Rev. Neurosci. 2015; 26(2): 129–142

Wen-Lin Chen, Ying-Ying Niu, Wei-Zheng Jiang, Hui-Lan Tang, Chong Zhang, Qi-Ming Xia and Xiao-Qing Tang*

Neuroprotective effects of hydrogen sulfide and the underlying signaling pathways Abstract: Hydrogen sulfide (H2S) is an endogenously produced gas that represents a novel third gaseous signaling molecule, neurotransmitter and cytoprotectant. Cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercaptopyruvate sulfur transferase with cysteine aminotransferase (3-MST/CAT) and 3-mercaptopyruvate sulfur transferase with d-amino acid oxidase (3-MST/DAO) pathways are involved in the generation of endogenous H2S despite the ubiquitous or restricted distribution of those enzymes. CBS, 3-MST/CAT and 3-MST/DAO can be found in the brain, while CSE is widely located in other organs. There also exist up-taking or recycling and scavenging mechanisms in H2S metabolism to maintain its persistence for physiological function. In recent years, investigating the role that H2S plays in the central nervous system and cardiovascular system has always been a hotspot. To date, effects of H2S are at least partially verified in multiple animal models or neuron cell lines of Alzheimer’s disease, Parkinson’s disease, cerebral ischemia, major depression disorders and febrile seizure, although subsequent studies are still badly needed. This article presents an overview of current knowledge of H2S focusing on its neuroprotective effects and corresponding signaling pathways, together with connections to potential therapeutic strategies in the clinic. Keywords: central nervous system diseases; hydrogen sulfide; neuroprotective; signaling pathways. DOI 10.1515/revneuro-2014-0051 Received July 25, 2014; accepted October 1, 2014; previously published online December 22, 2014 *Corresponding author: Xiao-Qing Tang, Institute of Neuroscience, Medical College, University of South China, 28 W Changsheng Road, Hengyang, Hunan, P. R. China, e-mail: [email protected] Wen-Lin Chen, Wei-Zheng Jiang, Hui-Lan Tang, Chong Zhang and Qi-Ming Xia: Institute of Neuroscience, Medical College, University of South China, Hengyang, 421001 Hunan, P.R. China; and Grade of 2009 in Clinical Medicine, Medical College, University of South China, Hengyang, 421001 Hunan, P.R. China Ying-Ying Niu: Institute of Neuroscience, Medical College, University of South China, Hengyang, 421001 Hunan, P.R. China; and Grade of 2011 in Clinical Medicine, Medical College, University of South China, Hengyang, 421001 Hunan, P.R. China

Introduction Hydrogen sulfide (H2S) is well known as a transparent, toxic gas with the characteristic strong smell of rotten eggs (Wang, 2010). Leading the trend by nitric oxide (NO) and extending the trudge by carbon monoxide (CO), here comes H2S, which builds up the momentum as the third gasotransmitter (Gadalla and Snyder, 2010). A multitude of experiments concentrating on the physiological and pathological effects of H2S have been conducted in the past two decades, and tremendous achievements have been made for the understanding of production (Enokido et al., 2005; Shibuya et  al., 2009a,b, 2013), catabolism (Lowicka and Beltowski, 2007; Whitfield et al., 2008; Kimura et al., 2012) and cytoprotective effects mediated by H2S via antioxidative (Kimura, 2010; Tang et  al., 2010a,b; Kimura et  al., 2012), anti-inflammatory (Zanardo et al., 2006; Lee et al., 2009; Fan et al., 2013), anti-apoptotic (Yin et al., 2013; Wei et  al., 2014) and pro-angiogenesis (Cai et  al., 2007) processes. Numerous molecules are involved in the signaling pathways underlying H2S-mediated effects, including ion channels (Dawe et al., 2008; Sun et al., 2008; Tay et al., 2010), protein kinases (Tay et al., 2010; Tang et al., 2012), transcriptional factors (Calvert et  al., 2009; Sutherland et al., 2013), growth factors (Schicho et al., 2006; Kimura, 2010; Jang et al., 2014) and inflammatory factors (Akiyama et al., 2000). There are three enzymes responsible for endogenous H2S generation: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase with cysteine aminotransferase (3-MST/CAT), and they share the same substrate – l-cysteine (Meister et  al., 1954; Abe and Kimura, 1996; Chiku et  al., 2009; Shibuya et al., 2009a,b). A recent study has unveiled that d-cysteine, another form of cysteine that is less toxic than l-cysteine, can serve as a new source of endogenous H2S generation through 3-mercaptopyruvate sulfur transferase with d-amino acid oxidase (3-MST/DAO) pathway in the kidney and the brain (Shibuya et al., 2013). In this review, we focus on the neuroprotective role H2S played in several central nervous system (CNS) diseases inclusive of Alzheimer’s disease (AD), Parkinson’s

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130      W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant disease (PD), cerebral ischemia, major depression disorders (MDD) and febrile seizure (FS). Discussion about the corresponding possible signaling pathways or mechanisms underlying neuroprotective effects of H2S and its potential therapeutic applications in those CNS diseases is included as well.

Generation, concentrations and catabolism of endogenous H2S Generation of endogenous H2S Three enzymes have been identified in the generation of endogenous H2S: CBS, CSE and 3-mercaptopyruvate sulfur transferase (3-MST) with cysteine aminotransferase (CAT) (Cavallini et al., 1962; Braunstein et al., 1971; Enokido et al., 2005). The main common substrate that those enzymes produce H2S from is deemed to be l-cysteine, whereas the most recent discovery of Shibuya has illustrated the limited existence of a 3-MST/DAO pathway of H2S generation from d-cysteine in the brain and the kidney (Shibuya et  al., 2013). However, the distribution of enzymes mentioned above varies a lot. In the brain, 3-MST/CAT rather than formerly recognized enzyme CBS functions as the main producer of H2S according to an updated report from Hideo Kimura (2014), because most H2S produced by 3-MST is bound as sulfane sulfur, one form in which endogenous H2S is stored in a considerable amount (Guo et al., 2012). CBS is found ubiquitous in the CNS as well, particularly in the hippocampus, cerebellum, cerebrum and brainstem. In the thoracic aorta, ileum, portal vein and uterus, CSE is the predominant source of H2S. However, regulation of the activities of these three H2S-producing enzymes is not well understood.

CBS CBS is a pyridoxal-5′-phosphate (PLP)-dependent enzyme. CBS is located in the hippocampus, cerebellum, cerebrum and brainstem using northern blot assays (Abe and Kimura, 1996). CBS produces H2S from cysteine via a β-elimination reaction, and the production of H2S becomes more efficient via a β-replacement reaction in which cysteine is condensed with homocysteine (Chen et  al., 2004). To some extent, CBS has its advantage over CSE in generating endogenous H2S, as CSE does not condense cysteine with homocystein. Expression of CBS mRNA or

the transcription of CBS could be upregulated by endogenous and exogenous compounds like epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and cyclic adenosine monophosphate (Schicho et  al., 2006; Kimura, 2010). CBS expression is abnormal in several diseases. In the brains of Down’s syndrome patients, CBS levels are found to be three times higher than the normal, while low expression levels of CBS alleles are found in children with a high intelligence quotient (Wisniewski et  al., 1985; Ichinohe et  al., 2005; Kimura, 2010). In cbs knockout mice, they display hyperhomocysteinemia and hypermethioninemia (Ishii et  al., 2010), thus suggesting that overexpression of CBS may impact cognitive functions, whereas absence of CBS could cause severe diseases. Therefore, maintaining CBS expressing balance may underlie the therapeutic strategies for neuropathology treatment.

CSE CSE is a pyridoxal-5′-phosphate (PLP)-dependent enzyme as well. CSE is mainly localized in the liver, kidney and vascular and nonvascular smooth muscle. The expression levels of CSE in vascular smooth muscle can be ranked as artery > aorta > tail artery > mesenteric artery (Szabo, 2007). The expression of CSE could be upregulated by S-nitrosoN-acetylpenicillamine (SNAP), which is a type of NO donor. Sodium nitroprusside, another NO donor, increases the activity of CSE as well. Intriguingly, H2S can act synergistically with NO in vasorelaxation, suggesting that H2S production in the cardiovascular system may be involved in the vasorelaxation effect of NO (Hosoki et al., 1997; Wang, 2009; Kimura, 2012). Although the effects of CSE in regulation of blood pressure are still controversial (Yang et al., 2008; Ishii et al., 2010), CSE is proposed to play a major role in modulation of vascular smooth muscle. The demonstration that blood pressure is unaffected by loss of CSE could be explained by at least two reasons: (a) 3-MST and CAT may be involved in the regulation of blood pressure, (b) increased generation of H2S by 3-MST and CAT may compensate for loss of CSE in the knockout mice (Kimura et  al., 2012). Specific evidence of the role CSE played in regulation of blood pressure is expected in the future.

3-MST/CAT The 3-MST/CAT pathway recently has been identified to produce H2S from substrates including both l-cysteine and d-cysteine in the brain (Kimura et al., 2010; Shibuya

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and Kimura, 2013). The location of 3-MST differs from that of CBS and CSE; CBS and CSE are localized to the cytoplasm, while 3-MST is mainly localized to the mitochondrial matrix of neurons in the brain and retina (Nagahara et  al., 1998; Shibuya et  al., 2009a,b; Mikami et  al., 2011a,b). 3-Mercaptopyruvate (3MP) is produced by CAT from l-cysteine, d-cysteine and α-ketoglutarate, but it needs participation of peroxisomal enzyme d-amino acid oxidase (DAO) when synthesizing 3MP in the mitochondria from cytoplasmic d-cysteine (Ubuka et al., 1978; Shibuya et al., 2013). A reducing substance such as dithiothreitol (DTT) is required for 3-MST to produce H2S. Thioredoxin and dihydrolipoic acid (DHLA) associated with 3-MST to release H2S has been recently found. Mitochondrial thioredoxin is approximately 20 μm, which is four times more potent than DTT in enhancing H2S production. The concentration of DHLA is approximately 40 μm in the brain, and DHLA enhances the H2S production as effectively as DTT (Mikami et  al., 2011a,b). The activity of CAT is suppressed by Ca2+ in a concentration-dependent manner. The production of H2S is maximal in the absence of Ca2+ and minimal at 2.9 μm Ca2+ (Mikami et  al., 2011a,b). Another source of H2S is bound sulfane sulfur, which may function as an intracellular storage of H2S. Cells expressing 3-MST and CAT contain twofold of bound sulfane sulfur than the control. The concentration of bound sulfane sulfur is low in cells expressing a mutant 3-MST that lack H2S-producing activity (Shibuya et al., 2009a,b). The concentration of bound sulfane sulfur in the brain is 1500 nmol/g protein, which is sufficient to release H2S to stimulate target molecules (Ishigami et  al., 2009). However, further studies are required to identify the specific proteins that contain bound sulfane sulfur and physiological or pathological signals that trigger the release of H2S from the storage form.

Catabolism of H2S In order to exert its physiological function, μm concentration of H2S has to be released from activated enzymes or intracellular storage, but it is enough in a short period of time in a restricted area. After the induction of responses, H2S may be restored or degraded to a steady state to stop the reactions (Kimura et al., 2012). But what are the catabolic pathways of H2S? In the brain, free H2S is released from bound sulfane sulfur upon neuron excitation or other stimulation, which afterward is mainly oxidized to thiosulfate, sulfite and finally sulfate by a cyanide sulfurtransferase called thiosulfate in the mitochondria. It can be methylated by the enzyme thiol-S-methyltransferase

to methanethiol and dimethylsulfid or get bound to methemoglobin, an oxidized form of hemoglobin as well (Lowicka and Beltowski, 2007). Free H2S clearance mediated by the kidneys, lung or liver is less likely due to the low concentration of free H2S in bloodstream and fast decaying speed (Whitfield et  al., 2008). Generation and catabolism of H2S are schematized in Figure 1.

Protective effects of hydrogen sulfide against nervous system diseases Alzheimer’s disease AD, the leading cause of dementia, is a devastating and progressive neurodegenerative disease characterized by microscopically neuropathological hallmarks of accumulation of senile plaques, neurofibrillary tangles, synaptic loss and neuronal death. Senile plaques are extracellular depositions of amyloid-β (Aβ) generated from a larger transmembrane amyloid precursor protein (GoldbergStern et al., 2014). The pathogenesis of AD involves both oxidative stress and neuroinflammation. The disturbance of H2S levels is significant in the serum of AD patients (Eto et al., 2002), indicating that endogenous H2S may be involved in the pathogenesis of AD. Our previous work suggests that physiological concentration of H2S may protect neurons by preserving mitochondrial membrane potential (MMP) and attenuating Aβ25–35-induced intracellular reactive oxygen species (ROS) generation using PC12 cells (Tang et al., 2008). PC12 cell line, a clonal rat pheochromocytoma cell line, is widely adopted as a well-established model for investigating neurological biology. Besides, Tang and coworkers have further confirmed that H2S can protect PC12 cells against homocysteine-induced cytotoxicity and apoptosis not only by reducing the loss of MMP but also by attenuating intracellular ROS through upregulation of Bcl-2 level (Tang et al., 2010a,b). Inflammatory cytokines overproduction in AD is also reported including IL-1α, IL-1β, IL-6, IL-8, IL-12 and particularly TNF-α (Akiyama et al., 2000). Activated immune cells, glial cells and neurons are present in neuroinflammation. Zhou et al. find that H2S dramatically suppresses the release of TNF-α, IL-1β and IL-6 and that H2S inhibits the upregulation of cycloxidase-2 (COX-2) and the activation of nuclear factor κB (NF-κB) in the hippocampus (Fan et  al., 2013). For instance, specific inhibition of COX-2 should theoretically exert anti-inflammatory

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132      W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant

Figure 1 Generation and catabolism of H2S. There are three pathways engaged in H2S generation from substrates of l-cysteine: cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase with cysteine aminotransferase (3MST/CAT). Additionally, d-cysteine can be transformed into 3-mercaptopyruvate (3-MP) by peroxisomal enzyme d-amino acid oxidase (DAO) which is then catalyzed by mitochondrial enzyme 3-MST to produce H2S. Interconvertibility between H2S and bond sulfur contributes to the yield of H2S via the 3-MST/CAT pathway, which makes 3-MST/CAT the dominating pathway in the brain. Expression of CSE can be upregulated by S-nitroso-N-acetylpenicillamine (SNAP). Similarly, epidermal growth factor (EGF), transforming growth factor-α (TGF-α) and cyclic adenosine monophosphate (cAMP) can promote H2S production by enhancing the expression of CBS. The catabolism of H2S flows primarily in three directions: (A) being oxidized into thiosulfate (S2O32-), thiocyanate (SCN-) and sulfate (SO42-) in the mitochondria; (B) being methylated into dimethylsulfide (CH3–S–CH3); and (C) binding to hemoglobin to form sulfhemoglobin. Free H2S clearance mediated by the kidneys, lung or liver is less likely due to the low concentration of free H2S in the bloodstream and fast decaying speed.

actions to sites where inflammation is ongoing, sparing unaffected regions where COX-produced prostaglandins are beneficial. And inhibition of NF-κB-mediated transcription of a diversity of proinflammatory and/or apoptosis-related genes by H2S can also present neuroprotective effects. Accumulating evidence signifies that endoplasmic reticulum (ER) stress plays a key role in development or pathology of AD, which is featured by an abnormal formation of inclusion bodies and aggregation of misfolded proteins (Hoozemans et  al., 2005, 2009; Stutzbach et  al., 2013). X.Q. Tang et  al. recently have proved that H2S inhibits homocysteine-induced ER stress and neuronal apoptosis in rat hippocampus via upregulation of the brain-derived neurotrophic factor/tropomyosinrelated kinase B (BDNF/TrkB) pathway. Sodium hydrogen sulfide (NaHS), a H2S donor, increases the endogenous H2S generation and BDNF expression dose-dependently, and markedly reduces Hcy-induced neuronal apoptosis and ER stress responses in the hippocampus. Treatment with k252a, a specific antagonist of TrkB (the receptor of BDNF), abolishes the protective effects of NaHS against Hcy-induced ER stress in the hippocampus, suggesting the neuroprotective effects of H2S in AD and its potential therapeutic use (Wei et al., 2014). Pathways underlying the neuroprotective effects of H2S in AD are shown in Figure 2.

Parkinson’s disease PD is characterized by progressive degeneration of dopaminergic neurons in the substantia nigra (SN) (Hirsch et  al., 1988). Several responsible mechanisms inclusive of abnormal protein handling, oxidative stress, mitochondrial dysfunction, excitotoxicity, neuroinflammation and apoptosis have been suggested (Hirsch and Hunot, 2009). It has also been reported that endogenous H2S level is markedly reduced in the SN in a 6-hydroxydopamine (6-OHDA)-induced PD rat model (Hu et al., 2010). The 1-methyl-4-phenylpyridinium ion (MPP+) is an active metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) that stimulates the production of superoxide radical in vitro and induces apoptosis in PC12 cells (Yin et al., 2009). MPP+ inhibited the expression and activity of CBS, leading to decreased endogenous H2S production. MPP+-induced cytotoxicity and ROS accumulation are extenuated by H2S and exacerbated by inhibition of endogenous H2S generation with amino-oxyacetate, an inhibitor of CBS (Tang et al., 2011). Ming Lu and coworkers have proved that H2S alleviates the loss of SN compacta Dopi amine (DA) neurons and protects against MPTP-induced neurodegeneration via a adenosine triphosphate-sensitive potassium (KATP) channel-independent but mitochondrial uncoupling protein 2 (UCP2)-dependent mechanism

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Figure 2 Neuroprotective effects of H2S in Alzheimer’s disease. H2S demonstrates its neuroprotective effects in Alzheimer’s disease mainly through anti-apoptosis, anti-oxidation and anti-inflammation. H2S preserves mitochondrial membrane potential (MMP) and attenuates endoplasmic reticulum (ER) stress via upregulation of the brainderived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) pathway to exert its anti-apoptotic effects. H2S can reduce reactive oxygen species (ROS) either directly or through upregulation of Bcl-2 level, and inhibition of endoplasmic reticulum (ER) stress with subsequential anti-apoptosis ensues. H2S dramatically suppresses the release of TNF-α, IL-1 and IL-6 and inhibits the upregulation of cycloxidase-2 (COX-2) as well as the activation of nuclear factor κB (NF-κB) to achieve its anti-inflammatory action.

(Lu et  al., 2012). UCP2 can reduce ROS production and acts upstream to the KATP channel in determining vulnerability of dopaminergic neurons. Extant studies have demonstrated that MPP+ suppresses generation of endogenous H2S (Tang et  al., 2011) and that H2S has protective effects against MPP+-induced cytotoxicity and apoptosis not only by reducing the loss of MMP but also by attenuating intracellular ROS (Yin et al., 2009; Tang et al., 2011). Recent investigation into the mechanisms underlying the neuroprotective effects of H2S in PC12 cells against MPP+induced cytotoxicity has revealed the involvement of adenosine triphosphate-sensitive potassium/phosphatidylinositol 3-kinase/protein kinase B/B-cell lymphoma 2 (KATP/PI3K/AKT/Bcl-2) pathway. Applications of glybenclamide, a KATP channel blocker, and LY294002, a specific PI3K-AKT pathway inhibitor, reverses the neuroprotective effects of NaHS against MPP+-induced cytotoxicity to PC12 cells. NaHS can upregulate the activity of AKT in PC12 cells, while it is abolished by blockade of KATP channels with glybenclamide. In addition, NaHS upregulates the expression of Bcl-2 and blocks MPP+-induced downregulation of Bcl-2; however, this augmentation of Bcl-2 expression is prevented by both glybenclamide and LY294002 (Tang et al., 2012). Hu and coworkers have proved that H2S specifically suppresses 6-OHDA-evoked nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and oxygen consumption as well as prevents the development of PD induced by rotenone. In addition, H2S inhibits microglial activation in the SN and accumulation of pro-inflammatory factors including TNF-α and NO (Hu et al., 2010). This work for the first time illustrated that H2S may serve as a

neuroprotectant via multiple mechanisms including antioxidative stress, anti-inflammation and metabolic inhibition in neurodegeneration model induced by neurotoxin. Likewise, in a classical animal model of PD induced by MPTP, the neuroprotective effects of H2S are associated with upregulation of genes encoding antioxidant proteins, including heme oxygenase-1 (HO-1) and glutamate-cysteine ligase, probably through a nuclear-factor-E2-related factor-2 (Nrf2)dependent signaling pathway. As a result, H2S enhances the antioxidant defense system and inhibits inflammation and apoptosis (Chen et al., 2009; Kida et al., 2011). Recently, an E3 ubiquitin ligase named parkin is thought to be neuroprotective, S-nitrosylation and inactivation of which is neurotoxic, whereas physiologic modification called sulfhydration of parkin mediated by H2S can enhance parkin’s catalytic activity. Interestingly, decline of parkin sulfhydration in the corpus striatum of PD patients is confirmed, which underlies the pathogenesis and therapeutic strategies of PD (Vandiver et al., 2013). A H2S-releasing l-DOPA hybrid compound demonstrates anti-oxidant, anti-inflammatory and monoamine oxidase B inhibitory effects, those neuroprotective properties of which make H2S a more promising agent in PD treatment (Lee et al., 2010). Pathways underlying the neuroprotective role of H2S in PD are illustrated in Figure 3.

Cerebral ischemia Cerebral ischemia is one of the primary causes of morbidity and mortality especially in aged groups in modern

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134      W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant

Figure 3 Neuroprotective effects of H2S in Parkinson’s disease. H2S can reduce the loss of mitochondrial membrane potential (MMP) to exert its anti-apoptotic action. The neuroprotective effects of H2S are associated with upregulation of genes encoding antioxidant proteins, including heme oxygenase-1 (HO-1) and glutamate-cysteine ligase, probably through a nuclear-factor-E2-related factor-2 (Nrf2)-dependent signaling pathway. H2S may alleviate MPTP-induced ROS accumulation and subsequential ER stress via a mitochondrial uncoupling protein 2 (UCP2)-dependent mechanism. H2S specifically suppresses 6-OHDA-evoked nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and oxygen consumption. And UCP2 acts upstream to the KATP channel in determining vulnerability of dopaminergic neurons. Besides, H2S can directly and indirectly reduce reactive oxidative species (ROS) through KATP/PI3K/AKT/Bcl-2 pathway. H2S can exert its anti-inflammatory action inhibiting microglial activation in the substantia nigra (SN) and diminishing the accumulation of pro-inflammatory factors including TNF-α and nitric oxide (NO).

society. Deprivation of blood and oxygen and consequential intracellular Ca2+ overload, oxidative stress together with a surge of glutamate released from synaptic vesicles, lipolysis, calpain activation and cerebral ischemia give rise to neural damages of various extent and compromise brain functions.. H2S has been reported to protect the embryonic brain from ischemia-reperfusion (I/R) injury (Yang et al., 2008; Ishii et  al., 2010). In addition, a H2S-releasing derivative of aspirin (ACS14) reduces glutamate-mediated oxidative stress via elevating the generation of glutathione in RGC-5 cells (Osborne et  al., 2012). H2S also decreases the activity of glutathione-catabolizing enzymes and promotes glutathione recycling. Therefore, the intracellular glutathione would be at least maintained at the normal level (Wang, 2012; Lee et al., 2014). Apparently, injured hippocampus leads to impairment of learning and memory. H2S ameliorates neural damage and improves spatial learning and memory deficits (Li et  al., 2011; Wen et  al., 2014). Administration of NaHS enhances synaptic plasticity in the hippocampus of brain-ischemic rats as well as inhibits post-ischemic edema around pyramidal neurons and nuclear shrink (Li et al., 2011). Possible molecular mechanisms may involve the following: (a) increasing phosphorylation of Akt and decreasing phosphorylation of apoptosis signal-regulating kinase 1 (ASK1) and c-Jun N-terminal kinase 3 (JNK3);

and (b) upregulating expression of growth-associated protein-43 (GAP-43) in the CA1 region. In a global cerebral I/R rat model, decreased level of H2S in hippocampus and cortex at 24 h of reperfusion after global cerebral ischemia is demonstrated, and low dose of NaHS can attenuate the neuronal injury (Ren et  al., 2010). H2S can directly prevent ischemia-induced neuronal death in cultured hippocampal neurons subjected to oxygen glucose deprivation/reoxygenation (OGD/R) (Shao et  al., 2011). Exogenous H2S is also reported to be neuroprotective against global cerebral I/R injury through anti-oxidative, anti-inflammatory and anti-apoptotic action (Yin et al., 2013). The underlying mechanisms may be as follows: (a) increasing of superoxide dismutase (SOD) activity in the brain; (b) suppression of mRNA levels of p47 (phox) and gp91 (phox) subunits of NADPH oxidase; (c) inhibition of pro-inflammatory factors TNF-α and monocyte chemotactic protein-1 (MCP-1) and induction of anti-inflammatory factor IL-10; (d) alleviation of pro-apoptotic protein Bax (Yin et al., 2013); and (e) activation of phosphatidylinositol 3-kinase/Akt/p70 ribosomal S6 kinase (PI3K/Akt/p70S6K) cell survival signaling pathways (Shao et al., 2011). NaHS enhances cyclic adenosine monophosphate (cAMP) concentration and expression of PI3K, Akt and p70S6K. Additionally, neuronal viability is increased and apoptotic neuronal numbers decreased after OGD/R in the rat hippocampus (Shao et  al., 2011).

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A.S. Tay and colleagues also find that H2S can protect neurons against hypoxic injury through stimulating ATPsensitive potassium channel/protein kinase C/extracellular signal-regulated kinase 1/2/heat shock protein 90 (KATP/PKC/ERK1/2/HSP90) pathways. Over-expression of CBS protects neurons against hypoxia-induced apoptosis. Blockade of KATP channels with glibenclamide and HMR1098, protein kinase C (PKC) with its three specific inhibitors (chelerythrine, bisindolylmaleide I and calphostin C), extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059 and heat shock protein 90 (HSP90) with geldanamycin and radicicol significantly abolishes the protective effects of NaHS (Tay et al., 2010). An experiment recently has revealed a novel role of astrocytes in protective act of H2S, which promotes angiogenesis and improves functional outcome after cerebral ischemia. Pathways may involve increased phosphorylation of AKT and ERK through PI3K/AKT signaling as well as expression of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) (Jang et al., 2014). Moreover, a preclinical research has demonstrated innovative application of gaseous H2S. H2S is adopted as a mild inhibitor of oxidative phosphorylation, and inhalation of a mixture of air and H2S leads to sustained, deep hypothermia. Then long-term hypothermia results in a 50% reduction in infarct size accompanying reduction in the number of phagocytes in focal

cerebral ischemia. A possible mechanism is decreased expression of caspase 12, NF-κB and grp78 in the periinfarcted region, suggesting an overall declined transcriptional activity concerning inflammation and apoptosis (Florian et  al., 2008). Pathways underlying the neuroprotective effects of H2S in cerebral ischemia are demonstrated in Figure 4. Expression of 3-MST located in cortex and striatum is downregulated in a permanent middle cerebral artery occlusion rat model (Zhao et al., 2013). Additionally, H2S production comes to a complete stop due to intracellular overload of Ca2+ in cerebral ischemia despite the intimate association of 3-MST/CAT with bound sulfane sulfur (Shibuya et  al., 2009a,b; Mikami et  al., 2011a,b). An in vitro experiment has demonstrated that neuro2a cells with 3-MST/CAT can notably resist oxidative stress induced by high-dose glutamate. However, they have not explored the effects of intracellular overload Ca2+ on 3-MST/CAT pathway and the protective role of H2S in the same cell lines (Kimura et al., 2010), while as mentioned above, both excessive Ca2+ and glutamate are involved in the pathogenesis of cerebral ischemia. Therefore, we would like to point out the possibility that even though the 3-MST/CAT pathway is the primal source of H2S in the brain, the alternative enzyme CBS may serve as a much more promising target for investigating the protective role of H2S in cerebral ischemia.

Figure 4 Neuroprotective effects of H2S in cerebral ischemia. H2S can spur the generation of glutamate, decrease the activity of glutathione-catabolizing enzymes and promote glutathione recycling. H2S increases superoxide dismutase (SOD) activity in the brain and suppresses mRNA levels of p47 (phox) and gp91 (phox) subunits of NADPH oxidase in a bid to reduce reactive oxidative species. Pro-apoptotic factors including apoptosis signal-regulating kinase 1 (ASK1), c-Jun N-terminal kinase 3 (JNK3), Bax and caspase 12 are alleviated by H2S. Additionally, anti-apoptotic factor such as growth-associated protein-43 (GAP-43) can be increased by H2S. H2S also exerts its anti-apoptotic effects through the following: (A) stimulating ATP-sensitive potassium channel/protein kinase C/extracellular signal-regulated kinase 1/2/heat shock protein 90 (KATP/PKC/ERK1/2/HSP90) pathways; (B) activation of phosphatidylinositol 3-kinase/Akt/p70 ribosomal S6 kinase (PI3K/Akt/p70S6K) cell survival signaling pathways; and (C) increasing phosphorylation of AKT and ERK through PI3K/AKT signaling as well as expression of vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1). Pro-inflammatory factors like monocyte chemotactic protein-1 (MCP-1) and TNF-α are diminished, while an anti-inflammatory factor IL-10 is increased by H2S. H2S can suppress activation of inflammatory transcriptional factor nuclear factor κB (NF-κB) as well.

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136      W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant Nevertheless, the effects of H2S can be protective or toxic in cerebral ischemia depending on different H2S concentrations; for example, high-concentration NaHS (11.2 mg/kg) aggravates infarct volume, whereas low dose (2.8 mg/kg) exerts its positive effects (Li et al., 2012). In a similar experiment, administration of 10.09 mg/kg NaHS causes a 50% increase of infarct volume. May be it is the adverse effects of sublethal doses of NaHS and overproduction of endogenous H2S in cerebral ischemia that lead authors to draw a conclusion that H2S is a mediator of cerebral ischemic damage (Qu et al., 2006). Therefore, we intend to propose a hypothesis that there exists a belllike curve that explicates the quantifiable relationship between indexes like infarct volume or brain functional indications and concentration of NaHS with the apex representing an optimal point or range of concentration(s) in cerebral ischemic protection. We believe that once this curve is verified, it will bring great noteworthy advancements in developing new agents of H2S donors for cerebral ischemia. The curve hypothesis is illustrated in Figure 5.

Major depression disorder MDD is a mental disorder characterized by a pervasive and persistent low mood causing disability and premature death (Denollet et  al., 2009). More than 16% of the adult population suffers from these conditions to various

extents (Schechter et  al., 2005). In actuality, the risk of depressive disorders has been reported to range from 25% to 79% among people suffering from cerebral ischemia, namely stroke, and post-stroke major depression prevalence ranges from 3% to 40% (Gordon and Hibbard, 1997; Spencer et  al., 1997). The specific mechanisms of depression are ambiguous at present, while there are two dominating hypotheses: (a) hippocampal neurogenesis (Hanson et al., 2011; Eisch and Petrik, 2012) and (b) brainderived neurotrophic factor and tropomyosin-related kinase B pathway (BDNF/TrkB) (Castren and Rantamaki, 2010; Cazorla et  al., 2010; Taliaz et  al., 2010). According to our preliminary experiments, H2S not only upregulates expression of BDNF but also alleviates inhibition of expression of BDNF in a chronic unpredictable mild stress animal model. We have also observed that H2S protects neurons against corticosterone stress insults via upregulating expression of BDNF in a dose-dependent manner, which potently indicates the neuroprotective effects of H2S. Our latest study has demonstrated that H2S inhibits homocysteine-induced ER stress and neuronal apoptosis in rat hippocampus via upregulation of the BDNF-TrkB pathway (Wei et al., 2014). Based on the neuroprotective effects of H2S in hippocampus and neural cell lines, we further investigate whether H2S can exert its positive role in anxiety and depression. And our recent findings for the first time speculate that H2S may be a novel therapeutic target for depression and anxiety. Notably, the innovative application of multiple testing methods including forced swimming test, tail suspension test (Gouret et  al., 1990), elevated plus-maze and locomotor activity test in rats or mice in this study provide significant parameters about anti-depressive like and anti-anxiolytic like effects of H2S (Chen et al., 2013). The study suggests that elevating H2S signaling in the brain may represent a novel approach for treatment of depressive and anxiety disorders in the clinic. Pathways underlying the neuroprotective role of H2S in MDD are shown in Figure 6A.

Febrile seizure Figure 5 Curve hypothesis of quantified indexes with concentrations of NaHS in cerebral ischemia. (A) We hypothesize that there is an optimal concentration at which NaHS can demonstrate the maximum function indexes in a bell-like curve. (B) We assume a bell-like curve that can depict the relationship between concentrations of NaHS and infarct volume because high concentrations of NaHS (11.2 mg/kg and 10.09 mg/kg) aggravate infarct volume, whereas a low dose (2.8 mg/kg) exerts its positive effects. Moreover, the apex stands for an optimal point or range of concentration(s) of NaHS with minimum infarct volume.

FS is the most common type of pathological brain activity in infants and children induced by high fever (Shinnar and Glauser, 2002). Little evidence supports that shortterm FS can cause adverse influence on children’s developing brains; Céline M. Dubé et  al. have proved that prolonged FS impairs children’s recognition memory in a clinic research. Acute hippocampal injury in children with prolonged FS is observed on magnetic resonance imaging.

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W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant      137

Figure 6 Neuroprotective effects of H2S in major depression disorder (A) and febrile seizure (B). (A) H2S promotes hippocampal neurogenesis and upregulates the brain-derived neurotrophic factor and tropomyosin-related kinase B pathway (BDNF/TrkB) followed by inhibition of ER stress. (B) Possible mechanisms for H2S protecting hippocampal neurons against excitatory toxicity may involve inhibition of synaptic transmission and normalization of the excitation/inhibition balance via reversing the loss of GABAB receptor 1 (GABABR1) and GABAB receptor 1 (GABABR2).

Decreased hippocampal volume with poor performance in recognition memory tests signifies that recognition memory impairments are closely associated with hippocampal injury in FS (Martinos et al., 2012). Ying Han and colleagues report that downregulation of H2S by an antagonist of CBS, hydroxylamine, leads to hippocampal hyperactivity in a FS rat model. Rats administered with NaHS show reduction in neural excitatory activity (Han et  al., 2005a,b). There is also evidence that H2S and CO exert protective effects in recurrent FS in a synergic way (Han et al., 2006). Inhibitory effects in the CNS mediated by γ-aminobutyric acid (GABA) are crucial because loss of GABAergic inhibition can give rise to seizures and neuronal hyperexcitability. H2S is found to protect against the damage to hippocampus caused by recurrent episodes of FS. Possible mechanisms for H2S protecting hippocampal neurons against excitatory toxicity may involve the following: (a) inhibiting of synaptic transmission (Abe and Kimura, 1996); (b) normalization of the excitation/inhibition balance via reversing the loss of GABAB receptor 1 (GABABR1) and GABAB receptor 2 (GABABR2) (Han et al., 2005a,b). Since FS is featured with high fever, H2S can function as a mild inhibitor of oxidative phosphorylation, and application of gaseous H2S leads to sustained, deep hypothermia (Florian et al., 2008). We believe that transplantation of this innovative application from cerebral ischemia to FS may represent a novel therapeutic clinic method in prevention of brain injury due to recurrent and long-term FS. Pathways underlying the neuroprotective role of H2S in FS are illustrated in Figure 6B.

Hypothesis of neuroprotective potential of H2S based on its interaction with various ion channels Apart from KATP, however, there are a lot of other ion channels in multiple systems mediating the protective effects of H2S. H2S gives rise to the upstroke of the cardiac action potential, and in human umbilical vascular endothelial cells, Nav could regulate the angiogenic effects of calcium signals (Andrikopoulos et  al., 2011; Strege et  al., 2011). Effects that H2S exerts in the regulation of vasodilation and cardioprotection involved KATP channel as a direct target. In fact, H2S S-sulfhydrates the subunit of KATP channels – Kir6.1, and DTT, a strong reducing agent, reverses H2S-mediated KATP S-sulfhydration (Mustafa et  al., 2011). H2S can inhibit both human recombinant Ca2+-activated K+ channels (BKCa) and native BKCa channels expressed in the carotid body in rats, which are widely localized in the CNS and vasculature. On the other hand, H2S may increase the activity of BKCa channels expressed in a rat pituitary cell line, leading to hyperpolarization and relaxation of smooth muscle cells (SMCs) (Telezhkin et al., 2010; Peers et  al., 2012). To date, NaHS has been proved to cause a negative chronotropic action in human atrial fibers by blocking l-type Ca2+ channels and an enhancement in the repolarization phase by opening KATP channels (Xu et al., 2011). Intriguingly, NaHS seems to exert a totally different effect on neurons. That is, NaHS (50–300 μm) induces cell death and Cai increase in cultured rat cerebellar granule neurons (Lipscombe et  al., 2004; Tang et  al., 2010a,b). Explanation for this phenomenon may be the variations

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138      W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant

Figure 7 Interaction of H2S with ion channels. Nav, voltage-dependent Na+ channels; KATP, ATP-sensitive potassium channel; SMC, smooth muscle cells; BKCa, big conductance Ca2+sensitive K+; DTT, dithiothreitol, a strong reducing agent; CNS, central nervous system; l-type Ca2+, l-type Ca2+channels; T-type Ca2+, T-type Ca2+channels; TRPV1, transient receptor potential vanilloid 1; dual, dual regulation; SP, substance P; ERK/NF-κB, ERK/NF-κB pathway; NF-κB, nuclear factor κB; ERK, extracellular signal-regulated kinases; TRPA1, transient receptor potential ankyrin 1; CSLV, capsaicin-sensitive lung vagal; HC-030031, a TRPA1 selective antagonist; Cl-, chloride channels.

of l-type Ca2+ channel complex nature between heart and CNS. NaHS enhances Ca2+ current through T-type channels in patch clamp studies using undifferentiated NG108-15 cells and promptly causes hyperalgesia in rats subjected to intraplantar and intrathecal NaHS administration through T-type Ca2+channel activation (Kawabata et  al., 2007; Maeda et al., 2009). T-type Ca2+ channel activation, in particular the Cav3.2 isoform by H2S, appears to regulate rhythmic neuronal activity and pain sensation (Maeda et  al., 2009; Nagasawa et  al., 2009). H2S may prevent ethanol-induced gastric lesions in mice by stimulating TRPV1 channels on the capsaicin-sensitive primary afferent neurons that innervate the gastric mucosa (Ang et al., 2011a,b). Consistently, animals pretreated with capsazepine reverse the gastroprotective action of either l-cysteine or NaHS. H2S induces a nonspecific sensitizing effect on capsaicin-sensitive lung vagal (CSLV) fibers to both chemical and mechanical stimulation in rat lungs, which is probably mediated by the TRPA1 receptors and ultimately leads to the development of airway hypersensitivity (Hsu et al., 2013). H2S may inhibit the chloride channels derived from the rat heart lysosomal vesicles incorporated into a bilayer lipid membrane via decreasing the channels opening in a concentration-dependent manner (Malekova et al., 2009) and activate cystic fibrosis transmembrane conductance regulator Cl- channels in HT22 neuronal cell lines, exerting its neuroprotective effects in oxidative stress (Kimura et al., 2006). And there is evidence that implies the pathophysiological involvements of both chloride channels and H2S (Geng et al., 2004; Johansen et al., 2006; Chen et al., 2007; Yang and Wang, 2007).

We would like to hypothesize that antagonists or activators of ion channels mentioned above are potential targets for the neuroprotective role of H2S in the CNS, in that the localization in body systems and associations with H2S of the ion channels are ubiquitous and yet to be discovered. For example, chloride channels play important functional roles in extensive processes, including blood pressure regulation, cell cycle and apoptosis, muscle tone, volume regulation, synaptic transmission and also cellular excitability (Nilius and Droogmans, 2003; Miller, 2006; Puljak and Kilic, 2006). Interaction of H2S with these ion channels and the effects are illustrated in Figure 7.

Conclusions and perspectives H2S exerts its neuroprotective effects primarily through anti-oxidant, anti-inflammatory and anti-apoptotic actions, which comprise multifarious underlying pathways involving heterogeneous molecules. Upregulation or downregulation of these pathways directed by endogenous or exogenous H2S can be manipulated by human intervention, for example, gene expression and agents’ administration in terms of H2S generation and metabolism. However, it is still a long journey to achieve the ultimate goal. Although hereby we provide an overview of the neuroprotective role of H2S, it is actually a double-faced molecule because there are both protective and pathogenic effects that H2S exerts in various systems. Even in the CNS, H2S is not always protective; for instance, there are

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W.-L. Chen et al.: Hydrogen sulfide as a neuroprotectant      139

experiments demonstrating dual effects with changes of infarct volume and function indexes in cerebral ischemia when animals or cells are given high- and low-dose NaHS. Based on the controversial effects of H2S, we propose a bell-like curve describing the relationships of infarct volumes or function indexes and concentration of H2S and the existence of an optimal point at which H2S can be neuroprotective at maximum extent excluding other microenvironmental factors in the brain. In conclusion, H2S draws a tempting blueprint for its application in cardiovascular diseases, neurological diseases, psychiatric diseases, metabolic syndromes and diabetes mellitus. Pharmacological blockers of H2Smetabolizing enzymes, inhalation of gaseous H2S and H2S-donors are promising agents remaining to be developed for therapeutic use against not only CNS diseases but also diseases in other organ systems. Acknowledgments: This work was supported by the National Natural Science Foundation of China (81371485 and 81200985), Natural Science Foundation of Hunan Province, China (11JJ3117 and 12JJ9032), Project of Research-based Learning and Innovative Experiment for Undergraduate Student in Hunan Province (2011-199) and the construct program of the key discipline in Hunan province.

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