Mechanisms of Endosome Trafficking in Neurons

描述（由申请人提供）： 任何细胞生物学的一个中心问题是膜蛋白如何被囊泡载体特异性地分选。这个问题与突触特别相关，突触是一个专门从事突触囊泡（SV）运输的神经元域。然而，突触囊泡蛋白进行分类决策的机制还不清楚。为了研究这个基本问题，我开发了一个适配器依赖的神经元囊泡模型。该机制能够使用衔接子复合物AP-3和两种酶（GTdR和磷酸化衔接子AP-3的AP-3相关激酶）从内体SV蛋白分选。虽然磷酸化已被认为是一种重要的细胞调节机制，但其在衔接子功能中的作用大多未被探索。指导这一提议的中心假设是AP-3相关激酶选择性地调节AP-3依赖的囊泡形成和分选功能。我们的目标是使用独特的无细胞重建试验加上完整神经元中突触囊泡蛋白运输的分析，以了解SV蛋白分选的基本机制，并测试磷酸化在控制适配器依赖性囊泡形成中的作用。这些研究将揭示第一个详细的模型中的作用，磷酸化的衔接依赖性囊泡形成过程。在这项提案中，我们将传达我们在PC 12细胞中表征的丰富的分子，机制和药物，以剖析神经元中新的内体SV生物遗传途径。在小鼠中，AP-3通路的消融导致色素稀释、癫痫、行为表型和SV蛋白分选缺陷，而在人类中产生Hermansky-Pudlak II综合征。对AP-3机制和调节的理解将为开发影响癫痫、行为障碍和Hermansky-Pudlak II综合征治疗的途径特异性药理学药物开辟途径。

DESCRIPTION (provided by applicant): A central question to the biology of any cell, is how membrane proteins are specifically sorted by vesicle carriers. This problem is particularly pertinent to the synapse, a neuronal domain specialized in the trafficking of synaptic vesicles (SV). However, the mechansisms by which synaptic vesicle proteins make their sorting decisions are not well understood. To study this fundamental problem I have developed an adaptor-dependent neuronal vesiculation model. This mechanism is capable to sort from endosomes SV proteins using the adaptor complex AP-3 and two enzymes, a GTPase and an AP-3-associated kinase that phosphorylates the adaptor AP-3. Although phosphorylation has been recognized as an important cell regulatory mechanism, its role in adaptor function is mostly unexplored. The central hypothesis guiding this proposal is that an AP-3-associated kinase selectively regulates AP-3-dependent vesiculation and sorting functions. Our goal is to use unique cell-free reconstitution assays plus the analysis of synaptic vesicle protein trafficking in intact neurons to understand the basic mechanisms that govern SV protein sorting and to test the role of phosphorylation in controlling adaptor-dependent vesicle formation. These studies will reveal the first detailed model for the role of phosphorylation in an adaptor-dependent vesiculation process. In this proposal, we will convey the wealth of molecules, mechanisms and drugs that we have characterized in PC12 cells to dissect a novel endosome SV biogenetic pathway in neurons. In mice ablation of the AP-3 pathway results in pigment dilution, epilepsy, behavioral phenotypes and defective SV protein sorting whereas in humans generates the Hermansky-Pudlak II syndrome. The understanding of the AP-3 mechanisms and regulation will open avenues for the development of pathway-specific pharmacological agents that will impact the treatment of epilepsy, behavioral disorders and the Hermansky-Pudlak II syndrome.