Adenoviral-TMBIM6 vector attenuates ER stress - induced apoptosis in a neonatal hypoxic-ischemic rat model

Abstract

Endoplasmic reticulum (ER) stress is a major pathology encountered after hypoxic-ischemic (HI) injury. Accumulation of unfolded proteins triggers the unfolded protein response (UPR), resulting in the activation of pro-apoptotic cascades that lead to cell death. Here, we identified Bax inhibitor 1 (BI-1), an evolutionarily conserved 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. The main objective of our study was to overexpress BI-1, via viral-mediated gene delivery of human adenoviral-TMBIM6 (Ad-TMBIM6) vector, to investigate its anti-apoptotic effects as well as to elucidate its signaling pathways in an in vivo neonatal HI rat model and in vitro oxygen-glucose deprivation (OGD) model. Ten-day-old unsexed Sprague Dawley rat pups underwent right common carotid artery ligation followed by 1.5 h of hypoxia. Rat pups injected with Ad-TMBIM6 vector, 48 h pre-HI, showed a reduction in relative infarcted area size, attenuated neuronal degeneration and improved long-term neurological outcomes. Furthermore, silencing of BI-1 or further activating the IRE1α branch of the UPR, using a CRISPR activation plasmid, was shown to reverse the protective effects of BI-1. Based on our in vivo and in vitro data, the protective effects of BI-1 are mediated via inhibition of IRE1α signaling and in part via inhibition of the second stress sensor receptor, PERK. Overall, this study showed a novel role for BI-1 and ER stress in the pathophysiology of HI and could provide a basis for BI-1 as a potential therapeutic target.

GW0742 activates miR‐17‐5p and inhibits TXNIP/NLRP3‐mediated inflammation after hypoxic‐ischaemic injury in rats and in PC12 cells

“…PC12‐rat pheochromocytoma cells (ATCC, American Type Culture Collection, Manassas) were grown as previously described 26 . Cells were cultured in the incubator at 37°C and 5% CO 2 in F12‐K medium supplemented with 2.5% (v/v) foetal bovine serum (FBS), 15% (v/v) horse serum (HS) and 1% (v/v) penicillin‐streptomycin solution (full growth complete medium).…”

“…Full growth complete medium was replaced with no‐glucose medium (pure FK12 medium without the addition of horse serum, FBS or antibiotics), and cells were placed in a hypoxic chamber and flushed with 1% oxygen for 3 hours. Complete growth media were then added, and cells were allowed to recover for 24 hours, after which the cells were prepared for cell viability assay and Western blotting 26 …”

“…PC12-rat pheochromocytoma cells (ATCC, American Type Culture Collection, Manassas) were grown as previously described. 26 Cells were cultured in the incubator at 37°C and 5% CO 2 in F12-K me-…”

“…Complete growth media were then added, and cells were allowed to recover for 24 hours, after which the cells were prepared for cell viability assay and Western blotting. 26…”

EPO and TMBIM3/GRINA Promote the Activation of the Adaptive Arm and Counteract the Terminal Arm of the Unfolded Protein Response after Murine Transient Cerebral Ischemia

“…To improve stroke outcome, new pharmacological approaches must be considered, such as boosting endogenous pro-survival pathways. Among them, the unfolded protein response (UPR) is a promising cellular mechanism, since the UPR maintains and restores endoplasmic reticulum (ER) function, which is critical for the survival of stressed cells in general and of hypoxic cells in particular [5,6,7,8].…”