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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之外，线粒体对于Ca 2+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量学失败的情况，如缺血再灌注损伤，创伤和毒性暴露的会聚点。然而，功能失调的线粒体导致这些病症的病理学的确切机制仍然未知。为了保持体内平衡并最大限度地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，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，我们称之为极长寿蛋白质（ELLP）。由于其持久性，ELLP可能会在较长时间内积累损害，从而产生先前被低估的细胞脆弱性层。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，并持续到小鼠的整个生命周期。我们推测这些新发现的线粒体ELLPs（mito-ELLPs）在神经退行性变和细胞对应激和损伤的反应过程中起着未被充分认识的作用。本研究的目的是鉴定和表征哺乳动物神经元中的线粒体ELLPs。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）的蛋白质组学分析和自定义生物信息学策略，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，确定线粒体ELLPs是否优先定位于受损、不健康或老化的线粒体。我们将进一步描绘亚细胞定位的mito-ELLPs的共定位研究使用标记的突触，ER，MAMs，和mtDNA翻译。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蛋白质组。从这些实验中获得的见解将大大推进我们对线粒体-ELLP在神经元中作用的理解，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。