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，在细胞的稳态和生存中起着关键的生化作用。除了产生ATP外，线粒体对Ca2+信号传导、神经递质代谢和活性氧（ROS）信号传导也很重要。线粒体功能障碍是几乎所有主要神经退行性疾病以及生物能量衰竭（如缺血-再灌注损伤、创伤和毒性暴露）的一个趋同点。然而，线粒体功能失调导致这些疾病的确切机制尚不清楚。为了维持体内平衡，尽量减少受损蛋白质和细胞器的影响，细胞蛋白质组不断被降解，为新的蛋白质组让路。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，我们发现了大脑中具有超长寿命的细胞内蛋白质，我们称之为极长寿命蛋白质（ellp）。由于它们的持久性，ellp可能会在较长时间内积累损害，从而产生先前被忽视的细胞脆弱性层。有趣的是，我们发现大脑线粒体蛋白质组的一小部分也很长寿，并在小鼠的整个生命周期中持续存在。我们假设这些新发现的线粒体ellp （mitto - ellp）在神经变性和细胞对应激和损伤的反应过程中发挥着未被充分认识的作用。本研究计划的目标是鉴定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踪标记，结合高分辨率霰弹枪质谱（MS）的蛋白质组学分析，以及定制的生物信息学策略，我们将定义小鼠脑提取物和原代神经元培养物中的线粒体长寿蛋白质组。此外，我们将通过结合先进的活细胞荧光技术和细胞器分选来确定mito- ellp是否优先定位于受损、不适应或老化的线粒体。我们将通过使用突触、ER、MAMs和mtDNA翻译标记物的共定位研究，进一步描述mito- ellp的亚细胞定位。最后，我们将研究已知影响线粒体功能的药物治疗是否会影响长寿的蛋白质组。从这些实验中获得的见解将大大促进我们对神经元中mito- ellp作用的理解，并可能为无数神经系统疾病的潜在治疗干预提供新的靶点。

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.