Single-cell transcriptomic and functional studies identify glial state changes and a role for inflammatory RIPK1 signaling in ALS pathogenesis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by motor neuron loss. Microglia and astrocyte-driven neuroinflammation is prominent in ALS, but the cell state dynamics and pathways driving disease remain unclear. We performed single-nucleus RNA sequencing of ALS spinal cords and identified altered glial cell states, including increased expression of inflammatory and glial activation markers. Many of these signals converged on the inflammation and cell death regulator receptor-interacting protein kinase 1 (RIPK1) and the necroptotic cell death pathway. In superoxide dismutase 1 (SOD1)G93A mice, blocking RIPK1 kinase activity delayed symptom onset and motor impairment and modulated glial responses. We used human induced pluripotent stem cell (iPSC)-derived motor neuron, astrocyte, and microglia tri-cultures to identify potential biomarkers that are secreted upon RIPK1 activation in vitro and modulated by RIPK1 inhibition in the cerebrospinal fluid (CSF) of people with ALS. These data reveal ALS-enriched glial populations associated with inflammation and suggest a deleterious role for neuroinflammatory signaling in ALS pathogenesis.