The length of time a protein persists in a cell or the extracellular matrix can be an important determinant of the protein's role in disease. Over time proteins can be damaged or modified and then must be removed from the cell by proteolytic processes involving the proteasome or autophagic systems within the cell or by turnover of the whole cell. Accordingly, protein lifetimes and maintenance are particularly important in post-mitotic cells such as neurons. Traditionally, pulse-chase techniques using radioactive isotopes have been a method to measure the expression and turnover of proteins. We have developed an in vivo pulse-chase method using 15N labeled animals to measure the lifetimes of proteins and using these methods we observed surprisingly that inner core nuclear pore proteins are unexpectedly long- lived proteins. These studies used a linear rate decay analysis model in rats starting at 3 months and extending through 12 months. In this research proposal, we will label the Borchelt Alzheimer's disease mouse model with 15N and chase the labeled animal with 14N food. Measurement of the 14N to 15N ratios will determine the lifetime of APP, abeta1-42 and other proteins present in aggregates. Our expectation is that protein found in the aggregates will be long lived because the normal processes that remove misfolded proteins have failed. A second goal of this R03 proposal is to develop a method using L-azidohomoalanine (AHA) to measure the expression of newly translated proteins in mice. As proof of principle we have successfully pulse labeled a mouse with L-azidohomoalanine to determine safety and incorporation levels. In the proposed research we will pulse AHA in the mouse's food at set timepoints in a Borchelt Alzheimer's mouse model of Alzheimer's disease. We will enrich and measure proteins incorporating AHA in the prefrontal cortex and in insoluble brain aggregates. Only newly translated proteins should have incorporated AHA and will be enriched using "click" chemistry. We will identify new proteins being translated and recruited to the prefrontal cortex and to aggregates. Our hypothesis is that new proteins are being expressed and recruited to aggregates to help remove them.
This proposal will measure the lifetime of APP, abeta1-42, and other proteins present in aggregates in the Borchelt Alzheimer's mouse model using a whole animal 15N pulse- chase labeling method. A new strategy to measure newly expressed proteins in response to aggregate formation in the Borchelt Alzheimer's mouse model will be developed using L-azidohomoalanine. This study will measure the longevity of proteins in and the arrival of new proteins to plaques associated with Alzheimer's disease.