脑缺血再灌注损伤中GOLPH3及其QT-Motif磷酸化对线粒体质量控制的调控机制研究

Increasing evidence suggests that the Golgi apparatus is a sensor and common downstream-effector of stress signals in cell death pathways. It is described by the term of “Golgi Apparatus Stress, GAS”, a stress repairing response analogous to ER or Mitochondrial stress. Our recent studies indicate that GOLPH3 (G3), a peripheral membrane protein of Trans-Golgi Network (TGN) involved in cell signal transduction, can potentially be mobilized by oxidative stress and act as a GAS marker. In patients with primary disorders of mitochondrial diseases－heritable mitochondrial gene defects such as CPEO (chronic progressive external ophthalmopelia) or MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), G3 expression was increased approximately two-fold compared to normal muscle fibers. Moreover, during the secondary mitochondria disorder induced by the depletion of mtDNA, prompt up-regulation of G3 was also found to increase the total mitochondrial mass and maintain the cellular oxidative homeostasis. Above evidence suggests that G3 is specificly sensitive to mitochondrial damage and may involve in mitochondria homeostasis, whereas the detailed mechanisms are poorly understood. Mitochondrial Quality Control (QC) is essential for the maintenance of mitochondria and cell survival especially for neurons during oxidative stress injury. The induction of mitochondrial biogenesis, mitochondrial autophagy (mitophagy), and/or the dynamic control of apoptosis is thought to be main strategies for the Mitochondrial Quality Control (QC). We thus hypothesize that G3 is a critical regulator of mitochondrial QC during ischemic oxidative injury. In order to confirm this hypothesis and further elucidate the specific mechanisms, we will use pharmacological and genetic intervention methods in the present study, to determine the role of G3 and its QT-Motif phosphorylation in both vitro and vivo models of Cerebral Ischemia Reperfusion (I/R) injury with Neuro-2a cells and SD rats.

研究表明高尔基体能够感知氧化应激损伤并参与应激，即“高尔基体应激 (Golgi Apparatus Stress, GAS)”。GOLPH3 (G3)来源于高尔基体，我们研究发现G3在缺血再灌注 (OGD/R)模型Neuro-2a细胞中表达显著上调，通过调控自噬、ROS生成以及高尔基体结构介导GAS的发生，是高尔基体应激标志蛋白。研究发现在线粒体DNA剔除及mtDNA异常疾病中G3表达特异性显著上调，提示G3介导的GAS可能参与线粒体损伤后稳态的维持，具体机制尚不明确。线粒体稳态维持对神经元存活至关重要，我们认为，G3其及QT-Motif磷酸化在介导GAS过程中能够通过调节线粒体自噬及线粒体相关凋亡参与应激后线粒体质量控制。本课题中我们将构建体外及在体缺血再灌注模型，采用基因及药物干预的方法，系统评价G3及其磷酸化在缺血/再灌注损伤中对线粒体质量控制的调控及具体机制。

我们课题组前期的研究阐明了来源于高尔基体的结构及转运蛋白GOLPH3 (G3) 可以通过调控自噬、ROS生成以及高尔基体结构参与到高尔基体应激 (Golgi Apparatus Stress, GAS)，是高尔基体应激的标志蛋白。最新研究发现，在线粒体DNA剔除及mtDNA异常疾病中观察到G3表达呈特异性显著性上调，提示G3介导的GAS可能参与线粒体损伤后稳态的维持，具体机制尚不明确。本课题中我们构建了体外缺血再灌注OGD//R损伤模型及DNA损伤药物干预模型，运用磷酸化抗体设计制备，基因位点突变转染，WB，RT-PCR等一系列技术，系统评价了G3及其TQ Motif中T143位点磷酸化对线粒体应激、线粒体自噬及线粒体结构蛋白表达的影响。经课题组研究证实，G3通过减轻线粒体应激，抑制线粒体自噬，维持高尔基体结构稳定这三个方面的作用，参与到线粒体的质量控制，且这一过程受G3 TQ Motif T143位点磷酸化调控。我们的研究进一步明确了高尔基体应激(GAS)与G3蛋白相关的具体机制，将为氧化应激损伤中线粒体质量控制(QC)、线粒体保护提供新的靶点和可能。