Amyotrophic lateral sclerosis
ICD-10 Code G12.21
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 1, 2.
While ALS can be both of sporadic (SALS) and of familial (FALS) origin, over 90% of ALS cases are sporadic. The global prevalence of this disease is estimated to be 3-9 cases per 100 000 people, depending on the region.
The clinical presentation of ALS is heterogeneous, which is believed to be the result of interaction of multiple genetic, lifestyle and environmental factors 3, 8, 9, 10. 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. In comparison with FALS, SALS appears to be more variable in symptoms and rates of progression. FALS has an earlier, 40-50 years of age at onset, while in SALS first symptoms typically appear later in adulthood, with an onset at 55 to 65 years of age in average.
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 4. In limb-onset ALS, LMN lesions are associated with muscle atrophy and flaccid paralysis (loss of muscle tone, muscle weakness, reduced reflexes), while UMN lesions present with hyperreflexia, poor dexterity, incoordination, and spastic paralysis (muscle spasms, involuntary jerky movements). In the less frequently occurring bulbar onset ALS, the most common symptoms are dysarthria (slurred speech) and dysphagia (difficulty swallowing). Respiratory-onset ALS, which occurs in around 5% of ALS cases, begins with progressive weakness of the diaphragm controlled by LMN, leading to shortness of breath during exertion and orthopnea (breathing difficulty when lying down).
In progressive muscular atrophy, most patients initially present with asymmetric LMN lesion symptoms that progress to UMN lesion symptoms. Patients with primary lateral sclerosis initially present with UMN lesion symptoms and the disease usually progresses to LMN lesion symptoms. Symptoms and their rates of progression vary according to the disease type. Patients with primary lateral sclerosis typically have a slower disease progression and do not show weight loss.
Cell types within affected regions include Betz cells in the primary motor cortex in UMN, anterior horn cells of the spinal cord in LMN, and lower cranial motor nuclei of the brainstem in LMN. Small eosinophilic intraneuronal inclusions, termed Bunina bodies, are common in degenerating neurons. The abnormal accumulation of TDP-43 in the motor cortex and spinal cord is present in up to 97% of cases of SALS, as well as in cases of FALS with TARDBP mutation 5, establishing TDP-43 inclusions as a major signature of ALS 6.
Up to half of ALS patients develop varying degrees of cognitive impairment and of change in behaviour. Notably, up to 75% of ALS patients that carry C9ORF72 mutation experience cognitive impairment, with up to 45% meeting criteria for frontotemporal dementia (FTD) 7. While the variability in survival time is high, individuals with ALS usually die of respiratory failure within 3 to 5 years in average following the onset of symptoms.
What do we know about the etiology of Amyotrophic Lateral Sclerosis?
The current knowledge on the causes and pathogenesis of ALS are mainly derived from human clinical, epidemiological, genetic and post-mortem data, as well as from animal modeling data. However, given the fact that ALS is a uniquely human disorder and given the multitude of below described inter-specific differences, it is unclear which, if any, ALS animal models enable human-relevant insights.
Based on epidemiological and clinical human data, ALS is a multifactorial disease involving a complex interplay of genetic, epigenetic, environmental and lifestyle triggering factors 2, 8. Although familial ALS (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 (heavy metals, pesticides, cyanotoxins, physical trauma) appear to be more prominent. The "multi-hit" hypothesis proposes that SALS arises from a combination of genetic vulnerability and environmental stressors, which would explain why only some individuals exposed to environmental toxins develop ALS.
The precise etiology of ALS is, nevertheless, not fully understood and is believed to involve multiple mechanisms, including genetic mutations, oxidative stress, excitotoxicity, defective RNA processing, intraneuronal protein aggregates, mitochondrial dysfunction, and neuroinflammation.
Environmental stressors are though 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. Age-related decline in protein autophagy and in DNA repair, that would have allowed to mitigate the ALS pathophysiology, are a factor of vulnerability, explaining the onset of ALS at an advanced age. The existence of distinct spatial and molecular patterns in ALS patient tissues supports this idea of multiple pathogenic pathways, with inter-individual variations in genetic background being a contributing factor 9.
SALS represents the overwhelming majority of ALS cases and only about 10–15% of ALS cases are considered as FALS - inherited in either an autosomal dominant, autosomal recessive, or X-linked mode. Over 120 genes have so far been implicated in ALS and this number keeps growing as new data from patient-population Genome-Wide Association Study (GWAS) and Whole Exome Sequencing (WES) keeps coming in. 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 10, 11, 12.
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 2, 10.
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 2, 10.
To date, more than 50 different mutations in fused in sarcoma (FUS) gene that encodes a multifunctional RNA binding protein involved in transcription, alternative splicing, mRNA transport, mRNA stability, and miRNA biogenesis, have been identified in patients with ALS, which together account for approx. 4% of FALS and fewer than 2% of SALS 2, 10.
Chromosome 9 open Reading frame 72 hexanucleotide G4C2 repeat expansions in the noncoding region of C9ORF72, involved in autosomal trafficking, immune regulation, and autophagy, are found in about 39% of FALS and 7% of SALS patients of European ancestry 2, 10.
Screening of a large ALS cohort demonstrated that 14% of FALS and 2.6% of SALS cases had more than one potential pathogenic mutation in a known ALS gene, and these cases had a significantly earlier onset of disease 10 FALS and SALS have a genetic architecture in which a few rare variants contribute to risk in each patient, rather than a polygenic architecture whereby the cumulative effects of many common variants increase risk 10, 11. In certain SALS cases, oligogenic combinations of several rare variants were observed, contributing collectively to disease onset and progression. Identified ALS-associated genetic mutations are thought to exert their pathogenic effects through a multitude of pathways that combine loss-of-function and toxic gain-of-function mechanisms 10, 13.
Among toxic gain-of-function mechanisms that drive ALS is misfolded protein response-mediated apoptosis. In this scenario, the motor neuron death is believed to be driven by a converging cascade of oxidative stress and persistent ER stress. ALS-associated genetic mutations that alter the protein structure, such as in mutant SOD1 and TDP-43, can produce protein misfolding and aggregation in the ER, activating the UPR. Persistent ER stress and UPR can increase ROS via oxidative folding and other enzymes (ERO1 and CHOP pathways). Progressively rising ROS levels intensify ER stress by damaging key components of its protein folding machinery, and disrupt the ER membrane through lipid peroxidation, adding to accumulation of unfolded proteins and activation of the UPR. If overwhelmed, the UPR machinery can trigger a shift from an adaptive to an apoptotic sequence of mechanisms (CHOP and JNK pathways) that also disrupt mitochondrial function and amplify ROS-induced oxidative stress.
Beyond SOD1 and TDP-43 mutations, several other ALS-associated mutations can contribute to protein misfolding, disruption of protein homeostasis and intensification of UPR by directly producing misfolded proteins, disrupting protein clearance systems or generating toxic proteins. For instance, FUS mutations cause cytoplasmic mislocalisation and aggregation that can activate UPR pathways. The C9ORF72 hexanucleotide repeat expansions produce toxic dipeptide repeat proteins (DPR) that interfere with RNA splicing, mitochondrial dysfunction, and nucleocytoplasmic transport of transcriptions factors, mRNA and other molecules, instigating ER stress, cellular dysfunction and motor neuron death.
Misfolded proteins act as damage-associated molecular patterns (DAMP) that activate microglia, the resident immune cells unique to the central nervous system, that clear misfolded proteins, apoptotic neurons and other cell debris 14. DAMP bind to microglia’s pattern recognition receptors (toll-like receptors, NOD-like receptors), triggering the NF-κB pathway and microglial release of cytokines IL-1β, TNF-α, and IL-6 that lock microglia into a M1 pro-inflammatory phenotype that sustains neuroinflammation. Cytokines signals that help microglia shift to a M2 reparative state are downregulated in ALS. In response to DAMPs, the complement cascade becomes aberrantly activated in ALS, leading to C3 and C1q marking of motor neurons and synapses for phagocytosis by microglia, participating in neuronal loss 15.
In ALS, tight junction proteins (ZO-1, occludin, claudin-5) are downregulated, leading to vascular leakage 16. Blood-spinal cord barrier disruption allows infiltration of CD4/CD8 T cells that produce IFN-γ and IL-4, further exacerbating the shifting of microglia to proinflammatory M1 state and astrocytes to A1 neurotoxic state that accelerate neuronal death 17, 18. Neuroinflammation triggers gliosis as a protective mechanism, in which proliferating astrocytes and microglia attempt to prevent further injury by forming a fibrotic barrier composed of extracellular matrix (ECM) proteins. However, as ALS progresses, chronic neuroinflammation entertains a chronic gliosis, in which over-reactive astrocytes form a dense glial scar that ultimately prevents tissue regeneration 19, 20.
Often linked to environmental or genetic factors, glutamate excitotoxicity is widely thought to be another major driver of ALS. Transcranial magnetic stimulation assessment of the functional integrity of the motor cortex and its corticomotoneuronal projections had shown cortical hyperexcitability both in patients with SALS and FALS 21. Possible causes of excitotoxicity include dysregulation of glutamate clearance by EAAT2 transporters, leading to accumulation of excitatory glutamate in the synaptic cleft, or change in AMPA and NMDA receptors, leading to sustained Ca2+ influx into post-synaptic neurons and subsequent mitochondrial dysfunction, oxidative stress, and apoptosis 22.
How similar are human and animal neuromuscular systems?
This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, and predictive validity of animal models.
Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance for amyotrophic lateral sclerosis (ALS) is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.
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 in humans and that cannot be recapitulated in animal models of ALS.
In human and non-human primates (NHP), the corticomotoneuronal (CM) system encompasses direct monosynaptic connections from cortical UMN, in the primary motor cortex, to LMN, in the spinal cord and brainstem, enabling fractionated and precise movements and other fine motor behaviours. These skilled behaviours, like speech articulation and complex gestures, are particularly developed in humans and cannot be recapitulated in rodent models 23. Unlike primates, rodents lack direct CM connections between UMN and LMN. Instead, their corticospinal neurons primarily influence movement indirectly through interneurons. The fact that humans have longer axons, and higher axonal transport of molecules, organelles, and proteins, also makes them uniquely vulnerable to mitochondrial dysfunction-reduced energy supply. The increased dependency on direct UMN-LMN connectivity in humans, makes dual UMN-LMN degeneration even more devastating, likely accounting for a more severe distal motor deficits in ALS patients than in ALS animal models 24.
Betz cells, a specialized subtype of L5 pyramidal excitatory neurons found in the UMN’s primary motor cortex, play a critical role in corticospinal motor control. Human Betz cells are larger in diameter, more numerous per hemisphere and have a more extensive dendritic arborization than their NHP or mouse counterparts 25. Larger Betz cells generate stronger action potentials and innervate multiple LMN, which amplifies both motor output and vulnerability to ALS-related excitotoxicity in humans. In Betz cells of ALS patients, TDP-43 accumulate more compared to mice, accelerating UMN loss 26.
Similarly to Betz cell, a specific subclass of excitatory neurons called Von Economo neurons, primarily located in the frontoinsular and anterior cingulate cortex, also show transcriptional dysregulation in ALS state, including in stress response, synaptic vesicle cycling, and autophagy 27. Von Economo neurons, believed to be linked to higher order cognitive and emotional functions, are present in humans but absent in many of commonly used model organisms, including mice, rats, rabbits, cats, and dogs 28.
Species-specific differences in genetics and gene expression
In humans, ALS risk is driven by rare highly penetrant genetic variants rather than common polygenic factors 10, 13. 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, while C9ORF72 gene has a murine ortholog, its repeat expansion mutation does not naturally occur in mice 29.
Comparative single-cell transcriptomics analysis of human and mouse cerebral cortex shows that despite basic transcriptomic similarities of cell types, there are numerous inter-species differences at the single-gene and gross-structural level. The most-divergent gene families include neurotransmitter receptors and ion channels 28, which play a part in glutamate excitotoxicity, cortical hyperexcitability and impaired inhibitory control associated with ALS.
Alternatively spliced exons help drive the molecular diversity, differentiation, and function of brain cell types 30. And yet, only about a quarter of alternatively spliced exons for a given transcript is conserved between humans and rodents 31, indicating inter-species differences is spliceosome machinery (small nuclear ribonucleoproteins, splicing factors, RNA-binding proteins).
Species-specific differences in protein-coding gene sequence, alternative splicing and post-transcriptional modification sites (phosphorylation, ubiquitination) can engender inter-species discrepancies in aggregation dynamics of SOD1, FUS and TDP-43 proteins, significantly influencing their loss of function and toxic gain of function.
Long noncoding RNA (lncRNA) can regulate gene expression in several manners, including by direct binding to pre-mRNA and interaction with transcription factors. Analysis of ALS patient-derived samples showed that expression of numerous lncRNA is dysregulated, sparking interest in lncRNA as disease progression biomarkers and therapeutic targets 32. Of crucial importance for the success of these therapeutic avenues, genome, transcriptome, and evolutionary analysis suggest that the same lncRNA mechanisms that have rewired gene expression in a human-specific manner, including in susceptibility to neurodegenerative diseases like ALS, are not likely to be found in model organisms 33.
In agreement with these findings, differential expression analysis between human and mouse UMN and LMN motoneurons, which degenerate in ALS, has confirmed human-specific enrichment in statistically significant ALS-associated genes 27, 34.
Species-specific differences in alternative splicing
There are ALS-associated genes that show variable mRNA splicing patterns across species, leading to species-specific protein diversity and function 35. One such example is inter-species variation in RNA-binding specificity of the TDP-43 protein, due to differences in TDP43 protein sequence, TDP43-binding RNA motifs, and cellular context 36. Inter-species differences in RNA binding motifs can arise as a result of variations across species in RNA sequence, in RNA secondary structure, and in RNA subcellular localisations. Implications for study of ALS are inter-specific differences in phenotype of experimental animal models of ALS, in disease mechanisms and in relevance of therapeutic targets 37.
Cryptic exons are silent intronic sequences that can be aberrantly included in mature mRNA in case of splicing errors or regulatory dysfunction, leading to mRNA degradation or reading frame alteration. Since TDP-43 protein regulates pre-mRNA splicing, TDP-43 dysfunction in ALS leads to misregulation of splicing, causing the inclusion of cryptic exons in mature mRNA transcripts derived from genes like STMN2 and UNC13A 38. The presence of cryptic exons in mature transcripts results in reduced levels of functional STMN2 and UNC13A proteins that are important for microtubule dynamics, axonal repair and synaptic communication, accelerating disease progression 39, 40. These splicing defects represent promising therapeutic targets for ALS that could potentially reverse neurodegeneration by restoring normal splicing. 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 38, 41. In ALS mouse models, the loss of STMN2 or UNC13A does not occur upon TDP-43 loss, contributing to inter-species differences in ALS phenotype and blocking opportunities to develop therapeutic strategies for splicing restoration in ALS patients.
Species-specific differences in neuromuscular vascular physiology
ALS patients often present with pathological alterations in their small cerebral blood vessels, which has detrimental effects on the integrity of the blood brain barrier (BBB), blood–spinal cord barrier (BSCB), and blood–cerebrospinal fluid barrier (BCSFB) 42. Endothelial cells, astrocytes and pericytes are essential for production of tight junction proteins, regulation of tight junction integrity, and stabilization of tight junctions. Decreased transcription of tight junction proteins that are essential for the integrity of BSCB was observed in spinal cords from ALS patients 43.
Brain vascular and perivascular cell types (endothelial cells, pericytes, smooth muscle cells) exhibit divergent transcriptional profiles between humans and mice, conferring human-specific perturbations across the brain vasculature that do not overlap with mouse models of human neurodegenerative diseases 44. The majority of genes that have been linked to risk of Alzheimer’s disease by GWAS are expressed in the human brain vasculature and are associated with endothelial protein transport, adaptive immunity, and ECM pathways. The current knowledge of disease mechanisms suggests that these human-specific pathways share commonalities with ALS pathophysiology, notably in neuroinflammation.
Brain pericytes (also known as Rouget cells), wrap around and communicate with the endothelial cells of capillaries and small blood vessels in the CNS, thus supporting the integrity of the blood brain/spinal cord barrier. Their loss or dysfunction is believed to contribute to ALS. Comparison of mouse and human pericytes transcriptomes showed that 206 orthologous genes were consistently differentially expressed between human and mouse pericytes, of which 91 genes were up-regulated in human and 115 in mouse pericytes 45. Genes that were expressed in human but not in mouse brain pericytes, such as MAGI2, DLG2, GPC5, and HTR1F, may indirectly contribute to ALS pathology through BSCB dysfunction, excitotoxicity, and neuroinflammation.
Vascular cells in the adult human brain exhibit well-established zonation - specialized gene expression and functional roles - of vascular cells along the arterial-capillary-venous axis. In contrast, adult mouse brain vascular zonation differs significantly in gene expression, reflecting species-specific vascular physiology 46. Owing to species-specific protective zonation genes, animal models of ALS may underrepresent the impact of BSCB dysfunction, neuroinflammation, and gliosis.
Species-specific differences in brain cells morphology, composition and function
In ALS, microglia and astrocytes are directly involved in pathological neuroinflammation and gliosis 14, 15, 17, 18. Astrocytes play many important roles in the brain, including supply in nutrients, removal of waste, mechanical support to neurons, signaling to endothelial cells, regulation of neurogenesis, and repair of injury. In human neocortex, astrocytes are 2.6-fold larger in diameter and extend 10-fold more glial fibrillary acidic protein-positive primary processes than in rodents. Several anatomically defined subclasses of astrocytes are not represented in rodents 47. Inter-species differences in morphology also extend to non-human primates, since in humans, the subclass of interlaminar astrocytes is more abundant in comparison to chimpanzee. Human-specific astrocytic complexity is likely to be involved in the increased functional competence of the human brain and increased vulnerability to neurodegenerative disorders such as ALS.
Microglia, the resident immune cells of the CNS, are particularly under scrutiny for their role in human neurodegenerative 48 and psychiatric disorders 49. Comparison of single-cell transcriptomics across ten species revealed a larger heterogeneity in human microglia transcripts 50. Species-specific gene expression pathways in microglia are associated with complement system, phagocytosis, and metabolic pathways, all of which play a crucial role in ALS progression.
Species-specific differences in neuromuscular junction molecular and cellular architecture
The human neuromuscular junction (NMJ) is composed of presynaptic motor neurons, postsynaptic muscle fibers, and glial cells, that engage in crosstalk to facilitate signal transmission from motor neurons to muscle fibers.
Super-resolution imaging, and proteomic profiling showed that human NMJ were significantly smaller, less complex, more fragmented, and more stable across the entire adult lifespan than mouse NMJ 51. In comparison to other mammalian species, human NMJ possess a distinctive distribution of active zone proteins and differential expression of core synaptic proteins and molecular pathways 52. These inter-species divergences in NMJ are likely to play a part in susceptibility to motoneurons degeneration in humans and suggest that mechanisms that underly motoneuron dysfunction in animal models of ALS may be different in ALS patients.
Species-specific differences in neurotransmission physiology
The neurotransmission physiology varies across species, likely due to both baseline inter-species differences and ALS-state inter-species differences in glutamate release, clearance, and uptake.
Since research into the brain physiology is typically executed in model organisms and since findings from animal studies are not systematically compared to findings from human studies, the exact extent of inter-species differences in neurotransmitter physiology remains unknown. Importantly, therapies that focus on modulating neurotransmission physiology have massively failed to translate into improved patient care, showing that the use of animals in preclinical research has resulted in a lack of understanding of the human brain physiology 53.
The following highlights some of the known human-specific features of post-synaptic glutamate dynamics in normal and ALS state, as well as plausible inter-species differences that are likely to have led to failure of EAAAT2 modulators, AMPA receptor antagonists, and NMDA receptor antagonists in clinical trials.
In humans, glutamate is the primary excitatory neurotransmitter in the CNS, playing a crucial role in synaptic connections between UMN and LMN. According to the "dying forward" hypothesis in ALS, hyperexcitable UMN drive excitotoxic damage to LMN 54. Owing to previously-mentioned human-specific direct CM connections and greater connectivity, the function spread of glutamate released from presynaptic neurons is amplified and the baseline excitability in human motoneurons is higher compared to model organisms 55. In ALS state, a variety of possible mechanisms (C9ORF72 mutations, SOD1 mutations, reduced GABAergic inhibition) are suspected to drive an increased glutamate release into synaptic cleft. Excessive glutamate release in combination with human-specific corticomotoneuronal features, makes humans more susceptible to downstream excitotoxicity than excessive glutamate release alone in SOD1 G93A mice.
In normal state, the EAAT2 glutamate transporter is highly expressed in astrocytes, clearing 90% of synaptic glutamate. In human ALS, EAAT2 expression is significantly reduced, through a variety of possible mechanisms (misfolded proteins, oxidative stress, neuroinflammation) 56. Based on studies in the SOD1-G93A mouse model of ALS, ceftriaxone was hypothesized to reduce glutamate excitotoxicity by upregulating EAAT2 expression. While ceftriaxone administered to SOD1-G93A mice slowed the disease course, preserved strength, and prolonged survival in mice 57, it did not significantly increase the survival time or significantly decrease the rate of decline in function in ALS patients 58. Judging from this discrepancy in outcomes between preclinical and clinical trials for ceftriaxone, it is possible that the reduction in EEAT2 expression was milder in mice than in ALS patients, which combined with other inter-species-differences, could have resulted in less severe excitotoxicity, leading to inflated drug efficacy expectations.
Postsynaptic ionotropic glutamate AMPA receptors are tetramers that assemble in various combinations of subunits. The presence of the GluR2 subunit renders in the AMPA receptor impermeable to Ca2+ and its absence enables receptor permeability to Ca2+. In normal state, a low-level Ca2+ influx via AMPA receptors supports mitochondrial function and sufficient ATP production, however, in disease state the excessive influx of Ca2+ leads to mitochondrial dysfunction, ROS production, ER stress, and apoptosis. In ALS state, the upregulation of GluR2-lacking AMPA receptors and the downregulation of GluR2 editing, enhance Ca2+ influx and increases the risk of downstream excitotoxicity 59. The AMPA receptor antagonist Talampanel failed to reduce ALS-related functional deterioration in clinical trials 60, despite improvement of motor function in SOD1-G93A mice 61. This mismatch in therapeutic outcomes could be explained by a higher baseline level of GluR2-containing AMPA receptors and/or fewer Ca2+-permeable AMPA receptors in mice which, combined with other inter-species-differences, may reduce Ca2+ influx, leading to a less severe and more therapeutically manageable excitotoxicity in ALS mice compared to ALS patients.
Postsynaptic NMDA receptors require both glutamate and depolarization to activate. GluN2B-rich NMDA receptors, that have higher Ca2+ permeability, add to the risk of excitotoxicity in humans 62. While treatment with the NMDA receptor antagonist Memantine significantly delayed disease progression in SOD1-G93A mice 63, it failed to slow ALS progression in patients 64. This disappointing outcome could be explained by a higher baseline prevalence of GluN2B-rich NMDA receptors in humans compare to mice which, in eventual combination with other inter-species-differences, may produce a more severe and more rapidly progressive motoneuron degeneration in ALS patients than in mouse models.
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 14, 15, 17, 18. Both types of immune responses differ across species, playing a part in overall poor predictive validity of ALS animal models.
Extensive differences between humans and mice were demonstrated in the structure of innate and adaptive immunity, including in balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, the NK inhibitory receptor families Ly49 and KIR, FcR, Ig subsets, the B cell (BLNK, Btk, and λ5) and T cell (ZAP70 and common γ-chain) signaling pathway components, Thy-1, γδ T cells, cytokines and cytokine receptors, Th1/Th2 differentiation, costimulatory molecule expression and function, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression 65. In addition, it was shown that transcriptional responses to acute inflammatory stresses of different etiologies in mouse models do not correlate with human acute inflammatory diseases 66. Major species-specific differences were also found in transcriptional regulation, chromatin state and higher order chromatin organization of immune system related genes, with cis-regulatory sequences showing the most divergence 67.
In confirmation of these findings, out of 7 therapeutics with primarily anti-inflammatory mechanism of action that demonstrated efficacy in SOD1 rodent models of ALS, none were approved in clinical trials for ALS 68.
It was suggested that using animal models with a humanized immune system might improve translatability to humans, however, such an approach would face persistent, insurmountable challenges: the role of the human immune system in ALS is complex and not fully understood, the equivalence of humanized animals to the human immune system was not demonstrated by objective measures 69, and the cross-talk between the human immune system and the rest of human organ systems cannot be recapitulated in animals.
Inter-specific differences in functional assessment
The ALS Functional Rating Scale-Revised (ALSFRS-R) is a validated rating instrument designed to assess the functional status and monitor the progression of disability in patients with ALS 70, 71. It typically assesses human functions like speech, salivation, swallowing, cutting food, handwriting, dressing, walking, hygiene, turning in bed and respiratory function. However, there is no equivalent of ALSFRS-R for animal models. Instead, researchers often use various behavioural and functional tests such as rotarod test, grip strength test, hindlimb extension reflex, and gait analysis. Such tests in animal models may not translate to function in ALS patients and can be difficult to analyse due to species-specific behaviours.
For instance, the rotarod test is one of the most commonly used tests to measure motor coordination in mice. Studies of inbred strains, selected lines, and transgenic animals have shown that rotarod performance in mice is highly influenced by the genetic background. In addition, there is little consensus on ideal parameters and test schedules to produce optimal results, despite wide use of rotarod tests in biomedical research. The variability in size of rod diameters, rotation rate, training regimens, experimenter effects etc. contribute to poor reproducibility and reliability of the test 72.
There are no standardized methods in animal neurobehavioral genetics, that would have allowed to understand how genetic variations influence behaviour, emotion, cognition and motor function in animals. Instead, most tests are done in a manner that is unique to each laboratory, making it difficult to compare findings and arrive to conclusions about the effects of ALS treatments in animals 73. Even if these assessment methods were harmonized and uniformly applied, they would still fall short of translating to human-specific behaviour, emotion, cognition and motor function.
Face validity - How well do animal models replicate the human disease phenotype?
Owing to human-specific genomic and physiological features, animal models of amyotrophic lateral sclerosis (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 74, 75, 76, 77. Knock-in mouse models of ALS carrying orthologous mutations in ALS associated genes (SOD1, TDP43, FUS) often develop much milder phenotypes than patients with ALS carrying the same mutations 78, likely due to species-specific differences described above. 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.
SOD1
Over a dozen SOD1 transgenic rodent models have been developed by overexpressing missense or truncated human ALS-associated gene variants, such as G93A, D83G, D85G, D86G, D90A, and G37R. The most widely used mouse model of ALS, the SOD1 G93A mouse, carries a point mutation in the SOD1 gene at position 93 that leads to toxic gain-of-function.
The SOD1 G93A mouse reproduces neuroinflammation, motor neuron degeneration in the spinal cord, muscle weakness, and limb paralysis. However, while in ALS patients microglia, astrocytes, and oligodendrocytes play a key role in neuroinflammation and gliosis, comprehensive study of glia in the cortex of the SOD1 G93A mouse, using single-cell RNA sequencing at four stages of the disease, showed minimal changes throughout the disease progression 79, indicating that the complexities of human neuroinflammation and gliosis are not recapitulated in SOD1 G93A mice.
While in SOD1 G93A mice, gait abnormalities, hindlimb tremors, and hindlimb weakness mimic human limb-onset, the progression is much faster than in humans, suggesting that early and intermediary features that precede overt symptoms, such as subclinical motor neuron loss, subtle coordination difficulties, fatigue, and impaired reflexes are not captured in this model, complicating identification of therapeutic targets to prevent ALS progression.
A further key limitation of the overwhelming majority of transgenic mouse models, including the SOD1 G93A mouse, is the absence of motor neuron degeneration in the cerebral cortex 76, 79. This is particularly problematic since cortical motor neuron degeneration is the fundamental feature of ALS and the basis for ALS diagnosis. Although the SOD1 D83G transgenic mouse shows some motor neuron degeneration in this brain region, it does not recapitulate the clinical paralytic phenotype 74. A subsequent repercussion of the limited cortical involvement in SOD1 mice is that cognitive impairments, observed in some SOD1-related human ALS cases, were not observed in these mouse models.
Despite existence of numerous features of blood-spinal cord barrier disruption (endothelial cell degeneration, capillary leakage, perivascular edema, downregulation of tight junction proteins, microhemorrhages), several other key features (pericyte degeneration, perivascular collagen IV expansion, white matter capillary abnormalities) were absent in SOD1 animal models 80, making it difficult to understand the role of vascular remodeling, cortical and subcortical vascular dysfunction, and fibrotic processes in ALS.
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 efficacy in SALS patients of drugs shown to be effective in SOD1 mouse models 5, 81.
TARDBP
On the basis of identified FALS-associated variants of TARDBP gene encoding TDP-43 protein, over 20 corresponding TDP-43 mouse models have been generated. Large animal models, such as transgenic SOD1 G93A/TDP-43 M337V pigs and TDP-43-overexpressing cynomolgus monkeys, were less frequently used as experimental models because of inefficiency of gene targeting in large animals, long reproductive cycles, complex maintenance, and high associated cost 74.
TDP-43 mice have shown inconsistent phenotypes depending on the genetic induction methods. In many cases it is not clear whether the genetically-induced phenotype in mice is related to the mutation itself or to overexpression of the mutated gene. Crucially, most TDP-43 mutant mice did not recapitulate the critical cytoplasmic mis-localization of TDP-43 protein, raising concerns about the validity of these ALS mouse models 74, 76.
For instance, the TDP-43 M337V mutation in the TARDBP gene has been identified in human FALS cases, where it is found in heterozygous state. However, knock-in mice carrying TDP-43 M337V mutation did not exhibit motor dysfunction and neurodegeneration, even in homozygous state 82.
Transgenic mouse models that overexpress exogenous human TDP-43 around twice the level of endogenous tdp-43, using Thy1.2 promoters that drive gene expression specifically in neuronal tissues, displayed accumulation of pathological aggregates of ubiquitinated proteins in specific neuronal populations, gliosis, varying degrees of spinal cord pathology, progressive paralysis and death. In contrast, the TDP-43 Q331K transgenic mice, that overexpresses human mutant TDP-43 Q331K up to 1.5 fold of endogenous TDP-43 mouse levels, displayed a limited period of progressive motor dysfunction and its ALS-like features did not result in death 75, 83. Another Thy1.2-TDP-43 line, with 2 to 5 fold overexpression of endogenous tdp-43, developed early onset tremor and abnormal hindlimb reflexes, with TDP-43 negative ubiquitinated cytoplasmic inclusions. Although a moderate loss of large caliber motor axons and abnormal neuromuscular junction morphology was reported in this mouse model, there was no loss of cortical and spinal cord motor neurons nor subsequent death. The TDP-43 A315T overexpression mouse model under mouse prion promoter control did not recapitulate neuromuscular deficits but instead showed gastrointestinal dysfunction, presumably due to site-specific overexpression of human TDP-43, highlighting the impact of promoter type and expression patterns on disease phenotype 84.
ALS patients who experience cognitive impairment also exhibit motor degeneration. However, the motor degeneration, which is the defining feature of human ALS, is not recapitulated in TDP-43 Q331Klow and G348C mutant mice. Instead, TDP-43 G348C mice developed learning/memory deficits but without overt motor neuron degeneration and without paralysis 85.
Designed to artificially induce mislocalization of TDP-43 from the nucleus to the cytoplasm, the rNLS8 mouse model showed extensive aggregation of TDP-43 in the neuronal cytoplasm, resulting in progressive brain and spinal cord motor neuron degeneration 86. In this doxycycline-modulable rNLS8 double transgenic construct, re-introduction of doxycycline to the diet reversed TDP-43 pathology and rescued motor impairment in rNLS8 mice. Rapid disease onset and progression in this mouse model does not allow to study early stages of ALS, limiting opportunity to explore ALS disease mechanisms and identify therapeutic targets.
C9ORF72
In C9ORF72-associated ALS, patients typically carry 30 to 1,600 repeats of the G4C2 hexanucleotide motif. In contrast to ALS patients, transgenic mice that carry approx. 500 and 1000 repeats of the G4C2 motif in the C9ORF72 gene did not show neurodegeneration, raising questions about the relevance of this mouse model for investigating human ALS 74.
To study the loss of function effects of C9orf72 protein Lopez-Herdioza et al. 87 generated miR-RNAi anti-C9orf72 mice to knock down the expression of the C9orf72 gene. Mild motor phenotypes and frontotemporal dementia-like behavioural changes were observed in this mouse model, however, without pronounced motor neuron degeneration.
In the knock-in C9orf72 mouse model 88, 400 codon sequences encoding poly-GR and poly-PR dipeptide repeat protein species were inserted into the C9orf72 locus under the control of the endogenous mouse promoter. The resulting mice showed mild spinal motor neuron loss but without cortical neuronal loss. While hyperexcitability was reported in superficial cortical layers in poly-GR mice, neuroinflammation, TDP-43 mislocalization and sustained motor deficits were absent.
Chemically-induced ALS animal models
Several chemical agents were used in the last decades to induce neurotoxicity, yet without succeeding in fully recapitulating ALS-like symptoms. For example, injection of aluminium was employed in rodents to induce neurotoxicity leading to ALS-like neuronal damage 77. However, this approach did not allow to recapitulate the degeneration of both upper and lower motor neurons.
The use of zebrafish as a model for ALS has a number of limitations, not least of which is the absence of upper motor neurons. The bisphenol A-induced motoneuron degeneration in zebrafish does not cause motoneuron-specific degeneration since other types of neurons also undergo cell death 75.
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 75.
The lack of mouse models that accurately replicate human-relevant TDP-43 proteinopathy, along with the inter-individual variability in ALS pathology, has recently prompted calls for urgent development of new animal models 89, 90. However, these calls fail to acknowledge the extensive evidence of above described species-specific differences. Given these inter-species discrepancies, there is no compelling evidence that newly proposed animal models would better reflect ALS than the currently inadequate ones. Moreover, the suggestion to combine multiple animal species with in vitro approaches introduces further complexity, making cross-species comparisons even more problematic, leading to further waste of time and resources and unnecessary risk for ALS patients.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
Incomplete recapitulation of amyotrophic lateral sclerosis (ALS) symptoms in animal models, and repeated clinical failures of drug candidates for ALS, challenge our understanding of its disease mechanisms.
Patient-derived data indicates that known rare genetic variants associated with both SALS and FALS may act in concert with modifier genes and modifying environmental factors. In SALS, environmental stressors are believed to function as triggers on a background of either single rare genetic variants or oligogenic combinations of multiple rare variants 8, 9, 10. Therefore, to truly recapitulate the complexity of ALS etiology in vivo, animal models would need to reflect the multi-variant, multi-hit and patient-specific nature of the 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.
Instead, most ALS animal models rarely go beyond single-gene transgenics on a C57BL/6 mouse genetic background that differs wildly from the patient-specific genetic background. Because of technical complexity, time and cost, only a handful out of several dozen pathogenic variants linked to FALS and SALS were used in overexpression and KI mouse models.
Researchers that rely on mouse experiments for insights on human diseases often argue that the mouse genome is similar to the human genome. This statement is, however, inaccurate and misleading.
Even though over 90% of human protein-coding genes have a counterpart in mice, these gene orthologs are known to diverge in structure and function across species 108, 109, 110. Additionally, the remaining up to 10% human-specific protein-coding genes are likely to play a crucial role in the human disease. While human and mouse protein coding genes may have 85% of DNA sequence in common, this is only on average – meaning that some genes may be highly conserved (up to 99%) while others may diverge significantly (as low as 60%) 111. Even if a given human protein-coding gene shared 99% of nucleotide sequence with its mouse ortholog, a single nucleotide difference could result in an amino acid substitution that may dramatically alter the protein’s structure and function 112.
Furthermore, protein-coding DNA represents just 1-2% of the human genome. 98-99% of DNA is composed of non-protein-coding elements, a number of which play a vital role in gene expression regulation 91. Notably, about 90% of disease-associated variants identified by GWAS reside in non-coding regions of the genome 92. The exact role of human non-protein-coding DNA has historically been understudied and remains yet to be fully elucidated in a human-based system.
It is important not to underestimate these inter-species differences in protein-coding genes and non-coding genetic elements, as these can lead to divergent loss of function and toxic gain of function effects, producing misleading hypotheses on disease mechanisms and repeated clinical trial failures 27, 33, 34, 41, 93.
To further complicate matters, the commonly used experimental induction methods introduce artefacts that further compromise the already poor construct validity of ALS animal models.
Genetically-induced ALS animal models
Knock-in ALS mouse models
In knock-in mouse models, the human ALS-associated mutation (TDP-43, FUS, SOD1 or other) is typically introduced into the endogenous locus of the mouse ortholog, under the control of the native mouse promoter and regulatory elements. Since knock-in ALS mouse models showed much milder motor deficit phenotypes than ALS patients, homozygous constructs were employed to amplify disease features 78. However, in ALS patients most mutations are heterozygous and homozygous cases are rare.
In ALS patients with G4C2 repeat expansions in the noncoding region of C9ORF72, the G4C2 is transcribed in both sense and antisense directions, producing toxic RNA foci and dipeptide repeat proteins (DPR). Out of the 5 distinct DPR translated through repeat-associated non-ATG (RAN) translation, poly-GR and poly-PR are particularly neurotoxic. In the knock-in C9orf72 mouse model, 400 codon sequences encoding poly-GR and poly-PR were inserted into the C9orf72 locus under the control of the endogenous mouse promoter in one allele of the mouse C9orf72 gene 88. This model mimics both the loss of function effect of C9orf72 disruption and the gain of function effect of poly-GR and poly-PR at physiologically-relevant levels comparable to those found in ALS patients. However, since G4C2 repeats are absent in this construct, this model does not replicate the mechanisms tied to toxic RNA foci and RAN translation intermediates.
In addition, human-specific regulatory elements of the human gene locus are not present in this model. Patient-specific genetic background containing modifier genes and eventual oligogenic combinations of multiple rare variants are also missing. The mouse orthologues of genes involved in human ALS may have a different structure and function, affecting their aggregation dynamics.
Overexpression ALS mouse models
In overexpression/transgenic ALS mice, the human transgene, that typically carries ALS-associated mutations expressed under the control of a strong non-native promoter (PrP, Thy1, CMV) driving high expression of the mutant gene, is often randomly integrated into the mouse genome 74, 77, 94. Overexpression mouse models are not representative of human-specific gene expression regulation and genetic background. In addition, random transgene integration into the mouse genome can disrupt endogenous mouse genes, alter expression patterns and produce variability in phenotype.
The fact that mutant genes usually need to be highly overexpressed in order for the animal to develop an ALS-like phenotype within its lifespan raises doubts as to the relevance for FALS and SALS patients of disease-triggering mechanisms employed in animals.
It is also noteworthy that the overexpressed transgene carrying mutations identified in ALS patients (TDP-43, FUS, SOD1, C9ORF72 etc.) does not replace or modify the mouse TDP-43, FUS, SOD1, or C9ORF72 locus. Therefore it does not replicate haploinsufficiency – loss of function, but toxic gain of function only.
Certain transgenic models, such as the rNLS8 mouse, do not represent mutations found in ALS patients 86. Designed to artificially reproduce the mislocalization of TDP-43 from the nucleus to the cytoplasm, the rNLS8 mouse carries a double transgenic - tetO-hTDP-43ΔNLS and NEFH-tTA - system in which overexpression of human TDP-43 with a mutated nuclear localization sequence (h-TDP-43 ΔNLS), is induced by doxycycline-modulated tTA synthetic transcription factor.
Moreover, in overexpression ALS models, excessive levels of the mutated protein are likely to produce artefacts (protein mislocalization, ER stress) that are due to a general overwhelming of cellular capacities (protein folding, autophagy) rather than to the pathogenic properties of the mutated protein.
The original SOD1-G93A mouse model (B6SJL-Tg(SOD1*G93A)1Gur), created by pronuclear injection of the human SOD1-G93A transgene that inserts randomly into the mouse genome, typically carry 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 74, 77, 95. Considered as the cornerstone of ALS animal research, the original high-copy SOD1-G93A transgenic mouse model has been used in the overwhelming majority of preclinical trials that supported IND applications for ALS therapies. However, since only about 10%-20% of FALS and 1% of SALS cases 2, 10 involve SOD1 mutations, the SOD1-G93A mouse model is not representative of the mechanisms that underly ALS in the majority of cases.
Moreover, 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. In confirmation of that concern, treatments by antibiotics minocycline and ceftriaxone, tested in SOD1-G93A mice, did not provide a survival benefit for ALS patients, suggesting that the SOD1-G93A mouse may be more susceptible to infections and other non-ALS related illnesses, and that it is this illness, rather than ALS that is alleviated by tested treatments 58, 97.
In addition to transgene overexpression, other confounding factors that can directly impact the disease phenotype in SOD1-G93A mice include the propensity of transgenic mice carrying mutant human SOD1 to spontaneously delete the copy number, disruption of endogenous mouse genes via random insertion of transgene into mouse genome, and the mouse strain unique genetic makeup.
The most widely used C9ORF72-G4C2 mouse models were created using BAC transgenesis, allowing to insert the pathogenic G4C2 hexanucleotide repeat expansion randomly into the mouse genome, together with its native human promoter and non-coding regulatory regions 74, 77, 96. Multiple copies of the G4C2 transgene may integrate during BAC transgenesis, resulting in overexpression of G4C2-transcribed toxic RNA foci and DPR. This C9ORF72-G4C2 mouse model can thus model gain of function toxicity only. By contrast, in ALS patients the G4C2 hexanucleotide repeat is located in the endogenous C9ORF72 gene and transcribed at normal level. ALS patients experience both loss of function (through G4C2 disruption of the endogenous C9ORF72 gene) and gain of function (normal transcription of G4C2 into toxic RNA/DPR).
Knock-down ALS mouse models
To the study the effects of loss of function of C9orf72 protein, Lopez-Herdioza et al. 87 used anti-C9orf72 RNA interference via microRNA to reduce its endogenous expression. Since this model does not recapitulate epigenetic silencing of C9orf72 by G4C2 repeat expansions and the G4C2-transcribed toxic RNA/DPR, it is not representative of combined loss- and gain-of-function effects in ALS patients. In addition, this model falls short of recapitulating human-specific spatiotemporal regulation of human C9ORF72 gene expression and lacks the patient-specific genetic background (modifier genes, combination of rare variants).
Chemically-induced ALS animal models
Certain experimental induction methods employ chemicals for which no direct causal link with ALS was established 77. For instance, ALS patients do not routinely present with aluminum toxicity and exposure to bisphenol A has not been associated with ALS. The mechanisms that underly neuronal damage in these chemically-induced ALS models are therefore likely to diverge from the disease mechanisms in human ALS.
Dietary exposure of mice to β-Sitosterol-β-d-glucoside (BSSG) is compatible with situation encountered by certain individuals with ALS 75, nonetheless, since this model does not incorporate the genetic susceptibility to ALS, it is not aligned with the multi-hit hypothesis which proposes that ALS arises from a combination of genetic vulnerability and environmental stressors.
The previously described species-specific differences will remain a significant barrier to understanding ALS pathophysiology and successfully translating preclinical findings, regardless of the degree of creativity by which animal modeling adjusts knock-in, overexpression, or other constructs to better match ALS.
The widespread belief that, despite of their shortcomings, distinct ALS mouse models can always be a source of insights into individual aspects of ALS pathophysiology, was repeatedly discredited in clinical trials, at the expense of ALS patients.
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 amyotrophic lateral sclerosis (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 68, 76. The range of mechanistic avenues explored include glutamatergic excitotoxicity, neuroinflammation, oxidative stress, and neuroprotection. 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. Sanofi's riluzol (Rilutek), approved by the FDA in 1995, is believed to modulate neuronal excitability and neurotransmission in ALS 68. Originally developed for treating neuronal excitability in conditions like epilepsy and anxiety disorder, riluzol did not obtain regulatory approval for these indications despite positive results in preclinical models. 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 modeling. First developed in the 80's to treat stroke, Mitsubishi Tanabe’s synthetic antioxidant edaravone (Radicava) helps preserve motor neurons by reducing oxidative stress 75. Real-world data suggests that riluzol and edaravone extend ALS patient survival by 6 months in average 98, 99.
The histone deacetylase inhibitor, sodium phenylbutyrate, was approved by the FDA in 1996 to treat urea cycle disorders. In 2022, the FDA approved the combination of sodium phenylbutyrate and taurursodiol (Relyvrio) for treatment of ALS. Relyvrio appeared to slow down neurodegeneration by reducing mitochondrial dysfunction and ER stress 2. However, this drug was pulled off the market in 2024, after failing to meet both primary and secondary endpoints in Phase 3 PHOENIX trial 100.
In 2023, the FDA granted accelerated approval for Biogen’s and Ionis’ antisense oligonucleotide (ASO) tofersen (Qalsody) that targets SOD1 mRNA 101, 102. Accelerated approval was based on the surrogate biomarker plasma neurofilament light chain (NfL), estimated to be reasonably predictive of a clinical benefit. Nevertheless, since NfL measures neuronal degeneration and not functional improvement, the long-term efficacy of tofersen remains to be demonstrated in further patient studies.
Biogen’s and Ionis’ ASO BIIB105, designed to reduce expression of the ATXN2 gene, did not demonstrate an improvement in motor function and respiratory function and was therefore discontinued in 2024 103. Two years earlier, it was Biogen’s and Ionis’ ASO BIIB078, targeting the C9orf72 mRNA, that was discontinued for the same reason. Earlier in 2024, Sanofi's receptor-interacting protein kinase 1 (RIPK1) inhibitor SAR443820 for ALS was discontinued after failing to show significant improvement in ALSFRS-R scores compared to placebo 104.
The example of Corcept's dazucorilant illustrates once again how great promise in animal models of ALS is virtually meaningless for ALS patients. While this selective cortisol modulator improved motor performance and reduced muscular atrophy in SOD1-G93A mice, it failed to slow ALS progress and caused gastrointestinal upset in Phase 2 trials 105. Validated in the TDP-43 rNLS8 mouse model 106, Denali’s Initiation Factor 2B complex (eIF2B) activator DNL343 was discontinued in 2025 after it failed to significantly slow ALS progression or improve muscle strength, respiratory function, or survival in phase 2/3 HEALEY-ALS platform trial 107.
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.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Humans assume the right to conduct scientific experiments on animals, despite the fact that animals clearly express non-consent through their behaviour (fleeing, vocalizing, defecating, defense).
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 113:
Table S13: Severity classification of genetically altered (GA) lines
Neurology and sensory organs - Paresis and paralysis, GA lines resulting in severe impairment of animal’s motility and weight loss due to inability to reach food/water, including development of paralysis like SOD ALS: Severe
Loss of coordination and gait - Abnormal gait impairing ability to move and function normally causing difficulties in eating and drinking: Up to severe clinical signs
Pain - GA lines resulting in long-term moderate pain or short-term severe pain: Severe
Emotional state - GA lines resulting in long-term moderate anxiety or short term severe anxiety: Up to severe clinical signs
Mortality - GA lines resulting in lethality (significantly earlier than WT strain) from 2 weeks post-partum on: Severe
Table S4: Severity classification of clinical signs
Weight loss and cachexia: Severe
Respiration - Reduced rate at rest and when active. Irregular rhythm. Appears to require effort: Up to severe clinical signs. Respiratory failure: Severe
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance safe and effective treatments for human diseases. However, statistics consistently show that efficacy and safety of drugs developed and tested in animals for amyotrophic lateral sclerosis (ALS) are very poor, raising the question of whether it is ethical to put ALS patients’ health and life at risk. For instance, although minocycline and topiramate had shown promising results in ALS mouse models, these drugs have led to degradation of motor function and severe adverse effects in ALS patients 114, 115.
The 3Rs principle rests on the unfounded assumption that animal models possess robust face, construct and predictive validity. It also implies that animal cruelty is acceptable unless alternative methods are applicable. In practice in vitro methods are used alongside, rather than in place of, animal models. Switching to 100% human-based research enables biomedical science without animal suffering, and invites a shift to a conceptually separate framework that excludes animal use entirely.
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 3, 4, 5, 7, 8, 9, 74, 75, 76, 77. This mismatch contributes to poor clinical translation, wasted resources and delayed progress.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, animal research enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible 116. Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 117. Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented 118, 119. As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted 120.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims 121.
While in vitro methods are not immune to issues of reproducibility, the moral weight of irreproducible animal studies is not the same. Animals are not inanimate, disposable objects - they are sentient beings who suffer. Their value lies not in how they might serve human interests, but in their intrinsic worth as living, feeling individuals.
In Summary
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 to inter-species differences in corticomotoneuronal system anatomy and physiology, neuromuscular vascular physiology, brain cells morphology and function, neuromuscular junction architecture, neurotransmission physiology, gene expression 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. Modeling 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, to identify new therapeutic targets, to developer safer and more effective treatments, and to bring to market personalized solutions for ALS patients.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Amyotrophic Lateral Sclerosis
*To model heterogenous FALS and SALS features, by using FALS/SALS patient iPSC-derived/primary 3D motoneuron tissues, motoneuron organoids, or motoneuron-on-chip 122, 123.
*To segment heterogenous ALS pathophysiology based on molecular subtypes, by non-negative matrix factorization and deep neural net classifier analyses of gene expression signatures in ALS patient-derived spinal cord and cortex tissues 124.
*To determine the impact of rare genetic variants on ALS pathogenesis, by gene editing (base editing, overexpression, knock-out, etc.) and gene perturbation (CRISP interference, siRNA) in iPSC-derived/primary 3D motoneuron tissues, organoids or motoneuron-on-chip 125, 126.
*To identify the epigenetic drivers of motor neuron degeneration and to test potential epigenetic therapies for ALS, through epigenetic editing (CRISPR activation/repression, lncRNA) in healthy human/ALS patient-derived motoneuron tissues, organoids or motoneuron-on-chip.
*To study the genotype-phenotype relationships in ALS patient iPSC-derived 3D tissues, organoids or neuromuscular junction-on-chip systems containing patient-specific genetic backgrounds 127.
*To identify the mechanisms underlying SALS, by studying the: -epigenetic modification patterns and sites in peripheral blood DNA of identical twin pairs discordant for ALS 128, 129. -role of microglia-to-astrocyte signaling in neurofilament accumulation in tissues derived from a set of identical twins discordant for ALS 130. -mechanisms of cell-to-cell propagation of pathogenic TDP-43 protein in SALS patient-derived cerebral organoids or motoneuron-on-chip 131. -mechanisms of pathogenic alterations in autophagy pathways and cytokine secretion in SALS patient-derived cerebral organoids or motoneuron-on-chip 132.
*To explore the cell-autonomous and non-cell autonomous mechanisms of FALS/SALS C9orf72 mutation, by coculturing mutant astrocytes with wild-type motoneurons and measuring neuronal firing patterns, ion channel activity, and neuronal excitability in ALS patient iPSC-derived astrocytes and motoneurons 133.
*To study the effects of environmental and lifestyle contributors to ALS (heavy metals, pesticides, smoke) and to model inter-individual differences in ALS pathophysiology, by exposing the culture medium of healthy human 3D tissues, genetically edited for GWAS-identified variants, to nutrients and to toxicants, and measuring resulting inflammation, oxidative stress, gene expression change, mitochondrial membrane potential, and other endpoints.
*To study human-specific corticospinal connections and ALS-related degeneration, by employing calcium imaging, optogenetics, and glutamate caging in healthy human/ALS patient-derived brain-spinal assembloids or multi-organ-on-chips (cortical organoids fused with spinal motor neuron organoids).
*To model ALS neuroinflammation, to dissect cell-autonomous/non-cell-autonomous effects, to study mechanisms of microglia & astrocyte neurotoxicity, and to test neuroprotective effects of drugs, by employing 3D spinal microtissues that combine patient iPSC-derived motor neurons, microglia and astrocytes carrying C9orf72 mutation 134.
*To study the mechanisms of adaptive immune response to C9orf72/TDP-43/SOD1 antigen and to develop precision immunotherapies targeting regulatory T cells or specific antigen epitopes, by stimulating in vitro ALS patients-derived peripheral blood mononuclear cells with synthetized peptides, followed by T cell response phenotyping (surface markers, cytokine profiling, intracellular cytokine staining & flow cytometry) and epitope mapping 135.
*To investigate excitotoxicity, by combining glutamate caging (glutamate overload) with real-time calcium-sensitive fluorescent staining (excessive Ca influx), and cell viability assays (neurodegeneration) in healthy human/ALS patient-derived NMJ organoids, brain-spinal assembloids or (multi)organ-on-chip.
*To study alterations in human-specific TDP-43-regulated alternative splicing, and to develop treatments that restore splicing, by using ALS patients-derived iPSC/post-mortem tissues that retain human RNA-binding motifs, human TDP43 protein sequence and human cellular machinery 41, 136, 137, 138.
*To determine the contribution of each cell type to ALS, by engineering chimeric bioprinted tissues containing one or more diseased cell types in the context of a healthy background of other cell types - astrocytes, microglia, oligodendrocytes, endothelial cells, motor neurons - in healthy/ALS patient-derived 3D tissues.
*To study the human-specific perturbations of the brain vasculature (BBB, BSCB, BCSFB), by measuring multi-omics, electrophysiology function, calcium imaging, barrier permeability, cytokines and other endpoints in healthy/ALS patient iPSC-derived co-culture vascular-motoneurons assembloids or (multi)organs-on-chip. To study the dysregulation of neurovascular interactions in ALS, by comparing the transcriptomes of vascular cells (endothelial cells, pericytes, smooth muscle cells) to transcriptomes of astrocytes, oligodendrocytes, immune, muscle, and neuron cells in healthy and in ALS state.
*To identify new biological markers of ALS for an improved accuracy of diagnosis, monitoring of disease progression, and response to treatment, by correlating data from patient-derived in vitro models to patients’ distinct phenotypes.
*To conduct reverse translational research by comparing safety and efficacy of candidate drugs for ALS both in clinical trials and in vitro 139.
*To identify broadly effective therapeutic targets to treat multiple forms of ALS, by leveraging FALS and SALS patient-derived tissues, organoids and organs-on-chip 140, 141.
*To test safety and efficacy of single and combination therapies, by high-throughput screening in ALS patient-derived organoids or organs-on-chip 142, 143, 144.
Last Updated: September 2025
Even though in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. As the above examples show, the full extent of inter-species differences is unknown and new species-specific features are discovered every day, often only once the drug has failed to show safety and efficacy in patients. Complementing in vitro methods with animal experiments is not effective for human patients, because species-specific differences prevent reliable integration and translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the complete human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!
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