A2-pancortins interact with Bcl-xL and WAVE1 to promote mitochondria-ER contact sites (MERCs) and exacerbate mitochondrial calcium elevation to mediate cell death in stroke.

Pancortin (PCT), a protein highly expressed in the cortex during neurodevelopment, comprises four isoforms (PCT1-4) characterized by a central B-region and N-termini (A1/A2) and C-termini (C1/C2). While PCT2, enriched in adult mouse cortex, mediates ischemic neuronal death via interactions with WAVE1 and Bcl-xL, perinatal isoforms (A2-PCTs, namely PCT3 and PCT4) remain poorly defined. Given the vulnerability of the developing brain to hypoxia-induced neurological deficits, here we asked whether A2-PCTs contribute to ischemic damage in the developing cortex. In primary cortical neurons, knockdown of PCTs mitigated cell death induced by oxygen-glucose deprivation. Consistently, using A2-PCTs knockout mice subjected to middle cerebral artery occlusion, we observed significantly reduced cortical damage in younger animals, implicating A2-PCTs as pro-death factors in hypoxic conditions. Mechanistically, A2-PCTs formed a tripartite complex with Bcl-xL and WAVE1. Overexpression of A2-PCTs in HEK293 cells resulted in elevated cell mortality, and as revealed by SPLICS (a split-GFP-based contact site sensor)-based imaging of mitochondria-ER contact sites (MERCs), increased its localization to MERCs. Furthermore, A2-PCTs interacted with GRP75, a MERCs-tethering protein, in a Bcl-xL/WAVE1-dependent manner. A2-PCTs/Bcl-xL/WAVE1 complex increased IP3R-mediated calcium transfer from the ER to mitochondria, leading to cytosolic and mitochondrial calcium overload, which was attenuated by IP3R inhibition. In Neuro2a cells subjected to oxygen-glucose deprivation, PCTs knockdown similarly suppressed pathological calcium flux. Our study identifies A2-PCTs as key regulators of MERCs-mediated calcium dysregulation in neonatal stroke, pointing to their potential as therapeutic targets for mitigating ischemic brain injury in the developing brain.