Abstract
Alzheimer’s disease (AD) is characterized by the progressive degeneration of neuronal cells. With the increase in aged population, there is a prevalence of irreversible neurodegenerative changes, causing a significant mental, social, and economic burden globally. The factors contributing to AD are multidimensional, highly complex, and not completely understood. However, it is widely known that aging, neuroinflammation, and excessive production of reactive oxygen species (ROS), along with other free radicals, substantially contribute to oxidative stress and cell death, which are inextricably linked. While oxidative stress is undeniably important in AD, limiting free radicals and ROS levels is an intriguing and potential strategy for deferring the process of neurodegeneration and alleviating associated symptoms. Therapeutic compounds from natural sources have recently become increasingly accepted and have been effectively studied for AD treatment. These phytocompounds are widely available and a multitude of holistic therapeutic efficiencies for treating AD owing to their antioxidant, anti-inflammatory, and biological activities. Some of these compounds also function by stimulating cholinergic neurotransmission, facilitating the suppression of beta-site amyloid precursor protein-cleaving enzyme 1, α-synuclein, and monoamine oxidase proteins, and deterring the occurrence of AD. Additionally, various phenolic, flavonoid, and terpenoid phytocompounds have been extensively described as potential palliative agents for AD progression. Preclinical studies have shown their involvement in modulating the cellular redox balance and minimizing ROS formation, displaying them as antioxidant agents with neuroprotective abilities. This review emphasizes the mechanistic role of natural products in the treatment of AD and discusses the various pathological hypotheses proposed for AD.
INTRODUCTION
All aerobic species produce oxygen-based radicals (called reactive oxygen species [ROS]), which trigger different important cellular pathways, but also cause several deleterious events in the biological system [1]. Aerobic organisms that use O2 as the final electron acceptor drive a constant supply of ROS in the body. Apart from their default production as byproducts of the mitochondrial respiratory chain, ROS are also produced by certain enzymatic (e.g., in macrophages to kill invaders) or non-enzymatic side reactions. As a measure to neutralize and control the excessive ROS burden, the cell encodes several enzymes and small molecules termed antioxidants. Antioxidants carry their activity of controlling these free radicals via diverse mechanisms, such as scavenging of ROS, quenching of ROS sources, and regeneration of endogenous antioxidants [2]. As ROS are involved in several signaling pathways of cellular proliferation and survival, they are maintained at low levels instead of being completely neutralized. However, excessive ROS accumulation drive the cell towards oxidative stress (OS), resulting in various damaging effects, primarily on macromolecules, eventually leading to cellular and tissue damage. OS may primarily arise from excessive ROS generation or inefficient neutralization of accumulated ROS in the system. However, this later forms a vicious cycle, with ROS yielding aberrant biomolecules. These biomolecules affect the organization and/or function of various organelles in different cell types unpredictably. Persistent OS affects the physiological mechanics of the entire body, leading to the onset of various diseases and disorders, including diabetes and cancer. In addition, it alters the neurological setup more deeply [3].
It is already known that approximately 20% of the total available oxygen concentration in the human body is consumed by the brain, making it the highest O2 consumer; therefore, it tends to be particularly sensitive to oxidative damage. Moreover, several reports have stated that OS-induced oxidative damage triggers many neurodegenerative diseases such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) [4–6]. This study by Cobley et al., explaining the high sensitivity of the neural system to OS discusses the “13 reasons (Fig. 1) why the brain is prone to OS” as follows:
Schematic representation of major reasons why the brain is prone to oxidative stress. Adapted and modified from Cobley JN, Fiorello M L, Bailey DM (2018) 13 reasons why the brain is susceptible to oxidative stress. Redox Biol
AD is the most common type of dementia in the elderly [7] and is mostly characterized by the abnormal accumulation of amyloid-β (Aβ) and hyperphosphorylated tau protein (pTau), the two hallmarks of AD. Tau stabilizes the microtubules of nerve cells (neurons) in the brain. This internal cytoskeleton has a tube-like shape through which nutrients and other essential substances travel to different parts of the neuron. In AD, an abnormal (hyperphosphorylated) form of tau (pTau) causes it to detach from microtubules and aggregate, resulting in disruption of microtubules. These aggregates form tangles that impact neuronal transport system, which harms synaptic communication between neurons thus affecting memory and cognition. In addition to tau, Aβ monomers aggregate into soluble oligomers (small aggregates of peptides) and higher aggregates. Aβ aggregates (oligomers, fibrils, and plaques) have been found in patients with AD. These aggregates can also disrupt communication between nerve cells and cause brain inflammation. Thus, Aβ plaques and tau tangles exhibit both direct and indirect cytotoxic effects that affect neurotransmission, axonal transport, signaling cascades, organelle function, and immune responses, leading to synaptic loss and dysfunction in neurotransmitter release. In addition to memory loss, these changes could have detrimental effects on self-autonomy, motor skills, and quality of life [7].
The establishment of memory decline often preceding subsequent neuropathological signs of AD has ignited interest in the pathology of AD, especially in the translational state between normal aging and AD, known as mild cognitive impairment (MCI) [8]. OS underlies MCI [9, 10] promoting the progression of AD [11]. Although biomarkers of oxidative damage in AD have been identified [10, 12–21], only limited data on their usefulness in the early detection of AD are available [22]. As far as therapeutic intervention is concerned, to date, no treatment has been found. Recently, the Food and Drug Administration-approved drug, aducanumab, to control and reduce Aβ plaques has shown efficacy, although it needs to be tested for a larger population to reach a decisive conclusion [23].
In the last few decades, several antioxidants have been proposed to prevent or alleviate oxidative damage in humans and non-human models of AD [24–27], with some being preventive and others touted to have therapeutic roles. There is active participation of the endogenous antioxidant system in the body, either through an enzymatic system (catalase [CAT], glutathione peroxidase, and superoxide dismutase [SOD]) for the direct reduction of free radical species or through cellular biomolecules (sodium selenite, uric acid, and glutathione) that work via protection of the cellular mechanisms [28].
In addition, when there is a heavy ROS load in our physiological system, we need an extra dose of antioxidants from external sources (exogenous form), such as diet (whole grains, cereals, millets, and essential oils) or supplements (vitamin C, vitamin E, and β-carotene). Therefore, the present review aimed to highlight the various pathophysiological mechanisms through which OS affects neural function, stimulating the onset of one of the most prevalent brain disorders (AD) and to analyze various pieces of evidence supporting the use of different natural antioxidants in the therapy and management of AD.
GENERATION OF ROS AND OS
By virtue of their functioning, nervous system cells (glia, neurons, and astrocytes) project an increased metabolic demand and a postmitotic nature that eventually augments their susceptibility to OS. This is accompanied by inadequate antioxidant potential of the brain, and the lower regeneration rate of brain cells that worsen the impact of OS and causes neurodegenerative diseases [29]. In addition to the amyloid cascade hypothesis, another important perspective related to the generation of ROS and the further progression of AD has emerged in the form of the mitochondrial cascade hypothesis [30]. Many studies state that in spontaneous, delayed-onset AD, age-related loss of mitochondrial function impairs the synthesis and metabolism of amyloid-β protein precursor (AβPP), causing Aβ to aggregate [30]. Various transgenic mouse models of familial AD have shown altered mitochondrial axonal transport, with additional abnormalities in molecular functioning before the appearance of amyloid plaques or memory loss [30].
Accumulation of ROS in general in cells is attributed to their generation in different components of the cell, including the plasma membrane, cytosol, endoplasmic reticulum (ER), and peroxisomes, with mitochondria being the primary source [30]. OS and synaptic disruptions that add to the initial pathophysiology before any clinical signs and symptoms appear are triggered by the presence of Aβ or tau aggregates and could also be primary drivers of the disease. ROS generation in different cellular components relevant to neurological disorders largely relies on reactions involving xanthine oxidases (XO), peroxisomal oxidases, NAD(P)H oxidases, and cP450 enzymes, which act on transition metal ions, thymidine, and polyamines [31, 32].
Evidence suggests that early phases of AD, which occur before the appearance of clinical signs, are influenced by mitochondrial dysfunction, leading to increased ROS generation [33]. The experimental results from human brain tissues and peripheral systems show high levels of glycosylated products, oxidized proteins, alcohols, lipid peroxidation, aldehydes, free carbonyls, cholestenone, and ketone formation [33]. In addition, the presence of unsaturated fatty acids in cortical varices provides easy access for free radical generation, leading to an increased rate of aerobic metabolic activities in neural cells. The increased iron content, which is an important prerequisite for brain development, is another add-on factor for OS generation by iron catalysis [33].
According to recent clinical research findings, it is been reported that oligomers and protofibrils, which are tiny soluble clusters/oligomers of Aβ, are more hazardous than Aβ fibers themselves [33]. These findings suggest a close relationship between spatial learning deficits and neurodegeneration that manifests in individuals with AD before the development of amyloid plaques [33]. In addition, the brain is a postmitotic tissue with neurons that have a limited regenerative rate and low self-restoration potential; thus, the build-up of OS damage is detrimental. Individuals with sporadic AD and older individuals without dementia have been found to have an elevated oxidative load in their brain tissues [33]. Furthermore, the amount of oxidized proteins and peptides directly corresponds to cognitive function of the human brain [33].
Mechanisms of mitochondria-independent ROS generation
XO is conventionally known as a generator of ROS which leads to hypoxic-reperfusion injury in tissues. It is a well-studied hydrolase enzyme that catalyzes ROS generation by oxidizing NADH [34]. Reversible modification of cysteine residues in xanthine dehydrogenase, an alternate form of XO originating from the same precursor, promotes its conversion back to NADPH-dependent XO [35].
NADPH is a major donor of reductive potential that promotes the ROS-detoxifying properties of antioxidants, such as glutathione reductase and thioredoxin reductase. NADPH also acts as a cofactor and tightly binds to CAT, thereby enhancing its activity. In contrast, NADPH oxidase (NOX), a family of seven proteins (NOX1-5, dual oxidase DOX-1, and DOX-2), transfers an electron from NADPH to flavin adenine dinucleotide, facilitating the reduction of oxygen and release of H2O2 and O2•– [36]. Furthermore, peroxisomes, the egregious oxidative cell organelles, are mainly involved in the metabolism of purines, polyamines, amino acids, and glyoxylates, as well as in setting the stage for α- and β-oxidation of fatty acids [30]. Despite the presence of CAT, which exhibits antioxidant activities, peroxisomal enzymes liberate significant amounts of H2O2, along with OH– and O2•– free radicals, consuming nearly 20% of the total cellular oxygen and generating approximately 35% of the total H2O2 in tissues [37, 38]. The production of O2•– in peroxisomes is driven by xanthine oxidoreductase and the electron transport chain in the peroxisomal membrane [39]. Accumulation of improperly folded proteins in the ER lumen imparts considerable stress that disrupts normal cell homeostasis by initiating a response from the unfolded proteins, further triggering ROS production and initiating feedback to increase the stress borne by the ER [40, 41]. Additionally, a microsomal monooxygenase system comprising microsomal cP450, NADPH-P450 reductase, and cytochrome b5 is another site of ROS generation in the ER that catalyzes the oxygenation of exogenous and endogenous compounds and substrates to produce O2•– and H2O2 [42, 43]. With its involvement in the microsomal electron transport system, cP450 catalyzes the oxidation of several organic substrates along with the reduction of molecular oxygen. cP450 utilizes the available H2O2 to carry its peroxidase activity, either to oxidize substrates directly or by acting as an oxygen donor, with the possibility of its reduction to •OH via the Fenton reaction [44].
Mechanisms of mitochondria-dependent ROS generation
Mitochondrial metabolism and homeostasis liberate the majority of ROS produced in the cell, facilitating their accumulation at potentially detrimental levels. Among all mitochondrial processes, autophagy is the most prominent source of ROS release [45, 46]. These mitochondrial ROS in turn cause oxidative damage to mitochondrial proteins, membranes, and DNA, ebbing the mitochondria’s ability to carry out ATP synthesis, in addition to their myriad metabolic functions critical to normal cellular operation [47]. This situation is worsened by the significant involvement of age-related mitochondrial dysfunction in AD progression due to excessive ROS production and reduced ATP production [48, 49]. O2•– is the primary free radical generated in mitochondria, and its production occurs at multiple sites [50]. The liberated radical is then released into both the mitochondrial matrix and the intermembrane mitochondrial space, with the conversion of O2•– to H2O2 via manganese SOD in the mitochondrial matrix and via copper SOD and zinc SOD in the intermembrane mitochondrial space or cytosol [51]. Furthermore, the Fenton reaction proceeds to liberate (•OH) using mitochondrial aconitase [52]. Another significant protein that is intensively involved in ROS generation is cytochrome c, which regulates redox signaling in mitochondrial oxidative phosphorylation and homeostasis and acts as an electron carrier in the mitochondrial electron transport chain [53].
NEUTRALIZATION OF ROS: ROLE OF NATURAL ANTIOXIDANTS
The role of antioxidants in neutralizing the deleterious effects of free radicals is immense as they avert free radical development before they can affect cellular physiology, help in repairing and cleansing impaired molecules, and avert mutations [54]. Furthermore, it has been observed that large quantities of polyunsaturated fatty acids in the brain are more susceptible to ROS attacks that promote the removal of membranous hydrogen atoms, augmenting the brain’s sensitivity to attack by free radicals [55]. The susceptibility of the brain to free radical damage necessitates an urge to attain a balance between oxidative and antioxidant species. Antioxidant species respond to OS via different mechanisms; for example, a specific set of individuals referred to as non-demented with AD neuropathology activate an efficient antioxidant response to cope with OS in symptomatic AD [56]. There are many natural, synthetic, endogenous, or diet-derived antioxidants, and detailed studies have shown that they effectively scavenge surplus ROS [57]. These antioxidants can adopt either of the two routes of action: 1) preventing, blocking, or capturing ROS generated through oxidative damage or 2) removing compromised biomolecules capable of aggregation during the antioxidant repair process [57]. These processes can either be nonenzymatic or enzymatically-catalyzed (Fig. 2).
Different mechanisms of reactive oxygen species (ROS) generation and neutralization.
Enzymatic scavenging of ROS
Primary enzymes involved in antioxidant catalysis include SOD, CAT, and glutathione peroxidase (GPx). They have been reported to extensively reduce O2•– and H2O2 ROS and are involved in the overhauling of damaged nucleic acids by nucleases, proteins by proteolytic systems, and oxidized lipids by phospholipases, peroxidases, or acyl transferases [58]. SODs exist in three isoforms in humans: SOD-1, SOD-2, and SOD-3, with their respective copper-zinc (SOD-1, SOD-3) and manganese (SOD-2) forms, catalyzing the conversion of toxic free radicals in cells to liberate H2O2 in a spatial manner [58]. In addition, CAT and GPx, with their respective presence in peroxisomes, mitochondria, and cytosol, catalyze the breakdown of noxious H2O2 to a much more acceptable form of H2O and O2. Additionally, CAT uses its heme group to react with H2O2 using Fe as a cofactor, and its ability to not reach saturation at any given H2O2 concentration makes it one of the most efficient enzymes [58]. It has been reported that both low expression and mislocalization of CAT may result in the buildup of H2O2 and other ROS and cause compromised neurological function. This indicates the significance of CAT in reducing OS levels in cellular system [59]. Finally, the GPx family of antioxidants is primarily involved in catalyzing the reduction of several hydroperoxides, including lipid peroxyl radicals, using its glutathione group in a localization-dependent manner. Compared with CAT, GPx activity is optimal only when low levels of OS are stimulated [58]. A study revealed that the deficiency of GPx4 in Gpx4KO transgenic mice negatively affects cognitive functions and mental processes, along with the initiation of hippocampal region degeneration, compared with the control group [60]. It has also been reported that antioxidant oxidoreductase proteins, thioredoxin and thioredoxin reductase, constitute the thioredoxin system, which catalyzes the transfer of an electron from NADPH to certain peroxiredoxins involved in the reduction of peroxides [61]. Another antioxidant system is the plasma membrane redox system, which is comprised of multiple redox enzymes, such as quinine reductase, NADH-cytochrome b5 reductase, NADH-quinone oxidoreductase 1, NADH-ferricyanide reductase, NADH coenzyme Q10 reductase, and NADH-cytochrome c reductase. This system collectively ignites plasma membrane-bound electron transport processes. This catalyzes the relocation of electrons from donors to the acceptor sites present extracellularly, thereby decreasing the membrane-bound OS condition [55].
Non-enzymatic ROS scavenging
While enzymatic antioxidants scavenge free radical species, non-enzymatic antioxidants act as oxidative enzyme inhibitors, singlet oxygen quenchers, peroxide decomposers, ultraviolet radiation absorbers, and metal chelators [62]. Non-enzymatic antioxidants are further classified based on their source, which could be metabolism-derived, such as glutathione, L-arginine, and α-lipoic acid, or diet-derived, such as vitamin E, vitamin C, flavonoids, β-carotene, cysteine, and phenolic compounds [55, 63]. Likewise, selenium, coenzyme Q10, and zinc, along with a few essential metal ions, also act as cofactors for enzymatic ROS scavenging [58]. Selenium aids ROS scavenging by modulating the influx of Ca2+ ions and stimulating antioxidative selenoproteins such as GPx, and its deficiency has been reported as a hallmark of OS-induced neurological diseases [64]. Furthermore, CoQ is another cofactor and antioxidant scavenger abundant in the brain and intestinal cells mitochondria, where it participates in the electron transport chain to reduce O2•– radicals to H2O during ATP production [65]. Cytosolic CoQ acts as a cofactor for thioredoxin reductase to enhance the antioxidant defense [66]. Augmented coenzyme Q10 levels have been shown to lower OS and improve antioxidant enzyme activity [67]. Zinc is redox inert, yet it does have some antioxidant properties. Zinc supplementation is linked to lower ROS generation, which has positive effects, notably in older adults and people with diabetes. The binding of redox-active transition metals, such as copper and iron, to the catalytic site is superseded by zinc, directing the hydrolytic polymerization of the former and prohibiting ROS production and initiation of lipid peroxidation [68].
In addition, vitamins A (β-carotene), E (α-tocopherol), and C (ascorbic acid) act as non-enzymatic free-radical scavengers to combat OS and its deleterious effects. The metabolized form of retinol, retinoic acid, confers neuroprotection against OS by eliminating glutathione and increasing SOD-1 and SOD-2 in brain cells [69, 70]. As discussed earlier, lipid-soluble vitamin E also acts as a potential scavenger of the free radicals generated during lipid oxidation to protect the cell membrane from OS-triggered damage. The most biologically active form of vitamin E, α-tocopherol, performs its activity by either reacting with singlet oxygen and OH• or donating labile hydrogens to oxidize radicals to prevent lipid peroxidation [71]. In contrast, vitamin C is a more direct scavenger of ROS and is involved in the repair of other scavengers in the oxidized states. Vitamin C primarily inhibits NOX and nNOS, which increases Ca2+ influx and helps restore the activity of active forms of vitamin E and repair glutathione [72]. Other systems reported are nuclear factor–erythroid-2–related factor 2 (Nrf2), which is a transcription factor involved in governing the pathways responsible for gene expression of endogenous antioxidant enzymes and antioxidant response element–dependent genes as a cellular response to OS [73]. This defensive pathway regulates the activation of oxidation/reduction factors, among other free radical scavengers, which involves approximately 500 genes that encode antioxidant proteins and factors (redox balancing factors, stress response proteins, detoxifying enzymes) and metabolic enzymes (NAD(P)H quinone oxidoreductase, SOD, glutathione S-transferases, GPx, carbonyl reductase, and glutamate-cysteine ligase) [58]. Recognition of these antioxidative strategies to combat AD has the potential to curb the involvement of OS in the progression of neurodegenerative diseases.
FREE RADICAL OXIDATIVE STRESS AND IMPAIRMENT OF THE BLOOD-BRAIN BARRIER
The BBB is a highly complex and selectively permeable structure that maintains overall cerebral homeostasis by separating peripheral blood from the central nervous system (CNS) [74, 75]. This barrier mainly comprises brain microvascular endothelial cells, pericytes, astrocytes, and a basement membrane [76, 77]. The selective permeability of the BBB can be attributed to the presence of the least permeable capillaries formed by physical barriers, that is, tight junctions (TJs), which are responsible for hindering the permeation of the majority of drug compounds through the BBB [78]. In association with AD pathology, BBB dysfunction may trigger neuroinflammation and OS, enhance the activities of β-secretase and γ-secretase, and promote Aβ generation. The progressive accumulation of Aβ in the brain and BBB dysfunction may become a feedback loop that gives rise to cognitive impairment and the onset of dementia. The breakdown of the BBB may also be considered a biomarker for normal aging and AD. Impairment of the BBB is driven by many factors such as OS, immune cells, neuroinflammation, and diverse pathogens [77, 80]. Furthermore, the interaction of these pathological elements results in the activation of matrix metalloproteinase (MMP)-mediated disruption of TJ connections between cerebrovascular endothelial cells [81]. Altogether, these result in the degradation of basement membranes and aggravation of BBB permeability, allowing the passage of large substances as well as toxins to reach the brain and induce damage [82, 83]. It has been evident from previous studies that OS plays a key role in the activation of BBB changes [84, 85]. Moreover, ROS-mediated pathways involved in triggering BBB impairment include mitochondrial dysfunction, giant cell/microglial induction, TJ modification, excitotoxicity, MMP induction, and extracellular transport [86]. Additionally, OS can lead to irreversible deleterious effects on cells such as pericytes, astrocytes, and brain microvascular endothelial cells, further disrupting the BBB [87]. Different mechanisms that highlight the role of OS in BBB impairment are discussed below (Fig. 3).
Schematic representation of different mechanisms through which reactive oxygen species (ROS) elevation leads to impairment of the blood-brain barrier.
Role of TJs and their associated proteins in BBB
TJs are junctional complexes formed between the endothelial cells of the paracellular space that barricade ions, drugs, systemic toxins, hydrophilic molecules, and other antigens from crossing the channels of the BBB [88]. The TJ chain of the brain endothelium is composed of claudins, intact membrane proteins, and JAMs (connecting adhesion molecules) [89], which are intricate in intercellular contacts and interactions with the cytosolic scaffold zonula occludens (ZO, an essential scaffold protein), actin cytoskeleton [90], and associated proteins such as small GTPase [91], VE-cadherin [92], heterotrimeric G protein [93], and protein kinases [94]. Numerous studies have indicated that TJ proteins are essential for maintaining the integrity of the BBB.
TJ proteins, including ZO-1, ZO-2, occludin, and claudin, expressed by pericytes have been documented to exhibit an association between pericytes and endothelial cells in maintaining BBB integrity [95]. It has been presented in in-vitro studies that, at the endothelial level, astrocytes are involved in regulating TJ tightness and the polarized distribution of transporters [96]. Therefore, any alteration in these proteins leads to changes in the BBB permeability. Occludin is the chief structural protein of TJs, and its expression levels represent the structural state of the BBB [97]. Claudin protein may act as a regulatory target of the BBB and can alter the selective opening of TJs. The generated ROS can control the activity of claudin-5, elevate solute leakage, and affect the integrity of the BBB [98–100]. ROS can also alter the BBB permeability by influencing the distribution of the ZO protein. It has been observed that upon exposure to H2O2, there is redistribution of ZO-1 from TJs to the cytosol, leading to a drop in trans-endothelial electrical resistance and an increase in the permeability of the BBB [101]. Many supportive studies have suggested an association between disruption of the BBB, OS, changes in TJ complexes, and the pathogenesis of several neurological disorders, including AD [92, 102].
Effect of nitric oxide and lipid peroxidation products on BBB
Nitric oxide (NO) is a potential signaling molecule produced by NOS, which plays a crucial role in disrupting the BBB and the infiltration of peripheral immune cells in the cerebellum [103, 104]. NOS is categorized into inducible NOS, neural NOS, and endothelial NOS, and the generation of NO from inducible NOS enzyme induction is a primary response factor. In addition, activation of NOS results in elevated production of NO, which along with ONOO may elevate BBB permeability by affecting TJ proteins or via the cyclic guanosine monophosphate-protein kinase G pathway [86]. In both humans and in-vitro models of AD, the presence of peroxynitrite has been documented in blood vessels, astrocytes, and neurons of AD brain specimens [105].
In conjunction with this, lipid peroxidation, which causes oxidative damage to polyunsaturated fatty acids through free radical chain reactions upon exposure to O2, is another player in addition to the OS load. It induces damage at various levels via the production of several reactive aldehydes, including 4-hydroxynonenal, which changes the arrangement of phospholipids around the membrane lipid bilayer and other outcomes of lipid peroxidation. This alteration further reacts with mitochondrial enzymes and facilitates dysfunctional mitochondrial energetics, thereby upregulating the release of free radicals to cause OS [106]. Skoumalova et al. (2012) reported that these intermediates may enter circulation and affect the red blood cell membrane [107, 108]. In a study, it was reported that lipid peroxidation stimulates MMP-2/9 production, which in turn activates RhoA (a small GTPase), resulting in phosphorylation of TJ proteins and further disruption of BBB [109].
Altered expression of transporters in BBB
Biomolecule transporters are essential components for ensuring the protective integrity of the BBB and the regulation of exogenous/endogenous substances through pores [110]. ATP-binding cassette (ABC) and solute carrier transporters are the most extensively studied transporters, with the former primarily being involved in influencing the permeability of various toxins and therapeutic agents [111]. The OS-activated signaling cascade affecting the expression of ABC transporters may contribute to the pathogenesis and treatment of CNS disorders. ROS are often associated with cytotoxicity and play an important role in the signaling of numerous transcription factors, including Nrf2, HIF-1, and NF-κB [112]. The expression of ABC transporters is regulated by these transcription factors. Along with ABC and solute carrier transporters, ROS may affect the permeability of the BBB by regulating the protein kinase, 5’ adenosine monophosphate-activated protein kinase (AMPK), which is involved in critical cell stress signaling responses around the BBB [113]. Many studies have confirmed that transporters, such as glucose transporter types 1 and 4 (GLUT-1 and GLUT-4), Na-K-Cl (NKCC2) co-transporters, and K+ and Cl-channels in the epithelium are targets of AMPK [114]. The association between pericytes and astrocytes is crucial for maintaining BBB integrity and AMPK activity. It is also important for regulating the expression of GLUT-1 and GLUT-4, which are involved in glucose uptake [115].
MMPs and BBB
MMPs are zymogens that are converted to their active form upon cleavage by other MMPs or proteases, such as tissue-type or urokinase-type plasminogen activators [116, 117]. MMPs are zinc-dependent endopeptidases that can affect BBB integrity by degrading the extracellular matrix and epithelial basement membrane [118]. Endothelial cells, TJs, and basement membranes are crucial for maintaining the integrity of the BBB. Thus, any alteration in the BBB can affect its integrity, resulting in the pathogenesis of various neurological diseases [119]. It has been proposed that inhibition of MMPs can prevent the digestion of TJs and basement membrane proteins, thereby preventing BBB impairment [120]. Thus, it can be suggested that MMP is a key player in maintaining the permeability of BBB [121, 122]. As discussed earlier, ROS are directly involved in the decreased expression of TJs, and they have been observed to indirectly induce activation of MMPs, promoting BBB opening [123–125]. OS-induced MMPs and aquaporin activation results in the detachment of the perivascular units and vasculature, which in turn stimulates vascular or cellular fluid edema, increases BBB leakage, and causes progression of neurodegenerative changes [126–128]. In addition, BBB integrity deficiency enables the entry of neurotoxic blood-derived debris, cells, and OS activation into the brain and is connected to inflammatory and immunological reactions that can initiate multiple neurodegenerative pathways [129].
Autophagy and BBB
As a pathway of cellular degradation and clearance, autophagy is involved in the transport of damaged/denatured proteins and impaired organelles to lysosomes for digestion and degradation [130]. ROS may primarily exhibit deleterious effects on the BBB by triggering lysosomal damage and autophagy of pericytes and astrocytes [131–133]. Studies have shown that hippocampal and the frontal cortex neurons are the most affected by OS load. Activation of the AKT/mTOR signaling pathway to control autophagy and suppress neural cell death may contribute to the mechanism underlying this damage [134]. During prolonged OS, inhibition of LC3 lipidation, aberrant autophagosome formation, and autophagic flux, despite concordant stimulation of various pro-autophagic signals, have been observed in astrocytes [135]. Under OS conditions, autophagy maybe induced by ROS as a synergistic interplay to minimize OS-induced damage and maintain the structural and functional integrity of brain cells and BBB [136, 137].
FREE RADICALS AND NEUROINFLAMMATION
At higher concentrations, ROS activate pathways related to cellular signaling, along with pro-inflammatory chemokines and cytokines [138]. Pro-inflammatory cytokines such as tumor necrosis factor, interleukin-6, interferons, and interleukin-1β stimulate NOX-mediated ROS generation in fibroblasts, renal mesangial cells, tubular cells, vascular smooth cells, and endothelial cells (non-phagocytic cells). NOX, the primary source of ROS within the CNS, is reported to be associated with ischemia, traumatic brain injury, and other neurodegenerative disorders [139, 140]. Evidence from different studies supports the role of NOX isoforms in brain-related damage and associated disorders, particularly NOX-1, NOX-2, and NOX-4. Studies have shown that aggregation of Aβ stimulates NOX-2-dependent ROS generation in microglia [141–143]. NOX-2 deficient animal models defended against the damage induced by Aβ, and there was no development of OS, cerebral dysregulation, or pathophysiology of AD [144].
Inflammation, a direct result of ROS generation, is initiated as a response to control or fight injury, infection, or other stimuli. Neuroinflammatory changes in the CNS involve the activity of resident glial cells, including oligodendrocytes, astrocytes, microglia, peripheral leukocytes, macrophages, and dendritic cells (non-glial resident myeloid cells) [145]. This response is generated as an initial pathological event in almost all types of neurological disorders, including ischemic, metabolic, traumatic, toxic, neurodegenerative, and neoplastic disorders. As neuroinflammation plays a vital role in triggering neural disorders including AD, it is crucial to acknowledge and manage the balance between the immunological and neurological systems to avert or attenuate disease progression. Activation of microglial cells is the chief component of neuroinflammation that follows a release of numerous inflammatory and cytotoxic elements including eicosanoids, chemokines, cytokines, excitatory amino acids, proteases, and ROS [146, 147].
Additionally, ROS-perturbed immune cells and their functions have been implicated in CNS diseases. The infiltration of T-cell into the CNS is among the most prevalent characteristics of inflammatory events in neural regions. It has been documented in various animal model studies that during the progression of neurodegenerative diseases, there is a surge in T cell count, causing a transformed phenotype [148]. These cells also contribute to the lytic and phagocytic activities of glial cells and mononuclear phagocytes [149]. Once CD4+T cells are activated, they can easily pass through the BBB. These cells mediate various actions depending on their phenotype once they invade the damaged site [150]. In case of excess OS, there is a loss of neuroinflammatory response, leading to cell damage followed by neurodegeneration [151]. It has been proposed that attenuation of the redox state of cells can be exploited with its therapeutic potential to regulate the activation and differentiation processes of CD4+T cell subpopulations [152].
ROLE OF BRAIN-DERIVED NEUROTROPHIC FACTOR: A CROSSTALK BETWEEN ROS AND OS
The presence of brain-derived neurotrophic factor (BDNF) in the cortical region is essential for the survival and progression of neural cells and neural plasticity, and BDNF also acts as a modulator of neural transmission. However, during a higher OS load, BDNF concentration decreases in the entire CNS region, eliciting the onset of neurodegeneration processes, and thereby initiating conditions such as AD and PD. Moreover, it has been observed that neurotrophin signaling plays an imperative role in learning and memory processes, preserving neurons and synaptic plasticity [153]. In the adult CNS, BDNF is the most abundant neurotrophin, binding to tropomyosin-related kinase B (TrkB) to control the PI3K/Akt pathway, mitogen-activated protein kinase pathway, and phospholipase C pathway activation [154, 155]. Activated Akt promotes cell survival by maintaining a balance between pro- and anti-apoptotic proteins [156]. The mitogen-activated protein kinase and phospholipase C pathways regulate the growth and survival of neurons by interacting with multiple genes in the cAMP-response element binding protein (CREB)-dependent pathway. CREB is accountable for BDNF and other proteins related to synaptic plasticity [157, 158].
The Nrf2-antioxidant responsive element (Nrf2-ARE) system is the main cellular defense against OS. It plays an important role in neuroprotection by regulating the expression of antioxidant molecules and enzymes [19]. ROS and dysregulation of the Nrf2-ARE system can damage important cellular components and cause the neuronal loss as well as loss of functional integrity [159, 160]. In contrast, TrkB signaling is a classic neurotrophic factor signaling pathway that regulates neural survival and synaptic plasticity and plays an important role in memory and cognition [161]. TrkB signaling, especially the TrkB/PI3K/Akt mediated pathway, promotes the stimulation of Nrf2 and nuclear translocation, thus providing neuroprotection against OS. However, the Trk signaling pathway is inhibited in brain diseases owing to a lack of neurotrophin support [162].
In particular, the neurotrophic factor (BDNF)-dependent brain TrkB signaling pathway is crucial for the survival and typical function of mature neurons and is threatened by the lack of BDNF [163], suggesting that the TrkB and Nrf2 signaling systems are potential targets for neural survival and the initiation of regeneration of damaged Neural structures and synaptic connections. Therefore, it appears that targeting the cellular antioxidant defense, and the BDNF/TrkB pathway can improve cognitive deficits after brain injury [164].
FREE RADICAL AND GLIAL CELL (ASTROCYTE AND MICROGLIA) ACTIVATION
ROS are widely regarded as harmful substances that can cause cell damage and control various pathological processes in the CNS. Because of extensive OS production in the brain regions, there is a well-designed neural counter-response wherein “microglia,” usually distinguished as “brain-resident macrophages,” are intricate mediators of cortical inflammation [165]. The cortical region is especially at risk of OS due to the triple cause of increased ROS production, moderate antioxidant effects, and restrained capability for regeneration. As phagocytes, microglia can conciliate OS insults, already compromised by AMPs, along with cell debris and protein accumulation [166]. Glial cells become phagocytic, forming a heterogeneous system that disrupts homeostasis and is intrigued by parenchyma responding to pathogen stimuli [167]. The involvement of immunological pathways in neurodegenerative disorders may enable us to understand the correlation between microglia and protein combinations, which leads to dementias [168]. Microglia in AD entangle with amyloid during dementing events, including the formation of amyloid plaques, and have a pro-inflammatory phenotype, as seen by the release of cytokines, which limits amyloidosis. Notably, the size and quantity of microglia are strongly correlated with plaque dimensions. The proliferation of microglia in the vicinity of plaques also permits the buildup of these cells near the edge of amyloid deposits; nonetheless, these cells may become overactive and neurotoxic [169]. The appearance of scavengers for amyloid proteins, along with CD36 receptors and their degrading enzymes, limits the abnormal inflammatory responses that cause AD conditions [170, 171]. Genetic factors such as rare variants of triggering receptor expressed on myeloid cells-2 (TREM2) strongly accelerate the risk of developing AD, confirming the role of microglia in AD pathogenesis [172]. Subsequently, this establishes a mechanized hyperlink among microglial cells and improves microglial activation in AD and tauopathies [172]. Malondialdehyde is a naturally occurring product of lipid peroxidation used as an indicator of the OS in neurodegenerative disorders such as AD [173]. Microglia play a key role in the pathophysiology of neurodegeneration, allowing us to harness their defensive capacity and constrain chronic damaging inflammation [166].
Moreover, ROS-mediated successive inflammatory reactions in neural cells (microglia and astrocytes) due to infection, neural dysfunction, protein accumulation, and toxicity have been linked to stress and pro-inflammatory characteristics. Activation of all such neural cells in the CNS is the primary cause of the inflammatory response via the neuroinflammatory mediator complex, which is primarily found in the cerebrospinal fluid, neurons, blood, brain, and serum of patients with AD [166, 173]. Depending on the specific immunotherapeutic scenario, antibody-induced phagocytosis of pathological protein deposits, neutralization of toxic soluble proteins, direct antibody-mediated disruption of aggregates, cell-mediated immune responses, a shift in equilibrium toward efflux of specific proteins from the brain, and other mechanisms may all play a significant role in the development of an efficient therapeutic option [174].
Immunotherapy-based approaches, such as immune checkpoint barriers and tricking immune responses through various active, passive, and cellular immunizations as well as their route of administration, are effective in combating AD. However, studies have suggested that innate immunity plays a significant role in the treatment of various neurodegenerative diseases by depicting efficient clearance of toxic proteins in cortical regions [174].
FREE RADICAL OXIDATIVE STRESS AND ALTERED MITOCHONDRIAL DYNAMICS IN BRAIN
Neurons are specialized cells that require an extremely high amount of energy for their function, which is fulfilled by cellular mitochondria via ATP production. Therefore, equilibrium between mitochondrial biogenesis and quality maintenance is essential for the development of efficient neural cells. The mitochondria are also involved in molecular processes, calcium homeostasis, and immunological triggers. Hence, neural cells are the most affected, as far as mitochondrial disruption is concerned. The demand for high ATP levels by neural cells is always accompanied by increased ROS production, which causes more damage to these cells. Although the brain carries different types of ROS-sensitive polyunsaturated fatty acids, it lacks antioxidant enzymes and glutathione. Moreover, neurons are terminally differentiated in nature; hence, the brain tissue is highly vulnerable to injury from ROS [175]. However, to reduce the OS and meet ATP demand, mitochondria undergo continuous fusion and fission processes, and the coordinated cycles of fission and fusion preserve the elemental characteristics of the mitochondrial population, referred to as mitochondrial dynamics. This term also includes maintenance of mitochondrial morphology, function, distribution, transportation, and selective degradation via mitophagy. These processes affect the viability and synaptic activity of neurons [176].
The fusion of mitochondria enables reduction in damage by replenishing the compromised mitochondria with healthy mitochondria, including the repair of mtDNA and uniform circulation of mitochondrial proteins and metabolites [177, 178]. By contrast, fission facilitates mitochondrial homeostasis by creating new and healthy mitochondria. Moreover, mitochondrial fission contributes to mitochondrial quality by selectively deleting damaged mitochondria via mitophagy or by completely killing the neuron via apoptosis under conditions of higher levels of cellular stress.
Mitochondrial fission and fusion are closely associated with mitochondrial motility and allocation of the mitochondria [179]. Additionally, altered fusion of neural mitochondria can cause swelling of these mitochondria and prevent them from entering the distal and smaller branches of neurons, which can lead to their degeneration in axonal and dendritic areas [180]. Similarly, altered fission reduces the isolation of damaged mitochondria; consequently, it promotes neural apoptosis [181]. Induction of mitophagy or selective degradation of damaged mitochondria via autophagic engulfment is crucial for protecting neurons against neurodegeneration and neural cell death [182]. Mitophagy is also essential for the regeneration of astrocyte mitochondrial networks during inflammation [183]. For example, ATG7 is an important gene that is required for autophagosome formation. ATG7 deletion leads to increased ROS production and cell death. Therefore, it is important to activate autophagy, which is critical for the protection of mitochondrial function in astrocytes during a response [183]. Moreover, mitophagy impairment in neurons has been reported in many neurodegenerative disorders such as AD, PD, and amyotrophic lateral sclerosis. A decrease in the rate of mitophagy in neural cells may lead to excessive ROS generation, followed by neurodegeneration. In contrast, uncontrolled (excessive) mitophagy may lead to the loss of healthy mitochondria and lower the ATP levels in neural cells. Therefore, tightly regulated mitophagy is important for avoiding neurodegeneration in neurons. The details of the role of impaired mitophagy in various neurodegenerative diseases have been reviewed by Mani et al. [184]. Aside from neurons, astrocytes are also an important component of the brain. Mitochondria in astrocytes lie within the closest proximity to the nearest neural bodies, and their dendrites are actively involved in fulfilling the demands of metabolites and ions at astrocyte-neuron points of contact. Motori et al. [183] suggested a more explicit linkage between altered mitochondrial framework functioning and neuroinflammation, specifically in astroglial cells, suggesting the controlled mechanics of astrocytes during any type of brain injury.
Since the generation of energy as well as ROS is high and antioxidant levels are lower in neurons, the chances of altered mitochondrial dynamics in neurons are higher. Overall, mitochondrial quality control is maintained through the balanced coordination of mitochondrial fission, fusion, and mitophagy, allowing regulated functioning of neural mitochondria. Recent studies have suggested the involvement of several mitochondrial proteins in the regulation of mitochondrial dynamics. For example, fusion of the outer mitochondrial membrane and inner mitochondrial membrane is facilitated by mitofusins Mfn1 and Mfn2 [185, 186] and optic atrophy 1 (OPA1) [187, 188], respectively. Homozygous mutations in OPA1 are correlated with fatal and severe infantile-onset encephalopathy [189]. Removing the genes contributing to mitochondrial dynamics can cause defects in the development of the brain as well as various neurodegenerative pathogeneses. For example, Mfn 2 gene encodes the Mfn 2 protein, and a study has shown that knocking out Mfn 2 in the cerebellum leads to a reduction in the size of the cerebellum and motor defects [180]. Similarly, dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1) are responsible for the fission reactions reported by Waterham et al. (2007), where there was a dominant-negative mutation in DRP1 gene, resulting in a defect in neural mitochondrial fission in a newborn girl with abnormal brain development, microcephaly, and optic atrophy [190]. Studies have suggested that oxidative damage blocks mitochondrial fission, leading to neurodegeneration. Sirtuin 3 (SIRT3), a protein enriched in the mitochondria, is activated by OS in neurons. Active SIRT3 deacetylates FOXO3, thus triggering the expression of BNIP3/NIX, LC3-1/LC3-II, DRP1, MNF2, and FIS1 and also activates PINK1–PARKIN mediated mitophagy [191]. This suggests that SIRT3 is important in governing the equilibrium of mitochondrial fission, fusion, and mitophagy in neurons. SIRT3 also improves OPA1 activity to calibrate the mitochondrial response in OS [192]. Recruitment and activation of DRP1 occur in response to OS [193]. Core fusion/fission proteins, such as DRP1 and OPA1, undergo post-translational modification in a redox-sensitive manner via S-nitrosylation, which can be induced by H2O2, *NO, or ONOO–. Studies have also shown that *NO and Aβ peptides induce mitochondrial fragmentation in neurons [194, 195]. Overall, free radicals counteract mitochondrial fission and fusion, resulting in altered mitochondrial dynamics. However, in normal brain neurons, altered mitochondrial dynamics are balanced by mitophagy to promote mitochondrial quality control for protecting and maintaining the health of the neural cells as well as the brain.
FREE RADICAL OXIDATIVE STRESS AND IMPAIRED ION CHANNELS IN THE BRAIN
Ion channels play a significant role in the nervous system because of the exclusive association between neurons and their electrical signals. A higher level of ROS also affects the regulation of ion channels, accounting for the inert movement of ions through the membrane [196]. Therefore, they produce and form electrical signals in cells and perform various functions, regardless of their ability to conduct ions. There is evidence that ROS act on ion channels through direct oxidation and indirect deregulation of their channels. Generally, oxidative interaction between ion channels leads to increased cell oxidation in Caenorhabditis elegans [197]. The sensory ability of these cells decreases with age [197–199], partly due to the oxidation of cysteine (cys113) in peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) protein [200]. Furthermore, voltage-gated sodium channels (Nav) are responsible for triggering neural action potentials [196]. The synthesis and/or modification of the Nav channel changes its surface expression; even if the conductivity characteristics of the channel permeability and selectivity remain unchanged, it affects the electrical excitability of neurons. Kim et al. showed that Navβ2 is a substrate for beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and γ-secretase, and processing of Navβ2 is parallel to the processing of amyloid-β protein precursor (AβPP) in the AD brain [201]. Moreover, there is evidence that BACE1 is activated in the aging brains of humans, monkeys, and mice [202–204].
A GRAND ALLIANCE BETWEEN FREE RADICAL OS AND PATHOPHYSIOLOGY OF AD
AD is a multifaceted syndrome predisposed by genetic risk factors, environmental exposure, age, mitochondrial haplotypes, and sex [205–207]. Dysfunctional mitochondria and enhanced apoptosis, followed by a meager antioxidant status, are the mechanisms linked to the etiology of AD. Numerous reports have highlighted the role of hydroxyl radicals, superoxide anions, NO, and H2O2 in OS-induced neurodegeneration in AD [208–210]. It was first reported by Alois Alzheimer that amyloid plaques are the “accumulation of unusual matter in the cortex.” These plaques are also recognized as senile plaques, mainly composed of peptides called Aβ, which accumulate and generally produce β-sheet-rich fibrils [211, 212]. Numerous cell culture and animal model studies have shown that Aβ accumulates within cells before the formation of extracellular plaques, where it typically disturbs synaptic function, resulting in extreme memory loss [213–215]. Various sites besides the plasma membrane, where AβPP may be present, include the ER, trans-Golgi network, and mitochondrial and lysosomal membranes [216], where Aβ can be produced through β- and γ-secretase cleavage. Moreover, secreted Aβ peptides can be incorporated through receptor-induced and/or receptor-independent endocytosis [217–219]. It has been widely studied and supported that soluble Aβ oligomers are the most noxious species that affect various primary molecular mechanisms triggering synapse loss in AD [220, 221].
The presence of intracellular neurofibrillary tangles in the brain [222] is the principal feature associated with AD [223], and these neurofibrillary tangles are composed of a microtubule-stabilizing protein (tau) [224]. When tau becomes hyperphosphorylated, it displaces the microtubules, resulting in their disruption thus disturbing the neural operating machinery [225]. The Aβ-mediated translocation of tau to neural spines is linked to primary synaptic dysregulation in AD pathophysiology [226]. Conclusive analysis of AD can only be carried out by exploring the autopsy of brain tissue, based on the presence of extracellular plaques produced by Aβ peptides, intracellular neurofibrillary tangles made up of pTau, Aβ deposits in the blood vessels, and substantial atrophy in definite brain regions (hippocampus, entorhinal, and frontal cortices) associated with cognitive dysfunction [225, 227].
Following the inception of clinical manifestations of AD including memory loss and the presence of Aβ pathology, it has been evident that mitochondrial damage leads to elevated ROS generation, triggering the primary stages of AD [63, 228]. In support of this fact, hallmarks of OS, such as elevated levels of glycosylated products; lipid peroxidation; generation of free carbonyls, ketones, alcohols, aldehydes, cholestenone, and oxidized proteins; and oxidative changes in RNA, nuclear, and mitochondrial DNA, are also observed in the autopsy of brain tissue and peripheral systems (cells and isolated mitochondria) of people with initial phases of AD [229, 230]. Mitochondrial ROS can disintegrate the mitochondrial membrane potential, hastening the intracellular production of ROS, eventually affecting the complete cell [30]. Another source of Aβ-induced ROS generation involves activation of microglia in the brain through an inflammatory response to the accumulation of extracellular amyloid plaques [231]. Furthermore, elevated Aβ levels can hasten ROS production by directly attaching to mitochondrial membranes, varying mitochondrial dynamics and function, eventually resulting in anomalous energy metabolism and synaptic function deficits [30, 232]. Membrane-linked OS, mediated via Aβ peptides, disturbs ceramide and cholesterol metabolism that, in turn, triggers the neurodegenerative cascade, leading to additional Aβ deposition and tau phosphorylation [233, 234]. Additionally, alterations in membrane components, such as fatty acids and cholesterol, regarding their characteristics, disposition, and distribution along the membranes, have been studied as indicators of cell membrane alterations in AD brain [235, 236]. Captivating data also stated that besides mitochondrial ROS generation, atypical homeostasis of bioactive metals such as iron, zinc, copper, manganese, aluminum, and magnesium could be associated with the production of free radicals and OS triggering aggregation of Aβ and tau [207, 237]. Specifically, zinc directly targets AβPP dispensation by attaching to the protein, and Zn, Al, Cu, and Fe unswervingly attach to Aβ, thereby promoting its accumulation. Similarly, redox metals can stimulate the phosphorylation of tau, its detachment from microtubules, and the generation of neurofibrillary tangles [4]. ROS generation is mediated by redox-active metals (Fe, Mn, and Cu) using catalytic reactions parallel to the Fenton reaction, where metals convert O2– and H2O2 to OH- species that are associated with lipid peroxidation [30]. In addition, a direct linkage between Aβ peptides and Cu or Fe has been demonstrated to produce H2O2 [4].
Hence, transition metals and Aβ could increase OS and extra-mitochondrial generation of ROS in a synergistic manner. Taken together, it can be suggested that varying mitochondrial function, elevated OS, poor antioxidant defense, and generation of Aβ and pTau, which additionally trigger mitochondrial dysfunction and ROS generation, could characterize a “malicious cycle” that gradually aggravates the etiology of disease, ultimately resulting in neural death [137]. Perturbed SOD-1 and SOD-2 gene expression levels in neurodegenerative diseases play a significant role in allowing the accumulation of ROS and thus leads to worsening the outcomes. Increased SOD-2 levels have also been shown to prevent neural death triggered by oxidative damage in AD and PD by mitigating membrane lipid peroxidation and protein nitration and managing mitochondrial dysfunction [238, 239]. The relationship between ROS levels and AD pathogenesis is shown in Fig. 4.
Schematic representation of the relationship between different factors elevating reactive oxygen species (ROS) level and Alzheimer’s disease pathophysiology. NFT, neurofibrillary tangles.
ANTIOXIDANTS: AN APPROACH FOR TREATMENT OF AD
Antioxidant therapies for AD have been widely implemented, and some traditional herbal antioxidants have exhibited therapeutic effects. Studies suggest that a diet rich in different types of antioxidants (natural/synthetic) reduce the risk as well as delay or prevent the progression of AD [240–242]. Table 1 summarizes the natural and synthetic antioxidants reported for their effects on AD model systems. Figure 5 also summarizes the available chemical structures of these antioxidants.
List of antioxidants investigated in preclinical and clinical studies for their effect on AD pathogenesis
AD, Alzheimer’s disease; Aβ, amyloid-β; AβPP, amyloid-β protein precursor; BBB, blood-brain barrier; ER, endoplasmic reticulum; iPSC, induced pluripotent stem cell; MCI, mild cognitive impairment; OS, oxidative stress; ROS, reactive oxygen species; SCD, subjective cognitive decline; STZ, streptozotocin.
Chemical structures of antioxidants as Alzheimer’s disease therapeutic agents.
Synthetic antioxidants
In the category of synthetic antioxidants, although there are not a large number of compounds reported to date, the selected compounds have been observed to exhibit antioxidant properties by different mechanisms in different AD model systems.
Recent studies have shown that plastoquinone-decyl triphenylphosphonium (SKQ) is a mitochondria-targeted antioxidant used for AD treatment [243, 244]. SKQ significantly improved behavioral activity in OXYS rats by reducing the hippocampal Aβ1 - 40 and Aβ1 - 42 protein levels, without affecting the levels of Aβ in the serum of OXYS rats [245]. Moreover, a similar experiment showed that treatment with SKQ improved the structural and functional state of the resulting decline in Aβ accumulation and tau hyperphosphorylation (markers of AD pathogenesis) in the hippocampus of the OXYS rat [246]. There are a limited number of recent studies on the effect of SKQ on AD; however, the effect of SKQ has recently been studied in a culture model of multiple sclerosis [247].
The Food and Drug Administration-approved drug UMI-77 binds the anti-apoptotic protein MCL-1, which has also been identified as a mitophagy receptor that networks with LC3A to activate mitophagy. The study suggested that UMI-77 activated mitophagy via the ATG5 pathway, independent of canonical mitophagy receptor proteins, such as BAX or PARKIN, and ameliorated cognitive decline and amyloid pathologies in the APP/PS1 mouse model of AD [248].
MitoQ is a mitochondria-targeting antioxidant that selectively accumulates in mitochondria at high concentrations. MitoQ has been reported to be effective in different AD models, as reviewed in recent studies [243, 249]. MitoQ reduced neurotoxicity induced by Aβ in cortical neurons and prevented elevated ROS production and loss of the mitochondrial membrane in a triple transgenic mouse model of AD (3xTg-AD mice) [250]. Moreover, MitoQ extended the lifespan and improved the health of a transgenic C. elegans model of AD by protecting against Aβ-induced toxicity and OS [251].
Although synthetic antioxidant agents have shown positive effects in the prevention of AD, they may act off-target and exhibit adverse effects on the disease. Therefore, the utilization of natural antioxidants may be a better choice for improving the efficacy of antioxidant agents in AD therapeutics [252, 253].
Natural antioxidants
Vitamins
Supplementation with antioxidant vitamins A, C, and E has been extensively studied for the treatment of AD [254]. Cell culture and animal model studies of vitamin E administration in APP transgenic mice (Tg2576) showed that vitamin E prevented the formation of plaques but was ineffective when treatment was given after the formation of amyloid deposits [255]. Another study showed that patients with mild to moderate AD treated with 2000 IU/d of vitamin E experienced a slow functional decline [256]. Such accumulating pieces of evidence of the potential of vitamin E to reduce Aβ-induced OS, alleviate Aβ toxicity, and improve cognitive ability in various AD models have extensively been reviewed in recent articles [254, 257–259]. A study has suggested that vitamin E scavenges the free radicals via transfer of hydrogen atom to liberate a non-radical product and a vitamin E radical, which can react with other radicals to yield steady products, damage lipids, or react with reducing agents (including vitamin C or ubiquinol) to restore vitamin E [260]. However, these aspects have not been fully demonstrated in AD model-based studies. Gugliandolo et al. (2017) in their review summarized that higher doses and prolonged vitamin E supplementation resulted in improved results in AD treatment [259]. Additionally, the National Institutes of Health factsheet also suggested that there were no adverse effects observed in the regulated consumption of vitamin E in food. However, excessive or high doses of vitamin E in the form of α-tocopherol may cause hemorrhagic effects, interrupt blood coagulation, and inhibit platelet aggregation [261]. In addition to α-tocopherol, the γ-tocopherol form of vitamin E has also been shown to possess potent antioxidant and anti-amyloid activities by reducing mitochondrial ROS and Aβ42 levels in SH-SY5Y cells expressing the mutated APP gene. This comparative study also showed that the anti-amyloid effect of γ-tocopherol was higher than that of α-tocopherol, suggesting the potential of γ-tocopherol to improve mitochondrial function by increasing the ATP level and activity of the complex V enzyme. Moreover, treatment with γ-tocopherol has been shown to reduce mitochondrial membrane permeability and subsequently prevent apoptosis, which was confirmed by the reduced expression of pro-caspase-3 protein; this was not observed in the case of treatment with α-tocopherol [262]. However, in vivo validation of the suggested in vitro potential of γ-tocopherol in AD models needs to be performed following optimization. In a cross-sectional study, the protective effect of γ-tocopherol on the oxidation of presynaptic proteins was investigated in 113 deceased participants in the Memory and Aging Project. Brain γ-tocopherol levels were associated with elevated levels of the SNARE protein composite, complex I, complex II, synaptotagmin synaptophysin composite, and septin-5 in the midfrontal cortex [263].
Moreover, the relations of α- and γ-tocopherol levels in the brain related to microglia density in 113 deceased participants from the Memory and Aging Project shows that higher α- and γ-tocopherol levels were associated with lower total and activated microglia density in cortical regions, but not in subcortical brain regions. These findings imply that there may be a connection between tocopherols and AD, which might be partly explained by the alleviating effects of tocopherols on microglia activation [264].
Further, a recent study showed that treatment with a combination of antioxidant coenzyme Q10 and vitamin E to endure cerebral hypoperfusion-mediated neurodegeneration in a rat model resulted in improved memory, neural cell viability, and antioxidant levels. Hence, it is advisable to use a combination of both vitamin E and CoQ10 for better protection against NDDs especially AD and PD [265]. This study also indicates the synergistic effect and enough antioxidant action without need of large and toxic doses of using a single antioxidant in the treatment of AD [265].
Vitamin C acts as an antioxidant by contributing two electrons, which prevents other compounds from being tarnished and modulates BBB integrity and mitochondrial morphology. High vitamin C intake may protect against AD-like pathologies. Supplementation with vitamin C reduces the burden of Aβ generation [266] and amyloid plaques [267]. Higher supplementation of vitamin C was reported to curtail amyloid plaque load in the cortex and hippocampus in KO-Tg mice, resulting in amelioration of BBB disruption and mitochondrial alteration [266]. Moreover, an insufficient supply of vitamin C can lead to late-onset AD [267]. A recent study has shown that deficiency of vitamin C (ascorbate) reduced the dopamine availability during synaptic transmission in APP/PSEN1 mice and gulo–/– mice (depend on dietary vitamin C to maintain its adequate levels in the brain and liver) [268]. Pretreatment with vitamin C protected cultured SH-SY5Y neuroblastoma cells from Aβ-induced programmed cell death [269]. Vitamin C acts as a reducing agent by reducing metals such as iron and copper in the body, and it can also be oxidized by ROS or reactive nitrogen species. A combinatory-based experiment investigating the amalgamation of vitamins E and C revealed a reduction in oxidative damage and improved memory in APP/PSEN1 mice. However, this observation was valid only for a combination with a medium dose of vitamin E and not for a higher dose [270].
Supplementation with vitamin A has also been shown to rescue marginal vitamin A deficiency-induced memory deficits and improve cognitive ability in an APP/PS1 mouse model of AD [271]. The potential antioxidant role of vitamin A in AD treatment has been extensively reviewed by Ono et al. (2012), with different forms of vitamin A, such as retinol, retinal, and retinoic acid involved in inhibiting the formation and destabilizing effect of Aβ fibrils in in vitro models. It also inhibited Aβ oligomerization. In vivo administration of vitamin A also decreased the accumulation of Aβ and tau phosphorylation in the brains of transgenic animal models of AD, resulting in attenuation of neurodegeneration and memory improvement [272].
Polyphenols
Polyphenols are secondary metabolites generally found in plants and are known for their antioxidant properties. Various polyphenols have been reported to exert substantial effects on the development of AD. Moreover, polyphenols with antioxidant effects have been shown to have positive effects on AD samples.
Curcumin (turmeric) is one of the most promising natural derivatives, with potent activity against several disorders. Curcumin is well known for its antioxidant properties and has potential roles in the management of AD. Curcumin exerts antioxidant, anti-inflammatory, and lipophilic effects, thereby improving cognitive function in patients with AD. The overall improvement in memory of patients with AD, including reduced Aβ plaques, delayed neurodegeneration, and reduced formation of microglia is suggested to be due to the various effects (metal-chelation, anti-inflammatory, antioxidant activities) of curcumin. The mechanisms by which curcumin prevents AD pathology have been discussed in a review by Mishra et al. [273]. Increasing evidence supports the role of curcumin in AD treatment. Supplementation with curcumin improved learning and cognitive ability in apolipoprotein E4 (ApoE4) transgenic mice by obstructing neuroinflammation via the ER stress pathway [274]. The underlying mechanism also suggested that curcumin reduces the elevated expression of ApoE4 and the release of inflammatory factors in ApoE4 mice [265].A recent report revealed the selection of curcumin as the “Cognition Supplement of the Year: 2021” [275].The synergistic effect of curcumin and berberine in the AD mouse model (i.e., B6C3-Tg (APPswePSEN1dE9)/Nju double transgenic mice) yielded an improved effect on enhancing the cognitive function of mice compared with a single-drug treatment, with the combinatory therapy reducing soluble Aβ peptides and declining inflammatory responses and OS in both cortices and hippocampi of AD mice. The combined treatment performed better than the single-drug treatment in reducing AβPP and BACE1 levels and expanding AMPKα phosphorylation and cell autophagy, which were suggested as the possible causal modality of action for the combined effects in reducing the symptoms in AD mice [276]. However, clinical trials of curcumin and its effects in humans with AD are restricted, and the results are less consistent, thereby confounding its interpretations [277]. Despite its advantages, curcumin has low bioavailability owing to its low solubility, low absorption, fast metabolism, and rapid removal [278]. Therefore, many attempts have been made to overcome these limitations and develop new formulations, such as liposomal encapsulation, nanoparticles, powder form, micellar form, emulsions, co-administration with other substances, or isolated administration of its elements [279–282].
Berberine, in recent years, has gained immense attention as a therapeutic intervention for mental health issues, including AD. A recent study revealed that berberine exhibited a neuroprotective effect in a 3xTg-AD mice by attenuating ER stress and OS. The study showed that berberine prevented the translation of BACE1 facilitated by PERK/eIF2α signaling, which in turn reduced the production of Aβ and the resulting neural apoptosis in 3xTg-AD mice [283]. In support of this, another study showed the protective effect of berberine on streptozotocin (STZ)-and Aβ25 - 35-induced AD diabetic rats by reducing the transcription of mRNAs and expression of proteins related to ER stress [284]. Berberine has also been shown to activate mitochondrial respiration and promote neural metabolism that switches from oxidative phosphorylation to glycolysis. This switch initiates AMPK and activates the clearance of tau via autophagy in induced pluripotent stem cell (iPSC)-derived neurons from patients with AD and tau-transgenic mice [285]. Berberine inhibits Aβ25 - 35-induced increase in pTau levels, inflammatory cytokine expression, and apoptosis via regulation of miR-107 and ZNF217 in AD model cells (PC12 cells) [286]. Another study showed that 3xTg-AD mice treated with berberine regained the memory and spatial learning ability associated with reduced pTau through the modulation of Akt/glycogen synthase kinase-3β and protein phosphatase 2A activity and elimination of tau via autophagy [287].
Resveratrol is a natural polyphenol that is prominently found in red wine as well as in other food sources such as grapes, bananas, blueberries, spinach, peanuts, and cocoa. Recent reviews have collectively highlighted that resveratrol is used to treat various neurodegenerative diseases because of its antioxidant potential [243, 288–290]. An in vivo study revealed that administration of resveratrol to AD and healthy mice (3xTg-AD and NonTg, respectively) protected against memory loss in the AD mouse model and enhanced cognitive ability in the healthy mouse model. This study suggested three key modes of action of resveratrol, which were as follows: 1) the reduced amyloid load via neprilysin activation and BACE1 downregulation, 2) enhanced ubiquitin-Proteasome System (UPS) leading to reduced levels of abnormal amyloids and tau proteins, and 3) upregulation of the AMPK/SIRT1 pathway leading to increased PGC-1α and CREB levels [291]. Resveratrol reduced amyloid load and increased the level of mitochondrial complex IV protein in a murine model of familial AD (i.e., AβPP/PS1) [292]. Recently, a novel bioavailable resveratrol formulation, known as JOTROL, was manufactured by Jupiter Orphan Therapeutics. JOTROL showed significantly better pharmacokinetic properties than non-formulated resveratrol; moreover, it decreased tau expression and increased mitochondrial biogenesis in triple-transgenic (APPSW/PS1M146V/TauP301L; 3xTg-AD) AD mice [293]. A recent study showed that resveratrol exhibited a neuroprotective role against oxidative damage in an in vitro AD model via activation of mitophagy. The activation of mitophagy was indicated by an increase in the number of acidic vesicular organelles, LC3-II/LC3-I ratio, PARKIN and Beclin-1 activity, and LC3 and TOMM20 levels in Aβ1 - 42-treated PC12 cells. Resveratrol also mitigated apoptosis, OS, and mitochondrial damage in Aβ1 - 42-treated PC12 cells [294]. However, the study also indicated that the underlying mechanism of resveratrol-induced mitophagy is not clear. In contrast to these benefits, a recent study by Jang et al. (2021) revealed that a moderate dose of resveratrol unexpectedly increased the production of Aβ by stabilizing AβPP, whereas a higher dose of resveratrol reduced Aβ production [295].
Extra virgin olive oil (EVOO) contains a substantially high number of polyphenols, which are known for their antioxidant properties and therapeutic contributions in the prevention of AD. A review by Rom
Oleuropein Aglycone (OA) and hydroxytyrosol are the major phenolic extracts found in olive oil. A study showed that pretreatment with OA protects neurons from neurotoxins and oxidative damage in male Wistar rats injected with colchicine to induce OS and cognitive impairment [300]. OA prevents amyloid aggregation and neurotoxicity by affecting different pathways, including AβPP processing, Aβ peptide accumulation, tau accretion, autophagy impairment, and neuroinflammation in the TgCRND8 mouse model of AD [301]. A recent molecular dynamics simulation-based study revealed that OA prevents Aβ aggregation and disrupts Aβ fibrils [302]. Hydroxytyrosol is the hydrolyzed form of oleuropein, which is also a constituent of olive oil and has higher bioavailability [303, 304]. Hydroxytyrosol is a powerful antioxidant and free radical scavenger that protects against amyloid-associated neurodegeneration and restores cognitive ability in TgCRND8 mice [305]. Moreover, a recent study revealed that hydroxytyrosol enhances mitochondrial energetics, as confirmed by the increased mitochondrial quantity and fusion in 7PA2 cells, which are one of the best cell models of Aβ toxicity and adequately dysfunctional mitochondria commonly observed in AD [306].
Anthocyanins are potent antioxidants that regulate the formation of amyloid peptides and generation of free radicals in the brain [307]. A previous study has shown that a combination of anthocyanins/anthocyanidins (MAF14001) protects against Aβ toxicity by preventing OS, mitochondrial dysfunction, and apoptosis in SK-N-SH cells. MAF14001 may directly affect Aβ to prevent Aβ accumulation and decrease tau phosphorylation induced by Aβ [308]. Moreover, a study showed that anthocyanins suppress Aβ toxicity in Neuro2a cells and divert Aβ aggregation to an alternate, non-toxic form [309].
Silibinin is the major active component of silymarin and prevents memory impairment and oxidative damage induced by Aβ25 - 35 in mice [310]. Moreover, silymarin and silibinin exhibited neuroprotective effects against OS induced by H2O2 in rat pheochromocytoma PC12 cells, with the latter being more effective [311]. Silibinin has also been shown to reduce Aβ plaques and soluble Aβ levels via downregulation of AβPP and BACE1 and upregulation of neurolysin in the hippocampi of APP/PS1 mice [312].
Carotenoids
Carotenoids are fat-soluble and highly unsaturated red, orange, or yellow pigments that are naturally present in plants, fungi, bacteria, and algae, and their color intensity is commonly linked to carotenoid levels. Carotenoids have been extensively studied for their antioxidant properties in AD treatment, and several studies have revealed that dietary supplementation with carotenoids ameliorates AD symptoms [254, 313]. Recent studies have suggested a potential role of carotenoids in AD models. Cara Cara is a type of red orange, categorized by a high carotenoid content. A previous study showed that pasteurized orange juice derived from Cara Cara reduced the levels of endogenous ROS and Aβ toxicity, thereby exhibiting an increased subsistence rate under normal and OS conditions in a C. elegans AD model [314]. Pasteurized orange juice induced the expression of gst-4:GFP and amplified resistance to stress, which depends on the SKN-1/Nrf2 transcription factor, a mandatory factor for the protective effect of pasteurized orange juice against Aβ toxicity [314]. Another carotenoid, β-carotene, which is used to treat STZ-induced cognitive deficits in mice, inhibits the decline in the concentration of acetylcholine by impeding the acetylcholinesterase enzyme [315]. Moreover, decreased Aβ plaques in the brains of β-carotene-treated mice enhanced cognitive function [315]. In another study, a total of 927 participants were enrolled to evaluate the prospective relationship between the level of carotenoids consumed and the risk of AD. Individual carotenoids, lutein-zeaxanthin and lycopene, were found to be inversely associated with global brain pathology. Furthermore, lutein-zeaxanthin showed additional inverse associations with the severity of neuritic plaques, AD diagnostic score, and density and severity of neurofibrillary tangles [208, 316].Therefore, β-carotene has been suggested for its significance in the improvement of memory and for its potent role in the treatment of many neurodegenerative diseases, including AD. Lutein is a carotenoid, and a study of a large cohort of older French participants maintaining higher concentrations of lutein showed a moderate decrease in the risk of dementia and AD [317].
Others
Urolithin A (UA) is a dietary metabolite that showed a neuroprotective effect in an AD mouse model (APP/PS1 mice) by targeting different processes, including reactive gliosis, inflammatory signaling, Aβ plaque formation, and apoptosis [318]. UA reduces Aβ deposition in the cortex and hippocampus of APP/PS1 mouse [318]. Recently, a study showed that UA reduced mitochondrial calcium influx, mitochondrial ROS accumulation induced by high glucose, AβPP processing enzyme BACE1 expression, and Aβ generation in SH-SY5Y and iPSC-derived neural differentiated cells [319]. In addition, a study on the effect of UA on diabetes-associated AD pathologies showed that UA reduced the expression of AβPP and BACE1, tau phosphorylation, Aβ accumulation, and damage to cognitive ability in a STZ-induced diabetic mouse model [319]. UA activates mitophagy in various neurodegenerative disorders. For example, a recent study showed that UA stimulated mitophagy and improved the health of mitochondria in phase I human clinical trials that included 60 older adults [320]. In the case of AD, supplementation of UA to AD iPSC-derived neurons, APP/PS1 transgenic mouse model of AD, and C. elegans AD model showed stimulation of mitophagy that reversed memory loss via PTEN-induced kinase-1-, Parkinson’s disease-related-1/Parkin RBR E3 ubiquitin-protein ligase-, or DAF-16/FOXO-controlled germline-tumor affecting-1-dependent pathways. Additionally, the study also showed that the enhanced mitophagy reduced the insoluble Aβ1 - 42 and Aβ1 - 40 in the APP/PS1 mouse model via microglial phagocytosis of extracellular Aβ plaques and inhibition of neuroinflammation and terminated the AD-associated tau hyperphosphorylation [321].
Nicotinamide adenine dinucleotide (NAD+) precursors have been studied for their potential as neuroprotective and bioenergetic stimulant agents for the treatment of various neurodegenerative diseases, including AD. Nicotinamide riboside (NR) is an NAD+precursor that reduced pTau pathology in 3xTg-AD and 3xTg-AD/Polβ+/– mice. Moreover, treatment with NR improved cognitive ability and restored synaptic plasticity in the hippocampi of 3xTg-AD mice. The study also revealed a reduction in DNA damage, neuroinflammation, and apoptosis in hippocampal neurons as well as an increase in SIRT3 activity in the brains of NR-treated 3xTg-AD/Polβ+/– mice [322]. As exemplified by the recent review by Hosseini et al. (2021), a limited number of recent studies strengthen the significant effect of NR in AD treatment [323, 324]. However, NR is in phase 1 clinical trials investigating the neurobiological mechanisms and clinical effects of NR in patients with MCI and mild AD [325].
Spermidine is a natural polyamine known for its anti-aging, anti-inflammatory, and antioxidant properties. A recent study showed that a higher intake of dietary spermidine is associated with reduced cognitive impairment in a Drosophila aging model, aged C57BL/6J male mice, and humans. The study suggested that spermidine increases mitochondrial respiratory capacity, which depends on the autophagy regulator ATG7 and mitophagy mediators, such as PARKIN and PINK1 [326]. A phase III clinical trial-based study showed that spermidine supplementation protected memory performance in 30 older adults with subjective cognitive decline (SCD). Older individuals with SCD exhibit increased Aβ accumulation, grey matter volume reduction, and neural impairments in the brain regions commonly affected in AD [327]. In support of this study, another study on 108 older adults with SCD also showed that dietary ingestion of spermidine is linked with larger cortical thickness in AD-vulnerable regions, the parietal and temporal lobes, and larger volume of the hippocampus [328]. The study suggested a neuroprotective effect of a higher dietary intake of spermidine in older human adults. Moreover, SCD generally occurs in the late preclinical stage of AD; hence, spermidine may be a potential therapeutic agent for AD. Another 3-month clinical trial-based study revealed a similar positive effect (i.e., improved cognitive performance) of spermidine in 92 older individuals with dementia [329]. Overall, spermidine is an important natural antioxidant agent with the potential to preserve brain health in older adults with various brain-associated problems, such as SCD, cognitive impairment, and dementia, which are closely related to AD.
CONCLUSION
Although ROS contribute critically to the pathogenesis of AD, it is challenging to devise treatment options. This is primarily because of the lack of a specific treatment target. To overcome the damaging effects of OS, various antioxidant defenses, either as nutritional supplements or vitamins, are recommended as supplementary therapies in AD. A large number of epidemiological, clinical, and fundamental studies have strongly recommended the use of antioxidants for the treatment of AD. Because OS is neutralized by a complex system of endogenous and exogenous antioxidants, using only a specific type of antioxidant may not be sufficient to defend against oxidative damage. Hence, a combinatorial approach is essential for the treatment of various neurodegenerative diseases, including AD. However, different in-vitro and animal-based studies have shown that nutrition rich in antioxidants may protect the brain from oxidative and inflammatory damage. However, limited data are available from different epidemiological studies and human clinical trials, indicating the effects of antioxidants on inflammation in AD. Moreover, although age is the most significant factor in AD pathophysiology, there is inadequate evidence regarding the possible risk factors for AD. Hence, AD research should focus on daily supplementary therapies, cognitive status, plausible risk factors, and genomic testing to form a full representation of the AD brain.
Footnotes
ACKNOWLEDGMENTS
The authors would like to thank their respective institutes for their support. We would like to thank Editage (
) for English language editing and BioRender (BioRender.com) for illustrations. Finally, the authors would like to express their gratitude to the editor and reviewers for their thorough evaluations and constructive criticism, which improved the scientific merit of this manuscript.
FUNDING
The authors acknowledge the research grant support #12R121 and 12R104 from UAE University, UAE.
CONFLICT OF INTEREST
The authors have no conflict of interest to report.