Rodriguez, Kassidy2024-01-052024-01-052020-08https://hdl.handle.net/11299/259588University of Minnesota M.S. thesis. August 2020. Major: Chemistry. Advisor: Joseph Johnson. 1 computer file (PDF); xii, 70 pages.Amyloid-β (Aβ) plaque formation in the brain is a major hallmark of Alzheimer’s disease (AD) and has been linked to known symptoms. According to the amyloid cascade hypothesis, Aβ is generated from the cleavage of amyloid-β precursor protein (APP) by BACE1 followed by the regulated intramembrane proteolysis (RIP) by γ-secretase. Aβ plaques are then formed through the extracellular deposition and aggregation of Aβ proteins. Current research suggests that BACE1 dimerizes in cells in a substrate-mediated manner, and that these dimers exhibit a much higher catalytic activity than their monomeric forms within acidic environments, such as endosomal compartments. It was also reported that the interaction between APP and BACE1 increased Aβ production. To date, there has been no research completed using HEK293 cells that compares the degree of co-localization of APP within specific endosomal compartments in the secretory pathway. The co-residence of APP and BACE1 in the same subcellular location would likely favor their interaction and facilitate the proteolysis of APP. Therefore, the study of their intracellular trafficking and localization should indicate where BACE1 is likely to process APP. BACE1 is an attractive therapeutic target for slowing the production of Aβ in the early stages of AD, further highlighting the importance of its study. In this contribution, we describe our efforts to examine where APP-GFP resides along the secretory pathway using fluorescently labeled Rab proteins that target distinct regions of the secretory pathway as points of reference. In addition, we observed an increase in the overall fluorescence intensity in cells co-transfected with APP-GFP and BACE1-YFP. HEK293 cells were transiently transfected with APP-GFP and either Rab5-DsRed, Rab7-DsRed, Rab11-DsRed, or BACE1-YFP fluorescent fusion proteins then imaged using widefield fluorescence and differential interference contrast (DIC) microscopy. The subcellular localization and distribution of each fluorescent fusion protein construct was determined by the colocalization of GFP and DsRed by evaluating Pearson’s correlation coefficients (PCC), Manders’ overlap coefficients (MOC), and Li’s intensity correlation quotients (ICQ). Fluorescence images were analyzed using the JACoP plug-in within FIJI/ImageJ. The results of this study provide evidence that APP-GFP has the highest degree of colocalization with Rab11-DsRed, suggesting that the APP concentration is highest within the recycling endosomes. HEK293 cells co-expressing APP-GFP and BACE1-YFP exhibited an increased fluorescence intensity compared to cells expressing only APP-GFP. These results establish baseline measurements for future cell-based studies of BACE1 with APP-GFP as its substrate.enAlzheimer's DiseaseAPPColocalizationFluorescenceMicroscopyThesis or Dissertation