Hydrogen Sulphide-Based Therapeutics for Neurological Conditions: Perspectives and Challenges

Central nervous system (CNS)-related conditions are currently the leading cause of disability worldwide, posing a significant burden to health systems, individuals and their families. Although the molecular mechanisms implicated in these disorders may be varied, neurological conditions have been increasingly associated with inflammation and/or impaired oxidative response leading to further neural cell damages. Therefore, therapeutic approaches targeting these defective molecular mechanisms have been vastly explored. Hydrogen sulphide (H2S) has emerged as a modulator of both inflammation and oxidative stress with a neuroprotective role, therefore, has gained interest in the treatment of neurological disorders. H2S, produced by endogenous sources, is maintained at low levels in the CNS. However, defects in the biosynthetic and catabolic routes for H2S metabolism have been identified in CNS-related disorders. Approaches to restore H2S availability using H2S-donating compounds have been recently explored in many models of neurological conditions. Nonetheless, we still need to elucidate the potential for these compounds not only to ameliorate defective biological routes, but also to better comprehend the implications on H2S delivery, dosage regimes and feasibility to successfully target CNS tissues. Here, we highlight the molecular mechanisms of H2S-dependent restoration of neurological functions in different models of CNS disease whilst summarising current administration approaches for these H2S-based compounds. We also address existing barriers in H2S donor delivery by showcasing current advances in mediating these constrains through novel biomaterial-based carriers for H2S donors.


Introduction
Neurological disorders may result from insult to the brain, spinal cord or peripheral nerves as well as congenital defects, degeneration or structural defects. These impairments are increasingly recognised as major burdens to the affected patient, families and health systems as they are listed as a major cause of disability and second leading cause of death [1]. Neurological disorders, especially those with a degenerative nature, including Alzheimer's disease (AD), Parkinson's disease (PD) and Huntington's disease (HD), are incurable, debilitating conditions resulting in progressive damage of nerve cells. In 2006, the Global Burden of Disease (WHO) estimated that combined, the prevalence of PD, AD and other dementias was estimated to reach 5.25 (per 1000 individuals) in 2015, however, these figures are projected to increase to 6.47 (per 1000) by 2030 [2] with all neurological-related cases predicted to triple in the coming decades to 152 million worldwide [1]. Therefore, improving therapeutic management options is of critical importance.
Hydrogen sulphide (H 2 S) is an endogenous gas which, along with nitric oxide and carbon monoxide, belongs to a family of transmitters known as gaseous transmitters [3]. H 2 S has increasingly gained recognition as a protective agent. Studies have shown that H 2 S is involved in a variety of physiological processes in the body, able to exert cytoprotective effects owing to its antioxidant properties and ability to modulate oxygen consumption [4]. However, H 2 S was once considered a toxic molecule. It has been demonstrated that H 2 S has a biphasic effect; low concentrations are beneficial, allowing enhanced mitochondrial respiration whereas higher concentrations inhibit the respiratory chain at level of mitochondrial complex IV, resulting in cytotoxicity [5].
In neurodegenerative conditions, the use of H 2 S-releasing compounds have demonstrated neuroprotective roles [6][7][8] when administered at physiological levels (approximately 20 to 300 µM) [9]. Although many molecular mechanisms have been described, our understanding of the potential for H 2 S-based compounds in the treatment or prevention of neurological disorders is not yet clear. Many of the challenges yet to be addressed include the rapid release of H 2 S and fast metabolism in vivo, the ability of H 2 S-related compounds to cross the blood brain barrier (BBB) and the optimisation of laboratory techniques to measure H 2 S levels in tissues or cells.
This literature review aims to highlight current evidence demonstrating the role of H 2 S in the central nervous system (CNS) whilst exploring the neuroprotective potential of H 2 S-donating compounds in different models of neurological disease, from in vitro to in vivo settings. Additionally, this review will explore challenges in H 2 S delivery to the nervous system and showcase potential approaches to mitigate these challenges.

H 2 S in the Central Nervous System
Synthesis of H 2 S H 2 S is produced enzymatically by the action of three enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate (3-MST) [10]. CBS catalyses a β-replacement reaction between homocysteine and cysteine, producing cystathionine and H 2 S [11]. While CSE employs a similar method, it can also catalyse a reaction that converts cystathionine into cysteine, which can then be used in further reactions to produce H 2 S [12]. Furthermore, 3-MST is a mitochondrial enzyme that produces H 2 S by transferring sulphur from 3-meracaptopyruvate (produced by cysteine aminotransferase) to sulphurous acid. This produces thiosulfate as one of the products, which is reduced into H 2 S [13].
In the CNS, CBS and 3-MST are the main enzymes involved in H 2 S production, with 3-MST being responsible for approximately 90% of the H 2 S produced in the brain [14]. While 3-MST is present mainly in neurons, CBS is present within astrocytes and microglia, suggesting that each enzyme may have a distinct role to promote H 2 S signalling [15]. Given that 3-MST is a located within the mitochondrial matrix [16], it highlights the potential role of mitochondrial H 2 S in regulating degenerative CNS processes whilst CBS has been found accountable for defective neurological functions such as neurogenesis, synaptogenesis and controlling BBB permeability [17]. Interestingly, CSE is hardly detected in brain tissue. Very low levels of CSE mRNA have been detected in the brain whilst CSE inhibitors have shown no significant suppression of H 2 S production [18], suggesting that CSE does not play a key role as a H 2 S producing enzyme in the brain (Fig. 1).
The importance of both CBS in generating H 2 S in brain cells (mainly astrocytes and microglia) suggest that their patterns of expression and/or activity may have an impact in the development of brain-related disorders. In neurodegenerative Fig. 1 Synthesis of H 2 S in CNS-related cells. Cytoplasmatic cystathionine-β-synthase (CBS) expressed in astrocytes and microglia, and mitochondrial 3-mercaptopyruvate (3-MST) expressed in neurons, are mainly involved in the endogenous generation of H 2 S. CBS catalyse the reaction between homocysteine and cysteine, producing cystathionine and H 2 S whereas 3-MST produces H 2 S by transferring sulphur from 3-meracaptopyruvate (3-MP) (produced by cysteine aminotransferase, CAT) to sulphurous acid disorders such as PD and AD, hyperhomocysteinemia has been regarded as risk factor for the development of these conditions. Although accumulation of amino acid homocysteine leading to hyperhomocysteinemia may occur due to defects in different enzymes, evidence shows that CBS 844ins68 mutation and VNTR polymorphisms of the CBS gene are independent risk factors for AD development in subjects aged 75 years or more [19]. Moreover, recent evidence by Bjørke-Monsen, et al. 2022 showed that severe hyperhomocysteinemia in a patient with PD associated with reduced availability of CBS cofactor, pyridoxal 5-phosphate (PLP) [20]. These observations suggest that deficient CBS signalling, due to deficient availability of cofactor PLP and polymorphisms of CBS may be considered risk factors for the development of neurodegenerative diseases.
There are also non-enzymatic routes for H 2 S synthesis. These routes are usually coupled to reducing reactions such as glycolysis, which produces glucose and reducing equivalents such as nicotinamide adenine dinucleotide. These reducing molecules can then participate in reducing numerous sulphur compounds into H 2 S [21,22].

Catabolism of H 2 S
H 2 S is mainly metabolised through oxidation reactions in the mitochondria known as the "sulphur oxidation unit" pathway [23]. A major enzyme in this pathway is sulphide quinone oxidoreductase (SQR), which oxidizes H 2 S to persulfide. SQR is found in many organs such as the liver and colon. SQR protein and mRNA are expressed in mouse primary microglia and astrocytes but not in primary neurons [24]. The patterns of SQR expression shows lower levels of SQR mRNA and protein in the brain when compared to other tissues, suggesting that SQR may have limited contribution to the breakdown of H 2 S in the brain [22,25].
Nevertheless, due to the remarkable importance of SQR in the metabolism of H 2 S in other tissues, researchers have investigated the presence of homolog forms of SQR in neuronal tissue. Ackermann et al. demonstrated the presence of a SQR homolog known as SQRDL (sulphur quinone oxidoreductase-like protein), which metabolises sulphur in rat brain [26]. This study also revealed that the SQRDL mRNA increased in rats with age, which may result in excess H 2 S degradation, contributing to age-related neurodegenerative disorders [26]. More research is required to better understand the existence of other relevant SQR variants that may play a significant role in the degradation of H 2 S.
Other routes of H 2 S metabolism suggest the neuroglobin may have a role in H 2 S oxidation in the brain. Neuroglobin is a member of the haemoprotein family which uses its Fe 2+ ion to bind to oxygen [27]. Neuroglobin binds to H 2 S and oxidises it to thiosulfate, however it has been suggested that this reaction is less efficient, compared to its haemoglobin and myoglobin counterparts [28]. In addition, as neuroglobin expression decreases with age in rodents, it has been suggested that it may play a role in age-related neurodegeneration [29] (Fig. 2). These association is still not yet well explored.
Altogether, the interplay between SQR and its homolog SQRDL, along with neuroglobin in the metabolism of H 2 S in the brain is not yet well established. Because SQR mRNA and protein expression is low in the brain, pathways involving neuroglobin and/or alternative SQR variants, warrant further investigation to better understand their role in sustaining H 2 S oxidation in the brain.

Neuroprotective Mechanisms of H 2 S
New avenues of research have resulted in the development and refinement of several H 2 S donor compounds. These donors have been useful to elucidate key molecular mechanisms mediated by H 2 S in the context of physiological responses. Moreover, they have also allowed the exploration of their potential to modulate impaired responses and act as potential therapeutics in a myriad of human conditions. Table 1 summarises identified molecular mechanisms of H 2 S in diverse models of neurological conditions.

H 2 S as ROS Scavenger
H 2 S is involved in a variety of physiological processes in the nervous system [10]. Kimura et al. demonstrated the protective effect of H 2 S in the nervous system, showing H 2 S is able to protect neuronal cells against oxidative stress (imbalance between oxidative molecules production and antioxidant mechanisms) by enhancing the synthesis of the ) from the reaction of H 2 S with ferrous neuroglobin (Fe III -Ngb) 2 antioxidant substrate glutathione (GSH) which functions as a storage for cysteine [8]. Importantly, GSH is maintained at high levels in astrocytes [8], where also CBS and SQR have been identified. In environments mimicking oxidative stress, such as in the presence of hydrogen peroxide (H 2 O 2 ), H 2 S has also demonstrated the ability to reduce oxidative damage in mouse brain neuroblastoma cells Neuro2a, by enhancing GSH levels reduced by H 2 O 2 [30]. It is possible that the regulation of sulphur/GSH metabolism in astrocytes is a key regulatory mechanism for the protection against oxidative molecules in the CNS. In addition to the enhanced production of GSH resulting from H 2 S, other mechanisms involving the direct scavenging of reactive oxygen species (ROS) by H 2 S have been reported. In this regard, H 2 S donor NaHS, has been identified as a direct scavenger of peroxynitrate, a reactive molecule derived from nitrogen in neuroblastoma cells SHSY-5Y [31].

Anti-apoptotic Role of H 2 S
Apoptosis, or programmed cell death, may occur through the intrinsic (mediated by the mitochondria) or extrinsic (associated with death receptor) pathways [32]. H 2 S has been shown to exert anti-apoptotic effects through the intrinsic pathway linked to its ROS neutralising properties, thus preventing oxidative stress to neuronal cells [33]. H 2 S can exert its anti-apoptotic effect via modulating nuclear translocation of nuclear factor kappa B (NF-κB) which is a transcription factor that can activate anti-apoptotic genes [34]. Additionally, the administration of NaHS has been observed to abrogate the generation of H 2 O 2 , reducing hippocampal neuronal apoptosis in mouse [35]. Studies by Shan et al., showed pre-treatment with H 2 S donors in a model of intracerebral haemorrhage in mice, significantly reduced caspase 3 and Bcl-2 (regulators of apoptosis) [36]. The molecular mechanisms linking anti-apoptotic function and ROS neutralising effects might work in parallel in the presence of H 2 S and this evidence suggest that H 2 S therapies may be effective to ameliorate a broad spectrum of pathophysiological mechanisms underpinning neurological conditions.

Role of H 2 S in Neuroinflammation
H 2 S has been shown to play a vital role in preventing neuroinflammation. Damage to neuronal cells can result in neuroinflammation by activation of pro-inflammatory cytokines that can aggravate the inflammation leading to neuronal death [37]. Several reports have demonstrated that H 2 S is able to prevent neuroinflammation by modulating the inflammatory response. In this regard, NaHS, has been observed to modulate inflammation by inhibiting the release of tumour necrosis factor-α (TNF-α) and reducing the expression of toll-like receptor 4 (TLR4), and NF-κB in the hippocampus of Sprague-Dawley rats subject to subarachnoid haemorrhage [38]. Furthermore, in rat models of AD established by injections of Amyloid-β1-40 into the hippocampus, H 2 S significantly reduced the release of inflammatory cytokines interleukin IL-1β and TNF-α whilst ameliorating spatial learning and memory impairment [39]. These observations suggest an interesting link between neuroinflammation and cognitive impairments that might be ameliorated by H 2 S.

Parkinson's Disease (PD)
PD is one of the most prevalent neurodegenerative disorders worldwide. Motor symptoms of Parkinson's include bradykinesia, resting tremor, and cogwheel rigidity whereas non-motor symptoms include sleep disturbance, cognitive decline, and depression [67]. The pathophysiology of PD involves the progressive destruction of the dopaminergic neurons in the nigrostriatal pathway in the midbrain [68].
Accumulation of ROS can damage the DNA leading to oxidative stress and cell death are associated with PD [69]. Several models of PD have been proposed in order to better understand this disorder. Examples include the use of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite 1-methyl-4-phenylpyridinium (MPP+). MPP + plays an active role in destroying the dopaminergic neurons that results in parkinsonism [70]. An in vitro study used the human neuroblastoma cells (SH-SY5Y) to investigate the role of H 2 S in attenuating the oxidative stress on the neurons in PD cell model [40]. This study used an MPP + -treated SH-SY5Y cell model showing that NaHS increased the cell viability, reduced cytotoxicity, and reduced oxidative stress-induced cell apoptosis in a dose dependent manner in the MPP + treated cells [40].
Another study investigated the neuroprotective role of H 2 S in a mouse model of PD. In line with the study carried out by Liu et al. [40], this study also showed that H 2 S elicited neuroprotective properties. MPTP was used in this study to generate a mouse model of PD and NaHS was used to investigate the effect of H 2 S. This study demonstrated that H 2 S prevents MPTP-induced neuronal damage and promotes neurogenesis through Akt/glycogen synthase kinase-3β (GSK-3β)/β-catenin pathways in adult neuronal stem cells [42]. Consistently, in a similar model of MPTP-induced PD in mice established by Lu et al., it was observed that NaHS reduced primary mesencephalic neurons cytotoxicity whilst explored molecular mechanism associated highlighting that H 2 S was able to enhance mitochondrial uncoupling protein 2 (UCP2) antioxidation resulting in abrogated ROS generation and reduced caspase 12-induced apoptosis [43]. Mitochondria are the main site of ROS generation thus may play a key role in the development of PD whilst compounds modulating mitochondrial-ROS, such as H 2 S, may be potential therapeutic candidates in the treatment of PD.
Although the molecular mechanisms of H 2 S-induced neuroprotection in PD may be broad (Fig. 3), these new avenues in PD research position H 2 S as a potential mitochondrial protectant. Thus, H 2 S may be a suitable therapeutic candidate for clinical use in treating Parkinson's in the future.

Alzheimer's Disease (AD)
AD is the most common neurodegenerative disorder and is the leading cause of dementia in the elderly population. Oxidative stress, neuroinflammation and damage to cholinergic neurons are some of the pathophysiological changes that are involved in AD [71].Furthermore, the accumulation of the microtubule-associated protein Tau [72] and β-amyloid peptides in neuronal cells [73] have been proposed as key molecular mechanism leading to this devastating disorder.
A research study investigated the neuroprotective role of H 2 S in an animal model of AD by using the 3xTg-AD mouse model which includes Tau protein mutations: PS1M146V, APPSwe, and Tau P301L [46]. This study showed that H 2 S prevents hyperphosphorylation of the Tau protein by S-sulfhydration (post-translational modification of proteins mediated by H 2 S) of GSK3β. Additionally, it was observed that the CSE catalytic effect was augmented by binding wild type Tau which resulted in decreased levels of Tau protein in the cell. In contrast, CSE could not bind the mutated Tau P301L. Consequently, CSE levels were decreased in the cortex and hippocampus of the brain in the 3xTg-AD mouse model compared to wild type. This interesting report demonstrated a direct relationship between decreased endogenous levels of H 2 S and AD-like condition in vivo. Interestingly, it was demonstrated that administrating H 2 S donor, GYY4137, by daily intraperitoneal injection for 12 weeks (100 mg/kg) to 3xTg-AD mice improved their motor and cognitive function. The level of Tau protein S-sulfhydration was higher in the group treated with GYY4137 compared to the control group [46].
Aligned with the study carried out by Giovinazzo et al. [46], a study by Vandini et al. [47] using 3xTg-AD transgenic mouse model, previously showed that intraperitoneal injection of sulphur water and NaHS daily for three consecutive months improved memory and cognitive functions in both young and aged animals Additionally, treatment with NaHS and sulphur water in 3xTg-AD mouse decreased the size of the β-amyloid plaques in the cortex and hippocampus. The molecular mechanisms implicated suggest the inhibition of c-Jun N-terminal kinases, extracellular signal-regulated kinases, and p38 protect against neuroinflammation and Tau protein hyperphosphorylation, leading to a decrease in accumulation of β-amyloid plaques in the cortical and hippocampal regions of the brain [47].
Moreover, a recently synthesised H 2 S donor targeted to the mitochondria, AP39, was probed against a model of AD in mice (APP/PS1 double-transgenic mice) [49]. AP39 injected intraperitoneally for 6 months enhanced cellular bioenergetic function and had mitochondrial protective effect on AD neurons and mice. Neurons treated with AP39 showed reduced ROS levels and increased ATP production. Within the AD neurons, it was observed that mitochondrial DNA was damaged, however AP39 treatment prevented these alterations by increasing mitochondrial DNA integrity. Furthermore, APP/PS1 double-transgenic mice treated with AP39 observed an improvement in their learning and memory impairments [49] suggesting that H 2 S, targeted to the mitochondria, could be a potential therapeutic candidate for AD. Given the fact that accumulation of Tau protein disturbs the neuronal mitochondrial respiration, molecular mechanism implied may include the restoration of the flow of electrons in the oxidative phosphorylation and/or antioxidant effects abrogating neurotoxicity induced by Tau, however, these theories warrant further exploration. Overall, H 2 S has proven to play a significant neuroprotective role in AD models (Fig. 4). However, the translation of these results to the clinical setting might be hindered by complex dosage regimen of the H 2 S donor, requiring multiple and extensive dose regimes (3 to 6 months) as observed in these studies [46,47,49]. This emphasises the necessity to further explore patient-friendly approaches including better delivery systems and/or more stable H 2 S donors to reduce frequency of administration.

Huntington's Disease (HD)
HD is an autosomal dominant progressive neurodegenerative disorder resulting from the toxic accumulation of mutant huntingtin protein in neurons [74]. The pathophysiology of HD involves the selective destruction of the corpus striatum in the brain which controls motor activities leading to involuntary and irregular muscle movements, behavioural changes and cognitive decline [75].
Paul et al. [76] explored the neuroprotective role of H 2 S in a mouse model of HD showing a direct correlation between reduction in CSE levels and increased neurodegeneration in the striatum. This study used both in vivo and in vitro models to demonstrate the impact of CSE depletion in the pathophysiology of HD. CSE depleted (CSE −/− ) mice model exhibited abnormal hindlimb clasping and clenching resembling HD. Additionally, this study demonstrated that CSE level was significantly decreased in the striatal cell line of HD cell model consisting of 111 glutamine repeats (STHdhQ 111/Q111 (Q111) compared to the control group [76]. These observations suggest the importance of H 2 S in HD and imply the potential application of H 2 S donors in halting the progression of the disorder.
In an attempt to explore the potential for NaHS in abrogating HD-like effects induced by 3-nitropropionic acid (3NP), a study in a rat HD model revealed that H 2 S improved overall cognitive and locomotor deficits whist provided antioxidant, anti-inflammatory and anti-apoptotic effects observed by reduced levels of oxidative stress marker malondialdehyde (MDA), TNF-α and caspase 3 activation [51]. Interestingly, this study also revealed that treatment with NaHS significantly enhanced CBS expression, suggesting that H 2 S availability may have a crucial effect in modulating endogenous H 2 S pathways. Nonetheless, these assumptions require further exploration to understand the potential molecular mechanisms implicated.

Cerebral Vascular Disease
Stroke is the second most prevalent cause of mortality worldwide. The two types of strokes include haemorrhagic and ischemic stroke [77]. Both lead to ischemia and consequent tissue damage/death.
Given the association between ischemic stroke and ROS, H 2 S as an antioxidant compound, may be a suitable candidate for management of these conditions. A study investigated the neuroprotective role of H 2 S in acute ischemic stroke using a model of cerebral ischemia/reperfusion injury in mice by administrating NaHS before reperfusion following an ischemic insult in the brain. By means of H-magnetic resonance spectroscopy/magnetic resonance spectroscopy (H-MRI/MRS) and immunohistochemistry, it was shown that the total infarct volume was reduced compared to sham, and administration of NaHS 1 min before perfusion exerted stronger effect that of 30 min [52]. Another study exploring the effect of NaHS on transient cerebral ischemia immediately after reperfusion, this, combined with mild Fig. 4 Protective mechanisms of H 2 S in AD. The neuroprotective effects of H 2 S in AD are associated with inhibition of anti-inflammatory cascade via reduction of TNF-β and IL-6 levels. Moreover, H 2 S has been linked to reduction of size of β-amyloid plaques and reduction in hyperphosphorylation of Tau. H 2 S has been observed to reduce levels of mitochondrial reactive oxygen species (ROS) leading to inhibition of apoptosis via caspase 3. H 2 S may also maintain the integrity of mitochondrial DNA whilst it may also regulate GSK3β protein functions via S-sulfhydration (persulfidation) hypothermia resulted in a decrease in ischemic-reperfusion injury via upregulation of N-methyl-D-aspartate receptor (NMDAR) [53].
To study mechanisms of haemorrhagic stroke in vivo, models such as the middle cerebral artery occlusion (MCAO) plus intravenous injection of tissue plasminogen activators (tPA), have been used [78]. Using this model, it was demonstrated that H 2 S reduces tPA-induced cerebral haemorrhage following MCAO. In this study, co-administration of two structurally distinct H 2 S donors, ADT-OH and NaHS reduced the tPA-induced cerebral haemorrhage through the inhibition of AKT-VEGF-MMP9 signalling cascade [55]. As one of the risk factors associated with tPA in the management of ischemic stroke, is the increased risk of cerebral haemorrhage, observations by Liu et al. [55] suggest that H 2 S donors may reduce the risk of haemorrhagic stroke in tPA-exposed subjects. Although more research is necessary to confirm this hypothesis, the co-administration of H 2 S and tPA may bring new avenues and may provide patients requiring treatment for ischemic stroke a better recovery profile.

Brain Tumours
Brain tumours carry a high morbidity and mortality rate which highlights their clinical significance and burden to health systems worldwide. The most malignant brain tumour is glioblastoma multiform (GBM) which can rapidly spread and invade brain parenchyma [79]. Despite the growing number of treatment options available for the patients, brain malignancies carry a poor prognosis [80].
An in vitro study investigated the anti-cancer properties of H 2 S in C6 glioma cells [58]. It was shown that NaHS induces C6 glioma cell apoptosis via upregulation of caspase 3 and Bax proteins and downregulation of Bcl-2 protein through p38 MAPK signalling pathway [58]. In contrast to this report, another in vitro study demonstrated that H 2 S promotes C6 glioma cell growth. However, when CBS activity was compromised, the enhanced cell proliferation was blunted [60]. This study showed that cell apoptosis was reduced resulting in increased proliferation and viability rates of C6 glioma cells when exposed to NaHS suggesting that p38 MAPK/ERK1/2-COX-2 pathways are involved in NaHS-induced cancer cell proliferation and anti-apoptosis in C6 glioma cells [60]. An in vivo study reported by Li et al. showed that the size of the tumour was significantly increased in the rats exposed to NaHS in GBM group compared to the GBM only group, and H 2 S promotes C6 glioma cell growth via augmenting neurovascular formation and increasing hypoxia-inducible factor-1alpha (HIF-1α) expression [61]. These contrasting results more investigation is required to better understand the role of H 2 S in promoting/ mitigating the expansion of brain tumours.

Epilepsy
Epilepsy is one of the most common neurological disorders affecting around 50 million of the global population. This condition is characterised by recurrent seizures which is the clinical manifestation of alerted neuronal electrical activities [81]. Additionally, despite antiepileptic drugs offering many patients symptomatic relief, effective management is not achieved in almost one third of patients with current pharmacotherapies. Hence, new medical therapies for epilepsy are needed [82].
Pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α play an important role in the pathophysiology of the epileptic seizures and the level of these cytokines is raised in the serum and cerebrospinal fluid of epileptic patients [83]. Increased level of pro-inflammatory cytokines results in increased excitability of neurons which may be crucial in the pathophysiology epilepsy [84]. As H 2 S promotes the release of anti-inflammatory cytokines and is involved in preventing neuroinflammation [85], it might have a potential clinical application in the treatment of epilepsy.
The role of H 2 S in pilocarpine-induced status epilepticus (SE) in vivo has been explored. Adult male C57BL/6 mice were exposed to pilocarpine to induce SE whilst treatment consisted of exposure to a novel carbazolebased H 2 S donor. This study demonstrated that SE + H 2 S group had a shorter duration of seizure compared to the SE group. Additionally, this group showed delayed onset of seizure, reduced damage to the hippocampus and reduced pro-inflammatory state in microglia compared to the SE control group [62].
In contrast, a study performed by Luo et al. [63] showed that H 2 S delivered as NaHS exacerbates Pentylenetetrazole (PTZ)-and pilocarpine-induced seizures in rats [63]. PTZ is a GABA-A receptor antagonist that when sequentially injected to animals, results in the development of chemical kindling, an epilepsy model [86]. Luo et al. [63] exposed rats to PTZ/pilocarpine and PTZ/pilocarpine + NaHS showing that the severity of seizures increased in the group treated with NaHS compared to the control group. Furthermore, the duration of seizure was longer in those rats exposed NaHS suggesting that H 2 S increased membrane excitability of entorhinal cortex neurones via facilitating the function of voltage-gated sodium channel, AMPAR, and NMDAR [63]. These observations are not consistent about the effect of exogenous H 2 S on the manifestation of epilepsy. However, they suggest a potential role of H 2 S signalling in the modulation and severity of epilepsy. Further research is warranted to better understand the potential of the modulation of H 2 S signalling in the management of epilepsy.

Multiple Sclerosis (MS)
MS is an autoimmune disease affecting the myelin structure in the CNS. Currently, 2.8 million people are affected worldwide, however, this number is on the rise [87]. MS results in a wide range of signs and symptoms that are variable depending on the severity and location of the lesions with optic neuritis being the most common [88]. Currently, the available immune-based therapies do not significantly improve MS prognosis, therefore, new treatment strategies, such as H 2 S donors, warrant exploration in the pursuit of better patient outcomes.
In physiological settings, the BBB protects the CNS by making it inaccessible to immune cells, while in MS, the BBB gets damaged due to endothelial cells' impairment [89]. This results in an exacerbated influx of inflammatory mediated cells into the brain tissue, which induces demyelination and axonal dysfunction. Recurrent inflammatory responses in MS irreversibly damage the CNS nerve cells with the involvement of ROS [90]. The pathophysiology of MS also involves chronic platelet activation that promotes differentiation of autoreactive T cells resulting in initiation and progression of autoimmune neuroinflammation [91]. Therefore, two potential molecular mechanisms by which H 2 S may show potential beneficial effects in MS include its antioxidant role as well as its ability to inhibit platelet activation and adhesion molecule-mediated aggregation [92].
An in vitro study by Talaei et al. [65] examined the effect of NaHS on peripheral blood mononuclear cells (PBM-NCs) obtained from healthy individuals to access its transmigration across an endothelial cells' barrier representing the BBB. This study showed that pre-treatment of human endothelial cells with NaHS decreased PBMNC transmigration of both control and serotonin-treated cells. Additionally, treatment with NaHS resulted in upregulation of IL-10 and downregulation of adhesion molecules: lymphocyte function-associated antigen 1 (LFA-1) and vascular cell adhesion protein 1 (VCAM-1). This suggests that whilst NaHS resulted in relaxation/expansion of endothelial cells it may also cause morphological changes to the BBB leading to increased BBB permeability during MS attacks [65].
Lazarević et al. [66] studied the in vitro effect of H 2 S donor GYY4137 on differentiated dendritic cells and T cells which are both involved in the pathogenesis of MS. GYY4137 enhanced transforming growth factor β (TGF-β) expression in dendritic cells, suggesting an anti-inflammatory effect of GYY4137 in an MS-like environment. Additionally, isolated lymph node and spinal cord T cells were obtained from mice and rats immunised with CNS antigens and treated with GYY4137. GYY4137 significantly reduced interferon gamma (IFN-γ) and IL-17 production and reduced the proportion of FoxP3 + regulatory CD4 + T cells in the lymph node and spinal cord T cells. Interestingly, when the expression of H 2 S-producing enzymes (CBS, CSE, and 3-MST) was assessed in immune cells from healthy donors and drug-naïve relapse-remitting MS patients, the authors reported no differences in the CSE and CBS expression but lower expression of 3-MST [66].
Given the relevance of 3-MST in the generation and availability of H 2 S within the mitochondria, this study further suggests a potential link between H 2 S signalling and mitochondria in the modulation of anti-inflammatory response in MS. However, this theory awaits more exploration.

Challenges in H 2 S Delivery to the Nervous System
Several limitations associated with the gaseous nature and rapid release of H 2 S from its various donor molecules hinder the translation of H 2 S-based therapeutics to the clinic. As shown throughout this review, it appears that a systemic increase in H 2 S availability may improve neurological function in certain neurological disorders such as PD and AD.
In conditions where H 2 S may have a therapeutic effect, desirable H 2 S-based compounds should bear long-circulating times with slow-releasing patterns. As Table 2 shows, current research evidence displaying the potential therapeutic effect of H 2 S donors in CNS related conditions, usually require extensive dosage regimes, ranging from weeks to months with daily administration of the donor, in order to observe therapeutic effect. This is consistent with previous observations that bolus administrations of H 2 S-donating compounds may result in spiked concentrations of H 2 S [93], leading to complex administration regimes. Understanding the burden that neurological conditions put on patients, families and healthcare professionals, patientfriendly approaches are necessary. This suggest that strategies to reduce administration of the compounds is of key importance. In an attempt to overcome this issue, several compounds with H 2 S-releasing properties have been developed which differ in their patterns of H 2 S release, including mechanisms triggered by hydrolysis, enzymatic reactions, pH variation, novel formulation strategies amongst others [94].
Another important limitation arises from the fact that to reach the brain, therapeutic compounds require to be able to cross the BBB. The BBB is a structure separating the blood and brain compartments. Its inner layer is composed of endothelial cells with tight junctions and its outer layer consists of pericytes and astrocytes [95]. The BBB allows hydrophobic substances (e.g., water and oxygen) to access the brain whilst preventing pathogens and large and hydrophilic substances (e.g., microorganisms and drugs) from entering the brain. For this reason, most drugs targeting the CNS cannot enter the brain, bringing difficulties [ 103] in developing new efficient treatments for neurological diseases.
In terms of sulphide donating salts, ions diffuse passively across the BBB and their flow can be accelerated by partial association between anions and cations to form neutral ion-pair species in solution [96]. Thiosulfate does not permeate across cell membranes freely as it is an anion, therefore it must be transported by a transport mechanism to get into cells. The transport mechanism for thiosulfate is an SLC13 protein called sodium sulphate cotransporter 2 (SLC13A4, NaS-2), which is found within the brain. The transport mechanism helps the transportation of thiosulfate across the cell membrane [97], therefore, approaches targeting these transport mechanisms may offer a feasible method to enhance H 2 S delivery, when thiosulphate is employed. Therefore, along with developing targeted formulations, understating the chemistry of H 2 S-donating compounds is key in the successful delivery of these drugs to the CNS.

Perspectives: Overcoming Limitations in H 2 S Delivery
Thus far, H 2 S donors have not been specifically targeted to the brain even when indicated in neurological dysfunction. Drugs with molecular weight below 500 Da and high lipophilic content usually diffuse the BBB easily [104] thus forth, H 2 S donors targeting the brain must possess such qualities. Carrier-mediated transport and receptor-mediated endocytosis/transcytosis have been explored and, more recently, biomaterial-mediated strategies have been envisaged, including polymeric-based systems, already approved by the Food and Drug Administration Agency. These polymeric systems include polylactide, polycaprolactum and polyglycolide that can be employed as hydrogels and nanoparticle formulations. Another approach has been the use of liposomes. These structures, due to their similarity with the lipid bilayer in cellular membranes along with CNS targeting potential offers an interesting approach when exploring CNS drug delivery. In particular, approaches embedding antibodies within liposome structure, also referred as immunoliposomes, have been explored in the treatment of AD [105].
Nanoparticle formulations based on polymer or liposomal structures may also assist drug movement across the BBB when CNS-related drugs cannot cross the BBB on their own. In other scenarios of human disease, preclinical studies of nanoparticle delivery of drugs have reported that nanoparticles could not only increase the compounds half-life time but also decrease the amount of drug being eliminated by the reticuloendothelial system, thus, improving stability and increasing drug exposure [106,107]. Recently, in our laboratory, we have explored nanoparticle approaches to encapsulate H 2 S donors, protecting from rapid degradation and release of H 2 S whilst exploring different routes of administration aiming to reduce administration regimes [108][109][110]. Whilst observations must be confirmed in an in vivo setting, it was noted that liposomes employed to carry ADT-OH across murine skin observed a delayed flux across the skin compared to formulations without liposomes suggesting liposomes may be beneficial in giving a controlled release of drug [108]. Furthermore, cationic liposomes used in the controlled release of H 2 S from sodium thiosulphate were able to retain its biological properties in hypoxia-like environment