Aging, Alzheimer?s disease (AD), and other neurodegenerative diseases have altered or impaired proteostasis. In healthy cells, proteostasis involves mechanisms for the stabilization of correctly folded proteins and the degradation of misfolded or damaged proteins. This balance maintains proteins in a specific conformation, concentration, and location to be functional, and the coordinated mechanisms prevent the accumulation of damaged proteins while permitting the continuous renewal of intracellular proteins. In aging, Alzheimer?s disease, and other neurodegenerative diseases, misfolded proteins aggregate into inclusions, which indicate that disruption of proteostasis has occurred. Effort has been put into determining what comprises the inclusions, but the cause of this altered or impaired proteostasis has never been determined. To improve our understanding of the causes of impaired proteostasis, we will examine the dynamic changes occurring in protein structure and protein turnover. We will use different types of samples and model systems to capitalize on the advantages of each.
In SPECIFIC AIM 1, we propose to target proteins involved in AD in human cerebrospinal fluid with limited proteolysis to assess differences in protease accessibility where there were minimal changes in protein levels. By measuring changes in protease accessibility, we will gain insight into protein conformational changes.
In SPECIFIC AIM 2, we will extend limited proteolysis to mouse brain to investigate differences in protease accessibility due to aging.
In SPECIFIC AIM 3, we will examine protein turnover in 5 regions of mouse brain to reveal proteins with abnormal renewal related to aging. Measuring protein turnover requires the use of stable isotope labeling, which precludes the use of human samples. A benefit of using proteomic methods in this project is that we can examine many features at once and return to the data with new hypotheses in the future. The feasibility of the project is supported by the preliminary data generated for SPECIFIC AIM 1 and 2 and previous work done in the lab using the method proposed for SPECIFIC AIM 3. This proposal will be the first use of limited proteolysis-mass spectrometry with cerebrospinal fluid and brain tissue and the first example of protein turnover being studied in more than 2 regions of brain tissue. The results from both Aims will improve our understanding of proteostasis and deepen our understanding of the aging brain, Alzheimer?s disease, and other neurodegenerative diseases. This project will occur in a collaborative, interdisciplinary research environment in the University of Washington Genome Sciences department under the mentorship of Dr. Michael J. MacCoss, and the project will provide training in aging, Alzheimer?s disease, neurodegenerative diseases, statistics, and quantitative proteomics.
Aging, Alzheimer?s disease, and other neurodegenerative diseases are known to have altered or impaired proteostasis, but the cause of the impaired system has never been determined. The proposed project uses proteomic techniques on cerebrospinal fluid and brain tissues to investigate the cause of impaired proteostasis by characterizing changes in protein structure and protein turnover.
