The long and (un)winding road: Defining the role of cytoskeletal pathways in ALS pathogenesis

Our ability to move with intention and precision depends on a specialized network of nerve cells called the motor system. At the center of this system are motor neurons, remarkably long and highly specialized cells that connect the brain, spinal cord, and muscles. Some of these neurons have axons that stretch up to a meter in length, making them among the most structurally demanding cells in the human body. To stay healthy and function properly, they rely on an internal framework known as the cytoskeleton, an internal scaffold which helps maintain their shape, transport materials across the cell, and support communication at their connections with other nerve cells (synapses) and muscles (specialized synapses, known as neuromuscular junctions). Because of these extreme demands, motor neurons are especially sensitive to any disruptions in their internal structure and transport systems. These vulnerabilities become more pronounced with aging and are a key factor in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). ALS is a fatal condition in which motor neurons gradually break down, leading to muscle weakness, paralysis, and eventually death. The overarching aim of this thesis was to investigate how the breakdown of the cytoskeleton and transport systems inside motor neurons contributes to their dysfunction ALS, and how these changes might be linked to other disease-related mechanisms. In Chapter 2, we identified STMN2, a regulator of microtubule stability, as a key TDP-43 target in human motor neurons and demonstrated that its cryptic splicing upon TDP-43 depletion leads to reduced expression, impairing axonal outgrowth and regeneration. Similar downregulation and altered splicing were found in ALS patient postmortem spinal motor neurons, positioning STMN2 as a crucial downstream effector of TDP-43 dysfunction in ALS. Building on these findings, Chapter 3 examined the in vivo role of STMN2 using a novel loss of function CRISPR mouse model. We showed that Stmn2 loss leads to NMJ denervation, muscle atrophy, and motor impairments, accompanied by impaired microtubule dynamics without motor neuron loss, gliosis or TDP-43 pathology. This work established STMN2 as a critical regulator of motor system integrity and reinforced its relevance as a contributor to motor neuropathy in ALS. In Chapter 4, we developed and optimized an in vitro system for studying human motor neuron biology in a reproducible and scalable manner across many iPSC lines. By combining small-molecule patterning with Ngn2-induced programming, we generated lower motor neurons (liMNs) that transcriptionally capture specific subtypes of embryonic human spinal motor neurons in seven days, offering a faster alternative to traditional differentiation methods. LiMNs were characterized through transcriptomic analyses, exhibited synaptic activity and could be co-cultured with other cell types, providing a well-defined platform to systematically study motor neuron function and disease mechanisms in ALS and related disorders. Extending this work, Chapter 5 examined the function of KIF5A, a critical molecular motor mutated in ALS and other late-onset motor neuron disorders (SPG10/CMT2), in liMNs. We demonstrated that KIF5A loss leads to axonal regrowth defects and time-dependent transport deficits, with reduced anterograde transport of mitochondria and SFPQ RNA granules occurring only at later developmental stages. These findings suggest that KIF5A plays a critical role in maintaining axonal integrity through supporting repair processes and axonal distribution of critical cargoes. Overall, this work combined in vitro and in vivo models to gain mechanistic insights into motor neuron dysfunction in ALS. By linking major disease pathways such as axonal cytoskeletal defects and disrupted RNA metabolism, this thesis identified potential convergent and divergent processes contributing to disease pathogenesis, offering a more complex understanding of the molecular underpinnings of ALS.