A mouse model for studying protein degradation in aging

A genetically altered mouse will be generated in which the selective degradation of specific cytosolic proteins in lysosomes could be modulated at wish. This mouse model will allow further analysis of the role of this lysosomal protein degradation pathway in preventing the intracellular accumulation of damaged proteins with age. Under stress conditions, such as nutrient deprivation, the selective degradation of cytosolic proteins in lysosomes by chaperone-mediated autophagy (CMA) is activated. Degradation of proteins by CMA requires their binding in a rate-limiting fashion to a receptor, the lysosome-associated membrane protein type 2a (lamp2a), in the lysosomal membrane. Activity of CMA is modulated by changes in the levels of lamp2a in the lysosomal membrane. CMA decreases with age in cultured cells and in different rodent tissues. Decreased levels of the receptor in the lysosomal membrane have been found in lysosomes from old tissues. We intend to prevent the decrease in receptor levels by conditionally overexpressing lamp2a in specific mouse tissues using the reverse tetracycline-controlled transactivator system (Tet-on). If, as demonstrated for cells in culture, overexpression of lamp2a in mouse increases CMA, the consequences of maintaining that increased CMA activity in several tissues on their ability to respond to different oxidative challenges will be analyzed. We will compare levels and rates of degradation of abnormally modified proteins in the cytosol of mice overexpressing or not lamp2a for different periods of time. If accumulation of damaged proteins after different challenges is prevented by improving their removal, the lamp2a-transgenic mice will become a very useful animal model to determine the contribution of the accumulation of abnormal proteins to the loss of cellular function with age. Using this system in old animals will allow us to test the reversibility of the protein accumulation and whether or not that results in improved function in old organisms.