Alzheimer's Disease Research - Current Award

	Gopal Thinakaran, Ph.D. University of Chicago Chicago, IL Title: Microdomain Localization And Trafficking Of BACE1 Non-Technical Title: Understanding The Mechanisms Of Alzheimer's Disease Amyloid Production Co-Investigator(s): Angele Parent, Ph.D. University of Chicago Duration: April 1, 2009 - March 31, 2012 Award Type: Standard Award Amount: $400,000
Summary: This proposal investigates BACE1, one of the enzymes critical for the production of Alzheimer's disease amyloid beta production. We are interested in deciphering the molecular principles of BACE1 trafficking in lipid rafts and non-raft domains using biochemical and live cell imaging approaches in order to elucidate the cell biology of BACE1 processing of amyloid precursor protein (APP). Our studies will uncover novel and significant basic insights into cellular processes that modulate amyloid beta production, thus contributing to the development of strategies aimed at reducing amyloid beta burden.

Details:

Introduction
Alzheimer's disease (AD) is the major cause of dementia in the elderly. Toxic amyloid beta peptides accumulate in the brains of individuals with AD. Production and accumulation of amyloid beta are central events in AD pathogenesis.

Hypothesis and Specific Aims
Our proposal seeks to investigate the regulation of amyloid beta production. Sequential cell division of amyloid precursor protein (APP) by BACE1 and gamma secretase generates amyloid beta peptides. Our investigation focuses on BACE1. We seek to investigate membrane localization and movement of BACE1 in cultured cells and hippocampal neurons. Our first aim is to characterize BACE1 association with specialized cholesterol-rich microdomains of cellular membranes, called lipid rafts, which play important roles in amyloid beta production. Our second aim is to explore the dynamics of BACE1 movement through the cells using live cell imaging strategies.

Long-term Goals
A better understanding of localization and dynamics of BACE1 movement in cells will shed more light on the mechanisms involved in amyloid beta production. Information stemming from our biochemical, molecular and cellular investigations will contribute to the development of novel and rational strategies for therapeutic intervention for AD aimed at reducing amyloid beta burden.

Progress Updates:

We are completing biochemical analyses using beta-secretase (BACE1) gene mutants and fusion proteins to understand how or whether cell membrane lipid raft association modulates amyloid precursor protein (APP) processing to generate beta-amyloid peptides. Beta-amyloid is deposited in brain plaques, a hallmark of Alzheimer’s disease. Lipid rafts are specialized parts of the cell membrane where many proteins are assembled to perform specific functions. From our previous studies it appears that lipid raft association per se is not required for beta-amyloid production. However, presence of BACE1 protein at different locations within the cell and along the cell membrane lipid rafts modulates the levels of beta-amyloid by changing the position in APP where BACE1 cleavage occurs. Cleavage at the +1 position of APP generates intact beta-amyloid peptides, whereas cleavage at the +11 position of APP truncates the beta-amyloid peptide by removing the first 10 amino acids. We are now following-up on our findings using APP mutants that localize to different organelles in the cells.

In a second line of investigation, we have used live cell imaging and high-speed video microscopy analysis to study how BACE1 is transported within cultured cell lines and cultured hippocampal neurons (brain cells). To accomplish this, we have attached a yellow fluorescence protein to BACE1, where we can visualize it using a special microscope with filters to detect different kinds of fluorescence emissions. Our studies show that BACE1 is largely localized in recycling endosomes, but remarkably, a significant fraction of BACE1 is transported in tubular carriers. [An endosome is essentially a “sorting sack” within the cell that will direct proteins either toward the lysosome to be broken down into parts or recycled back to the Golgi apparatus for repackaging and use in other parts of the cell. A tubular carrier is essentially a tube that continually provides protein(s) to the outer membrane of a cell (as opposed to a vesicular carrier that is in the shape of a round sack and dumps its contents by directly fusing with the cell membrane).] This is a novel finding and we are very excited about characterizing the cellular mechanisms and proteins that regulate BACE1 trafficking in tubular carriers.

We have also begun to study BACE1 trafficking in tubular and vesicular carriers in primary neurons. Thus far, we have confirmed co-localization of BACE1 in recycling endosomes that are marked by the presence of the transferrin receptor protein. Our future studies will determine the significance of BACE1 transport in tubular carriers.

The discoveries resulting from the support of this grant may provide a new method for treatment of Alzheimer’s disease.