Targeting the STING-NFκB Pathway via BTK Inhibition: A Dual-Mechanism Therapeutic Strategy Mitigating Neuronal and Microglial Dysfunction in Amyotrophic Lateral Sclerosis [scRNA-seq]

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with limited therapeutic interventions. We identified the cGAS-STING-NFκB pathway as a dual therapeutic target that combats both neuronal degeneration and neurotoxic glial activity in ALS. Utilizing SOD1-mutant human iPSC-differentiated motor neurons(hiPSC-MNs) and microglia(hiPSC-MGs), we demonstrated that ALS hiPSC-MGs displayed a pronounced proinflammatory phenotype, characterized by significant inflammatory marker elevation (NLRP3 increased 0.66-fold, secreted IL-1β elevated 2.85-fold compared to controls) and compromised phagocytic capacity (48% reduction in bioparticle uptake). Concurrently, ALS hiPSC-MNs exhibit DNA damage, activating STING-NFκB signaling and triggering caspase-3-mediated apoptosis. Notably, co-culture experiments revealed that inflammatory hiPSC-MGs substantially increased neuronal apoptosis, indicating a toxic intercellular communication mechanism mediated through STING-NFκB signaling. Pharmacological inhibition of Bruton's tyrosine kinase (BTK) - a critical upstream regulator of STING activation - effectively suppressed this inflammatory cascade. This intervention reduced DNA damage in ALS hiPSC-MNs by 61.4% and restored microglial phagocytic function to approximately 87.2% of normal levels. In vivo studies with SOD1-G93A ALS mice demonstrated that BTK inhibitor treatment significantly improved motor performance, extended median survival (183 days versus 158 days in untreated controls), and mitigated neuropathological progression. The treatment enhanced motor neuron survival, reduced microgliosis and astrogliosis, and modulated the PI3K-AKT-mTOR pathway without disrupting autophagy-lysosome dynamics. Our study presents a translational therapeutic strategy that simultaneously addresses neuron-intrinsic and glial-mediated mechanisms in ALS, providing a compelling rationale for repurposing BTK inhibitors in future ALS clinical trials.