What is the clinical spectrum of Amyotrophic Lateral Sclerosis?
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a progressive and lethal disorder that causes motor neuron degeneration, resulting in muscle weakness, paralysis, weight loss, and respiratory failure.
While ALS can be both of sporadic (SALS) and of familial (FALS) origin, over 90% of ALS cases are sporadic. The clinical presentation of ALS is heterogeneous, which is believed to be the result of interaction of multiple risk and contributing factors. Although most core ALS symptoms are shared between SALS and FALS, differences between the two disease forms were observed in age of onset, symptom variability, and progression rates.
The hallmark clinical feature of ALS is the coexistence of symptoms triggered by degeneration of upper motor neurons (UMN) in the cerebral cortex, and lower motor neurons (LMN) in the anterior horn of the spinal cord and the brainstem. In limb-onset ALS, LMN lesions are associated with muscle atrophy and flaccid paralysis, while UMN lesions present with hyperreflexia, poor dexterity, incoordination, and spastic paralysis. In the less frequently occurring bulbar onset ALS, the most common symptoms are dysarthria and dysphagia. Respiratory-onset ALS begins with progressive weakness of the diaphragm, leading to shortness of breath during exertion and orthopnea.
Up to half of ALS patients develop varying degrees of cognitive impairment and of change in behaviour. Patients with ALS who receive standard medications and supportive care typically survive an average of 3–5 years after symptom onset.
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What do we know about the etiology of Amyotrophic Lateral Sclerosis?
Based on epidemiological and clinical human data, ALS is a multifactorial disease involving a complex interplay of genetic, epigenetic, environmental and lifestyle triggering factors Although FALS is primarily driven by highly penetrant rare genetic mutations, environmental factors may still influence the age of onset, rate of progression and severity of symptoms. In SALS, there is no clear familial inheritance and environmental triggers appear to be more prominent.
Environmental stressors are thought to act through overlapping molecular pathways that involve damage to mitochondrial electron transport, excessive production of reactive oxygen species (ROS), endoplasmic reticulum (ER) damage, unfolded protein response (UPR) and apoptosis.
Over 120 genes have so far been implicated in ALS. While about 70% of FALS and 15% of SALS cases have mutations in known ALS genes, including SOD1, FUS, TARDBP, C9ORF72, and ATXN2, the distinction between the two disease forms is not clear-cut. For instance, mutations in superoxide dismutase 1 (SOD1), that regulates oxidative stress, lipid metabolism, and inflammation are associated with approximately 10%-20% of FALS and 1% of SALS cases. TARDBP gene, that encodes the transactive response DNA-binding protein 43 (TDP-43), that regulates various steps of RNA metabolism, including mRNA splicing, RNA transportation, translation, and miRNA biogenesis, is mutated in approx. 5% of FALS and 1-2% of SALS cases.
Identified ALS-associated genetic mutations are believed to exert their pathogenic effects through a multitude of pathways that combine loss-of-function and toxic gain-of-function mechanisms. For example, loss of TDP-43 nuclear function can occur with mutations (TARDBP, FUS) and/or under the influence of environmental stressors (oxidative stress, ER stress, inflammation), causing mislocalization of TDP-43 to the cytoplasm and thereby resulting in impaired splicing, cryptic exon inclusion and RNA instability. Among toxic gain-of-function mechanisms that drive ALS is the misfolded protein response-mediated apoptosis that can be caused by mutations that alter the protein structure (SOD1, TDP-43, FUS, C9ORF72) and/or through environmental stressors.
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How similar are human and animal neuromuscular systems?
Below are some of the examples of species-specific differences that are likely to have a negative effect on the face, construct, and predictive validity of animal models of ALS.
Species-specific differences in corticomotoneuronal system anatomy and physiology
The human corticomotoneuronal system has multiple human-specific features that play a key role in susceptibility to ALS and that cannot be recapitulated in animal models of ALS. For instance, in human and non-human primates the corticomotoneuronal (CM) system encompasses direct monosynaptic connections from cortical UMN to LMN enabling fractionated and precise movements and other fine motor behaviours. Unlike primates, rodents lack direct CM connections between UMN and LMN.
Species-specific differences in genetics and gene expression
Not all ALS-associated genes have orthologs in model organisms that have the same ALS-related function, and even when they do, their expression consistently differs across species, impacting the validity of the disease phenotype, relevance of insights on disease mechanisms, and applicability of therapeutic targets in model organisms. For example, TDP-43 dysfunction in ALS leads to misregulation of splicing, causing the inclusion of cryptic exons in mature mRNA transcripts of genes coding for STMN2 and UNC13A proteins, which supports disease progression. However, due to species-specific differences in splicing regulation, the cryptic exon inclusion event in STMN2 and UNC13A is not conserved in mice and appears to be human-specific.
Species-specific differences in immune system
Immune-mediated motoneuron degeneration in ALS involves both innate and adaptive immunity. The innate immune response manifests by microglia activation and complement system dysregulation, while the adaptive immune response includes CD4/CD8 T cell infiltration and production of autoantibodies by B cells. Both types of immune responses differ across species, playing a part in overall poor predictive validity of ALS animal models.
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Face validity - How well do animal models replicate the human disease phenotype?
Owing in great part to human-specific genomic and physiological features, animal models of ALS have severe drawbacks, including absence of several key pathophysiological and clinical features of ALS.
Genetically-induced ALS animal models - Identification in patient populations of genes associated with ALS had prompted development of rodent, zebrafish, pig, non-human primate and other animal species models that carry mutations found in ALS patients. Knock-in mouse models of ALS carrying orthologous mutations in ALS associated genes often develop much milder phenotypes than patients with ALS carrying the same mutations. ALS mouse models that overexpress the transgene carrying FALS/SALS-associated mutations tend to show stronger phenotypes but may introduce features that are not ALS-specific.
The most widely used mouse model of ALS, the transgenic SOD1 G93A mouse, reproduces neuroinflammation, motor neuron degeneration in the spinal cord, muscle weakness, and limb paralysis, but without motor neuron degeneration in the cerebral cortex. This is particularly problematic since cortical motor neuron degeneration is the fundamental feature of ALS and the basis for ALS diagnosis. Additionally, in sharp contrast to SALS patients, TDP-43 mislocalization and inclusion formation were absent in the spinal motor neurons of SOD1 G93A mice, which may play a part in the lack of clinical efficacy of drugs shown to be effective in SOD1 mice.
Chemically-induced ALS animal models - For example, injection of aluminium was employed in rodents to induce neurotoxicity leading to ALS-like neuronal damage. However, this approach did not allow to recapitulate the degeneration of both upper and lower motor neurons. Exposure of mice to β-Sitosterol-β-d-glucoside through diet showed degeneration of spinal motor neurons and persistent motor deficits. However, there was insufficient evidence of degeneration of upper motor neurons, and cytoplasmic aggregation or mislocalization of TDP-43 was not reported in these studies.
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Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
The impossibility of mirroring the complex combination of genetic, epigenetic, lifestyle and environmental factors on a patient-specific genetic background makes obtaining robust construct validity in ALS animal models virtually impossible.
Genetically-induced ALS animal models - Since knock-in ALS mouse models carrying human ALS-associated mutations showed much milder motor deficit phenotypes than ALS patients, homozygous constructs were employed to amplify disease features. However, in ALS patients most mutations are heterozygous and homozygous cases are rare. In certain cases, such as in knock-in C9orf72 mice without G4C2 repeats, the model does not replicate an ALS-relevant genomic construct. In addition, knock-in mice do not contain the human-specific regulatory elements of the human gene locus nor the patient-specific genetic background.
Overexpression mouse models are not representative of human-specific gene expression regulation and genetic background. In addition, the overexpressed transgene carrying mutations identified in ALS patients does not replace or modify the mouse locus. Therefore it does not replicate haploinsufficiency – loss of function, but toxic gain of function only.
Specifically, the original SOD1-G93A mouse model (B6SJL-Tg(SOD1*G93A)1Gur) typically carries 20–25 copies of the human SOD1 gene with G93A mutation under the control of its native human promoter and regulatory regions, resulting in high overexpression of mutant SOD1 protein. In contrast to the SOD1-G93A mouse, humans carry only one copy of the mutant gene. Mice carrying such a disproportionally high number of human SOD1 transgenes may suffer from a phenotype that is so overdriven that no therapy outside of the direct inhibition of SOD1 will ever affect ALS-related survival.
Chemically-induced ALS animal models - Certain experimental induction methods employ chemicals for which no direct causal link with ALS was established. For instance, ALS patients do not routinely present with aluminum toxicity and exposure to bisphenol A has not been associated with ALS. Dietary exposure of mice to β-Sitosterol-β-d-glucoside does not incorporate the genetic susceptibility to ALS, and is not aligned with the multi-hit hypothesis.
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Predictive validity - How well do animal models predict safety and efficacy of therapies in patients?
Just as it is easier to repair something when you know exactly how you have broken it, many therapy candidates for ALS have successfully reversed disease symptoms in experimentally-induced animal models of ALS - only to fail in ALS patients. Indeed, over the past 30 years more than 70 molecules investigated as possible treatments for ALS have failed to demonstrate effectiveness in clinical trials. The overwhelming majority of treatments that have obtained green light to proceed to clinical trials were validated in SOD1 G93A and TDP-43 mice.
Despite extensive research, as of 2025, only 2 treatments for ALS, riluzole and edaravone, were approved in the EU and the US. These treatments enable modest symptomatic relief or slowing of disease progression but do not reverse the disease course. It is also noteworthy that development and clinical trials of riluzol predate the creation of SOD1-G93A mouse. This underscores the value of mechanistic reasoning instead of dependence on animal modelling.
To date, effective treatments for ALS remain a significant unmet need, with substantial therapeutic improvement desperately awaited. The absence of models that faithfully recapitulate the ALS phenotype and pathophysiology continues to represent a major hurdle in clinical development. A shift to advanced human-based methods is needed to better understand ALS mechanisms, identify new therapeutic targets and reliably test efficacy of candidate treatments.
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Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical - Animal research is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment, and life purpose. In addition to this baseline, experiments inflict severe clinical harm in animals.
Clinical - Statistics consistently show that clinical success rates of drugs developed and tested in animals is very low, raising the question of whether it is ethical to put the health of patients at risk.
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Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Animal models of ALS are not representative of the heterogeneity in ALS etiology, genotype, clinical features, onset and progression patterns. This mismatch contributes to poor clinical translation, wasted resources and delayed progress.
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Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
In spite of significant investment in dissemination, various incentives and training of animal researchers, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines remain poorly implemented and the majority of animal experiments irreproducible. While in vitro methods are not immune to issues of reproducibility, the moral weight of irreproducible animal studies is not the same.
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Key takeaways
Amyotrophic lateral sclerosis (ALS) is a progressive and lethal neurodegenerative disorder, leading to muscle weakness and respiratory failure. As a result of a complex interplay of genetic, epigenetic, environmental and lifestyle factors, the clinical presentation of ALS is heterogeneous.
Owing in great part to inter-species differences in corticomotoneuronal system anatomy and physiology, genetics and immune system, animal models do not recapitulate some of the fundamental pathophysiological and clinical features of ALS. The most commonly used animal models of ALS are genetically-engineered mice, which do not take account of human causative and modifier factors. Reliance on modelling ALS in vivo causes severe suffering in animals, but also in ALS patients, since animal research has not translated to significant therapeutic benefits.
Human-based methods are needed to better understand the complex and heterogenous mechanisms of ALS, identify new therapeutic targets, developer safer and more effective treatments, and bring to market personalized solutions for ALS patients.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Amyotrophic Lateral Sclerosis
This section explores emerging ways in which human-based in vitro technologies can be used to advance scientific research on ALS. A list of over 15 examples outlines how complex human-based in vitro methods are leveraged to advance research on ALS while also offering ideas for expanding these approaches. These examples include the use of patient-derived 3D motoneuron tissues, organoids, bioprinted constructs and organ-on-a-chip systems to model heterogenous FALS and SALS features, segment ALS pathophysiology according to molecular subtypes, study the effects of environmental and lifestyle contributors to ALS, identify the mechanisms underlying SALS and discover broadly effective therapeutic targets to treat multiple forms of ALS.
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