GCN2 suppression attenuates cerebral ischemia in mice by reducing apoptosis and endoplasmic reticulum (ER) stress through the blockage of FoxO3a-regulated ROS production

https://doi.org/10.1016/j.bbrc.2019.05.181Get rights and content

Highlights

  • GCN2 expression is associated with I/R-induced ischemic brain damage.

  • GCN2 suppression inhibits FoxO3a-regulated oxidative stress, apoptosis and ER stress.

  • GCN2iB, a GCN2 inhibitor, inhibits oxidative stress and ER stress induced by cerebral ischemia in mice.

Abstract

Ischemic stroke is one of the leading causes of morbidity and mortality among human worldwide. Unfortunately, cerebral I/R still lacks effective therapeutic targets and strategies. In the study, we found that general control nonderepressible 2 (GCN2) expression was increased following ischemia in the ischemic penumbra in vivo and in vitro. GCN2 suppression using its significant inhibitor, GCN2iB, exhibited a protective role in cerebral I/R injury in mice, as evidenced by the improved neurological deficits and function. GCN2 inhibition with either GCN2iB or genetic knockdown led to significant reduction of pro-apoptotic protein expression, endoplasmic reticulum stress (ERS)-related protein and oxidative stress both in I/R-induced cerebral injury and oxygen-glucose deprivation and reoxygenation (OGD/R) stimulation in N2a cells. OGD/R-triggered apoptosis and ERS were significantly depended on oxidative stress in vitro. In addition, Forkhead box O 3a (FoxO3a), involved in the reactive oxygen species (ROS) production, was increased during OGD/R stimulation-regulated apoptosis and ERS, which could be abrogated by GCN2 suppression. Consistently, FoxO3a-regulated generation of ROS was markedly ameliorated upon GCN2 suppression with GCN2iB. Thereby, our findings indicated that GCN2 suppression alleviated apoptosis and ERS in cerebral ischemia through reducing FoxO3a-dependent ROS production, illustrating that GCN2 could be a promising target for the therapeutic interventions in cerebral ischemic stroke.

Introduction

Ischemic stroke is one of the leading causes of morbidity and mortality worldwide [1]. Despite rapid reperfusion is a significant therapeutic strategy for ischemic stroke. It can paradoxically result in elevated cerebral ischemia and reperfusion (IR) injury [2]. The molecular mechanisms of IR injury include multiple pathophysiological processes, such as abnormal apoptosis, oxidative stress and endoplasmic reticulum stress (ERS) [3,4]. As the importance of cerebral ischemic stroke is becoming increasingly evident, it is critical to develop new therapies to prevent cerebral injury.

General control nonderepressible 2 (GCN2) was first reported as a sensor of amino acid availability in yeast [5]. Under conditions of amino acid deprivation, GCN2 could phosphorylate eukaryotic initiation factor 2 alpha (eIF2α) to increase amino acid biosynthesis, and this process was tightly associated with the progression of ERS [6]. GCN2 elevates myocardial oxidative stress under stress conditions by promoting its down-streaming signals [7]. GCN2 ablation ameliorated transverse aortic constriction (TAC)-induced cardiac dysfunction associated with the inhibition of cardiomyocyte apoptosis [8]. GCN2 knockout protects against high-fat diet (HFD)-induced hepatic steatosis and insulin resistance [9]. Additionally, GCN2 deficiency was involved in the improvement of hippocampal synaptic plasticity and memory [10]. Thus, we hypothesized GCN2 inhibition might be implicated in the development of cerebral ischemia. However, if reducing GCN2 expression with its inhibitor GCN2iB can alleviate cerebral I/R injury and its possible mechanism remains unknown.

In this study, we explored if GCN2 suppression could regulate MCAO-induced cerebral I/R injury. We also investigated the potential molecular mechanisms implicated in the effects of GCN2 suppression on apoptosis, oxidative stress and ERS.

Section snippets

Experimental animals

The adult, male C57BL/6 mice (20–25 g) were obtained from the Laboratory Animal Center of the Shandong University (Shandong, China). All procedures and ethics guidelines were approved by the Committee for Experimental Animal Use and Care of the Shandong Provincial Hospital Affiliated To Shandong University, China. Efforts here were made to minimize the number of animals used and their suffering. The AAV9 vectors carrying GCN2 (AAV-GCN2) were constructed by GeneChem Co., Ltd. (Shanghai, China).

GCN2 expression was up-regulated after cerebral ischemia and GCN2iB treatment alleviated cerebral ischemia in mice following I/R-induced ischemic brain damage

Focal ischemia induced by MCAO led to increased brain damage with reperfusion time (12, 24, 48 or 72 h) following I/R, along with significantly up-regulated infarction area (Fig. 1A). RT-qPCR and western blot analysis indicated that GCN2 mRNA and protein levels in ischemic penumbra were markedly increased at different time points following I/R (Fig. 1B and C). Besides, OGD/R led to the increase of GCN2 in N2a cells as MCAO mice at different time points of reoxygenation (Fig. 1D and E). These

Discussion

In this study, we attempted to explore if targeting GCN2 could alleviate cerebral I/R injury and the underlying molecular mechanisms that controlled this process. The effect of GCN2 was first calculated using a classical animal model of I/R by MCAO operation. GCN2 over-expression markedly promoted the infarct size. However, GCN2 suppression alleviated MCAO-induced oxidative stress, apoptotic and ERS protein expression in vivo to improve cerebral ischemic injury. At the same time,

Acknowledgement

National Natural Science Foundation of China (81660210); The Key Research and Development Foundation of Shaanxi Province, China (2016SF-173, 2018SF-107); Science and Technology Project of Xi'an, Shaanxi Province, China (2017114SF/YX008(3)).

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