Mechanisms of autophagosome biogenesis and maturation in primary neurons

描述（由申请人提供）：自噬是一种重要的溶酶体降解途径，可从细胞中去除受损的细胞器和蛋白质聚集体。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出蛋白质毒素。事实上，神经元特异性自噬的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表现出明显的自噬缺陷。尽管有缺陷的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，自噬体优先在远端神经突产生。最初，它们表现出双向运动，但随后退出远端区域，并经历强大的逆行运输到细胞体。这种运动的转变伴随着与晚期内体/溶酶体的融合。当自噬体向细胞体移动时，它们继续酸化并成熟为可以更有效地降解货物的自噬体。潜在地，将自溶酶体递送到细胞体细胞确保降解产物快速有效地再循环到蛋白质合成的初级位点。基于初步数据，本研究将验证自噬体的发生和成熟是由原代神经元轴突在空间和时间上调控的假说。进一步，本研究也将检验假设

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.