bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒10‒25
33 papers selected by
Viktor Korolchuk
Newcastle University


  1. Autophagy. 2020 Oct 19.
    Kumar S, Jain A, Choi SW, Peixoto Duarte da Silva G, Allers L, Mudd MH, Peters RS, Anonsen JH, Rusten TE, Lazarou M, Deretic V.
      Macroautophagy/autophagy delivers cytoplasmic cargo to lysosomes for degradation. In yeast, the single Atg8 protein plays a role in the formation of autophagosomes whereas in mammalian cells there are five to seven paralogs, referred to as mammalian Atg8s (mAtg8s: GABARAP, GABARAPL1, GABARAPL2, LC3A, LC3B, LC3B2 and LC3C) with incompletely defined functions. Here we show that a subset of mAtg8s directly control lysosomal biogenesis. This occurs at the level of TFEB, the principal regulator of the lysosomal transcriptional program. mAtg8s promote TFEB's nuclear translocation in response to stimuli such as starvation. GABARAP interacts directly with TFEB, whereas RNA-Seq analyses reveal that knockout of six genes encoding mAtg8s, or a triple knockout of the genes encoding all GABARAPs, diminishes the TFEB transcriptional program. We furthermore show that GABARAPs in cooperation with other proteins, IRGM, a factor implicated in tuberculosis and Crohn disease, and STX17, are required during starvation for optimal inhibition of MTOR, an upstream kinase of TFEB, and activation of the PPP3/calcineurin phosphatase that dephosphorylates TFEB, thus promoting its nuclear translocation. In conclusion, mAtg8s, IRGM and STX17 control lysosomal biogenesis by their combined or individual effects on MTOR, TFEB, and PPP3/calcineurin, independently of their roles in the formation of autophagosomal membranes.
    Keywords:  Crohn’s disease; GABARAP; HIV; LC3; MTOR; Mycobacterium tuberculosis; TFEB; autophagy; lysosome; metabolism
    DOI:  https://doi.org/10.1080/15548627.2020.1837423
  2. Cells. 2020 Oct 18. pii: E2315. [Epub ahead of print]9(10):
    Baeken MW, Weckmann K, Diefenthäler P, Schulte J, Yusifli K, Moosmann B, Behl C, Hajieva P.
      Macroautophagy is a conserved degradative process for maintaining cellular homeostasis and plays a key role in aging and various human disorders. The microtubule-associated protein 1A/1B light chain 3B (MAP1LC3B or LC3B) is commonly analyzed as a key marker for autophagosomes and as a proxy for autophagic flux. Three paralogues of the LC3 gene exist in humans: LC3A, LC3B and LC3C. The molecular function, regulation and cellular localization of LC3A and LC3C have not been investigated frequently, even if a similar function to that described for LC3B appears likely. Here, we have selectively decapacitated LC3B by three separate strategies in primary human fibroblasts and analyzed the evoked effects on LC3A, LC3B and LC3C in terms of their cellular distribution and co-localization with p62, a ubiquitin and autophagy receptor. First, treatment with pharmacological sirtuin 1 (SIRT1) inhibitors to prevent the translocation of LC3B from the nucleus into the cytosol induced an increase in cytosolic LC3C, a heightened co-localization of LC3C with p62, and an increase LC3C-dependent autophagic flux as assessed by protein lipidation. Cytosolic LC3A, however, was moderately reduced, but also more co-localized with p62. Second, siRNA-based knock-down of SIRT1 broadly reproduced these findings and increased the co-localization of LC3A and particularly LC3C with p62 in presumed autophagosomes. These effects resembled the effects of pharmacological sirtuin inhibition under normal and starvation conditions. Third, siRNA-based knock-down of total LC3B in cytosol and nucleus also induced a redistribution of LC3C as if to replace LC3B in the nucleus, but only moderately affected LC3A. Total protein expression of LC3A, LC3B, LC3C, GABARAP and GABARAP-L1 following LC3B decapacitation was unaltered. Our data indicate that nuclear trapping and other causes of LC3B functional loss in the cytosol are buffered by LC3A and actively compensated by LC3C, but not by GABARAPs. The biological relevance of the potential functional compensation of LC3B decapacitation by LC3C and LC3A warrants further study.
    Keywords:  ATG8; GABARAP; LC3A; LC3B; LC3C; autophagy; sequestosome 1 (p62); sirtuin 1
    DOI:  https://doi.org/10.3390/cells9102315
  3. Redox Biol. 2020 Oct 12. pii: S2213-2317(20)30956-3. [Epub ahead of print]37 101751
    Jung SH, Lee W, Park SH, Lee KY, Choi YJ, Choi S, Kang D, Kim S, Chang TS, Hong SS, Lee BH.
      Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with various side effects, including cardiovascular and hepatic disorders. Studies suggest that mitochondrial damage and oxidative stress are important mediators of toxicity, yet the underlying mechanisms are poorly understood. In this study, we identified that some NSAIDs, including diclofenac, inhibit autophagic flux in hepatocytes. Further detailed studies demonstrated that diclofenac induced a reactive oxygen species (ROS)-dependent increase in lysosomal pH, attenuated cathepsin activity and blocked autophagosome-lysosome fusion. The reactivation of lysosomal function by treatment with clioquinol or transfection with the transcription factor EB restored lysosomal pH and thus autophagic flux. The production of mitochondrial ROS is critical for this process since scavenging ROS reversed lysosomal dysfunction and activated autophagic flux. The compromised lysosomal activity induced by diclofenac also inhibited the fusion with and degradation of mitochondria by mitophagy. Diclofenac-induced cell death and hepatotoxicity were effectively protected by rapamycin. Thus, we demonstrated that diclofenac induces the intracellular ROS production and lysosomal dysfunction that lead to the suppression of autophagy. Impaired autophagy fails to maintain mitochondrial integrity and aggravates the cellular ROS burden, which leads to diclofenac-induced hepatotoxicity.
    Keywords:  Diclofenac; Hepatotoxicity; Lysosomal dysfunction; Mitophagy; Nonsteroidal anti-inflammatory drugs; Reactive oxygen species
    DOI:  https://doi.org/10.1016/j.redox.2020.101751
  4. Cell Death Differ. 2020 Oct 21.
    Zhang Y, Xu X, Hu M, Wang X, Cheng H, Zhou R.
      Selective autophagic degradation of mitochondria (mitophagy) is important in maintaining proper cellular homeostasis. Here, we found that SPATA33 is a novel autophagy mediator for mitophagy in testis. The SPATA33 protein localizes on mitochondria via its binding of the carboxyl terminal with the outer mitochondrial membrane protein VDAC2. Upon starvation induction, SPATA33 is recruited to autophagosome by binding the autophagy machinery ATG16L1 via its N-terminal along with mitochondria. Notably, Spata33 knockout inhibited autophagy and overexpression can promote autophagosome formation for mitochondrial sequestration. Therefore, SPATA33 confers selectivity for mitochondrial degradation and promotes mitophagy in male germline cells.
    DOI:  https://doi.org/10.1038/s41418-020-00638-2
  5. Mol Cell Biochem. 2020 Oct 19.
    Chen S, Tang K, Hu P, Tan S, Yang S, Yang C, Chen G, Luo Y, Zou H.
      Silica-induced apoptosis of alveolar macrophages (AMs) is an essential part of silicosis formation. Autophagy tends to present a bidirectional effect on apoptosis. Our previous study found that the blockade of autophagy degradation might aggravate the apoptosis of AMs in human silicosis. We presume that targeting the autophagic pathway is regarded as a promising new strategy for silicosis fibrosis. As a main active component of the Atractylodes rhizome, Atractylenolide III (ATL-III) has been widely applied in clinical anti-inflammation. However, the effect and mechanism of ATL-III on autophagy in AMs of silicosis are unknown. In this study, we found that ATL-III might inhibit autophagy by mTOR-dependent manner, thereby improving the blockage of autophagic degradation in AMs. ATL-III alleviated the apoptosis of AMs in human silicosis. Furthermore, Rapamycin reversed the protective effect of ATL-III in AMs. These results indicate that ATL-III may be a potentially protective ingredient targeting autophagy for workers exposed to silica dust. These findings also suggest that inhibition of autophagy may be an effective way to alleviate the apoptosis of AMs in silicosis.
    Keywords:  Alveolar macrophages; Apoptosis; Atractylenolide III; Silicosis; mTOR-dependent autophagy
    DOI:  https://doi.org/10.1007/s11010-020-03946-w
  6. Sci Rep. 2020 Oct 20. 10(1): 17803
    Holczer M, Hajdú B, Lőrincz T, Szarka A, Bánhegyi G, Kapuy O.
      Autophagy is an intracellular digestive process, which has a crucial role in maintaining cellular homeostasis by self-eating the unnecessary and/or damaged components of the cell at various stress events. ULK1, one of the key elements of autophagy activator complex, together with the two sensors of nutrient and energy conditions, called mTORC1 and AMPK kinases, guarantee the precise function of cell response mechanism. We claim that the feedback loops of AMPK-mTORC1-ULK1 regulatory triangle determine an accurate dynamical characteristic of autophagic process upon cellular stress. By using both molecular and theoretical biological techniques, here we reveal that a delayed negative feedback loop between active AMPK and ULK1 is essential to manage a proper cellular answer after prolonged starvation or rapamycin addition. AMPK kinase quickly gets induced followed by AMPK-P-dependent ULK1 activation, whereas active ULK1 has a rapid negative effect on AMPK-P resulting in a delayed inhibition of ULK1. The AMPK-P → ULK1 ˧ AMPK-P negative feedback loop results in a periodic repeat of their activation and inactivation and an oscillatory activation of autophagy, as well. We demonstrate that the periodic induction of self-cannibalism is necessary for the proper dynamical behaviour of the control network when mTORC1 is inhibited with respect to various stress events. By computational simulations we also suggest various scenario to introduce "delay" on AMPK-P-dependent ULK1 activation (i.e. extra regulatory element in the wiring diagram or multi-phosphorylation of ULK1).
    DOI:  https://doi.org/10.1038/s41598-020-75030-8
  7. J Cell Sci. 2020 Oct 23. pii: jcs.246868. [Epub ahead of print]
    Xu X, Zhang C, Xu H, Wu L, Hu M, Song L.
      In our previous report, we demonstrated that one of the catalytic subunits of the I-κB kinase (IKK) complex, IKKα, performs an NF-κB-independent cytoprotective role in human hepatoma cells under the treatment of the anti-tumor therapeutic reagent arsenite. IKKα triggers its own feedback degradation by activating p53-dependent autophagy and therefore contributes largely to hepatoma cell apoptosis induced by arsenite. Interestingly, IKKα is unable to interact with p53 directly but plays a critical role in mediating p53 phosphorylation (at Ser15) by promoting CHK1 activation and CHK1/p53 complex formation. In the current study, we found that p53 acetylation (at Lys373/382) was also critical for the induction of autophagy and the autophagic degradation of IKKα in the arsenite responses. Furthermore, IKKα was involved in p53 acetylation through interaction with the acetyltransferases for p53, p300 and CBP, inducing CHK1-dependent p300/CBP activation and promoting p300/p53 or CBP/p53 complex formation. Therefore, taken together with the previous report, we conclude that both IKKα- and CHK1-dependent p53 phosphorylation and acetylation contribute to mediating selective autophagy targeting feedback degradation of IKKα in arsenite-induced proapoptotic responses.
    Keywords:  Apoptosis; Autophagy; IKKα; p300/CBP; p53 acetylation
    DOI:  https://doi.org/10.1242/jcs.246868
  8. Cells. 2020 Oct 18. pii: E2316. [Epub ahead of print]9(10):
    Larsen LJ, Møller LB.
      Hedgehog (Hh) signaling and mTOR signaling, essential for embryonic development and cellular metabolism, are both coordinated by the primary cilium. Observations from cancer cells strongly indicate crosstalk between Hh and mTOR signaling. This hypothesis is supported by several studies: Evidence points to a TGFβ-mediated crosstalk; Increased PI3K/AKT/mTOR activity leads to increased Hh signaling through regulation of the GLI transcription factors; increased Hh signaling regulates mTORC1 activity positively by upregulating NKX2.2, leading to downregulation of negative mTOR regulators; GSK3 and AMPK are, as members of both signaling pathways, potentially important links between Hh and mTORC1 signaling; The kinase DYRK2 regulates Hh positively and mTORC1 signaling negatively. In contrast, both positive and negative regulation of Hh has been observed for DYRK1A and DYRK1B, which both regulate mTORC1 signaling positively. Based on crosstalk observed between cilia, Hh, and mTORC1, we suggest that the interaction between Hh and mTORC1 is more widespread than it appears from our current knowledge. Although many studies focusing on crosstalk have been carried out, contradictory observations appear and the interplay involving multiple partners is far from solved.
    Keywords:  4E-BP1; GLI1; GLI2; GLI3; Mammalian target of rapamycin; S6K; TSC; autophagy; eIF4E; primary cilia
    DOI:  https://doi.org/10.3390/cells9102316
  9. Nat Commun. 2020 10 20. 11(1): 5311
    Yamaguchi H, Honda S, Torii S, Shimizu K, Katoh K, Miyake K, Miyake N, Fujikake N, Sakurai HT, Arakawa S, Shimizu S.
      Alternative autophagy is an Atg5/Atg7-independent type of autophagy that contributes to various physiological events. We here identify Wipi3 as a molecule essential for alternative autophagy, but which plays minor roles in canonical autophagy. Wipi3 binds to Golgi membranes and is required for the generation of isolation membranes. We establish neuron-specific Wipi3-deficient mice, which show behavioral defects, mainly as a result of cerebellar neuronal loss. The accumulation of iron and ceruloplasmin is also found in the neuronal cells. These abnormalities are suppressed by the expression of Dram1, which is another crucial molecule for alternative autophagy. Although Atg7-deficient mice show similar phenotypes to Wipi3-deficient mice, electron microscopic analysis shows that they have completely different subcellular morphologies, including the morphology of organelles. Furthermore, most Atg7/Wipi3 double-deficient mice are embryonic lethal, indicating that Wipi3 functions to maintain neuronal cells via mechanisms different from those of canonical autophagy.
    DOI:  https://doi.org/10.1038/s41467-020-18892-w
  10. J Cell Sci. 2020 Oct 19. pii: jcs.248336. [Epub ahead of print]
    Murakawa T, Kiger AA, Sakamaki Y, Fukuda M, Fujita N.
      Lysosomes are compartments for the degradation of both endocytic and autophagic cargoes. The shape of lysosomes changes with cellular degradative demands, however, there is limited knowledge about the mechanisms or significance that underlies distinct lysosomal morphologies. Here, we found an extensive tubular autolysosomal network in Drosophila abdominal muscle remodeling during metamorphosis. The tubular network transiently appeared and exhibited the capacity to degrade autophagic cargoes. The tubular autolysosomal network was uniquely marked by the autophagic SNARE protein, Syntaxin 17, and its formation depended on both autophagic flux and degradative function, with the exception of the Atg12 and Atg8 ubiquitin-like conjugation systems. Among ATG-deficient mutants, the efficiency of lysosomal tubulation correlated with the phenotypic severity in muscle remodeling. The lumen of the tubular network was continuous and homogeneous across a broad region of the remodeling muscle. Altogether, we revealed that the dynamic expansion of a tubular autolysosomal network synchronizes the abundant degradative activity required for developmentally regulated muscle remodeling.
    Keywords:  Atrophy; Autolysosome; Drosophila; Metamorphosis; Muscle; Syntaxin17
    DOI:  https://doi.org/10.1242/jcs.248336
  11. Mol Cancer Ther. 2020 Oct 21. pii: molcanther.0182.2020. [Epub ahead of print]
    Shaw JJ, Boyer TL, Venner E, Beck PJ, Slamowitz T, Caste T, Hickman A, Raymond MH, Costa-Pinheiro P, Jameson MJ, Fox TE, Kester M.
      Therapies for Head and Neck Squamous Cell Carcinoma (HNSCC) are, at best, moderately effective, underscoring the need for new therapeutic strategies. Ceramide treatment leads to cell death as a consequence of mitochondrial damage by generating oxidative stress and causing mitochondrial permeability. However, HNSCC cells are able to resist cell death through mitochondria repair via mitophagy. Through the use of the C6-ceramide nanoliposome (CNL) to deliver therapeutic levels of bioactive ceramide, we demonstrate that the effects of CNL are mitigated in drug-resistant HNSCC via an autophagic/mitophagic response. We also demonstrate that inhibitors of lysosomal function, including chloroquine (CQ), significantly augment CNL-induced death in HNSCC cell lines. Mechanistically, the combination of CQ and CNL results in dysfunctional lysosomal processing of damaged mitochondria. We further demonstrate that exogenous addition of Methyl Pyruvate rescues cells from CNL+CQ-dependent cell death by restoring mitochondrial functionality via the reduction of CNL- and CQ-induced generation of reactive oxygen species and mitochondria permeability. Taken together, inhibition of late-stage protective autophagy/mitophagy augments the efficacy of CNL through preventing mitochondrial repair. Moreover, the combination of inhibitors of lysosomal function with CNL may provide an efficacious treatment modality for HNSCC.
    DOI:  https://doi.org/10.1158/1535-7163.MCT-20-0182
  12. Autophagy. 2020 Oct 23. 1-2
    Metur SP, Klionsky DJ.
      Macroautophagy/autophagy is a complex process that involves over 40 proteins in Saccharomyces cerevisiae. How these proteins are organized, and their activities orchestrated to facilitate an efficient autophagic mechanism remain elusive. Sawa-Makarsha et al. reconstitute the initial steps of autophagosome biogenesis during selective autophagy using autophagy factors purified from yeast. Their results show that Atg9 vesicles serve as platforms for the recruitment of the autophagy machinery, and establish membrane contact sites to initiate lipid transfer for autophagosome biogenesis. Abbreviations: GUV, giant unilamellar vesicles; PAS, phagophore assembly site; PL, proteolipisomes.
    Keywords:  Membrane; phagophore; stress; vacuole; yeast
    DOI:  https://doi.org/10.1080/15548627.2020.1835231
  13. Mol Cell Biol. 2020 Oct 19. pii: MCB.00512-20. [Epub ahead of print]
    Payea MJ, Anerillas C, Tharakan R, Gorospe M.
      Senescence is a state of long-term cell-cycle arrest that arises in cells that have incurred sub-lethal damage. While senescent cells no longer replicate, they remain metabolically active and further develop unique and stable phenotypes that are not present in proliferating cells. On one hand, senescent cells increase in size, maintain an active mTORC1 complex, and produce and secrete a substantial amount of inflammatory proteins as part of the senescence associated secretory phenotype (SASP). On the other hand, these pro-growth phenotypes contrast with the p53-mediated growth arrest typical of senescent cells that is associated with nucleolar stress and an inhibition of rRNA processing and ribosome biogenesis. In sum, translation in senescent cells paradoxically comprises both a global repression of translation triggered by DNA damage and a select increase in the translation of specific proteins, including SASP factors.
    DOI:  https://doi.org/10.1128/MCB.00512-20
  14. Commun Biol. 2020 Oct 21. 3(1): 596
    Lobo MJ, Reverte-Salisa L, Chao YC, Koschinski A, Gesellchen F, Subramaniam G, Jiang H, Pace S, Larcom N, Paolocci E, Pfeifer A, Zanivan S, Zaccolo M.
      Programmed degradation of mitochondria by mitophagy, an essential process to maintain mitochondrial homeostasis, is not completely understood. Here we uncover a regulatory process that controls mitophagy and involves the cAMP-degrading enzyme phosphodiesterase 2A2 (PDE2A2). We find that PDE2A2 is part of a mitochondrial signalosome at the mitochondrial inner membrane where it interacts with the mitochondrial contact site and organizing system (MICOS). As part of this compartmentalised signalling system PDE2A2 regulates PKA-mediated phosphorylation of the MICOS component MIC60, resulting in modulation of Parkin recruitment to the mitochondria and mitophagy. Inhibition of PDE2A2 is sufficient to regulate mitophagy in the absence of other triggers, highlighting the physiological relevance of PDE2A2 in this process. Pharmacological inhibition of PDE2 promotes a 'fat-burning' phenotype to retain thermogenic beige adipocytes, indicating that PDE2A2 may serve as a novel target with potential for developing therapies for metabolic disorders.
    DOI:  https://doi.org/10.1038/s42003-020-01311-7
  15. Int J Mol Sci. 2020 Oct 16. pii: E7655. [Epub ahead of print]21(20):
    Roy M, Roux S.
      Small guanosine triphosphate hydrolases (GTPases) of the Rab family are involved in plasma membrane delivery, fusion events, and lysosomal and autophagic degradation pathways, thereby regulating signaling pathways and cell differentiation and function. Osteoclasts are bone-resorbing cells that maintain bone homeostasis. Polarized vesicular trafficking pathways result in the formation of the ruffled border, the osteoclast's resorptive organelle, which also assists in transcytosis. Here, we reviewed the different roles of Rab GTPases in the endomembrane machinery of osteoclasts and in bone diseases caused by the dysfunction of these proteins, with a particular focus on autophagy and bone resorption. Understanding the molecular mechanisms underlying osteoclast-related bone disease development is critical for developing and improving therapies.
    Keywords:  Rab GAP; Rab GTPases; autophagy; bone resorption; endomembrane machinery; osteoclasts
    DOI:  https://doi.org/10.3390/ijms21207655
  16. Eur J Med Chem. 2020 Oct 11. pii: S0223-5234(20)30889-8. [Epub ahead of print] 112917
    Yang G, Li Y, Zhao Y, Ouyang L, Chen Y, Liu B, Liu J.
      Atg4, a pivotal macroautophagy/autophagy-related cysteine protein family, which regulate autophagy through either cleaving Atg8 homologs for its further lipidation or delipidating Atg8 homologs from the autophagosome. There are four homologs, Atg4A, Atg4B, Atg4C, and Atg4D. Among them, an increasing amount of evidence indicates that Atg4B possessed superior catalytic efficiency toward the Atg8 substrate, as well as regulates autophagy process and plays a key role in the development of several human cancers. Recently, efforts have been contributed to the exploration of Atg4B inhibitors or activators. In this review, we comprehensively clarify the function of Atg4B in autophagy and cancer biology, as well as the relationship between pharmacological function and structure-activity of small molecule drugs targeting Atg4B. The development of novel drugs targeting Atg4B could be well applied in the clinical practice.
    Keywords:  Autophagy; Cancer; Drug target; Small molecule modulators; atg4B
    DOI:  https://doi.org/10.1016/j.ejmech.2020.112917
  17. J Biomed Sci. 2020 Oct 22. 27(1): 97
    Yamamoto YH, Noda T.
      Autophagy is a process in which a myriad membrane structures called autophagosomes are formed de novo in a single cell, which deliver the engulfed substrates into lysosomes for degradation. The size of the autophagosomes is relatively uniform in non-selective autophagy and variable in selective autophagy. It has been recently established that autophagosome formation occurs near the endoplasmic reticulum (ER). In this review, we have discussed recent advances in the relationship between autophagosome formation and endoplasmic reticulum. Autophagosome formation occurs near the ER subdomain enriched with phospholipid synthesizing enzymes like phosphatidylinositol synthase (PIS)/CDP-diacylglycerol-inositol 3-phosphatidyltransferase (CDIPT) and choline/ethanolamine phosphotransferase 1 (CEPT1). Autophagy-related protein 2 (Atg2), which is involved in autophagosome formation has a lipid transfer capacity and is proposed to directly transfer the lipid molecules from the ER to form autophagosomes. Vacuole membrane protein 1 (VMP1) and transmembrane protein 41b (TMEM41b) are ER membrane proteins that are associated with the formation of the subdomain. Recently, we have reported that an uncharacterized ER membrane protein possessing the DNAJ domain, called ERdj8/DNAJC16, is associated with the regulation of the size of autophagosomes. The localization of ERdj8/DNAJC16 partially overlaps with the PIS-enriched ER subdomain, thereby implying its association with autophagosome size determination.
    Keywords:  ATG9; Atg2; Autophagosome; Autophagy; CDIPT; COPII; ERdj8/DNAJC16; Endoplasmic reticulum; PIS; TMEM41b; VMP1
    DOI:  https://doi.org/10.1186/s12929-020-00691-6
  18. Alzheimers Dement. 2020 Oct 08.
    Hou X, Watzlawik JO, Cook C, Liu CC, Kang SS, Lin WL, DeTure M, Heckman MG, Diehl NN, Al-Shaikh FSH, Walton RL, Ross OA, Melrose HL, Ertekin-Taner N, Bu G, Petrucelli L, Fryer JD, Murray ME, Dickson DW, Fiesel FC, Springer W.
      INTRODUCTION: The cytoprotective PTEN-induced kinase 1 (PINK1)-parkin RBR E3 ubiquitin protein ligase (PRKN) pathway selectively labels damaged mitochondria with phosphorylated ubiquitin (pS65-Ub) for their autophagic removal (mitophagy). Because dysfunctions of mitochondria and degradation pathways are early features of Alzheimer's disease (AD), mitophagy impairments may contribute to the pathogenesis.METHODS: Morphology, levels, and distribution of the mitophagy tag pS65-Ub were evaluated by biochemical analyses combined with tissue and single cell imaging in AD autopsy brain and in transgenic mouse models.
    RESULTS: Analyses revealed significant increases of pS65-Ub levels in AD brain, which strongly correlated with granulovacuolar degeneration (GVD) and early phospho-tau deposits, but were independent of amyloid beta pathology. Single cell analyses revealed predominant co-localization of pS65-Ub with mitochondria, GVD bodies, and/or lysosomes depending on the brain region analyzed.
    DISCUSSION: Our study highlights mitophagy alterations in AD that are associated with early tau pathology, and suggests that distinct mitochondrial, autophagic, and/or lysosomal failure may contribute to the selective vulnerability in disease.
    Keywords:  Alzheimer's disease; PINK1; PRKN; Parkin; autophagy; granulovacuolar degeneration; lysosomes; mitochondria; mitophagy; tau; ubiquitin
    DOI:  https://doi.org/10.1002/alz.12198
  19. Cell Mol Life Sci. 2020 Oct 19.
    Romanello V, Sandri M.
      The dynamic coordination of processes controlling the quality of the mitochondrial network is crucial to maintain the function of mitochondria in skeletal muscle. Changes of mitochondrial proteolytic system, dynamics (fusion/fission), and mitophagy induce pathways that affect muscle mass and performance. When muscle mass is lost, the risk of disease onset and premature death is dramatically increased. For instance, poor quality of muscles correlates with the onset progression of several age-related disorders such as diabetes, obesity, cancer, and aging sarcopenia. To date, there are no drug therapies to reverse muscle loss, and exercise remains the best approach to improve mitochondrial health and to slow atrophy in several diseases. This review will describe the principal mechanisms that control mitochondrial quality and the pathways that link mitochondrial dysfunction to muscle mass regulation.
    Keywords:  Atrophy; Autophagy; FGF21; Fission; Fusion; Mitochondria; Mitochondrial proteostasis; Mitophagy; Myokines; Skeletal muscle
    DOI:  https://doi.org/10.1007/s00018-020-03662-0
  20. Cells. 2020 Oct 21. pii: E2333. [Epub ahead of print]9(10):
    Petit PX, Ardilla-Osorio H, Penalvia L, Nathan E R.
      Tafazzin is a phospholipid transacylase that catalyzes the remodeling of cardiolipin, a mitochondrial phospholipid required for oxidative phosphorylation. Mutations of the tafazzin gene cause Barth syndrome, which is characterized by mitochondrial dysfunction and dilated cardiomyopathy, leading to premature death. However, the molecular mechanisms underlying the cause of mitochondrial dysfunction in Barth syndrome remain poorly understood. We again highlight the fact that the tafazzin deficiency is also linked to defective oxidative phosphorylation associated with oxidative stress. All the mitochondrial events are positioned in a context where mitophagy is a key element in mitochondrial quality control. Here, we investigated the role of tafazzin in mitochondrial homeostasis dysregulation and mitophagy alteration. Using a HeLa cell model of tafazzin deficiency, we show that dysregulation of tafazzin in HeLa cells induces alteration of mitophagy. Our findings provide some additional insights into mitochondrial dysfunction associated with Barth syndrome, but also show that mitophagy inhibition is concomitant with apoptosis dysfunction through the inability of abnormal mitochondrial cardiolipin to assume its role in cytoplasmic signal transduction. Our work raises hope that pharmacological manipulation of the mitophagic pathway together with mitochondrially targeted antioxidants may provide new insights leading to promising treatment for these highly lethal conditions.
    Keywords:  Barth syndrome; apoptosis; autophagy; cardiolipin; electron transport; mitochondria; tafazzin
    DOI:  https://doi.org/10.3390/cells9102333
  21. Mol Cancer Res. 2020 Oct 23. pii: molcanres.0336.2020. [Epub ahead of print]
    Lobb IT, Morin P, Martin K, Thoms HC, Wills JC, Lleshi X, Olsen KC, Duncan RR, Stark LA.
      Elevated NF-κB activity is a contributory factor in many haematological and solid malignancies. Nucleolar sequestration of NF-κB/RelA represses this elevated activity and mediates apoptosis of cancer cells. Here we set out to understand the mechanisms that control the nuclear/nucleolar distribution of RelA and other regulatory proteins, so that agents can be developed that specifically target these proteins to the organelle. We demonstrate that RelA accumulates in intra-nucleolar aggresomes in response to specific stresses. We also demonstrate that the autophagy receptor, SQSTM1/p62, accumulates alongside RelA in these nucleolar aggresomes. This accumulation is not a consequence of inhibited autophagy. Indeed, our data suggest nucleolar and autophagosomal accumulation of p62 are in active competition. We identify a conserved motif at the N-terminus of p62 that is essential for nucleoplasmic-to nucleolar transport of the protein. Furthermore, using a dominant negative mutant deleted for this nucleolar localisation signal (NoLS), we demonstrate a role for p62 in trafficking RelA and other aggresome-related proteins to nucleoli, to induce apoptosis. Together, these data identify a novel role for p62 in trafficking nuclear proteins to nucleolar aggresomes under conditions of cell stress, thus maintaining cellular homeostasis. They also provide invaluable information on the mechanisms that regulate the nuclear/nucleolar distribution of RelA that could be exploited for therapeutic purpose. Implications: The data open up avenues for the development of a unique class of therapeutic agents that act by targeting RelA and other aberrantly active proteins to nucleoli, thus killing cancer cells.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-20-0336
  22. Nat Rev Mol Cell Biol. 2020 Oct 22.
    Song J, Herrmann JM, Becker T.
      Mitochondria contain about 1,000-1,500 proteins that fulfil multiple functions. Mitochondrial proteins originate from two genomes: mitochondrial and nuclear. Hence, proper mitochondrial function requires synchronization of gene expression in the nucleus and in mitochondria and necessitates efficient import of mitochondrial proteins into the organelle from the cytosol. Furthermore, the mitochondrial proteome displays high plasticity to allow the adaptation of mitochondrial function to cellular requirements. Maintenance of this complex and adaptable mitochondrial proteome is challenging, but is of crucial importance to cell function. Defects in mitochondrial proteostasis lead to proteotoxic insults and eventually cell death. Different quality control systems monitor the mitochondrial proteome. The cytosolic ubiquitin-proteasome system controls protein transport across the mitochondrial outer membrane and removes damaged or mislocalized proteins. Concomitantly, a number of mitochondrial chaperones and proteases govern protein folding and degrade damaged proteins inside mitochondria. The quality control factors also regulate processing and turnover of native proteins to control protein import, mitochondrial metabolism, signalling cascades, mitochondrial dynamics and lipid biogenesis, further ensuring proper function of mitochondria. Thus, mitochondrial protein quality control mechanisms are of pivotal importance to integrate mitochondria into the cellular environment.
    DOI:  https://doi.org/10.1038/s41580-020-00300-2
  23. Oncogenesis. 2020 Oct 22. 9(10): 94
    Meunier G, Birsen R, Cazelles C, Belhadj M, Cantero-Aguilar L, Kosmider O, Fontenay M, Azar N, Mayeux P, Chapuis N, Tamburini J, Bouscary D.
      Acute myeloid leukemia (AML) is an aggressive disease with a poor prognosis. Vacuolar protein sorting 34 (VPS34) is a member of the phosphatidylinositol-3-kinase lipid kinase family that controls the canonical autophagy pathway and vesicular trafficking. Using a recently developed specific inhibitor (VPS34-IN1), we found that VPS34 inhibition induces apoptosis in AML cells but not in normal CD34+ hematopoietic cells. Complete and acute inhibition of VPS34 was required for the antileukemic activity of VPS34-IN1. This inhibitor also has pleiotropic effects against various cellular functions related to class III PI3K in AML cells that may explain their survival impairment. VPS34-IN1 inhibits basal and L-asparaginase-induced autophagy in AML cells. A synergistic cell death activity of this drug was also demonstrated. VPS34-IN1 was additionally found to impair vesicular trafficking and mTORC1 signaling. From an unbiased approach based on phosphoproteomic analysis, we identified that VPS34-IN1 specifically inhibits STAT5 phosphorylation downstream of FLT3-ITD signaling in AML. The identification of the mechanisms controlling FLT3-ITD signaling by VPS34 represents an important insight into the oncogenesis of AML and could lead to new therapeutic strategies.
    DOI:  https://doi.org/10.1038/s41389-020-00278-8
  24. Front Cell Dev Biol. 2020 ;8 572182
    Joaquim M, Escobar-Henriques M.
      Mitochondria entail an incredible dynamism in their morphology, impacting death signaling and selective elimination of the damaged organelles. In turn, by recycling the superfluous or malfunctioning mitochondria, mostly prevalent during aging, mitophagy contributes to maintain a healthy mitochondrial network. Mitofusins locate at the outer mitochondrial membrane and control the plastic behavior of mitochondria, by mediating fusion events. Besides deciding on mitochondrial interconnectivity, mitofusin 2 regulates physical contacts between mitochondria and the endoplasmic reticulum, but also serves as a decisive docking platform for mitophagy and apoptosis effectors. Thus, mitofusins integrate multiple bidirectional inputs from and into mitochondria and ensure proper energetic and metabolic cellular performance. Here, we review the role of mitofusins and mitophagy at the cross-road between life and apoptotic death decisions. Furthermore, we highlight the impact of this interplay on disease, focusing on how mitofusin 2 and mitophagy affect non-alcoholic fatty liver disease.
    Keywords:  MFN2; NAFLD; apoptosis; mitochondria; mitofusins; mitophagy
    DOI:  https://doi.org/10.3389/fcell.2020.572182
  25. Sci Rep. 2020 Oct 19. 10(1): 17659
    Koller A, Bruckner D, Aigner L, Reitsamer H, Trost A.
      The retinal pigment epithelium (RPE), which is among the tissues in the body that are exposed to the highest levels of phagocytosis and oxidative stress, is dependent on autophagy function. Impaired autophagy and continuous cellular stress are associated with various disorders, such as dry age-related macular degeneration (AMD), a disease for which effective therapies are lacking. Cysteinyl leukotriene receptor (CysLTR) 1 is a potential modulator of autophagy; thus, the aim of this study was to investigate the role of CysLTR1 in autophagy regulation in the RPE cell line ARPE-19. The polarized ARPE-19 monolayer exhibited expression of CysLTR1, which was colocalized with β-tubulin III. In ARPE-19 cells, autophagic activity was rhythmically regulated and was increased upon CysLTR1 inhibition by Zafirlukast (ZK) treatment. H2O2 affected the proautophagic regulatory effect of ZK treatment depending on whether it was applied simultaneously with or prior to ZK treatment. Furthermore, mRNA levels of genes related to the leukotriene system, autophagy and the unfolded protein response were positively correlated. As CysLTR1 is involved in autophagy regulation under basal and oxidative stress conditions, a dysfunctional leukotriene system could negatively affect RPE functions. Therefore, CysLTR1 is a potential target for new treatment approaches for neurodegenerative disorders, such as AMD.
    DOI:  https://doi.org/10.1038/s41598-020-74755-w
  26. Cell Mol Immunol. 2020 Oct 23.
    Liu T, Wang L, Liang P, Wang X, Liu Y, Cai J, She Y, Wang D, Wang Z, Guo Z, Bates S, Xia X, Huang J, Cui J.
      Macrophage polarization to proinflammatory M1-like or anti-inflammatory M2-like cells is critical to mount a host defense or repair tissue. The exact molecular mechanisms controlling this process are still elusive. Here, we report that ubiquitin-specific protease 19 (USP19) acts as an anti-inflammatory switch that inhibits inflammatory responses and promotes M2-like macrophage polarization. USP19 inhibited NLRP3 inflammasome activation by increasing autophagy flux and decreasing the generation of mitochondrial reactive oxygen species. In addition, USP19 inhibited the proteasomal degradation of inflammasome-independent NLRP3 by cleaving its polyubiquitin chains. USP19-stabilized NLRP3 promoted M2-like macrophage polarization by direct association with interferon regulatory factor 4, thereby preventing its p62-mediated selective autophagic degradation. Consistent with these observations, compared to wild-type mice, Usp19-/- mice had decreased M2-like macrophage polarization and increased interleukin-1β secretion, in response to alum and chitin injections. Thus, we have uncovered an unexpected mechanism by which USP19 switches the proinflammatory function of NLRP3 into an anti-inflammatory function, and suggest that USP19 is a potential therapeutic target for inflammatory interventions.
    Keywords:  Autophagy; Deubiquitinating enzyme; Inflammasome; Macrophage polarization; NLRP3
    DOI:  https://doi.org/10.1038/s41423-020-00567-7
  27. J Biol Chem. 2020 Oct 21. pii: jbc.RA120.013716. [Epub ahead of print]
    Corum DG, Jenkins DP, Heslop JA, Tallent LM, Beeson GC, Barth JL, Schnellmann RG, Muise-Helmericks RC.
      Akt3 regulates mitochondrial content in endothelial cells through the inhibition of PGC-1α nuclear localization and is also required for angiogenesis. However, whether there is a direct link between mitochondrial function and angiogenesis is unknown. Here we show that Akt3 depletion in primary endothelial cells (EC) results in decreased uncoupled oxygen consumption, increased fission, decreased membrane potential and increased expression of the mitochondria-specific protein chaperones, HSP60 and HSP10, suggesting that Akt3 is required for mitochondrial homeostasis. Direct inhibition of mitochondrial homeostasis by the model oxidant paraquat results in decreased angiogenesis showing a direct link between angiogenesis and mitochondrial function. Next, in exploring functional links to PGC-1α, the master regulator of mitochondrial biogenesis, we searched for compounds that induce this process. We found that sildenafil, a phosphodiesterase 5 (PDE5) inhibitor induced mitochondrial biogenesis as measured by increased uncoupled oxygen consumption, mitochondrial DNA content and voltage-dependent anion channel protein expression.  Sildenafil rescued the effects on mitochondria by Akt3 depletion or pharmacological inhibition and promoted angiogenesis, further supporting that mitochondrial homeostasis is required for angiogenesis. Sildenafil also induces the expression of PGC-1 family member, PRC and can compensate for PGC-1α activity during mitochondrial stress by an Akt3-independent mechanism. The induction of PRC by sildenafil is dependent upon cAMP and the transcription factor CREB. Thus, PRC can functionally substitute during Akt3 depletion for absent PGC-1α activity to restore mitochondrial homeostasis and promote angiogenesis. These findings show that mitochondrial homeostasis as controlled by the PGC family of transcriptional activators is required for angiogenic responses.
    Keywords:  Akt PKB; Akt3; PRC; angiogenesis; mitochondria; peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1a)(PPARGC1A); phosphodiesterases
    DOI:  https://doi.org/10.1074/jbc.RA120.013716
  28. Cancers (Basel). 2020 Oct 17. pii: E3023. [Epub ahead of print]12(10):
    DeBlasi JM, DeNicola GM.
      The transcription factor NRF2 (nuclear factor-erythroid 2 p45-related factor 2 or NFE2L2) plays a critical role in response to cellular stress. Following an oxidative insult, NRF2 orchestrates an antioxidant program, leading to increased glutathione levels and decreased reactive oxygen species (ROS). Mounting evidence now implicates the ability of NRF2 to modulate metabolic processes, particularly those at the interface between antioxidant processes and cellular proliferation. Notably, NRF2 regulates the pentose phosphate pathway, NADPH production, glutaminolysis, lipid and amino acid metabolism, many of which are hijacked by cancer cells to promote proliferation and survival. Moreover, deregulation of metabolic processes in both normal and cancer-based physiology can stabilize NRF2. We will discuss how perturbation of metabolic pathways, including the tricarboxylic acid (TCA) cycle, glycolysis, and autophagy can lead to NRF2 stabilization, and how NRF2-regulated metabolism helps cells deal with these metabolic stresses. Finally, we will discuss how the negative regulator of NRF2, Kelch-like ECH-associated protein 1 (KEAP1), may play a role in metabolism through NRF2 transcription-independent mechanisms. Collectively, this review will address the interplay between the NRF2/KEAP1 complex and metabolic processes.
    Keywords:  KEAP1; NADPH; NRF2; amino acids; cancer metabolism; lipids; oxidative stress
    DOI:  https://doi.org/10.3390/cancers12103023
  29. Mol Neurobiol. 2020 Oct 22.
    Sinha P, Verma B, Ganesh S.
      Lafora disease (LD) is one of the progressive and fatal forms of a neurodegenerative disorder and is characterized by teenage-onset myoclonic seizures. Neuropathological changes in LD include the formation of abnormal glycogen as Lafora bodies, gliosis, and neuroinflammation. LD is caused by defects in the gene coding for phosphatase (laforin) or ubiquitin ligase (malin). Mouse models of LD, developed by targeted disruption of these two genes, develop most symptoms of LD and show increased susceptibility to induced seizures. Studies on mouse models also suggest that defective autophagy might contribute to LD etiology. In an attempt to understand the specific role of autophagy in LD pathogenesis, in this study, we fed LD animals with trehalose, an inducer of autophagy, for 3 months and looked at its effect on the neuropathology and seizure susceptibility. We demonstrate here that trehalose ameliorates gliosis, neuroinflammation, and endoplasmic reticulum stress and reduces susceptibility to induced seizures in LD animals. However, trehalose did not affect the formation of Lafora bodies, suggesting the epileptic phenotype in LD could be either secondary to or independent of Lafora bodies. Taken together, our results suggest that autophagy inducers can be considered as potential therapeutic molecules for Lafora disease.
    Keywords:  Autophagy; Carbohydrate; Chemical chaperone; Epilepsy; Neuroinflammation
    DOI:  https://doi.org/10.1007/s12035-020-02170-3
  30. Cells. 2020 Oct 20. pii: E2328. [Epub ahead of print]9(10):
    Wang F, Tasset I, Cuervo AM, Muller S.
      The phosphopeptide P140/Lupuzor, which improves the course of lupus disease in mice and patients, targets chaperone-mediated autophagy (CMA), a selective form of autophagy that is abnormally upregulated in lupus-prone MRL/lpr mice. Administered intravenously to diseased mice, P140 reduces the expression level of two major protein players of CMA, LAMP2A and HSPA8, and inhibits CMA in vitro in a cell line that stably expresses a CMA reporter. Here, we aimed to demonstrate that P140 also affects CMA in vivo and to unravel the precise cellular mechanism of how P140 interacts with the CMA process. MRL/lpr mice and CBA/J mice used as control received P140 or control peptides intravenously. Lysosome-enriched fractions of spleen or liver were prepared to examine lysosomal function. Highly purified lysosomes were further isolated and left to incubate with the CMA substrate to study at which cellular step P140 interacts with the CMA process. The data show that P140 effectively regulates CMA in vivo in MRL/lpr mice at the step of substrate lysosomal uptake and restores some alterations of defective lysosomes. For the first time, it is demonstrated that by occluding the intralysosome uptake of CMA substrates, a therapeutic molecule can attenuate excessive CMA activity in a pathological pro-inflammatory context and protect against hyperinflammation. This recovery effect of P140 on hyperactivated CMA is not only important for lupus therapy but potentially also for treating other (auto)inflammatory diseases, including neurologic and metabolic disorders, where CMA modulation would be highly beneficial.
    Keywords:  MRL/lpr mice; P140 peptide; chaperone-mediated autophagy; lupus; lysosomes
    DOI:  https://doi.org/10.3390/cells9102328
  31. J Cell Biol. 2020 Dec 07. pii: e202002144. [Epub ahead of print]219(12):
    English AM, Schuler MH, Xiao T, Kornmann B, Shaw JM, Hughes AL.
      Mitochondria are dynamic organelles with essential roles in signaling and metabolism. We recently identified a cellular structure called the mitochondrial-derived compartment (MDC) that is generated from mitochondria in response to amino acid overabundance stress. How cells form MDCs is unclear. Here, we show that MDCs are dynamic structures that form and stably persist at sites of contact between the ER and mitochondria. MDC biogenesis requires the ER-mitochondria encounter structure (ERMES) and the conserved GTPase Gem1, factors previously implicated in lipid exchange and membrane tethering at ER-mitochondria contacts. Interestingly, common genetic suppressors of abnormalities displayed by ERMES mutants exhibit distinct abilities to rescue MDC formation in ERMES-depleted strains and are incapable of rescuing MDC formation in cells lacking Gem1. Thus, the function of ERMES and Gem1 in MDC biogenesis may extend beyond their conventional role in maintaining mitochondrial phospholipid homeostasis. Overall, this study identifies an important function for ER-mitochondria contacts in the biogenesis of MDCs.
    DOI:  https://doi.org/10.1083/jcb.202002144
  32. FEBS J. 2020 Oct 22.
    Markaki M, Tavernarakis N.
      Autophagy is the main catabolic process by which cells recycle cytoplasmic components and superfluous or damaged organelles to preserve metabolic homeostasis under normal conditions and promote survival under stress. As a tightly regulated and dynamic process, autophagy has critical roles in development and cell differentiation, immune function, organismal health and lifespan. Accumulating findings suggest that defective or dysregulated autophagy accelerates ageing and increases susceptibility to diseases, such as neurodegenerative disorders and cancer, among others. This virtual issue of the FEBS Journal on Autophagy includes a collection of articles that present recent advances on the regulation of autophagy and provide a view of its complex roles in physiological and pathological contexts.
    DOI:  https://doi.org/10.1111/febs.15573
  33. PLoS Genet. 2020 Oct 21. 16(10): e1008844
    Terriente-Felix A, Wilson EL, Whitworth AJ.
      Balanced mitochondrial fission and fusion play an important role in shaping and distributing mitochondria, as well as contributing to mitochondrial homeostasis and adaptation to stress. In particular, mitochondrial fission is required to facilitate degradation of damaged or dysfunctional units via mitophagy. Two Parkinson's disease factors, PINK1 and Parkin, are considered key mediators of damage-induced mitophagy, and promoting mitochondrial fission is sufficient to suppress the pathological phenotypes in Drosophila Pink1/parkin mutants. We sought additional factors that impinge on mitochondrial dynamics and which may also suppress Pink1/parkin phenotypes. We found that the Drosophila phosphatidylinositol 4-kinase IIIβ homologue, Four wheel drive (Fwd), promotes mitochondrial fission downstream of the pro-fission factor Drp1. Previously described only as male sterile, we identified several new phenotypes in fwd mutants, including locomotor deficits and shortened lifespan, which are accompanied by mitochondrial dysfunction. Finally, we found that fwd overexpression can suppress locomotor deficits and mitochondrial disruption in Pink1/parkin mutants, consistent with its function in promoting mitochondrial fission. Together these results shed light on the complex mechanisms of mitochondrial fission and further underscore the potential of modulating mitochondrial fission/fusion dynamics in the context of neurodegeneration.
    DOI:  https://doi.org/10.1371/journal.pgen.1008844