Chaperone-mediated autophagy in protein quality control

Chaperone-mediated autophagy is a selective mechanism for degradation of soluble cytosolic proteins in lysosomes that distinguishes itself from other autophagic pathways by the selectivity with which CMA substrates are targeted for degradation. The recent molecular dissection of this autophagic pathway and the development of experimental models with compromised CMA have unveiled the important contribution of this pathway to protein quality control. In fact, CMA activation seems to be a common mechanism of cellular defense against proteotoxicity.     

Early cellular changes after blockage of chaperone-mediated autophagy

Cytosolic proteins can be selectively degraded in lysosomes by chaperone-mediated autophagy (CMA), an autophagic pathway maximally activated under stress. In previous works we have demonstrated the existence of a cross-talk between CMA and macroautophagy, the other stress-related autophagic pathway responsible for the "in bulk" degradation of whole regions of the cytosol and for organelle turnover. We found that chronic blockage of CMA, as the one described in aging cells, results in constitutive activation of macroautophagy, supporting that one pathway may compensate for the other. In this work we have investigated the series of early cellular events that precede the activation of macroautophagy upon CMA blockage and the consequences of this blockage on cellular homeostasis. Shortly after CMA blockage, we have found functional alterations in macroautophagy and the ubiquitin-proteasome system, that are progressively corrected as CMA blockage persists. Basal macroautophagic activity remains initially unaltered, but we observed a delay in its activation in response to serum removal, a well characterized inducer for this pathway. Slower degradation of short-lived proteins, and a transient decrease in some of the proteasome proteolytic activities are also evident in the first stages of CMA blockage. This global alteration of the proteolytic systems supports the coordinated functioning of all of them, and seems responsible for the intracellular accumulation of altered proteins. Based on the time-course of the cellular changes, we propose that a minimal threshold of these toxic products needs to accumulate in order to constitutively activate macroautophagy and thus return cellular homeostasis to normal.     

Constitutive activation of chaperone-mediated autophagy in cells with impaired macroautophagy

Three different types of autophagy-macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA)-contribute to degradation of intracellular components in lysosomes in mammalian cells. Although some level of basal macroautophagy and CMA activities has been described in different cell types and tissues, these two pathways are maximally activated under stress conditions. Activation of these two pathways is often sequential, suggesting the existence of some level of cross-talk between both stress-related autophagic pathways. In this work, we analyze the consequences of blockage of macroautophagy on CMA activity. Using mouse embryonic fibroblasts deficient in Atg5, an autophagy-related protein required for autophagosome formation, we have found that blockage of macroautophagy leads to up-regulation of CMA, even under basal conditions. Interestingly, different mechanisms contribute to the observed changes in CMA-related proteins and the consequent activation of CMA during basal and stress conditions in these macroautophagy-deficient cells. This work supports a direct cross-talk between these two forms of autophagy, and it identifies changes in the lysosomal compartment that underlie the basis for the communication between both autophagic pathways.     

Balance between autophagic pathways preserves retinal homeostasis

Aging contributes to the appearance of several retinopathies and is the largest risk factor for aged‐related macular degeneration, major cause of blindness in the elderly population. Accumulation of undegraded material as lipofuscin represents a hallmark in many pathologies of the aged eye. Autophagy is a highly conserved intracellular degradative pathway that plays a critical role in the removal of damaged cell components to maintain the cellular homeostasis. A decrease in autophagic activity with age observed in many tissues has been proposed to contribute to the aggravation of age‐related diseases. However, the participation of different autophagic pathways to the retina physiopathology remains unknown. Here, we describe a marked reduction in macroautophagic activity in the retina with age, which coincides with an increase in chaperone‐mediated autophagy (CMA). This increase in CMA is also observed during retinal neurodegeneration in the Atg5^flox/flox; nestin‐Cre mice, a mouse model with downregulation of macroautophagy in neuronal precursors. In contrast to other cell types, this autophagic cross talk in retinal cells is not bi‐directional and CMA inhibition renders cone photoreceptor very sensitive to stress. Temporal and cell‐type‐specific differences in the balance between autophagic pathways may be responsible for the specific pattern of visual loss that occurs with aging. Our results show for the first time a cross talk of different lysosomal proteolytic systems in the retina during normal aging and may help the development of new therapeutic intervention for age‐dependent retinal diseases.     

Lysosomal chat maintains the balance

The original idea that each protein follows a particular proteolytic pathway for its degradation is no longer supported. Instead, different proteolytic systems can simultaneously contribute to the degradation of a particular protein, or they can alternate in this task depending, for the most part, on the cellular conditions. It is thus reasonable to expect that some level of communication exists among different proteolytic systems to orchestrate these coordinated activities. Direct cross-talk between two forms of autophagy, macroautophagy and chaperone-mediated autophagy (CMA) has been recently demonstrated. Cells respond to blockage of CMA by upregulating macroautophagy. Although macroautophagy cannot completely substitute for the lack of CMA, the partial redundancy between both pathways allows some level of compensation, enough to maintain protein degradation and preserve cell homeostasis. Understanding the cross-talk among different autophagic pathways and with other proteolytic systems is important to predict the type of compensatory mechanisms that could be elicited in response to failure of one of these systems, and to understand the consequences that manipulating one of these pathways for therapeutic purposes could have on the activity of the other pathways.     

Lysosomal Chat Maintains the Balance

The original idea that each protein follows a particular proteolytic pathway for its degradation is no longer supported. Instead, different proteolytic systems can simultaneously contribute to the degradation of a particular protein, or they can alternate in this task depending, for the most part, on the cellular conditions. It is thus reasonable to expect that some level of communication exists among different proteolytic systems to orchestrate these coordinated activities. Direct cross-talk between two forms of autophagy, macroautophagy and chaperone-mediated autophagy (CMA) has been recently demonstrated. Cells respond to blockage of CMA by upregulating macroautophagy. Although macroautophagy cannot completely substitute for the lack of CMA, the partial redundancy between both pathways allows some level of compensation, enough to maintain protein degradation and preserve cell homeostasis. Understanding the cross-talk among different autophagic pathways and with other proteolytic systems is important to predict the type of compensatory mechanisms that could be elicited in response to failure of one of these systems, and to understand the consequences that manipulating one of these pathways for therapeutic purposes could have on the activity of the other pathways.

Addendum to:

Consequences of the Selective Blockage of Chaperone-Mediated Autophagy

A.C. Massey, S. Kaushik, G. Sovak, R. Kiffin and A.M. Cuervo

Proc Natl Acad Sci USA 2006; 103:5805-10     

Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function

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.     

Roles of Pichia pastoris Uvrag in vacuolar protein sorting and the phosphatidylinositol 3-kinase complex in phagophore elongation in autophagy pathways

Although it has been established that Atg6/Beclin 1, the phosphatidylinositol 3-kinase (PI3K) Vps34, and associated proteins have direct or indirect roles in autophagic pathways in both mammals and yeasts, the elucidation of these roles and the proteins required for them is ongoing. The involvement of the Beclin 1-binding protein, UVRAG, has been a particular source of disagreement. We found that PpAtg6 is required for all autophagic pathways that have been identified in the yeast Pichia pastoris, as well as for the carboxypeptidase Y (PpCPY) vacuolar protein sorting pathway. We localized PpAtg6 to the phagophore assembly site (PAS) and observed its continued presence at that site as the isolation membrane grew from it and matured into a pexophagosome. PpUvrag, however, was required for proper PpCPY sorting, but not for any autophagic pathway. Rather, the defects in all autophagic pathways observed when PpUvrag was overexpressed support its presence in a complex that competes with the PI3K complex required for autophagy.     

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats

Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.     

Manipulation of autophagy by bacteria for their own benefit

Autophagy is the host innate immune system's first line of defense against microbial intruders. When the innate defense system recognizes invading bacterial pathogens and their infection processes, autophagic proteins act as cytosolic sensors that allow the autophagic pathway to be rapidly activated. However, many intracellular bacterial pathogens deploy highly evolved mechanisms to evade autophagic recognition, manipulate the autophagic pathway, and remodel the autophagosomal compartment for their own benefit. Here current topics regarding the recognition of invasive bacteria by the cytosolic innate immune system are highlighted, including autophagy and the mechanisms that enable bacteria to evade autophagy. Also highlighted are some selective examples of bacterial activities that manipulate the autophagic pathways for their own benefit.     

Chaperone mediated autophagy contributes to the newly synthesized histones H3 and H4 quality control

Although there are several pathways to ensure that proteins are folded properly in the cell, little is known about the molecular mechanisms regulating histone folding and proteostasis. In this work, we identified that chaperone-mediated autophagy (CMA) is the main pathway involved in the degradation of newly synthesized histones H3 and H4. This degradation is finely regulated by the interplay between HSC70 and tNASP, two histone interacting proteins. tNASP stabilizes histone H3 levels by blocking the direct transport of histone H3 into lysosomes. We further demonstrate that CMA degrades unfolded histone H3. Thus, we reveal that CMA is the main degradation pathway involved in the quality control of histone biogenesis, evidencing an additional mechanism in the intricate network of histone cellular proteostasis.     

Chaperone-mediated autophagy: Molecular mechanisms and physiological relevance

Chaperone-mediated autophagy (CMA) is a selective lysosomal pathway for the degradation of cytosolic proteins. We review in this work some of the recent findings on this pathway regarding the molecular mechanisms that contribute to substrate targeting, binding and translocation across the lysosomal membrane. We have placed particular emphasis on the critical role that changes in the lipid composition of the lysosomal membrane play in the regulation of CMA, as well as the modulatory effect of other novel CMA components. In the second part of this review, we describe the physiological relevance of CMA and its role as one of the cellular mechanisms involved in the response to stress. Changes with age in CMA activity and the contribution of failure of CMA to the phenotype of aging and to the pathogenesis of several age-related pathologies are also described.     

Glucocerebrosidase deficits in sporadic Parkinson disease

Parkinson disease (PD) is a progressive neurodegenerative movement disorder characterized pathologically by abnormal SNCA/α-synuclein protein inclusions in neurons. Impaired lysosomal autophagic degradation of cellular proteins is implicated in PD pathogenesis and progression. Heterozygous GBA mutations, encoding lysosomal GBA/glucocerebrosidase (glucosidase, β, acid), are the greatest genetic risk factor for PD, and reduced GBA and SNCA accumulation are related in PD models. Here we review our recent human brain tissue study demonstrating that GBA deficits in sporadic PD are related to the early accumulation of SNCA, and dysregulation of chaperone-mediated autophagy (CMA) pathways and lipid metabolism.     

Osmotic stress induced toxicity exacerbates Parkinson's associated effects via dysregulation of autophagy in transgenic C. elegans model

The accumulation of aggregate-prone proteins is a major representative of many neurological disorders, including Parkinson's disease (PD) wherein the cellular clearance mechanisms, such as the ubiquitin-proteasome and autophagy pathways are impaired. PD, known to be associated with multiple genetic and environmental factors, is characterized by the aggregation of α-synuclein protein and loss of dopaminergic neurons in midbrain. This disease is also associated with other cardiovascular ailments. Herein, we report our findings from studies on the effect of hyper and hypo-osmotic induced toxicity representing hyper and hypotensive condition as an extrinsic epigenetic factor towards modulation of Parkinsonism, using a genetic model Caenorhabditis elegans (C. elegans). Our studies showed that osmotic toxicity had an adverse effect on α-synuclein aggregation, autophagic puncta, lipid content and oxidative stress. Further, we figure that reduced autophagic activity may cause the inefficient clearance of α-synuclein aggregates in osmotic stress toxicity, thereby promoting α-synuclein deposition. Pharmacological induction of autophagy by spermidine proved to be a useful mechanism for protecting cells against the toxic effects of these proteins in such stress conditions. Our studies provide evidence that autophagy is required for the removal of aggregated proteins in these conditions. Studying specific autophagy pathways, we observe that the osmotic stress induced toxicity was largely associated with atg-7 and lgg-1 dependent autophagy pathway, brought together by involvement of mTOR pathway. This represents a unifying pathway to disease in hyper- and hypo-osmotic conditions within PD model of C. elegans.     

BAG3 mediates chaperone-based aggresome-targeting and selective autophagy of misfolded proteins

Increasing evidence indicates the existence of selective autophagy pathways, but the manner in which substrates are recognized and targeted to the autophagy system is poorly understood. One strategy is transport of a particular substrate to the aggresome, a perinuclear compartment with high autophagic activity. In this paper, we identify a new cellular pathway that uses the specificity of heat-shock protein 70 (Hsp70) to misfolded proteins as the basis for aggresome-targeting and autophagic degradation. This pathway is regulated by the stress-induced co-chaperone Bcl-2-associated athanogene 3 (BAG3), which interacts with the microtubule-motor dynein and selectively directs Hsp70 substrates to the motor and thereby to the aggresome. Notably, aggresome-targeting by BAG3 is distinct from previously described mechanisms, as it does not depend on substrate ubiquitination.     

Linking lysosomal trafficking defects with changes in aging and stress response in Drosophila

Defects in pathways that direct cellular components to the lysosome for degradation are often linked with a decrease in viability and with progressive disorders. Previously we had shown that blue cheese (bchs: Drosophila homologue of human Alfy) mutations lead to reduced longevity and the accumulation of ubiquitinated neural aggregates. A genetic modifier screen based on overexpression of Bchs in the eye was used to identify several potential genetic interactions, which included autophagic and endocytic trafficking genes as well as cytoskeletal and motor proteins and members of the SUMO and ubiquitin signaling pathways. We found that mutations in several of the genes identified in the screen also result in bchs-like phenotypes, including a reduction in adult lifespan and changes in ubiquitinated protein profiles. In addition, we show here that Bchs modifiers belonging to the autophagic and trans-Golgi trafficking pathways also display defects in adult starvation response. Our data further support a role for Bchs/Alfy in the autophagic pathway and strongly indicate that autophagy plays an important role in aging and stress response.     

Autophagosome Formation Depends on the Small GTPase Rab1 and Functional ER Exit Sites

Autophagy is an important cellular degradation pathway present in all eukaryotic cells. Via this pathway, portions of the cytoplasm and/or organelles are sequestered in double‐membrane structures called autophagosomes. In spite of the significant advance achieved in autophagy, the long‐standing question about the source of the autophagic membrane remains unsolved. We have investigated the role of the secretory pathway in autophagosome biogenesis. Sar1 and Rab1b are monomeric GTPases that control traffic from the endoplasmic reticulum (ER) to the Golgi. We present evidence indicating that the activity of both proteins is required for autophagosome formation. Overexpression of dominant‐negative mutants and the use of siRNAs impaired autophagosome generation as determined by LC3 puncta formation and light chain 3 (LC3)‐II processing. In addition, our results indicate that the autophagic and secretory pathways intersect at a level preceding the brefeldin A blockage, suggesting that the transport from the cis/medial Golgi is not necessary for autophagosome biogenesis. Our present results highlight the role of transport from the ER in the initial events of the autophagic vacuole development.