Reactive oxygen species (ROS) result from normal daily cellular metabolism. Research conducted in the last two decades has clarified the role of ROS as secondary signaling molecules that regulate various biological and physiological processes, including proliferation, host defense, and gene expression. Furthermore, earlier reports have also indicated the role of ROS as a signal transduction mechanism.
Reactive oxygen species (ROS) result from normal daily cellular metabolism. Research conducted in the last two decades has clarified the role of ROS as secondary signaling molecules that regulate various biological and physiological processes, including proliferation, host defense, and gene expression . Furthermore, earlier reports have also indicated the role of ROS as a signal transduction mechanism. This allows adaptation to changes in environmental nutrients and the oxidative environment . In this respect, Kiley and Storz  have well-defined, in the prokaryotes, mechanisms whereby ROS directly activate transcription factors (TFs) for stress adaptation. On the contrary, oxidative stress (OS) refers to elevated levels of intracellular ROS, such as superoxide anion (O2•−), hydroxyl radical (OH•), and non-radical molecules, such as hydrogen peroxide (H2O2) and singlet oxygen (1O2), which further damage lipids, proteins, and DNA (Figure 1 A). A high-energy exposure or electron transfer reaction leads to the production of highly reactive ROS, which is a stepwise reduction of molecular oxygen (O2) as represented in equation (1). Moreover, ROS generation occurs at elevated rates in normal aging. It is an inevitable process in both acute and chronic pathophysiological conditions . Thus, OS is usually the result of excessive ROS production, mitochondrial dysfunction, and an impaired antioxidant system, or a combination of these factors. O2 → O2•− → H2O2 → OH • → H2O (1)
ROS are predominantly produced in mitochondria via mitochondrial enzymes. The electron transport chain (ETC) of mitochondria produces superoxide radicals at respiratory complexes I and III of the oxidative phosphorylation (OXPHOS) pathway through the single-electron leak . Nevertheless, the ROS production rate in complex I is much less than the Flavin-dependent enzymes in the mitochondrial matrix . Amongst various intracellular antioxidant enzymes, five have been mainly discussed in physiological conditions, i.e., (i) Cu/Zn-superoxide dismutase (Cu/Zn-SOD, SOD1) in the cytosol, (ii) manganese superoxide dismutase (Mn-SOD, SOD2) in the mitochondrial matrix, (iii) catalase (CAT), (iv) glutathione peroxidase (GPx), and (v) glutathione reductase. In Figure 1 B, SOD converts superoxide to O2 and H2O2, whereas CAT and GPx convert H2O2 into H2O and O2. Along with the primary antioxidant defense against ROS, secondary antioxidant and cellular detoxification programs are mainly regulated by NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1). Under normal conditions, Nrf2 is retained in the cytoplasm by the actin-binding protein Keap1; a substrate adaptor protein for the Cullin3-containing E3–ligase complex, which targets Nrf2 for ubiquitination and degradation by the proteasome . Keap1 is redox-sensitive since this protein can be modified by different oxidants and electrophiles . OS abrogates the Keap1-mediated degradation of Nrf2, which in turn accumulates in the nucleus . It heterodimerizes with a small musculoaponeurotic fibrosarcoma (Maf) protein on antioxidant response elements (AREs). Nrf2, along with ARE, further stimulates the expression of a wide array of phase II antioxidant enzymes, which includes NAD(P)H quinone oxidoreductase 1 (Nqo1), heme oxygenase 1 (Hmox1), glutamate-cysteine ligase, and glutathione S transferases (GSTs) . In addition, Nrf2 also contributes to cellular proteostasis by regulating the expression of molecular chaperones and various proteasomal subunits . Apart from antioxidant enzymes, small molecular weight and nonenzymatic antioxidants, such as vitamins, carotenoids, thiol antioxidants, and natural flavonoids, also protect intracellular components against ROS .
Aggregated proteins' deposition and spreading are the main characteristics of sporadic (s) and familial (f) forms of various neurodegenerative disorders, such as AD. This, in turn, results in excessive ROS production leading to OS, chronic neuroinflammation, and mitochondrial dysfunction, which altogether cause neuronal loss  and protein misfolding . ROS-induced protein misfolding/unfolding can result in gain/loss-of-function. The protein modification of the oxidized proteins is insufficient to achieve their actual shape, impacting stability, activity, and/or function . Several lines of evidence suggest that elevated ROS production initiates toxic amyloid-beta precursor protein (APP) processing and thereby triggers amyloid-beta (Aβ) generation . These elevations in ROS are the results of protein aggregation and corresponding neuronal damage, which in turn activates disease-associated microglia via damage-associated molecular patterns . These ROS are primarily generated via NADPH oxidase 2, which is well associated with DAMP signaling, inflammation, and amyloid plaque deposition . Additionally, ROS generated from mitochondria helps in the propagation of immune activation, leading to excessive OS and neurodegeneration. Interestingly, recent studies on postmortem AD brains and AD transgenic mice have shown that Aβ and APP are found in mitochondrial membranes to block protein transport and disrupt the ETC with final, irreversible cell damage . Moreover, these disruptions are further exacerbated by a defective repair system. Tamagno and colleagues reported that OS resulting from hydroxynonenal (HNE) or H2O2 leads to enhanced Aβ production in different cell models . In addition, HNE also modifies the γ-secretase substrate receptor nicastrin, which leads to enhanced binding of the γ-secretase substrate APP and likely results in elevated Aβ generation . Moreover, neurons contain a high amount of polyunsaturated fatty acids (PUFAs) that can interact with ROS, leading to a self-propagating cascade of lipid peroxidation and molecular destruction . Products of lipid peroxidation have also been shown to be elevated in blood samples and brains of AD patients at autopsy . Both nuclear and mitochondrial DNA and RNA also exhibit oxidative damage in the AD brain . Hence, understanding oxidative balance is regarded as an important event in understanding AD pathogenesis. OS might increase the aggregation and production of Aβ and assist polymerization and tau phosphorylation via the creation of a vicious cycle that stimulates the progression and even initiation of AD. Keeping this in mind, in this review, we sought to analyze the myriad interactions between oxygen radicals and toxic protein oligomers in the context of AD to understand their importance in disease pathogenesis. Furthermore, we also discuss the role of microbiota in altering the redox balance and its consequences concerning Aβ production and tau hyperphosphorylation.
ROS are oxygen-containing molecules that are chemically more reactive than O2 and, therefore, can damage cellular macromolecules. For example, ROS can react with nucleic acids (NA) by attacking nitrogenous bases and the sugar-phosphate backbone. Further, these can evoke single- and double-stranded DNA breaks, affecting the protein-coding region of mtDNA and influencing OXPHOS . mtDNA mutations can cause disturbances in the respiratory chain, and as a result, it loses control over ROS production . In addition, the modification in core DNA repair genes can result in an impaired recognition system and an inefficient repair of DNA damage, which in turn can accelerate aging and leads to age-related disruptions in cellular and tissue functions. This also results in the accumulation of ROS, which increases with age and intensifies OS. This elevation in OS damages mtDNA, leading to apoptosis, inhibition of mitochondrial respiratory chain transition, and increased mitochondrial membrane permeability in the absence of sufficient antioxidant capacity . Thus, pro-oxidative/antioxidative cellular imbalance between ROS production and the ability of the defense mechanisms of biological systems to eliminate ROS-mediated cellular stress disturbances results in a vicious cycle, since the OS reciprocally aggravates ROS production. ROS have also been reported to attack structural and enzymatic proteins via oxidation of residual amino acids, prosthetic groups, formation of cross-links and protein aggregates, and proteolysis . Lipid peroxidation (auto-oxidation) is a process in which PUFAs are oxidized due to several double bonds in their structure. This process involves producing peroxides (chemical compounds in which a single covalent bond links two oxygen atoms), ROS, and other reactive organic free radicals. Several markers of oxidative damage have been defined, including the following: 8-hydroxy-2-deoxyguanosine (8-OHdG) and 8-hydroxyguanosine, markers of oxidative DNA damage; 8-hydroxyguanine, a marker of RNA oxidation; protein carbonyls and nitrotyrosine, markers of protein oxidation; and malondialdehyde (MDA), thiobarbituric-acid-reactive substances, 4-hydroxy-2-nonenal (4-HNE), acrolein, isoprostanes, and neuroprostanes, markers of lipid peroxidation . Moreover, ROS and aging have also been linked to the promotion and accumulation of advanced glycation end products (AGEs). AGEs are insoluble in detergents, protease-resistant, and non-degradable protein, lipid, or NA aggregates generated by non-enzymatic glycation or glycoxidation after exposure to aldose sugar. AGEs have been reported to impair normal cellular/tissue functions directly or indirectly through the AGE/RAGE pathway after binding to specific receptors for advanced glycation end products (RAGEs) . Due to synergism with OS, the production of AGEs is promoted by OS, which eventually leads to ROS generation.
Furthermore, AGEs have been found to accumulate in numerous tissues throughout physiological aging, which leads to OS since the ability to respond to OS reduces with age. Due to this, many proteins lose their function, including those involved in gene transcription regulation . Thus, AGEs serve not only as proinflammatory molecules but also as potent neurotoxins . Protein glycation begins as a nonenzymatic process with a free amino acid group capable of producing a labile Schiff base. The process thus takes place along with the unconstrained condensation of aldehyde or ketone groups reportedly present in sugars. Furthermore, the phenomenon mentioned above also agrees with Maillard’s classical reaction in 1912 . Subsequently, a series of reactions occur that result in the generation of AGEs containing irreversibly cross-linked heterogeneous protein aggregates.
Recently, several pieces of evidence link the role of microbiota in brain biology and aging, being an essential factor involved in various physiological processes via interactive symbiotic network system with host . This interactive network between host and microbiota interconnects the gut track, epidermis, liver, and all other organs with the central nervous system generally referred to as the microbiota-gut-brain axis . The microbiota is composed mainly of bacteria that colonize all mucosal surfaces, with higher density in the gastrointestinal tract, approximately 100 trillion bacteria from nearly 1000 various bacterial species , thereby influencing and triggering various events associated with aging disorders such as AD . Recently, a line of evidence revealed an association of brain amyloidosis with pro-inflammatory gut bacteria in cognitively impaired patients  and various AD mouse models . These findings strongly highlight the association of microbiota and amyloid pathogenesis in AD. However, these fields lack crucial in-depth information and require more exploration.
OS's physiological levels have been generated in the microbiota, which can interfere with its composition and functionality . Furthermore, interactions between microbe-microbe or host-microbiota may also impact the CNS redox balance by elevating ROS levels or impairing the antioxidant system or both ; hence, serving not only as a cause but also a consequence of increased levels of oxidative injury in CNS , thus adding a new dimension to the interplay between the gut microbiota and the brain. Moreover, the microbiota can also produce a considerable amount of CNS neurotransmitters, including dopamine, serotonin, and gamma-aminobutyric acid, that can modulate the local activity of the enteric nervous system and can correlate with their respective levels within the CNS, which in turn depends on the intestinal and BBB permeability . The microbiota may also produce neurotoxic and potentially neurotoxic substances (such as lipopolysaccharides and amyloid proteins), which can also reach to CNS via the systemic circulation or the vagus nerve, promoting microglial activation and neuroinflammation, elevated ROS levels, and/or making neurons more susceptible to OS . Therefore, gut microbes were considered plausible triggering factors for several neurodegenerative disorders, considering the proximity of enteric nervous system neurons to the intestinal lumen .
However, the production of amyloid proteins helps in the formation of bacterial biofilms by promoting the binding of bacterial cells with each other, thus providing resistance from physical or immune factor-mediated destruction . However, in abnormal physiological conditions, bacterial amyloids may act as prion proteins and result in cross-seeding of amyloidogenic protein that elevates pathogenic Aβ formation both in vitro and in vivo . For instance, the interaction of cyanobacteria with synaptic receptors such as NMDA results in upregulation of β-N-methylamino- l -alanine (BMAA), an OS-inducing neurotoxin , in AD brains. Furthermore, BMAA has been linked with protein misfolding and resulting inflammatory consequences in the AD mice model . Numerous studies also suggested a link between activation of endogenous herpes simplex-1 (HSV-1) and amyloidogenesis in AD. This intimate relationship resulted in progressive neurodegeneration and cognitive impairment, contributing to AD pathogenesis . A possible reason for this could be the alteration in gut dysbiosis, which results in increased gut barrier permeability, which in turn hyper activates the innate immune response that leads to systemic inflammation, thus impairing the blood-brain barrier , which results in neuronal injury, protein misfolding, and neurodegeneration leading to cognitive impairment . In addition, overwhelmed microglial stimulation and NF-κB-mediated proinflammatory signaling and reactive oxidative and nitrosative stressors can result in neuronal and glial cell death, which can further impair phagocytosis, leading to the accumulation of Aβ42 . C/EBPβ/AEP signaling was activated in 3xTg mice 5xFAD mice due to gut dysbiosis, resulting in Aβ aggregates, OS, and tau hyperphosphorylation .
Furthermore, reduction in the relative abundance of Proteobacteria and the low levels of Bifidobacteria can reduce beneficial short-chain fatty acids, leading to lipid peroxidation . This, in turn, results in impaired APP processing and trafficking, thus impacting the production of Aβ. Studies conducted using germ-free mice have confirmed the impact of microbiota on microglia maturation, astrocyte activity, neuroinflammation, OS, protein misfolding, and cognitive impairment in AD pathogenesis . Modifying the gut microbiota composition with food-based therapy or supplementing with probiotics may be helpful as a new preventive and therapeutic option in both in vitro and in vivo AD models and clinical trials .
It is now evident that Aβ and tau pathologies are modulated by ROS and are also self-perpetuating concerning ROS formation . Hence, strategies involving inhibition of Aβ oligomerization or decreasing ROS production through the design of multitargeted compounds, such as antioxidants, have resulted in several promising approaches currently being tested in clinical trials. Antioxidants are a broad and heterogeneous collection of chemicals that work by inhibiting the production, detoxification, or scavenging of oxidant species. According to a different criterion, antioxidants can be classified into four different classes based on their chemical structure: vitamins (e.g., ascorbic acid, α-tocopherol, β-carotene, and retinol), synthetic compounds (e.g., butylated hydroxytoluene), natural compounds (e.g., plant-derived polyphenols), and inorganic compounds. Some antioxidants act as chain-breaking molecules, as they can prevent the propagation of or stop radical chain reactions (e.g., α-tocopherol). On the contrary, antioxidants, such as Gpx and catalase, can detoxify H2O2. This chemical reaction serves a vital role in cell biology as H2O2 can produce OH radicals in the presence of transition metals such as Fe2+, for which there is no detoxification system .
Several antioxidant studies in AD models have also been reported, demonstrating that antioxidants consistently positively affect the animals’ behavioral and amyloidotic phenotypes (Table 1). Vitamins are potent antioxidants that directly affect free radicals by reducing OS, inflammatory processes, and neuronal loss . Vitamin A (retinol) is essential for the neuronal formation and remains present in the nervous system across life. Along with β-carotene, vitamin A also protects regenerating neurons during the neurodegeneration process by preventing the development and aggregation of Aβ plaques both ex vivo and in vivo. It may also prevent impaired cognition in AD and improve memory performance and spatial learning in rodent models. Studies have shown that AD patients have lower vitamin A and β-carotene compared with healthy individuals . Early vitamin E (α-tocopherol) supplementation significantly reduced Aβ levels and deposition in the Tg2576 AD model . The same therapeutic regimen prevents a surge in amyloidosis . It improves cognitive function after experimental traumatic brain injury, a known risk factor for AD development in Tg2576 mice . Curcumin, a popular antioxidant and anti-inflammatory substance found in curry spices, substantially decreases OS and amyloid pathology in the Tg2576 mouse model .
Furthermore, curcumin is a potent inhibitor of Aβ fibrillization  and oligomerization  and promotes destabilization of pre-existing Aβ deposits in both cell culture models and animal models of AD . Curcumin and its derivatives also increase the uptake and clearance of Aβ by macrophages in AD patients . Furthermore, using LLC-PK 1 and NRK-52E cells, Balogun and colleagues reported that curcumin upregulates Aβ-induced SOD and catalase and can further activate Nrf2 by selectively binding to Keap1 . Luteolin has also been associated with activating the Nrf2 pathway, which increases endogenous antioxidative gene expression in neuronal cells . Melatonin, a drug with antioxidant properties, partially inhibits the expected time-dependent elevation in Aβ levels, reduces the abnormal nitration of proteins, and increases the survival of Tg2576 mice . Similarly, ferulic acid, rosmarinic acid, and nordihydroguaiaretic acid (NDGA) have also been reported to inhibit the fibrillization and/or oligomerization of Aβ into higher-order species in vitro .
|α-Tocopherol||Reduces Aβ and lipid peroxidation; delays development of tau pathology; reduction in learning deficits and motor weakness||Tg2576 mice||[156,157,167,168,169,170,173]|
|Ascorbic acid||Reduces Aβ oligomers, tau phosphorylation, and oxidative stress||hAPP-J20 mice|||
|Retinol||Reduces MDA levels; upregulates SOD activity; reduces Aβ pathology||APPswe/
tauP301L (3 × Tg) mice; in vitro enzymatic assay and in silico modeling
|CoQ10||Reduces MDA levels; upregulates SOD activity; reduces Aβ pathology||Tg19959 mice; APP/PS1 Tg mice||[176,177]|
|Mito Q||Prevents cognitive decline, oxidative stress, Aβ accumulation, synaptic loss, and caspase activation||3 × Tg mice|||
|Zeolite||Increases endogenous SOD; reduces Aβ levels and plaque burden||Randomized clinical trial|||
|β-Carotene||Improves cognitive impairment and oxidative stress||Streptozotocin-induced AD mice model|||
|Curcumin||Inhibits Aβ fibrillization and oligomerization; clearance of Aβ by macrophages; reduces Aβ40 and 42 and Aβ-derived diffusible ligands; increases Aβ-degrading enzymes; promotes destabilization||Tg2576 mice; APPSw mice; APPswe/PS1dE9 mice; in vitro enzymatic assay||[160,161,162,163,181]|
|Ferulic acid||Inhibits the fibrillization and/or oligomerization of Aβ||In vitro enzymatic assay||[167,168]|
|Rosmarinic acid||Inhibits the fibrillization and/or oligomerization of Aβ||Molecular docking analysis; Tg2576 mice; PC12 neuroblastoma||[168,169,170]|
|Nordihydroguaiaretic acid (NDGA)||Inhibits the fibrillization and/or oligomerization of Aβ||Tg2576 mice|||
|ApoE mimetic peptide Ac-hE18A-NH2||Reduces oxidative stress and ApoE secretion; inhibits Aβ plaque deposition||APP/PS1ΔE9 mice and U251 human astrocyte cells|||
|Catalase mimetic||Protects against oxidative stress, DNA, and protein oxidation; reduces Aβ and tau phosphorylation||3 × Tg-AD mice|||
|Melatonin||Inhibits time-dependent elevation of Aβ; reduces abnormal oxidation and nitration of proteins; increases survival; alleviates learning and memory deficits; decreases choline acetyltransferase activity and increases acetyltransferase activity; increases mitochondrial function||Tg2576 mice; APP 695 Tg mouse model; APP/PS1 mice; APPswePS1dE9 mice||[166,184,185,186,187]|
|N-Acetyl-l-cysteine||Reduces lipid peroxidation, oxidative stress, and glutathione peroxidase activity||APP/PS-1 knock-in mice|||
The significant outcomes of these studies are reductions in Aβ levels, phosphorylated tau, mitochondrial dysfunction, microglial activation, enhanced synaptic activity, and amelioration of cognitive decline. These results indicate that antioxidant treatment is beneficial in reducing and/or preventing AD progression. The findings also show that combination therapy positively impacts cognitive behavior and lowers AD pathology. The positive findings of these studies are promising. However, they warrant prospective studies (e.g., antioxidant treatment of elderly individuals without AD) and clinical trials (antioxidant treatment for patients with AD). Recent work has also highlighted the importance of a healthy and detoxified innate response by consuming diet precursors and enhancing responsiveness . For instance, the application of radiation health, such as UV radiation from the Sun, can prepare an individual for further UV exposure . Another example includes exposure to pro-oxidants such as H2O2, which can prepare the body for subsequent pro-oxidant exposure, which is similar to the formation of antibodies in vaccines.