Targeting MAPK-dependent pathways to modulate reactive astrocyte pathology in neurodegeneration.

Reactive astrocytes play a crucial role in the pathogenesis of neurodegenerative diseases; however, the mechanisms underlying glial cell interactions remain poorly understood. Here, we show that human primary astrocytes and iPSC-derived astrocytes in a tri-culture system adopt a reactive state with a distinct molecular phenotype overlapping with transcriptomic signatures observed in ALS astrocytes. Integrated proteomic and phosphoproteomic analyses revealed dysregulated cytoskeletal remodeling and kinase signaling in reactive astrocytes. Surface marker screening identified ICAM-1 as a novel in-vitro marker, validated in ALS spinal cord. Functionally, reactive astrocytes induced neurotoxicity and altered neuronal activity. To elucidate mechanisms regulating neurotoxicity, a phenotypic small molecule screen identified MAP kinase as a functional regulator, with subsequent single-nucleus RNA sequencing uncovering dysregulated signaling pathways modulated by MAPK inhibition in the tri-culture system. Together, these complementary approaches define the molecular landscape of reactive astrocytes, providing a systems-level framework for exploring disease mechanisms and therapeutic strategies in neurodegeneration.