Role of AP-3 in neuropeptide secretion from mammalian neurons

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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.