CRISPR-edited iPSCs reveal BSN gene mutations induce neuronal hyperexcitability via astrocyte lipid accumulation.

Abstract

Mutations in the BSN gene, encoding the presynaptic protein Bassoon, are implicated in epilepsy, but their impact on astrocytes remains unclear. Using CRISPR/Cas9, we introduced patient-derived BSN mutations (p.M1903V and c.5672insCG) into human induced pluripotent stem cells (iPSCs) and differentiated them into astrocytes. We found that mutant astrocytes exhibited significant lipid accumulation, evidenced by elevated free cholesterol, reduced arginase activity, and increased lipid droplets. Proteomic analysis revealed upregulation of lipid metabolism regulators, such as APOE and FASN. Electrophysiological recordings showed impaired Kir4.1 potassium channel function, depolarized resting membrane potential, and increased capacitance in mutant astrocytes following kainic acid stimulation. Co-culture experiments with neurons demonstrated that BSN-mutant astrocytes led to reduced neurite outgrowth, elevated neuronal apoptosis, increased pro-inflammatory cytokines (IL-1β, TNF-α), and neuronal hyperexcitability. These findings demonstrate that BSN mutations disrupt astrocyte lipid homeostasis and impair neurosupportive functions, thereby driving neuronal hyperexcitability. This study establishes astrocytes as critical mediators of epilepsy pathogenesis in BSN-related disorders and highlights lipid metabolism as a potential therapeutic target.