bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020–01–19
23 papers selected by
Viktor Korolchuk, Newcastle University
  1. Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons.
  2. mTOR at the nexus of nutrition, growth, ageing and disease.
  3. Autophagy in astrocytes and its implications in neurodegeneration.
  4. PI3KC2α-dependent and VPS34-independent generation of PI3P controls primary cilium-mediated autophagy in response to shear stress.
  5. Mitophagy in Alzheimer's Disease and Other Age-Related Neurodegenerative Diseases.
  6. miR-155 inhibits mitophagy through suppression of BAG5, a partner protein of PINK1.
  7. miR-218 Inhibits Mitochondrial Clearance by Targeting PRKN E3 Ubiquitin Ligase.
  8. IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover through a Conserved GSK-3β-Nrf2-BNIP3 Pathway.
  9. Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes.
  10. A proteomic atlas of senescence-associated secretomes for aging biomarker development.
  11. Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk.
  12. FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy.
  13. The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.
  14. Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Drosophila Model for Huntington's Disease.
  15. Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop to promote autophagy.
  16. Autophagy drives fibroblast senescence through MTORC2 regulation.
  17. Spatially and temporally defined lysosomal leakage facilitates mitotic chromosome segregation.
  18. Interaction between HuR and circPABPN1 modulates autophagy in the intestinal epithelium by altering ATG16L1 translation.
  19. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.
  20. Astrocyte senescence promotes glutamate toxicity in cortical neurons.
  21. SIRT3 Acts as a Positive Autophagy Regulator to Promote Lipid Mobilization in Adipocytes via Activating AMPK.
  22. Sestrin prevents atrophy of disused and aging muscles by integrating anabolic and catabolic signals.
  23. Therapeutic Targeting of mTORC2 in mTORopathies.
  1. Elife. 2020 Jan 14. pii: e50260. [Epub ahead of print]9
    Degradation of engulfed mitochondria is rate-limiting in Optineurin-mediated mitophagy in neurons.
    Chantell S Evans, Erika Lf Holzbaur.
      Mitophagy, the selective removal of damaged mitochondria, is thought to be critical to maintain neuronal homeostasis. Mutations of proteins in the pathway cause neurodegenerative diseases, suggesting defective mitochondrial turnover contributes to neurodegeneration. In primary rat hippocampal neurons, we developed a mitophagy induction paradigm where mild oxidative stress induced low levels of mitochondrial damage. Mitophagy-associated proteins were sequentially recruited to depolarized mitochondria followed by sequestration into autophagosomes. The localization of these mitophagy events had a robust somal bias. In basal and induced conditions, engulfed mitochondria remained in non-acidified organelles for hours to days, illustrating efficient autophagosome sequestration but delayed lysosomal fusion or acidification. Furthermore, expression of an ALS-linked mutation in the pathway disrupted mitochondrial network integrity and this effect was exacerbated by oxidative stress. Thus, age-related decline in neuronal health or expression of disease-associated mutations in the pathway may exacerbate the slow kinetics of neuronal mitophagy, leading to neurodegeneration.
    Keywords:  autophagy; cell biology; lysosome acidification; mitochondrial quality control; mitophagy; mouse; neurodegeneration; neuroscience; parkin; rat
    DOI:  https://doi.org/10.7554/eLife.50260
  2. Nat Rev Mol Cell Biol. 2020 Jan 14.
    mTOR at the nexus of nutrition, growth, ageing and disease.
    Grace Y Liu, David M Sabatini.
      The mTOR pathway integrates a diverse set of environmental cues, such as growth factor signals and nutritional status, to direct eukaryotic cell growth. Over the past two and a half decades, mapping of the mTOR signalling landscape has revealed that mTOR controls biomass accumulation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Given the pathway's central role in maintaining cellular and physiological homeostasis, dysregulation of mTOR signalling has been implicated in metabolic disorders, neurodegeneration, cancer and ageing. In this Review, we highlight recent advances in our understanding of the complex regulation of the mTOR pathway and discuss its function in the context of physiology, human disease and pharmacological intervention.
    DOI:  https://doi.org/10.1038/s41580-019-0199-y
  3. J Mol Biol. 2020 Jan 10. pii: S0022-2836(20)30023-1. [Epub ahead of print]
    Autophagy in astrocytes and its implications in neurodegeneration.
    Katherine Sung, Maria Jimenez-Sanchez.
      Autophagy is a major degradation pathway where double membrane vesicles called autophagosomes deliver cytoplasmic content to the lysosome. Increasing evidence suggests that autophagy dysfunction contributes to the pathogenesis of neurodegenerative diseases. In addition, misfolded proteins that accumulate in these diseases and that constitute a common pathological hallmark, are substrates for autophagic degradation. Astrocytes, a major type of glial cells, are emerging as a critical component in most neurodegenerative diseases. This review will summarize the recent efforts to investigate the role that autophagy plays in astrocytes in the context of neurodegenerative diseases. While the field has mostly focused on the implications of autophagy in neurons, autophagy may also be involved in the clearance of disease-related proteins in astrocytes as well as in maintaining astrocyte function, which could impact the cell autonomous and non-cell autonomous contribution of astrocytes to neurodegeneration.
    DOI:  https://doi.org/10.1016/j.jmb.2019.12.041
  4. Nat Commun. 2020 Jan 15. 11(1): 294
    PI3KC2α-dependent and VPS34-independent generation of PI3P controls primary cilium-mediated autophagy in response to shear stress.
    Asma Boukhalfa, Anna Chiara Nascimbeni, Damien Ramel, Nicolas Dupont, Emilio Hirsch, Stephanie Gayral, Muriel Laffargue, Patrice Codogno, Etienne Morel.
      Cells subjected to stress situations mobilize specific membranes and proteins to initiate autophagy. Phosphatidylinositol-3-phosphate (PI3P), a crucial lipid in membrane dynamics, is known to be essential in this context. In addition to nutriments deprivation, autophagy is also triggered by fluid-flow induced shear stress in epithelial cells, and this specific autophagic response depends on primary cilium (PC) signaling and leads to cell size regulation. Here we report that PI3KC2α, required for ciliogenesis and PC functions, promotes the synthesis of a local pool of PI3P upon shear stress. We show that PI3KC2α depletion in cells subjected to shear stress abolishes ciliogenesis as well as the autophagy and related cell size regulation. We finally show that PI3KC2α and VPS34, the two main enzymes responsible for PI3P synthesis, have different roles during autophagy, depending on the type of cellular stress: while VPS34 is clearly required for starvation-induced autophagy, PI3KC2α participates only in shear stress-dependent autophagy.
    DOI:  https://doi.org/10.1038/s41467-019-14086-1
  5. Cells. 2020 Jan 08. pii: E150. [Epub ahead of print]9(1):
    Mitophagy in Alzheimer's Disease and Other Age-Related Neurodegenerative Diseases.
    Qian Cai, Yu Young Jeong.
      Mitochondrial dysfunction is a central aspect of aging and neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease. Mitochondria are the main cellular energy powerhouses, supplying most of ATP by oxidative phosphorylation, which is required to fuel essential neuronal functions. Efficient removal of aged and dysfunctional mitochondria through mitophagy, a cargo-selective autophagy, is crucial for mitochondrial maintenance and neuronal health. Mechanistic studies into mitophagy have highlighted an integrated and elaborate cellular network that can regulate mitochondrial turnover. In this review, we provide an updated overview of the recent discoveries and advancements on the mitophagy pathways and discuss the molecular mechanisms underlying mitophagy defects in Alzheimer's disease and other age-related neurodegenerative diseases, as well as the therapeutic potential of mitophagy-enhancing strategies to combat these disorders.
    Keywords:  Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; aging; amyotrophic lateral sclerosis; lysosome; mitochondrial dynamics; mitochondrial quality control; mitophagosome; mitophagy
    DOI:  https://doi.org/10.3390/cells9010150
  6. Biochem Biophys Res Commun. 2020 Jan 13. pii: S0006-291X(20)30059-0. [Epub ahead of print]
    miR-155 inhibits mitophagy through suppression of BAG5, a partner protein of PINK1.
    Takatoshi Tsujimoto, Tatsufumi Mori, Kei Houri, Yuta Onodera, Toshiyuki Takehara, Kanae Shigi, Shinichi Nakao, Takeshi Teramura, Kanji Fukuda.
      Removal of dysfunctional mitochondria is essential step to maintain normal cell physiology, and selective autophagy in mitochondria, called mitophagy, plays a critical role in quality control of mitochondria. While in several diseases and aging, disturbed mitophagy has been observed. In stem cells, accumulation of damaged mitochondria can lead to deterioration of stem cell properties. Here, we focused on miR-155-5p (miR-155), one of the most prominent miRNAs in inflammatory and aged tissues, and found that miR-155 disturbed mitophagy in mesenchymal stem cells (MSCs). As a molecular mechanism of miR-155-mediated mitophagy suppression, we found that BCL2 associated athanogene 5 (BAG5) is a direct target of miR-155. Reduction of BAG5 resulted in destabilization of PTEN-induced kinase (PINK1) and consequently disrupted mitophagy. Our study suggests a novel mechanism connecting aging and aging-associated inflammation with mitochondrial dysfunction in stem cells through a miRNA-mediated mechanism.
    Keywords:  Aging; Bone marrow MSCs; Mitophagy; miR-155
    DOI:  https://doi.org/10.1016/j.bbrc.2020.01.022
  7. Int J Mol Sci. 2020 Jan 05. pii: E355. [Epub ahead of print]21(1):
    miR-218 Inhibits Mitochondrial Clearance by Targeting PRKN E3 Ubiquitin Ligase.
    Anthea Di Rita, Teresa Maiorino, Krenare Bruqi, Floriana Volpicelli, Gian Carlo Bellenchi, Flavie Strappazzon.
      The selective elimination of dysfunctional mitochondria through mitophagy is crucial for preserving mitochondrial quality and cellular homeostasis. The most described mitophagy pathway is regulated by a positive ubiquitylation feedback loop in which the PINK1 (PTEN induced kinase 1) kinase phosphorylates both ubiquitin and the E3 ubiquitin ligase PRKN (Parkin RBR E3 ubiquitin ligase), also known as PARKIN. This event recruits PRKN to the mitochondria, thus amplifying ubiquitylation signal. Here we report that miR-218 targets PRKN and negatively regulates PINK1/PRKN-mediated mitophagy. Overexpression of miR-218 reduces PRKN mRNA levels, thus also reducing protein content and deregulating the E3 ubiquitin ligase action. In fact, following miR-218 overexpression, mitochondria result less ubiquitylated and the autophagy machinery fails to proceed with correct mitochondrial clearance. Since mitophagy defects are associated with various human diseases, these results qualify miR-218 as a promising therapeutic target for human diseases.
    Keywords:  PARKIN/PRKN; miR-218; microRNA; mitochondria; mitophagy
    DOI:  https://doi.org/10.3390/ijms21010355
  8. Cells. 2020 Jan 08. pii: E147. [Epub ahead of print]9(1):
    IGF-1 Signalling Regulates Mitochondria Dynamics and Turnover through a Conserved GSK-3β-Nrf2-BNIP3 Pathway.
    Sarah Riis, Joss B Murray, Rosemary O'Connor.
      The Insulin-like Growth Factor I (IGF-1) signalling pathway is essential for cell growth and facilitates tumourogenic processes. We recently reported that IGF-1 induces a transcriptional programme for mitochondrial biogenesis, while also inducing expression of the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), suggesting that IGF-1 has a key mitochondria-protective role in cancer cells. Here, we investigated this further and delineated the signaling pathway for BNIP3 induction. We established that IGF-1 induced BNIP3 expression through a known AKT serine/threonine kinase 1 (AKT)-mediated inhibitory phosphorylation on Glycogen Synthase Kinase-3β (GSK-3β), leading to activation of Nuclear Factor Erythroid 2-related Factor 2 (NFE2L2/Nrf2) and acting through the downstream transcriptional regulators Nuclear Respiratory Factor-1 (NRF1) and Hypoxia-inducible Factor 1 subunit α (HIF-1α). Suppression of IGF-1 signaling, Nrf2 or BNIP3 caused the accumulation of elongated mitochondria and altered the mitochondrial dynamics. IGF-1R null Mouse Embryonic Fibroblasts (MEFs) were impaired in the BNIP3 expression and in the capacity to mount a cell survival response in response to serum deprivation or mitochondrial stress. IGF-1 signalling enhanced the cellular capacity to induce autophagosomal turnover in response to activation of either general autophagy or mitophagy. Overall, we conclude that IGF-1 mediated a mitochondria-protective signal that was coordinated through the cytoprotective transcription factor Nrf2. This pathway coupled mitochondrial biogenesis with BNIP3 induction, and increased the cellular capacity for autophagosome turnover, whilst enhancing survival under conditions of metabolic or mitochondrial stress.
    Keywords:  BNIP3; HIF-1α; IGF-1; IGF-1R; NFE2L2/Nrf2; NRF1; autophagy; cancer; cell death; mitophagy
    DOI:  https://doi.org/10.3390/cells9010147
  9. Autophagy. 2020 Jan 16. 1-15
    Cytosolic domain of SIDT2 carries an arginine-rich motif that binds to RNA/DNA and is important for the direct transport of nucleic acids into lysosomes.
    Katsunori Hase, Viorica Raluca Contu, Chihana Kabuta, Ryohei Sakai, Masayuki Takahashi, Naoyuki Kataoka, Fumihiko Hakuno, Shin-Ichiro Takahashi, Yuuki Fujiwara, Keiji Wada, Tomohiro Kabuta.
      RNautophagy and DNautophagy (RDA) are unconventional autophagic pathways where nucleic acids are directly transported through the lysosomal membrane, then degraded inside lysosomes. We have previously shown that bitopic protein LAMP2C and putative RNA transporter SIDT2, both lysosomal membrane proteins, mediate the direct transport of nucleic acids into lysosomes and that LAMP2C interacts with the nucleic acids and functions as a receptor during RDA. Because SIDT2-mediated RDA occurs in isolated lysosomes that lack LAMP2C, in this study, we tested the hypothesis that SIDT2 itself could also interact with the nucleic acids. Our results show that SIDT2 directly binds RNA and DNA through an arginine-rich motif (ARM) located within its main cytosolic domain, and disruption of this motif dramatically impairs SIDT2-mediated RNautophagic activity. We also found that SIDT2 interacts with exon 1 of HTT (huntingtin) transcript through the ARM in a CAG-dependent manner. Moreover, overexpression of SIDT2 promoted degradation of HTT mRNA and reduced the levels of polyglutamine-expanded HTT aggregates, hallmarks of Huntington disease. In addition, a comparative analysis of LAMP2C and SIDT2 functions at the cellular level revealed that the two proteins exert a synergistic effect on RNautophagic activity and that the ARMs which mediate the interactions of SIDT2 and LAMP2C with RNA are essential for the synergy. Together, our results point out the importance of nucleic acid-binding capacity of SIDT2 for its function in translocating nucleic acids through the lipid bilayer and suggests a potential application of RNautophagy activation to reduce the expression levels of disease-causing toxic proteins.Abbreviations: ACTB/β-actin: actin beta; ARM: arginine-rich motif; CBB: Coomassie Brilliant Blue; CD: cytosolic domain; COX4I1/COX4: cytochrome c oxidase subunit 4I1; E. coli: Escherichia coli; EGFP: enhanced green fluorescent protein; EtBr: ethidium bromide; FITC: fluorescein isothiocyanate; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GST: glutathione S-transferase; HRP: horseradish peroxidase; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; HTT: huntingtin; HTTex1: exon 1 of the HTT gene; LAMP2: lysosomal associated membrane protein 2; LMNA: lamin A/C; PAGE: polyacrylamide gel electrophoresis; PBS: phosphate-buffered saline; PEI: polyethyleneimine; polyQ: polyglutamine; qPCR: quantitative PCR; RAB5A: RAB5A, member RAS oncogene family; RDA: RNautophagy and DNautophagy; SCARB2/LIMP2: scavenger receptor class B member 2; SDS: sodium dodecyl sulfate; SID-1: systemic RNA interference deficient-1; SIDT2: SID1 transmembrane family member 2; WT: wild type.
    Keywords:  Arginine-rich motif; DNA; DNautophagy; LAMP2C; RNA; RNautophagy; SIDT2; autophagy; lysosome
    DOI:  https://doi.org/10.1080/15548627.2020.1712109
  10. PLoS Biol. 2020 Jan;18(1): e3000599
    A proteomic atlas of senescence-associated secretomes for aging biomarker development.
    Nathan Basisty, Abhijit Kale, Ok Hee Jeon, Chisaka Kuehnemann, Therese Payne, Chirag Rao, Anja Holtz, Samah Shah, Vagisha Sharma, Luigi Ferrucci, Judith Campisi, Birgit Schilling.
      The senescence-associated secretory phenotype (SASP) has recently emerged as a driver of and promising therapeutic target for multiple age-related conditions, ranging from neurodegeneration to cancer. The complexity of the SASP, typically assessed by a few dozen secreted proteins, has been greatly underestimated, and a small set of factors cannot explain the diverse phenotypes it produces in vivo. Here, we present the "SASP Atlas," a comprehensive proteomic database of soluble proteins and exosomal cargo SASP factors originating from multiple senescence inducers and cell types. Each profile consists of hundreds of largely distinct proteins but also includes a subset of proteins elevated in all SASPs. Our analyses identify several candidate biomarkers of cellular senescence that overlap with aging markers in human plasma, including Growth/differentiation factor 15 (GDF15), stanniocalcin 1 (STC1), and serine protease inhibitors (SERPINs), which significantly correlated with age in plasma from a human cohort, the Baltimore Longitudinal Study of Aging (BLSA). Our findings will facilitate the identification of proteins characteristic of senescence-associated phenotypes and catalog potential senescence biomarkers to assess the burden, originating stimulus, and tissue of origin of senescent cells in vivo.
    DOI:  https://doi.org/10.1371/journal.pbio.3000599
  11. Nat Commun. 2020 Jan 16. 11(1): 307
    Temporal inhibition of autophagy reveals segmental reversal of ageing with increased cancer risk.
    Liam D Cassidy, Andrew R J Young, Christopher N J Young, Elizabeth J Soilleux, Edward Fielder, Bettina M Weigand, Anthony Lagnado, Rebecca Brais, Nicholas T Ktistakis, Kimberley A Wiggins, Katerina Pyrillou, Murray C H Clarke, Diana Jurk, Joao F Passos, Masashi Narita.
      Autophagy is an important cellular degradation pathway with a central role in metabolism as well as basic quality control, two processes inextricably linked to ageing. A decrease in autophagy is associated with increasing age, yet it is unknown if this is causal in the ageing process, and whether autophagy restoration can counteract these ageing effects. Here we demonstrate that systemic autophagy inhibition induces the premature acquisition of age-associated phenotypes and pathologies in mammals. Remarkably, autophagy restoration provides a near complete recovery of morbidity and a significant extension of lifespan; however, at the molecular level this rescue appears incomplete. Importantly autophagy-restored mice still succumb earlier due to an increase in spontaneous tumour formation. Thus, our data suggest that chronic autophagy inhibition confers an irreversible increase in cancer risk and uncovers a biphasic role of autophagy in cancer development being both tumour suppressive and oncogenic, sequentially.
    DOI:  https://doi.org/10.1038/s41467-019-14187-x
  12. EMBO J. 2020 Jan 13. e102608
    FAM134B oligomerization drives endoplasmic reticulum membrane scission for ER-phagy.
    Xiao Jiang, Xinyi Wang, Xianming Ding, Mengjie Du, Boran Li, Xialian Weng, Jingzi Zhang, Lin Li, Rui Tian, Qi Zhu, She Chen, Liang Wang, Wei Liu, Lei Fang, Dante Neculai, Qiming Sun.
      Degradation of endoplasmic reticulum (ER) by selective autophagy (ER-phagy) is crucial for ER homeostasis. However, it remains unclear how ER scission is regulated for subsequent autophagosomal sequestration and lysosomal degradation. Here, we show that oligomerization of ER-phagy receptor FAM134B (also referred to as reticulophagy regulator 1 or RETREG1) through its reticulon-homology domain is required for membrane fragmentation in vitro and ER-phagy in vivo. Under ER-stress conditions, activated CAMK2B phosphorylates the reticulon-homology domain of FAM134B, which enhances FAM134B oligomerization and activity in membrane fragmentation to accommodate high demand for ER-phagy. Unexpectedly, FAM134B G216R, a variant derived from a type II hereditary sensory and autonomic neuropathy (HSAN) patient, exhibits gain-of-function defects, such as hyperactive self-association and membrane scission, which results in excessive ER-phagy and sensory neuron death. Therefore, this study reveals a mechanism of ER membrane fragmentation in ER-phagy, along with a signaling pathway in regulating ER turnover, and suggests a potential implication of excessive selective autophagy in human diseases.
    Keywords:  CAMK2B; ER stress; ER-phagy; FAM134B oligomerization; membrane fragmentation
    DOI:  https://doi.org/10.15252/embj.2019102608
  13. Nat Cell Biol. 2020 Jan 13.
    The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.
    Andrew M Leidal, Hector H Huang, Timothy Marsh, Tina Solvik, Dachuan Zhang, Jordan Ye, FuiBoon Kai, Juliet Goldsmith, Jennifer Y Liu, Yu-Hsin Huang, Teresa Monkkonen, Ariadne Vlahakis, Eric J Huang, Hani Goodarzi, Li Yu, Arun P Wiita, Jayanta Debnath.
      Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.
    DOI:  https://doi.org/10.1038/s41556-019-0450-y
  14. Cells. 2020 Jan 13. pii: E196. [Epub ahead of print]9(1):
    Glutamine Synthetase 1 Increases Autophagy Lysosomal Degradation of Mutant Huntingtin Aggregates in Neurons, Ameliorating Motility in a Drosophila Model for Huntington's Disease.
    Luisa Vernizzi, Chiara Paiardi, Giusimaria Licata, Teresa Vitali, Stefania Santarelli, Martino Raneli, Vera Manelli, Manuela Rizzetto, Mariarosa Gioria, Maria E Pasini, Daniela Grifoni, Maria A Vanoni, Cinzia Gellera, Franco Taroni, Paola Bellosta.
      Glutamine Synthetase 1 (GS1) is a key enzyme that catalyzes the ATP-dependent synthesis of l-glutamine from l-glutamate and is also member of the Glutamate Glutamine Cycle, a complex physiological process between glia and neurons that controls glutamate homeostasis and is often found compromised in neurodegenerative diseases including Huntington's disease (HD). Here we report that the expression of GS1 in neurons ameliorates the motility defects induced by the expression of the mutant Htt, using a Drosophila model for HD. This phenotype is associated with the ability of GS1 to favor the autophagy that we associate with the presence of reduced Htt toxic protein aggregates in neurons expressing mutant Htt. Expression of GS1 prevents the TOR activation and phosphorylation of S6K, a mechanism that we associate with the reduced levels of essential amino acids, particularly of arginine and asparagine important for TOR activation. This study reveals a novel function for GS1 to ameliorate neuronal survival by changing amino acids' levels that induce a "starvation-like" condition responsible to induce autophagy. The identification of novel targets that inhibit TOR in neurons is of particular interest for the beneficial role that autophagy has in preserving physiological neuronal health and in the mechanisms that eliminate the formation of toxic aggregates in proteinopathies.
    Keywords:  Drosophila model for neuronal degeneration; Huntington’s disease; TOR signaling; autophagy; glutamine synthetase 1; protein aggregates
    DOI:  https://doi.org/10.3390/cells9010196
  15. Autophagy. 2020 Jan 16.
    Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop to promote autophagy.
    Zhong-Qiu Yu, Ling-Ling Sun, Zhao-Di Jiang, Xiao-Man Liu, Dan Zhao, Hai-Tao Wang, Wan-Zhong He, Meng-Qiu Dong, Li-Lin Du.
      Macroautophagy (autophagy) is driven by the coordinated actions of core autophagy-related (Atg) proteins. Atg8, the core Atg protein generally considered acting most downstream, has recently been shown to interact with other core Atg proteins via their Atg8-family-interacting motifs (AIMs). However, the extent, functional consequence, and evolutionary conservation of such interactions remain inadequately understood. Here, we show that, in the fission yeast Schizosaccharomyces pombe, Atg38, a subunit of the phosphatidylinositol 3-kinase (PtdIns3K) complex I, interacts with Atg8 via an AIM, which is highly conserved in Atg38 proteins of fission yeast species, but not conserved in Atg38 proteins of other species. This interaction recruits Atg38 to Atg8 on the phagophore assembly site (PAS) and consequently enhances PAS accumulation of the PtdIns3K complex I and Atg proteins acting downstream of the PtdIns3K complex I, including Atg8. The disruption of the Atg38-Atg8 interaction leads to the reduction of autophagosome size and autophagic flux. Remarkably, the loss of this interaction can be compensated by an artificial Atg14-Atg8 interaction. Our findings demonstrate that the Atg38-Atg8 interaction in fission yeast establishes a positive feedback loop between Atg8 and the PtdIns3K complex I to promote efficient autophagosome formation, underscore the prevalence and diversity of AIM-mediated connections within the autophagic machinery, and reveal unforeseen flexibility of such connections.
    Keywords:  AIM; Atg38; Atg8; PtdIns3K complex I; autophagy; positive feedback loop
    DOI:  https://doi.org/10.1080/15548627.2020.1713644
  16. Autophagy. 2020 Jan 13. 1-13
    Autophagy drives fibroblast senescence through MTORC2 regulation.
    Monique Bernard, Bing Yang, Francis Migneault, Julie Turgeon, Mélanie Dieudé, Marc-Alexandre Olivier, Guillaume B Cardin, Mostafa El-Diwany, Katy Underwood, Francis Rodier, Marie-Josée Hébert.
      Sustained macroautophagy/autophagy favors the differentiation of fibroblasts into myofibroblasts. Cellular senescence, another means of responding to long-term cellular stress, has also been linked to myofibroblast differentiation and fibrosis. Here, we evaluate the relationship between senescence and myofibroblast differentiation in the context of sustained autophagy. We analyzed markers of cell cycle arrest/senescence in fibroblasts in vitro, where autophagy was triggered by serum starvation (SS). Autophagic fibroblasts expressed the senescence biomarkers CDKN1A/p21 and CDKN2A/p16 and exhibited increased senescence-associated GLB1/beta-galactosidase activity. Inhibition of autophagy in serum-starved fibroblasts with 3-methyladenine, LY294002, or ATG7 (autophagy related 7) silencing prevented the expression of senescence-associated markers. Similarly, suppressing MTORC2 activation using rapamycin or by silencing RICTOR also prevented senescence hallmarks. Immunofluorescence microscopy showed that senescence and myofibroblast differentiation were induced in different cells, suggesting mutually exclusive activation of senescence and myofibroblast differentiation. Reactive oxygen species (ROS) are known inducers of senescence and exposing fibroblasts to ROS scavengers decreased ROS production during SS, inhibited autophagy, and significantly reduced the expression of senescence and myofibroblast differentiation markers. ROS scavengers also curbed the AKT1 phosphorylation at Ser473, an MTORC2 target, establishing the importance of ROS in fueling MTORC2 activation. Inhibition of senescence by shRNA to TP53/p53 and shRNA CDKN2A/p16 increased myofibroblast differentiation, suggesting a negative feedback loop of senescence on autophagy-induced myofibroblast differentiation. Collectively, our results identify ROS as central inducers of MTORC2 activation during chronic autophagy, which in turn fuels senescence activation and myofibroblast differentiation in distinct cellular subpopulations.Abbreviations: 3-MA: 3-methyladenine; ACTA2: actin, alpha 2, smooth muscle, aorta; AKT1: AKT serine/threonine kinase 1; p-AKT1: AKT1 Ser473 phosphorylation; t-AKT1: total AKT serine/threonine kinase 1; ATG4A: autophagy related 4A cysteine peptidase; ATG7: autophagy gene 7; C12FDG: 5-dodecanoylaminofluorescein Di-β-D-Galactopyranoside; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; Ctl: control; DAPI: 4',6-diamidino-2-phenylindole, dilactate; ECM: extracellular matrix; GSH: L-glutathione reduced; H2O2: hydrogen peroxide; HLF: adult human lung fibroblasts; Ho: Hoechst 33342 (2'-[4-ethoxyphenyl]-5-[4-methyl-1-piperazinyl]-2.5'-bi-1H-benzimidazole); HSC: hepatic stellate cells; LY: LY294002; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MTORC1/2: mechanistic target of rapamycin kinase complex 1/2; N: normal growth medium; NAC: N-acetyl-L-cysteine; PBS: phosphate-buffered saline; PDGFA: platelet derived growth factor subunit A; PRKCA/PKCα: protein kinase C alpha; PtdIns3K: class III phosphatidylinositol 3-kinase; PTEN: phosphatase and tensin homolog; R: rapamycin; RICTOR: RPTOR independent companion of MTOR complex 2; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; SA-GLB1/β-gal: senescence-associated galactosidase beta 1; SGK1: serum/glucocorticoid regulated kinase 1; shRNA: short hairpin RNA; siCtl: control siRNA; siRNA: small interfering RNA; SQSTM1: sequestosome 1; SS: serum-free (serum starvation) medium; TP53: tumor protein p53; TUBA: tubulin alpha; V: vehicle.
    Keywords:  Autophagy; MTORC2; myofibroblast; rapamycin; senescence
    DOI:  https://doi.org/10.1080/15548627.2020.1713640
  17. Nat Commun. 2020 Jan 13. 11(1): 229
    Spatially and temporally defined lysosomal leakage facilitates mitotic chromosome segregation.
    Saara Hämälistö, Jonathan Lucien Stahl, Elena Favaro, Qing Yang, Bin Liu, Line Christoffersen, Ben Loos, Claudia Guasch Boldú, Johanna A Joyce, Thomas Reinheckel, Marin Barisic, Marja Jäättelä.
      Lysosomes are membrane-surrounded cytoplasmic organelles filled with a powerful cocktail of hydrolases. Besides degrading cellular constituents inside the lysosomal lumen, lysosomal hydrolases promote tissue remodeling when delivered to the extracellular space and cell death when released to the cytosol. Here, we show that spatially and temporally controlled lysosomal leakage contributes to the accurate chromosome segregation in normal mammalian cell division. One or more chromatin-proximal lysosomes leak in the majority of prometaphases, after which active cathepsin B (CTSB) localizes to the metaphase chromatin and cleaves a small subset of histone H3. Stabilization of lysosomal membranes or inhibition of CTSB activity during mitotic entry results in a significant increase in telomere-related chromosome segregation defects, whereas cells and tissues lacking CTSB and cells expressing CTSB-resistant histone H3 accumulate micronuclei and other nuclear defects. These data suggest that lysosomal leakage and chromatin-associated CTSB contribute to proper chromosome segregation and maintenance of genomic integrity.
    DOI:  https://doi.org/10.1038/s41467-019-14009-0
  18. Mol Cell Biol. 2020 Jan 13. pii: MCB.00492-19. [Epub ahead of print]
    Interaction between HuR and circPABPN1 modulates autophagy in the intestinal epithelium by altering ATG16L1 translation.
    Xiao-Xue Li, Lan Xiao, Hee Kyoung Chung, Xiang-Xue Ma, Xiangzheng Liu, Jia-Le Song, Cindy Jin, Jaladanki N Rao, Myriam Gorospe, Jian-Ying Wang.
      Intestinal epithelial autophagy is crucial for host defense against invasive pathogens and its defects occur frequently in patients with inflammatory bowel disease (IBD) and other mucosal disorders, but the exact mechanism that activates autophagy is poorly defined. Here, we investigated the role of RNA-binding protein HuR (human antigen R) in the posttranscriptional control of autophagy-related genes (ATGs) in the intestinal epithelium. We found that targeted deletion of HuR in intestinal epithelial cells (IECs) specifically decreased the levels of ATG16L1 in the intestinal mucosa. Intestinal mucosa from patients with IBD exhibited reduced levels of both HuR and ATG16L1. HuR directly interacted with Atg16l1 mRNA via its 3'-untranslated region and enhanced ATG16L1 translation, without affecting Atg16l1 mRNA stability. Circular RNA circPABPN1 blocked HuR binding to Atg16l1 mRNA and lowered ATG16L1 production. HuR silencing in cultured IECs also prevented rapamycin-induced autophagy, which was abolished by overexpressing ATG16L1. These findings indicate that HuR regulates autophagy by modulating ATG16L1 translation via interaction with circPABPN1 in the intestinal epithelium.
    DOI:  https://doi.org/10.1128/MCB.00492-19
  19. Science. 2020 Jan 17. pii: eaaz5357. [Epub ahead of print]367(6475):
    Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells.
    David P Hoffman, Gleb Shtengel, C Shan Xu, Kirby R Campbell, Melanie Freeman, Lei Wang, Daniel E Milkie, H Amalia Pasolli, Nirmala Iyer, John A Bogovic, Daniel R Stabley, Abbas Shirinifard, Song Pang, David Peale, Kathy Schaefer, Wim Pomp, Chi-Lun Chang, Jennifer Lippincott-Schwartz, Tom Kirchhausen, David J Solecki, Eric Betzig, Harald F Hess.
      Within cells, the spatial compartmentalization of thousands of distinct proteins serves a multitude of diverse biochemical needs. Correlative super-resolution (SR) fluorescence and electron microscopy (EM) can elucidate protein spatial relationships to global ultrastructure, but has suffered from tradeoffs of structure preservation, fluorescence retention, resolution, and field of view. We developed a platform for three-dimensional cryogenic SR and focused ion beam-milled block-face EM across entire vitreously frozen cells. The approach preserves ultrastructure while enabling independent SR and EM workflow optimization. We discovered unexpected protein-ultrastructure relationships in mammalian cells including intranuclear vesicles containing endoplasmic reticulum-associated proteins, web-like adhesions between cultured neurons, and chromatin domains subclassified on the basis of transcriptional activity. Our findings illustrate the value of a comprehensive multimodal view of ultrastructural variability across whole cells.
    DOI:  https://doi.org/10.1126/science.aaz5357
  20. PLoS One. 2020 ;15(1): e0227887
    Astrocyte senescence promotes glutamate toxicity in cortical neurons.
    Chandani Limbad, Tal Ronnen Oron, Fatouma Alimirah, Albert R Davalos, Tara E Tracy, Li Gan, Pierre-Yves Desprez, Judith Campisi.
      Neurodegeneration is a major age-related pathology. Cognitive decline is characteristic of patients with Alzheimer's and related dementias and cancer patients after chemo- or radio-therapies. A recently emerged driver of these and other age-related pathologies is cellular senescence, a cell fate that entails a permanent cell cycle arrest and pro-inflammatory senescence-associated secretory phenotype (SASP). Although there is a link between inflammation and neurodegenerative diseases, there are many open questions regarding how cellular senescence affects neurodegenerative pathologies. Among the various cell types in the brain, astrocytes are the most abundant. Astrocytes have proliferative capacity and are essential for neuron survival. Here, we investigated the phenotype of primary human astrocytes made senescent by X-irradiation, and identified genes encoding glutamate and potassium transporters as specifically downregulated upon senescence. This down regulation led to neuronal cell death in co-culture assays. Unbiased RNA sequencing of transcripts expressed by non-senescent and senescent astrocytes confirmed that glutamate homeostasis pathway declines upon senescence. Our results suggest a key role for cellular senescence, particularly in astrocytes, in excitotoxicity, which may lead to neurodegeneration including Alzheimer's disease and related dementias.
    DOI:  https://doi.org/10.1371/journal.pone.0227887
  21. Int J Mol Sci. 2020 Jan 07. pii: E372. [Epub ahead of print]21(2):
    SIRT3 Acts as a Positive Autophagy Regulator to Promote Lipid Mobilization in Adipocytes via Activating AMPK.
    Tian Zhang, Jingxin Liu, Qiang Tong, Ligen Lin.
      Obesity is increasing at an alarming rate worldwide, which is characterized by the excessive accumulation of triglycerides in adipocytes. Emerging evidence has demonstrated that macroautophagy and chaperone-mediated autophagy (CMA) regulate lipid mobilization and play a key role in energy balance. Sirtuin 3 (SIRT3) is an NAD+-dependent deacetylase, which is important in regulating macroautophagy and lipid metabolism. It is still unknown whether SIRT3 modulates macroautophagy and CMA in adipocytes. The current study found that macroautophagy was dynamically regulated during 3T3-L1 adipocyte differentiation, which coincided with SIRT3 expression. In mature adipocytes, overexpression of SIRT3 activated macroautophagy, mainly on lipid droplets (LDs), through activating the AMP-activated protein kinase (AMPK)-unc-51-like kinase 1 (ULK1) pathway, which in turn resulting in smaller LD size and reduced lipid accumulation. Moreover, SIRT3 overexpression induced the formation of perilipin-1 (PLN1)-heat shock cognate 71 kDa protein (HSC70)-lysosome-associated membrane protein 2 (LAMP2) complex, to activate CMA and cause the instability of LDs in adipocytes. In summary, we found SIRT3 is a positive regulator of macroautophagy and CMA in adipocytes, which might be a promising therapeutic target for treatment of obesity and its related metabolic dysfunction.
    Keywords:  AMPK; chaperone-mediated autophagy; lipid metabolism; macroautophagy; sirtuin 3
    DOI:  https://doi.org/10.3390/ijms21020372
  22. Nat Commun. 2020 Jan 13. 11(1): 189
    Sestrin prevents atrophy of disused and aging muscles by integrating anabolic and catabolic signals.
    Jessica Segalés, Eusebio Perdiguero, Antonio L Serrano, Pedro Sousa-Victor, Laura Ortet, Mercè Jardí, Andrei V Budanov, Laura Garcia-Prat, Marco Sandri, David M Thomson, Michael Karin, Jun Hee Lee, Pura Muñoz-Cánoves.
      A unique property of skeletal muscle is its ability to adapt its mass to changes in activity. Inactivity, as in disuse or aging, causes atrophy, the loss of muscle mass and strength, leading to physical incapacity and poor quality of life. Here, through a combination of transcriptomics and transgenesis, we identify sestrins, a family of stress-inducible metabolic regulators, as protective factors against muscle wasting. Sestrin expression decreases during inactivity and its genetic deficiency exacerbates muscle wasting; conversely, sestrin overexpression suffices to prevent atrophy. This protection occurs through mTORC1 inhibition, which upregulates autophagy, and AKT activation, which in turn inhibits FoxO-regulated ubiquitin-proteasome-mediated proteolysis. This study reveals sestrin as a central integrator of anabolic and degradative pathways preventing muscle wasting. Since sestrin also protected muscles against aging-induced atrophy, our findings have implications for sarcopenia.
    DOI:  https://doi.org/10.1038/s41467-019-13832-9
  23. Neuron. 2019 Dec 18. pii: S0896-6273(19)31035-9. [Epub ahead of print]104(6): 1032-1033
    Therapeutic Targeting of mTORC2 in mTORopathies.
    Brianne Dentel, Christine Ochoa Escamilla, Peter T Tsai.
      Dysregulated mTOR contributes to neurodevelopmental dysfunction. A new study (Chen et al., 2019) demonstrates that suppression of mTORC2, not mTORC1, ameliorates survival, seizures, and abnormal behaviors in a Pten mutant model, highlighting mTORC2 as a potential therapeutic target in mTORopathies.
    DOI:  https://doi.org/10.1016/j.neuron.2019.11.026
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