UC San Diego

Although macroautophagy (MA) has been shown to decline with age, whether these changes are manifest differently in a shorter-lived (BALB/c) vs. longer-lived (C57BL/6) mouse strain remains unclear. Several parameters of basal MA were measured in cardiac tissue from mice aged 2.5-3 and 24-25 months: LC3-II and a Triton-insoluble fraction containing p62/SQSTM1 and ubiquitinated proteins. We observed a decrease in LC3-II coupled with an increase in p62 and ubiquitinated proteins, confirming a decline in MA in aged C57BL/6 mice. In contrast, old BALB/c mice demonstrated increased LC3-II, reduced p62, and no change in ubiquitinated proteins. Further analysis in aged C57BL/ 6 revealed a significant decline in Atg5 and an increase in the upstream autophagy regulator, pAKT-Ser473. In contrast, aged BALB/c mice demonstrate an increase in the autophagy proteins, pULK1-Ser757, Atg3, Atg7, Atg5, Atg12, Beclin-1, Atg4D, and Parkin, and an increase in the upstream autophagy regulators, pAKT-Thr308, pAKT-Ser473, pAMPK-Thr172, pFoxO3a-Ser253, and SirT1. This demonstrates a dysregulation in autophagy since molecules that enhance as well as inhibit autophagy are altered with age. Importantly, as suggested by the data with aged mice, MA is manifest differently in these strains. Comparing young C57BL/6 and BALB/c mice confirmed differences in autophagy and its upstream regulatory proteins. Young BALB/c mice demonstrate higher levels of pULK1-Ser757, p62, Atg7, Atg12, Atg5/12, Beclin-1 (Atg6), Parkin, and pAMPK-Thr172 in addition to reduced expression of LC3-II, indicating a dysregulation in the autophagy pathway and depressed autophagy compared to its C57BL/6 counterpart. Moreover, when subjected to an acute stress (a fast), LC3-II levels increased in young and old mice of both strains. However, compared to young C57BL/6, maximal levels of LC3-II were attained later in aged C57BL/6 and in all BALB/c, suggesting defective flux. To confirm this, fasted mice were chloroquine-treated, which prevents autophagosome/ lysosome fusion and favors LC3-II accumulation. Whereas LC3-II accrued in young C57BL/6, we found no increase in the aged C57BL/6 or in young or old BALB/c. Thus, the aged can mount an acute response, albeit with reduced flux. In order to make autophagy detection clinically viable, we developed and validated a reagent that appears to detect autophagy in peripheral blood