Nrdp1 Increases Ischemia Induced Primary Rat Cerebral Cortical Neurons and Pheochromocytoma Cells Apoptosis Via Downregulation of HIF-1α Protein.

Yuan Zhang,Wenlan Liu,Xinchun Jin,Weiping Li,Ke Yang,Ting Wang

doi:10.3389/fncel.2017.00293

Abstract

Neuregulin receptor degradation protein-1 (Nrdp1) is an E3 ubiquitin ligase that targets proteins for degradation and regulates cell growth, apoptosis and oxidative stress in various cell types. We have previously shown that Nrdp1 is implicated in ischemic cardiomyocyte death. In this study, we investigated the change of Nrdp1 expression in ischemic neurons and its role in ischemic neuronal injury. Primary rat cerebral cortical neurons and pheochromocytoma (PC12) cells were infected with adenoviral constructs expressing Nrdp1 gene or its siRNA before exposing to oxygen-glucose deprivation (OGD) treatment. Our data showed that Nrdp1 was upregulated in ischemic brain tissue 3 h after middle cerebral artery occlusion (MCAO) and in OGD-treated neurons. Of note, Nrdp1 overexpression by Ad-Nrdp1 enhanced OGD-induced neuron apoptosis, while knockdown of Nrdp1 with siRNA attenuated this effect, implicating a role of Nrdp1 in ischemic neuron injury. Moreover, Nrdp1 upregulation is accompanied by increased protein ubiquitylation and decreased protein levels of ubiquitin-specific protease 8 (USP8) in OGD-treated neurons, which led to a suppressed interaction between USP8 and HIF-1α and subsequently a reduction in HIF-1α protein accumulation in neurons under OGD conditions. In conclusion, our data support an important role of Nrdp1 upregulation in ischemic neuronal death, and suppressing the interaction between USP8 and HIF-1α and consequently the hypoxic adaptive response of neurons may account for this detrimental effect.

Highlights

Cerebral ischemia initiates a cascade of cytotoxic molecules responsible for the death of neural cells as well as the damage of the blood brain barrier (BBB) at the injury site (Doyle et al, 2008)
To determine whether Nrdp1 plays a role in ischemic brain injury, we examined the change of Nrdp1 expression in the cerebral cortex isolated from the rats that were subjected to 3-h middle cerebral artery occlusion (MCAO) without reperfusion
The major findings include: (1) Nrdp1 is significantly upregulated in the ischemic brain tissue and in oxygen-glucose deprivation (OGD)-treated neuronal cells; (2) overexpression or knockdown of Nrdp1 enhances or attenuates OGD-induced apoptosis in neurons, respectively, and these changes are accompanied by the downregulation or upregulation of Nrdp1’s substrate ubiquitinspecific protease 8 (USP8); and (3) USP8 may directly interact with hypoxia inducible factor-1α (HIF-1α) to prevent its degradation, and under OGD conditions, Nrdp1 may interfere with HIF-1α stabilization via promoting USP8 degradation

Summary

Introduction

Cerebral ischemia initiates a cascade of cytotoxic molecules responsible for the death of neural cells as well as the damage of the blood brain barrier (BBB) at the injury site (Doyle et al, 2008). In more than one decade, ischemia-associated neuronal injury has been a topic of intensive investigation, which leads to the identification of several mechanisms accounting for cerebral ischemia injury, such as apoptosis, oxidative damage, inflammatory injury, mitochondrial dysfunction and dysregulated protein degradation (Caldeira et al, 2014; Kalogeris et al, 2014; Palencia et al, 2015). The ubiquitin-proteasome system (UPS) is the major intracellular machinery for protein degradation, which is responsible for maintaining cellular homeostasis by regulating several important processes such as cell death, cell division, cell signal transduction, cell cycle progression and transmembrane transport (Wagner et al, 2011). Emerging evidence has suggested a role of suppressed proteasome activity in contributing to neuronal death in ischemic brain injury. The exact role of Nrdp in ischemia-induced neuronal damage remains to be determined

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