*3.1. Reactive Nucleophilic Species*

Endogenous reductive stressors include nucleophilic free radical, inorganic, and organic molecules and antioxidative enzyme. (Figure 1b). Superoxide (O2 −•) anion is one of the reactive nucleophilic species and a powerful reducing agent under physiological conditions, which initiates reaction cascades generating another ROS, such as hydrogen peroxide (H2O2) and sulfur dioxide (SO2) derivatives. Hydrogen sulfide (H2S), thiolate (RS−), hydropersulfide (RSS−), and disulfide (RSSR) are reactive

nucleophilic species that can participate in nucleophilic substitution in vivo [133]. Selenium is more nucleophilic than sulfur due to its greater electron density. The selenol (RSeH) portion of selenocysteine (C3H7NO2Se) is ionized at physiological pH, making it more nucleophilic against oxidative species [164,165] (Table 2).

### *3.2. Antioxidative Enzymes*

Reductive stress is induced by excessive levels of reductive stressors that results from an elevation in GSH/GSSG ratio, NAD+/NADH, NADP+/NADPH and/or or overexpression of antioxidative enzymatic systems such as the GSH system, catalase, thioredoxin-peroxiredoxin (TRX-PRDX) system, α-ketoglutarate dehydrogenase (GPDH), and glycerol phosphate dehydrogenase [166,167]. The reductive stressors deplete reactive oxidative species and are harmful as oxidative stressors and implicated in pathological processes in AD, PD, and sporadic motor neuron disease, among others [168].

The GSH system consists of GSH, the enzymes for synthesis and recycling including gamma-glutamate cysteine ligase, glutathione synthetase, glutathione reductase (GSR), and gamma glutamyl transpeptidase, and the enzymes for metabolism and antioxidation including glutathione S-transferase and GPx [169]. The GPx is an enzyme containing four selenium-cofactors that catalyzes the reduction of hydrogen peroxide (H2O2) to water molecule (H2O) and organic hydroperoxide (ROOH) to alcohol (ROH) by converting reduced monomeric GSH to GSSG. Glutathione s-transferases show high activity with lipid peroxides [170]. Eight isozymes are in the cytosol, membrane, and plasma, protecting the organisms from oxidative stress [171].

Most studies on peripheral blood GPx activity reported nonsignificant results in a mixed population of MS [56,59,172–174]. However, lower mean GPx activity of erythrocyte lysates in remission and higher mean GPx were reported in acute relapse of RRMS [60]. GPx activity in CSF was found lower in MS patients [61]. The GSR activity of lymphocyte and granulocyte lysates were not significantly different in MS, compared to controls. However, a significant correlation of GPx and GRx was observed in controls, but not in MS [62]. Mean GRx activity of CSF was found significantly higher in MS patients [61] (Tables 3 and 4).

Catalases a tetrameric heme- or manganese-containing dismutase that catalyzes the conversion of two hydrogen peroxide (H2O2) molecules to water (H2O) in the presence of small amount of hydrogen peroxide. The cofactor is oxidized by one molecule of hydrogen peroxide and then regenerated by transferring the bound oxygen to a second molecule of substrate. The enzyme is located in the peroxisomes, the cytosol of erythrocytes, and the mitochondria, removing harmful hydrogen peroxides to prevent cellular and tissue damage [175].

Studies on the catalase activity of peripheral blood samples reported equivocal results in MS. The catalase activity of granulocyte lysates was found lower in MS patients, compared to controls [176]. The activities of CSF and plasma catalase were found increased in CIS and RRMS patients, compared to healthy controls, and MS patients with lower EDSS had higher plasma and CSF catalase activities [84] (Tables 3 and 4).

In TRX-PRDX system PRDXs catalyze the reduction of H2O2 to H2O. H2O2 oxidizes the peroxidatic cysteine of PRDXs to protein sulfenic acid (PSOH), which can react with the thiol (SH) group of the resolving cysteine to yield the formation of an inter-(typical) or intramolecular (atypical) disulfide bond. TRX/TRXR system mediates the reduction of the PRDX disulfide bond. TRX reduced state is maintained by the flavoenzyme TRXR in the presence of NADPH. When H2O2 exceeds the normal levels, PRDXs are overoxidized from PSOH to protein sulfinic acids (PSO2H). The latter can be reduced back to the native form of the enzyme by sulfiredoxin (SRX) in the presence of ATP. However, further oxidation of PRDXs to PSO3H is irreversible [177].

Serum Trx1 was significantly increased in the newly diagnosed MS patients, compared to controls. *TRX1* and *APEX1* mRNA expressions were significantly higher in the newly diagnosed MS patients, patients under INF-β treatment, and patients who received immunosuppressant azathioprine or betamethasone, compared to healthy controls [178]. *PRDX2* mRNA is upregulated and PRDX2

expression is higher in MS lesions white matter of autopsy tissue of patients its expression level is positively correlated with the degree of inflammation and oxidative stress [179] (Tables 3 and 4).

α-KGDH is a mitochondrial enzyme in Krebs cycle, which catalyzes α-ketoglutarate, coenzyme A and NAD<sup>+</sup> to succinyl-CoA, NADH and CO2, transferring an electron to the respiratory chain [180]. KGNH activity is sensitive to redox status. H2O2 reversibly inhibits KGNH by glutathionylation of lipoic acid cofactor, resulting reducing electron supply to the respiratory chain. A lipid peroxidation product 4-hydroxy-2-nonenal (4-HNE) reacts with lipoic acid cofactor, inhibiting α-KGDH activity [181]. The pyruvate tolerance test showed higher activity of α-KGDH in serum of MS patients [182]. However, reduced expression and activity of mitochondrial α-KGDH was observed in demyelinated axons that correlated with signs of axonal dysfunction (Table 3) [183].

α-GPDH catalyzes the reversible redox conversion of dihydroxyacetone phosphate to sn-glycerol 3-phosphate, linking carbohydrate and lipid metabolism. A loss of α-GPDH in oligodendrocytes were observed in chronic plaques of MS patients, suggesting the presence of antioxidant capacity impairment [63] (Table 3).

Nrf2 is a transcriptional factor of the antioxidative enzyme genes including catalase, GPx, GRx, glutathione S-transferase, and SOD. In response to oxidative stress, the Kelch-like ECH-associated protein 1 (KEAP1) inhibits the ubiquitin-proteasome system in the cytosol and facilitates the translocation of Nrf2 into the nucleus to bind to the *cis*-acting enhancer sequence of the promotor region, the antioxidant response elements [184,185]. Activation of the Nrf2-Keap1 pathway has been observed in various types of cancers, accompanied with reduced antioxidant capacity and elevated oxidative stress and inflammation [186]. The cytoplasmic and nucleic Nrf2 protein expression of PBMC was increased and correlated with clinical improvement in MS patients on 14-month course of natalizumab, an α4 integrin receptor blocker [110] (Tables 3 and 4).

Other transcriptional factors involved in energy metabolism have been investigated. Peroxisome proliferator-activated receptors (PPARs) are a transcriptional factor of the gene regulating energy metabolism including glucose metabolism, fatty acid oxidation, thermogenesis, lipid metabolism, and anti-inflammatory response [187]. PPARs have attracted growing attention as promising targets of many diseases such as diabetes and hyperlipidemia [188]. An isoform PPAR-gamma (PPAR-γ) was elevated in CSF samples of MS, compared to controls [64]. Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) 4 integrin receptor blocker is a transcriptional coactivator that regulates the genes involved in energy metabolism. Reduced PGC-1α expression was associated with mitochondria changes and correlated with neural loss in MS [189] (Tables 3 and 4).
