Mechanisms of Sensing and Responding to Lysosomal Stress in Neurons

神经元溶酶体应激的感知和响应机制

• 批准号: 10509979
• 负责人: Bettina R Winckler
• 金额: $ 43.46万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10509979
• 关键词: Acute; Affect; Alzheimer' s Disease; Axon; Axonal Transport; Behavior; Biogenesis; Biology; Calcium; Cells; Cessation of life; Chloroquine; Data; Degradation Pathway; Dendrites; Disease; Distal; Dominant-Negative Mutation; Drug Targeting; Drug usage; Endosomes; Equilibrium; Esters; Exposure to; Funding Mechanisms; Future; Genes; Hour; Image; Impairment; Incubated; Ischemia; Ischemic Stroke; Knowledge; Lead; Link; Lipids; Longevity; Lysosomes; Maintenance; Membrane; Microfluidic Microchips; Molecular; Monitor; Neurodegenerative Disorders; Neuroglia; Neuronal Dysfunction; Neurons; Niemann-Pick Diseases; Organelles; Organism; Oxidative Stress; Pathologic; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Protein Biosynthesis; Proteome; Protons; Regulation; Research; Role; Selective Serotonin Reuptake Inhibitor; Signal Transduction; Stress; Stroke; System; Testing; Time; Work; extracellular; first responder; improved; insight; late endosome; monomer; multicatalytic endopeptidase complex; nervous system disorder; neurological pathology; neuronal cell body; neurotoxic; novel; prevent; protein degradation; proteostasis; protonation; recruit; repaired; response; sensor; stressor; tau Proteins; tool; translational approach; unpublished works

Protein homeostasis in all cells is maintained by regulating the balance between protein synthesis and protein degradation. Since neurons are extraordinarily large and extremely long-lived, maintenance of the neuronal proteome is unusually challenging. Disruption of normal protein turnover can lead to accumulation of toxic aggregates, neuronal dysfunction, and death. Not surprisingly then, genes linked to degradative pathways are frequently linked to diseases of the nervous system. There is a big knowledge gap for how neurons monitor and handle proteostatic stress, and how axons and dendrites might have adapted different mechanisms to do this effectively. One cause of proteostatic stress is lysosomal damage which occurs much more frequently than previously realized. Agents of lysosomal damage include lysosomotropic drugs (such as LLOMe, chloroquine, SSRIs), neurotoxic aggregates (such as tau), and oxidative stress (as occurs after ischemic stroke). Lysosomal damage results in lysosomal membrane permeabilization (LMP) with immediate collapse of pH and calcium gradients, progressing to holes in the lysosomal membrane. Even though lysosomes are critical to neuronal function and often causally linked to neurological pathologies, the response of neurons to lysosomal damage on a mechanistic cellular level is poorly understood. This proposal focuses on new a damage response we discovered (late endosome rapid response “LERR”) for responding to and recovering from LMP. Current work in non-neuronal cells has discovered that cells respond to LMP by first trying to repair the damaged lysosome (in minutes). If repair fails, the cell disposes of damaged lysosomes via lysophagy (in hours) and initiates new lysosome biogenesis (24 hours). Our unpublished work discovered that undamaged compartments (especially LEs) rapidly change their dynamic behavior (in ~10 minutes). We pose the novel hypothesis that LEs mount a rapid response to LMP to maintain moderately degradative compartments in the short term. We propose two specific aims. Aim 1: Discover how endosomes in soma, dendrites, and axons respond to LMP. We will use vital sensors and multiplexing by live imaging of cortical neurons to determine the response of LEs and lysosomes in the soma, the dendrites, and the axon in order to elucidate how LMP responses are adapted to the great expanse of dendritic and axonal arbors. We hypothesize that dendritic compartments maintain moderate degradative capacity by halting fusion with damaged lysosomes in the soma. Aim 2: Discover if the neuronal “LE rapid response” is protective. Rab7 is the master regulator of LE maturation. We hypothesize that Rab7 effector cascades are required for the LE response to LMP and for return to normal after LLMOe washout. We will use pharmacological inhibition of key nodes of endosome maturation and transport in combination with acute approaches of Rab7 interference, including photoactivatable Rab7-dominant negative (DN) and degron tagged-Rab7-DN for rapid interference. Carrying out the proposed work holds the promise of establishing new paradigms for how protein turnover is spatially regulated in neurons.

所有细胞中的蛋白质稳态都是通过调节蛋白质合成和蛋白质合成之间的平衡来维持的。 降解由于神经元是非常大和非常长的寿命，维护神经元 蛋白质组学是一个非常具有挑战性的课题。正常蛋白质周转的中断可导致有毒物质的积累。 聚集、神经元功能障碍和死亡。毫不奇怪，与降解途径相关的基因 通常与神经系统疾病有关。对于神经元如何监测和控制， 处理蛋白质稳定压力，以及轴突和树突如何适应不同的机制来做到这一点 有效地蛋白质抑制应激的一个原因是溶酶体损伤，其发生频率远高于 以前实现的。溶酶体损伤剂包括亲溶酶体药物（如LLOMe、氯喹、氯喹、苯丙氨酸）。 SSRIs）、神经毒性聚集体（如tau）和氧化应激（如缺血性卒中后发生的）。溶酶体 损伤导致溶酶体膜透化（LMP），pH和钙立即崩溃 梯度，进展到溶酶体膜上的孔。尽管溶酶体对神经元的 神经元对溶酶体损伤的反应，通常与神经病理学有因果关系。 对细胞水平的机制了解甚少。这项建议的重点是新的损害反应， 发现（晚期内体快速反应“LERR”）用于响应LMP和从LMP恢复。 目前在非神经元细胞中的研究发现，细胞对LMP的反应是首先试图修复受损的细胞， 溶酶体（分钟）。如果修复失败，细胞会通过噬菌体（lysophagy）（数小时）处理受损的溶酶体， 启动新的溶酶体生物发生（24小时）。我们未发表的研究发现， （尤其是LE）快速改变其动态行为（在约10分钟内）。我们提出了一个新的假设， LE对LMP快速响应，以在短期内维持中度降解隔室。我们 提出两个具体目标。目的1：探索索马、树突和轴突中的内体对LMP的反应。 我们将使用重要的传感器和多路复用通过大脑皮层神经元的实时成像来确定LE的反应 以及索马、树突和轴突中的溶酶体，以阐明LMP反应如何适应 到广阔的树枝状和轴突乔木。我们假设树突状区室维持着 通过停止与索马中受损溶酶体的融合而具有中等降解能力。 目的2：发现神经元的“LE快速反应”是否具有保护作用。Rab 7是LE成熟的主要调节因子。 我们假设Rab 7效应级联是LE对LMP的反应和恢复正常所必需的 在LLMOe清洗之后。我们将使用药物抑制内体成熟的关键节点， 转运结合Rab 7干扰的急性方法，包括光活化Rab 7显性 阴性（DN）和降解决定子标记的Rab 7-DN用于快速干扰。开展拟议的工作， 有望为神经元中蛋白质周转的空间调控建立新的范式。