Current Perspective of Hydrogen Sulfide as a Novel Gaseous Modulator of Oxidative Stress in Glaucoma

Glaucoma is a group of diseases characterized by the progressive loss of retinal ganglion cells and their axons. Elevated intraocular pressure (IOP) is the main clinical manifestation of glaucoma. Despite being in the focus of the studies for decades, the characteristic and the exact pathology of neurodegeneration in glaucoma remains unclear. Oxidative stress is believed to be one of the main risk factors in neurodegeneration, especially its damage to the retinal ganglion cells. Hydrogen sulfide (H2S), the recently recognized gas signaling molecule, plays a pivotal role in the nervous system, vascular system, and immune system. It has also shown properties in regulating oxidative stress through different pathways in vivo. In this review, we summarize the distribution and the properties of H2S within the eye with an emphasis on its role in modulating oxidative stress in glaucoma.


Introduction
Glaucoma is a group of diseases characterized by a combination of progressive optic nerve damage and loss of visual function, with pathologically elevated intraocular pressure (IOP) being the main risk factor [1]. In clinical practice, controlling the IOP is the one and only treatment, but it usually does not halt the progression of glaucoma and the damage to the optic nerve [2]. This situation also reflects the fact that the pathogenesis of glaucoma is multifactorial and extremely complex. It remains to be thoroughly studied, however, certain pathological processes are identified to play a role in the progress of glaucoma, such as excessive oxidative stress, inflammatory reactions, and accumulation of protein mutations [3][4][5][6]. Aging is a well-known risk factor for neurodegenerative diseases as well as for glaucoma. The incidence of glaucoma worldwide increases three-fold within ageing population every decade [7] and the number of glaucoma patients is expected to be 112 million by 2040 [8]. Oxidative stress is proposed as a key modulator in ageing and in neurodegenerative diseases [9]. There is hypothesis that age-related neuronal functional losses are due to the accumulation of oxidative stress [10]. An overall increase of oxidative stress markers is also detected in glaucoma patients [11].
The reactive oxygen species (ROS) is essential for normal cellular function. When the production of ROS exceeds the antioxidant capacity of the cells, they pose a constant threat to the cell, a process known as oxidative stress [6]. The higher concentration and long-time exposure of oxidative stress cause damage to cellular macromolecules such as DNA, lipids, and proteins, ultimately resulting in necrosis and apoptotic cell death [9]. The characters of neurodegenerative diseases are apoptosis/necrosis and dysfunction of neuronal cells, resulting in neurodegenerative changes due to nerve cell dysfunction [9,[12][13][14]. In glaucoma, oxidative stress harms multiple ocular tissues via different mechanisms, which comprising stimulating apoptotic and inflammatory pathways at the trabecular meshwork level and promoting retinal ganglion cell apoptosis and glial dysfunction [15][16][17][18]. These studies contributed to our understanding of oxidative stress in glaucoma. However, the cellular mechanisms of oxidative damage in the eye and the retinal ganglion cells (RGCs), as well as the possible regulatory mechanisms in response to oxidative stress remain to be further elucidated.
H 2 S has been recently recognized to be a third gas signaling molecular of comparable importance to nitric oxide (NO) and carbon monoxide (CO) [19,20]. H 2 S plays a significant role in the human nervous system, vascular system, and immune system at a certain range of concentration [21][22][23]. The concentration of H 2 S in the peripheral blood is generally 30-300 Mm [24], while the physiological concentration of H 2 S in the brain is up to three times that in serum [25,26].
In the central nervous system (CNS), H 2 S is present in high concentrations and has shown protective effects on neurons by modulating oxidative stress and inflammatory responses, anti-apoptosis as well as acting as a vasculoprotective factor [27][28][29][30]. In cultured cells, H 2 S protects primary cortical neurons from oxidative stress induced by glutamate by increasing the production of antioxidant glutathione [31]. In the peripheral nervous system, H 2 S has been proven to protect RGCs, the neuron that is selected to die in glaucoma, in different pathological conditions, such as diabetic retinopathy, N-methyl-D-aspartic acid (NMDA)-induced neurotoxicity, and ischemic condition [16,19,20]. There has been an increasing interest in studying the role of H 2 S in neurodegeneration in glaucoma. We documented in our previous study that the administration of exogenous H 2 S protects RGC against different glaucomatous injuries in vitro and in vivo [21]. The underlying mechanism was partly attributed to its capability of vasorelaxation, anti-oxidative stress, neuroendocrine regulation, and inflammation suppression [22][23][24]. Although it is yet inconclusive as to through which mechanism exerts H 2 S its protective effects in glaucoma. By all counts, H 2 S plays a pivotal role in protecting RGCs and their axons against neurodegeneration.
This review aims to firstly summarize the generation and distribution of H 2 S in the eye, and secondly to further explore the interaction of H 2 S and oxidative stress in neurodegeneration in glaucoma, with a focus on the diverse underlying pathways.

Generation and Distribution of H 2 S in Ocular Tissues
There are two main production pathways of H 2 S in mammalian cells, enzymatic and non-enzymatic pathways [32]. Enzymatic pathways account for the central part. The enzymes known to participate in the enzymatic pathway of production of H 2 S are mainly cystathionine-γ-synthase (CSE), cystathionine-β-lyase (CBS), 3-mercapto-methylthio pyruvate aminotransferase (3MST), and cysteine aminotransferase (CAT) [32,33]. Generally, endogenous H 2 S is derived from the desulfurization of cysteine or homocysteine by the enzymes CSE and CBS. Different enzymes are expressed in different systems. CBS expression is significant in the brain as the primary physiological source of H 2 S in the central nervous system [34]. Robert et al. documented that CBS proteins are widely present in the adult rat brain and are most strongly expressed in the Purkinje cell layer and hippocampus [34]. CSE is abundantly expressed in the respiratory system and cardiovascular system [20]. Both CBS and CSE are only localized in the cytoplasm, while 3MST and CAT exist in both mitochondria and cytoplasm. Recently, it has also been demonstrated that H 2 S can also be produced from d-cysteine via d-amino acid oxidase (DAO) along with 3MST [35]. However, production through this pathway is limited as DAO exists exclusively in the brain and kidneys [35]. On the other hand, the non-enzymatic pathways of endogenous H 2 S are produced in erythrocytes through the glucose oxidation pathway [19]. Moreover, this pathway can be stimulated by increased oxidative stress and hyperglycemia to produce more H 2 S [32].
These H 2 S-productive enzymes are widely distributed in specific tissues in the eye, predominately in the cornea and retina [33,[36][37][38][39]. CBS is present in various ocular tissues, including conjunctiva, cornea, iris, lens, retina, and optic nerve, but not in vitreous humor [36,40]. CBS is abundant in anterior segments throughout the lifespan, and its abundance within the retina increases with age [33,36]. The presence of CBS and CSE has also been traced in all three layers of canine, non-human primate, and human retina: photoreceptors, outer plexiform layer (OPL), and notably in the ganglion cells layer/nerve fiber layer (GCL/NFL) [41]. 3MST/CAT pathway is the primary way to produce H 2 S in the mammalian retina as both 3MST and CAT are located in the retinal neurons [36]. Our previous study has shown that H 2 S produced by the 3-MST pathway increases after seven weeks of IOP elevation in a glaucoma animal model [42] (Figure 1).
Antioxidants 2021, 10, x FOR PEER REVIEW 3 o [36,40]. CBS is abundant in anterior segments throughout the lifespan, and its abunda within the retina increases with age [33,36]. The presence of CBS and CSE has also b traced in all three layers of canine, non-human primate, and human retina: photore tors, outer plexiform layer (OPL), and notably in the ganglion cells layer/nerve fiber la (GCL/NFL) [41]. 3MST/CAT pathway is the primary way to produce H2S in the mam lian retina as both 3MST and CAT are located in the retinal neurons [36]. Our prev study has shown that H2S produced by the 3-MST pathway increases after seven we of IOP elevation in a glaucoma animal model [42] (Figure 1).

Figure 1.
Endogenous H2S is produced through two main pathways, the non-enzymatic pathw and the enzymatic pathway. The non-enzymatic pathway is produced in erythrocytes via the g cose oxidation pathway. The enzymatic pathway promotes the production of H2S from cystein with four common enzymes (cystathionine-β-lyase (CBS), cystathionine-γ-synthase (CSE), 3-m capto-methylthio pyruvate aminotransferase (3MST), and cysteine aminotransferase (CAT)). T enzymes are distributed in various tissues of the body.

Reducing Intraocular Pressure (IOP)
Elevated intraocular pressure is the hallmark of the development of glaucoma, it is also considered one of the main causes of damage to retinal neurons and optic ner The production of aqueous humor (AH) in the ciliary body and the unobstru flow of various AH outflow pathways are important factors in determining stable IO The autonomic regulation of the blood vessels of the ciliary body and the ciliary thelium is crucial in the production of AH, while episcleral blood vessels are in the flow of AH [43]. In a study on isolated superfused bovine iris-ciliary bodies, all three producing substances (ACS67, a hybrid of latanoprost and an H2S-donating moiety cysteine, a substrate for endogenous production of H2S, and GYY4137, Morpho-lin-4i methoxyphenyl-morpholino-phosphinodithioate, a slow-releasing H2S donor) inhib sympathetic neurotransmission in the iris-ciliary bodies through the mediation of K channels [44]. Notably, the reduction of sympathetic neurotransmission in the ante uvea relaxes the iris ciliary muscle and diastolic ocular vascular smooth muscle, indire acting to lower IOP [40,45]. In the isolated porcine iris ciliary body, both H2S inhib norepinephrine release from sympathetic nerve terminals in the eye which promotes atrial outflow [40,46]. Its ability in modulating autonomic nerves indicates that H2S h critical influence on the anterior uveal tissue as well as the production and the outflow AH.
Other than the episcleral blood vessels, the regulatory processes of the trabec meshwork are also pivotal in regulating conventional aqueous humor outflow from Figure 1. Endogenous H 2 S is produced through two main pathways, the non-enzymatic pathway and the enzymatic pathway. The non-enzymatic pathway is produced in erythrocytes via the glucose oxidation pathway. The enzymatic pathway promotes the production of H 2 S from cysteine with four common enzymes (cystathionine-β-lyase (CBS), cystathionine-γ-synthase (CSE), 3-mercaptomethylthio pyruvate aminotransferase (3MST), and cysteine aminotransferase (CAT)). These enzymes are distributed in various tissues of the body.

Reducing Intraocular Pressure (IOP)
Elevated intraocular pressure is the hallmark of the development of glaucoma, and it is also considered one of the main causes of damage to retinal neurons and optic nerves.
The production of aqueous humor (AH) in the ciliary body and the unobstructed flow of various AH outflow pathways are important factors in determining stable IOP.
The autonomic regulation of the blood vessels of the ciliary body and the ciliary epithelium is crucial in the production of AH, while episcleral blood vessels are in the outflow of AH [43]. In a study on isolated superfused bovine iris-ciliary bodies, all three H 2 S producing substances (ACS67, a hybrid of latanoprost and an H 2 S-donating moiety, L-cysteine, a substrate for endogenous production of H 2 S, and GYY4137, Morpho-lin-4iummethoxyphenyl-morpholino-phosphinodithioate, a slow-releasing H 2 S donor) inhibited sympathetic neurotransmission in the iris-ciliary bodies through the mediation of K ATP channels [44]. Notably, the reduction of sympathetic neurotransmission in the anterior uvea relaxes the iris ciliary muscle and diastolic ocular vascular smooth muscle, indirectly acting to lower IOP [40,45]. In the isolated porcine iris ciliary body, both H 2 S inhibited norepinephrine release from sympathetic nerve terminals in the eye which promotes the atrial outflow [40,46]. Its ability in modulating autonomic nerves indicates that H 2 S has a critical influence on the anterior uveal tissue as well as the production and the outflow of AH.
Other than the episcleral blood vessels, the regulatory processes of the trabecular meshwork are also pivotal in regulating conventional aqueous humor outflow from the anterior chamber [43]. The trabecular meshwork (TM) is a key region for the initiation of glaucoma. Excessive ROS alters both TM motility and cytoarchitecture [18,47,48]; apoptotic TM cells and affected TM epithelial cells impair aqueous outflow [49][50][51], which leads to a failure to control IOP, a hallmark in the progress of glaucoma [52]. H 2 S is shown to facilitate the dynamic equilibrium of AH and stabilize IOP by increasing autonomic regulation in ocular anterior segments, reduce the cell volume of trabecular meshwork and relax the iris smooth muscle [33,45,53,54].
Elevated IOP leads to increased oxidative stress in the eye. Documented by Gericke et al., elevated levels of ROS were detected in the RGC layer and retinal vasculature in a mouse model with elevated IOP [55].
By regulating AH dynamic, H 2 S and its donors can effectively modulate the oxidative stress caused by elevated IOP.

Ocular Hemodynamic Changes
The retina, as an extension of the CNS, has an equally high demand for oxygen and other metabolites as the brain [56]. Lack of oxygen can lead to eye diseases such as diabetic retinopathy and glaucoma [57,58]. However, the vascular density in the inner retina is limited due to the optical function of the eye, which leads to an unstable vascular oxygen supply [59]. This feature renders the inner retina, especially the ganglion cell layer and the nerve fiber layer, extremely vulnerable to changes in ocular hemodynamic. It has been discussed over a decade, that hemodynamic change plays a role in glaucomatous neuropathy [60,61]. Studies have provided evidence that compared with healthy individuals, the blood flow in the eyes of glaucoma patients is significantly reduced, furthermore, the blood flow in the optic papilla region is reduced more obviously [62][63][64]. Furthermore, this was also demonstrated in our previous study in an in vivo glaucoma animal model, elevated IOP leads to shrinking of retinal vascular caliber [65], which limits the blood flow and oxygen supply in the retina and consequently promotes the production and accumulation of ROS [66]. Morphological and functional changes of the vascular play a central role in hemodynamic changes.
Like NO, H 2 S has similar effects on blood vessels. A low concentration of H 2 S relaxes the vascular smooth muscle [32]. However, it is demonstrated that NO mainly acts on large blood vessels, while H 2 S has a more significant effect on small blood vessels [32]. This difference may be related to H 2 S and blood oxygen concentration. According to several reports, H 2 S can exert a dilation effect on blood vessels at higher than physiological oxygen levels but lead to the opposite effect at lower than physiological oxygen levels [67]. The human eye is surrounded by small blood vessels and the partial pressure of oxygen in small blood vessels is low, therefore H 2 S may have a more substantial role in vasodilation in the ocular vasculature.
Slow releasing H 2 S donors, 4-methoxyphenyl)pyrrolidin-1-ylphosphinodithioc acid (AP67) and 4-methoxyphenyl)piperidin-1-ylphosphinodithioc acid (AP72) have been demonstrated to have vasodilatory effects on isolated bovine posterior ciliary arteries induced by adrenergic receptor agonists [68]. Moreover, this process may be dependent on the biosynthesis of endogenous NO and the action of K ATP channels [68]. In our previous study, we also observed that intravitreal injection of GYY4137 into mice increased the caliber of retinal vessels and significantly improved blood flow, which is beneficial in improving retinal perfusion and subsequently promotes RGC survival over the long term [42]. By expanding blood vessels, H 2 S improves blood flow and oxygen and metabolite to the retina as well as stabilizes retina hemodynamics, therefore modulates the oxidative stress in the retina in glaucoma.

Inhibition of Neurodegeneration in Glaucoma
The neurodegenerative changes in glaucoma are mainly manifested by progressive retinal ganglion cell death. The changes can be contributed to the absence of certain neurotrophic factors, intracellular and extracellular toxicity of glutamate, and stimulation of external factors [6,31]. Such neurodegenerative changes become more pronounced with age, accumulation of ROS, and reduction of antioxidant substances. In a mouse model of Alzheimer's disease (AD), H 2 S was found to interfere with the production of amyloid β-protein (Aβ), which affects the development of AD, and inhibits Aβ-induced neuronal apoptosis [69,70]. Similarly, in an animal model of Parkinson's disease (PD), H 2 S was found to act as an antioxidant to counteract the neurotoxic effects induced by 6-hydroxydopamine and to have a neuroprotective effect [71]. At the same time, in a clinical study that the H 2 S was significantly lower in the plasma of AD patients than in healthy controls [69,72,73]. These studies may infer that H 2 S plays a role in neurodegeneration in AD and protects neurological function and prevents neurodegeneration in animal models of PD and AD through anti-apoptotic, anti-inflammatory, and antioxidant pathways. However, the exact pharmacology of H 2 S in the clinical treatment of neurodegenerative diseases requires further research. In the eye, H 2 S has also been found to protect RGCs against glaucomatous damages caused by elevated IOP and oxidative stress both in vivo and in vitro in animal models [42].
Although the mechanism of H 2 S in protecting against glaucomatous optic nerve damage is to be thoroughly investigated, there are traces to follow, that H 2 S may regulate oxidative stress and protect against neurodegenerative changes in glaucoma by regulating iron homeostasis, changing mitochondrial dysfunction, and attenuate glutamate neurotoxicity. In the following chapters, we aim to summarize the established mechanistic link between H 2 S and oxidative stress in neurodegeneration from recently published studies (see Figure 2).
Alzheimer's disease (AD), H2S was found to interfere with the production of am protein (Aβ), which affects the development of AD, and inhibits Aβ-induced n apoptosis [69,70]. Similarly, in an animal model of Parkinson's disease (PD), H found to act as an antioxidant to counteract the neurotoxic effects induced b droxydopamine and to have a neuroprotective effect [71]. At the same time, in a study that the H2S was significantly lower in the plasma of AD patients than in controls [69,72,73]. These studies may infer that H2S plays a role in neurodegene AD and protects neurological function and prevents neurodegeneration in anima of PD and AD through anti-apoptotic, anti-inflammatory, and antioxidant pa However, the exact pharmacology of H2S in the clinical treatment of neurodege diseases requires further research. In the eye, H2S has also been found to prote against glaucomatous damages caused by elevated IOP and oxidative stress both and in vitro in animal models [42].
Although the mechanism of H2S in protecting against glaucomatous opt damage is to be thoroughly investigated, there are traces to follow, that H2S may oxidative stress and protect against neurodegenerative changes in glaucoma by re iron homeostasis, changing mitochondrial dysfunction, and attenuate glutamat toxicity. In the following chapters, we aim to summarize the established mechan between H2S and oxidative stress in neurodegeneration from recently published (see Figure 2). re 2. The anti-oxidative properties of H2S in glaucoma. The effect of H2S on glaucoma is mainly reflected in th cts. (1) By regulating the autonomic nerves and reducing the release of sympathetic nerves, it reduces the produc ueous humor (AH) and promotes the flow of AH to lowering intraocular pressure (IOP). (2) Through the relaxa scular smooth muscles, stabilize intraocular perfusion and reduce ischemia-reperfusion injury. (3) By regulating eostasis, regulating mitochondrial function, and reducing the toxicity of glutamate, it reduces the generation of R event nerve degenerative changes.

Regulation of Iron Homeostasis
Iron homeostasis plays a pivotal role in oxidative damage. Excess iron prom generation of damaging hydroxyl groups from oxidation reaction products, thus bating oxidative stress [74,75]. Current studies have found that iron metabolism i for neurotransmitter production and myelin synthesis [76]. In a number of studies

Regulation of Iron Homeostasis
Iron homeostasis plays a pivotal role in oxidative damage. Excess iron promotes the generation of damaging hydroxyl groups from oxidation reaction products, thus exacerbating oxidative stress [74,75]. Current studies have found that iron metabolism is critical for neurotransmitter production and myelin synthesis [76]. In a number of studies on neurodegenerative diseases, it has been found that increased levels of iron in the brains Antioxidants 2021, 10, 671 6 of 13 of animals and human produce significant cognitive impairment [77][78][79][80]. Furthermore, MRI scans have shown abnormal aggregation of iron in the hippocampus in the brains of post-mortem AD patients and AD mouse models [81,82]. It has also been shown in patients with PD, total iron concentrations in the substantia nigra are increased [71]. These evidences suggest that altered iron levels in the brain are associated with neuronal dysfunction and the pathology of neurodegenerative disease in CNS in both animal and human. Increased iron level has also been documented in not only various glaucoma models but also in AH of glaucoma patients [83,84]. As research continues to progress, alteration in iron homeostasis has been also connected to intracellular glutamate production and secretion, glutathione (GSH) synthesis, and hypoxia-inducible factor-1 (HIF-1) activity, yet all of these pathways are implicated in the pathogenesis of glaucoma [85]. These results indicate that iron homeostasis may also play a role in neurodegenerative changes in glaucoma.
The majority of iron in cells is located in the protoporphyrin ring of heme. The massive release of heme can lead to severe oxidative stress and promote apoptosis [74]. Maintaining heme release can effectively maintain iron homeostasis and control redox reactions. Under both hyperoxia conditions and ischemia-reperfusion injury, H 2 S has been shown to modulate the heme release, by interacting with heme oxygenase and biliverdin reductase A, respectively, while increasing the capacity of antioxidant production [76,86,87].
Another regulating iron homeostasis, H 2 S may also play a crucial role in the ironsulfide (Fe-S) cluster. Fe-S cluster is essential for retinal physiology and pathology [88,89]. Fe-S cluster is required for many essential processes in the cell, including catalysis, iron regulation, DNA repair, ribosome biogenesis, and tRNA modifications [90,91]. Fe-S clusters are also involved in many other vital processes in mitochondria. They not only donate electrons to the respiratory chain, but also constitute the respiratory protein complexes I, II, and III [92]. Defects in Fe-S cluster synthesis in mitochondria are generally not considered to be re-sponsible for all neurodegenerative diseases, but such defects can affect the normal function of the central nervous system, for instance, in X-linked sideroblastic anemia and mitochondrial encephalopathy, Fe-S cluster synthesis is believed to play a role in their pathogenesis [92][93][94]. Since both components (iron and sulfur) are toxic, Fe-S cluster assembly must therefore be both efficient and tightly regulated [95]. Glutaredoxin-3 (Glrx3) is an important factor in the assembly of Fe-S clusters [96]. H 2 S is shown to regulate intracellular Glrx3 levels, thereby maintaining iron homeostasis and regulating the redox state in cells [76].

Changes in Mitochondrial Function
Mitochondria are the most important energy-producing organelles in human cells. It produces energy through a variety of ways, which coordinate with each other to maintain the best energy state in the cell [97]. When mitochondria generate energy, they also generate many ROS, including hydrogen peroxide (H 2 O 2 ), superoxide (O 2 •− ), and hydroxyl ion ( • OH) [98]. Mitochondrial dysfunction leads not only to reduced ATP production but also to excessive ROS production, impaired calcium buffering, mitochondrial metal allostasis, and activation of mitochondria-dependent apoptosis [99]. In recent years, mitochondrial disorders have been considered to be an important factor in the development of age-related neurodegenerative diseases such as AD, PD, amyotrophic lateral sclerosis (ALS), and Freidriech ataxia (FRDA) [100]. This is mainly associated with DNA defects in the mitochondria, abnormal mitochondrial enzyme activity, and abnormal expression of mitochondrial genes. These abnormalities in mitochondrial function leading to the development of neurodegenerative diseases have been studied in many animal studies and in post-mortem autopsies of patients [101][102][103]. Worth noting, is that not all neuronal cells are susceptible to mitochondrial dysfunction. In Leber's hereditary optic neuropathy, a classical mitochondrial disease, only RGCs are selected to die while other neuronal population remains unaffected. Retinal ganglion cells have a high energy requirement, which can be reflected by the high cytochrome c oxidase activity [104], they are highly susceptible Antioxidants 2021, 10, 671 7 of 13 to mitochondrial dysfunction. Therefore, mitochondrial dysfunction is likely to play a role in glaucoma [97][98][99].
The intracellular production of ATP relies heavily on the high efficiency of the mitochondrial electron transport chain (ETC). However, the nature of its electron exchange leads to its susceptibility to side effects, such as with molecular oxygen, which reduces the production of ATP [105,106]. H 2 S is a "double-edged sword" in the mitochondrial energy production process and has different effects on the ETC at different physiological concentrations. At low concentrations, H 2 S is mainly a substrate for ETC, providing electrons to ETC at the ubiquinone level [107,108]. It stimulates oxidative phosphorylation and promotes the production of ATP. However, at higher concentrations, the inhibitory effect of H 2 S on cyclooxygenase (COX)is predominated, thus reducing ATP production and decreasing ROS generation [107,108]. Furthermore, H 2 S also modulates the production of ATP under different physio pathological conditions, thereby reducing ROS production by ETC and protecting cells from oxidative stress. H 2 S reduces ATP production by upregulating coupling protein (UCP)-2 and down-regulating protein expression of COX I and II subunits [109,110].
In research of altered protein expression in the retina in a mouse model of acute IOP H 2 S was found to limit ROS production by inhibiting mitochondrial oxygen consumption and leading to increased intracellular oxygen tension, possibly through upregulating HIF1α levels and decreasing mitochondrial oxidative phosphorylation levels [76]. This research also found that H 2 S promotes the production of ketone bodies, which can replace glucose as an energy source and enhance retinal resistance to oxidative stress, thereby reducing neuronal damage [76].
In addition, mutations in mitochondrial DNA (mtDNA) also lead to alterations in ETC [99]. Since mtDNA lacks protective proteins against oxygen radicals, it is susceptible to ROS and leads to mutations [111,112]. However, AP39, an H 2 S donor, is proven to prevent the destruction of mtDNA by glucose oxidase and plays an antioxidant and cytoprotective role [113]. It has also been shown that H 2 S maintains the transcriptional process of mtDNA by controlling the mitochondrial transcription factor A (TFAM) [114].

Effect on Glutathione Production Pathway
Glutathione is a tripeptide composed of glutamic acid, cysteine, and glycine. It is also one of the most common antioxidants in the human body. The sulfhydryl group on cysteine is the active group of glutathione, which is readily combined with free radicals induced through oxidative stress and has anti-neurotoxic effects, thus protecting neurons from oxidative damage [115][116][117]. It is shown that knockout of the gene for an enzyme affecting glutathione synthesis produced spontaneous apoptosis of RGC and damage to the optic nerve in mice without elevated IOP [118]. This demonstrates that glutathione has a pivotal role in the mechanism of RGC loss independent elevated IOP.
Glutathione synthesis is strongly influenced by extracellular glutamate. Cystine is the main source of cysteine (required for intracellular glutathione synthesis) and since glutamate and cystine share the same amino acid transport protein, high concentrations of extracellular glutamate compete with cystine to prevent intracellular glutathione synthesis [31,119,120]. However, H 2 S effectively prevents this competition, thus restoring normal cystine transport and promoting intracellular glutathione synthesis [31] Moreover, γ-glutamylcysteine synthase (γ-GCS) and γ-glutamylcysteine (γ-GC) are key enzymes in the synthesis of glutathione, and their activities directly affect the intracellular concentration of glutathione. Kimura et al. documented a two-fold increase in γ-GC levels in isolated rat neuronal cells after culturing with additional H 2 S donors for 2 h. Whereas in the control group, intracellular γ-GC showed a decreasing trend over time [31]. Promoting the intracellular levels of enzymes involved in glutathione synthesis might be part of the mechanism of how H 2 S increases intracellular glutathione.
At the same time, the gene expression of γ-GCS and GSH synthase can also be regulated by the nuclear factor erythroid 2-related factor 2 (Nrf2) [121]. It is reported that H 2 S donor promotes the synthesis of GSH through an Nrf2-dependent pathway [122]. In summary, H 2 S and its donors directly or indirectly promote the synthesis of glutathione in cells, thus achieving the effect of preventing neurodegenerative changes (see Figure 3). ntioxidants 2021, 10, x FOR PEER REVIEW 8 At the same time, the gene expression of γ-GCS and GSH synthase can also be r lated by the nuclear factor erythroid 2-related factor 2 (Nrf2) [121]. It is reported tha donor promotes the synthesis of GSH through an Nrf2-dependent pathway [122]. In mary, H2S and its donors directly or indirectly promote the synthesis of glutathio cells, thus achieving the effect of preventing neurodegenerative changes (see Figure   Figure 3. H2S promotes the synthesis of glutathione in cells through three pathways. (1) H2S s presses the extracellular competition between glutamate and cystine, therefore promotes cyst (the substrate of glutathione (GSH) synthesis) entering the cells. (2) H2S promotes the produc of a variety of glutathione synthase and increases the concentration of GSH in the cell. (3) H2S directly promotes the synthesis of GSH through the Nrf2-dependent pathway.

Conclusions
H2S is undoubtedly an attractive candidate of potential treatment in glaucoma shown in glaucoma animal models both in vitro and in vivo, H2S reduces intraocula dative stress and prevents neurodegeneration. However, the key roadblock to tra tional research in field of glaucoma is insufficient understanding of its pathophysio Therefore, there is a lack of good models. Using different glaucoma models goes way to reflect therapeutic potential of H2S in glaucoma. It has to be kept in mind tha results obtained from animal studies may not be fully translatable in human patients pharmacology of H2S in glaucoma remains to be further investigated.

Conclusions
H 2 S is undoubtedly an attractive candidate of potential treatment in glaucoma. It is shown in glaucoma animal models both in vitro and in vivo, H 2 S reduces intraocular oxidative stress and prevents neurodegeneration. However, the key roadblock to translational research in field of glaucoma is insufficient understanding of its pathophysiology. Therefore, there is a lack of good models. Using different glaucoma models goes some way to reflect therapeutic potential of H 2 S in glaucoma. It has to be kept in mind that the results obtained from animal studies may not be fully translatable in human patients. The pharmacology of H 2 S in glaucoma remains to be further investigated.