Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.

Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.

Recapitulation of anti-aging phenotypes by global overexpression of PTEN in mice.

The PTEN gene negatively regulates the oncogenic PI3K-AKT pathway by encoding a lipid and protein phosphatase that dephosphorylates lipid phosphatidylinositol-3,4,5-triphosphate (PIP3) resulting in the inhibition of PI3K and downstream inhibition of AKT. Overexpression of PTEN in mice leads to a longer lifespan compared to control littermates, although the mechanism is unknown. Here, we provide evidence that young adult PTENOE mice exhibit many characteristics shared by other slow-aging mouse models, including those with mutations that affect GH/IGF1 pathways, calorie-restricted mice, and mice treated with anti-aging drugs. PTENOE white adipose tissue (WAT) has increased UCP1, a protein linked to increased thermogenesis. WAT of PTENOE mice also shows a change in polarization of fat-associated macrophages, with elevated levels of arginase 1 (Arg1, characteristic of M2 macrophages) and decreased production of inducible nitric oxide synthase (iNOS, characteristic of M1 macrophages). Muscle and hippocampus showed increased expression of the myokine FNDC5, and higher levels of its cleavage product irisin in plasma, which has been linked to increased conversion of WAT to more thermogenic beige/brown adipose tissue. PTENOE mice also have an increase, in plasma and liver, of GPLD1, which is known to improve cognition in mice. Hippocampus of the PTENOE mice has elevation of both BDNF and DCX, indices of brain resilience and neurogenesis. These changes in fat, macrophages, liver, muscle, hippocampus, and plasma may be considered "aging rate indicators" in that they seem to be consistently changed across many of the long-lived mouse models and may help to extend lifespan by delaying many forms of late-life illness. Our new findings show that PTENOE mice can be added to the group of long-lived mice that share this multi-tissue suite of biochemical characteristics.

PTEN is both an activator and a substrate of chaperone-mediated autophagy.

PTEN is a crucial negative regulator of the INS/PI3K/AKT pathway and is one of the most commonly mutated tumor suppressors in cancer. Global overexpression (OE) of PTEN in mice shifts metabolism to favor oxidative phosphorylation over glycolysis, reduces fat mass, and extends the lifespan of both sexes. We demonstrate that PTEN regulates chaperone-mediated autophagy (CMA). Using cultured cells and mouse models, we show that PTEN OE enhances CMA, dependent upon PTEN's lipid phosphatase activity and AKT inactivation. Reciprocally, PTEN knockdown reduces CMA, which can be rescued by inhibiting class I PI3K or AKT. Both PTEN and CMA are negative regulators of glycolysis and lipid droplet formation. We show that suppression of glycolysis and lipid droplet formation downstream of PTEN OE depends on CMA activity. Finally, we show that PTEN protein levels are sensitive to CMA and that PTEN accumulates in lysosomes with elevated CMA. Collectively, these data suggest that CMA is both an effector and a regulator of PTEN.

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

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.