Mechanisms of autophagosome biogenesis and maturation in primary neurons

描述(申请人提供)：自噬是一种重要的溶酶体降解途径，它将受损的细胞器和蛋白质聚集体从细胞中移除。自噬在神经元等有丝分裂后细胞中是必不可少的，因为它们不能通过细胞分裂来稀释蛋白毒素。事实上，神经元特有的自噬丧失足以导致神经元细胞死亡。此外，以蛋白质过度聚集为特征的多种神经退行性疾病在自噬方面表现出明显的缺陷。尽管有缺陷的自噬在疾病中有明显的影响，但人们对驱动神经元自噬的基本机制知之甚少。初步数据表明，自噬小体优先在远端轴突中产生。最初，它们表现出双向运动，但随后离开远端区域，经历强劲的逆行运输到细胞体。这种运动性的改变伴随着与晚期内小体/溶酶体的融合。当自噬小体向细胞体移动时，它们会继续酸化并成熟为自溶体，从而可能更有效地降解货物。潜在地，将自溶酶体输送到细胞体可以确保降解产物快速有效地循环到蛋白质合成的初级位置。基于初步数据，这一提议将检验自噬小体的生物发生和成熟是沿初级神经元的轴突在空间和时间上受到调节的假设。此外，这项研究还将检验这一假设 自噬小体的功能与运输密切相关。为了验证这些假说，这一建议将：(1)确定原代神经元自噬小体生物发生的机制；(2)确定基础与应激条件下原代神经元自噬小体运输与成熟之间的关系；(3)确定基础条件与应激条件下原代神经元自噬降解货物的机制。综上所述，这一提议将确定自噬小体从出生到成熟成为可降解和功能细胞器的机制，以及这一途径如何改变以响应细胞应激因素，如线粒体损伤和蛋白质聚集。这些假说将使用从活细胞成像到生物物理技术的多学科方法在从野生动物和神经退行性疾病模型中分离出来的神经元中进行测试。在K99阶段，将建立神经生物学和生物物理学的新方法，以在机械水平上检验所提出的假说。这些方法随后将在R00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关于原代神经元自噬调节的新信息，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.