RIPK1-Regulated Neuroinflammation and Cell Death in Amyotrophic Lateral Sclerosis and Alzheimer’s Disease

Abstract

RIPK1 is a master regulator balancing cell death and pro-survival NF-κB signaling in response to inflammatory stimuli. RIPK1 activation has been demonstrated in postmortem patient samples from numerous diseases. Small molecule inhibitors of RIPK1’s deleterious kinase activity have shown efficacy in preclinical disease models and advanced into clinical efficacy studies for autoimmune and neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer’s disease (AD). As RIPK1 can regulate cell survival, death, or inflammation, cell-specific activation of RIPK1 can differentially impact disease pathogenesis. In neurodegenerative diseases, there is evidence for both neuronal-autonomous cell death and glial-mediated neuroinflammation. Here, I investigated cell-specific roles for RIPK1 activation in ALS and AD pathogenesis. ALS is characterized by death of motor neurons in the spinal cord and increased neuroinflammation, the source of which remains unclear. I used single cell RNA sequencing to identify which cell populations in the spinal cord were impacted by ALS-associated mutations SOD1G93A and Optn-/-. I identified significant alterations in microglial transcription, including a previously undescribed subcluster, RIPK1-Regulated Inflammatory Microglia (RRIM), which produce proinflammatory cytokines such as TNFα and IL1β. Treatment with the RIPK1 inhibitor Necrostatin-1s (Nec-1s) reduced RRIMs, consistent with prior findings that Nec-1s alleviates pathology in mouse models of ALS. This study provides a transcriptional signature for RIPK1-regulated microglial inflammation in ALS pathology which is distinct from Disease-Associated Microglia (DAM). Dysfunction of blood-brain barrier (BBB) endothelial cells (ECs) is seen early, often presymptomatically, in AD patients, however whether this is relevant to disease progression is unclear. Activated RIPK1 was seen in ECs in a mouse model of AD. Using single cell and bulk RNA sequencing, I identified alterations promoting the necroptotic death of ECs. Treatment with a cerebrovascular EC-specific AAV2 to block necroptosis rescued BBB damage, neuroinflammation, and cognitive defects in a mouse model of AD. This is the first study demonstrating that an EC-specific intervention can rescue cognition in a mouse model of AD. Taken together, my work elucidates clear cell-specific roles for RIPK1 activation in the context of neurodegenerative diseases, which may support understanding of biomarkers of clinical efficacy in ongoing trials for RIPK1 inhibitors.