Reduced membrane-associated neprilysin is associated with ETBR-skewed ET-1 signaling and mitochondrial fission after subarachnoid hemorrhage

Subarachnoid hemorrhage (SAH) triggers early brain injury and lasting cognitive dysfunction, but the upstream events that couple endothelin dysregulation to mitochondrial apoptosis in vulnerable hippocampal neurons remain unclear. Here we combined in vivo rat SAH and primary neuronal injury models to test whether reduced membrane-associated neprilysin (NEP) and altered NEP function are associated with endothelin-1 (ET-1)/endothelin B receptor (ETBR) signaling and mitochondrial fission-dependent apoptosis. At 24 h after SAH, neuronal apoptosis increased in both cortex and hippocampus, whereas caspase-9 activation was more prominent in hippocampal subregions, indicating stronger mitochondrial involvement. SAH decreased total and membrane-associated NEP, increased NEP phosphorylation, decreased hippocampal NEP enzymatic activity, elevated cerebrospinal fluid ET-1, downregulated endothelin A receptor, and upregulated ETBR. Co-immunoprecipitation showed reduced ET-1-NEP association but enhanced ET-1-ETBR association after SAH. Recombinant NEP restored membrane-associated NEP and hippocampal NEP enzymatic activity, lowered ET-1 abundance, rebalanced receptor expression, reduced the co-immunoprecipitated association between ET-1 and ETBR, alleviated neuronal apoptosis, preserved mitochondrial ultrastructure, and improved neurological recovery. In primary neurons, exogenous ET-1 induced NEP phosphorylation, reduced membrane-associated NEP, activated RhoA/ROCK1/DRP1 signaling, and increased apoptosis; these changes were reversed by NEP supplementation. Pharmacological ETBR activation re-established mitochondrial network fragmentation despite NEP treatment, whereas ETAR blockade had limited effects under the experimental conditions examined. These findings support a model in which reduced membrane-associated NEP and impaired hippocampal NEP activity are accompanied by ET-1 accumulation and preferential engagement of ETBR-associated RhoA/ROCK1/DRP1 signaling, contributing to mitochondrial fission and hippocampal neuronal apoptosis after SAH. NEP may represent a therapeutically actionable upstream regulator of early brain injury after SAH.