Viral-mediated gene delivery of TMBIM6 protects the neonatal brain via disruption of NPR-CYP complex coupled with upregulation of Nrf-2 post-HI

Abstract

Background Oxidative stress, inflammation, and endoplasmic reticulum (ER) stress play a major role in the pathogenesis of neonatal hypoxic-ischemic (HI) injury. ER stress results in the accumulation of unfolded proteins that trigger the NADPH-P450 reductase (NPR) and the microsomal monooxygenase system which is composed of cytochrome P450 members (CYP) generating reactive oxygen species (ROS) as well as the release of inflammatory cytokines. We explored the role of Bax Inhibitor-1 (BI-1) protein, encoded by the Transmembrane Bax inhibitor Motif Containing 6 (TMBIM6) gene, in protection from ER stress after HI brain injury. BI-1 may attenuate ER stress-induced ROS production and release of inflammatory mediators via (1) disruption of the NPR-CYP complex and (2) upregulation of Nrf-2, a redox-sensitive transcription factor, thus promoting an increase in anti-oxidant enzymes to inhibit ROS production. The main objective of our study is to evaluate BI-1’s inhibitory effects on ROS production and inflammation by overexpressing BI-1 in 10-day-old rat pups. Methods Ten-day-old (P10) unsexed Sprague-Dawley rat pups underwent right common carotid artery ligation, followed by 1.5 h of hypoxia. To overexpress BI-1, rat pups were intracerebroventricularly (icv) injected at 48 h pre-HI with the human adenoviral vector-TMBIM6 (Ad-TMBIM6). BI-1 and Nrf-2 silencing were achieved by icv injection at 48 h pre-HI using siRNA to elucidate the potential mechanism. Percent infarcted area, immunofluorescent staining, DHE staining, western blot, and long-term neurobehavior assessments were performed. Results Overexpression of BI-1 significantly reduced the percent infarcted area and improved long-term neurobehavioral outcomes. BI-1’s mediated protection was observed to be via inhibition of P4502E1, a major contributor to ROS generation and upregulation of pNrf-2 and HO-1, which correlated with a decrease in ROS and inflammatory markers. This effect was reversed when BI-1 or Nrf-2 were inhibited. Conclusions Overexpression of BI-1 increased the production of antioxidant enzymes and attenuated inflammation by destabilizing the complex responsible for ROS production. BI-1’s multimodal role in inhibiting P4502E1, together with upregulating Nrf-2, makes it a promising therapeutic target. Electronic supplementary material The online version of this article (10.1186/s12974-019-1559-4) contains supplementary material, which is available to authorized users.

Revisiting gene delivery to the brain: silencing and editing

“…48 Nevertheless, adeno-associated virus (AAV) and recombinant AAV vectors are commonly used to deliver various genes to brain mainly due to their favourable attributes such as capability to infect both dividing and non-dividing cells, as well as their minimal immunogenicity. 49 Along similar lines, transgenes that encode therapeutic proteins, [50][51][52][53][54][55] microRNAs (miRNAs), [56][57][58][59][60] antibodies, [61][62][63] and CRISPR-Cas9 guide RNA for gene editing [64][65][66][67][68] have been successfully delivered to the CNS with AAVs in mice and other species (Fig. 2A).…”

Fractalkine Enhances Hematoma Resolution and Improves Neurological Function via CX3CR1/AMPK/PPARγ Pathway After GMH

“…CX3CR1 CRISPR (Santa Cruz Biotechnology) or Control CRISPR was administered intracerebroventricularly using a Hamilton syringe (Microliter 701, Hamilton Company) into the right lateral ventricle (1.5 mm posterior, 1.5 mm lateral to the bregma and 1.7 mm deep from the surface) 24 hours before GMH induction in rat with 0.3 μL/min, as described previously. 13 , 14 After completing the injection, the needle was kept in situ for 10 min and then withdrawn slowly for 5 min to prevent backflow.…”

Activation of LRP6 with HLY78 Attenuates Oxidative Stress and Neuronal Apoptosis via GSK3β/Sirt1/PGC-1α Pathway after ICH

“…Dihydroethidium (DHE) staining was conducted to evaluate ROS production as previously described [ 32 ]. The brain sections were incubated with freshly prepared dihydroethidium (2 μ mol/L) (Thermo Fisher Scientific, USA) for 30 min at 37°C in the dark.…”