Adaptive autophagy in Alexander disease-affected astrocytes

The ubiquitin-proteasome and autophagy-lysosomal pathways are the two main routes of protein and organelle clearance in eukaryotic cells. The proteasome system is responsible for unfolded, short-lived proteins, which precludes the clearance of oligomeric and aggregated proteins, whereas macroautophagy, a process generally referred to as autophagy, mediates mainly the bulk degradation of long-lived cytoplasmic proteins, large protein complexes or organelles.(1) Recently, the autophagy-lysosomal pathway has been implicated in neurodegenerative disorders as an important pathway for the clearance of abnormally accumulated intracellular proteins, such as huntingtin, tau and mutant and modified alpha-synuclein.(1-6) Our recent study illustrated the induction of adaptive autophagy in response to mutant glial fibrillary acidic protein (GFAP) accumulation in astrocytes, in the brains of patients with Alexander disease (AxD), and in mutant GFAP knock-in mouse brains.(7) This autophagic response is negatively regulated by mammalian target of rapamycin (mTOR). The activation of p38 MAPK by GFAP accumulation is responsible for mTOR inactivation and the induction of autophagy. We also found that the accumulation of GFAP impairs proteasome activity.(8) In this commentary we discuss the potential compensatory relationship between an impaired proteasome and activated autophagy, and propose that the MLK-MAPK (mixed lineage kinase-mitogen-activated protein kinase) cascade is a regulator of this crosstalk.     

Adaptive autophagy in Alexander disease-affected astrocytes

The ubiquitin-proteasome and autophagy-lysosomal pathways are the two main routes of protein and organelle clearance in eukaryotic cells. The proteasome system is responsible for unfolded, short-lived proteins, which precludes the clearance of oligomeric and aggregated proteins, whereas macroautophagy, a process generally referred to as autophagy, mediates mainly the bulk degradation of long-lived cytoplasmic proteins, large protein complexes or organelles.¹ Recently, the autophagy-lysosomal pathway has been implicated in neurodegenerative disorders as an important pathway for the clearance of abnormally accumulated intracellular proteins, such as huntingtin, tau, and mutant and modified α-synuclein.^1-6 Our recent study illustrated the induction of adaptive autophagy in response to mutant glial fibrillary acidic protein (GFAP) accumulation in astrocytes, in the brains of patients with Alexander disease (AxD), and in mutant GFAP knock-in mouse brains.⁷ This autophagic response is negatively regulated by mammalian target of rapamycin (mTOR). The activation of p38 MAPK by GFAP accumulation is responsible for mTOR inactivation and the induction of autophagy. We also found that the accumulation of GFAP impairs proteasome activity.⁸ In this commentary we discuss the potential compensatory relationship between an impaired proteasome and activated autophagy, and propose that the MLK-MAPK (mixed lineage kinase–mitogen-activated protein kinase) cascade is a regulator of this crosstalk.

Addendum to: Tang G, Yue Z, Talloczy, Z, Hagemann T, Cho W, Sulzer D, Messing A, Goldman JE. Alexander disease-mutant GFAP accumulation stimulates autophagy through p38 MAPK and mTOR signaling pathways. Hum Mol Genetics 2008; In press.     

Does Huntingtin play a role in selective macroautophagy?

The accumulation of protein aggregates in neurons appears to be a basic feature of neurodegenerative disease. In huntington disease (HD), a progressive and ultimately fatal neurodegenerative disorder caused by an expansion of the polyglutamine repeat within the protein huntingtin (Htt), the immediate proximal cause of disease is well understood. However, the cellular mechanisms which modulate the rate at which fragments of Htt containing polyglutamine accumulate in neurons is a central issue in the development of approaches to modulate the rate and extent of neuronal loss in this disease. We have recently found that Htt is phosphorylated by the kinase IKK on serine (s) 13, activating its phosphorylation on S16 and its acetylation and poly-SUMOylation, modifications that modulate its clearance by the proteasome and lysosome in cells.¹ In the discussion here I suggest that Htt may have a normal function in the lysosomal mechanism of selective macroautophagy involved in its own degradation which may share some similarity with the yeast cytoplasm to vacuole targeting (Cvt) pathway. Pharmacologic activation of this pathway may be useful early in disease progression to treat HD and other neurodegenerative diseases characterized by the accumulation of disease proteins.Key words: Huntington disease, Huntingtin, polyglutamine, autophagy, IKKAn age-related reduction in protein clearance mechanisms has been implicated in the pathogenesis of neurodegenerative diseases including the polyglutamine (polyQ) repeat diseases, Alzheimer disease (AD), Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). These diseases are each associated with the accumulation of insoluble protein aggregates in diseased neurons. Huntington Disease (HD), caused by an expansion of the polyQ repeat in the protein Huntingtin (Htt), is one such disease of aging in which mutant Htt inclusions form in striatal and cortical neurons as disease progresses. Clarification of the mechanisms of Htt clearance is paramount to finding therapeutic targets to treat HD that may be broadly useful in the treatment of these currently incurable neurodegenerative diseases.     

Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.     

Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-superoxide dismutase proteins by macroautophagy and the proteasome

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause approximately 20% of familial cases of amyotrophic lateral sclerosis (fALS). Accumulating evidence indicates that a gain of toxic function of mutant SOD1 proteins is the cause of the disease. It has also been shown that the ubiquitin-proteasome pathway plays a role in the clearance and toxicity of mutant SOD1. In this study, we investigated the degradation pathways of wild-type and mutant SOD1 in neuronal and nonneuronal cells. We provide here the first evidence that wild-type and mutant SOD1 are degraded by macroautophagy as well as by the proteasome. Based on experiments with inhibitors of these degradation pathways, the contribution of macroautophagy to mutant SOD1 clearance is comparable with that of the proteasome pathway. Using assays that measure cell viability and cell death, we observed that under conditions where expression of mutant SOD1 alone does not induce toxicity, macroautophagy inhibition induced mutant SOD1-mediated cell death, indicating that macroautophagy reduces the toxicity of mutant SOD1 proteins. We therefore propose that both macroautophagy and the proteasome are important for the reduction of mutant SOD1-mediated neurotoxicity in fALS. Inhibition of macroautophagy also increased SOD1 levels in detergent-soluble and -insoluble fractions, suggesting that both detergent-soluble and -insoluble SOD1 are degraded by macroautophagy. These findings may provide further insights into the mechanisms of pathogenesis of fALS.     

Mutant ataxin-3 promotes the autophagic degradation of parkin

There is growing evidence that autophagy plays a key role in neurodegenerative diseases. For instance, stimulating autophagy is neuroprotective both in vitro and in vivo in models of trinucleotide-repeat diseases such as Machado-Joseph disease (MJD). Similarly, proteins associated with familial forms of Parkinson disease (PD) such as parkin and PINK1 converge on the autophagy pathway. Yet, despite these shared mechanisms, it is not clear whether or how these disorders are related at a molecular level. We reported that the mutant form of ataxin-3, the protein responsible for MJD, promotes the autophagic degradation of parkin. Given that the loss of parkin function leads to PD, we propose that the increased turnover of parkin triggered by mutant ataxin-3 may explain some of the parkinsonian features observed in MJD. Moreover, the findings suggest that an increased clearance of parkin in MJD could mitigate the otherwise beneficial effects of autophagy in neurodegeneration.     

Small molecule enhancers of autophagy for neurodegenerative diseases

Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, prion diseases and polyglutamine disorders, including Huntington's disease and various spinocerebellar ataxias, are associated with the formation of protein aggregates. These aggregates and/or their precursors are thought to be toxic disease-causing species. Autophagy is a major degradation pathway for intracytosolic aggregate-prone proteins, including those associated with neurodegeneration. It is a constitutive self-degradative process involved both in the basal turnover of cellular components and in response to nutrient starvation in eukaryotes. Enhancing autophagy may be a possible therapeutic strategy for neurodegenerative disorders where the mutant proteins are autophagy substrates. In cell and animal models, chemical induction of autophagy protects against the toxic insults of these mutant aggregate-prone proteins by enhancing their clearance. We will discuss various autophagy-inducing small molecules that have emerged in the past few years that may be leads towards the treatment of such devastating diseases.     

Enhanced degradation of mutant huntingtin by rho kinase inhibition is mediated through activation of proteasome and macroautophagy

Enhancing the degradation of mutant protein is one of the most investigated approaches in experimental therapy of the polyglutamine-related disorders such as Huntington disease (HD). Inhibition of rho-associated kinases (ROCKs) reduced the aggregation and levels of mutant huntingtin in cellular models of HD via activation of the ubiquitin proteasome system (UPS) and macroautophagy. This unique effect makes the Rho/ROCK pathway and its downstream effectors attractive therapeutic targets for polyglutamine-related diseases.     

Role of autophagy in the clearance of mutant huntingtin: a step towards therapy?

Macroautophagy (henceforth referred to simply as autophagy) is a bulk degradation process involved in the clearance of long-lived proteins, protein complexes and organelles. A portion of the cytosol, with its contents to be degraded, is enclosed by double-membrane structures called autophagosomes/autophagic vacuoles, which ultimately fuse with lysosomes where their contents are degraded. In this review, we will describe how induction of autophagy is protective against toxic intracytosolic aggregate-prone proteins that cause a range of neurodegenerative diseases. Autophagy is a key clearance pathway involved in the removal of such proteins, including mutant huntingtin (that causes Huntington’s disease), mutant ataxin-3 (that causes spinocerebellar ataxia type 3), forms of tau that cause tauopathies, and forms of alpha-synuclein that cause familial Parkinson’s disease. Induction of autophagy enhances the clearance of both soluble and aggregated forms of such proteins, and protects against toxicity of a range of these mutations in cell and animal models. Interestingly, the aggregates formed by mutant huntingtin sequester and inactivate the mammalian target of rapamycin (mTOR), a key negative regulator of autophagy. This results in induction of autophagy in cells with these aggregates.     

Insulin htts on autophagy

Macroautophagy has been shown to participate in the degradation and clearance of polyglutamine (polyQ) tract-containing proteins generated by trinucleotide repeat expansion mutations. Large expansions are the genetic cause of diseases such as Huntington's Disease that lead to neuronal dysfunction due to polyQ protein aggregates. Recently, a functional screen performed by Yamamoto et al to investigate proteins that regulate such autophagic processes revealed a novel role for insulin signaling in the promotion of autophagy of mutant protein aggregates. This suggests that insulin/insulin-like growth factor signaling pathways not only prevent the induction of autophagy, but paradoxically may promote autophagy of deleterious proteins in certain circumstances.     

E6-AP promotes misfolded polyglutamine proteins for proteasomal degradation and suppresses polyglutamine protein aggregation and toxicity

The accumulation of intracellular protein deposits as inclusion bodies is the common pathological hallmark of most age-related neurodegenerative disorders including polyglutamine diseases. Appearance of aggregates of the misfolded mutant disease proteins suggest that cells are unable to efficiently degrade them, and failure of clearance leads to the severe disturbances of the cellular quality control system. Recently, the quality control ubiquitin ligase CHIP has been shown to suppress the polyglutamine protein aggregation and toxicity. Here we have identified another ubiquitin ligase, called E6-AP, which is able to promote the proteasomal degradation of misfolded polyglutamine proteins and suppress the polyglutamine protein aggregation and polyglutamine protein-induced cell death. E6-AP interacts with the soluble misfolded polyglutamine protein and associates with their aggregates in both cellular and transgenic mouse models. Partial knockdown of E6-AP enhances the rate of aggregate formation and cell death mediated by the polyglutamine protein. Finally, we have demonstrated the up-regulation of E6-AP in the expanded polyglutamine protein-expressing cells as well as cells exposed to proteasomal stress. These findings suggest that E6-AP is a critical mediator of the neuronal response to misfolded polyglutamine proteins and represents a potential therapeutic target in the polyglutamine diseases.     

Autophagy,a guardian against neurodegeneration

Autophagy is an intracellular degradation process responsible for the clearance of most long-lived proteins and organelles. Cytoplasmic components are enclosed by double-membrane autophagosomes, which subsequently fuse with lysosomes for degradation. Autophagy dysfunction may contribute to the pathology of various neurodegenerative disorders, which manifest abnormal protein accumulation. As autophagy induction enhances the clearance of aggregate-prone intracytoplasmic proteins that cause neurodegeneration (like mutant huntingtin, tau and ataxin 3) and confers cytoprotective roles in cell and animal models, upregulating autophagy may be a tractable therapeutic strategy for diseases caused by such proteins. Here, we will review the molecular machinery of autophagy and its role in neurodegenerative diseases. Drugs and associated signalling pathways that may be targeted for pharmacological induction of autophagy will also be discussed.     

Macroautophagy in Immunity and Tolerance

Autophagy and proteasomal degradation constitute the two main catabolic pathways in cells. While the proteasome degrades primarily short-lived soluble proteins, macroautophagy, the main constitutive autophagic pathway, delivers cell organelles and protein aggregates for lysosomal degradation. Both the proteasome and macroautophagy are attractive effector mechanisms for the immune system because they can be used to degrade foreign substances, including pathogenic proteins, within cells. Therefore, both innate and adaptive immune responses use these pathways for intracellular clearance of pathogens as well as for presentation of pathogen fragments to the adaptive immune system. Because, however, the same mechanisms are used for the steady-state turnover of cellular self-components, the immune system has to be desensitized not to recognize these. Therefore, proteasomal degradation and macroautophagy are also involved in tolerizing the immune system prior to pathogen encounter. We will discuss recent advances in our understanding how macroautophagy selects self-structures in the steady state, how presentation of these on major histocompatibility complex class II molecules leads to tolerance and how macroautophagy assists both innate and adaptive immunity. This new knowledge on the specialized functions of the metabolic process macroautophagy in higher eukaryotes should allow us to target it for therapy development against immunopathologies and to improve vaccinations.     

Targeting autophagy in ALS: A complex mission

Several neurodegenerative diseases share a common neuropathology, primarily featuring the presence of abnormal protein inclusions in the brain containing specific misfolded proteins. Strategies to decrease the load of protein aggregates and oligomers are considered relevant targets for therapeutic intervention. Many studies indicate that macroautophagy is a selective and efficient mechanism for the degradation of misfolded mutant proteins related to neurodegeneration, without affecting the levels of the corresponding wild-type form. In fact, activation of autophagy by rapamycin treatment decreases the accumulation of protein aggregates and alleviates disease features in animal models of Huntington disease and other disorders affecting the nervous system. Recent evidence, however, indicates that the expression of several disease-related genes may actually impair autophagy activity at different levels, including omegasome formation, substrate recognition, lysosomal acidity and autophagosome membrane nucleation. A recent report from Zhang and co-workers indicates that treatment of an amyotrophic lateral sclerosis (ALS) mouse model with rapamycin actually exacerbates neuronal loss and disease progression, associated with enhanced apoptosis. This study reflects the need for a better understanding of the contribution of autophagy to ALS and other neurodegenerative diseases since this pathway may not only operate as a cleaning-up mechanism. Then, autophagy impairment may be part of the pathological mechanisms underlying the disease, whereas augmenting autophagy levels above a certain threshold could lead to detrimental effects in neuronal function and survival. Combinatorial strategies to repair the autophagy deficit and also enhance the activation of the pathway may result in a beneficial impact to decrease the content of protein aggregates and damaged organelles, improving neuronal function and survival.     

Targeting autophagy in ALS: a complex mission

Several neurodegenerative diseases share a common neuropathology, primarily featuring the presence of abnormal protein inclusions in the brain containing specific misfolded proteins. Strategies to decrease the load of protein aggregates and oligomers are considered relevant targets for therapeutic intervention. Many studies indicate that macroautophagy is a selective and efficient mechanism for the degradation of misfolded mutant proteins related to neurodegeneration, without affecting the levels of the corresponding wild-type form. In fact, activation of autophagy by rapamycin treatment decreases the accumulation of protein aggregates and alleviates disease features in animal models of Huntington disease and other disorders affecting the nervous system. Recent evidence, however, indicates that the expression of several disease-related genes may actually impair autophagy activity at different levels, including omegasome formation, substrate recognition, lysosomal acidity and autophagosome membrane nucleation. A recent report from Zhang and co-workers indicates that treatment of an amyotrophic lateral sclerosis (ALS) mouse model with rapamycin actually exacerbates neuronal loss and disease progression, associated with enhanced apoptosis. This study reflects the need for a better understanding of the contribution of autophagy to ALS and other neurodegenerative diseases since this pathway may not only operate as a cleaning-up mechanism. Then, autophagy impairment may be part of the pathological mechanisms underlying the disease, whereas augmenting autophagy levels above a certain threshold could lead to detrimental effects in neuronal function and survival. Combinatorial strategies to repair the autophagy deficit and also enhance the activation of the pathway may result in a beneficial impact to decrease the content of protein aggregates and damaged organelles, improving neuronal function and survival.     

Insulin htts on Autophagy

Macroautophagocytosis has been shown to participate in the degradation and clearance of polyglutamine (polyQ) tract-containing proteins generated by trinucleotide repeat expansion mutations. Large expansions are the genetic cause of diseases such as Huntington’s disease that lead to neuronal dysfunction due to polyQ protein aggregates. Recently, a functional screen performed by Yamamoto et al. to investigate proteins that regulate such autophagic processes revealed a novel role for insulin signaling in the promotion of autophagy of mutant protein aggregates. This suggests that insulin/insulin-like growth factor signaling pathways not only prevent the induction of autophagy, but paradoxically may promote autophagy of deleterious proteins in certain circumstances.

Commentary to:

Autophagy-Mediated Clearance of Huntingtin Aggregates Triggered by the Insulin-Signaling Pathway

A. Yamamoto, M.L. Cremona and J.E. Rothman

J Cell Biol 2006; 172:719-31     

Autophagy: PolyQ toxic fragment turnover

Recent studies have highlighted the importance of the lysosome in degrading proteins that misfold in neurodegenerative diseases. In this study we explore the role for autophagy in the clearance of an N-terminal caspase-7-generated fragment of ataxin-7, a protein with a pathogenic polyglutamine (polyQ) expansion in the neurodegenerative disease spinocerebellar ataxia 7 (SCA7). Using both cellular and transgenic mouse models of SCA7 we show that the stability of wild-type ataxin-7 is modified by macroautophagy, but not by proteasomal, inhibition, whereas both autophagy and proteasomal degradation have little effect on polyQ-expanded ataxin-7. We also create a post-translational modification-deficient ataxin-7 mutant that has increased protein turnover of both wild-type and polyQ-expanded ataxin-7, mediated through the autophagy pathway. Histological analysis reveals that wild-type ataxin-7 colocalizes with markers of chaperone-mediated autophagy (CMA) and macroautophagy, indicating that both of these mechanisms may play a role in the clearance of ataxin-7. Furthermore, there is an increase in LC3, a marker of autophagy initiation, in the cerebellum of SCA7 transgenic mice. Our findings indicate that the ataxin-7 fragment may be cleared via autophagy and that this process is altered in SCA7. Identification of the different types of autophagy involved in ataxin-7 turnover and the influence of post-translational modifications on these processes will be pursued in future studies.     

Ubiquilin at a crossroads in protein degradation pathways

Ubiquilin proteins are conserved across all eukaryotes and function in the regulation of protein degradation. We found that ubiquilin functions to regulate macroautophagy and that the protein is also a substrate of chaperone-mediated autophagy.Key words: autophagy, cell death, LC3, protein turnover, ubiquitinUbiquilin proteins are present in all eukaryotes and appear to function in protein degradation pathways. Humans contain four ubiquilin genes each encoding a separate protein. The proteins are approximately 600 amino acids in length and share extensive homology with one another. They are characterized by an N-terminal sequence that is very similar to ubiquitin, called the ubiquitin-like domain (UBL), followed by a longer, more variable central domain, and terminate with a conserved 50-amino-acid sequence called a ubiquitin-associated domain (UBA). This structural organization is characteristic of proteins that function to deliver ubiquitinated proteins to the proteasome for degradation. In accordance with this function, the UBL domain of ubiquilin binds subunits of the proteasome, and its UBA domain binds to polyubiquitin chains that are typically conjugated onto proteins that are marked for destruction. Indeed, we recently showed that ubiquilin is recruited to the endoplasmic reticulum where it binds and promotes the degradation of misfolded proteins to the proteasome during ER-associated degradation (ERAD).Remarkably, ubiquilin was also recently reported to be involved in macroautophagy. The finding was based on colocalization of ubiquilin with autophagosomal marker LC3 in cells, and because overexpression of ubiquilin-1 suppresses and silencing of its expression enhances, starvation-induced cell death. In our recently published paper we describe our evidence linking ubiquilin to autophagy. We demonstrate that ubiquilin is indeed present in different structures associated with macroautophagy and that it is required for a critical step in autophagosome formation. Additionally, we also demonstrate that ubiquilin is a substrate of chaperone-mediated autophagy. The findings suggest that ubiquilin might play an important, and perhaps a crucial, role in dictating the pathway of protein degradation in cells.In previous studies we found that ubiquilin proteins expressed in normal growing HeLa cells are very stable with a rate of turnover in excess of 20 h. Because most long-lived proteins are degraded by autophagy, we felt it was important to distinguish whether ubiquilin localization in autophagosomes was simply related to the expected route of degradation of the protein or whether it was related to some special function in autophagy. Accordingly, our experiments were designed to distinguish between these two possibilities.Using double immunofluorescence microscopy we found that endogenous ubiquilin and LC3 proteins are present in puncta in HeLa cells. To ensure this was not an artifact of the staining procedure, we cotransfected HeLa cells with ubiquilin-1 and LC3 expression constructs that were tagged with either mRFP or GFP proteins and again found that the two expressed proteins are colocalized in puncta, irrespective of which tag was fused to the proteins. Further evidence supporting ubiquilin localization to autophagosomes was obtained by showing strong enrichment of ubiquilin proteins upon purification of autophagosomes from mouse liver and by the strong immunogold staining of the protein in autophagosomes in mouse brains in a transgenic mouse model of Alzheimer disease.To determine if ubiquilin localization to autophagosomes is mediated by interaction with LC3 we conducted immunoprecipitation experiments to examine whether the two proteins coimmunoprecipitate with each other. Indeed, our results showed that the two proteins coimmunoprecipitate with one another, indicating that they bind together in a complex. However, we did not detect any strong binding between bacterially expressed forms of the proteins, suggesting that the interaction between the proteins in cells might be mediated by a bridging factor(s).We next used a pH-sensitive tandem-tagged mCherry-GFP-LC3 reporter that is used to monitor maturation of autophagosomes to autolysosomes to determine whether ubiquilin is present during the different steps of macroautophagy. Indeed, we found that anti-ubiquilin staining is present throughout the different structures involved in the process, and interestingly, we also noted that the structures are enriched for K48- and K63-ubiquitin linkages. Because ubiquilin contains a UBA domain that binds ubiquitin chains we examined whether proteins containing K48- and K63-ubiquitin linkages coimmunoprecipitate with ubiquilin. Indeed, our immunoblots indicated that proteins containing both of these types of linkages coprecipitate with ubiquilin, consistent with the idea that ubiquilin might target proteins with diverse ubiquitin linkages for degradation by autophagy.To determine if ubiquilin is required for autophagy, we knocked down the ubiquilin-1 and -2 proteins in HeLa cells (which mainly express these two ubiquilin isoforms) by siRNA transfection and examined if loss of the proteins altered LC3-I and LC3-II levels. Interestingly, we found that ubiquilin knockdown over a 72 h time period is associated with a progressive increase in LC3-I levels and a concomitant decrease in LC3-II levels. Furthermore, ubiquilin knockdown led to an ∼45% reduction in the number of cells containing five or more autophagosomes. Based on these results we propose that ubiquilin is required for maturation of LC3-I to LC3-II, which we speculate might be related to the requirement of the protein in macroautophagy.We next asked if ubiquilin protein is consumed during autophagy. We examined this by treating HeLa cells with puromycin to induce protein misfolding and macroautophagy. Immunoblot analysis of the protein lysates examined at 2 h intervals over a 7 h period of exposure to puromycin revealed a direct correlation between stimulation of macroautophagy and a time-dependent decrease in the ubiquilin and LC3-II protein levels. The time-dependent decline in the proteins is inhibited by treatment of cells with two different autophagy inhibitors, 3-methyladenine and bafilomycin A₁. The results suggest that ubiquilin protein is consumed during macroautophagy.The consumption of ubiquilin during macroautophagy prompted us to examine if ubiquilin might also be involved in chaperone-mediated autophagy (CMA), which involves the active transport of proteins into lysosomes. Support for this idea arose because ubiquilin proteins contain two sequences that conform to a pentapeptide motif involved in CMA. An in vitro CMA assay using recombinant GST-ubiquilin-1 fusion protein and purified lysosomes confirmed ubiquilin is an active CMA substrate. The results suggested that ubiquilin can be consumed by two different types of autophagy, macroautophagy and CMA. We speculate that this dual mode of consumption may provide a potential switch whereby changes in ubiquilin levels beyond a certain threshold might trigger execution of either macroautophagy or CMA. The idea that such a switch exists stems from previous work that showed inhibition of CMA can lead to activation of macroautophagy and vice versa.Several intriguing new questions emerge from this and previous works, including what exact function ubiquilin serves in autophagy, particularly in the execution of macroautophagy and CMA. Is there a signal that instructs ubiquilin to choose between its known functions in autophagy and ERAD or is the choice random? What role do its different domains play in these processes? The answers to these questions are likely to be important because in previous studies we showed that overexpression of ubiquilin protects cells against potentially toxic mutant huntingtin proteins containing polyglutamine expansions. In our new work we also found that ubiquilin overexpression protects cells against starvation-induced cell death caused by mutations in presenilin-2 proteins. The underlying conclusion from these studies is that ubiquilin appears to play important roles in regulating protein degradation pathways that are likely to have important implications in cell survival. Clearly, understanding ubiquilin function in different protein degradation pathways could lead to novel approaches to prevent diseases associated with protein misfolding.