Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive motor neuron degeneration and TDP-43 proteinopathy. While postmortem studies provide insights into end-stage pathology, they fail to capture early disease dynamics and cellular changes that precede neurodegeneration. To address this, we developed corticospinal assembloids by integrating cortical and spinal cord spheroids derived from TDP-43 mutant and isogenic control iPSCs. Although assembloid models remain relatively immature compared to adult human brain, they offer a unique opportunity to investigate early pathological events in a physiologically relevant context. Single-cell RNA sequencing identified a novel mutant-specific cellular cluster, suggesting transcriptional reprogramming associated with TDP-43 pathology. Longitudinal electrophysiological recordings further revealed progressive network dysfunction, synaptic deficits, and altered excitability in mutant assembloids. These findings highlight the potential of assembloids to model early-stage ALS pathology and provide a powerful platform for identifying disease-modifying targets and evaluating therapeutic interventions before irreversible neurodegeneration occurs.
