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

研究极长寿命线粒体蛋白在哺乳动物神经元中的作用

基本信息

批准号：
9611350
负责人：
Ewa Karolina Bomba-Warczak
金额：
$ 5.87万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9611350
关键词：
Address Affect Animals Antimycin A Autophagocytosis Axon Biochemical Bioenergetics Biogenesis Bioinformatics Biological Models Biology Brain Cell Death Cell division Cells Cellular Structures Characteristics Color Complex Custom Degradation Pathway Dendrites Economics Ensure Event Face Failure Goals Half-Life Health Homeostasis Hour Human Impairment In Vitro Individual Injury Isotope Labeling Label Lead Life Longevity Mammals Mass Spectrum Analysis Metabolic Methods Mitochondria Mitochondrial DNA Mitochondrial Proteins Mitotic Modernization Molecular Mus Nature Nerve Degeneration Neurodegenerative Disorders Neurodevelopmental Disorder Neurons Neurosciences Nuclear Nuclear Envelope Nuclear Pore Complex Nucleosomes Organelles Organism Oxidation-Reduction Pathology Pathway interactions Pharmacological Treatment Pharmacology Physiologic pulse Play Process Proteins Proteome Proteomics Quality Control Reactive Oxygen Species Reperfusion Injury Research Research Proposals Resolution Rodent Role S-nitro-N-acetylpenicillamine Shotguns Signal Transduction Sirolimus Site Societies Sorting - Cell Movement Stable Isotope Labeling Stimulation of Cell Proliferation Stress Synapses Techniques Therapeutic Intervention Time Toxicant exposure Translations Vesicle Yeasts aged base brain tissue cell injury cost discount experience experimental study fitness in vivo insight live cell imaging mitochondrial dysfunction mitochondrial fitness nervous system disorder neuronal cell body neurotransmitter metabolism protein degradation protein distribution response senescence stable isotope tandem mass spectrometry trauma exposure

项目摘要

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.

线粒体是动态的多功能细胞器，在细胞的稳态和生存中发挥着关键的生化作用。除了产生 ATP 之外，线粒体对于 Ca2+ 信号传导、神经递质代谢和活性氧 (ROS) 信号传导也很重要。线粒体功能障碍代表了几乎所有主要神经退行性疾病以及生物能学失败的病例，例如缺血再灌注损伤、创伤和有毒物质暴露。然而，线粒体功能障碍导致这些病症病理的确切机制仍然未知。为了维持体内平衡并尽量减少受损蛋白质和细胞器的影响，细胞蛋白质组不断降解，为新版本让路。有丝分裂后的长寿细胞，例如神经元，对这些过程特别敏感，因为它们无法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，我们发现大脑中的细胞内蛋白质具有极长的寿命，我们将其称为极长寿命蛋白质（ELLP）。由于其持久性，ELLP 可能会在较长时间内累积损伤，从而形成先前被低估的细胞脆弱层。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，并且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 (mito-ELLP) 在神经退行性变以及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。使用代谢稳定同位素脉冲追踪标记，结合基于高分辨率鸟枪质谱 (MS) 的蛋白质组分析和定制生物信息学策略，我们将定义小鼠大脑提取物和原代神经元培养物中的线粒体长寿命蛋白质组。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、不健康或老化的线粒体。我们将通过使用突触、ER、MAM 和 mtDNA 翻译标记的共定位研究，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。这些实验的见解将显着增进我们对 mito-ELLP 在神经元中的作用的理解，并可能为多种神经系统疾病的潜在治疗干预找到新的靶点。