Single-Nucleus Transcriptomics Identifies a C5AR1-Driven, Hypoxia-Responsive Microglial State in the Human ALS Spinal Cord

In this study, single-nucleus transcriptomic profiling of 62,711 nuclei from the human lumbar spinal cord was employed to resolve disease-specific immune heterogeneity in Amyotrophic Lateral Sclerosis (ALS). While neuroinflammation driven by microglial activation is a recognized driver of motor neuron attrition, the molecular checkpoints governing the transition of human microglia into a neurotoxic state remain poorly defined. Through unsupervised clustering, a discrete, ALS-exclusive microglial sub-population (Cluster 2) was identified, demonstrating an 87.7% enrichment in diseased tissue. This pathogenic cluster is uniquely characterized by the robust upregulation of the Complement 5a Receptor (C5AR1) (p < 10⁻²⁰, LogFC = 1.94), alongside a significant hypoxic stress signature defined by HIF1A and NAMPT expression. Crucially, ligand-receptor interaction mapping identified motor neurons as the primary source of the C5 ligand (p < 0.05), establishing a coordinated neuro-immune distress axis in the failing spinal cord. This C5-C5AR1 signature was validated as a consistent feature across all biological replicates n=6/6 ALS donors compared to n=6/6 neurologically normal controls; total N=12) through pseudobulk aggregation, confirming its robustness at the patient level. These findings identify the C5-C5AR1 signaling axis as a novel, hypoxia-fueled checkpoint in ALS pathology. Given that C5AR1 is a clinically targetable receptor with existing antagonists, these results provide a high-priority therapeutic window to arrest microglial-mediated neurotoxicity in human ALS.