Endolysosomal Function in Neuronal Maintenance

描述（由申请人提供）：溶酶体降解中的许多缺陷在影响其他器官之前影响神经系统，表明神经元对膜降解的需求增加或特化。我已经为果蝇中普遍存在的内溶酶体rab GTPase rab7产生了第一个零突变体。rab7的部分或完全缺失导致感觉神经元先于其他细胞类型出现神经病变样表型。此外，人类rab7的四个点突变通过部分功能丧失机制导致常染色体显性感觉神经病变Charcot-Marie-Tooth型2B （CMT 2B）。果蝇rab7零突变体和人类CMT 2B疾病都突出了神经元对内溶酶体降解能力缺陷的敏感性。基于我的初步发现，我假设神经元对内溶酶体降解有特殊的或增加的要求。我将通过结合果蝇遗传学和新的实时成像方法来验证这一假设，以定量测量与细胞体和其他细胞类型相比，神经元突触中rab7依赖性降解的货物特异性和动力学。接下来，我将利用基因相互作用实验结合成像和电生理读数来研究rab7在普遍自噬和神经元特异性内溶酶体降解机制下的细胞机制。最后，我将与我的共同赞助人一起，对rab7在小鼠神经元培养中的功能进行表征，以检验我在果蝇模型中发现的普遍性。总之，我的研究结果将阐明rab7依赖性降解的细胞机制及其在神经元维持和寿命中的作用，并在建立控制受内溶酶体功能障碍影响的神经元降解能力的方法方面具有潜在的治疗应用。

DESCRIPTION (provided by applicant): Many defects in lysosomal degradation affect the nervous system before other organs, suggesting an increased or specialized neuronal demand for endomembrane degradation. I have generated the first null mutant for the ubiquitous endolysosomal rab GTPase rab7 in Drosophila. Partial or complete loss of rab7 causes neuropathy-like phenotypes in sensory neurons before other cell types. Moreover, four point mutations in human rab7 cause the autosomal dominant sensory neuropathy Charcot-Marie-Tooth type 2B (CMT 2B) through a partial loss-of-function mechanism. Both the Drosophila rab7 null mutant and the CMT 2B disease in human highlight the neuronal sensitivity to defects in endolysosomal degradative capacity. Based on my preliminary findings, I hypothesize that neurons have a specialized or increased requirement for endolysosomal degradation. I will test this hypothesis by combining fly genetics and novel live imaging approaches to quantitatively measure cargo-specificity and dynamics of rab7-dependent degradation at neuronal synapses compared to cell bodies and other cell types. Next, I will use genetic interaction experiments combined with imaging and electrophysiological read-outs to investigate the cellular mechanism of rab7 function in the context of ubiquitous autophagy and neuron-specific endolysosomal degradation mechanism. Finally, together with my Co-Sponsor, I will characterize rab7 function in mouse neuronal culture to test the generality of my findings from the Drosophila model. Together, my findings will elucidate the cellular mechanism of rab7-dependent degradation and its role in neuronal maintenance and longevity, with a potential therapeutic application in the establishment of methods to manipulate the degradative capacity of neurons affected by endolysosomal dysfunction.