Exploring the mechanisms of dysfunctional mitochondrial quality control in cerebrovascular disease and the aging brain

项目摘要 线粒体功能障碍是许多主要残疾原因的一个突出因素，即脑血管疾病。 和神经退行性疾病。线粒体的功能和稳定性受到 线粒体动力学机制和质量控制（QC）。线粒体的动态本质是 由裂变和聚变的平衡力维持。这些线粒体动力学过程的运作， 保护线粒体网络的功能结构。线粒体QC的机制， 包括线粒体自噬、蛋白质稳态和生物发生，都是为了调节线粒体网络的组成部分 通过合成和降解。这些力量积极控制线粒体网络的功能 以确保高效的能源生产。在脑缺血/再灌注（I/R）损伤中，线粒体的过程 动力学和QC变得失调，导致代谢功能障碍和神经损伤。的 该提案的F99阶段旨在确定脑中线粒体动力学和QC中断的阶段。 I/R损伤及其分子机制。利用与机器相关的先进技术 学习，计算建模和活细胞成像，我已经创建了一个基于代理的线粒体模型， 这些调查的动力。该模型允许模拟个人的动态行为 线粒体单位达到顶峰的复杂模式通常在哺乳动物细胞中观察到。活细胞成像 来自新的转基因报道细胞系的小鼠原代皮层神经元（即，MitoTimer、MitoQC）和条件性 敲除系将用于观察单个动力学蛋白对细胞内蛋白质的各自贡献。 线粒体形态的模式。敲除神经元将暴露于氧葡萄糖剥夺（OGD）， I/R损伤的体外模型，和线粒体参数（即，形态学、氧化）将以真实的 时间来产生线粒体动力学的机械时间轴。这些活细胞记录将用于 优化和扩展我们的基于代理的模型，以允许在硅片上实验操作线粒体 proteins.我们的扩展模型将有能力测试关于基础率和病理率的假设 线粒体动力学和质量控制，以及为未来的实验提供信息，降低成本， 提高效率。在本提案的K 00阶段，我将从研究线粒体质量控制过渡到 在I/R中用于神经变性的研究。利用在博士前阶段获得的技术技能，我将 研究年龄相关的线粒体蛋白质稳态和关键的长寿线粒体蛋白质的变化， 突触K 00阶段旨在确定衰老如何影响突触线粒体蛋白的周转， 特别强调线粒体内蛋白质稳态和综合应激反应的作用。我打算 以确定线粒体蛋白质稳态对突触稳定性和相关神经变性的贡献。 这项工作在衰老和神经退行性变研究中具有重要意义，因为突触丢失和 线粒体功能障碍是与年龄相关的神经系统疾病的两个标志。

PROJECT SUMMARY Mitochondrial dysfunction is a prominent element of many leading causes of disability, namely cerebrovascular and neurodegenerative diseases. The function and stability of mitochondria are tightly regulated by the mechanisms of mitochondrial dynamics and quality control (QC). The dynamic nature of mitochondria is maintained by the balancing forces of fission and fusion. These processes of mitochondrial dynamics operate to preserve the functional architecture of the mitochondrial network. The mechanisms of mitochondrial QC, including mitophagy, proteostasis, and biogenesis, work to regulate the components of the mitochondrial network through synthesis and degradation. These forces actively control the functionality of the mitochondrial network to ensure efficient energy production. In cerebral ischemia/reperfusion (I/R) injury, the processes of mitochondrial dynamics and QC become dysregulated, contributing to metabolic dysfunction and neurological damage. The F99 phase of this proposal aims to identify the phases of disrupted mitochondrial dynamics and QC in cerebral I/R injury, and their respective molecular mechanisms. Utilizing advanced technologies related to machine learning, computational modeling, and live cell imaging, I have created an agent-based model of mitochondrial dynamics for these investigations. This model allows for the simulation of the dynamic actions of individual mitochondrial units to culminate in the complex patterns normally observed in mammalian cells. Live cell imaging of mouse primary cortical neurons from novel transgenic reporter lines (i.e., MitoTimer, MitoQC) and conditional knockout lines will be utilized to observe the respective contributions of individual dynamics proteins to the patterns of mitochondrial morphology. Knockout neurons will be exposed to oxygen glucose deprivation (OGD), an in vitro model of I/R injury, and mitochondrial parameters (i.e., morphology, oxidation) will be imaged in real time to generate a mechanistic timeline of mitochondrial dynamics. These live cell recordings will be used to optimize and expand our agent-based model to allow for in silico experimental manipulation of mitochondrial proteins. Our expanded model will have the ability to test hypotheses regarding the basal and pathological rates of mitochondrial dynamics and quality control, as well as inform future experiments with decreased costs and increased efficiency. In the K00 phase of this proposal, I will transition from studying mitochondrial quality control in I/R to its study in neurodegeneration. Utilizing the technical skills acquired in the predoctoral phase, I will investigate age-related changes in mitochondrial proteostasis and critical long-lived mitochondrial proteins at the synapse. The K00 phase aims to determine how aging affects the turnover of synaptic mitochondrial proteins, with specific emphasis on the roles of intramitochondrial proteostasis and the integrated stress response. I intend to determine the contribution of mitochondrial proteostasis to synaptic stability and related neurodegeneration. This work has significant implications in aging and neurodegeneration research, as synaptic loss and mitochondrial dysfunction are both hallmarks of age-related neurological diseases.