Investigating the Role of the Skeletal Muscle in Amyotrophic Lateral Sclerosis Neuromuscular Junction Dysfunction

Neuromuscular junction (NMJ) dysfunction has been identified as one of the earliest events in Amyotrophic Lateral Sclerosis (ALS) pathology. However, which tissue type induces NMJ disruption; be it the motoneurons (hMN), Schwann cells or skeletal muscle (hSKM) remains unresolved. While mechanisms by which ALS hMN contribute to NMJ dysfunction are well-described in literature, limited information exist on how the other tissue types in the tripartite synapse (hSKM and Schwann cells) induce and/or contribute to ALS NMJ disruption. A fair understanding of the role of each tissue type in NMJ dysfunction would help shape the trajectory of future ALS research and drug discovery. It is generally accepted that the observed ALS hSKM weakness and atrophy are a result of hMN axonal retraction. While recent findings postulate the active pathologic involvement of the hSKM in ALS onset via NMJ disruption, some scientists have questioned the validity of the transgenic model used in most studies and the translatability of their results. Contrarily, in vitro modeling of this phenomenon using patient hSKM samples has proved challenging due to the inability to reliably expand and maintain them before becoming senescent. Thus, this study sought to investigate the pathology of hSKM derived from patient ALS (ALS hSKM), its possible contribution to NMJ dysfunction, and whether hSKM-specific treatment confer any therapeutic benefit to the NMJ. To avoid the main challenges associated with human-based ALS hSKM studies i.e., tissue scarcity and established culturing challenges, patient iPSCs were utilized as the tissue source. This assured a single source for obtaining both cell types necessary for modeling the ALS NMJ. IPSC-derived wild type (WT) and ALS hSKM were differentiated using a serum-free, small molecule-directed protocol which centered on the concurrent modulation of the Wnt and bone morphogenetic protein pathways. To determine whether the ALS hSKM has intrinsic deficits independent of hMN, comparative assessment of WT and ALS hSKM were carried out. The myogenicity of resultant WT and ALS progenitors were confirmed via phase-contrast microscopy, immunocytochemistry, and flow cytometry, after which they were terminally differentiated in myotubes. WT and ALS hSKM were compared morphologically and functionally. The inner mitochondrial membrane potential (ΔΨM) and metabolic plasticity of both WT and ALS hSKM were also evaluated. After the hSKM-only characterization, both ALS and WT hSKM were co-cultured with either ALS or WT hMNs. This was to study how the previously outlined hSKM deficits affect NMJ formation, integrity and function in an effort to delineate the sole contribution to NMJ disruption. With knowledge of the pathologic contribution to NMJ dysfunction, hSKM-specific Creatine treatments were performed to investigate its therapeutic benefit to the ALS NMJ. This project advances the field\u27s knowledge on the hSKM\u27s role to ALS NMJ disruption and creates awareness about considering the hSKM in future research and drug discovery exploits. Additionally, this project resulted in the development of microphysiological platforms that recapitulate known phenotypic parameters of ALS while allowing the independent treatment and/or interrogation of each tissue type in the co-cultures