BTK inhibition suppresses neuroinflammation and neurodegeneration in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis(ALS) is a devastating neurodegenerative disorder with limited therapeutic interventions. Neuroinflammation represents a central pathogenic mechanism in ALS, yet the upstream molecular regulators that integrate multiple inflammatory cascades remain poorly understood. Here, we investigated whether Bruton's tyrosine kinase (BTK), which integrates DNA-sensing and Toll-like receptor signals upstream of the cGAS-STING-NF-κB cascade, serves as a key regulatory node in ALS pathogenesis. Public RNA-seq datasets of motor neurons and post-mortem tissues from ALS patients were utilized to identify BTK expression patterns. SOD1-mutant human induced pluripotent stem cells (hiPSC) were differentiated into motor neurons (hiPSC-MNs) and microglia (hiPSC-MGs). NF-κB dysregulation was profiled by scRNA-seq (hiPSC-MGs) and bulk RNA-seq (hiPSC-MNs). DNA damage (γH2AX), inflammatory signalling (western blot/ELISA) and phagocytosis (pH-rodo uptake) were quantified, and MG-conditioned medium was tested for MN toxicity. Monocultures and MN-MG co-cultures received zanubrutinib (3 µM, 12 h). SOD1-G93A mice were administered zanubrutinib (30 mg/kg, daily) from 2.5 months; motor performance, survival, spinal histology and PI3K-AKT-mTOR activity were assessed after 2 months of treatment. ALS spinal cord and cortex tissues of patients, as well as SOD1-mutant hiPSC-MGs and hiPSC-MNs, demonstrated elevated BTK phosphorylation with increased p-STING, p-TBK1, and nuclear NF-κB accumulation. ALS hiPSC-MGs exhibited inflammatory activation, NLRP3 induction, and impaired phagocytosis, while ALS hiPSC-MNs showed DNA damage and caspase-3-mediated apoptosis. Conditioned medium from inflammatory microglia amplified neuronal STING-NF-κB activity and apoptosis, demonstrating non-cell-autonomous toxicity. The STING inhibitor H-151 reduced neuronal p-STING/p-TBK1/NF-κB and apoptosis, confirming pathway causality. Pharmacological BTK inhibition reduced DNA damage in ALS hiPSC-MNs by 61.4% (p