Astrocyte-derived Exosomal GJA1-20 k Targets Pink1-mediated Mitophagy to Attenuate Traumatic Brain Injury

Connexin 43 (Cx43), particularly its truncated isoform GJA1-20 k, has shown promise in mitigating neuronal injury through mitochondrial regulation. This study aimed to investigate the therapeutic potential of astrocyte-derived extracellular vesicles (EVs) enriched with GJA1-20 k (Exo-GJA1-20 k) for treating traumatic brain injury (TBI). Primary astrocytes were isolated and transfected with an adeno-associated viral vector to overexpress GJA1-20 k. EVs were extracted and characterized using nanoparticle tracking analysis and Western blotting. A controlled cortical impact (CCI) model of TBI was established in mice, followed by daily administration of Exo-GJA1-20 k via tail vein injections. Mitochondrial function, neuroinflammation, pyroptosis, and cognitive outcomes were evaluated through molecular assays, histological staining, and behavioral tests, including the Morris Water Maze and open field tests. Exo-GJA1-20 k treatment significantly improved mitochondrial quality control by enhancing mitophagy and reducing mitochondrial dysfunction. Pyroptosis, driven by the NLRP3 inflammasome, was notably suppressed, with significant reductions in NLRP3, ASC, and IL-1β expression levels. Behavioral analyses revealed enhanced cognitive performance, as evidenced by shorter escape latencies in the Morris Water Maze and reduced anxiety-like behaviors in the open field test in Exo-GJA1-20 k-treated mice compared to controls. Importantly, the therapeutic effects of Exo-GJA1-20 k were diminished in Pink1-knockout mice, underscoring the dependence on Pink1-mediated mitophagy. This study demonstrates that Exo-GJA1-20 k exerts neuroprotective effects by modulating the mitophagy-NLRP3 inflammasome axis, alleviating neuroinflammation, and mitigating cognitive deficits in a TBI model. These findings propose a novel therapeutic strategy for addressing TBI-induced neuronal damage and underscore the potential of EV-based therapies for treating neurological disorders.