Mechanisms of Sensing and Responding to Lysosomal Stress in Neurons

所有细胞中的蛋白质平衡是通过调节蛋白质合成和蛋白质之间的平衡来维持的 退化。由于神经元非常大且寿命极长，因此神经元的维护 蛋白质组具有非同寻常的挑战性。破坏正常的蛋白质周转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 常与神经系统疾病有关。对于神经元如何监控和 处理蛋白平衡应激，以及轴突和树突如何适应不同的机制来完成这一任务 有效地。蛋白静止性应激的一个原因是溶酶体损伤，它比 之前意识到的。溶酶体损害的药物包括溶酶促性药(如LLOMe，氯喹， SSRIs)、神经毒性聚集体(如tau)和氧化应激(发生在缺血性中风后)。溶酶体 损伤导致溶酶体膜通透性(LMP)，pH和钙立即崩溃 梯度，逐渐形成溶酶体膜上的孔洞。尽管溶酶体对神经细胞是至关重要的 功能，通常与神经病理有关，神经元对溶酶体损伤的反应 人们对机械的细胞水平知之甚少。这项建议的重点是新的损害反应，我们 发现对LMP的反应和恢复的晚期内体快速反应(LERR)。 目前在非神经细胞中的研究发现，细胞对LMP的反应是首先尝试修复受损的 溶酶体(分钟)。如果修复失败，细胞通过溶血作用处理受损的溶酶体(在几个小时内) 启动新的溶酶体生物发生(24小时)。我们未发表的研究发现，未损坏的隔间 (尤其是LES)迅速改变它们的动态行为(约10分钟)。我们提出了一个新的假设 LES对LMP有快速反应，可在短期内维持中度降解性隔室。我们 提出两个具体目标。目的1：了解胞体、树突和轴突中的内小体对LMP的反应。 我们将使用生命感应器和皮层神经元的实时成像多路传输来确定LES的反应 以及胞体、树突和轴突中的溶酶体，以阐明LMP反应是如何适应的 到广袤的树枝状和轴突状乔木。我们假设树突室维持 通过停止与胞体中受损的溶酶体融合而具有中等的降解能力。 目的2：发现神经元的“快速反应”是否具有保护性。Rab7是LE成熟的主要调控因子。 我们假设，LE对LMP的反应和恢复正常需要Rab7效应器级联。 在LLMOe被淘汰之后。我们将利用药物抑制内容体成熟的关键节点和 转运结合Rab7干扰的急性途径，包括可光激活的Rab7显性 阴性(DN)和退化标记的Rab7-dN，用于快速干扰。实施拟议的工作符合 有望为神经元中蛋白质周转如何进行空间调控建立新的范例。

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.