Abstract TP322: Viral Mediated Gene Delivery of TMBIM6 Protects the Neonatal Brain

Abstract

Introduction: A wide variety of conditions may contribute to oxidative protein folding processes in the endoplasmic reticulum (ER) which cause ER stress and play a major role in the pathogenesis of neonatal hypoxic ischemic (HI) injury. ER stress triggers the unfolded protein response (UPR) resulting in the activation of pro-apoptotic signaling causing cells to undergo apoptosis. Here were explored the role of Bax Inhibitor-1 (BI-1) protein, an evolutionary protein encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, as a novel modulator of ER stress induced apoptosis after HI brain injury in a neonatal rat pup. BI-1 may modulate the UPR response via a direct physical interaction with the stress sensor receptor, IRE1α, and thus inhibiting it’s pro-apoptotic signaling. Hypothesis: Overexpression of BI-1, using viral-mediated gene delivery of human adenoviral-TMBIM6 (Ad-TMBIM6) vector, will protect the neonatal brain from HI by inhibiting ER stress induced apoptosis. Methods: Ten-day old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). To determine BI-1 confers protection via inhibition of IRE1α, we administered BI-1 siRNA, to silence BI-1, and an IRE1α CRISPR activation plasmid (AP), to counteract BI-1’s effects, at 48h pre-HI. Percent infarct area, immunofluorescent staining and western blot were performed at 72h post HI. Results: Overexpression of BI-1 showed to significantly reduce percent infarcted area, attenuated neuronal degeneration, reduced ER stress induced neuronal apoptosis and improved long-term neurological outcomes. Either silencing BI-1 or over activating IRE1α with a CRISPR AP significantly reversed BI-1’s protective effects. Conclusions: Overexpression of BI-1 attenuated the morphological and neurological consequences post neonatal HI via inhibition of the ER stress sensor, IRE1α. This study showed a novel role for BI-1 and ER stress in the pathophysiology of HI and provided a basis for BI-1 as a potential therapeutic target.