Impaired dynein function preserves spinal interneuron survival and positioning in an ALS-like mouse model

by Eleni Christoforidou, Jordan S. Rowe, Fabio A. Simoes, Raphaelle Cassel, Luc Dupuis, Peter Nigel Leigh, Majid Hafezparast

Impaired cytoplasmic dynein function has been implicated in amyotrophic lateral sclerosis (ALS) pathogenesis, yet the contributions of spinal interneurons to disease phenotypes remain unclear. We tested the hypothesis that hypomorphic dynein function in cholinergic neurons disrupts the development, survival, or positioning of inhibitory interneuron populations in the lumbar spinal cord. Using ChAT-Cre recombination, we generated four mouse genotypes with graded reductions in dynein activity in ChAT⁺ cells: Dync1h1^+/+ (wildtype), Dync1h1^−/+ (hemizygous wildtype), Dync1h1^+/Loa (heterozygous Loa mutation), and Dync1h1^−/Loa (hemizygous Loa). At 52 weeks of age, lumbar spinal cords (L3–L6) were harvested, cryosectioned, and immunostained for ChAT, GAD-67, Parvalbumin, and Calbindin. Cell counts were performed on confocal images from eight sections per mouse (N = 3 male mice/genotype), and radial distances from the central canal were normalised to gray matter width. Angular distributions were analysed via circular statistics. There were no significant genotype-dependent differences in the numbers of ChAT⁺, GAD-67⁺, Parvalbumin⁺, or Calbindin⁺ cells, nor in ChAT⁺ subpopulations (motor neurons versus interneurons) or double‐positive interneuron subsets (e.g., ChAT⁺–GAD-67⁺, Parvalbumin⁺–GAD-67⁺, Parvalbumin⁺–Calbindin⁺). Radial positioning relative to the central canal was similarly preserved across all markers and genotypes. Circular‐median tests revealed statistically significant shifts in mean angle for ChAT⁺, GAD-67⁺, and certain double‐positive cells, but these amounted to only 5–10° displacements, translating to lateral shifts of ~10–20 µm, well within single laminar bands, and are unlikely to impact circuit connectivity. Despite substantial motor deficits and hallmark TDP-43 pathology previously seen in these models, impaired dynein function does not precipitate interneuron loss or gross migratory defects in the lumbar spinal cord. Instead, our findings suggest that the primary contributions of dynein to ALS-like phenotypes likely arise from functional disruptions in axonal transport, synaptic maintenance, and neuronal physiology rather than from structural alterations or loss of interneuron populations.