (Taub et al., 1999). We found that CBS protein levels in Down's syndrome brains are approximately 3 times greater than those in the normal brains that is twice greater than those expected from the trisomy (Ichinohe et al., 2005). In addition, elderly adults of Down's syndrome are associated with an Alzheimer's type of dementia where CBS is localized to astrocytes that surround senile plaques in the brain (Ichinohe et al., 2005).

The greater levels of thiosulfate, a metabolite of H₂S, were detected in urine of DS patients compared to that of normal individuals (Kamoun, Belardinelli, Chabli, Lallouchi, & Chadefaux‐Vekemans, 2003). The increased production of H₂S by overexpressed CBS may be the cause of the neurological impairments in Down's syndrome patients (Figure 5a). In a mouse model of Down's syndrome, three copies of CBS gene are necessary to cause the Down's syndrome‐related recognition memory deficit (Marechal et al., 2019). Panagaki et al. (2019) showed that the levels of CBS and H₂S are markedly elevated in Down's syndrome fibroblast cells and the mitochondrial electron transport, oxygen consumption, and ATP generation are profoundly suppressed. They suggested the therapeutic potential of CBS inhibitors.

FIGURE 5.

Diseases caused by unbalance of H₂S and H₂S_n in the CNS. (a) Diseases caused by an excess amount of H₂S and H₂S_n. A trisomy of chromosome 21, on which cystathionine β‐synthase (CBS) is encoded, increases the levels of CBS in Down's syndrome. Sulfur dioxygenase, which is encoded in sulfur dioxygenase gene (ETHE1) and one of the enzymes metabolizing H₂S and H₂S_n, is defective in ethylmalonyl encephalopathy. Methylation of 3‐mercaptopyruvate sulfurtransferase (MPST) gene increases the production of MPST in schizophrenia. Glycolysis is decreased by the suppression of several enzymes such as triosephosphate isomerase, phosphoglycerate kinase, and phosphopyruvate hydratase. In these diseases, cytochrome c oxidase is suppressed by high concentrations of H₂S, resulting in the decreased production of ATP. (b) Diseases caused by a lack of H₂S or H₂S_n in the CNS. S‐sulfurated parkin in normal individuals is active, while parkin with the same cysteine residues being S‐nitrosylated in Parkinson's disease (PD) brains is inactive. Mutant huntingtin suppresses the transcription of cystathionine γ‐lyase (CTL), CTH gene, from specificity protein 1 (SP1), resulting in the decrease in the production of H₂S. In Huntingdon's disease (HD) animal model, the activity of cystine/glutamate antiporter xCT is suppressed, and GSH levels are decreased, while those of ROS increased. The animal model of Alzheimer's disease (AD) shows that H₂S suppresses PI3K/Akt, while enhances the activity of STAT3. Both effects result in the decreased production of Aβ1–42 and suppression of inflammation. In some report, the plasma levels of H₂S are lower in patients of schizophrenia than normal individuals

6.2. Ethylmalonyl encephalopathy

Ethylmalonyl encephalopathy is an autosomal recessive early onset and defective in cytochrome c oxidase in muscle and brain and excretes ethylmalonic acid in urine. In this disease, ETHE1, a gene encoding a β‐lactamase‐like iron‐coordinating metalloprotein or sulfur dioxygenase, is deficient. A great amount of thiosulfate, a metabolite of H₂S, is excreted in urine of Ethe1 knockout mice and patients with this disease (Tiranti et al., 2009). H₂S is mainly metabolized by mitochondrial enzymes, sulfur quinone oxidoreductase, sulfur dioxygenase, and rhodanese. In the brain and skeletal muslce deficiency of sulfur dioxygenase (ETHE1) inceases the basal levels of H₂S which suppress the cytochrome c oxidase that may lead to progressive neurological failure (Tiranti et al., 2009) (Figure 5a). In addition, acyl‐protein thioesterase, which hydrolyses fatty acids bound to cysteine and GSH transferases are suppressed in Ethe1 knockout mice probably due to the increased levels of H₂S or persulfides (Hildebrandt, Meo, Zeviani, Viscomi, & Braun, 2013).

The administration of metronidazole, an antibiotic, or N‐acetylcysteine prolonged the lifespan of Ethe1 knockout mice and marked clinical improvement in patients with ethylmalonyl encephalopathy (Viscomi et al., 2010). Metronidazole may be expected to decrease the levels of H₂S incorporated into blood from intestinal bacteria. N‐acetylcysteine is metabolized in cells to cysteine, which is a precursor of GSH being predicted to buffer H₂S, while it is also a substrate to produce H₂S. In this instance, the former may be a dominant mechanism of N‐acetylcysteine for the improvement of the disease.

H₂S decreased the activities of citrate synthase, aconitase, and creatine kinase in the brain of mouse model of ethylmalonyl encephalopathy (Fernandez Cardoso et al., 2017). H₂S also suppressed mitochondrial respiration, decreased mitochondrial membrane potential and induced swelling caused by calcium in brain mitochondria. Changes in mitochondrial membrane potential and the swelling caused by H₂S may be due to opening of mitochondrial permeability transition pore (Fernandez Cardoso et al., 2017). Bioenergetics disturbance, lipid peroxidation and mitochondrial permeability transition pore opening mediated by H₂S may be involved in the pathophysiology of brain damage observed in this disease (Fernandez Cardoso et al., 2017).

7. H₂S AND H₂S_n ARE BENEFICIAL

7.1. Parkinson's disease

Parkinson's diseas is a neurodegenerative disorder mainly causing motor dysfunction. Parkin and α‐synuclein are associated with pathophysiology of Parkinson's disease. α‐Synuclein is a major component of Lewy bodies associated with rare cases of Parkinson's disease, while parkin, which is an E3 ubiquitin ligase that ubiquitinates diverse substrates, is responsible for the clearance of misfolded proteins including α‐synuclein and its mutations cause autosomal recessive Parkinson's disease (Choi et al., 2001; Jęśko, Lenkiewicz, Wilkaniec, & Adamczyk, 2019). The activity of parkin is suppressed by S‐nitrosylation and thereby decreases its protective role in the brains of Parkinson's disease patients (Chung et al., 2004). In contrast, in the brains of normal individuals, it is active and S‐sulfurated. Cys95, Cys59, and Cys182 are S‐sulfurated in normal individuals, while the same cysteine residues are S‐nitrosylated in Parkinson's disease brains (Vandiver et al., 2013) (Figure 5b). Parkin is an example of complementary roles of the S‐sulfuration and S‐nitrosylation. H₂S may be involved in the conversion of S‐nitrosylated cysteine to S‐sulfurated one in parkin to improve its function, while NO may S‐nitrosylate cysteine residues to decrease the protective effect of parkin (Chung et al., 2004; Vandiver et al., 2013; Yao et al., 2004).

Systemic administration of sodium salt of H₂S, NaHS, dramatically reversed the progression of movement dysfunction and loss of dopaminergic neurons in the Parkinson's disease model rats (Hu et al., 2010). A similar effect was observed with an H₂S‐releasing l‐DOPA derivative compound (Xie et al., 2013). Overexpression of CBS increased the endogenous levels of H₂S, reversed the behaviour induced in Parkinson's disease model rats, and decreased apoptotic neuronal loss of the nigral dopaminergic neurons (Xie et al., 2013). The application of H₂S and the regulation of CBS activity have a therapeutic potential (Yin, Yin, Huang, & Wu, 2017).

7.2. Huntington's disease

Huntington's disease is a devastating neurodegenerative disorder characterized by the progressive development of involuntary movements, neuropsychiatric symptoms, and cognitive impairment (Cepeda & Tong, 2018). Huntington's disease belongs to triplet repeat diseases, in which elongated polyglutamine stretches affect the protein product, and the mutant huntingtin disrupts a number of vital cellular processes, including neuronal transmission, metabolism, and gene transcription (Chaganti, McCusker, & Loy, 2017).

Although the levels of CSE are very low in the brain compared to other tissues (Ishii et al., 2004), those are even much lower in mouse models of Huntington's disease and human Huntington's disease brain compared to a control mice and normal individuals, respectively (Paul et al., 2014). It is caused by the suppression of the transcription factor specificity protein 1, which regulates the transcription of CSE, by mutant huntingtin (Ishii et al., 2004; Paul et al., 2014) (Figure 5b). The expression of CSE is also regulated by transcription factor 4 (ATF4), which is dysfunctional in Huntington's disease, under endoplasmic reticulum (ER) stress or amino acid deficiency (Sbodio et al., 2016). Striatal cell lines derived from Huntington's disease model mice have decreased levels of cystine/glutamate antiporter xCT mRNA and protein expression, leading to the lower basal levels of GSH and higher basal levels of ROS (Wright et al., 2016) (Figure 5b). Administration of N‐acetylcysteine ameliorates Huntington's disease pathology in this model.

7.3. Alzheimer's disease

Alzheimer's disease is a chronic neurodegenerative disease that affects cognitive functions and memory formation. The cause of the disease is mostly sporadic with much less familial. In familial cases of Alzheimer's disease, the mutations in genes encoding amyloid precursor protein, presenilin 1 and 2, which constitute the catalytic subunits of γ‐secretase, have been identified (Selkoe & Hardy, 2016). In the process of metabolism in normal individual, amyloid precursor protein is digested by α‐secretase following by γ‐secretase, leading to the product which is easily eliminated, while in Alzheimer's disease brain the metabolism by β‐secretase (BACE) and γ‐secretase generates amyloid β‐protein (Aβ1–42), which aggregates and exerts neurotoxicity. Phosphorylated tau protein also causes deposition as neurofibrillary tangles (Reddy & Oliver, 2019).

Neuropolypeptide h3, also known as hippocampal cholinergic neuro‐stimulating peptide, up‐regulates the levels of ChAT whose activity is declined in patients with AD (Ojika, Tsugu, Mitake, Otsuka, & Katada, 1998; Reed, Pierce, Turner, Markesbery, & Butterfield, 2009). Nitration of tyrosine in this peptide decreases its neurotropic activity on cholinergic neurons that may lead to the decline in cognitive function (Reed et al., 2009).

S‐nitrosylation regulates the activity of proteins related to Alzheimer's disease in Alzheimer's disease patients and model animals. The administration of 0.1–100 μM of NO donors deactivated beta‐secretase 1(BACE1) by S‐nitrosylation in rat primary cortical neurons, and the levels of S‐nitrosylated beta‐secretase 1 are decreased in human Alzheimer's disease brains (Kwak et al., 2011). The activity of insulin degrading enzyme, which is a zinc metalloendopeptidase responsible for the metabolism of Aβ1–42 as well as insulin, is suppressed by S‐nitrosylation in Alzheimer's disease brain (Akhtar et al., 2016).

The levels of beta‐secretase 1, presenilin1 and pp38 MAPK are increased while those of disintegrin and metalloproteinase domain‐containing protein 17 (ADAM17) are decreased in the amyloid precursor protein/ presenilin1 transgenic mice (He et al., 2016). The administration of NaHS into the transgenic mice restores the changes in these factors characteristic for Alzheimer's disease, suggesting that H₂S inhibits the expression of beta‐secretase 1 and presenilin1 by activating PI3K/Akt pathway in Alzheimer's disease (He et al., 2016).

H₂S inhibited exogenous ATP‐induced inflammatory responses through reducing pro‐inflammatory cytokines, ROS, and activation of NF‐κB pathway. H₂S also suppressed the production of Aβ1–42, which was induced by exogenous ATP probably due to the augmented production of amyloid precursor protein and the activation of β‐ and γ‐secretase (Cao et al., 2018) (Figure 5b). As a mechanism for H₂S reducing inflammation and the production of Aβ1–42, they suggested that H₂S suppresses the activities of STAT3 by inhibiting ATP‐induced phosphorylation and decreases the activity of cathepsin S through S‐sulfuration of this enzyme (Cao et al., 2018) (Figure 5b).

8. H₂S AND H₂S_n ARE BENEFICIAL OR TOXIC

8.1. Schizophrenia

Schizophrenia is a chronic and severe mental disorder that affects a person's thinking, feeling, and behaviours. Symptoms, which typically come on gradually and begin in young adulthood, fall into three categories: positive symptoms (hallucinations, delusions, etc.), negative symptoms (flat affect, reduced feeling etc.), and cognitive symptoms (poor executive function, trouble focusing etc.) (American Psychiatric Association, 2013).

Although the excessive NO production has been shown to be involved in the pathology of this disease (Pitsikas, 2016), the potential beneficial effects have been reported for both NO donors and inhibitors on schizophrenia symptoms induced by amphetamine such as prepulse inhibition disruption and hyperlocomotion (Issy, Dos‐Santos‐Pereira, Pedrazzi, Kubrusly, & Del‐Bel, 2018), suggesting that the deviation of both decrease and increase of NO from the normal levels may be involved in the pathology of this disease.

Both excess and deficiency of H₂S and H₂S_n have also been proposed to be involved in the pathogenesis of schizophrenia. Plasma H₂S levels were significantly lower in patients with schizophrenia relative to healthy control subjects, and a positive association was observed between plasma H₂S levels and working memory, visual memory, or executive function in patients, suggesting that decreased H₂S is involved in the psychopathology and cognitive deficits of this disease (Xiong et al., 2018) (Figure 5b).

Untreated schizophrenia patients had significantly higher (disulfide/total thiol) and (disulfide/free thiol) ratio in blood and a significantly lower (free thiol/total thiol) ratio compared to those of healthy individuals (Figure 5b). Thiol homeostasis is disturbed by a shift to the disulfide bond formation (oxidized) in patients (Topcuoglu et al., 2017). Similar results were also obtained in schizophrenia patients using medication (Ünal et al., 2018).

Methylglyoxal, a highly reactive dicarbonyl compound, is a major precursor for advanced glycation end products, production of which is associated with various neurological disorders including schizophrenia (Ohnishi et al., 2019; Toyoshima et al., 2019). H₂S_n protect differentiated human neuroblastoma SH‐SY5Y cells from methylglyoxal‐induced cytotoxicity, suggesting that H₂S_n scavenge methylglyoxal and suppress the accumulation of advanced glycation end products and incidents induced by carbonyl stress (Koike, Ogasawara, Shibuya, Kimura, & Ishii, 2013).

Excess production of H₂S and H₂S_n has also been proposed to be involved in the pathogenesis of schizophrenia. Yoshikawa and colleagues found that C57BL/6N (B6) strain mice exhibited greater scores of prepulse inhibition, which is the normal suppression of a startle response, than C3H/HeN (C3H) mice did (Watanabe et al., 2007). The impaired prepulse inhibition is regarded as an endophenotype for schizophrenia (Braff, Geyer, & Swerdlow, 2001). By proteomic analysis, the same group found that the expression of MPST is increased in C3H mice compared to B6 (Ide et al., 2019). DNA methylation levels at MPST gene were highly enhanced in C3H mice, and the mean methylation levels of the sites were positively correlated with the expression levels of MPST. MPST levels in schizophrenia were positively correlated with symptom severity scores. In Mpst‐transgenic mice, the expression of genes for energy formation was decreased and mitochondrial energy metabolism was impaired (Ide et al., 2019) (Figure 5a).

Maternal immune activation model, which is induced by the injection of polyriboinosinic‐polyribocytidylic acid to mother, shows perturbed early neural development via inflammation and oxidative insults (Bundo et al., 2014; Giovanoli et al., 2013; Meyer & Feldon, 2012). In this model, the expression of MPST and other inflammatory and oxidative genes was elevated in the brain when pups grow to the adult (Ide et al., 2019). In human brains, catalase gene product was up‐regulated in schizophrenia samples compared to the controls (Ide et al., 2019). Inflammatory/oxidative insults in early brain development induce up‐regulation of H₂S/polysulfides production excessively as an antioxidative response that suppresses cytochrome c oxidase, leading to schizophrenia.

9. PERSPECTIVE

Neurotransmitters, cytokines and hormones diffuse to reach their targets to react with their receptors. An advantage of diffusion is that many targets localized in the area within its reach can be activated. After transmitting the signal, they are properly eliminated. For example neurotransmitters that are released from presynaptically and diffuse across the synaptic cleft to the postsynaptic receptors to exert their effects. For cessation of the responses the transmitters are recovered by uptake or degraded by enzymes to clear the synaptic cleft.

NO barely dissolves in water (5.6 mg·100 ml⁻¹ at 20°C), while H₂S does well (413 mg·100 ml⁻¹ at 20°C) (Kimura, 2015b). H₂S dissociates to H⁺ and HS⁻ (pK₁ = 7.04) and further to S²⁻ (pK₂ = 11.96). Because H₂S is also lipophilic, it readily passes through plasma membrane. These characteristics of H₂S have an advantage to transmit signals across the membrane by diffusion. HS⁻ channels or transporters, which have been identified in both bacteria and mammals (Czyzewski & Wang, 2012; Jennings, 2013), enable H₂S to pass through plasma membrane even more efficiently and selectively.

H₂S_n, which can pass through the plasma membrane, diffuse to the targets on nearby cells (Greiner et al., 2013; Kimura et al., 2017; Kimura et al., 2013; Nagai et al., 2006; Oosumi et al., 2010). For example, H₂S_n activate TRPA1 channels by passing through the plasma membrane to S‐sulfurate the amino terminus located in the cytoplasm (Kimura et al., 2013; Nagai et al., 2006; Oosumi et al., 2010). Although 23 out of 31 cysteine residues are localized to the amino terminus (Wang, Cvetkov, Chance, & Moiseenkova‐Bell, 2012), only Cys422 and Cys634 are responsible for regulating the activity of TRPA1 channels (Hatakeyama et al., 2015; Kimura, 2015a). The remaining 21 cysteine residues were not involved in the activation of channels whether or not they are S‐sulfurated. Signalling by diffusion may be less specific to target proteins than that by enzymatic modification. In this case, however, S‐sulfuration of the two specific cysteine residues by sulfur transferases, if any, may not have any advantage on specificity to activate the channels compared to the diffusion of H₂S_n.

S‐sulfuration is compared to phosphorylation, in which kinases incorporate phosphate to residues of serine, threonine and tyrosine (Kimura, 2020). H₂S and H₂S_n are able to S‐sulfurate cysteine residues of proteins even in cells surrounding the producing cells but do not S‐sulfurate the specific residues. In contrast, MPST transfers sulfur from 3MP to specific cysteine residues, but the reaction is restricted only inside the cells (Kimura et al., 2017; Shibuya et al., 2013; Shibuya et al., 2009). H₂S and H₂S_n endogenously exist (Kimura et al., 2015; Koike et al., 2017) and both their diffusion‐ and enzyme‐mediated mechanisms play a role in S‐sulfuration under physiological conditions. Bacteria use H₂S produced by MPST to protect themselves from antibiotics (Shatalin, Shatalina, Mironov, & Nudler, 2011). Because bacteria have channels specific to HS⁻ (Czyzewski & Wang, 2012), signalling by H₂S with other cells must have emerged in the early history of life on earth. Mammalian have developed HS⁻/Cl⁻ transporter on erythrocytes to rapidly and selectively exchange HS⁻ with the extracellular milieu (Jennings, 2013).

Patients exposed to high concentrations of H₂S suffer from cognitive impairment (Reiffenstein, Hulbert, & Roth, 1992) and the levels of neurotransmitters in the brain were affected in animals exposed to H₂S (Warenycia, Smith, Blashko, Kombian, & Reiffenstein, 1989). The expression of sulfur quinone oxidoreductase, which is the first step for the metabolism of H₂S, is very low in the brain (Linden et al., 2012). These observations suggest the vulnerability of the brain to excessive H₂S.

In Down's syndrome, ethylmalonyl encephalopathy and schizophrenia, the high levels of H₂S or its producing enzymes exert a toxic effect on cytochrome c oxidase, leading to neuronal dysfunction (Ichinohe et al., 2005; Ide et al., 2019; Linden et al., 2012; Panagaki et al., 2019; Taub et al., 1999; Tiranti et al., 2009; Viscomi et al., 2010). In addition, high concentrations of H₂S metabolized by sulfur quinone oxidoreductase must generate the toxic levels of polysulfides on neurons. The inhibitors of H₂S producing enzymes, which may also decrease the levels of polysulfides, have suggested to have therapeutic potential in these diseases. The accurate comparisons of the levels of H₂S and polysulfides between patients and normal individuals are required to examine the efficiency of these inhibitors for therapeutic uses.

S‐sulfuration makes parkin active in the brains of normal individuals, while S‐nitrosylation inactivates it in those with Parkinson's disease (Vandiver et al., 2013). It is intriguing to know whether or not exogenously applied H₂S or the enhancement of H₂S producing enzymes is able to convert S‐nitrosylated cysteine to S‐sulfurated one. The difference in the levels of H₂S between patients and normal individuals is not well understood in Parkinson's disease. The concentrations of polysulfides, which must be accompanied by the changes of H₂S levels, should also be clarified. Controlling the levels of these signalling molecules and the activities of enzymes related to S‐nitrosylation and S‐sulfuration may have a therapeutic benefit for diseases in the CNS.

9.1. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in the IUPHAR/BPS Guide to PHARMACOLOGY http://www.guidetopharmacology.org, and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

CONFLICT OF INTEREST

The author declared no conflicts of interest.

Kimura H. Hydrogen sulfide signalling in the CNS ‐ Comparison with NO. Br J Pharmacol. 2020;177:5031–5045. 10.1111/bph.15246