GSH is a major antioxidant that maintains the homeostasis of redox states in cells, and plays important roles in maintaining the physiological functions of all cells in vivo. The thiol (sulfhydryl, SH) residues play an important role in maintaining the redox state homeostasis intracellularly. In mammals, Cys and methionine (Met) are particularly important as thiol-containing amino acids [10], but GSH is the most abundant thiol-containing substance (derived from a non-protein) in all kinds of cells. The functions of GSH in living cells are diverse, and include roles in maintenance of the intracellular antioxidant system, redox balance, Cys transport/storage, cell signaling, regulation of some enzyme activities, gene expressions, and cell differentiation/proliferation [11]. GSH is especially abundant in the liver and kidney [12], both of which utilize the transsulfuration pathway to produce Cys from Met via homocysteine [13], while it is present at lower levels in the brain, where the regulatory system for GSH synthesis is independent of that in peripheral tissues. Therefore, the molecular mechanisms underlying GSH dysfunction in the brain differ from those in peripheral tissues.

All of the major biological processes of GSH involve the redox state of the thiol residue within the GSH molecule. Two molecules of GSH are oxidized to produce one molecule of GSH disulfide (GSSG) in order to eliminate reactive oxygen species (ROS)/reactive nitrogen species (RNS), or to maintain intracellular redox homeostasis, and GSSG can be reduced back to two GSH molecules via reaction with GSH reductase (GR). The intracellular GSH/GSSG ratio is 100 or more in the steady state, but decreases to 10 or less under oxidative stress conditions [14]. Proteins oxidized by ROS/RNS are reduced by glutaredoxins (Grxs) or thioredoxins (Trxs) [15] (Figure 1). Functionally, Grxs and Trxs share many common features; however, Grxs are more versatile than Trxs in terms of their substrate selectivity and reaction mechanisms [16]. The isoforms of Grxs and Trxs are known as cytosolic Grx1 and Trx1, and as mitochondrial Grx2 and Trx2 in mammals [16,17]. These isoforms are involved in controlling intracellular redox signaling for the cellular processes of apoptosis and proliferation [17,18]. Grx2 and Trx1 are also found in the nucleus. Many transcription factors are known to undergo reduction by Grxs and Trxs. In order for a transcription factor to bind to DNA, the thiol groups of Cys residues in the DNA-binding site should be in the reduced form. The thiol groups mainly exist in their oxidized forms, and are reduced in the process of becoming activated to enable DNA binding. Subsequently, the oxidized Grxs and Trxs are reduced back via reaction with GSH and Trx reductase (TrxR), respectively. GSH functions as an enzyme cofactor for Grxs, which are low-molecular-weight redox enzymes that are also known as thiol transferase, to maintain cellular redox homeostasis, and also acts as the primary reductant of the disulfide bonds of oxidized proteins [15] (Figure 1). However, excessive oxidative stress causes irreversible oxidation of the thiol residues and impairs cellular protein function [19,20]. In particular, Cys residues in active sites or functional motifs of intracellular proteins are important for their protein functions. Oxidative stress by ROS/RNS on the Cys residues in proteins can cause irreversible modifications that lead to critical dysfunction of the proteins [21]. GSH can also react with intracellular protein thiol residues to protect protein functions related to enzyme activities, DNA binding by transcription factors, and protein stability [22,23,24]. Therefore, under such oxidative stress conditions, GSH reversibly binds to the thiol residues to prevent irreversible changes in proteins due to oxidative stress. This post-translational modification, called “glutathionylation”, is reversible and protects the intracellular signal transduction system against oxidative stress [19] (Figure 1). The caspase family of Cys proteases, which induces cell apoptosis, would be a potential target for glutathionylation. Caspase-3, an important regulator of apoptotic responses, can undergo glutathionylation, leading to the inactivation of caspase-3 by GSSG in a dose- and time-dependent manner [25], suggesting that apoptosis can be regulated by glutathionylation. Once the cellular environment is free from oxidative stress, the disulfide bonds in the proteins are reduced back by Grx to function normally under physiological conditions [15]. Thus, the regulation of the redox state by intracellular GSH is extremely important for maintaining cellular functions under both physiological and pathological conditions. Especially in the brain, the regulatory mechanism of GSH function is more fragile in neurons than in glial cells, and intracellular GSH levels are also lower in neurons than in glial cells [26]. Under some pathological conditions, decreased GSH levels could have critical influences on neuronal activities, leading to neurodegeneration.

GSH occupies approximately 95% of non-protein thiol groups in vivo and is ubiquitously present in mammalian cells at concentrations of 0.5 to 10 mM, depending on the tissues [27,28]; these concentrations are 10 to 100 times higher than the concentrations of Cys in mammalian cells [29]. GSH is the major intracellular thiol compound, and is made from Glu, Cys, and Gly by two-step enzymatic reactions requiring ATP. The first step of these reactions is mediated by Glu-Cys ligase (GCL), and the second by GSH synthetase (GSS). In intracellular GSH synthesis, GCL can be the rate-limiting enzyme under the condition that all substrates are sufficiently present for the reactions, but the intracellular Cys concentration is much lower than those of Glu or Gly under physiological conditions, suggesting that the Cys availability is limiting for GSH synthesis [30,31]. GCL is comprised of both a catalytic (GCLc) and a modulatory (GCLm) subunit. GCLc is responsible for all of the enzyme activity of GCL, which is regulated via feedback inhibition by GSH [32]. Most GSH is present in the cytoplasm, where it is synthesized in mammalian cells [33]. Although mitochondria contain about 5–15% of all the GSH in the cell [34], they cannot synthesize GSH by themselves because they lack GCL [33]. The finding that GCLc- or GSS-deficient mice are non-viable in the embryonic period [35,36], while GCLm-deficient mice are viable and fertile with decreased GSH levels in the tissues compared to those of wild-type mice [37], suggest that GSH is essential for embryogenesis.

The highest GSH concentration in the body is in the liver (about 5 to 10 mM) [12], but hepatocytes can also produce Cys for GSH synthesis from Met via the transsulfuration pathway [38]. GSH in the liver is then released systemically, but it is decomposed in the blood, with the result that the blood GSH concentrations (approximately 2 to 20 µM) are hundreds to thousands of times lower than those in the liver [28]. In addition, GSH cannot directly enter the brain due to the existence of the blood-brain barrier (BBB). Moreover, extracellular GSH cannot be directly transported into the cells, and thus the three amino acids used as substrates for GSH synthesis should be taken up into the cells via transporters.

The brain tissue is generally rich in unsaturated fatty acids, which are targets of oxidative stress, and has relatively low levels of antioxidants or antioxidant enzymes. ROS, such as singlet oxygen (¹O₂), superoxide (O₂·⁻), and hydroxyl radicals (·OH), are endogenously produced by mitochondria, cytochrome P450 metabolism, peroxisomes, and inflammatory cell activation. Mitochondria generate most ROS, including O₂·⁻, into the matrix and the intermembrane space via the electron transport chain. The steady-state concentration of O₂·⁻ is about 5–10 times higher in the mitochondrial matrix than in the cytoplasm or nucleus [39], but the mitochondrial matrix contains Mn-superoxide dismutase (SOD), which can react with O₂·⁻ to form hydrogen peroxide (H₂O₂) (Figure 2). In addition, O₂·⁻ leaked into the cytoplasm reacts with Cu/Zn-SOD (SOD1) to form H₂O₂. H₂O₂ is toxic to eukaryotic cells at concentrations of 0.1 to 1 × 10⁻³ M, but the reaction with catalase or GSH peroxidase (GPx) can decompose H₂O₂ to oxygen and water. As a result, the concentrations of H₂O₂ in mitochondria are maintained in the range of 10⁻⁹ to 10⁻⁸ M [15]. Such high concentrations of H₂O₂ are unlikely to occur under physiological conditions in vivo. However, overproductions of both O₂·⁻ and H₂O₂ can be induced by mitochondrial dysfunction [40]. The increased H₂O₂ produces ·OH, which possesses the highest reactivity and the strongest oxidizing power among ROS, via the Fenton reaction (Figure 2). In addition, O₂·⁻ reacts with nitric oxide (NO) to generate peroxynitrite (ONOOˉ) (Figure 2), which targets DNA, proteins, and lipids, causing DNA damage, dysfunction of enzymes, receptors, transporters, and membrane channels, as well as protein aggregation, mitochondrial dysfunction, and lipid peroxidation [15]. ONOOˉ is produced approximately one million times faster, and can spread approximately 10,000 times farther over cells, than ·OH [41]. ONOOˉ is more globally toxic within tissues than ·OH, whose toxicities are limited to the local area inside the cells [42]. GSH acts protectively against oxidative stress by reacting directly with NO, O₂·⁻, H₂O₂, ·OH, and ONOOˉ (Figure 2). GSH also acts as an enzyme cofactor for GPx to degrade H₂O₂ and hydroperoxides (ROOH), and is involved in detoxifying electrophilic xenobiotics via GSH-S-transferase (GST) [43] (Figure 2). From these protective functions, GSH is considered to play an important role not only under physiological conditions but also under pathological conditions induced by oxidative stress in order to maintain the homeostasis of cell functions.