Abstract
Subarachnoid hemorrhage (SAH) is a devastating cerebrovascular disorder with high acute mortality and long-term neurological disability, and early brain injury (EBI) characterized by mitochondrial dysfunction, oxidative stress, and neuronal apoptosis is a pivotal determinant of poor prognosis. Mitochondria-endoplasmic reticulum contact sites (MERCs) are specialized membrane domains essential for maintaining cellular homeostasis via calcium trafficking and lipid exchange, but their regulatory mechanisms in SAH-induced EBI remain largely undefined. Here, we investigated the role and underlying mechanism of PDZD8, a core MERCs-stabilizing protein, in SAH pathogenesis using in vivo endovascular perforation models of male C57BL/6 mice and in vitro oxyhemoglobin (OxyHb)-challenged primary cortical neurons/HT22 cells, combined with PDZD8 overexpression, CRISPR/Cas9-mediated knockout, and C884A site-directed mutagenesis. Results demonstrated that PDZD8 was neuron-specifically downregulated at 48 h post-SAH, which closely correlated with MERCs structural disruption detected by transmission electron microscopy, impaired mitochondrial respiration analyzed via Seahorse assays, excessive reactive oxygen species production, and severe neuronal damage assessed by Nissl staining. PDZD8 overexpression preserved MERCs integrity, restored mitochondrial metabolic balance, mitigated oxidative stress, and ameliorated neurobehavioral deficits evaluated by modified neurological severity scores, rotarod, and open field tests. Mechanistically, SAH-induced PDZD8 downregulation was associated with enhanced global S-nitrosylation and post-translational regulation at cysteine 884 (C884), promoting its ubiquitination and proteasomal degradation, while C884A mutation abrogated this process. Our findings reveal a previously unrecognized mechanism involving S-nitrosylation-associated ubiquitination of PDZD8 in SAH-induced MERCs dysfunction and EBI, highlighting PDZD8 as a promising therapeutic target for SAH treatment.