Vesicular Mechanisms for Lysosomal Delivery of Synaptic Vesicle Proteins

描述(由申请人提供)：我的建议集中在内溶酶体运输机制在调节突触小泡蛋白水平中未被认识到的作用。内体适配器蛋白AP-3选择性地识别某些突触小泡蛋白，包括金属和神经递质转运蛋白。神经性AP-3缺陷小鼠体内这些转运蛋白水平的降低会导致癫痫。然而，这些转运蛋白在普遍存在的AP-3缺陷小鼠的突触小泡中增加，这表明AP-3亚型共同平衡了突触小泡蛋白的水平，以维持适当的神经传递。具体地说，我假设无处不在的AP-3将溶酶体融合机制整合到神经元AP-3衍生的突触小泡中，允许溶酶体递送和降解突触小泡蛋白。这一机制将防止与神经退行性疾病相关的突触小泡蛋白的异常积累。因此，我的研究计划致力于研究这种新的突触/溶酶体小泡的存在和膜转运动力学，作为一种将突触小泡膜蛋白运送到溶酶体进行降解的机制。为了支持AP-3衍生的突触/溶酶体囊泡，AP-3微囊蛋白质组鉴定了突触囊泡和溶酶体膜蛋白。为了进一步验证这一假设，第一个目的是研究AP-3分离的突触小泡和溶酶体膜蛋白是否存在于相同的小泡中，使用生化技术，包括亚细胞分离和小泡分离，以及定量去卷积显微镜和免疫金电子显微镜，在神经元细胞系和原代培养神经元中。第二个目的是研究-1)AP-3依赖的囊泡生物发生，利用AP-3缺乏的小鼠脑来检测囊泡内容物，以及2)通过AP-3分类的融合机制，即VAMP7和vps33b-与溶酶体融合的囊泡对突触囊泡蛋白半衰期的贡献；我将在原代神经元和神经细胞系中使用siRNA敲除AP-3分类的溶酶体融合机制VAMP7和vPS33b，并进行脉冲追逐实验来确定突触囊泡膜蛋白的半衰期。以往的研究表明，突触囊泡膜蛋白水平的维持对于突触的形成、神经传递和防止导致神经变性的异常蛋白积聚至关重要。与NINDS的使命“减轻神经疾病的负担”相一致，这一提议阐明了内溶酶体运输在维持神经传递的适当突触小泡蛋白水平方面的作用，同时防止神经退变基础上突触小泡蛋白的异常积累。

DESCRIPTION (provided by applicant): My proposal focuses on the unrecognized role of endo-lysosomal transport machinery in regulating synaptic vesicle protein levels. The endosomal adaptor protein AP-3 selectively recognizes certain synaptic vesicle proteins, including metal and neurotransmitter transporters. Decreased levels of these transporters in neuronal AP-3-deficient mice result in epilepsy. However, these transporters increase in synaptic vesicles from ubiquitous AP-3-deficient mice, suggesting that AP-3 isoforms together balance the levels of synaptic vesicle proteins to maintain proper neurotransmission. Specifically, I hypothesize that ubiquitous AP-3 incorporates lysosomal fusion machinery into neuronal AP-3-derived synaptic vesicles, allowing for lysosomal delivery and degradation of synaptic vesicle proteins. This mechanism would prevent the aberrant accumulation of synaptic vesicle proteins associated with neurodegenerative disorders. Therefore, my research proposal addresses the existence and membrane transport dynamics of this novel synaptic/lysosomal vesicle as a mechanism for delivery of synaptic vesicle membrane proteins to lysosomes for degradation. In support of an AP-3-derived synaptic/lysosomal vesicle, the AP-3 microvesicle proteome identified both synaptic vesicle and lysosomal membrane proteins. To further test this hypothesis, the first aim addresses whether AP-3-sorted synaptic vesicle and lysosomal membrane proteins reside in the same vesicles by using biochemical techniques, including subcellular fractionation and vesicular isolation, as well as quantitative deconvolusion microscopy and immuno-gold electron microscopy, in both neuronal cell lines and primary culture neurons. The second aim addresses the contribution of -1) AP-3-dependent vesicle biogenesis, by using AP-3-deficient mouse brains to examine vesicle content and 2) vesicle fusion with lysosomes through the AP-3-sorted fusion machinery, namely VAMP7 and vps33b - to the half-life of synaptic vesicle proteins; I will use siRNA knockdown of the AP-3-sorted lysosomal fusion machinery, VAMP7 and vps33b, in primary neurons and neuronal cell lines and perform pulse chase experiments to determine synaptic vesicle membrane protein half-life. Previous research shows that maintenance of synaptic vesicle membrane protein levels is critical for synapse formation, neurotransmission and prevention of abnormal protein accumulation leading to neurodegeneration. Consistent with the NINDS mission "to reduce the burden of neurological disease", this proposal illuminates the role of endo-lysosomal transport in maintaining proper levels of synaptic vesicle proteins for neurotransmission while preventing the aberrant accumulation of synaptic vesicle proteins underlying neurodegeneration.