Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function - PubMed (original) (raw)
Comparative Study
Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function
Cong Zhang et al. Nat Med. 2008 Sep.
Abstract
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 systems. 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 age, 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.
Figures
Figure 1
CMA activity is preserved in livers of aged Alb-Tet-off-L2A mice. (a) Schematic showing that administration of doxycycline prevents transcription of the gene encoding the extra copy of LAMP-2A in the Alb-Tet-off-LAMP-2A mouse. VP16, transactivation domain of the herpes simplex virus protein; tetO, tet operon operator; PhCMV*-1, tetracycline responsive promoter of the human cytomegalovirus. (b) Immunoblot for LAMP-2A (L-2A) or β-actin (β-Ac) in liver homogenates from 4-month-old wild-type (WT) or Alb-Tet-off-L2A (TL2A) mice from two different founder lines (F) maintained in the presence of doxycycline food (+Dox) or switched to doxycycline-free food (−Dox) for 4 weeks. (c,d) Immunoblot for LAMP-2A in liver and kidney homogenates (c) or for L-2A and LAMP-1 (L-1) in liver homogenates (d) from similar mouse groups as in b switched to doxycycline-free food for 4 weeks. (e) Schematic of the groups of mice used in this study. Graph shows the trend of changes in LAMP-2A abundance in liver with age (from our previous studies). TL2A-E, mice with early activation of LAMP-2A; TL2A-L, mice with late activation of LAMP-2A. (f) Immunoblot for L-2A in homogenates (Homog) and lysosomes from 6- or 22-month-old WT mice and 22-month-old TL2A mice maintained or not maintained on a doxycycline diet. (g) Uptake of GAPDH by lysosomes from the same mouse groups as in f. Values are the means ± s.e.m. of five or six experiments. (h) Immunoblot for L-2A and L-1 in lysosomes isolated from 22-month-old WT mice and 22-month-old TL2A-E or TL2A-L mice. (i) Degradation of [14C]GAPDH by intact (left) or broken (right) lysosomes isolated from the same mouse groups as in h. Values are the means ± s.e.m. of three different experiments with duplicate samples. Significant differences between experimental groups and young (*) or aged (#) WT mice were: *, P < 0.05; or ***, P < 0.001. β-Ac and L-1 are shown as loading controls for homogenates and lysosomes, respectively.
Figure 2
Livers of aged Alb-Tet-off-L2A mice accumulate fewer damaged proteins. (a) Two-dimensional electrophoresis and immunoblotting for carbonyl groups of cytosolic fractions from livers of 6- or 22-month-old WT mice, 22-month-old TL2A-E or TL2A-L mice or transgenic mice maintained on a doxycycline diet throughout their life span (TL2A-off). Loading controls are shown in Supplementary Figure 5a. Graph shows the mean ± s.e.m. value of the densitometric quantification of two to four immunoblots similar to the ones shown here for liver and in Supplementary Figure 5b for kidney. IP, isoelectric point; MW, molecular weight. (b) Representative immunoblot for 4-hydroxynonenal (4-HNE) in cytosolic fractions from the same groups of mice as in a (top). Actin is shown as a loading control. Densitometric quantification of two different experiments, expressed as means ± s.e.m. (bottom). (c) Aggregate proteins were visualized by fluorescence microscopy as round bright fluorescent puncta in liver sections from the same mouse groups as in a and b immunostained for polyubiquitin (profiles of red blood cells have been highlighted with a discontinuous line). An unstained (unst.) section is shown to account for the presence of autofluorescence. At right, the number of aggregates per microscopic field (top) and the percentage of cellular area occupied by the fluorescent puncta (bottom) are shown. Values are means ± s.e.m. of the morphometric analysis of 10–15 randomly taken pictures of liver sections from four different mice in each group. Significant differences between experimental groups and young (*) or aged (#) WT mice were: * or #, P < 0.05; ** or ##, P < 0.01; or *** or ###, P < 0.005.
Figure 3
Improved cellular homeostasis in livers of aged Alb-Tet-off-LAMP-2A mice. (a) Electron micrographs showing ultrastructure of livers from 6- or 220-month-old WT mice or 22-month-old TL2A-E or TL2A-L mice. LD, lipid droplets. Black arrows point to areas of accumulation of endoplasmic reticulum stacks. (b) Higher magnification electron micrographs of the same mouse groups as in a showing areas enriched in autophagosomes and autophagolysosomes (top panels) and representative examples of the two (bottom panels). The percentage of autophagosomes (APH, black asterisk) and autophagolysosomes (APHL, white asterisk) in livers from the different mouse groups (bottom) was calculated after morphometric analysis of seven to ten micrographs for each mouse. Values are expressed as percentage of the total number of autophagic vacuoles and are means ± s.e.m. of three to five mice in each group. (c) Lipofuscin abundance in livers from the same groups of mice as in a and b was detected as autofluorescent puncta when unstained liver sections were observed under a fluorescence microscope. Representative sections with higher magnification areas (insets) are shown at the top. Scale bars, 20 µm and 5 µm for main images and insets, respectively. The number of particles per cell (left) and the percentage of cellular area occupied by the fluorescent puncta (right) calculated in ten different fields from three different sections in each liver are shown at the bottom. Significant differences between the experimental groups and the young (*) or aged (#) WT mice were: ** or ##, P < 0.05; or *** or ###, P < 0.01.
Figure 4
Livers of aged Alb-Tet-off-L2A mice show lower levels of cellular damage and improved organ function. (a) Serum abundance of alanine aminotransferase (ALT) in 6- or 22-month-old WT mice or 22-month-old TL2A-E or TL2A-L mice. Values are means ± s.e.m. of six to eight mice in each group. (b,c) TUNEL staining (b) and activated caspase-3 immunofluorescence (c) of liver sections from the same mouse groups as in a. Quantification of the number of TUNEL-positive (b) or activated caspase-3–positive (c) cells per field (means ± s.e.m. of at least ten different liver sections) is shown in the graphs. Arrows indicate positive cells to differentiate them from the fluorescence of red blood cells in the section. Bottom images in c show examples of the two types of positive cells: cells with cytosolic staining (left) and condensed cells probably corresponding to apoptotic bodies (right). (d) Time of recovery from the paralysis induced after injection of the same mouse groups as in a–c with zoxazolamine. Values are means ± s.e.m. of the recovery times in six to eight mice in each group. Top images show recovery of muscle tone in transgenic aged mice 120 min after injection. The mice had turned from their backs and started crawling around, whereas the aged WT mice remained immobile, lying on their backs. Significant differences between the experimental groups and the young (*) or aged (#) WT mice were: #, P < 0.05; ** or ##, P < 0.01; or *** or ###, P < 0.001.
Comment in
- Live longer with LAMP-2.
Saftig P, Eskelinen EL. Saftig P, et al. Nat Med. 2008 Sep;14(9):909-10. doi: 10.1038/nm0908-909. Nat Med. 2008. PMID: 18776878 No abstract available. - Improve organ health by chewing up old proteins?
Guachalla LM, Rudolph KL. Guachalla LM, et al. Hepatology. 2009 Jan;49(1):330-2. doi: 10.1002/hep.22738. Hepatology. 2009. PMID: 19115216 No abstract available.
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