Mechanisms of autophagosome biogenesis and maturation in primary neurons

描述（由申请人提供）： 自噬是从细胞中清除受损细胞器和蛋白质聚集体的重要溶酶体降解途径。自噬在有丝分裂后的细胞如神经元中是必不可少的，因为它们不能通过细胞分裂稀释蛋白毒素。事实上，神经元特异性自噬丧失足以导致神经元细胞死亡。此外，以过度蛋白质聚集为特征的多种神经退行性疾病表现出明显的自噬缺陷。尽管缺陷性自噬在疾病中有明确的意义，但对神经元中驱动自噬的基本机制知之甚少。初步数据表明，自噬体优先产生于远端神经突。最初，它们表现出双向运动，但随后离开远端区域并经历强有力的逆行转运至细胞索马。这种运动性的转变伴随着与晚期内体/溶酶体的融合。当自噬体向细胞索马移动时，它们继续酸化并成熟为自溶体，自溶体可以更有效地降解货物。潜在地，将自体溶酶体递送至细胞索马确保降解产物快速且有效地再循环至蛋白质合成的主要位点。基于初步的数据，本研究将验证自噬体的生物发生和成熟是沿着原代神经元轴突在空间和时间上受到调节的假说。此外，本研究还将检验以下假设： 自噬体的功能和运输是紧密相连的。为了检验这些假设，本提案将（1）确定原代神经元中自噬体生物发生的机制（2）确定在基础与应激条件下原代神经元中自噬体运输和成熟之间的关系，以及（3）确定在基础与应激条件下原代神经元中自噬引起的货物降解机制。总之，该提案将确定自噬体生物合成从出生到成熟为降解和功能性细胞器的机制，以及该途径如何响应细胞应激源（如线粒体损伤和蛋白质聚集）而改变。这些假设将使用多学科方法进行测试，从活细胞成像到从野生型动物和神经退行性疾病模型中分离的神经元的生物物理技术。在K99阶段，将建立神经生物学和生物物理学的新方法，以在机制层面上检验所提出的假设。这些方法将在R 00阶段用于建立创新和独立的研究计划。这项研究的结果将揭示有关原代神经元自噬调控的新信息，并对理解神经退行性疾病的进展具有重要意义。

DESCRIPTION (provided by applicant): Autophagy is an essential lysosomal degradation pathway that removes damaged organelles and protein aggregates from the cell. Autophagy is essential in post-mitotic cells such as neurons since they are unable to dilute out proteotoxins by cell division. In fact, neuron-specific loss of autophagy is sufficient to cause neuron cell death. Further, multiple neurodegenerative diseases characterized by excessive protein aggregation exhibit pronounced defects in autophagy. Despite the clear implications of defective autophagy in disease, little is known about the basic mechanisms driving autophagy in neurons. Preliminary data indicate that autophagosomes are preferentially generated in the distal neurite. Initially, they exhibit bidirectional motility but then exit the distal region and undergo robust retrograde transport to the cell soma. This shift in motility is accompanied by fusion with late endosomes/lysosomes. As autophagosomes travel toward the cell soma, they continue to acidify and mature into autolysosomes that may more effectively degrade cargo. Potentially, delivery of autolysosomes to the cell soma ensures rapid and efficient recycling of degradation products to primary sites of protein synthesis. Based on the preliminary data, this proposal will test the hypothesis that autophagosome biogenesis and maturation are spatially and temporally regulated along the axon of primary neurons. Further, this study will also test the hypothesis that autophagosome function and transport are tightly linked. To examine these hypotheses, this proposal will (1) determine the mechanisms of autophagosome biogenesis in primary neurons (2) determine the relationship between autophagosome transport and maturation in primary neurons under basal versus stress conditions and (3) determine the mechanisms of cargo degradation by autophagy in primary neurons under basal versus stress conditions. Together, this proposal will determine the mechanisms of autophagosome biogenesis from birth to maturation into degradative and functional organelles and how this pathway becomes altered in response to cellular stressors such as mitochondrial damage and protein aggregation. These hypotheses will be tested using a multidisciplinary approach ranging from live-cell imaging to biophysical techniques in neurons isolated from wild type animals and models of neurodegenerative disease. During the K99 phase, new methodologies in neurobiology and biophysics will be established to examine the proposed hypotheses on a mechanistic level. These methodologies will then be utilized during the R00 phase to establish an innovative and independent research program. Results from this study will uncover novel information about the regulation of autophagy in primary neurons and have significant implications on understanding the progression of neurodegenerative disease.