Mechanisms of Endosome Trafficking in Neurons

描述(申请人提供)：突触前终末的一个明显特征是存在突触小泡(SVS)，即存储和分泌神经递质的分泌细胞器。SVS的功能是由细胞器膜蛋白组成决定的。因此，控制SV形成和组成的机制对突触功能至关重要。在这项建议中，我们专注于由适配器复合体AP-3控制的囊泡生物发生途径，AP-3是一种外壳复合体，负责将膜蛋白从早期的内体分类到SVS。SV蛋白的组成受接头复合体AP-3的两种亚型的调节，神经型和无处不在的，后者被认为只参与溶酶体的生物发生。神经元AP-3亚型的遗传缺陷阻碍了SV膜蛋白的靶向。令人惊讶的是，溶酶体分选途径中的零突变，即普遍存在的AP-3途径，会在SVS中触发SV特异性蛋白的积累。这些意想不到的结果使我们提出了一个新的概念，即存在于同一内体上的SV和溶酶体分选机制竞争将膜蛋白输送到两种不同的途径，SVS或溶酶体。这一概念与传统的溶酶体观点不同，溶酶体被视为参与处理正常和病理细胞成分的末端细胞器。此外，我们的模型提供了一种新的方法来理解溶酶体靶向机制对影响儿童和成人的家族性和零星形式的神经变性的贡献。我们的中心假设是：AP-3-异构体特异性机制将SV膜蛋白从一个共同的内体间隔靶向两条相互竞争的途径：要么到SV的生物发生途径，要么到晚期的内体-溶酶体途径。在这个提案中，我们将重点关注从我们的假设中得出的四个预测。这些预测将使用影响SV和内溶酶体靶向的小鼠缺陷模型、高分辨率免疫电子和活体成像显微镜以及分离的SVS和内小体的分子分析来系统地探索。从这项提议中获得的信息将有助于我们理解在生理和病理条件下，较晚的内切体-溶酶体分选过程如何影响突触。公共卫生相关性：我们提出了一个模型，该模型将提供知识，以了解溶酶体和突触是如何相互作用的，并有助于神经退行性和精神障碍。

DESCRIPTION (provided by applicant): A defining feature of presynaptic terminals is the presence of synaptic vesicles (SVs), secretory organelles that store and secrete neurotransmitters. SVs functions are specified by the organelle membrane protein composition. Thus, mechanisms controlling SV formation and composition are pivotal for synapse function. In this proposal we focus on the vesicle biogenesis pathway controlled by the adaptor complex AP-3, a coat complex that sorts membrane proteins from early endosomes to SVs. SV protein composition is regulated by two isoforms of the adaptor complex AP-3, neuronal and ubiquitous, the later thought to participate exclusively in lysosome biogenesis. Genetic defects in the neuronal AP-3 isoform hinder targeting of SV membrane proteins. Surprisingly, null mouse mutants in a lysosomal sorting pathway, the ubiquitous AP-3 route, trigger accruement of SV-specific proteins in SVs. These unexpected results lead us to propose the novel concept that SV and lysosomal sorting mechanisms present on the same endosome compete for membrane proteins to be delivered into two alternative routes, SVs or lysosomes. This concept departs from the traditional view of lysosomes, which are viewed as terminal organelles involved in the disposal of normal and pathological cellular components. Furthermore, our model provides a novel way to understand the contribution of lysosome targeting mechanisms to familial and sporadic forms of neurodegeneration that affect children and adult individuals. Our central hypothesis is that: AP-3-isoform-specific mechanisms target SV membrane proteins from a common endosomal compartment to two competing pathways: either to a SV biogenesis route or to a late endosome-lysosomal path. In this proposal, we will focus on four predictions derived from our hypothesis. These predictions will be systematically explored using a combination of mouse deficient models that affect SV and endo- lysosomal targeting, high-resolution immuno-electron and in vivo imaging microscopy, as well as the molecular analysis of isolated SVs and endosomes. Information gained in this proposal will illuminate our understanding of how late endosomes-lysosome sorting processes affect synapses under physiological and pathological conditions. PUBLIC HEALTH RELEVANCE: We propose a model that will contribute knowledge to understand how lysosomes and synapses interface and contribute to neurodegenerative and psychiatric disorders.