Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722716/

Chaperone-mediated autophagy (CMA), a selective mechanism for degradation of cytosolic proteins in lysosomes, contributes to the removal of altered proteins as part of the cellular quality-control systems1,2. We have previously found that CMA activity declines in aged organisms and have proposed that this failure in cellular clearance could contribute to the accumulation of altered proteins, the abnormal cellular homeostasis and, eventually, the functional loss characteristic of aged organisms. To determine whether these negative features of aging can be prevented by maintaining efficient autophagic activity until late in life, in this work we have corrected the CMA defect in aged rodents. We have generated a double transgenic mouse model in which the amount of the lysosomal receptor for CMA, previously shown to decrease in abundance with age3, can be modulated. We have analyzed in this model the consequences of preventing the age-dependent decrease in receptor abundance in aged rodents at the cellular and organ levels. We show here that CMA activity is maintained until advanced ages if the decrease in the receptor abundance is prevented and that preservation of autophagic activity is associated with lower intracellular accumulation of damaged proteins, better ability to handle protein damage and improved organ function.

Autophagy is a cellular process that mediates the degradation of intracellular components in lysosomes, thus contributing to maintenance of cellular homeostasis, intracellular clearance of damaged structures and adaptation to environmental challenges4. Defective autophagy has been linked to common human diseases4. A decrease in autophagic activity with age, described in almost all model organisms analyzed, has been proposed to contribute to age-dependent accumulation of damaged intracellular components that lead to altered cellular homeostasis and loss of function in aging5.
Three different autophagic pathways—macroautophagy, microautophagy and CMA—have been described in mammalian cells on the basis of their mechanisms for delivery of cargo to lysosomes4,6. Whereas in macro- and microautophagy complete regions of the cytosol are sequestered and delivered to lysosomes all at once, in CMA individual proteins cross the lysosomal membrane one by one for their degradation1,2. The substrates of CMA are a subset of cytosolic proteins with a motif recognized by the hsc70 chaperone7. The chaperone-substrate complex binds to the CMA receptor, the lysosomal-associated membrane protein-2A (LAMP-2A)8. After unfolding9, the substrate crosses the lysosomal membrane assisted by a lumenal chaperone (lys-hsc70)10 and is rapidly degraded. CMA is maximally activated during stresses such as prolonged starvation, mild oxidation and other conditions resulting in protein damage1,2. CMA activity decreases during aging3 and in some age-related disorders such as familial forms of Parkinson’s disease11.
We have proposed that reduced lysosomal abundance of LAMP-2A is responsible for the decline in CMA activity during aging3. To determine whether maintaining LAMP-2A abundance constant throughout the mouse life span prevents autophagic decline and delays aging features associated with poor handling of cellular damage, we generated a double transgenic mouse carrying a transgene encoding a Tet regulator (which is bound by tetracycline or a related antibiotic, doxycycline) under the control of the albumin promoter (Alb-Tet-off-L2A). In this mouse, expression of an exogenous copy of the gene encoding LAMP-2A can be regulated in liver—where the age-related CMA defect has been well characterized3,12—by addition of doxycycline to the diet (doxycycline diet; Fig. 1a). In young Alb-Tet-off-L2A mice, we verified that removal of the doxycycline diet increased LAMP-2A abundance two- to fourfold only in liver, that LAMP-2A was properly targeted to lysosomes and did not alter the levels of other LAMPs, and that the additional LAMP-2A was functional in CMA, as lysosomal-enriched fractions isolated from young transgenic mice exposed to mild oxidative stress (to maximally activate CMA) showed higher rates of CMA than those from wild-type littermates (Fig. 1b–d and Supplementary Fig. 1 online). Expression of the Tet regulator in liver was mainly restricted to hepatocytes (Supplementary Fig. 2 online).