NRF2 in Neurological Disorders: A Molecular Beacon for Therapeutics

Abstract

Neurological disorders represent a complex spectrum of conditions, ranging from neurodegenerative diseases to acute injuries, each posing unique challenges to treatment. This chapter delves into the emerging role of the NRF2 transcription factor as a molecular beacon with therapeutic implications in the realm of neurological disorders. NRF2, a master regulator of cellular defense mechanisms, orchestrates antioxidant responses and mitigates oxidative stress—processes intricately linked to neuroprotection. The chapter explores the intricate interplay between NRF2 and neurological pathologies, emphasizing its influence on the progression of conditions such as Alzheimer’s, Parkinson’s, and ischemic stroke. By dissecting the molecular pathways through which NRF2 modulates inflammation, oxidative damage, and apoptosis in the nervous system, we gain insights into potential therapeutic strategies. Exciting research on NRF2 activators, both natural and synthetic, offers promising avenues for drug development. Furthermore, the chapter navigates through preclinical and clinical studies, highlighting the potential of NRF2-targeted interventions in preserving neuronal function and promoting recovery. As we unravel the molecular intricacies of NRF2 in neurological contexts, this chapter serves as a guide to understanding its therapeutic potential, paving the way for innovative strategies aimed at mitigating the burden of neurological disorders.

1. Introduction

The Nuclear factor erythroid 2-related factor 2 (NRF2) is a known transcription factor encoded in humans by the NFE2L2 gene that plays an important role in cellular protection against oxidative stress (OS) by activating genes that protect cells. Oxidative stress (OS) is a condition of an imbalance of production of reactive oxygen species (ROS) and nitrogen species (RNS) ratio and the ability of cells to protect them with antioxidants. Excessive production of reactive oxygen species (ROS) leads to changes in important cellular macromolecules, including proteins, DNA, and lipids. Many studies have demonstrated an association between oxidative stress (OS) and various neurodegenerative diseases [1, 2], including Alzheimer’s disease (AD) [3], Huntington’s disease (HD) [4], Parkinson’s disease (PD) [5], multiple sclerosis (MS) [6], and amyotrophic lateral sclerosis (ALS) [7]. ROS/RNS are generated from different sources within cellular compartments, either from normal physiological processes or because of exposure to damage or disease in the environment [8]. Superoxide (O₂⁻) is the most severe form of reactive oxygen species (ROS) and is produced by the single electron reduction of O₂ in mitochondria. Superoxide can also be produced by a group of NADPH oxidases (NOX) using oxygen and NADPH as substrates, where superoxide is rapidly removed [9]. Hydroxyl radicals (•OH) are important by-products of mitochondrial oxidative phosphorylation. These chemicals exhibit instability, high reactivity, and produce more reactive aldehydes through the process of membrane lipid peroxidation (LP), ultimately leading to cell death. Additionally, superoxide dismutase 1 (SOD1) rapidly produces reactive oxygen species (ROS) hydrogen peroxide (H₂O₂) in the cytoplasm, while extracellular H₂O₂ is produced by extracellular superoxide dismutase 3 (SOD3). Consequently, H₂O₂ may be a product of beta-oxidation of fatty acids by cytochrome P450 enzymes. When H₂O₂ is exposed to Fe²⁺ or Cu⁺ (Fenton reaction), it can transform into hydroxyl radicals, although they are stable and less active [10]. The main reactive nitrogen species (RNS) is ONOO⁻, which rapidly decomposes into HO•, nitrogen dioxide radicals (NO₂•), and nitro cations (NO₂⁺) [11]. This drug is neurotoxic. Targeted therapy for neurodegenerative diseases involves addressing the various mechanisms involved in the formation of the operating system (OS) and imbalances in the endogenous cellular protection of neuronal cells [12, 13].

NRF2 plays an important role in the development and activation of blood cells, a group of enzymes responsible for metabolizing drugs [14]. Proteins whose expression increases as a result of NRF2 signaling include heme oxygenase-1 (HO-1), SOD1, catalase, and enzymes involved in glutathione (GSH) metabolism, such as glutathione S-transferase (GST), glutathione cysteine. Ligase modifier subunit and glutathione cysteine ligase catalytic subunit (GCLC) [15, 16].

The ability of NRF2 to regulate central metabolism and mitochondrial function suggests that NRF2 activation is a good and effective strategy for the treatment of neurodegenerative diseases [17]. This review explores the importance of oxidative stress in neurodegenerative diseases and the benefits of targeting the NRF2 pathway as an antioxidant and cytoprotective response. It also proposes new therapies designed to treat oxidative damage and neuroinflammation by activating the NRF2 signaling pathway.

2. Recent findings in specific disorders

2.1 Alzheimer’s disease

Forgetfulness and cognitive impairment are hallmarks of Alzheimer’s, the most common type of dementia. AD is an age related neurodegenerative disease. Without changes in treatment, the number of AD patients in the United States is expected to increase from currently more than 5 million to approximately 13.8 million by 2050 [18]. AD is characterized by a variety of diseases, including brain atrophy due to neuronal and synaptic loss, senile plaques composed primarily of fibrillar amyloid (Aβ) peptide, and neuronal plaques composed of hyperphosphorylated tau, a cytoskeletal protein fiber tangle (NFT). β- and γ-secretase cleave amyloid precursor protein (APP) to form Aβ. Small oligomers of self-assembled peptides are thought to contribute to brain damage in AD [19]. Aging is an important risk factor for AD, and although most cases of AD are unrelated, a small proportion have a familial basis. Early onset familial disease is associated with mutations in APP, presenilin 1 (PS1) and presenilin 2 [20]. In addition to plaques and NFTs, AD brains also feature reactive gliosis, mitochondrial dysfunction, and oxidative damage to proteins and lipids [21, 36].

2.1.1 NRF2-related genes in AD cells

Early studies have shown that the NAD(P)H:quinone oxidoreductase 1 (NQO1) content in AD tissues is higher than in control tissue [22, 23, 24] A. The same is true for both was also observed for oxygenase 1 (HO-1), glutathione reductase, and glutathione peroxidase [25, 26, 27]. Additionally, AD brains have higher levels of p62, HO-1, and glutamate cysteine ligase modifier subunit (GCLM). In addition, Tanji and colleagues [28] found co-localization of Keap1 with NFTs, evidence of interaction between them, and the presence of both p62 and Keap1 in both the soluble and insoluble protein fractions in AD cells. According to a study examining NRF2 by immunohistochemistry, NRF2 was detected in the cytoplasm and nucleus of healthy hippocampal neurons, but only in the cytoplasm in AD [29]. This suggests that NRF2 activation is lower in AD cells than in previous studies. In fact, studies have shown that some of the same NRF2-dependent genes described previously are downregulated or unchanged in AD [30, 31, 32]. These conflicting results are not surprising and may have much to do with the stage of disease at the time the tissues were collected. Additionally, according to our unpublished findings, most NRF2-dependent genes and proteins and glutathione (GSH) levels in the brain and spinal cord are NRF2-dependent. It is not reduced in −/− mice (Figure 1) [33, 34].

Figure 1.

The Keap1-NRF2 pathway is a signaling pathway involved in cellular responses to oxidative stress. The regulatory protein Keap1 regulates the activity of the transcription factor NRF2. Under conditions of oxidative stress, NRF2 is released from Keap1 and translocates to the nucleus, where it activates the expression of genes encoding antioxidant enzymes. This pathway plays an important role in cell defense against oxidative damage and redox maintenance of homeostasis.

2.2 Parkinson’s disease

Parkinson’s disease (PD) is the most common neurodegenerative disease. It is characterized by a variety of non-motor neuropsychiatric problems including motor symptoms such as tremor, bradykinesia, rigidity, and postural instability [35]. Loss of dopaminergic neurons in the substantia nigra pars compacta (SN), which reduces the number of dopamine nerve terminals projecting to the striatum, is one of the symptoms of Parkinson’s disease (PD). Another notable feature is the formation of intracellular inclusions or Lewy bodies (LB) [36, 37, 38] composed of ubiquitin-linked α-synuclein (SYN). Similar to AD, PD is generally rare, with familial onset accounting for approximately 15% of cases [39]. α-SYN, parkin, leucine-rich repeat kinase 2 (LRRK2), putative PTEN-induced kinase 1 (PINK1), and DJ1 are among many genes associated with familial Parkinson’s disease (PD) [40]. Just as Alzheimer’s has both sporadic and familial features, Parkinson’s disease tends to run in families. Therefore, the majority of familial PD and AD are early-onset, progressive diseases.

Studies have shown that NQO1 expression is increased in astrocytes, endothelial cells, and dopaminergic neurons in people with Parkinson’s disease [41]. HO-1 and peroxidase in the substantia nigra of Parkinson’s disease patients show a similar pattern [42]. Immunostaining demonstrated the presence of NRF2.

2.2.1 NRF2-related genes in PD cells

Nuclear localization of NRF2 was observed in dopaminergic neurons in the Parkinson’s disease brain [29]. Oxidative stress, mitochondrial dysfunction, protein carbonylation, and oxidative damage to substantia nigra DNA have been implicated in Parkinson’s disease [43, 44]. Glutathione concentrations are reduced in the brain of patients with Parkinson’s disease [45, 46]. In addition, Keap1 colocalizes with SYN and p62 in Lewy bodies in the Parkinson’s disease brain [28]. New research on induced pluripotent stem cells (iPSCs) from patients with Parkinson’s disease (PD) and control subjects provides further evidence of the role of NRF2 in Parkinson’s disease. Neurons derived from Parkinson’s disease-induced pluripotent stem cells (PD iPSCs) show decreased glutathione (GSH), increased oxidative stress, increased NRF2 activation, and increased NQO1 levels [47].

2.3 Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a serious motor disease in adults and is characterized by the deterioration of slow motor neurons [48]. Motor neurons have intracellular inclusions that are ubiquitin-positive, hyaline, and have a twisted appearance [49]. These include SOD1, which colocalizes with p62 [50]. ALS has also been shown to have TDP-43 (TAR DNA binding protein) and FUS-positive inclusions [51, 52]. Like AD and PD, most ALS patients have a variant form (sALS); Approximately 10–15% of cases are associated with a familial variant (fALS). The first genetic mutation found in familial amyotrophic lateral sclerosis (fALS) was in copper/zinc superoxide dismutase 1 (SOD1), which causes disease by increasing its activity [53]. More than 150 mutations have been identified in the SOD1 gene. Mice with an extreme hSOD1 mutant often have an ALS-like phenotype [54, 55]. The specific molecular mechanism responsible for the increase in dysfunction is currently unknown. However, toxicity to motor neurons requires expression of mutant SOD1 in both non-neuronal cells and motor neurons [56]. Over the last few years, researchers have discovered 24 additional genes associated with amyotrophic lateral sclerosis (sALS) and familial amyotrophic lateral sclerosis (fALS) [57]. One of these genes is p62, which directly affects Keap1/NRF2 and autophagy [58].

2.3.1 NRF2-related genes in ALS cells

The presence of NRF2 and Keap1 in the primary motor cortex and spinal cord of ALS patients was investigated [59]. NRF2 mRNA and protein levels were reduced in ALS patient tissues compared with control tissues, but Keap1 mRNA and protein levels were unchanged. NRF2 dependent genes were not analyzed. Furthermore, Keap1 has been shown to co-localize with intracellular inclusions in post-mortem ALS spinal motor neurons [28].

2.4 Huntington’s disease

Huntington’s disease (HD) is a neurological disease caused by the expansion of CAG repeats in the gene responsible for producing the Htt protein [60]. This leads to elongation of the N-terminal glutamine residue, and the intensity and duration of the disease are related to the length of the polyglutamine repeat. In addition, structural changes in mutant Htt lead to increased self-aggregation, which is associated with the appearance of intracellular inclusions [61]. Huntington’s disease (HD) is defined by pathological degeneration of the neostriatal regions (caudate and putamen) and cerebral cortex. This degeneration is thought to be the main cause of mobility, cognitive impairment, and psychiatric symptoms that worsen as the disease progresses. Oxidative stress due to mitochondrial dysfunction has been shown to be responsible in human patients [62]. According to data obtained from postmortem brain examination, dysfunction of mitochondrial complex II, III, and IV was detected in the striatum of deceased Huntington’s disease patients [63, 64].

2.4.1 NRF2-related genes in HD cells

Surprisingly, very little work has been done on HD tissues and NRF2. Only one study investigated the presence of NRF2 or NRF2-dependent genes in post-mortem HD cells [65]. This study demonstrated HO-1 immunohistochemical staining in HD cells.

2.5 Multiple sclerosis

Approximately 400,000 people in the United States and more than 2.3 million people worldwide are thought to be affected by multiple sclerosis (MS). Multiple sclerosis (MS) is a chronic disease characterized by inflammation of the nervous system. It begins with the activation of CD4+ T lymphocytes in the peripheral zone, which then cross the blood-brain barrier and initiate an autoimmune attack on myelin and oligodendrocytes in the central nervous system (CNS) [66]. Neurological disorders occur in a variety of conditions, including vision loss, coordination problems, fatigue, hearing loss, cognitive impairment, and bladder and bowel problems. Multiple sclerosis (MS) disease occurs as a result of the destruction of the immune system, and autoreactive CD4+ Th1 and Th17 cells (myelin sheath), which are the protective covering of the brain, emerge and myelin-producing cells (oligodendrocytes) emerge. This attack is also accomplished by infiltrating macrophages and resident microglia. As a result, nerve cells decrease and the number of stellate cells (a type of glial cell) increases in response to the damage. Activated microglia and stellate cells secrete inflammatory substances such as cytokines and chemokines, as well as reactive oxygen species and nitrogen. These drugs exacerbate oxidative stress and further exacerbate the deleterious tissue damage associated with neuroinflammation, leading to disease progression [67, 68]. Currently, the FDA has approved eight drugs to treat relapsing-remitting MS, the most common form of MS, affecting 87 percent of people with MS. The main purpose of this medication is to prevent the immune system from crossing the blood-brain barrier. However, due to the complexity and diversity of diseases, the evolution of treatment over time, and the general belief that oxidative stress plays a role in disease, further research is required to improve and/or supplement existing treatments [69].

2.5.1 NRF2-related genes in MS cells

Damage to brain tissue in multiple sclerosis causes extensive oxidative damage resulting from the production of reactive oxygen species and nitrogen species resulting from inflammation. Antioxidants and enzymes, as well as NRF2 protein levels, have been shown to serve as markers of cellular stress in active disease compared to normal white matter (NAWM) and normal tissue in the brain that are not neuronally controlled [70, 71, 72]. Therefore, initiation of NRF2-mediated antioxidant response may indicate the existence of cellular defense mechanisms that prevent damage from oxidative stress caused by long-term inflammation. Previous studies have shown that NQO1, an enzyme putatively regulated by NRF2, is increased in multiple sclerosis (MS). This stimulation was particularly evident in hypertrophic stellate cells and myelin-laden macrophages. Oligodendrocytes sometimes show NQO1 immunoreactivity, but neuronal NQO1 labeling is rare [70]. Additional studies have shown that the disease in multiple sclerosis (MS) exhibits strong resistance to markers of oxidative stress, including 4-HNE, 8-hydroxydeoxyguanosine, and nitrotyrosine. It has also been found that these active bacteria contain antioxidant enzymes such as HO-1, SOD1, SOD2, and catalase. Like NQO1 manifestations, this staining is seen in hypertrophic stellate cells and myelin-rich macrophages. Rare staining was observed in NAWM. Upregulation of NRF2 in MS lesions can be seen in the nuclei and cytoplasm of invasive macrophages and reactive stellate cells, with little expression in oligodendrocytes around active lesions [72].

3. Therapeutic scope of the activation or inhibition of the transcriptional factor NRF2 in neurological disorders

NRF2 activation mitigates multiple pathogenic processes involved in these neurodegenerative disorders through upregulation of antioxidant defenses, inhibition of inflammation, improvement of mitochondrial function, and maintenance of protein homeostasis.

3.1 Oxidative stress and Nrf2

NRF2 controls the production of many antioxidant enzymes and proteins that provide protection to cells. Studies show that enhancing NRF2-dependent antioxidant activities may enhance brain functions in illness models. The synaptic degeneration and neuronal death in Alzheimer’s disease are a result of various disrupted processes such as heightened oxidative stress, persistent inflammation, impaired mitochondrial function, buildup of amyloid-β (Aβ) 1–42 peptides from the amyloid precursor protein (APP), proteasome inhibition, mutations in APP, presenilin-1, and presenilin-2 genes, and accumulation of hyper-phosphorylated tau protein [73]. Oxidative stress is partly caused by oligomeric Aβ peptides and occurs before other metabolic changes, leading to persistent inflammation due to oxidative damage.

3.2 Neuroinflammation and Nrf2

It is a feature of neurodegenerative diseases and is caused primarily by activated microglia (inhabitants of the immune system). When activated, microglia release cytotoxic reactive oxygen species and nitrogen, which can affect neurons, especially in cases of excessive microglial activation and dysregulation. Additionally, the presence of dead or injured neurons leads to activation of microglia (known as reactive microgliosis) through recognition receptors such as Toll-like receptors. This causes a continuous cycle of neuronal cell death. The density of microglia in the adult brain varies by region and can range between 0.5 and 16.6%. They are usually found in a dormant state with branching-like patterns in the gray matter. Regions with the most cells include the hippocampus, olfactory telencephalon, basal ganglia, and substantia nigra [74, 75]. Therefore, it is not surprising that certain regions of the brain can be affected by microgliosis, as seen in many neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), and amyotrophic lateral multiple sclerosis (ALS) [76]. The main source of reactive chemicals produced by microglia is the activation of the NADPH oxidase (NOX) system, which produces more superoxide anions (•O₂⁻). The NOX enzyme complex in the cell membrane normally enhances the elimination of pathogens when triggered by different stimuli such as pathogen-associated molecular patterns (PAMPs), inflammatory cytokines, and neurotoxins. It also causes changes in cell shape and movement and regulates gene activity. Sustained activation of NOX leads to oxidation of lipids and other macromolecules and causes apoptosis, ultimately leading to neuronal cell death and degeneration. Similarly, enzyme-inducible nitric oxide synthase (iNOS) is stimulated, and its activation produces nitric oxide (NO•), similar to •O₂⁻ which can produce neurotoxic effects when consistently present at high concentrations. An increase in the level of NO• can disrupt the balance of redox homeostasis, and NO• can also react with •O₂⁻, leading to the formation of more harmful compounds called peroxynitrite (ONNO⁻) [50]. At rest, microglia constantly monitor its environment for bacteria, abnormal cells, or cellular debris. They also help reorganize and maintain synapses. When activated by risk, microglia become activated and subsequently release anti-inflammatory substances such as TNF, interleukin (IL)-1, IL-6, IL-12, and IL-23. This phase of microglial activation is often referred to as the classical proinflammatory phase and is referred to as M1. M2 is the stage after the M1 stage and is characterized by a long period and immunity or change. During this time, microglia are activated in response to inflammatory cytokines, including IL-4, IL-10, and IL-13. The body then produces the same cytokines while stimulating genes involved in wound healing, including arginase 1 (ARG1), mannose receptor (MMC), and chitinase-like 3 in domain 1 (FIZZ1)-1 (Ym1 in mice). In fact, the antioxidant properties of microglia maintain cellular homeostasis and keep microglia constantly alert. NF-kappaB (NF-kß) is a master regulator of the brain’s response to infection, the environment, and cellular damage. The redox state of microglia plays an important role in regulating the nuclear level of NF-kß as well as the flux of NF-kß kinases [74]. Microglia function well when the body’s antioxidant defense system maintains oxidative stress (OS) at normal levels. However, when OS or neuroinflammation (NS) persists, the NF-kß signaling pathway remains active. This causes more inflammation in microglia, making them more active and affecting the nervous system. Transcription of NRF2 is responsible for the activation of antioxidant genes and maintenance of redox homeostasis. It is a master regulator of microglia fate, protecting microglia from oxidative stress and preventing hyperactivation of the M1 phenotype. Activation of NRF2 specifically leads to increased expression of genes involved in various processes, including elimination of oxidative stress (SOD3, GPx, and Prx), production of reducing agents (GCLM/C or NADPH), and regeneration of cofactors. Proteins (GR, Trx-TrxR, Prx/thioredoxin), transport of redox molecules (xCT), metal chelation (MT1/2 or ferritin), and antioxidant production enzymes (HO-1, NQO1 and Trx). In addition, NRF2 activation also inhibits the expression of the Trx inhibitor TXNIP. Many studies have identified NRF2 as a useful target for the treatment of neurological diseases associated with neuronal protection secondary to neurodegenerative pathologies. In one study, mice lacking the NRF2 gene (NRF2−/−) were injected with lipopolysaccharide (LPS), a substance known to cause inflammation. These animals show sensitivity to lipopolysaccharide-induced neuroinflammation, especially in the hippocampal region. NRF2−/− mice have protein and mRNA levels of the microglial marker F4/80 in the hippocampal gyrus compared to WT animals. In addition, the inflammatory markers iNOS, IL-6, and TNF-α were also improved. Additionally, SFN-treated mice showed a two- to threefold increase in HO-1 levels, decreased microglia numbers, and decreased production of specific markers in response to LPS [77]. Mice treated with SFN after spinal cord injury showed an increase in NRF2 and GCL levels as well as a decrease in the cytokines IL-1ß and TNF-α. As a result, these mice had decreased volume and improved coordination [78]. In a mouse model of Parkinson’s disease (PD), MPTP injection into animals lacking the NRF2 gene (NRF2−/−) showed strong oxidative stress (OS). These mice had significantly more impaired dopaminergic activity in the basal ganglia than control mice with normal NRF2 genes (WT littermate controls). Although there was no significant difference in the number of Cd11b-positive/CD45 advanced cells between WT and NRF2−/− mice (indicating peripheral macrophage infiltration), NRF2−/− mice showed more astrocyte hyperplasia and microgliosis. This is determined by higher expression and protein expression of GFAP and F4/80 [79]. This study confirmed previous studies showing that NRF2−/− mice exposed to MPTP showed increased neuroinflammation caused by activation of astrocytes (GFAP) and microglia (Iba-1) [80]. In addition, levels of inflammatory markers associated with microglial activation (such as COX2, iNOS, IL-6, and TNF-α) were also increased in the M1 state. In contrast, markers associated with M2 status microglia, such as FIZZ-1, YM-1, arginase-1, and IL-4, were found to be decreased. Findings indicate that NRF2 plays an important role in regulating neuroinflammation after neurotoxic injury [78]. A mouse model of tauopathy was induced in the mouse hippocampus by stereotaxic injection of an adeno-disrupting vector expressing TAUP301L. In this model, NRF2−/− animals showed high levels of microgliosis and astrocytosis. Validation included measuring TNF and IL-6 mRNA levels in the hippocampus and immunohistochemical staining for Iba-1 and GFAP. In contrast to WT mice, NRF2−/− animals did not show an increase in hippocampal HO-1 and GCLC mRNA levels and did not show cellular staining for HO-1. These findings suggest that TAUP301L-induced disease activates the NRF2 signaling pathway as a regulatory response. In line with these observations and previous studies showing that neuronal production of the chemokine fractalkine (CX3CL1) affects TAU-mediated neuroinflammation, the authors of this study showed that CX3CL1 secreted by injured neurons was exclusively associated with an increase in microglia. Receptors are found in cells. This interaction activates the NRF2-ARE signaling pathway and reduces the incidence of TAUP301L-induced microglial proliferation. Subsequent studies were supported by the finding that mice lacking CX3CR1 had increased hippocampal microgliosis in response to TAUP301L compared with normal controls. Additionally, NRF2-dependent HO-1 immunohistochemical staining was absent in these animals. So, when neurons are injured, they produce a substance called CX3CL1, which then interacts with microglia through the CX3CR1 receptor. This interaction activates the NRF2-ARE pathway, which reduces the neuroinflammatory response to injury [81].

In a study with co-culture experiments using microglial conditioned media and mouse primary neurons, degenerating neurons exposed to neurotoxic oligomeric amyloid ß (oAß) were found to induce microglia to release milk fat globule-EGF factor 8. It was found to emit signal (MFG-E8). Release of MFG-E8 increases the phagocytic activity of microglia. Additionally, microglia treated with MFG-E8 was found to activate the NRF2 signaling pathway and HO-1. However, when examining neuronal-microglia co-cultures, retention of MFG-E8 was counteracted by application of the HO-1 inhibitor tin (IV)-mesoporphyrin IX dichloride (SnMP). This suggests that increased HO-1 in microglia is important for protecting neurons from toxicity [82]. These studies suggest that message exchange between neurons and microglia and stimulation of the NRF2 signaling pathway may be a new and effective way to protect neurons, especially in the context of neurodegenerative diseases.

3.3 Mitochondrial function and Nrf2

NRF2 controls cellular defense by adjusting mitochondrial function. NRF2 activation offsets the generation of reactive oxygen species by mitochondria and protects against toxins generated by mitochondria. NRF2 function is reduced in mitochondrial illnesses such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Friedreich’s ataxia (FRDA). Research conducted with isolated mitochondria and cultured cells has shown that a lack of NRF2 results in reduced mitochondrial fatty acid oxidation, respiration, and ATP generation. Chemical substances that activate NRF2 help maintain mitochondrial health by either stimulating mitophagy or providing protection against oxidative stress-induced damage to the mitochondrial permeability transition pore [83].

4. Association between NFE2L2 and KEAP1 haplotypes and neurodegenerative disorders

Genomic studies were performed to determine whether there were genetic differences in the genes encoding NRF2 and its repressor protein Keap1 (called NFE2L2 and KEAP1, respectively). As discussed in this review, this is based on significant evidence that the neurodegenerative effects of OS/NS in diseases such as PD, AD, and ALS can be inhibited or prevented by increasing NRF2 activity. The aim is to investigate whether genetics plays a role in the development or susceptibility to these diseases. Based on analysis of published SNPs and other genetic variants, the mutation frequency of human NFE2L2 was estimated to be 1 in every 72 base pairs. This suggests that genes may have many variations, called polymorphisms [84]. Von Otter et al. Initial data analysis in patients with Parkinson’s disease (PD) identified a protective NFE2L2 haplotype, GAGCAAAA. This haplotype contains a variant in the NRF2 promoter that is active in Swedish and Polish samples and is associated with high activity [85]. In a Swedish study, the presence of GAAAA haplotype alleles was associated with a higher estimated age of onset (AAO) of Parkinson’s disease (PD) by approximately 5 years per allele (p = 0.001). In the Polish study, individuals with the protective entire haplotype GAGCAAAA had a lower risk of developing PD; There was a difference of 0.4 and 0.2 for heterozygous and homozygous carriers, respectively (p = 2 × 10⁻⁶). Additionally, studies in Sweden found that GAGGG and GAAAG haplotypes were associated with an increased risk of Parkinson’s disease, with odds of 2.4 and 3.7 for each allele, respectively. Importantly, the promoter haplotype AGA tended to increase the risk of PD, whereas the entire promoter haplotype GAGAAGGG showed an overall risk (difference between each haplotype allele was 2.8). However, studies have shown that the entire haplotype allele GAGCAAAAA causes a delay in the age of onset of Parkinson’s disease for all haplotype alleles. Neither the Swedish nor the Polish samples found a significant effect on Parkinson’s disease risk for specific SNPs alone. Genetic polymorphisms of KEAP1 were also examined. However, there was no association with age at onset (AAO) or likelihood of Parkinson’s disease [85]. A recent meta-analysis investigated the genetic association of NFE2L2 variants encoding NRF2 with Parkinson’s disease (PD). The review included the literature mentioned above, as well as four new independent European patient-controlled studies from Malta, Germany and Italy, and a second Swedish study. The results of this analysis showed an association between the GAGCAAAAA haplotype, including the high-activity promoter haplotype AGC, and reduced risk of PD (odds ratio (OR) of none per allele 0.8, p = 0.012). Additionally, this haplotype is associated with delayed onset of PD (+1.1 years per allele, p = 0.048) [84]. The results of this study confirm the first haplotype study linking genetic variation in the NFE2L2 gene to the development of Parkinson’s disease. This latest study also reported four single nucleic acid polymorphisms (SNPs) found in haplotypes associated with age of onset (AAO) in Parkinson’s disease (PD). Three of the SNPs (rs7557529 G > A, rs35652124 A > G, rs2886161 A > G) were found to accelerate the onset of PD and each allele was associated with a reduction in AAO at 1.0, 1.1, and 1.2 years, respectively. The P values of these relationships were 0.042, 0.045, and 0.021, respectively. On the other hand, the fourth SNP (rs1806649 G > A) was shown to delay the onset of PD, with each allele being associated with an increase in AAO at 1.2 years. It is worth noting that the single nucleotide polymorphism (SNP) rs35652124 is functional and is located in the promoter region of the NFE2L2 gene [86]. Unlike the Parkinson’s disease (PD) studies mentioned above, analysis of the NFE2L2 promoter in a control group of Taiwanese patients did not reveal any genetic changes. Three single nucleotide polymorphisms (SNPs) in the NFE2L2 promoter were examined: rs35652124, rs6706649, and rs6721961. These SNPs can be detected alone or in combination with a genetic haplotype associated with susceptibility to Parkinson’s disease (PD). However, there was no association between these SNPs or haplotype variants and susceptibility to PD. Researchers have suggested that differences between studies in Taiwan and Europe may be due to differences in ethnic and environmental variables in different regions [87]. Previous studies using AD Swedish case-control data found no evidence supporting single SNPs or haplotypes in NFE2L2 or KEAP1 as genes that increase AD risk. However, genetic analysis in the NFE2L2 gene shows a significant association between the GAAAA haplotype and the onset of Alzheimer’s disease 2 years earlier than average (p = 0.013). This means that some common mutations in the NFE2L2 gene may affect Alzheimer’s disease and therefore the onset of the disease. In studies, the presence of NFE2L2 or KEAP1 gene mutations was not associated with Mini-Mental State Examination (MMSE) results or total tau and amyloid ß 1–42 (Aß42) levels in cerebrospinal fluid (CSF) [88]. A Swedish case study of NFE2L2 and KEAP1 in ALS disease showed that a specific NFE2L2 haplotype (GGGAC) was associated with a reduced risk of ALS (or one allele = 0.62, p = 0.015). Additionally, haplotypes in KEAP1 (CGC) were found to be associated with delayed onset of ALS (+3.4 years per allele, p = 0.015). Furthermore, the distribution of ALS revealed the existence of a specific group characterized by the NFE2L2-related haplotype GAGCAGA. This subgroup reveals three function-promoting SNPs associated with increased NRF2 protein expression. Each additional allele of this haplotype was associated with a 4.0-year delay in the onset of ALS (p = 0.008). Each additional allele of this haplotype was associated with a 4.0-year delay in the onset of ALS (p = 0.008) [89]. In a separate case-control study in Italian ALS patients, three NFE2L2 promoter polymorphisms (i.e., SNPs −653 A/G, −651 G/A, and −617 C/A) were tested. However, there was no significant difference between ALS cases and controls. The frequency of the −653 A/G promoter polymorphism was slightly higher in patients than in controls but did not reach significance. Additionally, there was no association between the NFE2L2 promoter polymorphisms tested and the oxidative stress biomarkers tested [84]. Similar studies have not been conducted in patients with Huntington’s disease (HD) or multiple sclerosis (MS).

5. Conclusion

The NRF2-ARE pathway is an important target for therapeutic intervention in neurodegenerative diseases, as demonstrated by the facts presented. Current data provide the highest level of evidence for Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). However, future trials in Alzheimer’s disease (AD) and Huntington’s disease (HD) are needed to better understand the importance of NRF2 in these diseases. Cell and in silico high-throughput screens have identified new drugs that activate NRF2 [90]. Effective strategies may include the identification of non-covalently linked compounds with interactions between DLG or ETGE motifs and Keap1 [91, 92]. Now the US Food and Drug Administration (FDA) has approved Biogen Idec’s DMF (BG-12) for the treatment of multiple sclerosis (MS). As described previously, DIF has been evaluated in previous HD models. However, it is important to note that in previous models of the above diseases, only CDDO drugs were analyzed. Reata Pharmaceuticals developed the first CDDO molecule, bardoxolone methyl (also known as CDDO methyl ester), and initiated a Phase III study for the treatment of kidney disease. Unfortunately, this study was discontinued in 2012 due to a higher incidence of cardiovascular disease in the bardoxolone methyl group compared to the placebo group [93]. A new CDDO derivative called RTA 408 has been developed and is currently being tested in several non-neurodegenerative diseases [94, 95, 96, 97]. A clinical trial investigating the use of RTA 408 to treat Friedreich’s ataxia, a neurodegenerative disease that causes cerebellar ataxia, has recently begun. This condition is characterized by a decrease in the protein frataxin, resulting in a defect in mitochondrial respiration (ClinicalTrials.gov identifier NCT02255435). The inclusion of these future CDDO drugs in the major neurodegenerative diseases mentioned in this review may be an important first step, supported by the findings of previous CDDO history.

A new aspect of treatment strategies for this system is that some cells are more effective in protecting neurons from degeneration. Compared to the protection obtained by overexpression of NRF2 in stellate cells in models of ALS and PD, the lack of protection in vivo by overexpression of NRF2 in neurons or muscle cells suggests that not only the pharmacological target but also the cell specificity is still affected. Targetability may be important when considering NRF2-dependent therapeutic approaches.