LAMP2A, and other chaperone-mediated autophagy related proteins, do not decline with age in genetically heterogeneous UM-HET3 mice

Abstract

Chaperone-mediated autophagy (CMA) selectively degrades proteins that are crucial for glycolysis, fatty acid metabolism, and the progression of several age-associated diseases. Several previous studies, each of which evaluated males of a single inbred mouse or rat strain, have reported that CMA declines with age in many tissues, attributed to an age-related loss of LAMP2A, the primary and indispensable component of the CMA translocation complex. This has led to a paradigm in the field of CMA research, stating that the age-associated decline in LAMP2A in turn decreases CMA, contributing to the pathogenesis of late-life disease. We assessed LAMP2A levels and CMA substrate uptake in both sexes of the genetically heterogeneous UM-HET3 mouse stock, which is the current global standard for the evaluation of anti-aging interventions. We found no evidence for age-related changes in LAMP2A levels, CMA substrate uptake, or whole liver levels of CMA degradation targets, despite identifying sex differences in CMA.

Keywords: aging; autophagy; chaperone-mediated autophagy.

Figure 1

Age does not decrease LAMP2A levels in UM-HET3 or C57BL/6J mice. (A) Representative western blots and quantifications of LAMP2A and HSPA8 are shown in whole livers lysates from ad libitum fed male and female UM-HET3 mice of ages 4, 14, and 24 months. H3 and ENO1 are loading controls. n = 6 animals per group. (B) Representative western blots and quantifications of LAMP2A and HSPA8 are shown in whole kidney lysates from ad libitum fed male and female UM-HET3 mice of ages 4, 14, and 24 months. H3 is a loading control. n = 6 animals per group. (C) Representative western blots and quantifications of LAMP2A and HSPA8 are shown in whole brain lysates from ad libitum fed male and female UM-HET3 mice of ages 4, and 24 months. H3 and ACTB are loading controls. n = 6 animals per group. (D) Representative western blots and quantifications of LAMP2A and total LAMP2 are shown in whole livers lysates from ad libitum fed male C57BL/6J mice of ages 2, 8, and 24 months. ACTB is a loading control. n = 9 for 2- and 24-month groups, n = 8 for 8-month group. Statistical analysis was performed in GraphPad Prism 9. Lines are drawn at each mean, with error bars showing S.E.M. p-values derived from 2-way ANOVAs of both “full models” and “main effects models” are shown beneath each graph. “Estimation plots” are shown to the right of each graph; error bars on estimation plots show the 95% C.I. for the difference between the means of the indicated groups. p values displayed directly on the graphs are derived from unpaired t tests.

Figure 2

CMA differs by sex, but not by age in UM-HET3 mouse liver lysosomes. (A) Representative western blots and quantifications are shown for the indicated proteins in 4 μg of the light (CMA+) lysosome fraction from the livers of ad libitum fed male and female mice of ages 4 and 24 months. (B) Representative western blots and quantifications are shown for the indicated proteins in 4 μg of the light (CMA+) lysosome fraction from the livers of male and female mice of ages 4 and 24 months that were fasted for 18 hours before euthanasia. (C) Representative western blots and quantifications are shown for a substrate binding and uptake assay using the light (CMA+) lysosome fraction from male and female mice of ages 4 and 24 months that were fasted for 18 hours before euthanasia. The right panel shows the fraction of broken lysosomes. In each case, fewer than 10% of lysosomes were broken. n = 3 for each group in every experiment. Statistical analysis was performed in GraphPad Prism 9. Lines are drawn at each mean, with error bars showing S.E.M. p-values derived from 2-way ANOVAs are shown beneath each graph. “Estimation plots” are shown to the right graphs for LAMP2A and HSPA8 (the two proteins most important for CMA activity) Error bars on estimation plots show the 95% C.I. for the difference between the means of the indicated groups. p values displayed directly on the graphs are derived from unpaired t tests.

Figure 3

Age does not modify the effect of fasting on CMA target protein abundance in UM-HET3 liver. Representative western blots and quantifications are shown for the indicated proteins in whole liver lysates from male and female UM-HET3 mice of ages 4 and 24 months. Mice were either fed ad libitum (AL; green circles) or fasted (F; purple circles) for 18 hours prior to euthanasia. n = 6 for every group. Age was not found to modify the effects of fasting on CMA-sensitive proteins, by 3-way ANOVA. Statistical analysis was performed in GraphPad Prism 9. p-values derived from 3-way ANOVAs are shown beneath each graph.