Amino acid transporters in neurological disorders and neuroprotective effects of cysteine derivatives

For most diseases and disorders occurring in the brain, the full causes behind them are yet unknown, but many show signs of dysfunction of amino acid transporters or abnormalities in amino acid metabolism. The blood-brain barrier (BBB) plays a key role in supporting the function of the central nervous system (CNS). Because of its unique structure, the BBB can maintain the optimal environment for CNS by controlling the passage of hydrophilic molecules from blood to the brain. Nutrients, such as amino acids, can cross the BBB via specific transporters. Many amino acids are essential for CNS function, and dysfunction of these amino acid transporters can lead to abnormalities in amino acid levels. This has been linked to causes behind certain genetic brain diseases, such as schizophrenia, autism spectrum disorder, and Huntington ’ s disease (HD). One example of crucial amino acids is L-Cys, the rate-limiting factor in the biosynthesis of an important antioxidant, glutathione (GSH). Deficiency of L-Cys and GSH has been linked to oxidative stress and has been shown as a plausible cause behind certain CNS diseases, like schizophrenia and HD. This review presents the current status of potential L-Cys therapies and gives future directions that can be taken to improve amino acid transportation related to distinct CNS diseases.


Introduction
Genetic brain diseases are a group of diseases and disorders that can greatly affect a person's cognitive and physical functions and are often linked to genetic mutations, either occurring randomly or due to environmental exposure (Hickman et al., 2022;Sawada et al., 2022).Neurodegenerative diseases, in turn, are broadly classified by specific protein accumulations as pathological markers, including amyloidosis, tauopathies, synucleinopathies, and transactivation response DNA binding protein 43 (TDP-43) proteinopathies (Hansson, 2021;Dugger and Dickson, 2017).Synergistic interactions of several protein depositions, such as β-amyloid (Aβ), tau-protein, and α-synuclein are likely to advance aggregation processes that lead to more complex degeneration and excessive accumulation (e.g., Aβ-plagues and neurofibrillary tangles), which ultimately results in neurodegenerative disorders (Clinton et al., 2010).Their underlying pathologies share numerous basic mechanisms that lead to progressive neuronal deterioration and death, including oxidative stress, neuroinflammation, and proteotoxic stress.Nevertheless, many neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS), have also genetic underpinnings.
The central nervous system (CNS) is isolated and protected by several barriers, including the blood-brain barrier (BBB) (Davson, 1976;Ballabh et al., 2004).The BBB acts as a diffusion barrier to upkeep an optimal environment and molecule concentrations for a healthy function of the brain.The BBB endothelial cells (ECs) are unique and are connected by tight junctions (TJs), limiting paracellular movement of compounds (van Meer and Simons, 1986;Oldendorf and Brown, 1975).Besides the TJs, the BBB consists of the capillary basement membrane covering the capillaries, astrocyte end-feet surrounding the basement membrane, and pericytes embedded within the basement membrane.Since the BBB tightly controls the passage of hydrophilic and larger molecules to the brain, nutrients such as amino acids, need to cross the barrier via specific transport mechanisms (van Meer and Simons, 1986).
Amino acid transporters are responsible for the exchange of amino acids to and from the brain and have a key role in maintaining the appropriate concentration levels for the vital brain functions (Hawkins et al., 2006).The amino acid transporters can be divided into facilitative Na + -independent or active Na + -dependent transporters, and they can be located either on the luminal or abluminal sides of the ECs or on both sides.Amino acids are crucial for brain function, and they play a role in the synthesis of proteins and signaling molecules, like neurotransmitters and peptide hormones, as well as in other key functions, like protection against oxidative stress (Fernstrom, 1994).
Thus, amino acid deficits can play a vital role in genetic CNS diseases and neurodegenerative diseases.Dysfunction of transporter systems either at BBB or neurons, especially when coupled with other factors hindering the regulation of amino acid concentrations, can be the cause of severe neurological symptoms.This review gives an overview of different CNS diseases and disorders associated with amino acid deficiencies caused by abnormal expression and/or function of amino acid transporters and discusses the possibilities of how to improve amino acid brain drug delivery as a viable treatment option.

Transport of amino acids across the blood-brain barrier
The CNS requires several amino acids to function properly.Certain amino acids, such as L-Tyr, L-Trp, L-His, L-Phe L-Thr, L-Met, and L-Arg act as precursors for neurotransmitters and neuromodulators (Dalangin et al., 2020).The brain's ability to synthesize amino acids differs from the rest of the body.For example, L-Tyr is generally considered a non-essential amino acid, but it has been suggested that the brain cannot synthesize L-Tyr sufficiently enough to meet its needs, making L-Tyr essential in the brain.Due to their polar structure, amino acids, need a specific transport mechanism to passive diffusion in order to access the brain.The protein complexes in the cellular membrane of the BBB controlling the cell-cell attachment polarize the membrane (Hawkins et al., 2006;van Meer and Simons, 1986).Due to this, the membrane can be divided into the luminal (blood-facing) and the abluminal (brain-facing) sides.Each molecule crossing the BBB needs to get through both the luminal and abluminal sides.There are several known amino acid transporter systems and distinct solute carrier (SLCs) with different distributions located on either side or both sides of the BBB (Zaragozá, 2020;Cesar-Razquin et al., 2015).Some of them are Na + -independent transporters that are responsible for ensuring the availability of amino acids in the brain and facilitating their bidirectional transport across ECs.In addition to the facilitative systems, there are also several Na + -dependent transporters, which transport amino acids against their concentration gradient together with the movement of Na + ions (Table 1).These systems are crucial in maintaining the optimal amino acid concentrations in the brain and controlling its homeostasis.Throughout the literature, amino acid transporters have had different types of classifications.The most recent is the classification by the Human Genome Organization (HUGO) Gene Nomenclature Committee (HGNC), in which all SLCs are grouped into different families based on their amino acid sequence.Members of the same family typically share about 20-25 % of their amino acid sequence as well as similar substrate (s) (Schlessinger et al., 2010).However, another older classification system is based on the transport characteristics and substrates selectivity, including system A, system N, system L, and system ASC, co-exists.

Excitatory amino acid transporters (EAATs)
Three Na + -dependent L-Glu transporters are expressed on the abluminal membrane of brain capillary ECs (O'Kane et al., 1999).These transporters belong to the excitatory amino acid transporter (EAAT) family and namely are EAAT1 (SLC1A3), EAAT2 (SLC1A2), and EAAT3 (SLC1A1).The relative activity of EAAT1-3 in the brain has been found with a ratio of 1:3:6 for EAAT1, EAAT2, and EAAT3, respectively (Table 2).The EAAT family has been described as the most powerful Na + -dependent amino acid transporters as they exhibit the greatest affinity to amino acids at low concentrations and are an important part of maintaining the L-Glu gradient in the extracellular fluid (Hawkins et al., 2006).In addition to the BBB, EAAT1 and 2 are expressed in glial cells and have the highest role in clearing up the excess of L-Glu in and around the glutamatergic synapses, and thus, they prevent the excitotoxicity and the death of the surrounding neurons.Contrarily, EAAT3 is mainly expressed in neurons and is responsible for the reuptake of the neurotransmitter L-Glu from the synapses into the neurons (Kanai et al., 2013).Moreover, EAAT3 has been indicated as the primary route of neuronal L-Cys uptake (Chen and Swanson, 2003).

Alanine, serine, and cysteine transporters (ASCTs, system ASC)
Alanine, serine, and cysteine transporters (ASCTs) have very close structure similarities with the L-Glu transporters of the EAATs family (40% amino acid sequence identity) (Kanai et al., 2013;Utsunomiya--Tate et al., 1996;Scopelliti et al., 2014).Both ASCT1 (SLC1A4) and ASCT2 (SLC1A5) are expressed in the brain, preferentially in astrocytes, but also to some extent in neurons (Table 2) (Kanai et al., 2013).ASCT1 and ASCT2 are also expressed on the abluminal side of mouse BBB but ASCT2 with a higher degree (Tetsuka et al., 2003).When ASCTs were first identified, the name ASC was given due to their preference for neutral amino acids, i.e., L-Ala, L-Ser, and L-Cys (Christensen et al., 1967).Distinct from EAATs, they carry neutral amino acids in exchange for intracellular amino acids (as antiporters), although in a Na + -dependent manner (Scopelliti et al., 2013).Despite the huge sequence similarity with EAATs, ASCTs are different in their function and even in their substrate selectivity.ASCT1 provides neuronal cells with their metabolic needs but also helps in releasing L-Ser from glial cells, where it is synthesized and stored, in exchange for L-Ala, L-Ser, or L-Cys (Sakai et al., 2003).In turn, ASCT2 has broader substrate selectivity with a high affinity towards L-Gln and a low affinity towards D-Ser, L-Met L-Leu, and L-Asn (Utsunomiya-Tate et al., 1996).L-Gln is one of the most abundant amino acids found in the CNS and plays a major role as a precursor to neurotransmitters, the amino acids L-Glu and L-Asp, as well as γ-amino butyric acid (GABA) (Albrecht et al., 2010).

Solute carrier 7 family
2.2.1.Cationic amino acid transporters (CATs) SLC 7 family consists of two subfamilies; one is the cationic amino acid transporters (CATs) that facilitate the diffusion of basic amino acids; and the other is the heterodimeric amino acid transporters (HATs) that consist of functional light subunit (from SLC7A-family) and regulatory heavy subunit (from SLC3A-family; rBAT, SLC3A1 or 4F2hc, SLC3A2) linked via disulfide bond (Fotiadis et al., 2013).The transport of cationic amino acids, such as L-Lys, L-Arg, and L-Orn, across the BBB is less known, but two protein families have been described; cationic amino acid transporters (CAT1-3; SLC7A1-3; system y + ) and broad scope amino acid transport systems, which includes b 0,+ (SLC7A9), and y + LAT1-2 (SLC7A6-7), as well as ATB 0,+ (SLC6A14) from the SLC 6-family (Dev ÉS and Boyd, 1998).From these, CATs are selective for basic amino acids, while the other group of transporters also carry neutral amino acids.However, there is no evidence for the presence of systems ATB 0,+ , b 0,+ , or y + LAT1-2 in either the luminal or abluminal membranes of the BBB, and therefore, the brain transport of cationic amino acids is thought to be facilitated solely by CAT1, highly expressed at the BBB (Table 2) (Tachikawa et al., 2018;O'Kane et al., 2006).System ATB 0,+ is the only Na + -dependent transporter of cationic amino acids and since no evidence of its existence at the BBB has been found, this suggests there is no Na + -dependent transport of cationic amino acids across the BBB, making an exception from all other amino acids that have active transporters on the abluminal membranes (O'Kane et al., 2006).CAT1 has been found on both membranes with greater activity on the abluminal side, and in addition to transporting cationic amino acids, CAT1 has been shown to exhibit weak affinity to neutral amino acids in the presence of Na + (O' Kane et al., 2006).Although CAT1 may transport many essential and non-essential neutral amino acids, its contribution to the transport of these amino acids is significantly less than that of LAT1 (discussed below).Moreover, while CAT1 has an essential role in the transportation of all cationic amino acids, its affinity for L-Arg is greater compared to the other CATs (Zaragozá, 2020).Complexing together with endothelial nitric oxide synthase (eNOS), this suggests that CAT1 has an important role in the synthesis of nitric oxide requiring L-Arg.In addition to CAT1, CAT3 has been found in the neurons, and CAT2B splice variant in the neurons, astrocytes, and oligodendrocytes (Table 2) (Hosokawa et al., 1999;Braissant et al., 2001;Stevens et al., 1996).However, their function in the brain is to date less well understood.

Heterodimeric amino acid transporters (HATs)
Neutral amino acids are transported across the cell membranes in addition to SLC1, also via SLC7, SLC38, and SLC43.From these, SLC7 and SLC43 belong to system L-amino acid transporter (system LAT) and SLC38 is divided into 2 classes; system A and system N (discussed below).In vivo studies have shown that Na + -independent transport of neutral amino acids into the brain is facilitated mainly by the large neutral amino acid transporter 1 (LAT1; SLC7A5) (Boado et al., 1999).LAT1 is highly expressed at both, the abluminal and luminal sides of ECs, in a ratio of 1:2, respectively (Table 2) (Sánchez Del Pino et al., 1992;Sánchez Del Pino et al., 1995).The transporter consists of two subunits, the light chain subunit (LAT1) and the heavy chain subunit 4F2hc linked via a disulfide bond (Kanai et al., 1998;Yan et al., 2019).The light subunit handles the exchange of amino acids, while the heavy subunit acts to localize LAT1 on the plasma membrane.The preferred substrates of LAT1 are large branched and neutral amino acids, including L-Phe, L-Trp, L-Leu, L-Ile, L-Met, L-His, L-Tyr, L-Val, and L-Thr, and it exchanges these essential amino acids to counter substrates, such as L-Gln, with 1:1 stoichiometry (Meier et al., 2002).As LAT1 requires another transporter to sustain its counter ions inside the cells, such as ASCT, it can also be called as a secondary active transporter.Curiously, LAT1 has been shown to exhibit a higher affinity for intracellular amino acids than extracellular ones (Meier et al., 2002).
System X C − has also been identified to be composed of the 4F2hc heavy chain (SLC3A2) and xCT light chain (SLC7A11), linked by a disulfide bridge (Sato et al., 1999).Similar to LAT1, the light chain xCT acts as the functional unit while the heavy chain 4F2hc is responsible for trafficking and localization on the cell surface.xCT complex is expressed in neurons as well as in cells separating the brain from the periphery, such as endothelial cells (Table 2) (Burdo et al., 2006).It is an antiporter that mediates simultaneous efflux of L-Glu from ECs to plasma.xCT also carries cystine in the luminal membrane of ECs in a 1:1 ratio (Sato et al., 1999;Bridges et al., 2012).Cystine, an oxidized and disulfide derivative of L-Cys, is essential for glutathione (GSH) production and the prevention of oxidative stress, since cystine is quickly converted into L-Cys, a rate-limiting substrate needed for the synthesis of GSH.This L-Glu/cystine exchange makes xCT a potential contributor to many CNS diseases.

System A
Since the concentration of most amino acids, excluding L-Gln, in cerebrospinal fluid (CSF) and brain extracellular fluid is significantly lower than in plasma, amino acids transferring from the brain to the circulation are moving against the concentration gradient and need active transporters.From the Na + -dependent L-Gln transporting systems that also include ACSTs, system A was the first to be characterized and shown to facilitate the active transport of small nonessential neutral amino acids (Sánchez Del Pino et al., 1992).System A, which consists of sodium-coupled neutral amino acid transporters 1 (SNAT1, ATA1, SAT1; SLC38A1) and 2 (SNAT2, ATA2, SAT2; SLC38A2), differs from other Na + -dependent L-Gln carriers by accepting a unique substrate called N-methylamino-isobutyric acid (MeAIB).The system is named for its preference for L-Ala as a substrate, but it carries also other small amino acids, such as L-Pro, L-His, L-Ser, L-Asn, and L-Gln.From system A transporters, SNAT2 is the only one found on the abluminal membranes of the BBB, while both SNAT1 and SNAT2 are expressed in neurons (Table 2).SNAT2 has a key role in the brain efflux of L-Gln and it accounts for approximately 20 % of Na + -dependent abluminal L-Gln transport (Lee et al., 1998).

System N
Several nitrogen-rich amino acids, such as L-His, L-Gln, and L-Asn, as well as L-Ser can be transported via system N, a transporter system that works as a symport with Na + and an antiport with H + (Kilberg et al., 1980).System N, including sodium-coupled neutral amino acid transporters 3 (SNAT3, SN1, SLC38A3) and 5 (SNAT5, SN2, SLC38A5), is mainly expressed in glial cells and it accepts the substitution of Na + by Li + (Table 2) (O' Kane et al., 2004).SNAT3 was originally described only on the luminal membranes of rat BBB (Ennis et al., 1998).More recent studies however have detected it also on the abluminal side in mouse BBB (Ruderisch et al., 2011).Nevertheless, unlike system A and other amino acids transporting SLCs, the functions of system N are currently less well understood.

CNS diseases and disorders associated with abnormal amino acid transport
As discussed above, amino acid availability is vital for cellular functions, and their supply to the brain is highly regulated at the BBB and parenchymal cells via specific transport mechanisms.Nevertheless, abnormalities in amino acid transporter expression or function have been associated with many CNS diseases and disorders (Nguyen et al., 2021;Yahyaoui and Pérez-Frías, 2019), although it needs to be acknowledged that overall, the diseases and disorders occurring in the brain are complex and the causes are multifactorial.Furthermore, in many cases, the etiologies of the diseases are still not fully understood.Due to the complexity, it is also highly unlikely, that a single drug or supplement will cure or halt the progression of these diseases, but given together with other drugs or supplements, amino acid supplementation in a way or another, may have a potential to improve the outcomes of the therapies.

Schizophrenia
Schizophrenia is a chronic mental disorder that is characterized by a complex behavioral and cognitive phenotype (Afshari et al., 2015).The cause of schizophrenia has not been fully identified, but research has suggested an interaction between genes and several environmental factors as a probable cause.Glutamatergic systems are strongly involved in the pathophysiology of schizophrenia (Afshari et al., 2015).While research has initially been focused on the N-methyl-D-aspartate (NMDA) -type glutamate receptors and their disturbances in neurotransmission, the "glutamate hypothesis" has recently been expanded to also include the dysfunction of other types of glutamate-related proteins.For example, L-Ser is a precursor for neuromodulators, such as L-Gly and D-Ser, which are important for NMDA receptor activation (de Koning et al., 2003;Foster et al., 2016).Therefore, a contribution of ASCT1, a D-Ser carrier, to the pathophysiology of schizophrenia has also been suggested.
A study conducted with multiple members of a 5-generation family depicting schizophrenia-like symptoms has described a hemi-deletion of the SLC1A1 (Myles- Worsley et al., 2013).SLC1A1 coding EAAT3 plays an important role in maintaining the extracellular levels of L-Glu concentrations and keeping them below neurotoxic levels, regulating glutamate-mediated neuroplasticity as well as the neuronal uptake of L-Cys (Afshari et al., 2015, Afshari et al., 2017).Animal studies with EAAT3-null mice have implicated the importance of EAAT3 function compared to EAAT1 and EAAT2 (Afshari et al., 2017).These mice presented increased indicators of neuronal oxidative stress, age-dependent neurodegeneration, cognitive impairment as well as behavioral abnormalities associated with decreased neuronal GSH levels.
GSH acts as one of the main nonprotein antioxidants and redox regulators and thus plays a significant role in protecting nervous tissues from oxidative stress (Lu, 2013).Evidence points to oxidative stress and NMDA-receptor hypofunction as a probable cause of schizophrenia in both directions; NMDAR hypofunction can activate the superoxide producing enzyme, NADPH-oxidase 2 (NOX2), and on the other hand, NMDAR is regulated by redox state via redox-sensitive cysteine residues whose disulfide bond formation decreases NMDAR currents polymorphic inactivation of the GSH-synthesizing enzyme (Hardingham and Do, 2016).Moreover, polymorphism in glutamate-cysteine ligase has also been interlinked with schizophrenia (Tosic et al., 2006) and the studies examining have revealed that schizophrenia patients' CSF, striatum post-mortem tissues, and prefrontal cortex in vivo tissues display decreased GSH levels (Yao et al., 2006;Do et al., 2000).As mentioned above, L-Cys is crucial for the intracellular synthesis of GSH and thus, important in protecting tissues from oxidative reactions (Paul et al., 2018), and its precursor, cystine, is carried by xCT (Hung et al., 2021).Interestingly, altered SLC3A2 and SLC7A11 gene expressions has been less abundant in patients with schizophrenia (Lin et al., 2016).This suggests that xCT impairment may be implicated in the pathogenesis of schizophrenia through regulating GSH and extracellular L-Glu.

Autism spectrum disorder
Autism spectrum disorder (ASD) is a developmental neurological genetic disorder characterized by repetitive behaviors and impairment in social interactions and verbal communication.Several studies of ASD have shown that genetics play a significant role in the development of the disease (Folstein and Rutter, 1977;Bailey et al., 1995;Sandin et al., 2017).However, the genetic architecture of ASD is complex and mutations in single genes are rarely responsible for a significant proportion of cases.Studies have found a connection between ASD and abnormalities in amino acid metabolism, specifically L-Trp and branched-chain amino acids, like L-Leu, L-Ile, and L-Val (Cascio et al., 2020;Randazzo et al., 2023;Zheng et al., 2017).A group of aromatic amino acids (L-Trp, L-Phe, L-Tyr) and branched-chain amino acids, defined together as large neutral amino acids, play an important part in neurodevelopment.These have a key role in upholding protein homeostasis, and they act as precursors of neuroactive molecules, such as serotonin, hence abnormalities in the metabolism of these amino acids may have critical consequences (Louros and Osterweil, 2016;Muller et al., 2016).
A study by Tȃrlungeanu et al. (2016) found that LAT1 encoded by the SLC7A5 gene is important for maintaining normal branched-chain amino acid levels in the brain (Tȃrlungeanu et al., 2016).Lack of LAT1 expression at the BBB can lead to significant reductions in brain branched-chain amino acids, particularly the decrease in L-Leu and L-Ile concentrations.The study was conducted using slc7a5− /− mouse models and it detected reduced levels of L-Leu and L-Ile in mice expressing autism-related phenotype that was rescued by intracerebroventricular injection of branched-chain amino acids.The group also screened whole exome sequencing data from families with children with various neurological diseases.They found two independent families that had multiple children with ASD as well as a homozygous missense mutation in SLC7A5.By mapping these two genetic variants onto a homology model of SLC7A5, they found these mutations very likely affected the structure and function of the transporter.This effect was confirmed with transport assays by the group.
Nine likely pathogenic variants in SLC3A2, SLC7A5, and SLC7A8 (LAT2) have also been detected in another study conducted with ASD patients (Cascio et al., 2020).Coding variants in the LAT genes were found in 17 of 97 tested patients, and all 17 of these patients were diagnosed with non-syndromal ASD.All tested cell lines showed abnormalities in the utilization of large neutral amino acids.Although no mutation hotspots were detected, some variants seem to cluster in certain domains of the proteins encoded by the LAT genes.All four of the variants in SLC3A2 are located within the α-amylase catalytic domain, which plays an important role in the translation and binding of amino acids.
Findings in the metabolic responses of amino acids suggest that abnormalities in the LAT genes have greater effects on the metabolism of branched-chain amino acids and L-His than they do with aromatic amino acids (Cascio et al., 2020).This is thought to be due to the different sites and types of the variants and their effect on the binding affinity for smaller amino acids, as well as alternative transporters compensating for the cellular intake of aromatic amino acids.Overall, the findings from different studies suggest that certain heterozygous variants in the LAT genes are suffice to cause perturbation of the aromatic and branched-chain amino acid transportation and thus can be considered as a possible cause for ASD.

Huntington's disease
Huntington's disease (HD) is an inherited neurodegenerative disease that causes neurons in certain parts of the brain to break down and die.A mutation in the huntingtin gene impacts several diverse cell processes, although oxidative stress has been shown to play also a crucial role in HD.Even though the mechanisms for the origin of oxidative stress in HD are not clear, studies have suggested L-Cys deficit as one possible cause.Since neurons cannot uptake extracellular GSH, L-Cys levels are crucial for enabling its de novo synthesis (Aoyama et al., 2008).Studies have found that in HD, all pathways that are normally responsible for sustaining L-Cys levels and preventing oxidative stress, are compromised by L-Cys deficiency.This includes the impairment of L-Cys metabolism as well as abnormal uptake of cystine and L-Cys (Li et al., 2010;Frederick et al., 2014;Paul et al., 2014;Sbodio et al., 2016).The involvement of L-Cys deficiency in HD pathogenesis has indirect support, since treatments with L-Cys and its derivatives have shown improvement in neuron survivability in rodent models (discussed below) (Paul et al., 2014).
The studies of abnormal L-Cys transporter function have focused on the neuronal L-Cys and L-Glu transporter EAAT3 and its possible role in decreased GSH levels in HD.A study by Aoyama et al. (2006) found not only reduced GSH levels, but also increased oxidant levels and susceptibility to oxidant injury of hippocampal neurons in brain slices collected from EAAT3-null mice (Aoyama et al., 2006).They also found decreased thiol levels in the hippocampal neurons and thus, concluded that these findings together suggest the impaired function of EAAT3 leads to oxidative stress and neurodegeneration as EAAT3 seems to act as the primary route for neuronal L-Cys uptake.
In addition to EAAT3, another important indirect L-Cys transporter is the xCT.This transporter has also been shown to play a key role in L-Glu homeostasis, as well as ensuring sufficient amounts of L-Cys (in the form of oxidized cystine) for GSH synthesis.A study by Frederick et al. (2014) seeking to characterize the abnormalities in L-Glu homeostasis in HD using the STHdh Q111/Q11 neuronal cell line found that both the function and expression of xCT are abnormal in the cells expressing mutant huntingtin (Frederick et al., 2014).The study found that the xCT protein and mRNA levels were decreased in STHdh Q111/Q11 cells, compared to the wild-type STHdh Q7/Q7 cells, as well as in the striatum of the R6/2 HD mice.In accordance with the dysfunction of xCT, the study found decreased cellular GSH levels as well as increased oxidative stress in the STHdh Q111/Q11 cells.

Other diseases
The dysfunction of L-Glu transporters and the elevated levels of extracellular L-Glu is one of the major causes of epilepsy (Barker-Haliski and White, 2015).Thus, the role of EAATs and their function has also been connected to epilepsy (Green et al., 2021).However, it has been suggested that the role of EAAT3 in epilepsy might not be connected to L-Glu removal but rather to its other activities, including L-Cys uptake.Oxidative stress is a major factor behind the brain damage caused by epileptic activity and oxidative stress has been observed in epilepsy models as well as epileptic human brains (López et al., 2007;Shin et al., 2011).This suggests that EAAT3-mediated uptake of L-Cys and its role in preventing oxidative stress may play a part in preventing neuronal cell death in epilepsy.
L-Ser has also been reported to play a pivotal role in neuronal development and function.Many neurological disorders, such as schizophrenia, depression, and epilepsy, as well as some neurodegenerative diseases have been linked to reduced L-and D-Ser function in the brain (MacKay et al., 2019;Phone Myint and Sun, 2023).ASCT1 carries L-Ser across the BBB and into neurons, and therefore, the role of ASCT1 in those diseases has been speculated (Savoca et al., 1995).Biallelic mutations in the SLC1A4 gene encoding ASCT1 have also been implicated as a rare cause behind neurodevelopmental disorders affecting a small number of individuals of Ashkenazi Jewish descent (Nadirah et al., 2015).The phenotypes seen in the individuals with this SLC1A4 gene mutation match those reported in L-Ser deficiency (El-Hattab, 2016).
Curiously, amino acid transporter expression has also been noticed to be upregulated in CNS diseases.For example, EAAT1 aberrant expression has been detected in patients with dementia cases showing Alzheimer-type pathology (Scott et al., 2002).In addition, xCT has been suggested to promote neurodegeneration if disrupted.An example has been observed in the normal-appearing white matter of MS patients and the brain, spinal cord, and spleen of the experimental autoimmune encephalomyelitis (EAE) mouse model of MS (Merckx et al., 2017).

Neuroprotective effects of cysteine and its derivatives
As mentioned above, L-Cys plays an important role in detoxification, collagen production, and protein synthesis, as well as acts as an antioxidant (Paul et al., 2018).L-Cys is also the rate-limiting factor in the synthesis of GSH, which gives L-Cys a significant role in preventing oxidative stress and other issues caused by GSH deficiency.In the brain, L-Cys can be obtained from various sources; from the diet, or it can be synthesized endogenously via the reverse transsulfuration pathway from L-Met (Fig. 1) (Paul et al., 2018).The biosynthesis of L-Cys is a multistep process starting with L-Met and requiring the enzymes cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE).CBS acts by condensing homocysteine, converted from L-Met, with L-Ser to form cystathionine. Cystathionine in turn is converted into cysteine by CSE.While the transsulfuration pathway is shown to be important for GSH synthesis in the liver, in the brain it is thought to be much less significant and the RNA expression and enzymatic activity relating to the pathway were much lower in the brain than those in the liver (Ishii et al., 2004).The synthesis of GSH from L-Cys, L-Glu, and L-Gly has two enzymatic steps involving ATP (Fig. 2) (Lu, 2013).The first rate-limiting step is a reaction between L-Cys and L-Glu to form γ-glutamylcysteine.This step is mediated by glutamate-cysteine ligase (GCL).Then, γ-glutamylcysteine reacts with L-Gly to form GSH in a reaction catalyzed by glutathione synthetase (GSS).
In addition to GSH, L-Cys is also a substrate for the generation of H 2 S, an important gasotransmitter (Paul et al., 2018).H 2 S has a key role in various physiological processes in the brain, and dysregulation in the generation of H 2 S has been found in several neurogenerative diseases.In mammals, the synthesis of H 2 S is mediated by three enzymes, namely CSE, CBS, and 3-mercaptopyruvate sulfurtransferase (3-MST).CSE generates H 2 S from L-Cys or homocysteine while CBS utilizes a combination of these two.3-MST generates H 2 S from 3-mercaptopyruvate, formed from the metabolism of L-Cys and α-ketoglutarate by cysteine aminotransferase.All three enzymes are found in the central nervous system, but CSE mainly generates H 2 S in the peripheral tissues while CBS is the major generator in the brain (Paul and Snyder, 2015).

N-acetylated L-Cysteine and other prodrugs
Since brain delivery of L-Cys seems to be very important for healthy brain functions and the abnormalities related to L-Cys transportation have been associated with many CNS diseases, several different attempts have been reported to increase the levels of L-Cys in the brain.One of these is N-acetyl-L-cysteine (NAC), which is a N-acetylated derivative of L-Cys and widely known as a mucolytic agent, paracetamol toxicity antidote, and an antioxidant (Fig. 2).It has been proposed that NAC elicits its antioxidant properties by acting as an L-Cys precursor (or a prodrug that is bioconverted by acetylase I), which increases the levels of L-Cys and consequently, the synthesis of GSH.NAC also activates extracellular signal-related kinase pathways, thus protecting cells from apoptosis (Soo et al., 2020).Moreover, several reports have shown in vitro and in vivo neuroprotective benefits of NAC.For example, a study using the rodent phencyclidine (PCP) model of schizophrenia has shown that NAC reversed the psychotomimetic effects via activation of L-Cys/L-Glu antiporters, whose decrease may account for many pathological events thought to underline schizophrenia (Baker et al., 2008).On the other hand, a treatment of transgenic R6/2 mice (Huntington's disease model) with NAC alongside an L-Cys-enriched diet has shown a delay in the onset of motor abnormalities as well as enhanced survivability.Cystamine, a decarboxylated derivative of cystine, has also been shown to increase L-Cys levels in the mouse brain and to have neuroprotective properties (Fox et al., 2004).Nevertheless, overall, there have not been human trials with adequate sample sizes needed to determine the reliability of NAC as a treatment in different CNS diseases, such as AD and PD, toward which it has attracted a lot of attention (Monti et al., 2016;Hsiao et al., 2008).
Another example is N-acetylcysteine ethyl ester (NACET) which is a more lipophilic derivative or prodrug of L-Cys compared to NAC (Fig. 2) (Giustarini et al., 2012).It has been reported to have a high antioxidant potential and higher bioavailability than NAC due to its greater ability to permeate into the tissues and cells, including the brain.Subsequently, NACET is hydrolyzed to more hydrophilic NAC and eventually to L-Cys which are, as ionic compounds, more easily trapped in the cells.To improve the stability of NAC, L-2-oxothiazolidine-4-carboxylic acid (OTC) has also been reported (also called procysteine), with strong antioxidant and anti-inflammatory functions.The advantage of OTC is that it avoids the spontaneous disulfide linkage formation of free sulfhydryl groups and it increases L-Cys levels and subsequently GSH productions after a bioconversion reaction mediated via 5-oxo-L-prolinase (Fig. 2).Being a more stable L-Cys derivative, OTC has greater chances to be delivered across the BBB and elicit its neuroprotective effects site-selectively (Ni et al., 2022;Park et al., 2004;Önen Bayram et al., 2016).

S-alkylated and sulfonylated L-Cysteine derivatives
S-Methyl-L-cysteine (SMC), S-ethyl-L-cysteine (SEC), S-propyl-Lcysteine (SPC), and S-allyl-L-cysteine (SAC) are organosulfur compounds found in abundant amounts from garlic (Allium Sativum) (Fig. 2) (Hsu et al., 2006).All of them are known to possess extremely high antioxidant properties and they are highly soluble in water.SEC has been used against pulmonary tuberculosis already in the 1950s, however, the effects of these derivatives in the brain are less well known.It has been shown that SAC exerts significant protective effects against endoplasmic reticulum (ER) stress-induced neurotoxicity and cell death that are involved in various neurological diseases, including brain ischemia, AD, PD, ALS, and HD.Recently, it has been shown that there is a probable target molecule for S-alkylated L-cysteine derivatives; Calpain, a cytoplasmic cysteine protease, an enzyme responsible for the cleavage and activation of caspase-12 (Kosuge, 2020).The proposed mechanism of exertion in neuroprotective effect against ER stress signal is direct inhibition of Calpain enzyme, ultimately diminishing caspase-12 activation and subsequent induction of apoptosis (Colín-González et al., 2015).In addition, SAC, SEC, and SPC have directly decreased the production of Aβ-peptide in the brains of mice with D-galactose-induced aging and exerted neuroprotective effects against dopaminergic neuron injury in a murine model of Parkinson's disease (induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPTP).
S-Sulfo-L-cysteine (SSC) is another S-derivatized L-cysteine compound that is an effective NMDAR agonist (Fig. 2) (Plate et al., 2019).SSC is transported via xCT, but curiously, it has been associated with an induction of seizure-like behavior in sulfite oxidase (SOX) deficient Zebrafish, resulting in overstimulation of ionotropic glutamatergic receptors.In humans, this SOX deficiency can lead to seizures, severe brain damage, and even early childhood death.Thus, the role of SSC in neuroprotection is questionable and creates a shadow also over the other S-derivatives of L-Cys.Therefore, all the effects, both neuroprotective, but also neurotoxic ones of all S-derivatized L-cysteines with different patient groups need to be carefully explored.

Cysteine String Protein (CSP)
Cysteine String Protein (CSP) is a member of the DnaJ/Hsp40 family of co-chaperones that are widely distributed mainly at neuronal synaptic vesicles in the presynaptic terminal, membranes, and lysosomes.The name derives from a chain containing 12-15 L-Cys residues (Burgoyne and Morgan, 2015).The existence of CSP contributes to the normality of protein folding in the presynaptic terminal.The major mechanism in neuroprotection is maintaining the synaptosome-associated protein of 25-kDa (SNAP-25) and facilitating the entry to the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNARE) complex.The declination of SNAP-25 and SNARE complex has been shown to reduce the lifespan of mice and increase the rate of neurodegeneration in AD, PD, and HD (Huang and Zhang, 2022).Thus, therapies that can support normal CSP function in the brain, may have a potential in the prevention of neurodegeneration in the future.

Future prospects
One way to improve the brain transportation of selected amino acids is to convert them into substrates of another "surrogate" transporter, e. g., via the prodrug approach.However, as described above, many essential amino acids are substrates of several distinct transporters, rather than only one specific transporter.Therefore, amino acid supplements and the use of more stable amino acid derivatives have shown improvements in cognitive symptoms in diseases, where a specific amino acids transporter is downregulated or its function is abnormal.Perhaps, more attention should be paid to how the downregulated/ abnormal transporters could be upregulated/stabilized.Many amino acid transporters are upregulated in cancer cells that have a higher demand for precursors of protein synthesis.For example, hypoxiainducible factor (HIF) 1α or 2α, a translational regulator, is known to induce the expression of LAT1 and SNAT2 under hypoxic conditions (Morotti et al., 2019;Zhang et al., 2021).Therefore, in normoxic conditions, in which HIF is rapidly degraded, stabilizing HIF complex, could be one option to upregulate the expression of the above-mentioned amino acid transporters.Depending on the transporter, post-translational modifications may also be involved affecting the protein stabilization and/or specific transportation of these carriers to the plasma membrane, which could be also used as a target in future therapies.Nevertheless, not only the expression but also the function of amino acid transporters need to be considered.For example, the timing of the amino acid supplementation may be very crucial for specific amino acids, not to compete with other endogenous or exogenous transporter substrates.However, aberrant transport function may be more difficult to normalize if the reason lies in the polymorphic difference of the given transporter, and hence, utilizing a surrogate transporter and prodrug approach could be a feasible strategy to improve the transportations of related amino acids into the brain.
A clever prodrug design of amino acids requires a detailed understanding of the surrogate transporter structure and function.The current technologies in structural biology have enabled the detailed understanding of different conformers of transporters, including inward-open stages, outward-open stages, and everything between those.Structural biology with cryogenic electron microscopic (Cryo-EM) structures, has also revealed potential ligand binding sites.Together with computational approaches, such as molecular dynamics simulations (MDS), the dynamicity of transport processes can be better understood in the future, which is expected to improve the success rate of drug development processes (Majumder et al., 2018;Colas et al., 2016).
Via the prodrug approach, amino acids could also be coupled with a specific drug used for the treatment of the selected disease and gain dual effects.For example, utilization of L-Trp, a precursor of the monoaminergic neurotransmitter serotonin (5-hydroxytryptamine, 5-HT), could have potential in depressive disorders and other mood disorders, including anxiety disorders, schizophrenia, addiction, attention deficit hyperactivity disorder (ADHD), and autism.L-Trp could be coupled with drugs that are used to treat these diseases, like benzodiazepines, β-blockers, or D 2 antagonists, to form a prodrug (e.g., LAT1utilizing one), similarly to L-Tyr that has been successfully used in the past (Gynther et al., 2016).However, as with amino acid supplements, also with novel prodrugs and amino acid derivatives, the selectivity of transporter expression in the CNS vs. peripheral tissues may create challenges that are difficult to control.Many amino acid transporters are also expressed throughout the body and as mentioned, many amino acids are carried by several transporters.Moreover, variations, not only between the species but also between different patients complicate the situation and hinder successful drug development.Therefore, pharmacoproteomics should be implemented more effectively in personalized medicine approaches in the future.
As highlighted in this review, the GSH synthesis in the brain seems to be impaired in many CNS diseases.Therefore, one practical strategy would also be the development of GSH prodrugs or GSH or L-Cys precursor prodrugs, such as prodrugs of γ-glutamylcysteine, cystathione, or homocysteine.Since the half-life of GSH itself is relatively short, only ca. 90 min in vivo (Baudouin-Cornu et al., 2012), the latter approach, delivering GSH precursors could be a more feasible strategy to achieve higher GSH into the brain, rather than carrying GSH across the BBB.Moreover, since the intracellular GSH concentration does not regulate GSH synthesis rate, supplying sufficient amounts of GSH precursors is crucial in maintaining sufficient GSH levels in cells.Nevertheless, as mentioned above, oxidative stress in the brain is not the only mechanism behind various distinct CNS diseases, and therefore, a single anti-oxidative therapy will not cure and halt the progression of these diseases.However, as an adjuvant therapy to other disease-modifying drugs, it may still hold great potential.

Conclusions
The BBB has long been identified as an important barrier in protecting the brain from unwanted compounds while allowing essential substances, such as amino acids, to reach the brain via specific transport mechanisms.Dysfunction of amino acid transporters either at BBB or in neurons, especially when coupled with other factors hindering the regulation of amino acid homeostasis, can exacerbate the neurological symptoms related to various CNS diseases and disorders (Fig. 3).The role of L-Cys in the brain is interesting, since oxidative stress caused by lack of GSH is often present in many CNS diseases/disorders.Therefore, the importance of normal expression and/or function of the amino acid transporters carrying the GSH precursor, L-Cys, is critical.Notably, xCT (SLC7A11) and EAAT3 (SLC1A1) are already known to be involved in the pathogenesis of schizophrenia, epilepsy, and HD.Moreover, treatment of schizophrenia and HD with L-Cys derivatives, such as NAC, has decreased the typical symptoms of these diseases.This suggests that L-Cys and its derivatives or prodrugs might offer a viable therapeutic option to treat CNS diseases that suffer from GSH deficiency in the future.However, these therapies seem to be in their infancy and more efforts should be paid to the development of transporter expression-or function-modifying therapies.

Fig. 3 .
Fig. 3. Amino acids and their derivatives play a key role in neurological disorders.

Table 1
Amino Acid Transporting System Expressed in the Brain.The transporters can be Na + or pH-dependent (+) or Na + or pH-independent (− ).

Table 2
Brain expression patterns of selected amino acid transporters.