Effect of blockage of chaperone-mediated autophagy in vivo

Abstract

Chaperone-mediated autophagy (CMA) degrades a select pool of cytosolic proteins in lysosomes and contributes to proteostasis. Our group previously found that CMA activity decreases with age but the consequences of this functional decline in vivo remained unknown.;The goal of this thesis research was to elucidate how systemic and organ-specific failure of CMA impacts protein homeostasis, cellular metabolism, and ultimately health- and life-span. In order to study the consequences of CMA blockage in vivo, we developed a tissue-specific conditional knockout mouse model for LAMP-2A, the limiting component of CMA.;In the first section of this thesis, we used mice with impaired CMA specifically in the liver to explore how CMA loss influences hepatic and whole-body metabolism. We found that CMA blockage caused hepatic glycogen depletion and hepatosteatosis. Comparative lysosomal proteomics revealed that enzymes involved in carbohydrate and lipid metabolism are degraded by CMA and that impairment of their regulated degradation contributes to the metabolic abnormalities observed in CMA-defective mice. These findings highlight the involvement of CMA in modulating metabolism and suggest that the age-related decline of CMA may underlie the metabolic dysfunction characteristic of old organisms.;In the second part of this study, we studied the changes in proteostasis attributable to reduced CMA activity with age. We found that other proteolytic systems can compensate for the loss of CMA in young mice but these compensatory responses were not sufficient for protection against different types of cellular stress. Physiologic aging caused deterioration in the compensatory responses for loss of CMA, leading to altered proteostasis, increased liver dysfunction, and cell death. Age-dependent failure of protein quality control systems led to enhanced susceptibility to oxidative stress and was accompanied by alterations in hepatic and adipose lipid storage with age. We postulate that the loss of CMA contributes to the gradual decline in proteostasis and stress resistance observed with age.;In the last section of this work, to better understand the extent of compensation for CMA failure and whether these responses are sustained with age and in a tissue-specific manner, we developed a temporally-inducible total-body LAMP-2A knockout mouse model. Blocking CMA at different ages allowed us to test the consequences of CMA disruption early in life (when compensatory mechanisms are presumably intact) versus later in life (when compensation is less likely to occur). We found that certain organs showed signs of upregulation of macroautophagy in response to CMA deficiency when animals were young, but lack of compensation when aged. Collectively, these data support that compensation among different types of autophagy exists in vivo and that there are tissue-specific and age-dependent differences in how mice are able to cope with loss of CMA activity.;Overall, our studies uncovered novel physiological functions for CMA in modulating protein quality control and energy metabolism in vivo . The knowledge obtained from this work on the effects of CMA blockage paves the way for future development of anti-aging strategies based on modulation of this type of selective autophagy.