Dietary lipids and aging compromise chaperone-mediated autophagy by similar mechanisms

Abstract

Chaperone-mediated autophagy (CMA) is a selective form of autophagy whose distinctive feature is the fact that substrate proteins are translocated directly from the cytosol across the lysosomal membrane for degradation inside lysosomes. CMA substrates are cytosolic proteins bearing a pentapeptide motif in their sequence that, when recognized by the cytosolic chaperone HSPA8/HSC70, targets them to the surface of the lysosomes. Once there, substrate proteins bind to the lysosome-associated membrane protein type 2 isoform A (LAMP2A), inducing assembly of this receptor protein into a higher molecular weight protein complex that is used by the substrate proteins to reach the lysosomal lumen. CMA is constitutively active in most cells but it is maximally activated under conditions of stress.

Defective CMA activity has been described in different neurodegenerative diseases, in lysosomal storage disorders, diabetes and kidney diseases. A gradual decline in CMA activity also occurs with age due, for the most part, to an age-dependent decrease in lysosomal levels of LAMP2A. Previous analysis of the reasons behind this reduced content of LAMP2A in lysosomes from old organisms revealed no changes in the rates of LAMP2A synthesis or trafficking to lysosomes. Instead, the half-life of LAMP2A in lysosomes is markedly reduced with age due to accelerated degradation of this receptor protein in this compartment. We have previously described our finding that lysosomal levels of LAMP2A are modulated through its tightly regulated cleavage at the lysosomal membrane. Molecules of LAMP2A destined for degradation are mobilized to regions of the lysosomal membrane of discrete lipid composition that are enriched in the pair of proteases responsible for the cleavage and release of the lumenal portion of LAMP2A into the lysosomal matrix for complete degradation (Fig. 1A). CMA activity is consequently highly dependent on the ability of LAMP2A to move laterally at the lysosomal membrane, either to organize into translocation units, when CMA is activated, or to enter the membrane microdomains for degradation, when CMA is suppressed. We hypothesized that changes in the lipid composition of the lysosomal membrane could thus have a marked impact on CMA activity and contribute to CMA malfunctioning in aging and disease. In fact, we have previously shown that dietary lipids can modify the dynamics of vesicular intracellular compartments as, for example, they exert an inhibitory effect on macroautophagy by reducing the fusion between autophagosomes and lysosomes. Similarly, changes in cholesterol metabolism and trafficking are also the basis for reduced macroautophagy in the metabolic syndrome or lysosomal storages disorders such as Niemann-Pick type C disease.

Figure 1. Effect of dietary lipids on the dynamics of the CMA receptor at the lysosomal membrane. (A) LAMP2A organizes into multimeric complexes at the lysosomal membrane required for the translocation of cytosolic substrate proteins into the lysosomal lumen. A small fraction of LAMP2A is recruited into lipid microdomains where it undergoes degradation upon cleavage by cathepsin A (CTSA). A high percentage of LAMP2A present in multimeric complexes, and a lower LAMP2A content in microdomains are the signature of activated CMA. (B) High-lipid content diets reduce CMA activity by increasing the extension of lipid-enriched microdomains and thus augment the fraction of lysosomal LAMP2A undergoing degradation at a given time. Cholesterol is the main lipid detected in these microdomains in mice subjected to a high-cholesterol diet, whereas an increase in membrane ceramide is observed in animals maintained on a high-fat diet. A combination of these changes in lipid composition occurs at the lysosomal membrane with age and contributes to the reduced CMA activity observed in this condition.

To directly analyze the consequences on CMA of changes in the lipid composition of the lysosomal membrane, we subjected mice to diets with either a high content of cholesterol (Chol diet) or one enriched with vegetable oil and lard (high-fat diet). We found that both diets markedly decreased CMA activity in the liver of these animals due, for the most part, to their reduced content of LAMP2A. Decreased levels of this CMA receptor were a consequence of its accelerated degradation in lysosomes, which was further supported by the increased presence of LAMP2A in the lysosomal microdomains where the selective proteases reside.

To better understand the reasons for the increased partitioning of LAMP2A into the lipid microdomains, we performed a comparative lipidomic analysis of the lysosomal membranes from mice maintained on a regular diet and those under either of the high-lipid content diets. We found striking quantitative and qualitative changes in the lipid composition of the lysosomal membranes from the animals exposed to the lipid challenges. Interestingly, although each of the diets induced changes in different specific lipid species, both had an overall similar outcome, which was an increase in lipid-enriched microdomains with lower mobility than the surrounding membrane (Fig. 1B).

In the case of the animals exposed to a high cholesterol diet, almost 45% of the lipids in their lysosomal membranes are contributed by cholesterol. Interestingly, studies in artificial membranes have demonstrated that the maximum concentration of cholesterol allowed in a bilayer is 66% and that concentrations of cholesterol over 50% result in membranes formed exclusively by liquid ordered microdomains (the so-called detergent-resistant microdomains). Consequently, after administration of the cholesterol diet, the majority of the lysosomal membrane is organized into cholesterol-enriched microdomains that are sites of LAMP2A degradation. Most of the other changes in the lipid composition of these membranes, such as, for example, the enrichment in the monounsaturated forms of sphingolipids, seem to be oriented to preserve membrane fluidity to allow for lateral mobility. However, the fact that we found a marked decrease in the percentage of LAMP2A organized into translocation complexes suggests that lateral mobility, at least for LAMP2A, is likely compromised in these membranes.

The lipid profile of lysosomal membranes in the case of mice subjected to a high fat diet is somehow different, as the increase in the relative concentration of cholesterol is no longer observed. In contrast, we found that levels of ceramide have almost doubled and the levels of short saturated forms of both sphingolipids and ceramides are also increased. These changes have been shown to favor formation of membrane domains even more rigid than the cholesterol-enriched domains. The changes adopted by these membranes to maintain fluidity are a preferential increase of levels of polyunsaturated fatty acids in the acyl chains of the phospholipids, as these lateral chains exclude cholesterol, favoring their concentration in separated membrane domains. As in the case of animals on a high cholesterol diet, the increase in membrane microdomains leads to a higher percentage of LAMP2A undergoing degradation in these regions.

We then performed similar lipid analysis of lysosomal membranes isolated from livers of old mice. Interestingly, the aged membranes recapitulated a combination of the membrane lipid changes promoted by both diets. We found both an increase in cholesterol, although lower than that observed in the animals under a high cholesterol diet, and an increase in short saturated forms of ceramides and sphingolipids, comparable to that observed in animals on the high fat diet (Fig. 1B). The fact that, in contrast to young animals, the degradation of LAMP2A in old animals is not further increased when subjected to the high-fat diets, supports the notion that the main cause of the reduced stability of LAMP2A in lysosomes from old organism is the changes in the lipid composition of their membranes.

Our studies have primarily focused on the consequences that changes in the lipid composition at the lysosomal membrane have on CMA, but, evidently, the implications of the modified lipid profiles can affect many other aspects of lysosomal physiology. Changes in the membrane lipid composition could affect the stability of the multiple membrane transporters that control flux of ions and macromolecules in and out of the lysosome and that ultimately determine the composition of the lysosomal milieu. Likewise, impaired lateral mobility could alter the recruitment of the protein complexes involved in the fusion events between lysosomes and autophagic and endocytic compartments.

An additional implication of our findings is the fact that high-lipid diets do not only lead to alterations in lipid metabolism, but can also have severe consequences for protein catabolism, as demonstrated here by the low rates of CMA. Failure to degrade proteins could, on the one hand, affect protein synthesis when nutrients are scarce by limiting the replenishment of the pool of available free amino acids. On the other hand, selective degradation of proteins by mechanisms such as CMA also exerts important regulatory functions by modulating their intracellular levels. Consequently, compromised CMA activity by high-lipid diets may have important consequences in the cellular proteome.

Future studies should be aimed at determining whether the observed changes in the lipid profile of the lysosomal membrane are reproduced also in other organelles or are unique for these degradative compartments. From a practical standpoint, we have previously shown that genetic manipulations aimed at restoring levels of LAMP2A in the lysosomal membrane of old organisms result in marked improvement in cellular homeostasis and organ function. Consequently, the negative impact that changes in the lysosomal membrane lipidome have on LAMP2A stability now reveal a possible use of lipid-modifying interventions to stabilize this receptor and contribute to the restoration of normal CMA activity in old organisms.

Rodriguez-Navarro JA, Kaushik S, Koga H, Dall’Armi C, Shui G, Wenk MR, et al. Inhibitory effect of dietary lipids on chaperone-mediated autophagy. Proc Natl Acad Sci U S A. 2012;109:E705–14. doi: 10.1073/pnas.1113036109.