Vesicular Mechanisms for Lysosomal Delivery of Synaptic Vesicle Proteins

突触小泡蛋白溶酶体递送的囊泡机制

基本信息

批准号：
7330088
负责人：
Karen A Litwa
金额：
$ 2.73万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-08-01 至 2010-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7330088
关键词：
Adaptor Signaling Protein Address Affect Autophagocytosis Biochemical Biogenesis Brain Brefeldin A Cell Fractionation Cell Line Cells Chromosome Pairing Complement Complex Data Dependency Electron Microscopy Endosomes Epilepsy Equilibrium Failure Gold Half-Life Hippocampus (Brain)Lead Localized Lysosomes Maintenance Mammalian Cell Membrane Proteins Metals Microscopy Mission Mus Mutation Nerve Degeneration Neurodegenerative Disorders Neurologic Neurons Neurotransmitters Organelles PC12 Cells Pathogenesis Pathway interactions Phenotype Physiologic pulse Population Postsynaptic Membrane Presynaptic Terminals Prevention Protein Binding Protein Isoforms Proteins Proteome Pulse taking Rattus Recycling Research Research Proposals Resolution Role Signal Transduction Small Interfering RNA Sorting - Cell Movement Staining method Stains Synapses Synaptic Vesicles Synaptosomes System Techniques Testing Transmembrane Transport Ubiquitin Vesicle Wild Type Mouse genetic analysis in vivo lysosomal proteins multicatalytic endopeptidase complex nervous system disorder neuron loss neurotransmission neurotransmitter release novel presynaptic prevent protein aggregation protein degradation research study size synaptogenesis therapeutic target

项目摘要

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.

描述（由申请人提供）：我的提议着重于内溶体传输机制在调节突触囊泡蛋白水平中的未认识的作用。内体衔接蛋白AP-3有选择地识别某些突触囊泡蛋白，包括金属和神经递质转运蛋白。这些转运蛋白在神经元AP-3缺陷型小鼠中的水平降低导致癫痫。但是，这些转运蛋白在无处不在的AP-3缺陷型小鼠中增加突触囊泡，这表明AP-3同工型共同平衡突触囊泡蛋白的水平以维持适当的神经传递。具体而言，我假设普遍存在的AP-3将溶酶体融合机械纳入神经元AP-3衍生的突触囊泡中，从而允许溶酶体递送和突触囊泡蛋白的降解。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，我的研究建议介绍了这种新型突触/溶酶体囊泡的存在和膜转运动力学，作为将突触囊泡膜蛋白递送到溶酶体降解的机制。为了支持AP-3衍生的突触/溶酶体囊泡，AP-3微丝蛋白组鉴定出突触囊泡和溶酶体膜蛋白。为了进一步检验该假设，第一个目的解决了通过使用生化技术（包括亚细胞分级分离和囊亚囊泡分离），以及定量的解次识别显微镜和免疫神经元培养物和NE NERON ELERON和NEEROR和NEEROR和NEEROR，在同一囊泡中是否存在于同一囊泡中AP-3分配的突触囊泡和溶酶体膜蛋白是否存在于同一囊泡中。第二个目的是通过使用AP-3缺陷型小鼠大脑检查囊泡含量和2）通过AP-3分融合融合机器的囊泡融合来解决-1）AP-3依赖性囊泡生物发生的贡献。我将在原代神经元和神经元细胞系中使用AP-3分别的溶酶体融合机械，VAMP7和VPS33B的siRNA敲低，并执行脉冲追逐实验，以确定突触囊泡膜蛋白蛋白半寿命。先前的研究表明，维持突触囊泡膜蛋白水平对于突触形成，神经传递和预防异常蛋白的积累至关重要。该提案与NINDS的任务“减轻神经疾病的负担”一致，该提案阐明了内溶剂体转运在维持适当水平的突触囊泡蛋白中用于神经传递的作用，同时又可以防止异常的突触囊泡蛋白质蛋白质蛋白质蛋白质下属神经变性。