Oxidative Stress in the Pathogenesis of Neurodegenerative Diseases

Abstract

Neurodegenerative diseases are devastating conditions with a rising incidence and prevalence due to the aging of the population for which we currently do not have efficient therapies. Despite compelling evidence provided by basic research on the involvement of oxidative stress in their pathogenesis, most trials with antioxidants have failed. The reasons may relate to the low bioavailability of the used compounds or to starting therapy late, when the pathogenic cascades have already induced irreversible damage. The current review discusses the sources of oxidative stress in the central nervous system, the involvement of reactive oxygen species in the pathogenesis of Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis, and the importance of further research on improved delivery methods of antioxidants as well as the search for biomarkers that could help in early diagnosis in the hope of finding more efficient therapies for these diseases.

1. Introduction

The complex and fascinating human brain contains roughly 86–100 billion neurons and 10 times more glial cells, with approximately 0.15 quadrillion (0.15 × 10¹⁵) synaptic connections in the neocortex [1,2]. Due to their terminally differentiated post-mitotic state, which develops very early during development and keeps neurons alive and functional for decades, neurons cannot properly renew themselves [3]. Evidence supports adult hippocampal neurogenesis, although it suggests that neurogenesis may occur in healthy individuals but decreases in those with neurodegenerative conditions [4]. Therefore, efficient molecular mechanisms are necessary for the cells to maintain their integrity and repair themselves if needed [5]. The human brain experiences minor changes as we age, such as the collapse of neuronal and glial cell structures and functions, and changes in metabolism, biochemistry, and cell arrangement also play a role in the progressive decrease of cognitive performance [6]. Under typical circumstances, these modifications also include changes in gene expression and synaptic function, memory deterioration, a reduction in brain volume, and cholinergic system failure. In contrast, neurodegeneration causes more significant cellular and molecular impairments that jeopardize the brain’s overall integrity and function. The collapse of neural networks due to the brain’s continual degradation results in abnormal motor function, changed behavior, memory impairment, and cognitive decline [5]. Several neurodegenerative disorders are common, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) [7]. Dementia is a common outcome of many neurodegenerative diseases. It is expected to impact 150 million people worldwide in 2050, costing patients and their families immeasurable amounts of money and an estimated USD 10 trillion in economic burden [8]. The main risk factor for the development of these ailments is age.

Moses Gomberg discovered free radicals (FRs) in the early years of the twentieth century (1900) [9]. Following their discovery in biological systems in the early 1950s by Commoner and coworkers [10], FRs were almost immediately linked to aging [11] and a variety of diseases [12]. Later on, Helmut Sies presented the idea of oxidative stress in 1985 in the first chapter of his book Oxidative Stress, where he also provided the first explanation of the phenomenon [13]. Several studies focusing on oxidative stress were sparked by this groundbreaking work, to the point that the notion of oxidative stress has since become commonly used in both basic and applied domains of biology and medicine [14].

2. Oxidative Stress (OS)

2.1. Definition

The necessity of oxygen for life, despite its reactive and chemically aggressive nature [15], poses a risk to biomolecules in all aerobic organisms living on earth due to the potential for oxidative modifications caused by the uncontrolled generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [16]. ROS, including superoxide (O₂⁻), hydroxyl radicals (HO•), peroxyl radicals (RO₂•), hydrogen peroxide (H₂O₂), organic peroxides (ROOH), and peroxynitrite (ONOO–), along with RNS, like nitric oxide (NO), nitrogen dioxide (NO₂•), nitrous acid (HNO₂), and peroxynitrite, are constantly produced in living organisms and serve important signaling functions [17]. In physiologic conditions, when they are not overproduced and are promptly dealt with by the organism after fulfilling their role, ROS/RNS contribute to a state called oxidative eustress, also called “positive stress”. This condition is marked by low to moderate levels of oxidants that play a key role in regulating biochemical processes, such as carboxylation, hydroxylation, and peroxidation, as well as modulating signaling pathways, including Nuclear Factor-κB (NF-κB), the Mitogen-Activated Protein Kinase (MAPK) cascade, phosphoinositide-3-kinase (PI3K), and nuclear factor erythroid 2-related factor 2 (Nrf2) [18].

To mitigate oxidative damage, aerobic organisms have developed highly efficient antioxidant defense mechanisms. However, many metabolic disorders and conditions characterized by chronic low-grade inflammation exhibit elevated levels of oxidative stress biomarkers [19]. A substantial body of evidence links increased oxidative stress to the pathogenesis of cardiovascular, neurodegenerative, metabolic, and inflammatory diseases [16].

2.2. ROS Generation

It is widely known now that ROS can be generated in cells through the action of endogenous and exogenous factors. Examples of exogenous sources are different types of radiation, such as ultraviolet (UV) and ionizing radiations, environmental toxins, and drugs that use ROS as mediators in their mechanism of action [20]. ROS can also originate from multiple cellular sources in the cerebral parenchyma.

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The main generator appears to be the mitochondrial activity. Around 1% of a healthy brain cell’s mitochondrial electron flow produces O₂⁻, preponderantly via complex I (NADH dehydrogenase) and complex III (ubiquinone cytochrome c reductase) [21,22]. Superoxide is neutralized by superoxide dismutases (SOD1, SOD2, SOD3), thus resulting in H₂O₂ molecules. Hydrogen peroxide is less toxic than superoxide, but its danger lies in its potential to create even more harmful byproducts, e.g., hydroxyl radicals (–OH) by reacting with Fenton’s reagent or peroxynitrite anions (ONOO–) by reacting with NO [23]. Various metabolic factors can influence mitochondrial ROS production, such as a shift of the NADH/NAD⁺ balance toward reduction of NADH [24], increases in succinate levels [25], or alterations in the mitochondrial membrane potential, as occurs in conditions of hypoxia [26].

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Monoamine oxidases (MAOs), enzymes located on the outer mitochondrial membrane, metabolize serotonin, epinephrine, and dopamine [27]. MAO-A is expressed in neurons, and glial cells express both MAO-A and -B [28]. They use flavin adenine dinucleotide (FAD) for metabolizing monoamines, and hydrogen peroxide results from the FAD-FADH₂ cycle [29].

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Several other mitochondrial enzymes can produce significant amounts of ROS, such as α-ketoglutarate dehydrogenase, glycerol phosphate dehydrogenase, and p66shc [30].

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Peroxisomes participate in the beta-oxidation of fatty acids, a process leading to the generation of H₂O₂. However, other enzymes, such as xanthine oxidase, acyl CoA oxidases, D-amino acid oxidase, D-aspartate oxidase, and L-α-hydroxy oxidase, may contribute to the generation of superoxide, hydroxyl radicals, nitric oxide, and hydrogen peroxide [31].

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Several nicotinamide adenine dinucleotide phosphate oxidases (NADPH oxidases) are expressed in brain cells and microglia, and they are involved in the regulation of cell survival/apoptosis, neuroinflammation, migration, differentiation, the proliferation of brain cells, and synaptic plasticity [32]. NOX isoforms localize to the mitochondria, nucleus, endoplasmic reticulum, and plasma membrane. NOX4 mainly produces hydrogen peroxide, while NOX2 activity generates superoxide [33]. Moreover, NOX-generated ROS can lead to mitochondrial dysfunction or even depolarize the mitochondrial membrane, open the mitochondrial permeability transition pore, and ignite apoptosis [34].

2.3. Antioxidant Defenses

Antioxidants can be classified into exogenous and endogenous antioxidants. Several cellular enzymes can directly neutralize FRs, prevent FR formation, or repair FR-induced damage [35].

2.3.1. Superoxide Dismutases (SODs)

Multiple isoforms fit into the category of SODs, namely SOD1, SOD2, and SOD3, each of them having a different function. For them to be active and work properly, they need to associate with metals, specifically copper/zinc (SOD1), iron, or manganese (Mn) (SOD2). Each of them works in different cellular spaces.

• SOD1 is active in cytosol and organelles.
• SOD2 is active in mitochondria.
• SOD3 is an extracellular enzyme with a comparatively restricted expression in only a few types of cells [36].

These enzymes catalyze the breakdown of the O₂ radical into the less reactive hydrogen peroxide (H₂O₂) and oxygen [37].

2.3.2. Catalase

This enzyme facilitates the breakdown of H₂O₂ into water and oxygen, utilizing either iron or Mn as a cofactor [38]. It is primarily found in peroxisomes, but it is also present in the cytoplasm and the mitochondria. While catalase plays a minor role at low concentrations of H₂O₂, its importance significantly increases at higher concentrations [39].

2.3.3. Glutathione Peroxidase (GPx)

GPx refers to an enzyme family with multiple isoenzymes that catalyze the reduction of hydrogen peroxide and lipid peroxides using two molecules of GSH as electron donors [37,38]. GPx can be found in both the cytosol and the mitochondria of the cell. Eight isoforms were found in mammals, classified into two categories: selenium-dependent and selenium-independent [40] (

Table 1. The repartition of the eight isoforms of mammalian GPx based on selenium dependence. In the case of selenium-dependent forms, they are selenoproteins that contain a selenocysteine molecule in their catalytic center. Selenium-independent GPx isoforms contain a cysteine molecule in their catalytic center [40].

Categorization of GPx Isoenzymes Based on Selenium Dependence
Selenium-dependent	Selenium-independent
GPx1
GPx2	GPx5
GPx3	GPx7
GPx4	GPx8
GPx6

). The antioxidant function of each family member depends on the isoform and its location. GPx1 can be found universally in the mitochondria and the cytosol, GPx2 in the epithelium of the intestine, and GPx3 in the plasma. The current literature suggests that GPx1 may be one of the responses that helps protect against neuronal injury [41].

2.3.4. Glutathione (GSH)

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine that has a crucial role in cell survival against oxidative stress [37,38]. In the brain, in vivo, the tripeptide is synthesized through the sequential action of two enzymes: γ-glutamylcysteine synthetase (γ-ECS) and GSH synthetase (GS). First, γ-glutamylcysteine is formed from glutamate and cysteine under the action of γ-ECS. The resulting γ dipeptide is then combined with glycine through the catalyzing action of GS to synthesize the final GSH [42].

GSH takes part in two types of reactions. First, in its reduced form, GSH can non-enzymatically react with O₂ and hydroxyl radicals to remove ROS. Second, it acts as an electron donor during peroxide reduction, as mentioned above [43]. Following the reaction with ROS, which oxidizes GSH, glutathione disulfide is formed. GSH regeneration is possible by reacting glutathione disulfide with glutathione reductase, which transfers electrons from NADPH to glutathione disulfide [43,44]. Multiple studies have reported that GSH inhibits apoptotic cell death [45] and cellular DNA damage after oxidative stress [46].

2.3.5. Vitamins C and E

Vitamin C (ascorbic acid) is a water-soluble antioxidant that participates in clearing FRs through electron transfer. It also acts as a cofactor for some antioxidant enzymes [37,38].

Vitamin E is a fat-soluble antioxidant that can minimize the effects of peroxide and help prevent lipid peroxidation in cellular membranes [47].

2.3.6. Trace Elements

A series of trace elements can also be regarded as exogenous antioxidants. Selenium, naturally found in water, foods, and soil, is incorporated into a variety of selenoproteins important for their antioxidant properties [48], including glutathione peroxidase and thioredoxin synthase [49]. As already mentioned, copper, zinc, and manganese are required by superoxide dismutases, and they require certain amounts of daily intake [50]

However, a series of characteristics render the brain particularly vulnerable to oxidative stress.

• Action potentials propagated in the CNS cause calcium influx, with raised intracellular calcium leading to the activation of neuronal nitric oxide synthase (nNOS) and the production of nitric oxide [51].
• Mitochondria attempt to buffer the excess intracellular calcium, but mitochondrial calcium overload leads to dysfunction of the organelles and impairs energy homeostasis [52].
• Activated microglia produce high amounts of ROS, mainly superoxide, and increase the transcription of SOD2 that converts superoxide into H₂O₂ [53].
• ROS are also generated via the metabolization or auto-oxidation of neurotransmitters, such as dopamine, serotonin, or adrenaline [54].
• The relatively high contents of redox-active transition metals of the brain, such as Fe²+ and Cu⁺, act as catalyzers in the Fenton reaction and promote the generation of ROS [55].
• The brain has low antioxidant defenses. For example, cytosolic glutathione is 50% lower in neurons than in other cells [56], whereas the catalase content is 50 times less in brain cells than in hepatocytes [57].
• The cellular membranes are rich in polyunsaturated fatty acids and very susceptible to lipid peroxidation. The high membrane surface/cytoplasmic volume ratio of the brain cells creates the premises for the chain propagation of peroxidation reactions following ROS attack [57].
• The synaptic plasticity relies on non-coding RNAs [58] that lack protective histones and are vulnerable to oxidation, leading to the production of mutated or truncated proteins prone to misfolding [59].

3. Pathways Through Which ROS Promote Neurodegeneration

Research has shown that OS is significantly involved in the pathophysiology of neurodegenerative diseases, characterized by an extensive loss of neurons from specific locations [33,60].

3.1. Oxidation of Proteins

Free radicals, such as superoxide, the hydroxyl radical, and the alkoxyl, peroxyl, or hydroperoxyl radicals, are potent oxidizers of cellular proteins [61]. Protein oxidation leads to structural (protein–protein cross-linkages) and functional changes (loss of enzymatic activity, functional impairments of receptor and transport proteins) [62]. The altered proteins are cleared by two complementary systems: the ubiquitin–proteasomal system (UPS) and the autophagy–lysosome pathway [33]. Because the UPS is overwhelmed under stress, the autophagy–lysosome system compensates for the increased protein damage [63]. Autophagy receptors, such as p62, NDP52, and NBR1, target damaged proteins of the autophagosome by binding to Atg8/LC3/GABARAP [64]. The expression of genes involved in protein degradation is regulated by FOXO3 (forkhead box O3), which is activated by oxidative stress [65]. Parkin, ubiquitin ligases, and the C-terminus of HSC70-interacting protein (CHIP) additionally contribute to the degradation of proteins [66].

3.2. Lipid Peroxidation

Oxidative species have a high affinity for attacking the C-C double bond in polyunsaturated fatty acids, generating a lipid radical that will react further with molecular oxygen to form peroxyl radicals. The latter initiates a self-sustained chain reaction that is terminated only by the intervention of antioxidant molecules or by the interaction of the lipid peroxides with lipid radicals and the generation of stable species [67]. 4-hydroxynonenal (4-HNE) interacts with cysteine thiols in Keap1 (Kelch-like ECH-associated protein 1), leading to the release of Nrf2 (Nuclear factor erythroid 2-related factor 2), the master transcription factor regulating the expression of endogenous antioxidants [68]. Moreover, 4-HNE inhibits IκB (inhibitor of κB) kinase, preventing the phosphorylation of IκB and the release of NF-κB, one of the main transcription factors regulating the production of pro-inflammatory cytokines [69].

However, lipid peroxidation results in the alteration of membrane permeability, and intracellular accumulation of 4-HNE activates both intrinsic and extrinsic apoptosis. 4-HNE upregulates the expression of p53, leading to the activation of p21, JNK (c-Jun NH2-terminal kinase), Bax (Bcl-2-like protein 4), and caspase-3 [70]. 4-HNE can also initiate the binding of death-associated proteins to the Fas ligand, followed by extrinsic apoptosis via the downstream signaling proteins JNK and ASK1 (apoptosis signal-regulating kinase 1) [71].

3.3. DNA Oxidative Damage

ROS can induce base oxidation, single-strand breaks (SSBs), double-strand breaks (DSBs), DNA crosslinks, or deoxyribose modifications [72]. Delays in repairing these alterations via base excision repair, nucleotide excision repair, and single- and double-strand repair mechanisms can lead to genomic instability and promote aging [73] or induce signaling cascades resulting in cell death [74].

Mitochondrial DNA is even more prone to oxidative damage because it lacks protective histones and because it is close to mitochondria-generated ROS [75]. Deletions of mitochondrial DNA significantly impair both mitochondrial function and biogenesis. Research has proven the involvement of oxidative stress in the pathogenesis of neurodegenerative diseases by showing higher levels of 8-oxoguanine DNA glycosylase 1 (OGG1) in PD [76] and higher levels of oxidized DNA bases in nuclear and mitochondrial DNA in AD [77].

3.4. RNA Oxidative Damage

RNA is more abundant in cells, and it is vulnerable to oxidative damage due to its single-stranded structure, lack of RNA repair mechanisms, and reduced protection by proteins [78]. The hydroxyl radical reacts with guanine and forms 8-hydroxyguanosine, used as a marker of RNA oxidation. Oxidation of ribosomal RNAs, transfer RNAs, and messenger RNAS results in altered protein synthesis, which tends to accumulate [79].

4. Oxidative Stress Is Intricately Linked to Other Pathogenic Cascades in Neurodegeneration

Autophagy is an essential step in promoting cellular health by maintaining the balance between protein synthesis and clearance and organelle biogenesis and degradation [80]. The signaling protein mTOR (mammalian target of rapamycin) has important roles in modulating autophagy. In the presence of abundant cellular nutrients and growth factors, mTOR inhibits autophagy by phosphorylating Atg13 and preventing the activation of the initiation complex. Conversely, in starvation, mTOR colocalizes with LC3 and initiates autophagy [33]. Oxidation of mTOR inhibits its activity and thus impairs cellular autophagy [81].

One must not overlook that the most important risk factor for neurodegenerative diseases, aside from the identified genetic mutations, is age. Among the hallmarks of aging, loss of proteostasis and disabled macroautophagy are prominent, leading to the accumulation of various protein aggregates (β amyloid and neurofibrillary tangles in AD, α-synuclein in PD, and huntingtin aggregates in Huntington’s disease) [73,82].

Soluble Aß oligomers and amyloid precursor protein associated with the mitochondrial membrane import channels blocking the entry and function of the electron transport chain and OXPHOS enzymes, further augmenting the generation of ROS in a vicious cycle [83]. The increased ROS levels enhance amyloid precursor protein cleavage and Aβ generation [84].

In PD, mitochondria are shortened and fragmented [85], a phenotype partly caused by α-synuclein binding to the mitochondrial outer membrane, which leads to a decrease in the mitochondrial fusion rate [86].

Excess ROS can also activate microglia and trigger neuroinflammation, a pathogenic cascade increasingly shown to contribute to neurodegeneration. Again, chronic inflammation and neuroinflammation are aging features [73]. In turn, activated microglia secrete proinflammatory cytokines and produce supplemental amounts of ROS in a vicious cascade [17]. The microglia-released cytokines activate further glial cells and stimulate ROS-induced apoptosis of pericytes, resulting in the break-down of the blood–brain barrier and creating the premises for supplemental macrophage recruitment and allowing toxic compounds produced in the gut to gain access into the CNS [73]. Increased levels of Il-1β enhance the neuronal production of Aβ and induce tau phosphorylation [87]. ROS and mitochondrial dysfunction also activate inflammasomes, multiprotein oligomers formed by the inflammasome adaptor protein ASC, caspase-1, and components of the inflammasome, such as nucleotide-binding oligomerization domain-pyrin domain-containing-3, -1 (NLRP3, NLRP1), NOD-like receptor (NLR) family CARD domain containing 3 (NLRC3), or absent in melanoma-2 (AIM2), which cleave pro-IL-1β to IL-1β or produce IL-18 [88]. Gut dysbiosis, another hallmark of aging, leads to activation of the immune system and increases oxidative stress, resulting in enhanced permeability of the intestinal epithelium for bacterial products that spread to the CNS via the weakened blood–brain barrier. In the CNS, these molecules induce microglial activation and mitochondrial dysfunction and promote oxidative stress and neuroinflammation [89].

5. Oxidative Stress in Specific Neurodegenerative Diseases

5.1. Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most common and well-known neurodegenerative diseases. The pathological hallmarks, which are obvious since the original description of the condition, consist of neurofibrillary tangles (formed of hyperphosphorylated tau protein) and extracellular amyloid β plaques (Aβ), which lead to the impairment of neuronal homeostasis, synaptic dysfunction, and neuronal loss [90,91]. One of the important mechanisms in its pathogenesis is thought to be excessive ROS generation [92].

One of the earliest impairments in AD is the lipoxidation of ATP synthase, described in entorhinal cortical (EC) neurons already in Braak stages I–II [93]. Due to its location inside of the inner mitochondrial membrane, the enzyme is easily targeted by ROS generated by complexes I and III [94], igniting a pathogenic cascade that enhances ROS generation and causes the energetic failure of neurons. The bioenergetic defects impair the import of nuclear-coded mitochondrial subunits, further enhancing mitochondrial dysfunction and dynamics [95]. Excessive mitochondrial fragmentation, expressed as an increased number and a decreased size of mitochondria, is enhanced by hyperphosphorylated tau and intracellular build-up of Aβ, which both increase the GTPase (guanosine triphosphatase) activity of Drp1 (dynamin-related protein 1) [96]. Moreover, Aβ oligomers inhibit the axonal transport of mitochondria and cause the depletion of Parkin and PINK1, leading to impairments in the autophagy of damaged mitochondria [97].

Table 2. Proteins that suffer from oxidative alterations in AD.

Protein	Function	Reference
Glutamate dehydrogenase 1	TCA cycle	[98]
Malate dehydrogenase	TCA cycle	[99]
Subunit Va of cytochrome c oxidase	ETC	[100]
Ubiquinone (NADH dehydrogenase)	ETC	[101]
Core protein 1 of ubiquinol-cytochrome c reductase complex	ETC	[102]
ATP synthase	OXPHOS	[103]

Abbreviations: TCA—tricarboxylic acid cycle; ETC—electron transport chain; OXPHOS—oxidative phosphorylation.

presents the oxidized proteins in AD and their function (adapted from ref. [33]).

The oxidation of membrane lipids causes altered membrane fluidity and thickness and interferes with the function of membrane-bound proteins, favoring the amyloidogenic processing of amyloid precursor protein [104].

The hyperphosphorylated state of tau protein also interferes with the ability of tau to bind to chromatin and protect the genome from oxidative damage, thus enhancing the oxidative damage of DNA [105].

The accumulation of Aβ aggregates and neuronal destruction, along with the release of intracellular compounds into the extracellular space, activate microglia, causing excessive production of pro-inflammatory cytokines and further production of ROS via upregulation of the NF-κB pathway activated by the upregulated expression of complement receptors, as well as increased expression of toll-like receptors [106].

Atrophy of the entorhinal cortex (EC), mainly of neurons in layer II (ECII) and the hippocampus, particularly in the CA1 region, causes the characteristic cognitive decline and memory loss seen in AD patients [107]. Studies have indicated that neurons in these regions have high energy needs and are highly sensitive to drops in oxygen and glucose availability [108]. Furthermore, CA1 and EC II pyramidal neurons are glutamatergic, which makes them more susceptible to NMDA (N-methyl-D-aspartate) excitotoxicity and the harmful effects of elevated intracellular calcium concentrations [109] in contrast to neocortical inhibitory neurons that possess high levels of Ca²⁺-binding proteins [110]. Recently, several molecular features of the susceptible neurons have been identified. For example, there is an impaired activity of a tau splicing regulator in EC II pyramidal neurons, which is probably related to abnormal microtubule dynamics [111]. This may help tau spread to other brain regions through CA1 neurons [112]. It has been demonstrated that subpopulations of selectively vulnerable excitatory neurons express RORB (RAR-related Orphan Receptor B) while also displaying differences in the expression of genes encoding synapse- versus axon-localized proteins, subunits of potassium channels, G-protein signaling molecules, and neurotransmitter receptor signaling molecules [113]. Nevertheless, further research is needed to fully characterize the relationship between RORB expression, the accumulation of phosphorylated tau, and neural degeneration [33].

5.2. Parkinson’s Disease

The second most common neurodegenerative disorder, Parkinson’s disease, exhibits an increasing prevalence worldwide [114]. It is characterized by selective neuronal death of dopaminergic (DA) neurons located in the substantia nigra pars compacta (SNc) and decreased DA concentrations in the nigrostriatal pathway of the brain [115]. These factors determine the motor symptoms seen in PD patients. Histopathologically, the nigral neurons show α-synuclein aggregates in the form of Lewy bodies.

The study of a severe parkinsonian syndrome induced by exposure to 1-methyl-phenyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in the 20th century highlighted the involvement of mitochondrial dysfunction and oxidative stress in the pathogenesis of PD because MPTP interferes with the function of mitochondrial complex I [115]. Further research confirmed a reduction in the activity of mitochondrial complex I in PD due to a decreased rate of production of the complex subunits, destruction, and oxidative damage [116,117]. The degenerating neurons exhibit an accumulation of oxidized lipids, proteins, and DNA [118], while the glutathione is deficient [119]. Moreover, dopamine auto-oxidation with metal ions, such as Fe³⁺, as catalysts generates dopamine- and DOPA-quinones, which are highly reactive molecules that enhance oxidative stress [120].

A series of genetic mutations, such as PINK1 (PARK6), PRKN (PARK2), DJ-1 (PARK7), SNCA (PARK1), FBXO7 (PARK15), CHCHD2 (PARK22) and VPS13C (PARK23), lead to familial or early-onset forms of PD. Many of these mutated genes code for proteins involved in mitochondrial function, the degradation of altered proteins, and cell survival pathways. PINK1 and PRKN (coding for Parkin) regulate autophagy. DJ1 is important for mitochondrial dynamics. Overexpression of DJ1 reduces the mitochondrial fragmentation seen in PD [121]. LRRK2 (leucine-rich repeat kinase 2) mutations increase α-synuclein levels, which target mitochondria and supplementally decrease complex I activity [122]. In addition, mutant LRRK2 interacts with Drp1 [123] and impairs mitochondrial fission, and it prevents the removal of MIRO1 (mitochondrial Rho GTPase 1), the protein that binds the mitochondria to the microtubule motors dynein and kinesin [124]. The Ca²⁺-buffering capacity of nigral dopaminergic neurons is low despite high activity-dependent Ca²⁺ loads, which may cause mtDNA damage [125,126] and increase OXPHOS and ROS formation [127].

Regarding the structure of nigrostriatal neurons, they possess very long and branched axons that can reach up to 4.5m in length. These axons can connect to many neurons, forming up to 2.4 million synapses [128,129]. Thus, a high density of axonal mitochondria is required, challenging mitochondrial bioenergetics [130]. However, the question of whether the degeneration of dopaminergic neurons is initiated by the bioenergetic failure in the axonal arbor or rather in the cell body is still open and subject to research [120].

5.3. Amyotrophic Lateral Sclerosis

With a worldwide prevalence of 46 cases/100,000 inhabitants and an average survival rate of 2–3 years from diagnosis, ALS (also known as Lou Gehrig’s disease) [131] is one of the most fatal neurodegenerative disorders [132]. The disease is clinically characterized by muscle weakness and atrophy, leading to dysphagia, respiratory failure, and, ultimately, death. It is caused by the loss of motor neurons from the cortex, the brainstem motor nuclei, and the spinal cord.

The main genetic mutations that increase susceptibility to ALS—C9orf72 (chromosome 9 open reading frame 72), SOD1 (superoxide dismutase 1), TARDBP (transactive response DNA binding protein 43, TDP-43), and FUS (fused in sarcoma/translocated in liposarcoma or heterogenous nuclear ribonucleoprotein P2)—all increase oxidative stress, highlighting its prominent involvement in the pathogenesis of the disease [131].

The SOD1 mutation alters the antioxidant activity of Cu/ZnSOD by reducing its affinity for Zn [133], thereby favoring a higher load of DNA damage [134]. Alternative hypotheses state that mutant SOD1 acts as a peroxidase [135] or could react with peroxynitrite to cause tyrosine nitration [136]. SOD1 also influences the Nrf2 pathway, the master regulator of the antioxidant response. Mutant SOD1 decreases the expression of Nrf2 [137].

In C9orf72-related ALS, there is an expansion of the GGGGCC (G4C2) hexanucleotide in the first intron of the C9orf72 gene, leading to the synthesis of dipeptide repeat proteins, of which poly-glycine-arginine may increase oxidative stress [138].

Although still under study, the role of FUS mutations appears to derive from their involvement in DNA double-strand repair mechanisms, rendering the cells more vulnerable to oxidative stress via the accumulation of DNA double-strand breaks [139].

Mutant TDP-43 protein associates with the nuclear factor erythroid 2–related factor 2 (Nrf2) via interaction with members of the family of heterogeneous nuclear ribonucleoproteins. Studies in transgenic mice have revealed decreased levels of glutathione, which is an antioxidant downstream of the Nrf2-ARE (antioxidant response element) pathway [132].

In addition, the cortical neurons of transgenic mice exhibited decreased glucose metabolism and reduced ATP generation even before the clinical picture of ALS [140]. Consequently, neurons upregulate glycolysis at the expense of increasing oxidative stress [141].

The particular configuration of motor neurons, with very long axons requiring proper mitochondrial function and trafficking, may explain the selective vulnerability of these neurons in ALS [106]. Another factor that further increases the high energetic demands of spinal motor neurons is their large motor unit size, mitochondrial trafficking, and normal function, which are essential for maintaining neurotransmission and muscle contraction [142]. The relative preservation of function in the oculomotor, trochlear, and abducens nerve territories may be explained by their reduced size at the motor unit, which places less energetic demands on the axonal terminal [33].

Many antioxidant strategies that have been evaluated in preclinical and clinical trials (vitamin E, N-Acetyl-L-cysteine, Coenzyme Q10, Nrf2 modulators, curcumin, melatonin, and NAD⁺ modulators) have failed. However, both approved treatments—riluzole and edaravone (FDA approved since May 2017 [143])—exhibit antioxidant effects. Edaravone is a free radical scavenger [144], while riluzole has a direct antioxidant effect against OS but not against nitrosative stress [145]. Several mitochondria-targeted antioxidants are currently in preclinical evaluations [114].

6. Antioxidant Therapeutic Strategies in Neurodegenerative Diseases

Given the convincing evidence of the involvement of oxidative stress in the pathogenesis of neurodegenerative diseases, a series of molecules with antioxidant properties have been evaluated in the early phases of these conditions or as add-on therapies to approved treatments to slow down the progression of these diseases. Some of these approaches have even escalated to clinical trials.

6.1. Antioxidant Therapeutic Strategies in Alzheimer’s Disease

Currently approved therapies for Alzheimer’s disease are those designed to inhibit the action of acetylcholinesterase in the brain (Donepezil, Rivastigmine, and Galantamine), those designed to prevent glutamate-induced excitotoxicity (Memantine), and the recently developed monoclonal antibodies against amyloid beta (Aducanumab, Donanemab, and Lecanemab). Aside from these drugs, antioxidant therapy has been widely studied and shown to have beneficial effects in the prevention and slowing of AD progression [54].

Vitamin E reduces oxidative and nitrosative damage in AD [146], acting mainly against peroxyl radicals [147] and suppressing tau-induced neurotoxicity in animal models [148].

The endogenous antioxidant melatonin, a hormone synthesized by the pineal gland, scavenges oxygen and nitrogen-based free radicals and enhances the expression and activity of other antioxidants, such as superoxide dismutase and glutathione peroxidase [149].

A series of naturally occurring, plant-derived molecules (nutraceuticals) have beneficial effects via multiple mechanisms of action, with antioxidant effects included. Resveratrol, a naturally occurring polyphenol, directly neutralizes free radicals and enhances the expression of natural antioxidant enzymes, thereby mitigating oxidative damage to proteins, lipids, and nucleic acids [150]. Alpha-lipoic acid shows antioxidant, antiapoptotic, anti-inflammatory, and metal chelating properties in both in vivo and in vitro studies [151]. Flavonoids activate the Nrf2 pathway [152]. Carotenoids (lycopene, lutein, zeaxanthin, astaxanthin) are strong scavengers of natural singlet oxygen and neutralize ROS and various other free radicals [153].

However, because the mitochondria are the main sources of ROS, several mitochondria-targeted compounds have emerged and have been tested. Mitoquinone (MitoQ), resulting from the conjugation of a modified ubiquinone with triphenylphosphonium as a carrier, scavenges superoxide, peroxyl, and peroxynitrite ROS once it reaches the mitochondria [154]. Interestingly, oxidized MitoQ can be recycled by the electron transport chain [155]. In vitro, MitoQ improved the mitochondrial membrane potential and reduced the hydrogen peroxide levels [156], while in transgenic mouse models of AD, MitoQ led to lower levels of lipid peroxidation and improved behavioral phenotype and cognitive performance [157]. MitoVitE is a mitochondrially targeted Vitamin E able to reduce lipid peroxidation, caspase activation, and apoptosis after peroxide exposure [158]. MitoTEMPO is composed of (2,2,6,6-Tetramethylpiperidin-1-yl)oxyl (TEMPO, piperidine nitroxide) conjugated to triphenylphosphonium. In primary mouse cortical neurons exposed to amyloid β, MitoTEMPO effectively mitigated ROS-induced damage through the reduction of ROS production and Aβ-induced lipid peroxidation [159]. Moreover, in cultured tauopathy mice neurons, MitoTEMPO led to a reduction in tau oligomers and ROS and an amelioration of OXPHOS [160].

Table 3. Antioxidant and mitochondrially targeted drugs in AD.

Drug	Mechanisms of Action	Outcomes	References
Vitamin E	Maintains membrane integrity in mitochondria	Antioxidant properties in AD	[161]
Alpha-lipoic acid	Scavenges the toxic byproducts of lipid peroxidation	Antioxidant properties in AD	[161]
Curcumin	Suppresses TNF-α activity	Antioxidant and amyloid disaggregating properties in AD	[162]
Epigallocatechin-3-gallate (Camellia sinensis)	Inhibits oxidative stress via the Keap1/Nrf2 signaling pathway	Antioxidant effects in AD	[163]
Ginsenosides	Suppress Aβ-associated generation of ROS by enhancing the activity of endogenous antioxidants and reducing the expression of NOX2	Inhibit Aβ and neurofibrillary tangle formation	[164]
Melatonin	Direct scavenger of many ROS species	Protective role against H-89-induced memory impairment in mouse brain	[165]
MitoQ	Scavenges peroxyl, peroxynitrite, and superoxide ROS	Lower levels of lipid peroxidation	[157]

provides an overview of antioxidant compounds and their effects on AD.

6.2. Antioxidant Therapeutic Strategies in Parkinson’s Disease

Observational studies support the roles of vitamin E and C in reducing the risk of PD [166], although earlier observational studies yielded contradictory results [167].

Coenzyme Q10 has been shown to slow the progression of PD if administered in large doses [168], although a later study showed no benefit [169].

Melatonin was shown to prevent damage to the nigrostriatal pathway resulting from oxidative stress in an MPTP-induced mouse model of PD [170], with similar results obtained in a 6-OHDA animal model of PD [171]. Moreover, in a rotenone-induced mouse model of PD, melatonin attenuated glutathione depletion and increased SOD and catalase activity [172].

MitoQ exerted protective activity in MPTP and 6-OHDA (6-hydroxydopamine hydrochloride)-induced PD models [173]. However, MitoQ failed in a clinical trial [174], possibly due to poor brain penetration or the severity of neuronal damage at the time of patient recruitment [159].

MitoTEMPO was able to reduce H₂O₂ production and cell apoptosis in rat PC12 cells and primary murine neurons exposed to 6-OHDA, MPTP, and rotenone [175].

SkQ1, composed of triphenylphosphonium conjugated to plastiquinone instead of ubiquinone, exhibits similar antioxidant activity to MitoQ. In 8-week-old mice with MPTP-induced PD, SkQ1 induced higher levels of tyrosine hydroxylase and dopamine, leading to improved motor and sensorimotor abilities [176,177].

Table 4. Antioxidant strategies in PD.

Drug	Mechanisms of Action	Outcomes	References
Vitamins E and C	Maintain the integrity of mitochondrial membranes	Antioxidant and neuroprotective activities in PD	[161]
P7C3 (aminopropyl carbazole)	Acts by protecting mitochondria	Stabilized mitochondrial membrane potential in PD (dopaminergic cell lines), reduced ROS production, inhibited GSK3β activation and p53 activity, Bax upregulation, cytochrome c release exposed to MPTP, and prevented neuronal loss in the substantia nigra (mouse brain)	[178]
Terpene lactones and flavonoids from Ginkgo biloba	Stabilize mitochondrial functions and interact with the mitochondrial electron transport chain	Antioxidant effects in PD	[161]
Triterpene saponin and phenol from Glycyrrhiza	Reduces oxidative stress and damage to brain cells	Antioxidant and neuroprotective effects in PD	[161]
MitoQ	Scavenges peroxyl, peroxynitrite, and superoxide ROS	Antioxidant effects in PD	[179]

shows the effects of various antioxidant strategies in PD

6.3. Antioxidant Therapeutic Strategies in Amyotrophic Lateral Sclerosis

Many antioxidant strategies have been evaluated in preclinical and clinical trials for ALS.

Vitamin E supplementation and higher plasma levels of vitamin E were shown to be protective against the onset of ALS [180]. Patients on combined riluzole and alpha-tocopherol (vitamin E) therapy had predominantly mild disease courses [181]. However, systematic reviews and meta-analyses failed to show any beneficial effect [182].

Coenzyme Q10 prolonged the survival of SOD1G93A-transgenic mice [183], and a case report emphasized the improvements in muscle strength and the reduction in muscle wasting following coenzyme Q10 treatment [184]. Unfortunately, dietary supplementation of coenzyme Q10 proved futile in ALS [185]. MitoQ, a formulation targeted to the mitochondria, slowed the decline of muscle function, reduced the markers of oxidative stress, and increased the lifespan of transgenic mice [131,186].

Melatonin delayed disease progression and extended survival in transgenic mouse models of ALS via inhibition of the caspase-1/cytochrome c/caspase-3 pathways [187], while an increase in motor neuron loss and 4-hydroxynonenal levels and an upregulation of toxic SOD1 expression were reported by other researchers [188].

The NAD⁺/SIRT1 pathways are involved in the maintenance of oxidative balance and mitochondrial metabolism [189]. As such, administration of resveratrol, an SIRT1 activator, was shown to improve ALS symptoms in mouse models [190].

N-acetyl-L-cysteine replenishes plasma levels of cysteine, a glutathione precursor. Administration of N-acetyl-cysteine to SOD1G93A transgenic mice resulted in improved motor symptoms and extended survival [191]. However, a randomized, double-blind, placebo-controlled clinical did not reduce the rate of disease progression and did not increase the 12-month survival of randomized patients [192], an outcome that may be related to reduced bioavailability [193].

A series of Nrf2 activators showed promising results in preclinical setting, while clinical trials are ongoing [194]. Such molecules are curcumin (ongoing clinical trial NCT04499963), resveratrol (trial NCT04654689), trehalose (trial NCT05136885), rapamycin (evaluated in trial NCT03359538), tideglusib (trial NCT05105958), and lithium, for which the clinical trial NCT00818389 showed slowed ALS progression [195].

Both approved treatments, riluzole and edaravone (FDA approved since May 2017) [143], exhibit antioxidant effects. Edaravone is a free radical scavenger [144], while riluzole has a direct antioxidant effect against OS but not against nitrosative stress [145]. More recently, in 2022, AMX0035, a combination of sodium phenylbutyrate and taurursodiol (trade name Relyvrio), was FDA approved as well. However, only riluzole is approved in Europe [196].

Nonetheless, because about 10% of ALS cases are familial, with a known genetic defect, antisense oligonucleotides (ASOs) are being developed for ALS, which will interfere with the translation of the mutant protein [197]. Tofersen (also known as BIIB067, trade name Qalsody) was approved in April 2023 [198] for the treatment of patients with ALS with mutations in SOD1, and the medical community is actively researching ASO-based strategies for patients with sporadic ALS, as well [196].

6.4. The Road to Finding Efficient Therapies for Neurodegenerative Diseases Is Paved with Many Trial Failures

However, despite preclinical studies pointing to antioxidant substances as potential therapeutic agents for the treatment of neurodegenerative diseases, their translation into clinical therapeutic strategies has not yet led to significant advances [199]. Several reasons for these failures could be discussed.

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Insufficient dose of the chosen antioxidant.

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Inappropriate timing and insufficient duration of the treatment.

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Poor solubility and blood–brain penetration of the antioxidant. Antioxidant drugs are usually polar molecules with high molecular weights and poor absorption, which, together with their quick metabolism, limit their bioavailability [200].

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Antioxidant supplementation might affect the natural redox equilibrium between pro-oxidant and antioxidant species and reduce the natural antioxidant response, further increasing the redox homeostasis failure in neurodegenerative diseases [201].

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It may be that we have a poor understanding of the antioxidant effect exerted by a particular antioxidant compound. For example, glutathione can act via post-translational modifications to mediate protective effects, which may be confused with an antioxidant effect [200]

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The animal models used are mainly transgenic animals, which do not recapitulate the complexity of the human brain or the complex pathogenic cascades of human disease. These limitations could be overcome by studies performed on brain organoids [202].

In addition, given the complex link between the various pathogenic cascades involving oxidative stress, neuroinflammation, and impaired proteostasis, the “one disease-one target-one drug” approach is unlikely to yield impressive results. Compounds targeting multiple pathways could be more promising. For example, targeting the Nrf2 pathway can reduce oxidative stress, neuroinflammation, and protein aggregates characteristically found in neurodegenerative diseases [203]. Targeting all of these processes simultaneously with an Nrf2 inducer may have a greater impact on the complex pathophysiology of neurodegeneration, exhibit antioxidant effects, and promote autophagy and clearance of the altered cellular components [204]. The link between these two effects is mediated by p62/SQSTM1, a ubiquitin-binding autophagy receptor protein [205]. Phosphorylation of p62 enhances its affinity for Keap1, which leads to the release of Nrf2 by its inhibitor, Keap1. The p62-Keap1 heterodimer recruits LC3 and mediates the permanent degradation of Keap1 in the selective autophagy pathway, while Nrf2 translocates into the nucleus and activates the transcription of downstream genes that encode antioxidant enzymes. Nrf2 also upregulates the expression of the p62 gene, creating a positive p62-Keap1-Nrf2 feedback loop [206]. Because Nrf2 negatively controls the NF-κB signaling pathway through multiple mechanisms (decreasing intracellular ROS levels, preventing IκB-α proteasomal degradation and the nuclear translocation of NF-κB), activation of the Nrf2 pathway also results in reduced inflammation [207].

Another important step forward in the development of more efficient therapeutic strategies for neurodegenerative disorders is to be able to diagnose the disease early by using reliable biomarkers. Currently, the diagnosis of these diseases is based on a defined set of symptoms and signs, which usually manifest after 10–15 years of subclinical progression of the disease. Parkinson’s disease is diagnosed based on motor signs, which emerge after more than 50% of the nigrostriatal neurons are lost. It is unrealistic to expect that an antioxidant (with uncertain penetration into the CNS) could reverse these massive cell losses and improve the patient’s clinical picture. Unfortunately, if in familial cases of AD, PD, or ALS genetic testing could identify the diseases in the “silent” stage, large-scale screening methods with invasive or expensive techniques are currently not applicable. Similarly, a robust set of biomarkers to quantify the modulation of the various pathways (such as the Nrf2 signaling pathway) [208] would allow for a more personalized approach in individual patients.

7. Conclusions and Future Perspectives

Reactive oxygen species (ROS) are signaling molecules that, when kept at low levels, serve as signaling molecules and contribute positively to organism survival by aiding in the production of certain cellular structures. Nonetheless, when levels exceed a specific threshold (for example, due to lower antioxidant defense or higher production), ROS become harmful to both the cell and the organism [209]. The detrimental effects of elevated ROS levels during OS are due to the oxidative damage of proteins, nucleic acids, and lipids, as well as abnormal redox signaling [210]. Aside from its recognized function in aging [211], oxidative stress is linked to various age-related illnesses, including neurological disorders [212].

Neurodegenerative disorders are becoming increasingly prevalent, and by 2040, they will be the second leading cause of mortality [213]. This group of diseases causes a loss of neurons in the central nervous system and memory, motor, and cognitive deficits, and even a combination of the three. Many neurodegenerative diseases share the common characteristic of disordered proteins being present, which lead to the formation of toxic aggregates. Studies have consistently shown the link between ROS and protein aggregation in these illnesses. Although it is unclear whether ROS cause aggregation or aggregates produce ROS, these studies undoubtedly highlight the significance of oxidative stress and demand more research on its role in neurodegeneration [214] (Figure 1).

Nonetheless, a large number of trials with exogenous antioxidants in neurodegenerative diseases have failed to reach their pre-specified end-points, which is a significant setback in the discovery of an effective drug candidate [215]. However, those trials reporting significant changes derived from exogenous antioxidant intake show promising findings, including changes in cognitive function and clinical prognosis [216], and even significant improvement in molecular biomarkers, including, mainly, mitochondrial-related activity and oxidative stress parameters [217].

Lifestyle factors, such as exercise and diet, have been convincingly shown to reduce oxidative stress levels and exhibit neuroprotective effects [218,219]. Phytochemicals have long been shown to exert beneficial effects via diverse mechanisms. Their main setback is their reduced bioavailability [220]. Improved delivery methods (with nanoparticles or nanocarriers) [221] and starting treatment early in the disease course could help us delay the progression of these devastating diseases [222]. However, the toxicity of nanoparticles, which depends on the size, the surface charge, ionic dissolutions, and the shape, should be considered when developing nanoparticle-based drug-delivery systems [223]. Moreover, this strategy calls for identifying biomarkers that could diagnose neurodegenerative conditions in the presymptomatic stage [224].

Gene-based therapy is a rapidly expanding field of therapeutics, and it can be used in combination with genome-manipulation tools in the future clinical management of both inherited and sporadic neurodegenerative diseases after a rigorous evaluation of their safety [225]. In addition, cell therapy is emerging following a greater understanding of the contributions of the various cell types to health and disease and the identification of promising approaches to modulate them. Although there are challenges, human-stem-cell-based products could produce disease-altering therapies to improve the lives of patients with currently incurable neurodegenerative diseases [7].