3.1.2. Catalase

Catalase (CAT), a heme-containing tetrameric protein is a common antioxidant enzyme naturally produced by the body when exposed to oxygen. H2O2 generated inside the cell is enzymatically catabolized in aerobic organisms by catalase and the activity of several peroxidases. CAT is one of the most efficient enzymes that cannot be saturated by H2O2 at any concentration. It reacts with H2O2 to form water/alcohol and oxygen using Fe as a cofactor [55,56]. CAT protects cells by detoxification of the generated H2O2 and plays an important role in the acquisition of tolerance to oxidative stress as an adaptive response [57]. CAT can maintain the concentration of O2 either for repeated rounds of chemical reduction or for direct interaction with the toxin [58] (Figure 2). Furthermore, inhibition of CAT activity results in enhanced cytotoxicity and increased ROS, indicating an important role of CAT in maintaining the oxidative balance [59]. Specifically, mislocalized CAT is associated with accumulation of H2O2 [60] and other ROS in the cells leading to compromised neurological function [61]. Thus, deficiency or malfunction of CAT is postulated to be related to the pathogenesis of many age-associated degenerative diseases.

CAT treatment reduces H2O2 levels and improves neuronal survival following Aβ-induced toxicity in neuronal culture [62]. In Aβ toxicity, CAT–SKL (serine-lysine-leucine) treatment reduced the pathology of microglial activation, and rat brains did not show long-term memory impairments via the reduction of H2O2 levels [63]. In the PD model, mutant α-synuclein inhibited the expression and activity of CAT and induced low catalase activity and high H2O2 production [64]. This antioxidant enzyme plays an important role in maintaining oxidative balance.

#### 3.1.3. Glutathione Peroxidase

Glutathione peroxidase (GPx) catalyzes the reduction of a variety of hydroperoxides (ROOH and H2O2) to water or the corresponding alcohols using GSH to protect mammalian cells against oxidative damage [65] (Figure 2). GPx has four identical subunits containing one selenocysteine in each residue for essential enzyme activity, which are classified as selenium-containing GPxs (GPx1–4 and 6) and their non-selenium congeners (GPx5, 7, and 8) [30]. The glutathione redox cycle is mostly active during low levels of oxidant stress, while CAT is more significant in protecting against severe oxidant stress. GPx isoenzymes appear to have antioxidant functions at different locations and cellular compartments, and the levels of expression of each isoform vary depending on the type of tissue, even though their expression is omnipresent. Furthermore, GPx is responsible for the conversion of GSH to oxidized glutathione disulfide (GSSG) and glutathione reductase (GR) reduces GSSG back to GSH [66]. GPx1 and GPx4 are found in most tissues and the predominant forms of GPx are found in brain tissue. GPx1 reduces H2O2 and organic hydroperoxides and is expressed both in neurons and astrocytes [67]. GPx4, the phospholipid hydroperoxide glutathione peroxidase, is located in the cytosol and a membrane fraction that can directly reduce phospholipid hydroperoxides, fatty acid hydroperoxides, and cholesterol hydroperoxides [68]. In the brain, mitochondrial and cytosolic GPx4 isoforms have been detected in neurons of the cerebral cortex, hippocampus, and cerebellum. In contrast, GPx4 in glial cells is barely activated under normal physiological conditions, whereas reactive astrocytes upregulate the expression of GPx4 following selective brain injury [31]. Recently, GPx4 has become well known as a key regulatory factor in ferroptosis which is a non-apoptotic and iron-dependent programmed cell death pathway that causes a rapid elevation of oxidative stress [69]. As the function of GPx is related to normal development and cellular metabolism via the regulation of oxidative stress, GPx4 function is potentially the key for cell survival.

GPx activity decreased significantly in PC12 cells during oxygen-glucose deprivation [70], and a two-fold increase in GPx and a three-fold increase in CAT activities were observed during a time course that reflected the temporal increase in nerve growth factor (NGF) in a brain contusion model [71]. The ferroptotic potential of neurons in the forebrain regions of the hippocampus and cerebral cortex is severely affected in AD patients, with increased vulnerability to ferroptosis. The role of GPx4 as a key regulator of ferroptosis was observed in Gpx4BIKO transgenic mice, a mouse model with a conditional deletion of GPx4 in forebrain neurons. Gpx4BIKO transgenic mice showed significant deficits in spatial learning and memory function and exhibited hippocampal neurodegeneration compared to control mice [72]. In human PD tissue biopsies, GPx4 expression was upregulated and ferroptosis-related events were observed [73].

#### 3.1.4. Thioredoxin

The thioredoxin (Trx) system consists of two types of antioxidant oxidoreductase proteins, Trx and thioredoxin reductase (TrxRs) with NADPH as an electron donor. Trx has a conserved active site such as Cys-Pro-Gly-Cys, that has an important function of acting as an active oxidoreductase and electron donor of some peroxiredoxins (Prx) which are crucial for the reduction of peroxides [74]. Trx is an important regulator of cellular function that can respond to redox balance by modulating signaling pathways, transcription factors, and immunological responses for cell survival in many conditions, including neurodegenerative diseases [75]. There are three isoforms in the Trx family such as Trx1 in the cytosol, Trx2 in the mitochondria, and a testis-specific Trx3 in mammalian cells. Trx1 exists in cell compartments such as the nucleus and the plasma membrane or as a secreted protein, depending on its localization and function in different cell types [76]. Trx1 is coupled with Prx 1/2 and methionine sulfoxide reductase and is essential role in the control of growth and apoptosis. TrxR 1 and Trx 1 are observed in neuronal synaptic vesicles. In addition, Trx 1 and 2 expressions are also present mainly in rat brain neurons. Neurons with mitochondrial dysfunction by complex IV inhibition show low levels of Trx and are thus, more vulnerable to H2O2. Reduced Trx is a powerful reductase that acts through a disulfide–dithiol exchange mechanism to catalyze the conversion of disulfide bonds into thiols with high efficiency [77]. The disulfides in the oxidized Trx are converted to thiols by the consumption of NADPH [78] (Figure 2). TrxR, a homodimer, catalyzes the reduction of the disulfide at the Trx active site and is encoded by three distinct genes: the cytosolic TrxR (*TrxR1*), mitochondrial TrxR (*TrxR2*), and thioredoxin-glutaredoxin reductase (*TrxR3*). TrxR can directly reduce substrates such as peroxides, including lipid hydroperoxides, H2O2, and protein disulfide isomerase [76,79]. TrxR is also involved in the regeneration of other antioxidant molecules such as dehydroascorbate, lipoic acid, and ubiquinone [80–82]. TrxR is very important when other selenoproteins including GPx lose most of their activity and it effectively donates electrons during DNA synthesis [83]. TrxR activation in the rat brain can be maintained at a certain level under severe selenium (Se)-deficient conditions. Both Trx and TrxR are widely expressed in tissues and organs, including the brain. In a central nervous system (CNS) study, it has been suggested that increased expression of Trx and TrxR is closely associated with cell damage due to oxidative stress [84,85]. In addition, NADPH and human TrxR are efficient electron donors to human plasma peroxidase and reduce hyperoxides, even with low GSH capacity [86].

The upregulation of Trx was observed as a neuroprotective effect on retinal ganglion cells against oxidative stress-induced neurodegeneration [87]. Overexpression of Trx1 attenuated endoplasmic reticulum stress by regulating the activation of the molecular mechanism for neuroprotection in an in vitro and in vivo model of PD [88]. In AD brains, TrxR activity was increased in the cerebellum and amygdala [89]. It has been implied that TrxR activation increases a compensatory mechanism during increased oxidative stress that is limited by the substrate Trx and the subsequent neurodegeneration seen in AD. Another evidence for the peripheral response to oxidative stress during neurodegeneration is the reduction of Trx1 and TrxR1 in the plasma and erythrocytes in blood samples

from patients with Huntington's disease (HD) [90]. Exogenously administered human recombinant Trx attenuates the generation of ROS involved in cytotoxic mechanisms, ameliorates neuronal damage, and augments neurogenesis following brain ischemia/reperfusion injury in rats [91]. In the ischemic brain, administration of Prx3 and Trx2 shows substantial neuroprotective effects by reducing oxidative stress [92].

Important enzymatic antioxidants such as SOD, CAT, GPx, and Trx remove superoxide and peroxides before they react with metal catalysts to form more reactive species (Figure 2). Peroxidative chain reactions initiated by reactive species that escape enzymatic degradation are terminated via chain-breaking antioxidants, including water-soluble ascorbate, lipid-soluble vitamin E, and ubiquinone. To enhance the antioxidative effect, oxidative stress should be controlled by supplying all the antioxidant nutrients and by minimizing the effect of substances that stimulate reactive oxygen metabolites (ROM) [93].

**Figure 2.** Enzymatic antioxidant defense system against the production of reactive oxygen species (ROS). Various pathways of cell death caused by ROS and its transformation are observed in brain injury. NADPH oxidase and mitochondrial respiratory transport chain are known as major cellular sources of the superoxide radical anion (O2 −). The superoxide radical anion reacts with nitric oxide (NO) to form the peroxynitrite anion (ONOO−) which mediates oxidative modification of protein residues via an interaction with NO. The superoxide radical is dismuted by the superoxide dismutase enzyme (SOD) to form hydrogen peroxide. In addition, manganese containing superoxide dismutase (Mn-SOD) reduces the superoxide radical anion generated during the electron transport chain in the mitochondrial matrix. Catalase (CAT) and/or glutathione peroxidase (GPx) decomposes hydrogen peroxide to water and oxygen by enzymatic reactions. Hydrogen peroxide is decomposed into reactive hydroxyl radicals by reaction with catalytically active redox metals such as (copper and iron). Hydroxyl radicals can react with oxygen to form peroxyl or alkoxyl radicals which can lead to lipid peroxidation and react with DNA and primarily cause damage to DNA. Peroxiredoxins (Prx) and thioredoxin (Trx) act as redox-regulated proteins to additional redox relay bases.
