Vitamin E

Vitamin E is a major group of lipid-soluble antioxidants called tocopherols and tocotrienols, of which the most biologically active isoform is α-tocopherol [154]. It is a major chain-breaking antioxidant and exists in a low molar ratio compared to unsaturated phospholipids. The most important function of vitamin E is its antioxidant activity, which protects the integrity of cellular membranes from polyunsaturated fatty acid-generated oxygen free radicals and to act as a direct scavenger of superoxide and hydroxyl radicals [154,155]. Based on studies of brain capillary endothelial cells, the mechanism of entry of α-tocopherol into the CNS correlated to α-tocopherol and scavenger receptor class B type 1 (SRB1) levels. α-Tocopherol uptake occurs via a selective high-density lipoprotein (HDL) pathway, which modulates the expression of SRB1 receptor [156–158]. Once α-tocopherol passes through the BBB, it may be directly delivered to specialized astrocytes. Astrocyte-synthesized apolipoprotein E (ApoE) moves through the cerebral spinal fluid transporting α-tocopherol between various cell types in the CNS cell [154,159]. ApoE lipoprotein particles in astrocytes are secreted through membrane proteins and interact with low-density lipoprotein receptor-related protein (LRP) on the neurons [160]. The neurons take up these ApoE particles and distribute them throughout the body, axon, and dendrites to preserve the membrane from lipid peroxidation (Figure 3). The expression of α-tocopherol transfer protein is enhanced by an increase in ROS, and it is useful for combating neuronal damage.

**Figure 3.** Schematic representation of vitamin uptake and protective mechanisms by exogenous vitamins as antioxidants. **Vitamin C:** During uptake in the CNS, ascorbate passes through the BBB to enter directly through the SVCT-2 and/or possibly DHA through GLUT1s. Moreover, the neuronal uptake of ascorbate occurs through SVCT-2 and DHA via the GLUT1s. In the neuron, DHA can be reduced to ascorbate or released back into the extracellular space by GLUT1. Ascorbate free radicals convert to form DHA and ascorbate. Ascorbate recycles both the ascorbate free radical and DHA by cellular metabolism. Astrocytes contain ascorbate from recycling of DHA which is taken up through GLUT1s. Neurons directly acquire ascorbate via SVCT-2. **Vitamin E:** With respect to vitamin E uptake, HDL particles can pass through SRB1 receptors expressed on endothelial cells. Astrocytes that exist adjacent to the BBB take up vitamin E into the inner cell membrane. Synthesized ApoE lipoproteins take up vitamin E that is left out of an ABC transporter, for transport into neurons through LRP1 requiring vitamin E for maintenance or during conditions of oxidative stress. **Vitamin A:** In cellular retinoid signaling pathways, retinol is metabolized to all-trans-retinoic acid (ATRA). Vitamin A (retinol, ROL) binds to plasma retinol binding protein (RBP4) and circulates; RBP4 protein binds to the membrane receptor STAR6 to promote cellular absorption of retinol from the cells. A chylomicron remnant (CMRE), as a form of circulating vitamin A, can serve as a source of vitamin A for the cells and retinol is esterified and stored by lecithin: retinol acyltransferase (LRAT) and is reversibly oxidized to retinaldehyde (RAL) by retinol dehydrogenase (RDH/ADH). In addition, retinol is further oxidized to RA in an irreversible manner by retinaldehyde dehydrogenase (RALDH). ATRA regulates gene transcription through retinoic acid receptors (RAR) and/or retinoid X receptors (RXRs) which are bound to retinoic acid response elements (RARE) in the nucleus. These representative schematics are modified from [130,154,161]. Abbreviations: VC, vitamin C; VE, vitamin E; VA, vitamin A; ASC, ascorbate; ASF, ascorbate free radical; DHA, dehydroascorbic acid; GLUT1, glucose transports; SVTC-2, sodium-dependent transporters; LRP, lipoprotein receptor-related protein; GSH, glutathione; ApoE, apolipoprotein E; HDL, density lipoprotein; CMRE, chylomicron remnant; CRABP, cellular retinoic acid-binding protein; LRAT, lethicin: retinol acyltransferase; RBP, retinol binding protein; RAL, retinaldehyde; RDH/ADH, retinol dehydrogenase; RALDH, retinaldehyde dehydrogenase; RXR, retinoid X receptors; RAR, retinoic acid receptors; ATRA, all-trans-retinoic acid; BBB, blood–brain barrier.

The antioxidant ability of vitamin E is continuously restored via vitamin E recycling by other antioxidants such as vitamin C, ubiquinols, and thiols. The half-life of vitamin E in the brain tissue is slower than that of other vitamins, and it is also actively retained and protected from auto-oxidation in the brain [162]. The distribution of α-tocopherol is significantly different in the brain. These CNS-regional disparities are suggestive of the specific protective antioxidant effect of α-tocopherol. Specifically, the concentrations of α-tocopherol were relatively higher in the nuclear membranes than in the other membranes of the brain. It has been suggested thatα-tocopherol may play a role in nuclear-associated functions in the cerebellum and striatum, wherein preferential accumulation of α-tocopherol in the membrane was most apparent [163]. Vitamin E, similar to other radical scavengers/trappers, influences the flux of lipid hydroperoxide (LOOH), which is derived from both spontaneous and enzymatic formation of lipid peroxyl radicals (LOO•) on the cellular membrane [164]. The effects of vitamin E on peroxidation activity appear to involve both the radical scavenging mechanism such as the H atom donor activity and a physical interaction with the polyunsaturated lipid substrate. Tocotrienols, another form of vitamin E that are highly metabolized, show more potent inhibition of the phospholipase A2/lipoxygenases pathway as compared to tocopherols, and have different cellular bioavailability, distribution, and protein interaction in the saturated and unsaturated form of vitamin E in the brain [165]. Deficiency of vitamin E showed increased biochemical and histological markers of oxidative stress, including total glutathione and lipid peroxidation in the CNS.

Therapeutic effects of α-tocopherol by application of vitamin E in neurological lesions caused by neuronal excitotoxicity and the conditional activation of neuroglial cells have also been reported. In KA treatment-induced oxidative stress [166], delayed neuronal death was detected in the hippocampal CA3 region and ROS formation and lipid peroxidation were also increased. Both co-treatment and post-treatment with α-tocopherol (100 μM) or α-tocotrienol (100 μM) significantly increased cell survival and reduced the number of TUNEL-positive cells in the CA3 region. Increased dichlorofluorescein (DCF) fluorescence and thiobarbiturate reactive substance (TBARS) levels were decreased by drug treatment (Figure 4). In AD patients, long-term administration of vitamin D and E alone or in combination could inhibit morphological changes of neurons and improve learning and memory [167]. Vitamin E prevented the memory impairment associated with post-traumatic stress disorder (PTSD)-like behavior in rats. Significant decreases in oxidative stress biomarkers were detected with reduced glutathione/oxidized glutathione (GSH/GSSG) ratio [168]. In PD patients, omega-3 fatty acid and vitamin E co-supplementation had favorable effects on the UPDRS score and increased the total antioxidant capacity (TAC) and GSH concentration compared to placebo [169].

**Figure 4.** Neuroprotective effect of vitamin E. (**A**): Representative propidium iodide (PI) images. When hippocampal slices were exposed to 5 μM KA for 15 h, PI uptake in the CA3 region was significantly higher than the CA1 region. Co-treatment using ATPH (100 μM) or ATTN (100 μM) with KA significantly reduced PI uptake in the CA3 region compared with KA treatment alone. (**B**): Quantification of PI intensity. \* *p* < 0.05, # *p* < 0.05; one-way ANOVA followed by Dunnett's post hoc comparison (\* *p* < 0.05 vs. normal, # *p* < 0.05 vs. KA-treated cultures). This present data is a part of our previous research showing neuronal rescue after oxidative stress by alpha-tocopherol and tocotrienol treatment [166]. Abbreviations: ATPH, alpha-tocopherol; ATTN, alpha-tocotrienol; OHSC, organotypic hippocampal slice culture; KA, kainic acid; PI, propidium iodide; DCF, dichlorofluorescein.

### Vitamin A

Vitamin A, carotenoids including retinol and beta-carotene, are fat-soluble chemicals synthesized by plants and some microorganisms and have many functions in human growth, development, and health [170,171]. Vitamin A is available in the human diet as pro-vitamin A carotenoids and preformed vitamin A (retinol-alcohol form, retinal-aldehyde form, retinoic acid-carboxylic acid form, and retinyl ester-ester form). The preformed vitamin A from animal-derived food and pro-vitamin A carotenoids from plant-derived foods are converted to all-trans-retinol as vitamin A alcohol by a series of reactions in the intestine. Carotenoids, mainly via dietary intake, can function directly as antioxidants by quenching ROS through energy transfer [172]. Vitamin A deprivation was investigated in the deficiency of cognitive function in adult mice and rats, which highlights the importance of adequate vitamin A status by the retinoid signaling pathways [161]. Carotenoids are classified into pro-vitamin A carotenoids such as β-carotene and β-cryptoxanthin, which are capable of converting to retinal, and non-pro-vitamin A carotenoids such as lycopene and lutein, which cannot be converted to retinal [173]. Carotenoids act through several pathways and interact with free radicals in the plasma, mitochondria, and nuclear membranes of cells via electron transfer, hydrogen abstraction, and physical quenching [174]. Carotenoids indirectly react with cell signaling cascades, including the nuclear factor elytroid 2 (NF-E2)-related factor 2 (Nrf2), NF-κB, or mitogen-activated protein kinase (MAPK) [175,176]. The antioxidant action of carotenoids involves singlet oxygen quenching and trapping of peroxyl radicals. Retinol-binding proteins observed in the BBB regulate the access of retinol into the brain [177]. High concentrations of retinol and carotenoids have been observed in the postmortem human frontal lobe cortex [178]. β-carotene, a precursor of retinol and retinoic acid, is reported to be a potent free radical quencher, singlet oxygen scavenger, and lipid antioxidant in tissues and plasma. Therefore, β-carotene acts in a hydrophobic environment such as the lipid core of the membranes and is used

faster than α-tocopherol, implying that β-carotene is more favorable than α-tocopherol to quench lipophilic radicals in the membrane. The most efficient synergistic inhibition during oxidative stress was observed with a combination treatment of α-tocopherol and ascorbic acid [179].

Retinoids, compounds structurally related to vitamin A, are considered vitamin A derivatives that contribute to regular cellular morphogenesis, proliferation, and differentiation. Retinoids are involved in normal signaling cascades in modulating brain functions [180]. Retinoids modulate the availability of glucocorticosteroids in the brain, an important biological mechanism that can be explored in many stress-related pathologies to prevent alterations in the plasticity of the hippocampus [181]. Retinol metabolic pathways have shown that retinol can be stored intracellularly as retinyl esters and metabolized into all-trans-retinoic acid (ATRA) as a bioactive derivative. ATRA induces cellular differentiation and growth by reacting to retinoic acid receptors (RARs). Cellular retinol-binding proteins (CRBP-I and II) and cellular retinoic acid-binding proteins (CRABP-I, II) are distributed in the adult CNS. Furthermore, CRBP-I distribution parallels that of ATRA with expression in the meninges, hippocampus, amygdala, and olfactory bulb [182] (Figure 3). Under oxidative stress conditions such as metal exposure and production and accumulation of ROS, retinoids protect the cells against this imbalance through multiple mechanisms, including interference with ROS production, scavenging free radicals directly, upregulation of antioxidant enzymes, and signaling pathways involved in defense system such as Nrf2 signaling [183]. It has also been observed that retinoic acid has a protective effect on neuronal apoptosis and oxidative damage by reducing glutathione [184] and restoring SOD-1 and SOD-2 in the hippocampal cells [185]. The role of retinoid signal transduction in the control of dopaminergic neurotransmission was observed in the presence of high levels of retinoic acid-synthesizing enzymes [186] and RAR, which may play a critical role in controlling the survival, adaptation, and homeostatic regulation of the dopaminergic system [187]. Retinoid signaling play a physiological role in synaptic plasticity and learning and memory behaviors [188].

Retinoic acid supplementation upregulated μ-type opioid receptor 1 (MOR1) and its signaling and alleviated dyskinetic movements, which is a known consequence of prolonged administration of L-DOPA, in Pitx3ak/ak mice [189]. Moreover, RA triggered the neuroprotective effect on DA neurons in MPTP-treated mice model of PD. Administration of RA-loaded polymeric nanoparticle significantly reduced the loss of DA neuron in the substantia nigra as well as their neuronal fiber/axonal innervations in the striatum [190]. Moreover, oral administration of lycopene (5–20 mg/kg), a carotenoid with unique pharmacological properties, attenuated oxidative stress in mice with PD, which was induced with intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Lycopene supplementation also inhibits apoptosis in PD mice by decreasing Bax and caspases, and contrarily increasing Bcl-2 [191]. Dietary supplementation with astaxanthin, another carotenoid family member, significantly decreased intracellular ROS accumulation in a hippocampal neuronal cell line after exposure to glutamate and induced antioxidant mediators, such as heme oxygenase-1 (HO-1) and nuclear Nrf2 expression in vitro [192]. In multiple sclerosis (MS) patients, vitamin A supplementation had a significant effect in the treatment group for fatigue and depression. In addition, when a synthetic retinoid was tested, it showed reduction in inflammation, Aβ burden, and tau phosphorylation with associated cognitive benefits in AD patients [193,194].

Aside from elucidating the efficiency of endogenous antioxidants, several studies have increasingly accredited the role of various exogenous antioxidants. Limited clinical studies are reported herein and are illustrated in Table 1. Table 1 also shows several examples of previously mentioned exogenous antioxidant reactions that have worked in clinical settings. Although the administration of exogenous antioxidants showed positive effects, there are some inconsistencies in clinical trials. The clinical trials performed multivariable analyses with various factors, such as sample size, replication or validation studies using the same agent and outcome measures, assessment of vitamins, different conditions of endogenous antioxidants, long-term monitoring, etc., which caused variability because of inconsistencies. In general, several common antioxidants and their clinical effects are described in Table 1.


**Table 1.** Clinical trials of antioxidant in neurodegenerative diseases.

#### 3.2.3. Nrf2 Antioxidant System

The transcription factor Nrf2 is characterized as a regulator of redox homeostasis and antioxidant defense mechanisms. This protective pathway also encompasses the activation of a detoxification network such as oxidation/reduction factors (Phase I), metabolizing enzymes (Phase II), efflux transporters (Phase III), and free radical scavengers [201]. Oxidation/reduction factors consisting of nearly 500 genes encoding proteins, including redox balancing factors, stress response proteins, detoxifying enzymes, and metabolic enzymes such as NAD(P)H quinone oxidoreductase (NQO1), HO-1, SOD, GST, GSR, GSH-Px, carbonyl reductase (CR), and glutamate-cysteine ligase (GCL) play important roles in antioxidant and pro-survival effects and detoxification of xenobiotics. Nrf2 is generally targeted for ubiquitin-mediated degradation by its endogenous inhibitor Keap1, but oxidative modification of Keap1 inhibits the Nrf2 degradation process during conditions of redox imbalance [202]. To maintain cellular redox homeostasis, basal Nrf2 accumulation increases to mediate the normal expression of antioxidant response element (ARE)-dependent genes in the nucleus. The mitochondrial membrane directly interacts with Nrf2 which can respond to mitochondrial oxidative stressors that can collapse cellular bioenergetics leading to cell apoptosis [203]. Thus, the Keap1/Nrf2 system is another good homeostatic regulator of intrinsic cellular antioxidant defense and mitochondrial health. The suppression of Nrf2 activity increases the susceptibility of the brain to the damaging effects of oxidative stress and inflammatory stimuli [204]. Nrf2 activation promotes neuroprotective effects in both in vitro and in vivo neurodegenerative models. Nrf2 activity diminishes with age, and consequently, the effect of antioxidant enzyme activity decreases. Nrf2/ARE system impairment leads to higher susceptibility to oxidative injury, abnormal protein aggregation and neurodegeneration in the brain. Many studies have demonstrated the importance of the Nrf2/ARE pathway in the pathogenesis and control of neurological disorders, including PD, AD, ischemia, and other neurodegenerative diseases.

After middle cerebral artery occlusion (MCAO) in rats, Keap1 levels are decreased and this loss is correlated with an increase in Nrf2 and its downstream proteins, such as thioredoxins, GSH synthases, and HO-1 [205]. Stroke models of Nrf2-deficient mice exhibit higher levels of ROS than the wild-type littermates, which supports the natural compensatory mechanism of Nrf2 [206]. In AD transgenic mice, Nrf2-deficiency brains presented increased marker of oxidative stress and exhibited deficits in spatial learning and memory [204]. Nrf2 knockout and wild-type mice can be administered MPTP with doses ranging from 20 to 60 mg/kg for an animal model of PD. Nrf2−/<sup>−</sup> mice exhibited increased sensitivity to the dopaminergic toxins MPTP and 6-OHDA [207]. Nrf2 deficiency increased the MPTP sensitivity by 30 mg/kg administration of MPTP, but astrocytic Nrf2 overexpression showed the amelioration of MPTP toxicity in ARE-hPAP mice [208]. Astrocytic Nrf2 reduced the MPTP neurotoxicity by β-lapachone treatment in PD models [209].
