Vesicular Mechanisms for Lysosomal Delivery of Synaptic Vesicle Proteins

描述（由申请人提供）：我的建议集中在未被认识的作用内溶酶体运输机制在调节突触囊泡蛋白水平。内体衔接蛋白AP-3选择性识别某些突触囊泡蛋白，包括金属和神经递质转运蛋白。神经元AP-3缺陷小鼠中这些转运蛋白水平的降低导致癫痫。然而，这些转运蛋白在普遍存在的AP-3缺陷小鼠的突触囊泡中增加，表明AP-3同种型共同平衡突触囊泡蛋白的水平以维持适当的神经传递。具体来说，我假设无处不在的AP-3将溶酶体融合机制纳入神经元AP-3衍生的突触囊泡，允许突触囊泡蛋白的溶酶体传递和降解。这种机制将防止与神经退行性疾病相关的突触囊泡蛋白的异常积累。因此，我的研究计划解决了这种新型突触/溶酶体囊泡的存在和膜转运动力学作为突触囊泡膜蛋白递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微囊泡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，第一个目标解决是否AP-3排序突触囊泡和溶酶体膜蛋白驻留在相同的囊泡通过使用生化技术，包括亚细胞分级分离和囊泡分离，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。第二个目的在于：1）AP-3依赖性囊泡生物发生，通过使用AP-3缺陷小鼠脑检查囊泡含量，和2）囊泡通过AP-3分选的融合机制（即VAMP 7和vps 33 b）与溶酶体融合，对突触囊泡蛋白的半衰期的贡献;我将在原代神经元和神经元细胞系中使用AP-3分选的溶酶体融合机制VAMP 7和vps 33 b的siRNA敲低，并进行脉冲追踪实验以确定突触囊泡膜蛋白半衰期。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“减轻神经系统疾病负担”的使命一致，该提议阐明了内-溶酶体转运在维持神经传递的突触囊泡蛋白的适当水平同时防止神经变性基础的突触囊泡蛋白的异常积累中的作用。

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.