Department of Pharmaceutical Sciences (Pharmacology Division), School of Applied Sciences & Technology, University of Kashmir-J&K, India-190006.
Amyotrophic lateral sclerosis (ALS) is a grievous neurodegenerative disease whose survival is limited to only a few years. It is an intractable disease that causes respiratory failure leading to mortality. Several theories have been proposed, involving genetic factors, molecular dysfunctions, and cellular abnormalities. Growing evidence has linked oxidative stress (OS) and genetic mutations to disease progression, particularly mutations in the superoxide dismutase 1 (SOD1) gene. SOD1 plays a key role in regulating reactive oxygen species (ROS) generated by the electron transport chain and promotes the activation of genes that defend against oxidative stress. This review provides a comprehensive overview of ALS etiology, highlighting the molecular and cellular mechanisms implicated in its pathogenesis besides the treatment approaches for ALS and the physiological role of SOD1 in cellular defense against oxidative stress and its regulation within different cellular compartments. Currently, multiple FDA-approved therapies are available for ALS which are designed either to partially address the effects of the SOD1 mutation or to inhibit cellular and neurological pathways involved in motor neuron degeneration. By integrating insights from genetics, molecular biology, and environmental studies, this review aims to deepen the understanding of SOD1-related mechanisms in ALS and identify potential avenues for future therapeutic intervention.
Amyotrophic lateral sclerosis (ALS), commonly referred to as Lou Gehrig's disease and front temporal dementia (FTD) are two debilitating neurodegenerative disorders that exist within a shared disease spectrum, characterized by similar clinical, genetic, and pathological features. Jean-Martin Charcot initially documented ALS in 1869, but a significant advancement in understanding the origins of ALS occurred in 1993. This breakthrough came with the identification of mutations in the copper/zinc superoxide dismutase (SOD1) gene as causative factors for ALS, coupled with the development of dependable transgenic animal models [1]. ALS, specifically, impacts the upper motor neurons (UMNs) located in the motor cortex, along with the lower motor neurons (LMNs) situated in the brainstem and spinal cord. Motor symptoms encompass muscle weakness, fasciculation’s, cramps, along with spasticity and brisk reflexes that lead to significant limb and bulbar impairment progressively, culminating in eventual respiratory insufficiency [2]. Over time, in individuals afflicted by ALS, the brain gradually loses its capacity to instigate and govern voluntary actions like walking, speaking, chewing, and other bodily functions, including breathing. The cause of ALS still unknown, but several genetic, environmental, and pathological factors play a key role in its development. Notably, 5% to 10% of patients appear to have inherited ALS in an autosomal dominant pattern. Among the patients who suffer from ALS, 2% possess a mutation in a gene located on chromosome 21 known as Cu, Zn superoxide dismutase (SOD1), which typically plays a role in neutralizing superoxide free radicals [3]. ALS is mostly sporadic, however, familial ALS is linked to monogenic causes, such as mutations in C9orf72, SOD1, TDP-43, FUS or other genes [4, 5]. A missense mutation in the D-amino acid oxidase (DAO) gene has been documented in multiple families grappling with ALS. These DAO mutations result in a decrease in cell viability, an increase in ubiquitinated aggregates, and a heightened level of apoptosis among primary motor neurons cultured in vitro [6, 7]. ALS follows a progressive course, signifying that the symptoms deteriorate as time passes. As respiratory muscles weaken gradually, respiratory failure approaches, and this marks the impending inevitability of death. Typically, the majority of patients succumb to the disease within three years from the onset. Roughly 10% of patients manage to extend their survival beyond eight years [8]. The discovery of TAR DNA-binding protein 43 (TDP-43) positive ubiquitinated cytoplasmic inclusions is a significant finding observed in nearly all ALS patients and more than half of individuals with fronto-temporal dementia (FTD). This finding has positioned ALS within the "ALS-FTD continuum," emphasizing the substantial overlap in clinical presentation, pathophysiology, and neuroimaging between these two neurodegenerative disorders [9].
Etiology of ALS
The etiology is completely unknown. It is suggested that it arises from a combination of multiple factors, with both genetic predisposition and environmental influences contributing to its development [10]. The "multi-hit" hypothesis offers insight into why ALS presents with varying onset, progression, and treatment outcomes. Although substantial research has been conducted, a definitive cause remains unidentified, emphasizing the importance of further studies on the interaction between genetic factors and environmental triggers [11]. Based on genetic factors, ALS is classified into two forms.
Familiar Form
This type is an inherited form of the disease, most commonly following an autosomal dominant pattern. Although autosomal recessive and X-linked types exist, they are relatively rare. A primary risk factor for familial ALS is a positive family history, with its onset primarily driven by underlying genetic mutations [12]. More than 50 gene mutations have been linked to ALS, with the most extensively studied involving SOD1, TARDBP (encoding TDP-43, an RNA-binding protein), FUS, and C9ORF72. Familial ALS (fALS) is associated with the description of over 20 genes. Nevertheless, these gene products exhibit substantial diversity in their functions, rendering the quest for the origins of ALS complex [13-15]. These genetic alterations collectively account for approximately 75% of familial ALS cases. Among them, C9ORF72 mutations are the most prevalent, found in 45–50% of familial instances [16]. About 20% of familial ALS cases and around 1–2% of sporadic cases are associated with SOD1 gene mutation [17]. The genetic factor contributing to ALS is the presence of expanded hexanucleotide repeats (GGGGCC) within the non-coding region of the C9ORF72 gene.
Sporadic Form
Sporadic ALS, which represents approximately 90% of cases, develops without a family history and emerges unpredictably. Due to the absence of clearly defined triggers, it is thought to arise from a multifactorial process involving environmental exposures acting on underlying genetic, immune, and neural susceptibilities. Genetic alterations alone do not initiate the disease, instead they indicate a vulnerability that, when combined with external factors, contributes to the development of this severe neurodegenerative condition [18, 19]. Mutations in the FUS gene are identified in roughly 3% of familial ALS cases and about 1% of sporadic ones [20]. This particular neuropathological variant is marked by basophilic cytoplasmic inclusions within neurons of the motor cortex and anterior horn of the spinal cord [21]. Exposure to harmful chemicals found in substances like pesticides, heavy metals, and cigarette smoke has been suggested as a contributing factor in neuronal injury and degeneration. Some studies also point to the role of electromagnetic radiation in promoting motor neuron death. Another hypothesis involves viral infections, such as those caused by human endogenous retrovirus K (HERV-K), which is regulated by the TDP-43 protein and has been associated with cellular toxicity [19, 22].
SOD1 in Normal Physiology and ALS
The primary function of SOD1 is to act as an antioxidant enzyme, safeguarding the cell against the toxicity of reactive oxygen species. It was over two decades ago when SOD1 was first identified as the initial gene associated with the development of ALS, a neurodegenerative disease [23]. Mutations in the SOD1 gene lead to ALS, constituting roughly 20% of familial cases. A distinctive pathological characteristic involves the depletion of motor neurons coupled with an increased presence of intracellular misfolded SOD1. Mutation of the this gene found in ALS interrupts the cellular detoxification and leads to free radical toxicity and cell death [24]. The SOD1 protein, is a highly conserved gene found in the nucleus, cytoplasm, and the mitochondria's membrane. Comprising five exons, it encodes a 153-amino acid metalloenzyme that binds to copper and zinc ions, creating a dismutase enzyme responsible for eliminating free radicals in cells and converting them into oxygen and hydrogen peroxide [25]. The SOD1 gene is situated on chromosome 21 and encodes the enzyme known as Cu, Zn superoxide dismutase. The typical role of the SOD1 protein involves the removal of reactive oxygen species within the cellular cytosol and mitochondria, providing neuroprotective effects [23, 26]. The predominant mutations linked to ALS in SOD1 typically involve alterations in the protein's structure, particularly affecting specific amino acid positions including A4V (16), G93A (17), L84F [27, 28]. The A4V mutation involves the substitution of alanine with valine at codon 4 in exon 1 and is linked to more aggressive forms of ALS, accounting for approximately 50% of reported SOD1 mutation cases in North America [29]. G93A is a well-examined genetic mutation of the SOD1 protein, involving the replacement of glycine with alanine at codon 93, resulting in a change in its conformation. This mutation is responsible for approximately 20% of familial ALS cases [30]. The L84F mutation involves the alteration of the amino acid at position 84, replacing leucine with phenylalanine. It is linked to a milder form of ALS compared to mutations like G93A. This mutation can result in both protein instability and misfolding, potentially leading to the formation of protein accumulations [28] According to a study conducted by the Chinese Pharmaceutical Association in 2023. Oxidative stress plays a significant role in ALS pathology, leading to various observed markers in patients, including glutamate excitotoxicity, dysfunction at multiple levels such as mitochondria due to calcium influx, and axon, as well as protein oxidation [31]. These changes have been observed in SOD1-G93A mice. Numerous animal studies have demonstrated that the presence of the SOD1-G93A mutation alone is sufficient to induce motor neuron degeneration [32, 33]. The enzyme binds both copper and zinc ions, which play a direct role in neutralizing harmful superoxide radicals. Mutations in the SOD1 gene can lead to both gain-of-function and loss-of-function alterations [34].
Significance of Oxidative Stress and Mitochondrial Impairment
Oxidative stress is considered a key initiating factor in the progression of ALS. It arises when the production of free radicals surpasses the ability of motor neurons to neutralize them through antioxidant defences. This imbalance leads to cellular damage, including disruption of motor neuron membrane integrity through lipid peroxidation, impaired mitochondrial function, modifications in protein and DNA handling, and excitotoxic effects, ultimately culminating in neuronal death [35]. At the intracellular level, the mitochondrial respiratory chain is the primary source of free radical generation. Consequently, any disruption in mitochondrial function plays a significant role in the underlying mechanisms of ALS. Damage to mitochondria intensifies free radical formation and lipid peroxidation, leading to membrane destabilization, reduced ATP production, compromised DNA repair processes, and a further decline in mitochondrial efficiency [36]. Alterations in RNA-binding proteins like TDP-43 and FUS have been suggested to contribute to mitochondrial dysfunction, which, in turn, enhances free radical accumulation and sustains elevated levels of oxidative stress [37]. A few preliminary studies have suggested melatonin as a potential therapeutic agent for addressing oxidative stress and excitotoxicity in ALS. Its antioxidant properties have been evaluated in both SOD1-mutant mouse models and patients with sporadic ALS. In mice, melatonin treatment resulted in up to a 25% reduction in disease severity and extended survival duration following symptom onset, compared to untreated controls [16]. The electron transport chain includes five protein complexes that facilitate electron transfer to oxygen, resulting in the generation of reactive oxygen species (ROS) like hydrogen peroxide (H?O?), superoxide anions (O??), and hydroxyl radicals (HO?) [38]. Mitochondrial Complex I (NADH: ubiquinone oxidoreductase) facilitates the transfer of electrons from NADH to coenzyme Q (ubiquinone). Additionally, Complex II (succinate dehydrogenase) contributes electrons to ubiquinone. The reduced form of ubiquinone then passes these electrons to Complex III (cytochrome bc1), which subsequently transfers them to cytochrome C. Complex IV (cytochrome C oxidase) enables the final electron transfer to molecular oxygen, leading to the formation of water. Among these, Complexes I, II, and III are especially prone to premature electron leakage, which significantly contributes to the generation of ROS
Therapeutic approaches for ALS
ALS is a highly complex neurodegenerative disease characterized by the involvement of multiple pathways, proteins, and cellular damages contributing to disease progression [39]. Despite over three decades of research on ALS, presently only few drug, riluzole has remained in clinical use since its approval. The therapeutic approaches for ALS are shown in Figure1.
Figure 1: Different Therapeutic Approaches in ALS
Targeting SOD1
ALS is a highly complex neurodegenerative disease characterized by the involvement of multiple pathways, proteins, and cellular damages contributing to disease progression [39]. Despite over three decades of research on ALS, presently only few drug, riluzole has remained in clinical use since its approval. Riluzole functions primarily as an antioxidant, helping to lower levels of reactive oxygen species [39, 40]. However, it is considered a palliative option, as it offers only a modest extension of survival, typically by a few months [41]. Riluzole functions through a multimodal mechanism, primarily by reducing glutamatergic neurotransmission, blocking voltage-gated sodium channels, and exerting neuroprotective and antioxidant effects [42]. Rasagiline has demonstrated neuroprotective effects in Parkinson’s disease models, and studies using SOD1 Gly93Ala mouse models have shown that treatment with rasagiline, either alone or in combination with riluzole, results in increased survival in the mice [43, 44]. It is an irreversible and selective inhibitor of monoamine oxidase B (MAO-B), with additional neuroprotective, antioxidant, and anti-apoptotic properties that operate independently of its MAO inhibition [45, 46]. Ozanezumab is a monoclonal antibody developed for the treatment of ALS. In studies using SOD1 Gly93Ala mouse models, treatment with this drug led to improved skeletal muscle strength, a noticeable delay in symptom onset, and extended survival. However, phase II clinical trials in ALS patients did not show any significant difference between those receiving ozanezumab and those given a placebo [47]. It targets Neurite Outgrowth Inhibitor (NOGO-A), a protein known to suppress neuronal growth. This protein is found at elevated levels in patients with ALS [48]. Edaravone (Radicava) did not demonstrate highly significant results, it was approved in 2014 as a "disease-modifying drug" for ALS treatment in United States and Japan [39, 49, 50]. It is a drug that aims to slow down the loss of motor function and the progression of ALS, ultimately, aiming to decrease the mortality rate from ALS as well [51] Tofersen (Qualsody) recently approved by FDA as a treatment for patients with familial ALS (fALS) who carry mutations in the SOD1 gene [52]. It binds both mutant SOD1 and SOD1 WT mRNA, but it has shown serious adverse effects [53]. Moreover, a major challenge in creating effective ALS therapies lies in the limited availability of therapeutic biomarkers and the difficulty in delivering drugs to the brain and spinal cord [46, 54].
Gene Therapy
Gene therapy in ALS primarily aims to replace faulty genes, deliver protective or therapeutic genetic material, or inhibit the expression of genes that play a role in the onset and progression of the disease. Gene therapy is viewed as a promising approach, as it enables targeted intervention on specific genes associated with the disease [55, 56].
Adeno?Associated Virus
These vectors are employed to deliver genetic material to the central nervous system. One of their key advantages is their ability to transduce fully differentiated cells and form stable nuclear episomes without the risk of insertional mutagenesis. However, studies involving adeno-associated viruses (AAV) targeting SOD1 in animal models like mice and monkeys have yielded limited success [46]
Adeno?Associated Virus?Mediated Gene Delivery
It is based on the delivery of therapeutic transgenes using AAV. The aim is to decrease the production of certain key neurodevelopmental factors and neurotrophins, including insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), glial cell line-derived neurotrophic factor (GDNF), neuromuscular junction protein DOK7, the cytosolic chaperone, hepatocyte growth factor (HGF) and macrophage migration inhibitory factor (MIF) [46, 57].
RNA Interference
It is a well-studied strategy for reducing the damage caused by the toxic gain of function of SOD1 In mouse models carrying the Gly93Ala mutation, treatment led to a 39% increase in survival. However, the effectiveness of the therapy was found to be age-dependent, with a noticeable decline in efficacy as the mice aged. Clinical trials involving two patients with SOD1-associated familial ALS (fALS) treated with iRNA demonstrated a reduction in SOD1 levels; however, this outcome was accompanied by significant adverse effects [57]
Antisense Oligonucleotides
Antisense oligonucleotide (ASO) therapy has gained increasing attention in the treatment of SOD1-associated familial ALS (fALS). This approach works by targeting and degrading specific cytoplasmic and nuclear mRNA, thereby reducing the production of the corresponding protein [57]. Studies involving mouse models with the SOD1 Gly93Ala mutation treated with various types of ASOs, delivered through different administration routes such as continuous infusion into the lateral ventricle or lumbar spine demonstrated substantial ASO distribution throughout the brain, along with strong tissue penetration [58]. Furthermore, studies using mouse models with the same SOD1 mutation, treated before the onset of symptoms, showed a considerable decrease in SOD1 mRNA and protein levels in the brain and dorsal spinal cord [59].
Other Treatments
A recent review highlights pharmacological and genetic strategies targeting copper homeostasis as potential interventions in SOD1-associated ALS [60]. Among these interventions, the copper complex diacetylbis [N(4)-methylthiosemicarbazonato] copper (II) [CuII (atsm)] has shown notable effects in ALS mouse models by delaying disease onset and prolonging survival when given orally. Additionally, treatment with CuII (atsm) has been shown to enhance the activity of mutant SOD1 in ALS-model mice with SOD1 overexpression [61-63]. The CuII (atsm) complex is known for its high membrane permeability, allowing it to efficiently cross the blood-brain barrier [64]. Indeed CuII(atsm) is in continuation study for participants from an earlier trial (NCT04313166).
CONCLUSIONS AND FUTURE PERSPECTIVES
ALS is a multifactorial neurodegenerative disorder characterized by progressive motor neuron loss and limited therapeutic options. Advances in understanding the disease’s etiology have underscored the critical role of superoxide dismutase 1 (SOD1), both in its physiological function and its pathological involvement through toxic gain-of-function mutations. The resulting oxidative stress, mitochondrial dysfunction, and cellular degeneration form key targets for therapeutic intervention. Current treatment approaches targeting SOD1, including small molecules, antisense oligonucleotides, and gene-silencing technologies, have demonstrated promise in preclinical and early clinical studies. Although FDA-approved therapies like riluzole and edaravone offer only modest clinical benefits, ongoing research is steadily expanding the therapeutic landscape. Gene therapy has also emerged as a transformative strategy, offering the potential for long-term modulation of disease-associated genes. Despite some progress, continued research into the molecular mechanisms of ALS and the development of targeted, multi-pathway treatments may pave the way toward improved disease management. Although ALS remains a formidable clinical challenge, the growing understanding of its underlying mechanisms offers renewed hope for more effective, targeted, and patient centered therapeutic approaches in the near future.
Abbreviations
ALS: Amyotrophic lateral sclerosis
OS: Oxidative stress
SOD1: Superoxide dismutase 1
ROS: Reactive Oxygen Species
UMNs: Upper Motor Neurons
LMN: Lower Motor Neurons
TDP-43: TAR DNA-binding protein 43
FTD: Fronto-temporal Dementia
VEGF: Vascular Endothelial Growth Factor
HGF: Hepatocyte Growth Factor
REFERENCES
Mohd Umar Sofi*, Nahida Tabassum, Role of SOD1 on the Etiology, Pathophysiology and Therapeutic approaches for Amyotrophic Lateral Sclerosis, Int. J. of Pharm. Sci., 2025, Vol 3, Issue 7, 2478-2488. https://doi.org/10.5281/zenodo.16072698
10.5281/zenodo.16072698
