Regulation of DRP1-Mediated Mitochondrial Fragmentation in Ischemic Brain Injury

脑血管疾病引起的急性损伤是导致显著发病率和死亡率的原因。在缺血性脑损伤过程中，线粒体发生分裂，导致促凋亡因子的释放，促凋亡因子导致细胞死亡。线粒体分裂机制，特别是动力蛋白相关蛋白1（Drp 1），已经被认为是细胞死亡的介质。Drp 1受一系列复杂的翻译后修饰的调控;然而，对Drp 1调控的全面了解仍有待开发。我的长期目标是描述钙依赖性蛋白磷酸钙调磷酸酶（CaN）和Drp 1之间的相互作用。这是我的假设，在缺血期间钙内流激活CaN去磷酸化Drp 1，这反过来又碎片线粒体，促进凋亡和坏死的神经元死亡。该提案的具体目的是开发一个全面的模型CaN调节Drp 1和研究急性缺血性损伤后神经元这种调节的病理生理后果。目的1将确定CaN与Drp 1结合和去磷酸化的分子决定因素。重组CaN将用于磷酸盐测定和等温滴定量热法，以表征Drp 1上的Can对接位点的修饰如何影响Can反应循环。此外，CaN：：Drp 1复合物形成的分子决定因素将在完整细胞中通过使用共免疫沉淀研究和蛋白质印迹分析与磷酸化状态特异性Drp 1的Drp 1上的CaN对接位点的遗传操作进行研究。这些研究的目的是首先表征Can活性如何通过靶底物上的Can对接位点进行调节，其次对缺血性损伤中钙诱导的线粒体片段化进行详细的机制理解。目的2将表征海马神经元中CaN调节Drp 1的路径生理影响。为了模拟中风后CaN对Drp 1的调节，将培养的海马神经元暴露于毒性水平的谷氨酸或在CaN：：Drp 1复合物的受控形成和活性的背景下模拟缺血。神经元损伤后，先进的荧光显微镜技术将用于探测CaN调节Drp 1在决定线粒体形态，细胞内钙管理和细胞存活中的作用。这些研究的目的是评估潜在的CaN：：Drp 1复合物作为一种新的分子靶点治疗干预神经元损伤。

Acute injury due to cerebrovascular disease is responsible for significant morbidity and mortality. During ischemic brain injury mitochondria undergo fission resulting in the release of proapoptotic factors which contribute to cell death. The mitochondrial fission machinery, specifically dynamin-related protein 1 (Drp1), has been implicated as a mediator of cell death. Drp1 is regulated by a complex series of posttranslational modifications; however, a full understanding of Drp1 regulation remains to be developed. My long term goal is to characterize the regulatory interaction between the calcium-dependent protein phosphates calcineurin (CaN) and Drp1. It is my hypothesis that calcium influx during ischemia activates CaN to dephosphorylate Drp1, which in turn fragments mitochondria to promote apoptotic and necrotic neuronal death. The specific aims of this proposal are designed to develop a comprehensive model of CaN regulation of Drp1 and investigate the pathophysiological consequences of this regulation in neurons following acute ischemic injury. Aim 1 will identify the molecular determinants for CaN binding to and dephosphorylation of Drp1. Recombinant CaN will be used for phosphates assays and isothermal titration calorimetry to characterize how modification of the Can docking site on Drp1 influences the Can reaction cycle. Further, the molecular determinants of CaN::Drp1 complex formation will be investigated in intact cells by genetic manipulation of a CaN docking site on Drp1 using co-immunoprecipitation studies and western blot analysis with a phospho-state specific Drp1. The objective of these studies will be to first characterize how Can activity is modulated by the Can docking site on target substrates and second to develop a detailed mechanistic understanding of calcium-induced mitochondrial fragmentation in ischemic injury. Aim 2 will characterize the path physiologic impact of Drp1 regulation by CaN in hippocampal neurons. To model the regulation of Drp1 by CaN following stroke, cultured hippocampal neurons will be exposed to toxic levels of glutamate or simulated ischemia in the background of controlled formation and activity of the CaN::Drp1 complex. Following neuronal injury, advanced fluorescence microscopy techniques will be used to probe the role of CaN regulation of Drp1 in dictating mitochondrial morphology, intracellular calcium management and cell survival. The objective of these studies is to assess the potential of the CaN::Drp1 complex as a novel molecular target for therapeutic intervention following neuronal injury.