Abstract 2473: Ubiquitin Ligase Huwe1 Modulates Prongf/p75ntr In Monkey Cerebral Ischemia-reperfusion Injury

Abstract

Background: Ischemic neuronal cell apoptosis is a principal neuropathological feature of stroke. The p75 neurotrophin receptor (p75NTR) induces apoptosis associated with JNK-p53-BAX pathway, p53 is the substrate of the HECT domain-containing E3 ubiquitin ligase Huwe1. Recent studies suggest that the precursor form of NGF (proNGF) binds to p75NTR, and withhold the interaction of proNGF/p75NTR is efficacious in reducing neuronal apoptosis. Studies on tumor and phylogeny, show that Huwe1 highly expressed in CNS, playing a role in the regulation of cell apoptosis and a variety of injury types. Our aim is to examine whether Huwe1 modulates proNGF/p75NTR in cerebral ischemia-reperfusion injury. Methods: Eight male rhesus monkey were randomly divided into two groups: sham(n=2) and model group(n=6). The model group was administered equal volume of PBS, or silencing huwe1 Lentiviral Vector or empty Vector in right caudatum and putamen using brain stereotaxic technology and subjected to transient right middle cerebral artery occlusion (MCAO) a month later. A battery of neurological evaluation and magnetic resonance imaging (MRI) were employed to evaluate animals. Animals were sacrificed 3 days after MCAO and brains were processed for testing transfection efficiency using GFP fluorescence and evaluating cell apoptosis using TUNEL staining. The related factors in caudatum, putamen, temporal lobe and hippocampus was analyzed with QPCR, western blotting with loading control GADPH, and Immunohistochemistry. Results: The model group showed significant functional deficit than sham group with neurological evaluation (p<0.05), whereas the silencing Huwe1 group’s was the most serious. In right caudatum and putament, ischemia-reperfusion injury increased the number of TUNEL+cells(p<0.05 vs sham group) and upregulation of huwe1, proNGF and p75NTR in protein and nucleotides level (p<0.05 vs sham group), but silencing Huwe1 group increased TUNEL+cells most significantly, produced profound modulation with decreased expression of Huwe1 and obvious upregulation of proNGF and p75NTR(p<0.05 vs PBS or empty Vector group) ( Figure 1 ). However, there is no significant difference in other positions (data not show). Conclusions: Huwe1 modulates proNGF/p75NTR in the cerebral ischemia-reperfusion injury, and p53 may be as a indirect fator involved in this process. Our findings provide a novel mechanism in regulating proNGF/p75NTR signaling, suggesting its potential therapeutic target in ischemic stroke.