Abstract
ABSTRACTEndoplasmic 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.
Highlights
Temporal changes in the expression levels of endogenous Bax inhibitor 1 (BI-1), IRE1α, X-boxbinding protein 1 (XBP1) and CHOP post-HI In ipsilateral hemispheric brain tissue samples from 10-day-old neonatal rats subjected to HI, BI-1 expression levels increased over time, peaking at 24 h and returning to sham levels by 72 h postHI (Fig. 1A,B)
The results showed that, for animals treated with Ad-transmembrane BAX inhibitor motif-containing 6 (TMBIM6), both BI-1 siRNA and IRE1α CRISPR activation plasmid (AP) significantly increased the relative size of infarcted area compared to the Ad-TMBIM6-treated group, reversing the protective effects of BI-1 (Fig. 4A,B)
A variety of disturbances and stresses, such as ischemic events, can modify endoplasmic reticulum (ER) function and result in the accumulation of unfolded proteins, triggering the unfolded protein response (UPR) (Rissanen et al, 2006; Xu et al, 2005). Under prolonged stress, such as after HI injury, accumulation of unfolded proteins leads to the activation of stress sensor receptors that trigger pro-apoptotic signaling, leading to cell death (Oyadomari and Mori, 2004; Madeo and Kroemer, 2009; Chaudhari et al, 2014; Zhang et al, 2015; Carloni et al, 2014; Xu et al, 2005)
Summary
The endoplasmic reticulum (ER) is responsible for correct protein folding and protein function (Zhang et al, 2015) It is crucial for cell survival; under pathological conditions, such as HI, there is an accumulation of unfolded proteins that exceeds the capacity of the chaperones to fold the proteins, leading to ER stress (Zhang et al, 2015; Xu et al, 2005; Barrett et al, 2007). This event triggers the unfolded protein response (UPR) resulting in the activation of pro-apoptotic cascades (Sano and Reed, 2013; Madeo and Kroemer, 2009). Among the three stress sensor receptors, IRE1α is the most conserved receptor (Ron and Walter, 2007)
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