Alzheimer’s Disease (AD) is a neurodegenerative disorder that results from the formation of beta-amyloid (Aβ) plaques in the brain, which triggers known symptoms in AD patients such as memory loss. Aβ plaques are formed via the β-secretase pathway, in which cleavage of the amyloid precursor protein (APP) at the beta site by the proteases BACE1 and γ-secretase. This enzyme-facilitated cleavage leads to the production of Aβ fragments that are believed to form plaques, which cause neuronal cell death. Recent detergent protein extraction studies suggest that the native BACE1 protein forms a dimer that has significantly higher catalytic activity than its monomeric counterpart. Currently, however, there are no <i>in vitro</i> studies that support the dimerization of BACE1 in living cells. In this contribution, we describe our ongoing effort to examine the dimerization hypothesis of BACE1 in cultured HEK293 cells using a complementary fluorescence spectroscopy and microscopy methods. Cells transfected with a BACE1-GFP fusion protein construct will first be imaged using confocal and DIC microscopy for monitoring labeled BACE1 localization within the cell morphology. Subsequently, single-molecule fluctuation analysis will allow us to test the dimerization hypothesis of the labeled BACE1, using direct measurements of the size and the molecular brightness, as a function of substrate binding. Complementary two-photon fluorescence lifetime imaging will help probe any changes of BACE1 conformation and its local environment as a function of substrate binding. These studies will help elucidate the function of BACE1 dimerization on its in vivo activity.

Fluorescence microspectroscopy assessment of the in vitro dimerization of BACE1-GFP fusion protein in cultured cells

249th National Meeting (2015)

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