Transsulfuration pathway: a targeting neuromodulator in Parkinson ’ s disease

: The transsulfuration pathway (TSP) is a metabolic pathway involving sulfur transfer from homocysteine to cysteine. Transsulfuration pathway leads to many sulfur metabolites, principally glutathione, H 2 S, taurine, and cysteine. Key enzymes of the TSP, such as cys-tathionine β -synthase and cystathionine γ -lyase, are essential regulators at multiple levels in this pathway. TSP metabolites are implicated in many physiological processes in the central nervous system and other tissues. TSP is important in controlling sulfur balance and optimal cellular functions such as glutathione synthesis. Alterations in the TSP and related pathways (transmethylation and remethylation) are altered in several neurodegenerative diseases, including Parkinson ’ s disease, suggesting their participation in the pathophysiology and progression of these diseases. In Parkinson ’ s disease many cellular processes are comprised mainly those that regulate redox homeostasis, in ﬂ ammation, reticulum endoplasmic stress, mitochondrial function, oxidative stress, and sulfur content metabolites of TSP are involved in these damage processes. Current research on the transsulfuration pathway in Parkinson ’ s disease has primarily focused on the synthesis and function of certain metabolites, particularly glutathione. However, our understanding of the regulation of other metabolites of the transsulfuration pathway, as well as their relationships with other metabolites, and their synthesis regulation in Parkinson´s disease remain limited. Thus, this paper highlights the importance of studying the molecular dynamics in di ﬀ erent metabolites and enzymes that a ﬀ ect the trans-sulfuration in Parkinson ’ s disease.


Introduction
The transsulfuration pathway (TSP) is a process by which the Hcy produced by transmethylation is irreversibly converted to cystine (Cys). The enzyme cystathionine β-synthase (CBS) catalyzes a condensation reaction of the amino acids Hcy and serine to form cystathionine; then, cystathionine γ-lyase (CSE) catalyzes a hydrolyzation reaction to generate Cys and α-ketobutyrate (Paul 2021). Cys is a building block amino acid for protein synthesis and is related to multiple pathways to generate sulfur-containing molecules with neuroprotective properties such as hydrogen sulfide (H 2 S), glutathione (GSH), and taurine. Cys participates in GSH synthesis by the enzyme ɣ-glutamylcysteine synthetase (GCS). Or to taurine synthesis by the enzyme cysteine dioxygenase (CDO). On the other hand, the gasotransmitter (H 2 S) is generated by the action of the enzymes CSE, CBS, and 3-mercapto pyruvate sulfurtransferase (3-MPST) having as intermediaries: Hcy, cystathionine, and Cys (Hensley and Denton 2015;Paul 2021;Sbodio et al. 2019).
TSP is a crucial part of sulfur metabolism and cellular redox regulation and is linked to providing methyl groups essential for optimal cell function (Sbodio et al. 2019). In the human brain, all disturbances of this pathway are associated with disorders like homocystinuria, Huntington's disease, Alzheimer's disease, PD, and aging (Paul 2021;Sbodio et al. 2019). In this review, we focused on the status of different molecules of the TSP and related pathways, including remethylation and transmethylation in PD.

Parkinsons disease
Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuronal loss in the substantia nigra (SN) (Dickson 2018). Symptoms and signs of PD include rapid eye movement, sleep behavior disorder, and decreased smell in early pathology; motor symptoms, like bradykinesia, rigidity, rest tremor, and postural instability, become present in the late stages. Cognitive impairment and hallucinations can also be found in patients with advanced PD (Armstrong and Okun 2020). PD affects 1 % of the population above 60 years, and the peak incidence predominates between 70 and 79 years (Twelves et al. 2003;Tysnes and Storstein 2017). To this date, PD's cause is uncertain; however, there is a strong association between environmental and genetic factors (5-10 %) (Olanow and Tatton 1999). Mutations can occur in different genes, including the α-synuclein (SNCA) gene, Parkin, PTEN-induced putative kinase one gene loci (PINK1), and DJ-1. The remaining cases are idiopathic (Nuytemans et al. 2010;Repici and Giorgini 2019;Simon et al. 2020). However, exposure to chemical compounds such as pesticides, solvents, metals, and toxicants in the laboral environment or diet increases the risk of PD (Simon et al. 2020).
Compounds such as volatile organic compounds, manganese derivates, and iron can cross the blood-brain barrier (BBB) and enter the brain. Once inside, they can trigger various responses in the central nervous system, including the generation of oxidative stress and the promotion of a pro-inflammatory environment through the activation of glial cells (Ogbodo et al. 2022;Verina et al. 2013;Zheng and Monnot 2012). When these insults occur alongside impaired clearance of SNCA, they can sustain chronic microglial activation. This sustained activation leads to increased production and release of pro-inflammatory and pro-oxidative mediators such as reactive oxygen species (ROS), interleukin-1 beta (IL-1β), tumor necrosis factoralpha (TNF-α), and nitric oxide (NO). Thus, this sustained activation, in turn, contributes to the aggregation of SNCA aggregation and Lewy bodies formation (Li et al. 2010;Pathak and Sriram 2023). In PD, lewy bodies formation is associated with mitochondrial dysfunction (Mullin and Schapira 2013). These molecular events in the SN ultimately result in the loss of dopaminergic neurons (Henchcliffe and Beal 2008;Jankovic and Tan 2020;Olanow and Tatton 1999;Poewe et al. 2017).
All these dynamic processes and several repairing mechanisms could lead to pathophysiological processes and the irreversible cellular damage observed in PD (Jankovic and Tan 2020). PD can be approached pharmacologically and non-pharmacologically. Some of the pharmacological treatments include the dopamine precursor L-DOPA, complemented by catechol o-methyltransferase (COMT)-inhibitors extending the duration of L-DOPA effects; otherwise, the administration of MAO-B inhibitors (monoamine oxidase type B) are used to increase and extend synaptic dopamine concentrations (Poewe et al. 2017). Other pharmacological treatments include N-methyl-D-aspartate (NMDA) antagonists, serotonin re-uptake inhibitors, and tricyclic antidepressants. Non-pharmacological approaches (physical, speech, and occupational) and surgical treatments like deep brain stimulation are used in PD. Currently available therapy for PD helps many patients, though these drugs is linked to an increased risk of adverse events (Calne 1993). 3 Remethylathion, transmethylathion, and homocysteine in PD Remethylation and transmethylation are two pathways closely interconnected to the TSP (Sbodio et al. 2019). During transmethylation, dietary methionine is adenylated to S-adenosylmethionine (SAM) in a reaction catalyzed by S-adenosylmethionine synthase . SAM is a methyl donor to biomolecules like DNA, RNA, proteins, and monoamine neurotransmitters (Al Mutairi 2020). Demethylation of SAM by methyl transferases (MT) generates S-adenosylhomocysteine (SAH), which then is finally hydrolyzed by SAH hydrolase to form homocysteine (Hcy) and adenosine Hoffman et al. 1979). The Hcy can be re-methylated back to methionine via remethylation, where 5-methyltetrahydrofolate participates as a methyl donor to Hcy, yielding methionine and tetrahydrofolate in a reaction catalyzed by methionine synthase (MTR), using vitamin B 12 as a cofactor. In the human brain, the enzyme methylenetetrahydrofolate reductase (MTHFR) transforms 5-10-methylenehydrofolate into 5-methyltetrahydrofolate (Martignoni et al. 2007;Selhub 1999). Hcy, generated during transmethylation, is a sulfhydryl-containing amino acid derived from methionine (Al Mutairi 2020) that can be found in plasma, either free, reduced, or linked to proteins. The oxidized forms of Hcy are bound to amino acids such as Cys via disulfide linkages. Hcy, synthesized via the transmethylation pathway, can be either exported to the extracellular space, undergo a catabolic pathway, or even be remethylated or enter the TSP Stead et al. 2004).
Hcy homeostasis is disrupted by genetic defects, age, lifestyle, nutritional deficiencies, and demographics, among other factors. The augmented concentration in the plasma of Hcy is known as hyperhomocysteinemia (HHcy) (McCully 2015). The etiology of HHcy is diverse, including gene defects and nutritional deficiencies (Kayadibi et al. 2014). According to different authors, HHcy leads to different anomalies in the organism's functions, including in the central nervous system. For instance, many potential mechanisms are suggested for HHcy-induced brain damage, including oxidative stress, hypo-methylation of DNA and proteins, reticulum endoplasmic stress, cerebrovascular damage, neuroinflammation, and blood-brain barrier disruption. These are risk factors for neurovascular and neurodegenerative diseases like Alzheimer's and PD (Kamat et al. 2016;Longoni et al. 2018;Tawfik et al. 2021;Yoon et al. 2014).
HHcy is a significant risk for vascular disease, cognitive decline, and dementia in older people (Yap 2003). Increased ischemic heart disease and cerebrovascular disease ratios have been reported in treated PD patients (Ben-Shlomo and Marmot 1995;Kim et al. 2021). HHcy was found in approximately 30 % of PD patients, related to the onset, progression, and development of cognitive decline of PD (Allain et al. 1995;Fan et al. 2020;Kuhn et al. 1998;Licking et al. 2017;Muller et al. 1999). Likewise, different animal studies have demonstrated that intracerebroventricular administration of Hcy decreases locomotor activity in rats and reduces striatal dopamine levels (Lee et al. 2005). Vasculopathies linked to Hhcy in PD include endothelial dysfunction and arterial stiffness, promoting neurovascular disintegration such as blood-brain barrier (BBB) breakdown in rodents and humans (Beard et al. 2011;Yoon et al. 2014). In this context, Hcy has been proposed to increase BBB permeability via activation of NMDAr regulation of tight junctions (Beard et al. 2011). Extracellular Hcy excitotoxicity via NMDARs increases calcium inflow, causing ROS and nitric oxide (NO) production. The increase in ROS production promotes a proinflammatory environment through nuclear factor κB (NF-κB), leading to neuronal injury ( Figure 2) (Djuric et al. 2018;Mattson and Shea 2003). Experimental evidence demonstrates that HHcy sensitizes dopaminergic neurons to dysfunction and death by oxidative stress, mitochondrial dysfunction, and apoptosis in PD models and human neurons exposed to iron and rotenone (Duan et al. 2002). Hcy also promotes DNA damage preceding Hcy-induced oxidative stress and mitochondrial dysfunction. In cultured neurons treated with Hcy, ATP levels are depleted to repair Hcy-induced DNA damage (Kruman et al. 2000). The reduction of intracellular ATP levels is a crucial factor in the development of PD (Streck et al. 2003). Studies in humans and rats have supported L-DOPA treatments as the cause of elevated Hcy plasma levels (Miller et al. 2003;Yoon et al. 2014), The methylation of L-DOPA and dopamine to 3-omethyldopamine and 3-methoxytiramine respectively, depletes the SAM pool and of the available methyl groups, necessary for the remethylation of hcy to methionine, that leads to HHcy (Todorovic et al. 2006). In L-DOPA-treated PD patients, HHcy is reversed by supplementing folate and vitamin B 12 , which reduces Hcy concentrations (Lamberti et al. 2005a;Miller et al. 2003;Paul and Borah 2016). Vitamin B 12 diet supplementation can increase Hcy catabolism, lowering the risk for cerebrovascular diseases, cognitive impairment, and dementia (Boushey et al. 1995;He et al. 2004). Likewise, COMT-inhibitors (COMT-I) prevent the L-DOPA-associated HHcy (Muller and Muhlack 2009). The methylation of L-DOPA by COMT produces SAH, which is converted to Hcy by SAH hydrolase. The chronically increased synthesis of SAM and Hcy exceeds the capacity of cells to metabolize Hcy, leading to HHcy (Lamberti et al. 2005b;Zoccolella et al. 2005). Thus L-DOPA-induced HHcy could be controlled by administering COMT-I or supplementation with B-vitamins (Lamberti et al. 2005a;Zoccolella et al. 2005).
HHcy is related to DNA hypomethylation and is highly correlated to increased plasma levels of SAH (Yi et al. 2000). HHcy inhibits SAH's catalysis, enhancing SAH levels (Fan et al. 2020). SAH inhibits the methyltransferase, slowing down the methylation processes in the brain and leading neurons to apoptosis (Lin et al. 2008). Higher methylation potential (High SAM or low SAH levels) is related to better cognitive function in patients with PD; an inverse correlation between SAM and α-synuclein plasma levels has been described (Obeid et al. 2009). In this context, PD patients' brains or peripheral blood show reduced DNA methylation in the promoter region and intron 1 of α synuclein-encoding gene (SNCA), indicating that this hypomethylation could contribute to elevated SNCA expression as seen in PD patients' brains (Ai et al. 2014;Jowaed et al. 2010;Matsumoto et al. 2010). Also, within the cell, Hcy can be converted to Hcy thiolactone, triggering protein aggregation by incorporating Hcy residues into proteins, enhancing the probability of forming aggregates. Although it remains to be verified if Hcy could enhance α-synuclein aggregation as seen in PD (Jakubowski 2000). Table 1 shows all the associated metabolites to PD discussed in this paper.
Specific genetic polymorphisms in genes encoding the MTHFR and MTR enzymes of the remethylation pathway have been associated with neurodegenerative diseases, including PD (Diao et al. 2019;Fong et al. 2011;Lionaki et al. 2022). Studies have described the relationship between PD and different MTHFR genotypes. In the Chinese population, it has been reported that the TT genotype of the rs1801133 variant and the AT haplotype of rs18011131-rs1801133 in the MTHFR gene are protective factors for PD; as well as the genotype AA in the rs13306560 variant in Mexican population (Garcia et al. 2017;Yuan et al. 2016). Thus, it is hypothesized that the reduced activity (TT genotype) or concentration (AA genotype) of MTHFR might be neuroprotective in brain when methionine levels are high, generation of GSH is compromised, or by a disruptive TSP (Garcia et al. 2017). In contrast, the reduced activity of TT genotype rs1801133 in L-DOPA treated PD patients, combined with low folate ingestion, impairs the remethylation causing HHcy in PD patients (Yasui et al. 2000). Meanwhile, the CC of the rs1801133 genotype was associated with sporadic Parkinson'ss disease (SPD) in the Mexican and Italian populations; this genotype has the highest decrease of DNA methylation in comparison to the TT genotype in folate depletion conditions (Garcia et al. 2015;Valleet al. 2014).

Cysteine
The Cys produced during TSP, is a semi-essential amino acid that contains sulfur and a polar group, which provides hydrophilic properties to the molecule. This amino acid can be found inside the cell as Cys, but extracellular in an oxidative environment, is predominantly present in a dimeric form known as cystine (Cys 2 ) Rehman et al. 2020). The Endogenous Cys synthesis is by TSP. Initially, Hcy condensates with serine by the enzymatic action of cystathionine β-synthase (CBS) to form cystathionine, then this molecule reacts with cystathionine γ-lyase (CSE), to finally produce Cys (Singh et al. 2009).
Cys functions occur in different processes, essential in building fatty acids, enabling the synthesis of cell membranes and the myelin sheaths of neural cells to protect neurons from oxidative stress and its repercussions (Weimbs and Stoffel 1992). Cys is a rate-limiting substrate of the synthesis of GSH, the precursor of taurine, and coenzy-meA (CoA). It is relevant to mention that Cys is also a proteinogenic amino acid that takes part in up to 2 % of proteins in the body and is involved in different posttranslational modifications like sulfhydration, palmitoylation, glutathionylation, guanylation, cysteinylation, sumoylation, farnesylation, and nitrosylation .
Increased levels of Cys are considered neurotoxic in the central nervous system via the overactivation of NMDAr receptors evoking neuronal death and potentiating the Ca 2+ influx (Janaky et al. 2000). Elevated plasma levels of Cys to sulfate ratios were found in patients with PD and Alzheimer's disease (Heafield et al. 1990;Stipanuk et al. 2006). The primary enzymatic route for cysteine breakdown is catalyzed by cysteine dioxygenase (CDO), in which molecular oxygen is added to the sulfhydryl group of Cys. This reaction keeps Cys levels below the toxicity threshold and forms less toxic products than Cys, such as sulfate and taurine (Galvan et al. 2012). In this sense, it has been suggested a reduction in CDO activity in PD patients (Heafield et al. 1990). Studies in rats have shown that CDO is present in the substantia nigra of rats and is degraded by the ubiquitin-26 S proteasome system. The 26 S proteasome recognizes and degrades the ubiquitin-protein conjugates Parsons et al. 2001). A characteristic feature of PD is the presence of Lewy bodies containing free and ubiquitinated proteins. Lewy bodies' formation is linked to the ubiquitin-proteosome system failing, possibly leading to aberrant cysteine oxidation by CDO (Jameson 2011;McNaught et al. 2001).
Also, Cys neurotoxicity has been linked to the formation of many neurotoxic compounds (Janaky et al. 2000). Cys may generate oxygen-free radical species by Haber-Weiss-type reactions when Cys interacts with transition metal ions like Fe, which are highly present in the substantia nigra of post-mortem PD patients (Sofic et al. 1988). Catecholamines, like dopamine, also form neurotoxic compounds by the nucleophilic addition of Cys to oxidize catechols, forming cysteinylcatechols (Janaky et al. 2000). Cysteinylcatechols are described as contributors to excitotoxic neurodegeneration, principally via mitochondrial dysfunction, increasing sensitivity to endogenous NMDAr agonists (Montine et al. 1997). Cysteine can also form adducts with L-DOPA and 3,4-dihydroxyphenylacetic acid (DOPAC, metabolite of dopamine degradation). The levels of cysteinyl adducts of L-DOPA, dopamine, and DOPAC, significantly increase in the substantia nigra of PD patients (Spencer et al. 1998). Contributing to oxidative stress damage by inhibiting mitochondrial complex I, a hallmark of PD    (Danielson and Andersen 2008). Cys is metabolized into three main compounds: hydrogen sulfide (H 2 S), taurine, and glutathione, as mentioned below.

Hydrogen sulfide
H 2 S is a gas molecule known to be toxic, colorless, and a smell similar to rotten eggs. Numerous studies have revealed the role H 2 S plays in physiological and pathological regulation in the brain of mammals. The metabolism of H 2 S is involved in neurodegenerative disorders, including PD. Evidence suggests that H 2 S production is impaired in the progression of PD (Cao et al. 2018). Hu et al. have shown that H 2 S levels were lower in rats lesioned unilaterally in the striatum with 6-hydroxydopamine (a PD rat model) in contrast with sham-operated rats (Hu et al. 2010).
Currently, H 2 S is considered a signaling molecule, a gasotransmitter, an antiapoptotic agent, an antioxidant, and a neuroprotector in the nervous system (Kumar et al. 2018). Different studies have demonstrated the neuroprotective properties of H 2 S in different rodent and cellular models of PD. Some neuroprotective effects of H 2 S are a consequence of the support it provides to the mitochondria function because, in lower concentrations, H 2 S stimulates mitochondrial energetics, enhancing adenosine triphosphate (ATP) production and activation of protein kinase A (PKA) (Paul and Snyder 2015). H 2 S can also prevent neuronal damage produced by endoplasmic reticulum stress, which can result in endothelial cell apoptosis (Zhong et al. 2020). Depending on the scenario, H 2 S stimulates or inhibits autophagy, a process essential for the degradation and recycling of cellular components. Autophagy disturbances are involved in pathological changes in many neurodegenerative disorders (Wu et al. 2018).
Furthermore, H 2 S can protect neural cells from oxidative stress. H 2 S reduces Cys 2 into Cys in the extracellular space for GSH production and makes cells more efficient in Cys transport into neurons (Kimura et al. 2010). Alternatively, the direct effects of H 2 S are as a scavenger of reactive oxygen species (ROS) as well as peroxynitrites (ONOO − ) and as a reducing agent of glutathione disulfide (GSSG) (Xiao et al. 2018).
Different studies have demonstrated that inhalation and intraperitoneal administration of H 2 S prevents the death of dopaminergic neurons, ameliorates movement disruption, and alleviates astrocytic activation in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD-mouse model, MPTP is a prodrug of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), causing permanent symptoms of PD in rodents. This protection is conferred by the activation of heme oxygenase and gcs genes, by the uncoupling protein 2 (UCP2)-dependent mechanism, reducing ER stress and apoptosis, and by the attenuation in the increases of ROCK2 (Rho-associated protein kinase 2) through miR-135a-5p by MPTP. ROCK2 is implicated in neurodegenerative processes, including axonal degeneration and neuronal death in the brain (Kida et al. 2011;Liu et al. 2016;Lu et al. 2012). Administration of the NaHS and H 2 S donor reversed the progression of movement dysfunction and loss of tyrosinehydroxylase in the substantia nigra of a 6-hydroxidopamine (6-OHDA) and rotenone rat PD models. The proposed mechanism for NaHS-protection is by inhibiting NADPH oxidase activity and oxygen consummation induced by 6-OHDA. Already that NaHS reduce the expression and translocation to the cytoplasmatic membrane of the p47phox, a subunit of NADPH oxidase (Chen et al. 2015;Hu et al. 2010). NaHS also has anti-inflammatory properties through the inhibition of the microglial activation in the substantia nigra and accumulation of pro-inflammatory factors, such as NO and TNFα in the striatum, via NF-кB (Chen et al. 2015;Hu et al. 2010). Moreover, in vitro studies with PC12 and SH-SY5Y cells also demonstrate the protection of H 2 S against MPP+, rotenone, and 6-OHDA-induced neuronal damage by enhancing mitochondrial functions, mitochondrial potential membrane channel, attenuating intracellular ROS production, apoptosis, and endoplasmic reticulum stress Tiong et al. 2010;Xie et al. 2012;Yin et al. 2009). H 2 S also modifies many proteins in a mechanism that involves the transformation of protein Cys residues to form protein persulfides, also known as S-sulfhydration (Filipovic 2015). S-sulfhydration (S-SH) modification is a posttranslational process involved in many physiological and pathological processes, like the inflammatory response, cell differentiation, oxidative stress, or neuroprotection . Aberrant sulfhydration patterns occur in neurodegenerative conditions such as PD (Paul and Snyder 2015). The E3 ubiquitin ligase, Parkin, suffers sulfhydration by H 2 S, which occurs mainly in Cys95, Cys59, and Cys182; Parkin sulfhydration enhances its catalytic activity, whereas nitrosylation (oxidative modification of Cys by nitric oxide) inactivates Parkin Vandiver et al. 2013). The nitrosylation of Parkin inhibits its E3 ligase responsible for the clearance of toxic misfolded proteins (Kida and Ichinose 2015;Vandiver et al. 2013). Parkin nitrosylation is enhanced, and Parkin sulfhydration is decreased in PD brains, indicating a possible reduction of endogenous H 2 S production and enhanced nitrosative stress in the progression of PD (Chung et al. 2004;Vandiver et al. 2013).
Other strategies have also been designed to evaluate the effects of H 2 S. Xie et al. observed that the H 2 S-releasing L-DOPA derivate, ASC84, this compound attenuates oxidative stress by activating the Nrf2 transcription factor in a 6-OHDA-PD induced rat model (Xie et al. 2013). The proposed mechanism for the activation of Nrf2 described is by the S-sulfhydration of Keap1 (Nrf2 inhibitor) at Cys-151, releasing Nrf2 from Keap1 inhibition, activating Nrf2 in the 6-OHDA PD-induced rat model (Yang et al. 2013). Compound GYY4137, another H 2 S releaser, is neuroprotective and reduced nitrated α-synuclein in an MPTP mouse PD model. α-synuclein tyrosine residues can be nitrated by peroxynitrite when excessive NO is generated, thus facilitating α-synuclein protein aggregation (Hou et al. 2017).

Enzymatic H 2 S production in PD
Four enzymes are responsible for H 2 S biosynthesis in the brain, including CBS, CSE, 3-mercaptopyruvate sulfur transferase, which acts together with cysteine aminotransferase (3-MPST/CAT) or with D-amino acid oxidase (3-MPST/ DAO) (Chen et al. 2015). CBS and CSE generate H 2 S in the cytosol and are pyridoxal-5′ phosphate (PLP)-dependent enzymes, while 3MST produces H 2 S in mitochondria and is PLP-independent (Cao et al. 2018).
The enzyme CBS has the property to transform Cys and Hcy into H 2 S and can either catabolize Hcy to the Cys precursor cystathionine (Miles and Kraus 2004). In contrast, CSE can generate Cys from cystathionine but favors the enzymatic production of H 2 S from Cys or Hcy (Sbodio et al. 2019). 3-mercaptopyruvate (3 MP) is the substrate of the enzyme 3MPST producing H 2 S under reducing conditions; 3 MP is provided from the metabolism of Cys and α-ketoglutarate due to the action of the enzyme Cysteine aminotransferase (CAT) or by the enzymatic activity of D-amino acid oxidase (DAO) using D-cysteine (Chen et al. 2015;Shibuya et al. 2009;Zhong et al. 2020). On the other hand, the production of H 2 S by 3-MPST is accompanied by the production of polysulfides, which are an endogenous reservoir of sulfur in the absence of GSH and Cys (Augsburger and Szabo 2020; Tabassum and Jeong 2019).
In general, CBS is recognized as the dominant H 2 Sproducing enzyme in the brain, while CSE may not contribute to H 2 S generation in this organ since CSE inhibition does not alter the production of H 2 S (Abe and Kimura 1996). Elevated plasma levels of cystathionine produced by CBS are accumulated over time in PD, while Cys levels were not modified over time (Hertel et al. 2019). Studies from Diwakar in 2007 have demonstrated that even though the activity of the CSE in the mouse brain represents only 1 % of the liver, CSE inhibition by oxidative stress alters GSH levels since CSE is a rate-limiting enzyme for Cys synthesis from cystathionine; thus CSE activity maintains GSH homeostasis in the brain and preserves mitochondrial function, which is altered in PD patients (Diwakar and Ravindranath 2007). In addition, H 2 S is also produced in the brain by the activity of 3-MPST (Kimura et al. 2015). Shibuya et al. (2009) have demonstrated that brain homogenates of CBS-knockout mice can produce H 2 S via 3-MPST enzyme in similar levels as wildtype mice (Shibuya et al. 2009).
CBS and CSE are present in astrocytes and microglial cells, while 3-MPST is localized in neurons (Lin et al. 2021). H 2 S production in glial cells attenuates inflammatory responses, protecting neurons from PD in rodent models where activated glial cells were induced by inflammation. In vivo studies have demonstrated a significant decrease in CBS expression in the MPTP PD mouse model (Yuan et al. 2018). Striatal CBS gene overexpression in 6-OHDA and MPTP PD-induced rodent models enhances H 2 S production and is neuroprotective, preventing dopaminergic cell death (Yin et al. 2017;Yuan et al. 2018). Also, a significant decrease in CBS expression was observed in MPP +− exposed astrocytes and microglia but not in dopaminergic neurons (Yuan et al. 2018). Likewise, DJ-1 knockout astrocytes have reduced H 2 S and CBS production (Bae et al. 2013). CBS transcription and protein expression are also decreased in rotenone-treated microglia. While the overexpression of CBS in microglia reduces the expression of pro-inflammatory genes and enhances anti-inflammatory markers in response to rotenone stimulation (Du et al. 2014). As mentioned, reduced sulfhydrated parkin has been observed in PD post-mortem patients compared to healthy post-mortem subjects . Overexpression of CBS in neuronal cells increases Parkin sulfhydration related to enhanced Parkin activity and H 2 S production (Vandiver et al. 2013). The role of the H 2 S-generating enzymes in the development of PD is not yet fully elucidated.

Glutathione
Glutathione (GSH) is a linear thiol-containing peptide present in every cell, particularly abundant in the liver and kidney (Commandeur et al. 1995). In the brain, GSH concentration ranges from 1 to 2 mM, and astrocytes appear to have higher GSH levels than neurons (Dringen 2000;Rae and Williams 2017). In the presence of oxide radicals and during the enzymatic detoxification of peroxides, GSH is oxidized to GSSG (GSH disulfide) . Under normal conditions, GSH is the most prevalent in the brain (97 %) (Iskusnykh et al. 2022).
Thanks to numerous studies, GSH is known to have a large range of functions. GSH can function as a physiological reservoir for Cys and glutamate (Aoyama 2021;Koga et al. 2011). In the brain, it is an essential antioxidant able to protect brain cells from damage produced by ROS, RNS, and other reactive species like hydroxyl radicals, peroxynitrites, and superoxide radicals (Asanuma and Miyazaki 2021;Bjorklund et al. 2021;Dringen and Hirrlinger 2003). The proportion of reduced and oxidized GSH (GSH/GSSG) is used as a biomarker of cellular redox homeostasis (Bjorklund et al. 2021). As well, GSH can protect the organism from inflammatory pathologies and xenobiotic factors (Ghezzi 2011;Sipes et al. 1986). Also participates in the transport of some amino acids and ensures the thiol group in proteins (Griffith et al. 1979;Iskusnykh et al. 2022).
De novo synthesis of GSH is dependent on the "rate limiting precursor," Cys. This amino acid enters the cell from plasma or through the transmethylation and transsulfuration pathways (Asanuma and Miyazaki 2021;Bjorklund et al. 2021;Iskusnykh et al. 2022). Since GSH cannot cross the brain-blood barrier (BBB), neurons and astrocytes need to produce their own GSH (Aoyama 2021). Extracellular Cys is easily autooxided to Cys 2 and is principally transported into astrocytes via Cys 2 /glutamate (xCT) antiporter. Cys 2 is taken via xCT into the cell and then is reduced by GSH or thioredoxin to Cys for GSH synthesis (Asanuma and Miyazaki 2021;Mandal et al. 2010). In general, GSH synthesis depends on the activity of the enzyme ɣ-glutamylcysteine synthetase (ɣ-GCS), glutathione synthetase (GS), and through pathways that include its catabolism. GSH catabolism occurs extracellularly through the activity of a membrane ectoenzyme by the Ɣ-glutamyltranspeptidase (ɣ-GT), which hydrolyzes the gamma-glutamyl bond of extracellular GSH or GSH conjugates (Franco et al. 2007). ɣ-GT catalyzes transfers of the ɣ-glutamyl moiety from GSH, or its glutathione conjugates, onto an acceptor; the product of this reaction is L-cysgly or L-cysgly conjugate. Then L-cysgly is hydrolyzed to Cys and glycine, which can be taken up by neurons via excitatory amino-acid carrier 1 (EAAC1) and used as precursors for GSH synthesis; thus, GSH synthesis in neurons depends on the GSH synthesis by surrounding astrocytes (Asanuma and Miyazaki 2021;Valdovinos-Flores and Gonsebatt 2012).
Evidence has demonstrated that oxidative stress plays an important role in the pathophysiology of PD. Post-mortem studies of PD patients reveal lower levels of the antioxidant GSH only in the substantia nigra (40 %) in comparison to control patients, and no depletion in other brain regions was observed (Sian et al. 1994). In addition, the decrease in GSH levels is accompanied by a reduction in the GSH/GSSG ratio in brain tissue and blood of PD patients in comparison to healthy subjects (Bjorklund et al. 2021). The reduction of GSH levels in PD patients is not at all well understood. In different studies, no failure has been found in GSH synthesis since ɣ-GCS activity, like glutathione S-transferase in the substantia nigra, is normal in PD patients. Nevertheless, changes in ɣ-GT and glutathione peroxidase (GPx) were observed in the tissue and blood of patients with PD (Power and Blumbergs 2009;Sian et al. 1994). The enhanced activity of ɣ-GT in PD patients might reflect a compensatory requirement of GSH precursors for dopaminergic neurons. If Cys is not used for GSH synthesis, it forms cisteynil adducts with L-DOPA, dopamine, and DOPAC. These conjugates easily form cytotoxic compounds, which are irreversible complex I mitochondrial inhibitors (Li and Dryhurst 1997;Shen and Dryhurst 1996). Secondly, GPx catalyzes the reduction of H 2 O 2 to water, using GSH to protect cells against oxidative damage. Lower levels of GPx were detected in the blood cells of PD patients (Vida et al. 2019). In contrast, studies from Power and Blumges in 2009 have demonstrated in human brain tissue that GPx1 is highly expressed in microglia and has lower levels in neurons. In the same research, GPx-1 positive microglia were hypertrophied and more abundant in PD tissues, making contacting multiple neurons, proposing that the upregulated levels of the antioxidant GPx-1 protect neurons from oxidative stress (Power and Blumbergs 2009). Different studies have demonstrated that increased oxidative stress and decreased GSH/GSSG activity in the brain of PD patients might boost chronic inflammatory reactions, mitochondrial superoxide, as well as oxidative damage to biomolecules (Dias et al. 2013;Hauser and Hastings 2013).
In the dopaminergic cell, dopamine oxidation occurs in an enzyme-dependent or enzyme-independent manner to produce dopamine quinones and active radicals. Enzymatic oxidation of dopamine by monoamine oxidase leads to the formation of H 2 O 2 and metabolites such as 3,4-dihydroxybenzoic acid (DOPAC) and homovanillic acid (HVA). The H 2 O 2 is inactivated by catalase or GPx, which uses GSH as a co-substrate. Nevertheless, H 2 O 2 may form highly reactive hydroxyl radicals (OH • ) in excess iron. OH • are directly neutralized with GSH in a non-enzymatic reaction (Figure 1) (Fiser et al. 2013).
In contrast, spontaneous dopamine oxidation leads to neuromelanin formation and can generate dopaminequinone, semiquinone species, and superoxide anion (O 2 − ) (Bjorklund et al. 2018;Zhang et al. 2019). Dopamine-quinones exert their toxic effects by binding to sulfhydryl residues on functional proteins, such as tyrosine hydroxylase (TH), dopamine transporter, and parkin, declining protein function and evoking dopaminergic toxicity (Asanuma and Miyazaki 2021;Kuhn et al. 1999;LaVoie et al. 2005;Whitehead et al. 2001). GSH binds to DA quinones via its thiol group, preventing dopaminergic toxicity. Therefore, GSH is critical for the protection of DA neurons in the substantia nigra pars compacta from dopamine-quinone and free radicals neurotoxicity. The loss of the neuroprotective functions of GSH in dopaminergic neurons in the substantia nigra pars compacta may lead to the neurodegenerative processes observed in PD (Andersen 2001;Liddell and White 2018;Smeyne and Smeyne 2013). Figure 1 summarizes the main functions of glutathione and cysteine in astrocytes and dopaminergic neurons in PD. Taurine (Tau) is a semi-essential amino acid that contains a sulfur group, endogenously produced from Cys (Chen et al. 2019;Jakaria et al. 2019). The body has different alternatives to get Tau; one is by dietary intake, and from de novo synthesis through the catabolism of Cys which however is limited by the oxidation of hypotaurine to Tau (Wojcik et al. 2010). The enzymes cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSAD) are in charge of producing hypotaurine from Cys (Rafiee et al. 2022). At the same time, cysteamine dioxygenase (ADO) synthesizes Tau from cysteamine instead of Cys as substrate (Dominy et al. 2004). In the brain, ADO expression predominates from CDO expression (Dominy et al. 2007). Tau is mainly produced in the liver and kidney; however, it has also been found in other tissues, including the brain (El Idrissi 2019; Park et al. 2014). In the rat brain, Tau is expressed in all regions (Chen et al. 2019). Tau content in the brain is mainly transported from the periphery and by the local synthesis from de novo catabolism of Cys, both in neurons and astrocytes (Vitvitsky et al. 2011). Tau concentration levels in the brain vary in the different developmental stages. A high concentration of Tau has been described in the developing brain, decreasing levels in adulthood even more in older stages of different brain species (Baliou et al. 2021;Rafiee et al. 2022), proposing Tau as an essential player in neurodevelopment, Figure 2: Association of pro-inflammatory and oxidative events in Parkinson's disease and different compounds of the transsulfuration pathway. Chemical compounds such as manganese (Mn), iron (Fe), and volatile organic compounds (VOC) can cross the blood-brain barrier (BBB). Once inside the brain, along with impaired clearance of α-synuclein (SNCA), they induce microglia to release inflammatory and pro-oxidative mediators, including tumor necrosis factor (TNFα), interleukin 1β (IL1β), transforming growth factor β (TGFβ), nitric oxide (NO), and reactive oxygen species (ROS). This leads to SNCA aggregation and stimulates astrocytes to release TNFα, IL-1β, NO, ROS, and interleukin 6 (IL-6), promoting dopaminergic injury and death. Fe additionally induces ROS generation and SNCA fibrillation and aggregation. Compounds of the transsulfuration pathway (TSP) are closely related to the events described above. Homocysteine (Hcy) increases BBB permeability, NO, ROS, and proinflammatory cytokines through N-methyl-D-aspartate receptor (NMDAr) activation, favoring dopaminergic neuron death. Reduced levels of hydrogen sulfide (H 2 S), taurine (Tau), and glutathione (GSH) promote oxidative stress and inflammation in neurons, inducing SNCA aggregation and dopaminergic neuron damage.
including neurite growth and synaptogenesis (Mersman et al. 2020). Studies in mice reported that tissue depletion of Tau leads to a shorter lifespan (Ito et al. 2014). In the brain, Tau is involved in numerous processes in brain cells, acting as an anti-inflammatory, neuromodulator, and osmoregulator (Chen et al. 2019;Niu et al. 2018;Rafiee et al. 2022). Tau has been reported as a neuroprotective agent against excitotoxicity, apoptosis death, reticulum endoplasmic stress, mitochondrial stress, and oxidative stress (Leon et al. 2009;Pan et al. 2010;Pan et al. 2012;Rafiee et al. 2022;Wu et al. 2005).
Levels of Tau in the plasma of PD patients are decreased compared to control patients and are negatively associated with motor severity (Zhang et al. 2016). Dawson et al. (1999) have demonstrated that age-related reductions of striatal Tau and dopamine are correlated in aged rats, suggesting neuronal dopaminergic degeneration (Dawson et al. 1999).  (Hcy), which in turn is synthesized from methionine during transmethylation, having as intermediates S-adenosylhomocysteine (SAH) and the methyl donor S-adenosyl methionine (SAM). Hcy can be remethylated to methionine by the activity of the enzyme methylenetetrahydrofolate reductase (MTHFR). Then Hcy is converted to cysteine (Cys) by the activity of cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), having cystathionine as an intermediate. Likewise, Cys is a precursor of different molecules, including glutathione (GSH), taurine, and hydrogen sulfide (H 2 S). GSH is synthesized from Cys by the activity of ɣ-glutamylcysteine synthetase (ɣ-GCS) and glutathione synthetase (GS); taurine by the activity of cysteamine dioxygenase (ADO), cysteine dioxygenase (CDO) and cysteine sulfinate decarboxylase (CSAD); and finally, H2S by the activity of CBS, CSE, and 3-mercaptopyruvate sulfur transferase (3-MPST) in conjunction with cysteine aminotransferase (3-MPST/CAT) or (3-MPST/DAO). The down black arrow or up red arrow denotes the downregulation and upregulation respectively, in metabolites or enzymes of the TSP in PD. Association of pro-inflammatory and oxidative events in Parkinson's disease and different compounds of the transsulfuration pathway. Chemical compounds such as manganese (Mn), iron (Fe), and volatile organic compounds (VOC) can cross the blood-brain barrier (BBB). Once inside the brain, along with impaired clearance of α-synuclein (SNCA), they induce microglia to release inflammatory and prooxidative mediators, including tumor necrosis factor (TNFα), interleukin 1β (IL1β), transforming growth factor β (TGFβ), nitric oxide (NO), and reactive oxygen species (ROS). This leads to SNCA aggregation and stimulates astrocytes to release TNFα, IL-1β, NO, ROS, and interleukin 6 (IL-6), promoting dopaminergic injury and death. Fe additionally induces ROS generation and SNCA fibrillation and aggregation. Compounds of the transsulfuration pathway (TSP) are closely related to the events described above. Homocysteine (Hcy) increases BBB permeability, NO, ROS, and proinflammatory cytokines through N-methyl-D-aspartate receptor (NMDAr) activation, favoring dopaminergic neuron death. Reduced levels of hydrogen sulfide (H 2 S), taurine (Tau), and glutathione mmm(GSH) promote oxidative stress and inflammation in neurons, inducing SNCA aggregation and dop.
Different studies have demonstrated that taurine exerts neuroprotection in cells and murine models of PD through the inactivation of microglia-mediated neuroinflammation Hou et al. 2018;Tian et al. 2020;Wang et al. 2021). In the dopaminergic neuron, high levels of iron and dopamine oxidation make neurons more vulnerable to oxidative stress and, in specific ferric iron induces aggregation of SNCA (Jellinger 1999;Li et al. 2010). Dopamine autoxidation is catalyzed by metals such as iron and manganese. Studies in vitro have demonstrated that Tau significantly reduced ferric iron, and manganese stimulated dopamine oxidation in vitro, thus protecting dopaminergic neurons from ROS-induced oxidative stress ( Figure 2) (Dawson et al. 2000).
Tau is a neuromodulator of gamma-aminobutyric acid (GABA), glycine, and NMDA receptors and is found in high concentrations in the substantia nigra and striatum, mainly observed in GABAergic terminals coming from the striatum to the substantia nigra pars reticulate (Bianchi et al. 1998;Dray and Straughan 1976;Palkovits et al. 1986). Studies in rat brain slices have demonstrated that Tau acts as a neuroprotector agent against MPP + neurotoxicity via the activation of GABAA receptors (O'Byrne and Tipton 2000). Studies in rat brain slices reported that inhibition by Tau in the substantia nigra pars compacta is mediated by GABA receptors (Ye et al. 1997). Thus Tau could be inhibiting the PD-induced hyperactive projection from the subthalamic nucleus to substantia nigra pars reticulata, disinhibiting the motor thalamus and generating movement (Dray and Straughan 1976;Menzie et al. 2014). Likewise, Tau plays a vital role in the modulation of the release and metabolism of striatal dopamine by the glycine activation, in turn enhancing the activity of dopaminergic neurons of the ventral tegmental area, enhancing dopamine levels in the striatum (nucleo accumbens) (Ericson et al. 2013;Ruotsalainen and Ahtee 1996). Figure 2 Associates the pro-inflammatory and oxidative events in Parkinson's disease and different compounds of the transsulfuration pathway. Figure 3 shows the transsulfuration pathway in PD in which the previously described molecules are involved (Figure 3).

Concluding remarks
In this review, we highlighted and offered a summary of the role of different intermediates, products, and enzymes related to TSP. This pathway maintains redox homeostasis, integrates stress responses, and synthesizes various molecules with many functions in the brain (antioxidant, antiapoptotic, neuromodulator, structural, reducing power, etc.). Low methylation potential (SAM/SAH) is found in PD patients and is influenced by the transmethylation and remethylation pathway, ingesting folates and vitamin B12, and the pharmacological treatment for PD. This methylation potential regulates TSP, since it inversely regulates Hcy levels and directly regulates CBS activation for H 2 S synthesis. The TSP products, H 2 S and Cys, are downregulated and upregulated in PD. Although Cys levels are elevated, the products of its metabolism (GSH, taurine, and H 2 S) are decreased in PD, possibly due to the down-regulation of enzymes related to its synthesis and greater consumption of these molecules due to the increase in the generation of ROS in PD (Dias et al. 2013;Heafield et al. 1990). GSH, taurine, and H 2 S are molecules with anti-inflammatory and antioxidant properties in the brain, so their decrease can lead to neuronal death, as occurs in dopaminergic neurons in PD and other neurodegenerative processes (Mosley et al. 2006). Therefore, metabolites of TSP can potentially predict the development and progression of PD, and can be proposed as predictive biomarkers.
Despite the increased knowledge about the molecular mechanisms of the TSP and its relationship with PD, it is crucial to consider that it is only a tiny part of the extensive network of interconnected pathways. Additionally, more in vivo, and in vitro studies are needed to elucidate the connection between TSP and other more complex pathways involved in PD to propose possible targets and biomarkers against dopaminergic neuronal damage.