Investigating the role of extremely long-lived mitochondrial proteins in mammalian neurons

线粒体是一种动态的多功能细胞器，在细胞的内稳态和生存中发挥着关键的生化作用。除了产生ATP外，线粒体还对钙信号、神经递质代谢和活性氧(ROS)信号起着重要作用。线粒体功能障碍是几乎所有主要神经退行性疾病的汇合点，也是生物能量学失败的案例，如缺血再灌注损伤、创伤和中毒暴露。然而，功能失调的线粒体对这些疾病的病理作用的确切机制仍不清楚。为了保持动态平衡并将受损的蛋白质和细胞器的影响降至最低，细胞蛋白质组不断降解，为新的版本让路。有丝分裂后的长寿命细胞，如神经元，对这些过程特别敏感，因为它们不能通过细胞分裂来稀释大分子损伤。然而，在我们之前的研究中，我们在大脑中发现了具有极长寿命的细胞内蛋白质，我们称之为极长寿蛋白质(ELLP)。由于它们的持久性，ELLP可能会在更长的时间内积累损害，从而创建以前折扣的细胞脆弱性层。有趣的是，我们发现大脑线粒体蛋白质组的一小部分也是长寿的，并持续到老鼠的整个一生。我们推测，这些新发现的线粒体ELLP(mito-ELLP)在神经退化和细胞对应激和损伤的反应过程中发挥的作用被低估。这项研究计划的目标是识别和表征哺乳动物神经元中的线粒体ELLP。利用代谢稳定同位素脉冲追逐标记，结合基于高分辨率鸟枪质谱仪(MS)的蛋白质组学分析，以及定制的生物信息学策略，我们将在小鼠脑提取液和原代神经元培养中定义线粒体长寿命蛋白质组。此外，我们将结合先进的活细胞荧光技术和细胞器分类来确定mito-ELLP是否优先定位于受损、不合适或老化的线粒体。我们将通过使用突触、内质网、MAMs和mtDNA翻译的标记进行共定位研究，进一步描述mito-ELLP的亚细胞定位。最后，我们将调查已知的影响线粒体功能的药物治疗是否会影响长寿命的蛋白质组。来自这些实验的见解将极大地促进我们对mito-ELLP在神经元中作用的理解，并可能为无数神经疾病的潜在治疗干预带来新的靶点。

Mitochondria are dynamic multifunctional organelles that play pivotal biochemical roles in cell's homeostasis and survival. In addition to generating ATP, mitochondria are also important for Ca2+ signaling, neurotransmitter metabolism, and reactive oxygen species (ROS) signaling. Mitochondrial dysfunctions represent a point of convergence for almost all major neurodegenerative disorders, as well as cases of bioenergetics failures, such as ischemia-reperfusion injury, trauma, and toxic exposures. Yet, the exact mechanisms via which dysfunctional mitochondria contribute to the pathology of these conditions remains unknown. In order to maintain homeostasis and minimize the effect of damaged proteins and organelles, the cellular proteome is constantly degraded to make way for the new versions. Post-mitotic long-lived cells, such as neurons, are particularly sensitive to these processes since they cannot dilute the macromolecular damage though cell division. However, in our previous research we identified intracellular proteins in the brain with exceptionally long-life spans that we termed extremely long-lived proteins (ELLPs). Due to their persistence, ELLPs are likely to accumulate damage over longer periods and thus create a previously discounted layer of cell's vulnerability. Intriguingly, we found that a small subset of brain's mitochondrial proteome is also long-lived and persists for the entire lifetime of a mouse. We hypothesize that these newly identified mitochondrial ELLPs (mito-ELLPs) play an underappreciated role in the processes of neurodegeneration and cell's response to stress and injury. The goal of this research proposal is to identify and characterize mitochondrial ELLPs in mammalian neurons. Using metabolic stable isotope pulse-chase labelling, together with high-resolution shotgun mass spectrometry (MS)-based proteomic analysis, and custom bioinformatics strategies, we will define the mitochondrial long-lived proteome in mouse brain extracts and primary neuron cultures. Furthermore, we will determine whether mito-ELLPs localize preferentially to damaged, unfit, or aged mitochondria by combining advanced live cell fluorescent techniques and organelle sorting. We will further delineate subcellular localization of mito-ELLPs by co-localization studies using markers of synapses, ER, MAMs, and mtDNA translation. Lastly, we will investigate whether pharmacological treatments known to affect mitochondrial function affect the long- lived proteome. Insights from these experiments will significantly advance our understanding of the role of mito- ELLPs in neurons and could lead to new targets for potential therapeutic interventions for a myriad of neurological disorders.