Mitochondrial Fidelity in Mammalian Neurons

摘要 线粒体是多方面的细胞器，在包括能量在内的多种细胞功能中发挥着重要作用。 产生、新陈代谢、钙稳态和细胞死亡。人们普遍认为， 线粒体质量是线粒体功能障碍、衰老的关键因素，并代表着 几种神经性疾病的融合。然而，功能失调的线粒体究竟是如何导致 这些条件仍然难以捉摸。线粒体被认为通过协作不断恢复活力 有丝分裂、裂变-融合过程和多层次质量控制机制。因此，平均 据估计，线粒体蛋白质在大脑中的半衰期不到3周。最近，我发现了一个 在小鼠脑内持续至少四个月的线粒体长寿命蛋白(mt-LLP)的离散数量 心。这些长寿命线粒体蛋白(mt-LLP)包括OxPhos复合体和几个线粒体 冠状突起相关蛋白，类似于其他结构稳定和寿命长的结构(即核 孔道复合体)因其高度定义和精细的超微结构而被公认。因此，我们假设 Mt-LLP的超长寿命可以在经济的长期稳定中发挥重要作用 长寿的有丝分裂后细胞中的线粒体脊。 这项研究计划的目标是描绘mt-LLPs在神经元线粒体中的定位， 检查它们的时间动力学和与新合成的蛋白质的整合，并研究它们的潜力 对线粒体的适合性和长期的脊稳定性的贡献。在目标1中，使用脉冲追逐的组合 蛋白质标记方法，超分辨荧光成像和质谱学我建议(1) 初级神经元轴突和体树突域内mt-LLPs的时空动态。在AIM 2，我们建议通过研究配位作用将我们的分析扩展到包括线粒体DNA(MtDNA) Mt-LLPs的丰富与mtDNA长寿神经元之间的关系。在《目标3》中，我将探讨这一机制(S) 利用针对线粒体脊的遗传操作参与mt-ELLPs在神经元中的持久性 稳定性。最后，在目标4中，我们将开始研究核和线粒体之间的协调 神经元的基因组表达。总而言之，从拟议的实验中获得的见解将显著提高 我们对神经元线粒体蛋白质组长期稳态和基因组完整性的理解，这是 可以为调控线粒体网络动态过程提供新的分子靶点 神经退行性变。

Summary Mitochondria are multifaceted organelles that play vital roles in a myriad of cellular functions, including energy production, metabolism, calcium homeostasis, and cell death. It is generally accepted that a decline in mitochondria quality is a key contributor to mitochondrial dysfunction, aging, and represents a key point of convergence for several neurological disorders. Yet, precisely how dysfunctional mitochondria contribute to these conditions remains elusive. Mitochondria are thought to be constantly rejuvenated via collaborative processes of mitogenesis, fission-fusion, and multi-level quality-control mechanisms. Accordingly, the average half-life of mitochondrial proteins in the brain has been estimated at less than 3 weeks. Recently, I identified a discrete number of mitochondrial long-lived proteins (mt-LLPs) that last at least four months in mouse brain and heart. These long-lived mitochondrial proteins (mt-LLPs) include OxPhos complexes and several mitochondrial cristae associated proteins, which similarly to other architecturally stable and long-lived structures (i.e. nuclear pore complexes) are recognized for their highly defined and elaborate ultrastructure. Therefore, we hypothesized that the exceptional longevity of mt-LLPs could play an essential role in the long-term stabilization of the mitochondrial cristae in long-lived, post-mitotic cells. The goal of this research proposal is to delineate the localization of mt-LLPs within mitochondria in neurons, examine their temporal dynamics and integration with newly synthesized proteins, and investigate their potential contribution to mitochondrial fitness and long-term cristae stability. In Aim 1, using a combination of pulse-chase protein labeling methods, super-resolution fluorescent imaging and mass spectrometry I propose to (1) examine the spatio-temporal dynamics of mt-LLPs in axonal and somato-dendritic domains of primary neurons. In Aim 2, we propose to extend our analysis to include mitochondrial DNA (mtDNA) by investigating the coordination between mt-LLPs enrichment and mtDNA longevity neurons. In Aim 3, I will investigate the mechanism(s) involved in persistence of mt-ELLPs in neurons using genetic manipulations targeting mitochondrial cristae stability. Lastly, in Aim 4 we will begin the investigation into the coordination between nuclear and mitochondrial genome expression in neurons. In summary, insights from the proposed experiments will significantly advance our understanding of long-term of mitochondrial proteome homeostasis and genome integrity in neurons, which could provide with new molecular targets for modulating the mitochondrial network dynamics in the processes of neurodegeneration.