Effects of Aging on Neuronal Lysosomal Damage Responses Driven by CMT2B-linked Rab7

项目摘要/摘要内体-溶酶体系统负责细胞蛋白质降解， 并因此用于所有细胞中的蛋白质稳态。神经元是空间复杂的大细胞类型， 必须在生物体的一生中存活。这使得神经元蛋白质组的维持异常 挑战性溶酶体蛋白质降解的破坏可导致毒性聚集体、神经元聚集体和神经元聚集体的积累。 功能障碍和死亡正常的蛋白质降解可以通过对溶酶体的直接损伤而被破坏， 导致腔溶酶体内容物在称为溶酶体膜的过程中渗漏到胞质溶胶中 透化作用（LMP），其可促进细胞死亡。LMP通常与神经病理学有因果关系 不成比例地影响老龄化人口。尽管溶酶体对神经元蛋白质稳态至关重要， 神经元对LMP的反应在机制水平上知之甚少。此外，尽管亲密 由于核内体和溶酶体功能之间的关系，核内体在LMP应答中的作用尚不清楚。 小GTdR ab 7是晚期内体和溶酶体的主要调节物。这也是至关重要的， 自噬Rab 7的突变导致Charcot Marie-Tooth 2B（CMT 2B）疾病，一种进行性外周血管疾病， 神经病变我的初步数据表明Rab 7是LMP的急性反应者。在LMP之后，Rab 7 聚集在各种扩大的囊泡隔室上，包括晚期内体（LE）和溶酶体（Lys）， 处于过度活跃的GTP结合状态在神经元内，这种反应只局限于索马。在这 根据该提案，我的目标是发现（1）Rab 7激活下游LMP反应的要求，例如 半乳糖凝集素-3在赖氨酸上的积累和LE的扩大，（2）CMT 2B突变等位基因对已知LMP的影响 LMP诱导后Rab 7的反应和激活，以及（3）年龄对Rab 7介导的神经元细胞凋亡的影响。 降解和LMP响应。我认为Rab 7激活在协调早期反应中至关重要， LMP中的内体损伤控制。 我将检验这一假设，即LMP后Rab 7的激活是介导LMP的一种重要反应。 神经元中的保护途径，其在CMT 2B突变等位基因中被破坏。在目标1中，我将探讨 Rab 7在（1）半乳糖凝集素-3介导的噬菌体和（2）响应于LMP的LE空泡化中的活化。在目标2中， 本人将测试在CMT 2B中，LMP对Lys/LE反应和Rab 7激活的反应是否受到影响。在目标3中， 我将确定老化是否破坏Rab 7介导的（1）货物降解和（2）野生型中的LMP反应。 型和突变型CMT 2B神经元。总之，这些实验将确定体内平衡机制， 解决溶酶体损伤的神经元以及这是否在疾病中受到影响。我的长期目标是发现 并表征LMP中溶酶体破坏的代偿性内体反应机制， 广泛适用于神经系统疾病的最终设计更好的治疗。

Project Summary/Abstract The endosomal-lysosomal system is responsible for cellular protein degradation, and thus for protein homeostasis (proteostasis) in all cells. Neurons are spatially complex, large cell types, and must survive for the lifetime of an organism. This makes maintenance of the neuronal proteome unusually challenging. Disruption of lysosomal protein degradation can lead to accumulation of toxic aggregates, neuronal dysfunction, and death. Normal protein degradation can be disrupted via direct damage to lysosomes, which leads to leakage of luminal lysosomal contents into the cytosol in a process known as lysosomal membrane permeabilization (LMP), which can facilitate cell death. LMP is often causally linked to neurological pathologies that disproportionately affect aging populations. Even though lysosomes are critical to neuronal proteostasis, the response of neurons to LMP on a mechanistic level is poorly understood. Further, despite the intimate relationship between endosomal and lysosomal function, the role of endosomes in LMP responses is unknown. The small GTPase Rab7 is a master regulator of late endosomes and lysosomes. It is also critical for autophagy. Mutations in Rab7 lead to Charcot Marie-Tooth 2B (CMT2B) disease, a progressive peripheral neuropathy. My preliminary data suggest that Rab7 is an acute responder to LMP. Following LMP, Rab7 accumulates on various enlarged vesicular compartments, including late endosomes (LEs) and lysosomes (Lys), in a hyperactive, GTP-bound state. Within neurons, this response is exclusively localized to the soma. In this proposal, I aim to discover (1) the requirement of Rab7 activation for downstream LMP responses, such as galectin-3 accumulation on Lys and enlargement of LEs, (2) the impact of CMT2B mutant alleles on known LMP responses and Rab7 activation post LMP induction, and (3) the impact of age on Rab7 mediated neuronal degradation and LMP responses. I posit that Rab7 activation is essential in coordinating early responses for endosomal damage control in LMP. I will test the hypothesis that Rab7 activation following LMP is an essential response that mediates a protective pathway in neurons, which is disrupted in CMT2B mutant alleles. In Aim 1, I will explore the necessity of Rab7 activation in (1) galectin-3 mediated lysophagy and (2) LE vacuolization in response to LMP. In Aim 2, I will test whether Lys/LE responses and Rab7 activation in response to LMP are impacted in CMT2B. In Aim 3, I will determine whether aging disrupts Rab7 mediated (1) cargo degradation and (2) LMP responses in wild- type and mutant CMT2B neurons. Together, these experiments will determine homeostatic mechanisms in neurons that address lysosomal insults and whether this is impacted in disease. My long-term goal is to discover and characterize compensatory endosomal response mechanisms to lysosomal disruptions in LMP that will be broadly applicable to neurological disorders for the ultimate design of better therapeutics.