eSymposia | Autophagy: Mechanisms and Disease

Oct 5, 2020 ‐ Oct 8, 2020



Sessions

ATG14 and RB1CC1 play essential roles in maintaining muscle homeostasis

Sep 30, 2020 4:00pm ‐ Sep 30, 2020 4:00pm

Speaker(s):

Autophagy-enhancing drugs boost HIV-1 restriction and suppress viral replication ex vivo

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Autophagy-enhancing drugs boost HIV-1 restriction and suppress viral replication ex vivo Alexandra P.M. Cloherty 1,*, Nienke H. van Teijlingen 1, Tracy-Jane T.H.D. Eisden 1,2, John L. van Hamme 1, Anusca G. Rader 1, Teunis B.H. Geijtenbeek 1, Renée R.C.E. Schreurs 1, Carla M.S. Ribeiro 1 1 Department of Experimental Immunology, Amsterdam Infection and Immunity Institute, Amsterdam University Medical Centers, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. 2 Department of Medical Oncology, Amsterdam UMC, Vrije Universiteit Amsterdam, Cancer Center Amsterdam, 1081 HV Amsterdam, The Netherlands * Presenting Many human pathologies are associated with autophagy dysregulation, and amongst these is chronic HIV-1 infection and pathogenesis. Although current direct-acting antiviral therapies are highly effective in suppressing HIV-1 replication, HIV-1 persists in long-lived tissue-derived dendritic cells (DCs) and CD4+ T cells of treated patients. In addition, mucosal inflammation undermines current prophylactic antiviral treatment efficacy. Notably, previous research has also shown that HIV-1 infected individuals who remain clinically stable in the absence of antiretroviral therapy have significantly higher levels of autophagy vesicles. We and others have shown that viral components escape or inhibit different stages of the autophagy pathway in human DCs and CD4+ T cells, impeding autophagic destruction of HIV-1. Here, we have investigated the impact of pharmaceutically enhancing autophagy on HIV-1 acquisition and residual viral replication. To this end, we developed a human tissue infection model permitting concurrent analysis of HIV-1 cellular targets ex vivo. Prophylactic treatment with FDA-approved autophagy-enhancing drugs targeting host factors such as mTORC and IP3 boosted HIV-1 restriction in skin-derived CD11c+ DCs and CD4+ T cells. An mTORC inhibitor decreased susceptibility to multiple strains of HIV-1 in skin-derived and vaginal Langerhans cells, which are an epidermal DC subset. Notably, we observed cell-specific effects of therapeutic treatment with autophagy drugs. Only therapeutic treatment with mTORC inhibitors suppressed HIV-1 replication in skin-derived dermal CD11c+ DCs, while all selected drugs limited HIV-1 replication in CD4+ T cells. Strikingly, these results were replicated in primary human intestinal CD4+ T cells. Our findings highlight that manipulation of host autophagy is a relevant target for HIV-1 therapies. Furthermore, this study illustrates that our novel human tissue infection model, which permits discrimination of pharmaceutical effects in distinct immune cell types, is an exciting new option for screening drugs for limitation of mucosal virus replication. We show that that repurposing clinically-approved autophagy drugs can limit mucosal HIV-1 acquisition and suppress ongoing replication in a cell-specific manner. Finally, we propose that combinatory use of autophagy-based and direct-acting antivirals may potentiate the efficacy of direct-acting antivirals by optimizing host protective immune defenses, thereby accelerating treatment efficacy and reducing likelihood of antiviral resistance.

Speaker(s):

Measuring autophagic flux by quantitative mass spectrometry.

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Measuring autophagic flux by quantitative mass spectrometry. Bertina Telusma1, Jean-Claude Farre2, Suresh Subramani2, Joey Davis1 1Department of Biology, Massachusetts Institute of Technology; 2Molecular Biology, Division of Biological Sciences, University of California, San Diego. Autophagy is an essential, well-conserved catabolic process known to deliver a variety of proteins and organelles to the lysosome/vacuole for degradation. Despite extensive study, a comprehensive profile of autophagic substrates has not been reported and the relative contributions of non-specific (bulk) autophagy vs. selective autophagy to such degradation is underexplored. Here, we develop and employ a novel autophagic assay that utilizes stable isotope pulse-labeling and mass spectrometry to quantify bulk or selective autophagic degradation on a protein-by-protein basis. Using this assay, we assess: 1) what fraction of the proteome is degraded through autophagy upon a change in growth conditions; 2) what are the relative contributions of bulk and selective autophagy to this degradation; and 3) whether the degradation of some substrates is prioritized relative to that of others. Using the methylotrophic yeast P. pastoris, we find that enormous swaths of the proteome are degraded in an Atg9- and Atg11-dependent fashion as the microbe transitions between growth on different carbon sources. This result suggests that autophagy plays an outsized role in facilitating this nutrient adaptation, and our method directly identifies both the bulk and selective autophagic substrates targeted. Additionally, we observe that canonical selective autophagy substrates are degraded at different rates and in a growth-media dependent manner, suggesting a form of substrate prioritization. In sum, our study establishes a novel autophagy assay that could be readily extended to other model organisms, enumerates autophagic substrates in the yeast P. pastoris, and highlights the relative contributions of bulk and selective autophagy to nutrient adaptation in this methylotrophic yeast.

Speaker(s):

Autophagy and lysosomal degradation ensure accurate chromosomal segregation to prevent genomic instability

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Autophagy and lysosomal degradation ensure accurate chromosomal segregation to prevent genomic instability Authors: Eugènia Almacellas (1, 2), Joffrey Pelletier (2), Charles Day (3, 4), Santiago Ambrosio (5), Albert Tauler (1, 2), Caroline Mauvezin (2) Affiliations: 1: Department of Biochemistry and Physiology, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain. 2: Metabolism and Cancer Laboratory, Molecular Mechanisms and Experimental Therapy in Oncology Program (Oncobell, Institut d'Investigació Biomèdica de Bellvitge - IDIBELL, L'Hospitalet de Llobregat, Spain. 3: Hormel Institute, University of Minnesota, Austin, MN, USA. 4: Neuro-Oncology Program, Mayo Clinic, Rochester, MN, USA. 5: Department of Physiological Sciences, Faculty of Medicine and Health Sciences, University of Barcelona, Barcelona, Spain. ABSTRACT Lysosomes are cytosolic organelles responsible for the degradation of substrates coming from different converging pathways, including macroautophagy. To date, the degradative function of the lysosomes has been mainly studied in interphase cells, while their role during mitosis remains controversial. Indeed, some studies propose that they are inactive during cell division to protect the genetic material from degradation, and others indicate certain activity of selective autophagy at specific mitotic phases. Mitosis dictates the faithful transmission of genetic material among generations, and perturbations of mitotic division lead to chromosomal instability, a hallmark of cancer. Heretofore, correct mitotic progression relies on the orchestrated degradation of mitotic factors, which was mainly attributed to ubiquitin-triggered proteasome-dependent degradation. We undertook to study different cancer cell lines and found that lysosomes and autophagy are active during mitosis and are necessary for the process. We showed that mitotic transition also relies on lysosome-dependent degradation, as impairment of lysosomes increased mitotic timing and led to mitotic errors, thus promoting chromosomal instability. Furthermore, using proteomic approach, we identified more than 100 novel putative lysosomal substrates in mitotic cells. Among them, WAPL, a cohesin regulatory protein, emerged as a novel SQSTM1-interacting protein for targeted lysosomal degradation. Understanding the role of lysosomes and autophagy in mitotic progression led to another discovery. Indeed, cells that have suffered errors during mitotic progression, either due to alterations of lysosomal function or by other stresses, present a nucleus with a toroidal morphology, with the appearance of a perforated nucleus, after they divided. Until now, the only biomarker used for the detection of chromosomal instability was the micronucleus. We propose that the toroidal nucleus represents a complementary new biomarker for the identification of cells with chromosomal instability, inherent in cancer cells. Our results establish a connection between two influential fields in cancer research: autophagy and chromosomal instability. Our findings serve as precedent for the characterization of the regulating mechanisms involving autophagy and lysosomes to maintain chromosomal stability.

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mt-Keima detects PINK1/Parkin-mitophagy in vivo; mito-QC does not

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

mt-Keima detects PINK1/Parkin-mitophagy in vivo; mito-QC does not Yi-Ting Liu1†, Danielle A. Sliter2†, Mario K. Shammas1, Xiaoping Huang1, Hannah Calvelli1, Chunxin Wang2, Derek P. Narendra1 PINK1 and Parkin, which cause Parkinson’s disease when mutated, form a quality control mitophagy pathway that is well-characterized in cultured cells (1). The extent to which PINK1/Parkin contributes to mitophagy in vivo, however, is controversial. This is due in large part to conflicting results from studies using one of two mitophagy reporters: mt-Keima or mito-QC (2–6). Studies using mt-Keima have generally detected PINK1/Parkin mitophagy in vivo, whereas those using mito-QC generally have not. Here, we directly compared the performance of mito-QC and mt-Keima in cell culture and in mice subjected to a PINK1/Parkin activating stress. We found that mito-QC was less sensitive than mt-Keima for mitophagy, and that this difference was more pronounced for PINK1/Parkin-dependent mitophagy. These findings suggest that mito-QC’s poor sensitivity may account for conflicting reports of PINK1/Parkin mitophagy in vivo and caution against using mito-QC as a reporter for PINK1/Parkin mitophagy. References 1. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol 2011; 12:9–14. 2. Katayama H, Kogure T, Mizushima N, Yoshimori T, Miyawaki A. A sensitive and quantitative technique for detecting autophagic events based on lysosomal delivery. Chem Biol 2011; 18:1042–52. 3. Sun N, Yun J, Liu J, Malide D, Liu C, Rovira II, Holmström KM, Fergusson MM, Yoo YH, Combs CA, et al. Measuring In Vivo Mitophagy. Mol Cell 2015; 60:685–96. 4. Allen GFG, Toth R, James J, Ganley IG. Loss of iron triggers PINK1/Parkin-independent mitophagy. EMBO Rep 2013; 14:1127–35. 5. McWilliams TG, Prescott AR, Allen GFG, Tamjar J, Munson MJ, Thomson C, Muqit MMK, Ganley IG. mito-QC illuminates mitophagy and mitochondrial architecture in vivo. J Cell Biol 2016; 214:333–45. 6. McWilliams TG, Prescott AR, Montava-Garriga L, Ball G, Singh F, Barini E, Muqit MMK, Brooks SP, Ganley IG. Basal Mitophagy Occurs Independently of PINK1 in Mouse Tissues of High Metabolic Demand. Cell Metab 2018; 27:439-449.e5. Funding source This work was supported by the Intramural Research Program of the NINDS, National Institutes of Health.

Speaker(s):

Atg4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Atg4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system Thanh Ngoc Nguyen1, Benjamin Scott Padman1, Susanne Kraft2, Louise Uoselis1, Marvin Skulsuppaisarn1, Christian Behrends2 and Michael Lazarou1 1Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Melbourne, Australia 2Munich Cluster for System Neurology, Medical Faculty, Ludwig‐Maximilians‐University München, Munich, Germany The mammalian family of Atg8 proteins (LC3/GABARAPs) are crucial for autophagosome-lysosome fusion during starvation-induced autophagy and PINK1/Parkin mitophagy. Although also important for autophagosome expansion, these ubiquitin-like proteins are dispensable for autophagosome biogenesis and cargo sequestration. Atg8s become functional during canonical autophagy upon conjugation to the lipid phosphatidylethanolamine (PE) on autophagosomal membranes, which occurs through a ubiquitin-like conjugation system. However, prior to conjugation, Atg8s must be processed by the Atg4 family of cysteine proteases to expose their C-terminal Glycine which is required for lipidation. The Atg4 family also plays a role in removing Atg8s from PE by a process termed de-lipidation or de-conjugation. In yeast, de-lipidation is proposed to release Atg8 that has been non-specifically conjugated to intracellular membranes in order to supply Atg8 for autophagosome biogenesis. In mammals, the purpose of de-lipidation remains unclear. Nevertheless, it is widely accepted that the role of Atg4s in autophagy is completely dependent on their ability to cleave Atg8 family members. We find that HeLa cells lacking all four human Atg4s (Atg4A, Atg4B, Atg4C and Atg4D) fail to build autophagosomes during PINK1/Parkin mitophagy. This phenotype is unexpected given that autophagosomes can form in both hexa KO cells lacking LC3/GABARAPs, and in Atg5 KO cells lacking LC3/GABARAP lipidation. To address whether Atg4s play an LC3/GABARAP-independent role in autophagosome formation, the Atg4 family was further knocked out in Atg8 hexa KO cells (Atg4/Atg8 deca KO). In contrast to the parental Atg8 hexa KO line, Atg4/Atg8 deca KO cells were incapable of forming mitophagosomes during PINK1/Parkin mitophagy. Our analyses of autophagosome formation revealed that Atg4s drive phagophore growth independently of their protease activity and of Atg8s. Analysis of phagophore ultrastructure using artificial intelligence-directed 3D electron microscopy revealed a key role for the Atg4 family in driving phagophore progression. Collectively, we have uncovered a novel Atg8-independent function of the Atg4 family in regulating autophagosome biogenesis during PINK1/Parkin mitophagy.

Speaker(s):

De-phosphorylation of Atg13 by Cdc14 functions as a molecular switch for meiosis-specific autophagy oscillation

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

De-phosphorylation of Atg13 by Cdc14 functions as a molecular switch for meiosis-specific autophagy oscillation Authors: Wenzhi Feng, Fei Wang* Affiliations: UT Southwestern Medical Center, Department of Internal Medicine, Center for Autophagy Research, Dallas, TX Abstract Autophagy is a conserved eukaryotic lysosomal degradation pathway, on which the signals from various signaling pathways can merge for cell to respond to dynamic changes of internal and external environments. Autophagy regulation is linked to literally every aspect of biology, and remains unknown in the field of meiosis, a specialized cell cycle in all sexual reproducing organisms. Using S. cerevisiae as model system, we have recently reported that autophagy is essential for meiosis exit. With combined genetic, biochemical and cell biological approaches, we reported here a striking pattern of autophagy during meiotic cell division: it oscillates, with peaks at Anaphase I (meiosis I exit) and Anaphase II (meiosis II exit). Importantly, we identified that Cdc14, an essential and conserved phosphatase that is periodically released from nucleus into cytosol at Anaphase I & Anaphase II to counteract Cdc28 (yeast CDK1) kinase activity so that meiosis exit and cytokinesis can be achieved, is responsible for autophagy oscillation. To elucidate the underlying molecular mechanism, we reconstituted in vitro three sequential events: the Cdc14 mediated Atg13p de-phosphorylation, de-phosphorylated Atg13 binding to Atg1, and Atg1 kinase activation. We conclude that Cdc14 stimulates autophagy at least through Atg1 activation. We further demonstrated that Cdc14 is a meiosis-specific autophagy regulator. These findings illustrate a novel meiotic pathway of Cdc14 that enables autophagy oscillation, while the physiological role(s) of autophagy oscillation remains to be explored.

Speaker(s):
  • Fei Wang, PhD, UT Southwestern Medical Center

Investigation of mechanisms involved in Apolipoprotein E autophagic degradation and endocytosis

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Investigation of mechanisms involved in Apolipoprotein E autophagic degradation and endocytosis Gianna M. Fote1, Nicolette R. Geller2, Nikolaos Efstathiou3, Demetrios G. Vavvas3, Jack C. Reidling4, Leslie M. Thompson1,2,4,5, Joan S. Steffan*2,4 1UC Irvine Department of Biological Chemistry, 2UC Irvine Department of Psychiatry and Human Behavior, 3Harvard Medical School Department of Opthalmology, 4UC Irvine MIND Institute, 5UC Irvine Department of Neurobiology and Behavior The Apolipoprotein E4 allele (APOE4) is the greatest genetic risk factor for late-onset Alzheimer’s disease (AD). Compared to the more common APOE3, APOE4 expression results in enlarged endosomes and reduced autophagic flux. Impaired endolysosomal trafficking has been proposed as one potential mechanism contributing to AD. Levels of APOE4 are reduced relative to APOE3 in human tissue, and APOE4 is believed to be degraded rapidly, but the mechanism of degradation is unknown. We have found that APOE is degraded by the lysosome, and have investigated autophagic mechanisms of APOE degradation. Lysosomal de-acidification with bafilomycin A1 (Baf) causes APOE accumulation in transiently transfected immortalized neuronal cells (St14a). Both Baf and Brefeldin A (BFA), which disrupts the golgi apparatus, prevent pH-sensitive dual-tagged APOE-mCherry-SepHluorin from entering acidic compartments and fluorescing red. To investigate the mechanism of APOE lysosomal degradation, we knocked down autophagy proteins Lamp2, Atg7, Stx17, and non-canonical autophagy protein Rubicon. In mouse brain tissue, Lamp2 knockout results in accumulation of mouse APOE in vivo. In transfected St14a cells, dual-tagged APOE3 and APOE4 significantly accumulate with Lamp2A knockdown, but APOE2, which is protective for AD, does not. Imaging of APOE3-mCherry shows accumulation around the periphery of Lamp2A knockdown lysosomes, and more APOE3 was found to immunoprecipitate with lysosomes in Lamp2A knockdown cells. In human hepatic HepG2 cells, knockdown of Lamp2A, Atg7 or Stx17 all significantly increase endogenous APOE3 levels. Atg7 knockdown also impairs endocytosis of APOE, as does knockdown of Rubicon. Endocytosis of APOE3 significantly increases LC3 lipidation in St14a and HepG2 cells, suggesting that APOE internalization may be LC3-dependent. GABARAPL1, another member of the Atg8 family, co-localizes with endocytosed APOE3 following treatment with the fusion inhibitor chloroquine. Imaging of the endocytosis of fluorescent APOE suggests that APOE is endocytosed in an isoform-dependent manner, with APOE4 endocytosed more robustly than APOE3, and APOE2 endocytosed the least. In conclusion, our data suggest that APOE is degraded by the lysosome through autophagy that may require Lamp2A, Atg7, and Stx17, and may enter the cell through LC3-dependent endocytosis. APOE4, the AD risk allele, appears to be degraded by the lysosome through autophagy and to be endocytosed more robustly than APOE2, suggesting a possible mechanism for rapid APOE4 degradation observed in tissue, which may contribute to AD pathogenesis. Understanding how APOE traffics through the endolysosomal system may lead to insight into mechanisms that contribute to AD progression, and may be targeted to develop novel therapies for AD. Funding sources: NIH AG016573, F30 AG060704

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Role of autophagy in the maintenance of definitive hematopoiesis using zebrafish platform

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Role of autophagy in the maintenance of definitive hematopoiesis using zebrafish platform Autophagy is a dynamic and evolutionary conserved lysosomal degradation pathway for cellular remodeling, development and homeostasis. It is also essential for the maintenance of different hematopoietic lineages including erythroid, myeloid, and lymphoid ones, yet its function in definitive hematopoiesis is not clear. Here, we inhibited autophagy in zebrafish (Danio rerio) embryos via ulk1b (homologous to human ULK1) knock-out to observe hematopoiesis. Taken advantages of the optically clear and externally fertilized zebrafish embryos together with the genetic tractability and pharmacological approaches, here we reported that, zebrafish lacking the key autophagy gene ulk1b inhibit autophagy at the earlier stage by blocking the autophagosome formation or activation process. Dysfunction of the ulk1b gene in zebrafish embryos resulted in minimal cytosolic microtubuleassociated protein 1A/1B-light chain 3 (Lc3I) conjugation to phosphatidylethanolamine (PE) to form LC3-PE conjugate (LC3-II) followed by decreased autophagosomal membrane recruitment. Consequently, in-vivo live imaging showed that inhibition of autophagy further suppress autophagosomes and autolysosomes numbers. However, this autophagy inhibition modulated the hematopoietic lineage specific concomitant upregulation of myeloid progenitors (spi1b), pan-leukocytes (lcp1), macrophages (mpeg1.1) and neutrophils (mpo). Conversely, erythroid progenitors (gata1), embryonic hemoglobin (hbae1.1) and hematopoietic stem cell (cmyb) numbers significantly decreased. Treatment with autophagy inducer calpeptin was not sufficient to ameliorate the defective hematopoiesis in mutant embryos. These findings indicated that lack of autophagy throughout definitive hematopoiesis incorporate with myeloproliferation and anemia in zebrafish embryos which further warrant important role of autophagy to maintain normal HSC functions.

Speaker(s):

Dissecting a neuron-to-liver crosstalk to modulate lipid metabolism in Batten disease

Oct 5, 2020 12:00am ‐ Oct 5, 2020 12:00am

Dissecting a neuron-to-liver crosstalk to modulate lipid metabolism in Batten disease García-Macia M, Bolaños JP Instituto de Investigación Biomédica de Salamanca (IBSAL), Universidad de Salamanca, Institute of Functional Biology and Genomics (IBFG), Salamanca. Neuronal ceroid lipofuscinoses (NCL), known as Batten disease, are the most common of the rare neurodegenerative disorders in children. To date, defects in thirteen different genes have been identified in NCL patients. Despite the genetic heterogeneity, Batten diseases are grouped together based on clinical similarities and broadly uniform neuropathological features, including accumulation of lipofuscin in lysosomes, as well as profound neurodegeneration and widespread gliosis. Amongst these, the incidence of Cln7 disease, caused by mutation in MFSD8 gene, is the highest in southern and Mediterranean Europe. CLN7/MFSD8 encodes a lysosomal membrane glycoprotein with unknown function. Lysosomes are the only organelles able to hydrolyse triacylglycerols, which fuels the mitochondria for energy generation. The autophagic machinery provides TGs to the lysosomes through a process known as lipophagy. Although defective autophagy has been related with Cln7 disease, Cln7 role in lipid metabolism is unknown, particularly in lipophagy. Here, we hypothesized that disruption of lipophagy links neuronal death and Cln7-mediated Batten disease. To address this, we have studied lipophagy in Cln7 knockout (Cln7-KO) mice and investigated whether Cln7 loss in the hypothalamus disrupts liver lipophagy. We have obtained experimental data from different techniques in Cln7-KO mice fibroblasts, liver, brown fat and brain, to stablish a connection between the brain and the metabolism of the peripheral tissues. Thus, our data show that Cln7 deficiency in the hypothalamus damages liver lipophagy resulting in fat accumulation. Ongoing work is being developed to validate these observations using robust metabolic and in vivo uncoupling approaches. Acknowledgments: We thank Resch M., Prieto E. and Carabias M. for their excellent technical assistance in animal housing and genotyping. This work was supported by the Fundación BBVA and the Fundación Ramon Areces.

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