Search

Disrupted homeostasis of the microtubule binding protein tau is a shared feature of a set of neurodegenerative disorders known as tauopathies. Acetylation of soluble tau is an early pathological event in neurodegeneration. In this work, we find that a large fraction of neuronal tau is degraded by chaperone-mediated autophagy (CMA) whereas, upon acetylation, tau is preferentially degraded by macroautophagy and endosomal microautophagy. Rerouting of acetylated tau to these other autophagic pathways originates, in part, from the inhibitory effect that acetylated tau exerts on CMA and results in its extracellular release. In fact, experimental blockage of CMA enhances cell-to-cell propagation of pathogenic tau in a mouse model of tauopathy. Furthermore, analysis of lysosomes isolated from brains of patients with tauopathies demonstrates similar molecular mechanisms leading to CMA dysfunction. This study reveals that CMA failure in tauopathy brains alters tau homeostasis and could contribute to aggravate disease progression., The tau protein has been implicated in neurodegenerative disorders and can propagate from cell to cell. Here, the authors show that tau acetylation reduces its degradation by chaperone-mediated autophagy, causing re-routing to other autophagic pathways and increasing extracellular tau release.

18 p.-8 fig., Chaperone-mediated autophagy activity, essential in the cellular defense against proteotoxicity, declines with age, and preventing this decline in experimental genetic models has proven beneficial. Here, we have identified the mechanism of action of selective chaperone-mediated autophagy activators previously developed by our group and have leveraged that information to generate orally bioavailable chaperone-mediated autophagy activators with favorable brain exposure. Chaperone-mediated autophagy activating molecules stabilize the interaction between retinoic acid receptor alpha - a known endogenous inhibitor of chaperone-mediated autophagy - and its co-repressor, nuclear receptor corepressor 1, resulting in changes of a discrete subset of the retinoic acid receptor alpha transcriptional program that leads to selective chaperone-mediated autophagy activation. Chaperone-mediated autophagy activators molecules activate this pathway in vivo and ameliorate retinal degeneration in a retinitis pigmentosa mouse model. Our findings reveal a mechanism for pharmacological targeting of chaperone-mediated autophagy activation and suggest a therapeutic strategy for retinal degeneration., This work was supported by National Institutes of Health grants AG054108, AG021904, and AG017617 (to AMC) and AG038072 (to AMC, FM, and EG), the JPB Foundation (to AMC), the Michael J. Fox Foundation (to AMC and EG), the Rainwater Charitable Foundation (to AMC and EG) and the Backus Foundation (to AMC). Chemical synthesis, NMR and MS data were in part supported by NIH awards P30 CA013330 and 1S10OD016305. Research in PB and RGS lab is funded by Ministerio de Ciencia, Innovación y Universidades (MCIU), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) PGC2018-098557-B-I00 to PB and RTI2018-098990-J-I00 to RGS. CAR was supported by T32 GM007491 and MM by T32 AG023475.

Obesity is associated with changes in the immune system that significantly hinder its ability to mount efficient immune responses. Previous studies have reported a dysregulation of immune responses caused by lipid challenge; however, the mechanisms underlying that dysregulation are still not completely understood. Autophagy is an essential catabolic process through which cellular components are degraded by the lysosomal machinery. In T cells, autophagy is an actively regulated process necessary to sustain homeostasis and activation. Here, we report that CD4(+) T cell responses are inhibited when cells are challenged with increasing concentrations of fatty acids. Furthermore, analysis of T cells from diet-induced obese mice confirms that high lipid load inhibits activation-induced responses in T cells. We have found that autophagy is inhibited in CD4(+) T cells exposed in vitro or in vivo to lipid stress, which causes decreased autophagosome formation and degradation. Supporting that inhibition of autophagy caused by high lipid load is a key mechanism that accounts for the effects on T cell function of lipid stress, we found that ATG7 (autophagy-related 7)-deficient T cells, unable to activate autophagy, did not show additional inhibitory effects on their responses to activation when subjected to lipid challenge. Our results indicate, thus, that increased lipid load can dysregulate autophagy and cause defective T cell responses, and suggest that inhibition of autophagy may underlie some of the characteristic obesity-associated defects in the T cell compartment. Abbreviations: ACTB: actin, beta; ATG: autophagy-related; CDKN1B: cyclin-dependent kinase inhibitor 1B; HFD: high-fat diet; IFNG: interferon gamma; IL: interleukin; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; MAPK8/JNK: mitogen-activated protein kinase 8; LC3-I: non-conjugated form of MAP1LC3B; LC3-II: phosphatidylethanolamine-conjugated form of MAP1LC3B; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MS: mass spectrometry; MTOR: mechanistic target of rapamycin kinase; NFATC2: nuclear factor of activated T cells, cytoplasmic, calcineurin dependent 2; NLRP3: NLR family, pyrin domain containing 3; OA: oleic acid; PI: propidium iodide; ROS: reactive oxygen species; STAT5A: signal transducer and activator of transcription 5A; TCR: T cell receptor; T(H)1: T helper cell type 1

Autophagy delivers cytosolic components to lysosomes for degradation and is thus essential for cellular homeostasis and to cope with different stressors. As such, autophagy counteracts various human diseases and its reduction leads to aging-like phenotypes. Macroautophagy (MA) can selectively degrade organelles or aggregated proteins, whereas selective degradation of single proteins has only been described for chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI). These 2 autophagic pathways are specific for proteins containing KFERQ-related targeting motifs. Using a KFERQ-tagged fluorescent biosensor, we have identified an eMI-like pathway in Drosophila melanogaster. We show that this biosensor localizes to late endosomes and lysosomes upon prolonged starvation in a KFERQ- and Hsc70-4- dependent manner. Furthermore, fly eMI requires endosomal multivesicular body formation mediated by ESCRT complex components. Importantly, induction of Drosophila eMI requires longer starvation than the induction of MA and is independent of the critical MA genes atg5, atg7, and atg12. Furthermore, inhibition of Tor signaling induces eMI in flies under nutrient rich conditions, and, as eMI in Drosophila also requires atg1 and atg13, our data suggest that these genes may have a novel, additional role in regulating eMI in flies. Overall, our data provide the first evidence for a novel, starvation-inducible, catabolic process resembling endosomal microautophagy in the Drosophila fat body.

Summary Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule‐based minus‐end‐directed motor proteins, the well‐characterized dynein, and the less‐studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti‐aging purposes.

Macroautophagy is a cellular process that mediates degradation in the lysosome of cytoplasmic components including proteins and organelles. Previous studies have shown that macroautophagy is induced in activated T cells to regulate organelle homeostasis and the cell's energy metabolism. However, the signaling pathways that initiate and regulate activation-induced macroautophagy in T cells have not been identified. Here, we show that activation-induced macroautophagy in T cells depends on signaling from common γ-chain cytokines. Consequently, inhibition of signaling through JAK3, induced downstream of cytokine receptors containing the common γ-chain, prevents full induction of macroautophagy in activated T cells. Moreover, we found that common γ-chain cytokines are not only required for macroautophagy upregulation during T cell activation but can themselves induce macroautophagy. Our data also show that macroautophagy induction in T cells is associated with an increase of LC3 expression that is mediated by a post-transcriptional mechanism. Overall, our findings unveiled a new role for common γ-chain cytokines as a molecular link between autophagy induction and T-cell activation.

Chaperone-mediated autophagy (CMA), a selective form of degradation of cytosolic proteins in lysosomes, contributes to maintenance of proteostasis and to the cellular adaptation to stress. CMA substrates are delivered by a cytosolic chaperone to the lysosomal surface, where, upon unfolding, they are internalized through a membrane translocation complex. The molecular components that participate in CMA substrate targeting and translocation are well characterized, but those involved in CMA regulation remain mostly unknown. In this study, we have identified that CMA is under the positive control of the phosphatase PHLPP1 that associates with the lysosomal membrane and counteracts the inhibitory effect of mTORC2 on CMA. Lysosomal Akt, a target of the mTORC2/PHLPP1 kinase-phosphatase pair, modulates CMA activity by controlling the dynamics of assembly and disassembly of the CMA translocation complex at the lysosomal membrane. The lysosomal mTORC2/PHLPP1/Akt axis could become a target to restore CMA dysfunction in aging and disease.

Chaperone-mediated autophagy (CMA) is a multistep process that involves selective degradation and digestion of a pool of soluble cytosolic proteins in lysosomes. Cytosolic substrates are selectively identified and targeted by chaperones to lysosomes where they are subsequently translocated into the organelle lumen through a dedicated CMA-associated lysosomal membrane receptor/translocation complex. CMA contributes to maintaining a functional proteome, through elimination of altered proteins, and participates in the cellular energetic balance through amino acid recycling. Defective or dysfunctional CMA has been associated with human pathologies such as neurodegeneration, cancer, immunodeficiency or diabetes, increasing the overall interest in methods to monitor this selective autophagic pathway. Here, we describe approaches used to study CMA in different experimental models.

Selective macroautophagy is an important protective mechanism against diverse cellular stresses. In contrast to the well-characterized starvation-induced autophagy, the regulation of selective autophagy is largely unknown. Here, we demonstrate that Huntingtin, the Huntington disease gene product, functions as a scaffold protein for selective macroautophagy but it is dispensable for non-selective macroautophagy. In Drosophila, Huntingtin genetically interacts with autophagy pathway components. In mammalian cells, Huntingtin physically interacts with the autophagy cargo receptor p62 to facilitate its association with the integral autophagosome component LC3 and with Lys-63-linked ubiquitin-modified substrates. Maximal activation of selective autophagy during stress is attained by the ability of Huntingtin to bind ULK1, a kinase that initiates autophagy, which releases ULK1 from negative regulation by mTOR. Our data uncover an important physiological function of Huntingtin and provide a missing link in the activation of selective macroautophagy in metazoans.

Chaperone-mediated autophagy (CMA), a cellular process that contributes to protein quality control through targeting of a subset of cytosolic proteins to lysosomes for degradation, undergoes a functional decline with age. We have used a mouse model with liver-specific defective CMA to identify changes in proteostasis attributable to reduced CMA activity in this organ with age. We have found that other proteolytic systems compensate for CMA loss in young mice which helps to preserve proteostasis. However, these compensatory responses are not sufficient for protection against proteotoxicity induced by stress (oxidative stress, lipid challenges) or associated with aging. Livers from old mice with CMA blockage exhibit altered protein homeostasis, enhanced susceptibility to oxidative stress and hepatic dysfunction manifested by a diminished ability to metabolize drugs, and a worsening of the metabolic dysregulation identified in young mice. Our study reveals that while the regulatory function of CMA cannot be compensated for in young organisms, its contribution to protein homeostasis can be handled by other proteolytic systems. However, the decline in the compensatory ability identified with age explains the more severe consequences of CMA impairment in older organisms and the contribution of CMA malfunction to the gradual decline in proteostasis and stress resistance observed during aging.

Summary Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signaling motivated us to explore the possible functional interactions between this signaling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signaling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. While abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signaling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie the basis of some common ciliopathies.

Author manuscript; available in PMC 2017 June 14.-- et al., Calorie restriction (CR) is the most robust non-genetic intervention to delay aging. However, there are a number of emerging experimental variables that alter CR responses. We investigated the role of sex, strain, and level of CR on health and survival in mice. CR did not always correlate with lifespan extension, although it consistently improved health across strains and sexes. Transcriptional and metabolomics changes driven by CR in liver indicated anaplerotic filling of the Krebs cycle together with fatty acid fueling of mitochondria. CR prevented age-associated decline in the liver proteostasis network while increasing mitochondrial number, preserving mitochondrial ultrastructure and function with age. Abrogation of mitochondrial function negated life-prolonging effects of CR in yeast and worms. Our data illustrate the complexity of CR in the context of aging, with a clear separation of outcomes related to health and survival, highlighting complexities of translation of CR into human interventions., This work was supported in part by the Intramural Research Program of the National Institute on Aging, NIH, and by NIH grants R01 AG043483 and R01 DK098656 (J.A.B.), NIH grant AG031782 (A.M.C.), the Proteostasis of Aging Core AG038072 (A.M.C.). J.M.M. was supported by a postdoctoral fellowship from the American Diabetes Association, grant 1-15-MI-03. P.C. was supported by NIH grants (1P01AG034906, 1R01GM090311, 1R01ES 020812). F.M. is grateful to the FWF for grants LIPOTOX, I1000, P 27893, P 29203 and P24381-B20 and the BMWFW for grants “Unconventional research” and «Flysleep (80.109/0001 -WF/V/3b/2015). JMV was supported by the Spanish Ministerio de Economía y Competitividad (grants BFU2011-23578 and BFU2015-64630-R). The authors thank the personnel from the Servicio Centralizado de Apoyo a la Investigación (SCAI; University of Córdoba) for technical support.

Poorly differentiated and anaplastic thyroid carcinomas are very aggressive, almost invariably lethal neoplasms for which no effective treatment exists. These tumors are intrinsically resistant to cell death, even when their driver oncogenic signaling pathways are inhibited. We have undertaken a detailed analysis, in mouse and human thyroid cancer cells, of the mechanism through which Obatoclax, a pan-inhibitor of the anti-apoptotic proteins of the BCL2 family, effectively reduces tumor growth in vitro and in vivo. We demonstrate that Obatoclax does not induce apoptosis, but rather necrosis of thyroid cancer cells, and that non-transformed thyroid cells are significantly less affected by this compound. Surprisingly, we show that Obatoclax rapidly localizes to the lysosomes and induces loss of acidification, block of lysosomal fusion with autophagic vacuoles, and subsequent lysosomal permeabilization. Notably, prior lysosome neutralization using different V-ATPase inhibitors partially protects cancer cells from the toxic effects of Obatoclax. Although inhibition of autophagy does not affect Obatoclax-induced cell death, selective down-regulation of ATG7, but not of ATG5, partially impairs Obatoclax effects, suggesting the existence of autophagy-independent functions for ATG7. Strikingly, Obatoclax killing activity depends only on its accumulation in the lysosomes, and not on its interaction with BCL2 family members. Finally, we show that also other lysosome-targeting compounds, Mefloquine and LLOMe, readily induce necrosis in thyroid cancer cells, and that Mefloquine significantly impairs tumor growth in vivo, highlighting a clear vulnerability of these aggressive, apoptosis-resistant tumors that can be therapeutically exploited.

Connexins modulate intercellular communication when assembled in gap junctions. Compromised macroautophagy increases cellular communication due to failure to degrade connexins at gap junctions. Nedd4-mediated ubiquitinylation of the connexin molecule is required to trigger its autophagy-dependent internalization and degradation., Different pathways contribute to the turnover of connexins, the main structural components of gap junctions (GJs). The cellular pool of connexins targeted to each pathway and the functional consequences of degradation through these degradative pathways are unknown. In this work, we focused on the contribution of macroautophagy to connexin degradation. Using pharmacological and genetic blockage of macroautophagy both in vitro and in vivo, we found that the cellular pool targeted by this autophagic system is primarily the one organized into GJs. Interruption of connexins' macroautophagy resulted in their retention at the plasma membrane in the form of functional GJs and subsequent increased GJ-mediated intercellular diffusion. Up-regulation of macroautophagy alone is not sufficient to induce connexin internalization and degradation. To better understand what factors determine the autophagic degradation of GJ connexins, we analyzed the changes undergone by the fraction of plasma membrane connexin 43 targeted for macroautophagy and the sequence of events that trigger this process. We found that Nedd4-mediated ubiquitinylation of the connexin molecule is required to recruit the adaptor protein Eps15 to the GJ and to initiate the autophagy-dependent internalization and degradation of connexin 43. This study reveals a novel regulatory role for macroautophagy in GJ function that is directly dependent on the ubiquitinylation of plasma membrane connexins.

Huntington disease (HD) is caused by a unique mutation, an abnormal expansion of a polyglutamine (polyQ) tract in the HTT (huntingtin) protein. It has long been hypothesized that in addition to gained toxicity from the expanded polyQ, loss of HTT's normal cellular functions also contributes to HD pathogenesis. Consistently, wild-type HTT has a well-documented neuronal protective activity and is essential for postnatal neuronal survival. However, despite its relatively simple genetic cause, the etiology of HD remains unclear, due in part to the poor understanding of the physiological cellular functions of HTT. The perplexing HTT is a large ∼3,144 amino acid-long protein with ubiquitous localization, but it lacks any known functional domain that could provide clues about its cellular activities. The phenotypes of HTT knockout (KO) mice are equally puzzling: homozygote HTT KO mice die by day 7.5, but this early embryonic lethality is due to a critical role of HTT in extraembryonic membranes, not in the embryo per se, as it can be rescued if wild-type HTT is provided in extraembryonic tissues. HTT has a vast number of reported interacting partners that have been used to infer some of the growing list of cellular pathways that HTT could participate in. However, it is still uncertain how HTT achieves its well-documented neuroprotective role. Using both Drosophila and mammalian experimental models, we recently demonstrated that HTT has an essential function in selective autophagy where it serves as a scaffold by modulating the activities of the cargo receptor SQSTM1/p62 and the autophagy initiation kinase ULK1. We previously created a null deletion mutant (htt-ko) of the single HTT homolog (htt) in Drosophila and showed that in contrast to mouse, htt-ko flies, which develop ex utero, are fully viable with only mild aging-related defects. Unexpectedly, ectopic expression of a truncated form of the mammalian MAPT induced severe defects in htt-ko flies, suggesting that htt protects against pathogenic MAPT toxicity. Further, in genetic screens using this MAPT-induced phenotype as a functional readout, we detected dosage-sensitive genetic interaction between htt-ko and several components of the autophagy pathway, including atg8a (MAP1LC3A homolog), atg1 (ULK1 homolog) and ref(2)P (SQSTM1/p62 homolog), thus unveiling a functional link between HTT and autophagy. HTT is not a foreigner to the autophagy field, although the main emphasis has been on the toxic effect of mutant HTT on this cellular clearance mechanism. In fact, our earlier work in HD demonstrated that reduced autophagy in the disease context, also reported by many other groups, originated from diminished ability to “trap” cytosolic cargo. We attributed the “empty autophagosomes” phenotype in HD cells to defective cargo recognition due to the presence of mutant polyQ-HTT in the inner part of the closing autophagosomes. However, our genetic studies ablating the wild-type HTT made us reconsider this proposed gain-of-toxicity function. In this recent study, we have found that in both htt-ko flies and HTT-depleted mammalian cells, autophagic abnormalities are present but surprisingly, starvation-induced autophagy is largely normal. However, loss of HTT compromised autophagic induction when challenged by several other stresses, including proteotoxicity, lipotoxicity, and mitochondria damage. In all these instances, mammalian cells depleted of HTT also showed an “empty autophagosomes” defect, suggesting that wild-type HTT is required for efficient cargo recognition by the autophagosome. We found that HTT and SQSTM1/p62, as well as their Drosophila counterparts Htt and Ref(2)P, physically interact and that proteotoxic stress promotes this interaction. Depletion of HTT reduces the association of SQSTM1 with MAP1LC3A and also compromises the binding of SQSTM1 with proteins with lysine-63-linked ubiquitin (K63-Ub) chains, which are preferential substrates of autophagy. However, loss of HTT does not affect self-polymerization of SQSTM1 or its association with K48-Ub-modified proteins mainly cleared by the proteasome. Collectively, these findings support the idea that HTT facilitates cargo recognition by modulating the assembly of the cargo receptors and autophagy proteins. We also found that the dependence on HTT for the initiation of stress-induced selective autophagy is related to its physical interaction with ULK1, whose kinase activity is essential for autophagy initiation. HTT depletion does not affect starvation-induced activation of ULK1 kinase, but compromises ULK1 activation upon proteotoxicity challenge, revealing a contribution of HTT to differentially regulating autophagosome biogenesis under basal or stress conditions. We found that the regulation of HTT on ULK1 is at the level of its interaction with the MTORC1 complex, another main regulator of ULK1 that binds to and inactivates ULK1 under nutrient-rich conditions. We showed that (1) ULK1 exists in 2 mutually exclusive complexes, ULK1-HTT and ULK1-MTORC1; (2) HTT does not affect the kinase activity of MTORC1; (3) HTT competes with MTORC1 for binding with ULK1; (4) stresses that induce selective autophagy (e.g., proteotoxicity, lipotoxicity, or mitophagy), but not starvation, result in an increased association of ULK1 with HTT at the expense of MTOR, thus freeing ULK1 from its inhibition by MTORC1. Collectively, HTT promotes selective autophagy by activating and bringing together SQSTM1/p62 and ULK1 to assure spatial proximity between the cargo and autophagy initiation components, thereby orchestrating 2 major autophagy steps: cargo recognition and autophagy induction (Fig. 1). Figure 1. Model of HTT in promoting selective autophagy. HTT serves as a scaffolding for selective autophagy by bringing together cargo bound through SQSTM1 and an initiator of autophagy, ULK1 kinase. Basal autophagy: under basal conditions, binding of HTT to the ... Increasing evidence supports a tangled relationship between HTT, HD, and autophagy. Thus, after the first report of altered autophagy in HD by DiFiglia, the Holzbaur lab showed that HTT facilitates axonal trafficking of autophagosomes, a function that is compromised by polyQ-HTT; Hayden's group reported an autophagy-inducing domain within HTT; Steffan with Thompson and colleagues also recently reported physical interactions between HTT and several autophagy proteins including SQSTM1 and ULK1. Our finding places HTT in the core of the autophagic process, bringing together components of the cargo recognition and autophagy initiation complexes, and raising many new interesting questions. How is HTT differentially activated upon different stress challenges? Does polyQ expansion directly compromise the endogenous activities of HTT, as implicated by the very similar “empty autophagosomes” phenotypes in both HD and HTT-depleted cells, and if so, will this effect be partially responsible for the toxicity of mutant polyQ-HTT in long-lived neurons? Last, how can we exploit HTT's role in selective autophagy to modulate this process in the fight against this devastating brain disease called HD?

The plasma membrane contributes to the formation of autophagosomes, the double-membrane vesicles that sequester cytosolic cargo and deliver it to lysosomes for degradation during autophagy. In this study, we have identified a regulatory role for connexins (Cx), the main components of plasma membrane gap junctions, in autophagosome formation. We have found that plasma-membrane-localized Cx proteins constitutively downregulate autophagy through a direct interaction with several autophagy-related proteins involved in the initial steps of autophagosome formation, such as Atg16 and components of the PI(3)K autophagy initiation complex (Vps34, Beclin-1 and Vps15). On nutrient starvation, this inhibitory effect is released by the arrival of Atg14 to the Cx-Atg complex. This promotes the internalization of Cx-Atg along with Atg9, which is also recruited to the plasma membrane in response to starvation. Maturation of the Cx-containing pre-autophagosomes into autophagosomes leads to degradation of these endogenous inhibitors, allowing for sustained activation of autophagy.