自噬相关基因ATG7在缺氧缺血性脑损伤中的作用与机制研究

Hypoxic-ischemic encephalopathy (HIE) remains an incurable disease. Despite decades of research there are currently no interventions to reverse the cellular consequences of HIE in patients. In our previous work, we made two important discoveries. First, we found autophagy-related gene 7 (Atg7) levels significantly increased after 3 hours in HIE patients and showed clear gender difference; Second, we developed a new model of asphyxial cardiac rest (ACA) in rats that produce region specific neurodegeneration with apoptotic and non-apoptotic phenotypes, and functional outcome deficits proportional to the duration of asphyxia in patients, most importantly, autophagic stress-a novel cell death/survive pathway in a sex-dependent manner. Although the role of autophagy is controversial, autophagy mostly plays a pro-survival role after ischemia. Classic autophagy is initiated by nutrient and amino acid deprivation, glucagon, and cAMP. Phosphorylation of pre-autophagosomal membranes, conjugation to Atg12-Atg5 and Atg8/light chain 3(LC3) complexes regulated by Atg7. Here we hypothesize that global cerebral hypoxia-ischemia initiates sex-specific neuronal autophagic pathway; Atg7 plays a significant role during this pathway which can be regulated by estrogen. In order to address this hypothesis, we set up the following specific aims: 1. Fully characterize autophagy and Atg7 after ACA in male and female rats and after oxygen-glucose deprivation (OGD) in sex segregated neurons; 2.Investigate the role of estrogen during autophagy after experimental models. We will apply both in vivo and in vitro models, using siRNA and cDNA technology for Atg7 to establish the role of autophagy, specially Atg7, in male and female models. The primary objective of this research proposal is to identify efficacious and sex-specific therapeutic strategies and the role of autophagy, specially Atg7, to serve as the foundation for clinical trials designed to improve outcome in HIE patients.

缺氧缺血性脑损伤严重危害患者的健康，目前尚无有效的治疗方法。我们在临床患者血清中首次发现该疾病导致细胞自噬基因7(ATG7)早期显著提高并呈现性别差异。我们根据这一现象并结合在体动物模型和体外细胞模型预实验结果提出：细胞自噬在缺氧缺血性脑损伤中的作用因损伤程度、演变时间和不同部位而异，并且Atg7在上游调控该细胞自噬现象；雌激素则通过调节Atg7参与调控细胞自噬小体的成熟（Atg7/Atg5-Atg12/LC3-I）从而参与受损神经元的保护作用。为了验证这一假说，我们拟采用qPCR、Western blot、腺病毒载体转染、RNA干扰等手段，从分子、细胞、组织以及动物整体水平等多方面探讨Atg7以及性别差异在缺氧缺血性脑损伤所致细胞自噬中的重要作用。本研究将从Atg7和细胞自噬这个新视点为揭示缺氧缺血性脑损伤的发生机制奠定基础，为治疗该类疾病提供新的靶向位点。

缺氧缺血性脑损伤（hypoxic-ischemic encephalopathy）是由多种原因引起脑缺氧和脑血流减少或暂停而导致的脑损伤，多发于胎儿或围生期新生儿，也常见于心肺复苏和颅内血管破裂或阻塞后的成年患者。目前该类疾病的临床治疗主要是支持治疗，即使在医学相对发达的美国，该疾病的致死率仍高达87%，并且在幸存者中，约50%留有严重的神经系统后遗症。近年来还没有有效的治愈该疾病的方案或方法，因此探索缺氧缺血性脑损伤的关键途径，寻找保护神经元的有效分子靶标，对正确干预这类脑损伤以及新型靶向药物的开发具有重要意义。缺氧缺血性脑损伤首先表现为大脑对营养和能量的缺乏，这一重要的生理病理变化是由细胞自噬调控的，且缺氧缺血性脑损后性别差异在自噬中的作用尚不清楚。我们在临床患者血清中首次发现该疾病导致细胞自噬基因7(ATG7)早期显著提高并呈现性别差异以及我们开发了一种新的缺血缺氧模型（asphyxial cardiac rest ACA），这种模型产生区域特异性的细胞凋亡和非凋亡表型的神经退行性疾病，且功能缺陷与缺血缺氧病人病人相似，最重要的是，细胞自噬应激性死亡/生存路径是性别依赖性的。细胞中敲掉ATG7基因在缺血缺氧情况下能显著减少神经元的死亡并呈性别差异。我们还发现脑缺血缺氧引发的性别特异性神经元自噬途径；Atg7起着重要的作用，这一通路可被雌激素调节,并且雌激素起着保护作用。本研究将从Atg7和细胞自噬这个新视点为揭示缺氧缺血性脑损伤的发生机制奠定基础，为治疗该类疾病提供新的靶向位点。