Investigating Motor Neuron Function and Pathology using 2D and 3D cell culture systems

Motor neurons (MNs) are among the most structurally complex and physiologically demanding cells in the nervous system. Their extreme size, long axons, and reliance on sustained vesicle trafficking and degradative clearance make them intrinsically vulnerable to perturbations in intracellular homeostasis. These vulnerabilities are central to neuromuscular and neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This thesis investigates the developmental, molecular, and genetic mechanisms that shape MN maturation and stress susceptibility by combining physiologically relevant 3D neuromuscular models with mechanistic studies of key regulators of the endosomal–lysosomal and autophagy pathways. The first part of the study established a temporal map of human neuromuscular junction (NMJ) development using a 3D co-culture system composed of iPSC-derived spinal MNs and primary human myoblast-derived skeletal muscle. Bulk RNA-seq at weeks 1, 3, and 6 identified three major transcriptional phases that parallel in vivo NMJ maturation: (1) early myogenesis driven by cell- cycle activity and ECM remodeling, (2) synapse assembly marked by coordinated induction of presynaptic cytoskeletal and vesicle-related genes, and (3) functional maturation defined by enhanced ion handling, contractile specialization, and postsynaptic structural organization. These trajectories were highly reproducible across independent iPSC lines. Although the cultures displayed an embryonic-like AChR profile, the dataset provides a robust developmental framework for interpreting MN vulnerability within a human neuromuscular context. The second part of the study investigated how the lysosomal regulator TMEM106B, a genetic modifier of ALS and FTD, influences autophagy–lysosomal homeostasis in MNs. Using CRISPR/ Cas9-generated knockout lines across risk versus protective TMEM106B haplotypes, we observed striking allele-dependent effects. In the risk background, TMEM106B loss induced widespread dysregulation of synaptic, trafficking, and lysosomal gene networks, with downregulation of autophagy initiation genes and compensatory upregulation of degradative components. Functional assays showed impaired autophagic flux and diminished lysosomal acidification specifically in risk-haplotype MNs. In contrast, protective-haplotype MNs exhibited minimal transcriptional disturbance and maintained degradative competence. These results demonstrate that TMEM106B haplotype strongly shapes neuronal sensitivity to lysosomal stress and clarifies why TMEM106B variation modifies neurodegenerative disease risk. The third part of the study examined how perturbation of the ESCRT-II component VPS25 affects MN survival and intracellular homeostasis. VPS25 knockdown in both mouse primary MNs and human iPSC-derived MNs led to rapid neurite degeneration, elevated caspase activation, and widespread cell death. Mechanistically, VPS25 loss severely impaired BDNF–TrkB survival signaling by reducing full-length TrkB and attenuating downstream AKT and MAPK activation. In parallel, VPS25 depletion disrupted lysosomal function, producing reduced acidity, impaired Cathepsin D processing, and LC3-II accumulation consistent with stalled autophagic flux. These findings highlight VPS25 as a key regulator of endosomal maturation, degradative capacity, and trophic signaling fidelity—processes essential for MN resilience. Notably, MNs carrying ALS- linked SOD1 mutations expressed reduced endogenous VPS25, suggesting that compromised ESCRT-II function contributes to disease-associated lysosomal stress. Across all three parts, this thesis shows that MN development, survival, and stress tolerance depend on the integrity of interconnected vesicle trafficking and autophagy–lysosomal pathways. The 3D NMJ model provides the developmental and physiological context in which MNs acquire synaptic function; mechanistic disruption of VPS25 and TMEM106B reveals that degradative and trafficking systems are central determinants of MN resilience. Together, these findings provide a unified framework for understanding MN vulnerability and establish a foundation for future work aimed at dissecting the molecular determinants of MN resilience in neuromuscular and neurodegenerative disease.