Netrin-1超量表达对缺血再灌注后神经元线粒体钙超载的影响及分子机制

Ischemia reperfusion induces penumbra neuronal damages after cerebral vascular occlusion/reperfusion, likely initiated from calcium overload of mitochondria that leads to neuronal apoptosis, and there is no effective prevention and cure method at present. Previous studies have shown that Netrin-1 overexpression promotes the recovery of penumbra neuronal function after ischemia reperfusion. Recent studies have also shown that Netrin-1 has mitochondrial protection. Here, we hypothesized that Netrin-1 may protect the structure and function of mitochondria through regulation of the mitochondrial Ca2+ concentration in penumbra neurons and suppression of the calcium overload. As a result, it can play the role of brain protection by alleviation of the damages on penumbra neurons after ischemia reperfusion. In this study, we aim to investigate the role and mechanism of Netrin-1 overexpression in the protection of penumbra neurons after ischemia reperfusion by regulating the concentration of mitochondrial Ca2+ in neurons, and to explore the experimental methods of non-invasive targeted intervention on the overexpression of Netrin-1 in the brain with adenovirus transfection and investigation into APPL1, PI3K/Akt pathway and MCU in the OGD/R cell model, MCAO rats and knockout mice. The purpose of this study is to provide a new theoretical basis and method for the clinical treatment of cerebral ischemia reperfusion injury.

半暗带神经元缺血再灌注可触发钙超载引起线粒体损伤导致神经元凋亡，是脑血管闭塞再通后引发进一步损伤的主要原因，目前尚无有效防治方法。本课题组前期研究发现，Netrin-1过量表达可促进半暗带神经元缺血再灌注后功能恢复。最新研究也表明Netrin-1具有线粒体保护作用。在此基础上，我们提出Netrin-1可通过调节半暗带神经元线粒体Ca2+浓度，抑制钙超载发生，保护线粒体结构和功能，减轻缺血再灌注后半暗带神经元损伤，进而发挥脑保护作用的假说。本课题利用腺病毒转染过表达Netrin-1，在OGD/R细胞模型、MCAO大鼠及基因敲除小鼠中针对APPL1、PI3K/Akt通路和MCU，研究超量Netrin-1对神经元线粒体Ca2+浓度调节在缺血再灌注后半暗带神经元保护中的作用及机制，并探讨非创伤性靶向干预脑Netrin-1表达的实验方法。本研究旨在为脑缺血再灌注损伤的临床治疗提供新的理论依据和方法。

缺血性脑卒中后血运重建导致的脑缺血再灌注(I/R)损伤与线粒体功能障碍、氧化应激和神经元丢失有关。在本研究中，我们使用氧剥夺/再氧化(OGD/R)模型来确定Netrin-1、AKT和线粒体AAA蛋白酶AFG3L2之间的相互作用是否会影响I/R后神经元线粒体功能。我们发现Netrin-1可以保护初级皮质神经元免受OGD/R诱导的细胞死亡，并调节线粒体活性氧(ROS)和Ca2+水平。采用免疫印迹法检测细胞中线粒体钙单转运体(MCU)亚单位的积累情况。虽然调控亚基MICU1和MICU2相对不受影响，但MCU调节(EMRE)必需亚基的聚集受到损害。在OGD/ R诱导的细胞中，EMRE的11 kDa形式显著减少。Netrin-1通过上调AFG3L2和AKT活化抑制EMRE和线粒体Ca2+水平的积累。AFG3L2缺失或AKT抑制增加了11 kDa EMRE水平。此外，AKT的过表达增加了Netrin -1敲低神经元在OGD/R后AFG3L2的表达。我们的研究结果表明，Netrin-1通过激活AKT增强AFG3L2蛋白的表达。我们还观察到，在I/R诱导的大鼠脑损伤模型中，Netrin-1的过表达显著减少了梗死面积，但AKT被抑制时则没有效果。我们的数据表明AFG3L2是AKT的蛋白底物，并表明Netrin-1通过激活AKT磷酸化和AFG3L2限制线粒体ROS和Ca2+水平来减轻脑I/R损伤。