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Title page for ETD etd-07232018-142806


Type of Document Dissertation
Author DelBove, Claire Elise
Author's Email Address clairedelbove@gmail.com
URN etd-07232018-142806
Title Visualizing APP trafficking and processing reveals its functional relationship with neuronal membrane cholesterol
Degree PhD
Department Pharmacology
Advisory Committee
Advisor Name Title
Christine Konradi Committee Chair
Charles Sanders Committee Member
Kevin Currie Committee Member
Qi Zhang Committee Member
Randy Blakely Committee Member
Keywords
  • Alzheimers disease
  • choleserol
  • protein trafficking
  • amyloid precursor protein
Date of Defense 2018-06-20
Availability restricted
Abstract
Amyloid precursor protein (APP) is a type I transmembrane protein and has been studied extensively due to its relevance to Alzheimer’s disease. However, its subcellular distribution and the mechanisms that regulate its concentration in neuronal membranes remain unclear. To address those questions, we generated pHluorin-APP-BFP2 by adding a pH-insensitive BFP2 to the C-terminal of an APP fusion protein that contains a pH-sensitive pHluorin at its N-terminal. By so doing, we can measure APP fractions in the surface or intracellular membranes and estimate APP cleavage at the same time. A pH-sensitive synaptic vesicle marker, Synaptophysin-pHTomato, is co-expressed to monitor synaptic vesicle proteins for comparison. We made the following discoveries. First, we confirmed that the trafficking and processing of both reporters resemble their endogenous counter parts. We found that the concentration of plasma membrane APP is controlled by dynamin-mediated endocytosis more than by alpha-cleavage, and there is a limited correlation between APP trafficking and synaptic activities. We also discovered that membrane cholesterol is inversely correlated to synaptic surface APP distribution, and that point mutations abolishing APP’s cholesterol-binding without affecting its cleavage not only significantly increase surface APP distribution but also make synapses more vulnerable to membrane cholesterol loss. Collectively, our results suggest that APP trafficking between surface and intracellular membranes is modulated by membrane cholesterol and reciprocally influences synaptic vesicle turnover and synaptic membrane integrity.

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