Role of AP-3 in neuropeptide secretion from mammalian neurons

神经肽是行为和生理的强大调节剂。为了释放神经肽，神经肽被包装成致密的核心囊泡（DCVs），并经历钙依赖的胞外作用。神经元DCV形成的机制，特别是高极化细胞（如神经元）中跨膜DCV货物的运输途径，目前尚不清楚。接头蛋白复合物3 （Adaptor protein complex 3, AP-3）是运输囊泡外壳的一个组成部分，介导跨膜蛋白向溶酶体、溶酶体相关细胞器、神经内分泌dcv和突触囊泡的运输。神经元AP-3突变可导致神经发育迟缓和癫痫。本项目旨在探讨AP-3在哺乳动物神经元dcv形成和神经肽分泌中的作用。为了解决这个问题，我们将使用cas9介导的AP-3表达沉默的小鼠原代神经元。利用该模型系统，我们将首先研究AP-3的缺失是否以及如何影响神经元dcv的数量、形态和胞吐。接下来，我们将对神经元AP-3进行无偏筛选，并研究它们在神经元DCV成熟、稳定和胞吐中的潜在作用。最后，我们将研究这些货物到成熟DCVs的运输路线，并阐明AP-3对这些货物在特定神经元区室（体细胞、轴突、树突）运输的影响。该项目的预期结果是加深对神经元DCV形成的理解，这将促进对大脑中这一过程的特定操作工具的设计。

Neuropeptides are powerful regulators of behavior and physiology. For their release, neuropeptides are packaged into dense core vesicles (DCVs) that undergo calcium-dependent exocytosis. Mechanisms of neuronal DCV formation, in particular trafficking routes of transmembrane DCV cargos in highly polarized cells like neurons, are poorly understood. Adaptor protein complex 3 (AP-3) is a component of a transport vesicle coat that mediates trafficking of transmembrane proteins to lysosomes, lysosome-related organelles, neuroendocrine DCVs, and synaptic vesicles. Mutations in neuronal AP-3 result in neurodevelopmental delay and epilepsy. The aim of this project is to investigate the role of AP-3 in formation of neuronal DCVs and neuropeptide secretion in mammalian neurons. To address this question, we will use primary mouse neurons with Cas9-mediated silencing of AP-3 expression. Using this model system, we will first examine whether and how loss of AP-3 affects numbers, morphology and exocytosis of neuronal DCVs. Next, we will perform an unbiased screen for neuronal AP-3 cargos and investigate their potential roles in neuronal DCV maturation, stability and exocytosis. Finally, we will examine delivery routes of these cargos to maturing DCVs and elucidate the impact of AP-3 on trafficking of these cargos in specific neuronal compartments (soma, axons, dendrites). The expected outcome of this project is a deepened understanding of neuronal DCV formation, which will promote the design of tools for specific manipulation of this process in the brain.