Amyotrophic Lateral Sclerosis (ALS), a Motor Neuron Disease (MND) subtype, is a debilitating neurodegenerative disorder affecting the upper and lower motor neurons (UMNs/LMNs), brain stem and spinal cord. This leads to progressive muscular weakness and atrophy, paralysis, and eventually death, usually within three to five years after the onset of symptoms. Decades of failed drug development mean that MND/ALS is still incurable; only two FDA-approved drugs exist (riluzole, and more recently, edaravone), but these only slow down disease progression.

A recent drive towards improving proof-of-principle drug screening assays suggests MND/ALS drug discovery is taking a turn for the better. This is in large part due to recent advances in stem cell biology and the development of motor neurons derived from human induced pluripotent stem cells (hiPSCs) . In vitro culture of these human iPSC-derived motor neurons (from both healthy and patient donors) now offer physiologically relevant models for in vitro assays due to their potential to replicate the altered neurophysiological pathways underpinning MND/ALS in humans, which until now had not been available.

Below we explore how past research efforts have led us up to this revolutionary turning point. We look at how traditional animal models expressing genetic mutations causing MND/ALS (e.g. mutations in SOD1 and C9orf72 genes) poorly recapitulate the neuronal pathophysiology of MND/ALS in humans, instigating doubts about their validity as drug screening assays. We also explore how this is spurring the development of advanced assays that incorporate physiologically relevant hiPSC-derived motor neurons, to enhance and accelerate MND/ALS drug discovery.

Studying Motor Neuron Disease: From mice to neurons

About 25 years ago, an important discovery identified SOD1 gene mutations in ALS patients . The subsequent development of the SOD1 mouse model (high copy number hSOD1) and its recapitulation of many aspects of human pathology (e.g., progressive decline in motor function, muscle wasting, and LMN loss) made it a staple tool for drug screening in research. Based on this model, only when a compound extended the lifespan of hSOD1 mice was it progressed to human clinical trials, otherwise it was thought to be ineffective and discarded as a candidate.

But as research progressed, it became apparent that this approach was flawed. SOD1 mutations are actually rare in ALS patients , and with >150 genes identified as causative or associated with ALS, the one-gene-one-mutation hSOD1 model could not represent many other underlying causes, leading to a high failure rate in clinical trials. New efforts to find other, more representative model systems included transgenic mouse models expressing other SOD1 mutations and mutations in other genes that were considered causative or associated with ALS (e.g., C9orf72, Alsin, prolifin and TDP43 mice).

But the key turning point was yet to come. Over the years, the research community realized that, instead of focusing on extending the lifespan of mouse models (which does not directly translate to lifespan extension in patients), the focus should be on studying vulnerable and diseased motor neurons at the cellular level. Extending the survival and health of diseased neurons, and using them as a cellular target for therapeutic intervention, has now become a key aim that could enhance MND/ALS translational research and drug discovery .

Studying the role of motor neuron health in Motor Neuron Diseases

It is now understood that during the early stages of ALS, there is significant corticospinal motor neuron or upper motor neuron (CSMN/UMN) degeneration, according to evidence corroborating the 'dying-forward' hypothesis and the 'independent degeneration' hypothesis . Numerous studies using advanced imaging technologies have revealed early cortical hyperexcitation before disease onset , suggesting a crucial role of cortical dysfunction. This has been linked to the disintegration of the apical dendrites of Betz (UMN) cells , which disrupts essential neural circuitry, leading to impaired overall motor function typically seen in ALS patients.

Despite the evidently significant role of motor neuron health in MND/ALS, it has been historically difficult to study the biology of a distinct neuron population due to the complexity and heterogeneity of the cerebral cortex. One suggested approach is to study UMNs derived from well-characterized mouse models that recapitulate the pathophysiology of diseased human UMNs. For example, apical dendrites of CSMNs in ALS mouse models have a similar biology and pathophysiology to that seen in Betz cells of ALS patients . It has been suggested that CSMNs derived from ALS mouse models (e.g., from C9orf72, Alsin, hSOD1 and TDP43 mice) could be used as a tool to reveal the biology and cellular pathology of Betz cells in ALS patients.

Many doubt that mice are a good model for the study of motor neuron diseases , mainly because UMNs and the corticospinal tract play much more important roles in connecting neural circuitry in humans than they do in mice . Drug discovery researchers are now turning to more physiologically relevant human iPSC-derived motor neurons to revolutionize the development of novel therapies for MND/ALS.

How can human iPSC-derived Motor Neurons enhance research and drug discovery?

The ability of hiPSC-derived motor neurons to recapitulate the specific altered cellular pathways underlying MND/ALS in a dish is causing great excitement in drug discovery. As the cells are of human origin, they are more physiologically relevant than animal models of ALS/MND. They are derived from clinically and/or genetically well-defined patients to ensure reproducibility and reliability. Assays using disease-specific hiPSC-derived motor neurons also offer the exciting possibility of a targeted personalized approach to drug development and could be used to investigate or reverse physiological changes associated with MND/ALS.

Axol has developed hiPSC-derived Motor Neuron Progenitors from two healthy donors (one with C9orf72 extension) , and has imminent plans to make them available from an ALS patient, also with C9orf72 extension. Applying these to your drug discovery efforts could expedite your search for the next breakthrough compound and enhance the translatability of your research. HiPSC-derived motor neurons with C9orf72 (and other) mutations have already helped to make great progress in identifying novel therapeutic targets.

For example, C9orf72 hiPSC-derived motor neurons have helped to further examine the pathogenesis of ALS and confirmed RNA toxicity as a major contributing factor and promising therapeutic target . Using the inhibitor kenpaullone to block the activity of HGK (an enzyme that sets off a chain of reactions that leads to motor neuron death in ALS patients) has been found to promote the survival of SOD1 hiPSC-derived motor neurons . Other recent in vitro data have emphasized the importance of hiPSC-derived motor neurons not only as a tool for high-throughput screening of potential targets, but also for furthering our understanding about the mechanisms involved in disease pathogenesis (e.g., see this recent review ).

The search for novel MND/ALS therapeutics has undoubtedly been challenging, but we are now entering an era of renewed hope. With screening assays now incorporating both pathogenetic and neurophysiological changes that underlie these diseases, we are moving towards a more translational approach to identifying therapeutic targets. HiPSC-derived Motor Neurons that recapitulate disease-specific pathophysiology are also enabling more targeted, reliable, and informative assays, which could uncover all-important compounds to effectively treat these debilitating diseases.

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