An integrated single-nucleus ribonucleic acid sequencing and spatial transcriptomic atlas reveals stage-specific neuronal and glial trajectories in a mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a progressive multifocal neurodegenerative condition involving motor neurons and other cell types. To analyze spatiotemporal cellular dynamics in amyotrophic lateral sclerosis, we performed single-nucleus ribonucleic acid sequencing and spatial transcriptomics analysis of cervical spinal cords from wild-type control mice and SOD1-G93A transgenic mice in the pre-symptomatic (d50), early symptomatic (d90), and late-stage (d130) phases of disease. Single-nucleus ribonucleic acid sequencing identified 17 cell clusters and showed that progressive neuronal loss occurred over time, paralleled by glial expansion. Spatial transcriptomics mapped these clusters anatomically onto oligodendrocytes in white matter, neurons in horns, and diffuse astrocytes/microglia. Subcluster analysis demonstrated neuronal heterogeneity, with early mitochondrial stress in ventral motor neurons evolving into synaptic dysfunction, transient maturation peaks in interneurons, and amplified age-related decline in amyotrophic lateral sclerosis. Astrocyte and oligodendrocyte subclusters, which were originally misclustered due to spot-level contamination, were reinterpreted to highlight A1-reactive states and progenitor expansions, validated by immunohistochemistry detection of serum/glucocorticoid regulated kinase 1. Temporal profiles tracked the transition from compensatory to inflammatory gliosis, while gene signatures were linked to human amyotrophic lateral sclerosis cohorts, including complement activation and mitochondrial dysfunction. This study provides a high-resolution spatiotemporal cellular map of amyotrophic lateral sclerosis pathogenesis through the integration of single-nucleus and spatial transcriptomics, uncovering early mitochondrial impairment in neurons, delineating the trajectory of neurotoxic glial states, and identifying compensatory progenitor responses, to highlight the highly intricate interaction between glial reactivity and neuronal susceptibility that drives the pathogenesis of ALS.