NADPH oxidase 4 (NOX4) as a biomarker and therapeutic target in neurodegenerative diseases

Abstract

Diseases like Alzheimer's and Parkinson's diseases are defined by inflammation and the damage neurons undergo due to oxidative stress. A primary reactive oxygen species contributor in the central nervous system, NADPH oxidase 4, is viewed as a potential therapeutic touchstone and indicative marker for these ailments. This in-depth review brings to light distinct features of NADPH oxidase 4, responsible for generating superoxide and hydrogen peroxide, emphasizing its pivotal role in activating glial cells, inciting inflammation, and disturbing neuronal functions. Significantly, malfunctioning astrocytes, forming the majority in the central nervous system, play a part in advancing neurodegenerative diseases, due to their reactive oxygen species and inflammatory factor secretion. Our study reveals that aiming at NADPH oxidase 4 within astrocytes could be a viable treatment pathway to reduce oxidative damage and halt neurodegenerative processes. Adjusting NADPH oxidase 4 activity might influence the neuroinflammatory cytokine levels, including myeloperoxidase and osteopontin, offering better prospects for conditions like Alzheimer's disease and Parkinson's disease. This review sheds light on the role of NADPH oxidase 4 in neural degeneration, emphasizing its drug target potential, and paving the path for novel treatment approaches to combat these severe conditions.

Introduction

Oxidative stress is increasingly understood to play a pivotal role in aging and the development of various neurological conditions. It is a significant driver behind neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD) (Olufunmilayo et al., 2023). Oxygen, indispensable for both energy metabolism and normal function, undergoes limited reduction during its movement through the respiratory chain. This produces superoxide, a primary free radical, which can further evolve into other reactive oxygen species (ROS) (Li et al., 2013). Contemporary studies suggest that ROS and reactive nitrogen species at physiological concentrations are not just destructive entities; they also facilitate standard operations. These functions encompass signal transduction regulation, the initiation of growth responses, and protection against pathogens (Li et al., 2013; Korovesis et al., 2023). Reactive nitrogen species, which belongs to the nitrogen-based free radical family, can also be generated by cell metabolism. Oxidative stress plays a dichotomous role in biology. On one hand, physiological oxidative stress, or eustress, is utilized for redox signaling and regulation (Sies et al., 2022), while ROS production and antioxidant defenses imbalance can lead to excessive accumulation of ROS (Shields et al., 2021). ROS can become detrimental when this threshold is exceeded (Murphy et al., 2022; Sies et al., 2022). Maintaining this balance is crucial, as it determines the manifestation of both beneficial and harmful effects (Shields et al., 2021). Under oxidative stress conditions, free radicals can penetrate the plasma membrane, resulting in detrimental effects such as lipid peroxidation (Angelova et al., 2021), misfolding and aggregation of structural and signal proteins (Michalska and León, 2020), and interruption of transcription through the oxidation of RNA/DNA (Juan et al., 2021), eventually leading to gene mutations. The oxidation of various amino acid residues (depending on the oxidants involved) can lead to a range of oxidative modifications in proteins. These modifications have the potential to regulate cellular function by affecting the stability, activity, subcellular localization, and protein-protein interactions of oxidized proteins (Murphy et al., 2022; Sies et al., 2022).

ROS plays a significant role in cellular oxidative stress and redox regulation, contributing to the development of neurodegenerative diseases, as shown in Figure 1 (Lee et al., 2021b). Mitochondria and NADPH oxidase (NOX), of which there are seven isoforms (NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2), are the primary sources of ROS (Cáceres et al., 2020). The NOX family plays a pivotal role in various physiological settings, including the regulation of redox signaling, gene expression, and the maintenance of normal physiological functions, while also contributing to the pathogenesis of various conditions (Panday et al., 2015). In the central nervous system (CNS), NOX, which produces superoxide anion (O•⁻₂) and hydrogen peroxide (H₂O₂), is a major source of ROS (Tarafdar and Pula, 2018). Among the various types of oxidant generated, the superoxide anion radical (O•⁻₂) emerges as a vital redox signaling molecule, prominently created by both the NOX enzyme family and the mitochondrial electron transport chain (Pecchillo Cimmino et al., 2023). Subsequently, O•⁻₂ is converted to H₂O₂ by spontaneous dismutation or by superoxide dismutase (SOD) (Bedard and Krause, 2007; Lee and Jose, 2021; Pecchillo Cimmino et al., 2023).

Figure 1.

Source of ROS in neurodegenerative diseases.

Mitochondrial dysfunction, glial activation, protein misfolding, DNA mutation, and different environmental stressors such as smoking, UV light, and air pollution mainly induce ROS production in neurodegenerative diseases. Excessive ROS leads to apoptosis and ultimately to neurodegenerative disorders. Created with BioRender.com. ROS: Reactive oxygen species; UV: ultraviolet.

NOX4 possesses a unique characteristic: it can produce both H₂O₂ and O•⁻₂, whereas other NOX enzymes predominantly generate only O•⁻₂. Notably, NOX4 is known for its constitutively active H₂O₂ production, with H₂O₂ as its primary output, and its continuous role in generating this specific oxidant (Block et al., 2009; Lyle et al., 2009; Nisimoto et al., 2014; Brendel et al., 2020). The regulation of NOX4 activity primarily occurs at the mRNA level, and the NOX4 protein is localized to various subcellular sites. NOX4 is found in various cell membranes, including those of the mitochondria, endoplasmic reticulum, and nucleus (Sarkar, 2021). Recent studies have linked the generation of mitochondrial O•⁻₂ to the regulation of glial inflammation, providing evidence that the activation of the NOX4 catalytic unit may drive such inflammation, given its localization in the mitochondria (Graham et al., 2010; Canugovi et al., 2019). NOX4 has been implicated in various brain diseases (Gola et al., 2023). Evidence suggests that manipulating NOX4 activity can alter the expression of proinflammatory genes, highlighting the possibility of using NOX4 as a therapeutic target (Lozhkin et al., 2017; Sarkar et al., 2017). Furthermore, there is a growing interest in the significant involvement of non-neuronal cells in the onset, advancement, and pathology of various neurodegenerative diseases (D'Ambrosi et al., 2022). The exacerbation of astrocytic dysfunction has been causally linked to the advancement of the aging process (Griffiths et al., 2020). Considering the crucial function of astrocytes in these disorders, possible approaches to focus on these cells as potential therapeutic pathways in the future have been proposed (Brandebura et al., 2023; Chen et al., 2023).

In this review, we summarize the specific role of NOX4-induced ROS in the CNS, particularly in astrocytes, in the progression of neurodegenerative diseases. We further discuss the putative targets of NOX4 inhibitors in physiological and pathological functions to provide a new direction for future interventions in neurodegenerative diseases, especially AD and PD.

Search Strategy

We employed a variety of search engines, including PubMed, Google Scholar, Scopus, and Web of Science, along with keywords like “NADPH oxidase 4 (NOX4)”, “neurodegenerative diseases”, “myeloperoxidase (MPO)”, “osteopontin (OPN)”, “reactive oxygen species (ROS)”, “mitochondrial dysfunction”, “glucose transporter 1 (GLUT1)”, “oxidative stress”, and “NOX inhibitors”. Our work aims to examine NOX4's close relationship with neurodegenerative diseases and its potential use as a target for neurodegenerative disease diagnosis and treatment. Search Dates: The search was conducted between May 1, 2023, and September 27, 2023. Limits Used: Publication Year (1997–2023).

Role of Astrocyte-Related Oxidative Stress in Neurodegenerative Diseases

Astrocytes, the predominant cells in the CNS, are crucial for preserving brain equilibrium and executing a myriad of physiological roles within the CNS. Their responsibilities span maintaining blood-brain barrier integrity, generating trophic factors, influencing neuronal synaptic activity, overseeing extracellular ion balance, aiding in lactate and glutamate transfers to neurons, and metabolite processing (Giovannoni and Quintana, 2020). Furthermore, the collaboration between astrocytes, neurons, and the blood-brain barrier's endothelial cells is vital in brain energy metabolism. They jointly support neuronal metabolism via neurometabolic coupling mechanisms such as aerobic glycolysis (Yoo et al., 2020), glutamate (Chen et al., 2020), Na⁺-K⁺-ATPase activation (Sweeney et al., 2018), and lactate release (Li et al., 2022). Contemporary research has continually underscored the significance of astrocytes in the CNS (Chen et al., 2020). A comprehensive antioxidant response within astrocytes promotes the degradation and elimination of free radicals generated by neurons and other cellular subtypes in the CNS, thereby protecting the CNS from the detrimental effects of oxidative stress. In contrast, under specific pathological circumstances, astrocytes may serve as one of the primary elements of detrimental ROS and reactive nitrogen species, and excessive free radicals can stimulate microglial activation or trigger direct neural damage. Extensive research has been conducted on oxidative stress in microglia; however, few reviews have specifically examined the role of oxidative stress in the CNS from the perspective of astrocytes.

The concept of the astrocyte-neuron lactate shuttle is widely accepted, and this pathway has been suggested to play a major metabolic role in the brain (Xue et al., 2022). Furthermore, the uptake of glutamate by astrocytes stimulates increased glucose uptake from the surrounding capillaries via glucose transporters (GLUT1), as well as increased aerobic glycolysis (Ardanaz et al., 2021; Beard et al., 2022). Under stressful conditions, high ROS levels in the brain contribute to astrocyte dysfunction, which consequently compromises neurotoxicity (Chen et al., 2020). Moreover, oxidative stress in reactive astrocytes has the potential to cause significant long-term effects on specific proteins, including connexins, glutamate transporters, and enzymes, thereby affecting the interplay between astrocytes and neurons (Lee et al., 2021a).

The release of cytokines, neurotransmitters, growth factors, and ion channels is modulated by astrocytic function, which affects neuronal death when affected by acute stress or chronic inflammatory/degenerative changes. Neurodegenerative diseases include a series of CNS disorders that result from the progressive degeneration of neurons, leading to neuronal dysfunction (Dugger and Dickson, 2017). Astroglial atrophy and asthenia are significant contributors to the disruption of CNS homeostasis in many neuropsychiatric, neurodevelopmental, and neurodegenerative diseases (Verkhratsky et al., 2014). Additionally, astrocyte dysfunction subsequently reduces the ability of neurons to support energy supply and consequently promotes neuronal death (Escartin et al., 2021). Further, it is important to note that aberrant reactive astrogliosis can lead to the chronic production of ROS and thus contribute to the progression of neurodegenerative diseases (Rizor et al., 2019). Therefore, the pivotal role of astrocytes in various neurodegenerative diseases is being increasingly acknowledged.

AD is the most prevalent neurodegenerative disorder and the principal cause of dementia worldwide. The etiology and pathogenesis of AD are not yet fully understood; however, oxidative damage is a crucial element that plays a significant role in the disease (Jiang et al., 2016). AD primarily develops due to the buildup of neuritic plaques outside cells and the presence of neurofibrillary tangles inside them. These structures emerge as a result of amyloid-β (Aβ) and irregular tau protein phosphorylation, respectively (Long and Holtzman, 2019). Numerous studies have demonstrated that oxidative stress plays a significant role in the pathogenesis of AD (Butterfield and Halliwell, 2019) and that the pathology of AD is intricately linked to both oxidative stress and astrocytes. In vitro studies have suggested that Aβ aggregation increases astrocyte oxidant production, thereby exacerbating neural impairment (Schubert et al., 2009). Cytokines, notably insulin-like growth factor-1, play a role in modulating astrocytic mitochondrial functions, which are correlated with memory and learning processes (Logan et al., 2018). In specific oxidative stress scenarios, astrocytes release nitric oxide (NO) near Aβ plaque formations (Wallace et al., 1997). Additionally, lipoproteins from AD patients have been suggested to amplify the production of both peroxynitrite and NOS in these cells (Nanetti et al., 2005). We have previously reported that astrocytic dysfunction is implicated in the progression of AD and PD (Park et al., 2021; Boonpraman et al., 2023).

PD is recognized as the second most common neurodegenerative disease globally (Rizor et al., 2019). The pathogenesis of this disease is attributed to misfolding of α-synuclein, an essential protein associated with the pathological process (Lee et al., 2010; Valdinocci et al., 2017). Studies have indicated that in PD models, α-synuclein can migrate from neurons to astrocytes, making it a crucial protein in disease progression. When human α-synuclein is directly administered to the basal ganglia of mice, there is a pronounced activation of both astrocytes and microglia (Sznejder-Pachołek et al., 2017). In laboratory settings, α-synuclein has been observed to stimulate abundant O•⁻₂ and pro-inflammatory compounds in astrocytes via the TLR4 receptor activation (Fellner et al., 2013). Furthermore, a rise in α-synuclein levels in astrocyte-neuron combined cultures can intensify oxidative stress, leading to lipid damage and eventual neuron loss (Rizor et al., 2019).

Finally, amyotrophic lateral sclerosis (ALS) has been observed in approximately 90% of individuals with familial ALS possessing mutations in the superoxide dismutase 1 (SOD1) gene, which encodes a vital antioxidant enzyme (Lee et al., 2016). Research on ALS has shown that astrocytes with the mutant SOD1 gene adversely affect healthy motor neurons (Ferraiuolo et al., 2011). These astrocytes release more transforming growth factor-β, show increased presence of inflammasomes like NLRP3, and trigger the nuclear factor-κB signaling route, amplifying the inflammation seen in ALS models (Lee et al., 2016). Furthermore, a distinct study indicated that astrocytes with mutant SOD1 and TDP43 genes intensified nitroxidative stress, leading to an increased occurrence of motoneuron fatalities (Rojas et al., 2014).

Overall, the results highlighted suggest that astrocytes could be potential key players in managing oxidative stress, offering hope for enhanced outcomes in neurodegenerative conditions.

NADPH‐Derived Oxidative Stress in Astrocytes

Studies on the vulnerability of the nervous system to oxidative stress have highlighted the role of NOX expression and ROS in various neurodegenerative disorders, notably PD and AD. These factors are also associated with a rise in inflammation-related markers (Barua et al., 2019; Ganguly et al., 2021). This leads to neuronal damage and cell death during neurodegenerative processes (Rostami et al., 2021). NOX is involved in neurodegeneration, with NOX2 and NOX4 being the most commonly expressed isoforms in various brain cell types, including the microglia, neurons, and astrocytes (Sorce et al., 2017; Hernandes et al., 2022). Increasingly, research findings suggest that as individuals age, there is a notable elevation in NOX activity and superoxide levels in astrocytes, underscoring the significant role these cells play in the progression of AD and PD (Barua et al., 2019; Chen et al., 2020). The latest research points out that a slight NOX presence in astrocytes can have a considerable impact on managing oxidative stress within the CNS. Furthermore, as astrocytes age, there is a noted rise in NOX activity and superoxide concentrations, linking it closely to diverse pathological states (Bellaver et al., 2017; Sharma and Nehru, 2018). Taken together, these results indicate that NOX significantly affects the physiological function of astrocytes. Additionally, several studies have shown that both deficiency and overexpression of NOX4 cause severe damage to the brain microenvironment (Jiranugrom et al., 2020; Park et al., 2021; Boonpraman et al., 2023; Pecchillo Cimmino et al., 2023). In particular, maintaining the function of NOX4 in the brain is thought to be very influential in maintaining the function of GLUT1 (Pecchillo Cimmino et al., 2023), which has already been mentioned earlier (in the concept of the astrocyte-neuron lactate shuttle), and abnormal expression of NOX4 debilitates the regular operation of GLUT1, ultimately affecting energy transfer between astrocytes and neurons (Benarroch, 2005; Porras et al., 2008). Since many studies have already reported how vital GLUT1 is for the smooth interaction between astrocytes and neurons (Beard et al., 2022), abnormal expression of NOX4 must be a risky signal for bracing brain function. Therefore, we propose that greater attention should be devoted to astrocytic NOX stress to explore novel approaches for regulating NOX activity in the CNS.

NADPH Oxidase 4 in Neurodegenerative Diseases

Interestingly, NOX4 has been reported to be the most highly expressed isoform in astrocytes and has been suggested to be localized to the mitochondria, unlike NOX1, 2, and 3 (Block et al., 2009; Ago et al., 2010). Activation of NOX4 (heterodimerized with p22phox), which does not require classical cytosolic subunits or their homologs, predominantly produces H₂O₂ (Lyle et al., 2009). NOX4 is widely expressed in various tissues, including the brain (Xie et al., 2020), and some brain regions have been investigated in relation to NOX4 elevation and neurodegenerative diseases. It has further been established that NOX4 expression is upregulated in the hippocampus of animal models and patients with both AD (Zhu et al., 2020; Tao et al., 2021; Luengo et al., 2022) and PD (Choi et al., 2019; Boonpraman et al., 2023), as well as in the cortex of individuals with AD (Bruce-Keller et al., 2011; Fragoso-Morales et al., 2021; Park et al., 2021). Additionally, in Braak stage 6 PD, which indicates a widespread distribution of α-synuclein pathology in the brain, NOX4 expression is elevated in the nuclei of dopaminergic neurons in the substantia nigra (Zawada et al., 2015). Furthermore, studies have demonstrated increased NOX4 expression in the cerebellum in animal models of neurodegenerative diseases, such as spinocerebellar ataxia (Weyemi et al., 2015).

With this understanding of the neurodegenerative progression mechanism, our recent studies have demonstrated that AD and PD are associated with mitochondrial dysfunction induced by NOX4 upregulation (Park et al., 2021; Boonpraman et al., 2023). Furthermore, the elevation of NOX4 was shown to lead to mitochondrial impairment by mediating redox imbalance and oxidative stress in serial reports (Park et al., 2021; Boonpraman et al., 2023). NOX4 has further been shown to promote ferroptosis in astrocytes in the cortex during AD (Park et al., 2021). Moreover, in a recent investigation into this cellular mechanism, we discovered a new finding in our research that NOX4 is also involved in PD progression in hippocampal astrocytes (Boonpraman et al., 2023).

Notably, we reported elevated levels of myeloperoxidase (MPO) and osteopontin (OPN) owing to the induction of NOX4 in hippocampal astrocytes in PD patients (Boonpraman et al., 2023). In this study, we reported for the first time, that MPO and OPN levels are dependent on NOX4 expression in hippocampal astrocytes. Interestingly, several studies have previously demonstrated a robust correlation between MPO and OPN elevations in neurodegenerative diseases (Cappellano et al., 2021). MPO is an oxidant-generating enzyme that produces the potent oxidant, hypochlorous acid, by reacting with H₂O₂ to oxidize chloride. MPO has occasionally been detected in microglia and neurons in AD and PD brains (Maki et al., 2009; Smyth et al., 2022). Aberrant huMPO expression in astrocytes has also been shown to result in AD, with the causative factors contributing to neuronal dysfunction and phospholipids (Maki et al., 2009).

Our prior investigation also identified a consistent impact of increased MPO expression in hippocampal astrocytes in a PD model, leading to apoptosis through the downregulation of mitochondrial metabolism and an increase in oxidant production and lipid peroxidation. The expression of MPO was further found to correlate with elevated levels of lipid peroxidation, resulting in outcomes similar to those observed by other researchers. The pathological circumstances of cognitive dysfunction include key features of ferroptosis, such as iron dysregulation, lipid peroxidation, and inflammation (Hambright et al., 2017). OPN is a glycosylated phosphoprotein that exhibits multifunctional properties as both an immobilized extracellular matrix protein and a secreted cytokine in body fluids. Furthermore, there is a suggestion that OPN may potentially trigger self-injury by inducing inflammatory responses in neurodegenerative diseases, such as AD, multiple sclerosis, and PD (Cappellano et al., 2021). OPN expression has also been shown to be upregulated during the injury/inflammation process, which is related to reduced mitochondrial activity in PD or elevated OPN expression in the hippocampal neurons of both patients with AD and AD mouse models during autopsy (Rentsendorj et al., 2018). However, to date, few studies have investigated the contribution of OPN expression to the progression of neurodegeneration in astrocytes. OPN has also been implicated in neuroinflammation in the brain (Riew et al., 2019), and may be associated with oxidants in neurodegenerative diseases. Further extensive investigations are required to determine the role of OPN in various disease phases and its potential as a therapeutic target owing to its participation in various biological pathways and networks. Indeed, we found that NOX4 directly intercommunicates with MPO and OPN, strongly suggesting that the hippocampal expression of these factors is upregulated during PD progression in a NOX4-dependent manner. Mitochondrial dysfunction was observed, as indicated by the upregulation of MPO and OPN, which are the mechanisms associated with astrocyte death. NOX4 characteristically leads to a significantly increased impairment of mitochondrial metabolism in astrocytes and represents the result of oxidative stress-induced lipid peroxidation. Owing to these characteristics, it is believed that NOX4 plays a pivotal role in mitochondrial oxidant production and NOX4 has been hypothesized to be a key mediator in the induction of astrocytic oxidative stress by mitochondrial oxidants during neurodegenerative progression.

NADPH-induced oxidative stress significantly affects the physiological functions of astrocytes. We expect to contribute to the understanding of the role of NOX4 in the astrocytic CNS, leading to the regulation of pathological progression in neurodegenerative diseases, including AD and PD. Pathological symptoms such as AD and PD are thought to be caused by an increase in NOX4 in astrocytes. The general direction of the pathological development of dementia symptoms conforms to the stages of PD. The discovery of an increase in NOX4 in the hippocampus in PD is believed to have considerable diagnostic and therapeutic importance in this pathological progression. Key studies highlighting the role of NOX4 in neurodegenerative diseases, ischemia, and brain injury are presented in the supplementary materials (Additional Table 1).

Overall, current research indicates that NOX4 may be a key participant in the increased NOX activity repeatedly observed in the progression of AD, PD, and other neurodegenerative diseases (Bruce-Keller et al., 2011; Ansari and Scheff, 2011; Kim et al., 2017) in various affected brain regions such as the hippocampus, cortex, substantia nigra, and cerebellum. The modulation of NOX4 activity balance through NOX4 inhibition in the brain holds substantial promise as a therapeutic approach for treating neurodegenerative diseases.

NADPH Oxidase 4 Inhibitors Exhibit Therapeutic Effects in Neurodegenerative Diseases

In the early 1980s, the NOX inhibitor apocynin was reported to exhibit promising neuroprotective effects by blocking the migration of p47phox to the membrane, thereby inhibiting the function of MPO to alleviate oxidative stress (Kim et al., 2017; Hou et al., 2019). The inhibition of NOX by apocynin has since been reported to effectively inhibit impairments in hippocampal neurodegeneration, and α-synuclein pathology through the suppression of oxidative stress and neuroinflammation in a PD mouse model (Hou et al., 2019). However, it should be noted that apocynin has limited efficacy in inhibiting vascular NOX and primarily serves as an antioxidant in the vascular system (Heumüller et al., 2008). In recent years, significant advancements have been made in the development of NOX4 inhibitors in the brain (such as GLX351322, GKT137831, and GKT36901), as promising therapeutics for neurodegenerative diseases. GKT137831 (also known as setanaxib) and GKT136901 are dual NOX1 and NOX4 inhibitors. A previous study demonstrated the ability of GKT137831, to suppress oxidant production, reduce lipid peroxidation, and attenuate inflammatory responses through its inhibition of NOX4 activity (Liu et al., 2022; Khayrullina et al., 2023), as well as its potential as a therapeutic approach for neurodegenerative diseases by targeting NOX4 activity in astrocytes (Liu et al., 2022; Khayrullina et al., 2023). Additionally, it exerts a broad impact in various contexts, including neural stem/progenitor cells, vascular response, repair of peripheral myelin profile in Schwann cells, and the reduction of oxidant production (Yoshikawa et al., 2019; Eid et al., 2020). Similarly, GKT136901, identified in 2010, shows high specificity for NOX1 and NOX4 and partial inhibition of NOX2. This drug is orally bioavailable (Laleu et al., 2010; Sedeek et al., 2010), and can greatly affect the signaling pathways associated with inflammation, which are integral to the implementation of cellular death (Schildknecht et al., 2014).

Overall, NOX4 inhibitors have shown promise in preclinical studies for the treatment of neurodegenerative diseases, such as AD and PD (Barua et al., 2019). Recently, a newly developed NOX4 inhibitor, GLX351322, demonstrated the potential to protect against synaptic and memory dysfunction (Tao et al., 2021), and was further shown to reduce amyloid levels and oxidative stress in the hippocampus of APP/PS1 mice (Zhu et al., 2020). These findings highlight the ability of NOX4 inhibition to reverse oxidative stress in the hippocampi of APP/PS1 mice (Zhu et al., 2020).

Neuronal NOX4 knockdown effectively reduced neurotoxicity and prevented cognitive decline, suggesting its direct causal role for NOX4 in neurons (Luengo et al., 2022). The amelioration of neuronal mitochondrial function following intracerebral hemorrhage can be achieved by downregulating NOX4 expression and reducing mitochondrial H₂O₂ production, which can be accomplished by NOX4 knockdown (Ding et al., 2023). On the other hand, NOX4 also serves a neuroprotective function in hyperexcitability, as NOX4 deficiency can lead to a lack of NOX4-derived oxidants, resulting in calcium overload and mitochondrial dysfunction, which are associated with neuronal death (Gola et al., 2023). In addition, Jiranugrom et al. (2020) reported that the NOX4 deficient mice have shown to have impairment in the hippocampal neurogenesis. It is a crucial consideration to the balance of NOX4 activity. Further research is necessary to comprehensively understand the beneficial and detrimental effects of NOX4 inhibitors, highlighting the need for ongoing investigation in this area. According to Tao et al. (2021), treatment of APP/PS1 mice with GLX351322 resulted in decreased levels of oxidant and H₂O₂, as well as reduced levels of biomarkers for oxidative stress, including 4-HNE, 3-NT, and 8-OHdG. The same study demonstrated that NOX4 mediates Aβ_1–42-induced neuronal death and that the inhibition of NOX4, either by miR-204-3p or GLX351322, rescued memory, and synaptic deficits, leading to memory improvement (Fragoso-Morales et al., 2021; Tao et al., 2021). Accordingly, the precise function of miR-204-3p in neurodegenerative diseases remains unknown; however, Tao et al. (2021) revealed that NOX4 is a direct target of miR-204-3p and that overexpression of miR-204-3p in the hippocampus of APP/PS1 mice has the potential to alleviate both synaptic and memory impairments, as well as decrease amyloid accumulation and oxidative stress.

Meanwhile, limited clinical trials or commercialized NOX4 inhibitors are available for the detailed evaluation of their mode of inhibition, resulting in incomplete characterization. Many NOX inhibitors lack specificity for a single NOX isoform, including NOX4 (Altenhöfer et al., 2014). This lack of specificity challenges our understanding of their precise effects on NOX4 and modes of action. Based on the general principles of drug development and potential mechanisms, the expression of NOX4 in various tissues and cell types suggests that its inhibition may have distinct effects depending on the specific context (Dao et al., 2020). Additionally, the potential impact on the normal physiological functions of NOX4 in non-diseased tissues remains an important consideration. Off-target effects and the lack of specificity of NOX4 inhibitors further complicate their development. Further, the long-term safety profile of NOX4 inhibitors remains poorly understood, and their efficacy in the clinical setting for treating neurodegenerative diseases or other conditions associated with NOX4 dysregulation is yet to be determined. Further research, clinical trials, and thorough evaluations are necessary to assess the disadvantages, weaknesses, and potential side effects of specific NOX4 inhibitors.

In summary, NOX4 inhibition is widely recognized as a promising therapeutic strategy for neurodegenerative diseases, providing advantages, such as reduced oxidant production, lipid peroxidation, inflammation, and synaptic/memory dysfunction. NOX4 inhibitors have therapeutic potential as they selectively target NOX4 in astrocytes, neural stem/progenitor cells, and other specific cell types, thereby offering broad applicability in a variety of contexts. Notably, these findings are based on general considerations related to drug development and are not specific to any other NOX4 inhibitor that may emerge in the future. Further studies are required to explore the precise functions of NOX4 and its inhibitors in the pathophysiology of neurodegenerative diseases.

Conclusions and Future Directions

Neurodegenerative diseases pose significant challenges to the global healthcare system, emphasizing the need for effective preventative and therapeutic strategies. Once considered solely supportive cells, astrocytes have emerged as key players in maintaining neuronal homeostasis and the pathogenesis of neurodegenerative diseases. Understanding the intricate interplay between astrocytes and neurons is crucial in elucidating the mechanisms underlying neuronal disorders. The NOX4 enzyme, which is predominantly expressed in astrocytes, represents a major source of specific oxidants (H₂O₂ and O•⁻₂) generation within the central nervous system; however, the precise role of NOX4-derived oxidants in astrocytes and its contribution to neuronal disorders remain poorly understood. Herein, we reviewed the current literature exploring the involvement of NOX4-derived ROS in astrocytic signaling and their potential impact on neuronal function. Overall, we show that targeting astrocytic oxidants through selective NOX4 inhibition is an attractive therapeutic strategy for preventing neuronal disorders and ameliorating the progression of neurodegenerative diseases (Figure 2).

Figure 2.

NOX4 in the brain participates in neurodegenerative diseases (including AD and PD).

NOX4 dominates astrocytes, other brain cells (neurons and microglia), and endothelial cells. Aß, tau, α-syn, proinflammatory cytokines, chemokines, and activated astrocytes are concerned with linking in the pathological process of neurodegeneration. The activation of NOX4 and NOX4-derived mtROS contribute to the progression of neurological diseases. The potential of NOX4 inhibitors has been indicated. Created with BioRender.com. AD: Alzheimer's disease; Aβ: amyloid-β; mtROS: mitochondrial reactive oxygen species; NOX4: NADPH oxidase 4; PD: Parkinson's disease; α-syn: α-synuclein.

There are ample data indicating elevated NOX subunit levels and activity across multiple neurodegenerative conditions. Insights from genetic elimination and drug-based suppression propose that focusing on NOX subunits might be a viable treatment method for these disorders. Yet, discerning the outcomes of particular NOX interventions remains a complex task. Gaining a more profound understanding of neurodegenerative complexities while curbing adverse impacts and fostering a conducive environment for neurogenesis would be beneficial. Despite the inherent complexities in addressing these issues, the steady surge of technological advancements instills confidence that achieving this goal could soon be plausible.

By elucidating the role of NOX4-derived oxidants in astrocytes, this study provides valuable insights into the underlying mechanisms and proposes avenues for future research to address existing knowledge gaps. A comprehensive understanding of astrocytic ROS-mediated signaling holds promise for the development of novel therapeutic interventions targeting neurodegenerative diseases. In conclusion, we suggest that NOX4 upregulation is a potential common denominator in the pathological molecular mechanisms of AD and PD. While NOX4 dysregulation has been observed in the hippocampus and cortex of patients with AD and PD, it is crucial to acknowledge that the current knowledge regarding NOX4 elevation in other brain regions is limited. Ongoing research and further studies are required to fully understand the mechanisms and implications of NOX4 dysregulation in these brain regions and diseases. However, translational research and emerging concepts regarding NOX4 balance are essential for the prediction and development of future medicines and diagnostics for neurodegenerative diseases.

Additional file:

Additional Table 1: Involvements of NADPH oxidase 4 (NOX4) in the neurodegenerative diseases, ischemia, and brain injury.

Supplementary 1.

Involvements of NADPH oxidase 4 (NOX4) in the neurodegenerative diseases, ischemia, and brain injury

Brain Disease	Related references
Alzheimer's Disease	(Zhu et al., 2020; Park et al., 2021; Tao et al., 2021; Luengo et al., 2022; Boonpraman et al., 2023)
Parkinson's Disease	(Weyemi et al., 2015; Zawada et al., 2015; Choi et al., 2019; Boonpraman et al., 2023)
Amyotrophic lateral sclerosis (ALS)	(Carter et al., 2009)
Ischemic Stroke	(Kleinschnitz et al., 2010; Li et al., 2014; Kuroda et al., 2015; Zhang et al., 2015; Casas et al., 2017)
Multiple Sclerosis	(Doğan and Yildiz, 2019)
Traumatic Brain Injury	(Cooney et al., 2013; Li et al., 2015; Ma et al., 2018; Xie et al., 2020)

Funding Statement

Funding: This work was supported by the National Research Foundation of the Republic of Korea 2018R1D1A3B07047960 and the Soonchunhyang University Research Fund (to SSY).