Protein sorting and trafficking by neuronal adaptor proteins: characterization of a conserved dendritic targeting motif on Kv4 channels and endocytic sorting of amyloid precursor protein by Mint2/X11L

Thesis (Ph.D.)--Boston UniversityPLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Precise spatial and temporal localization of signaling proteins is critical for proper neuronal function. A major contributor to these protein distribution patterns are multi-domain adaptor proteins. Adaptor proteins facilitate the assembly of multi-protein complexes to direct the delivery or internalization of key signaling components. This dissertation explores the mechanisms behind two distinct adaptor protein-mediated trafficking events by investigating the targeted delivery of the Kv4 potassium channel and the endocytic sorting and proteolytic processing of the amyloid precursor protein (APP). Kv4 is a member of a large family of voltage-gated potassium channels that serve as key determinants of neuronal excitability. Mammalian Kv4 channels display a somatodendritic distribution pattern that relies upon an evolutionarily conserved di-leucine motif. We used an in vivo assay to determine the distribution of Drosophila Kv4 channels in an identified cell type, and confirmed the role of the di-leucine motif in the somatodendritic localization of the Kv4 channel. Interestingly, we discovered that there are multiple mechanisms underlying Kv4 distribution in vivo, including a di-leucine motif independent mechanism that has yet to be explored. APP is widely studied for its role in the generation of neurotoxic amyloid beta (AB) peptides that comprise the amyloid plaques found in brains of Alzheimer's disease (AD) patients. Mint adaptor proteins bind APP and affect the production of AB by regulating APP cleavage. Loss of each of the three Mint proteins delays the age-dependent production of amyloid plaques in transgenic mouse models of AD. The aim of this study was to investigate the molecular mechanism by which Mint proteins influence APP production. We found that APP endocytosis was dramatically attenuated in cultured Mint knockout neurons. We show that Mint2 can be phosphorylated by Src kinase and that this phosphorylation regulates the endocytic sorting and processing of APP. Phospho-mimetic Mint2 mutants increase co-localization of APP with the autophagy marker, LC3, and reduce AB secretion. Conversely, phosphoresistant Mint2 mutants increase AB production by targeting internalized APP for retrograde transport to the Golgi. This study identifies a novel regulatory signaling pathway involved in AB secretion that may provide insight into the molecular mechanisms contributing to AD pathology.2031-01-0