bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2021–10–03
47 papers selected by
Viktor Korolchuk, Newcastle University
  1. Selective Autophagy as a Potential Therapeutic Target in Age-Associated Pathologies.
  2. Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy.
  3. Advances in ER-Phagy and Its Diseases Relevance.
  4. Aberrant Stress Granule Dynamics and Aggrephagy in ALS Pathogenesis.
  5. ULK1 phosphorylation of striatin activates protein phosphatase 2A and autophagy.
  6. Autophagy and the Hallmarks of Aging.
  7. A Review of APOE Genotype-Dependent Autophagic Flux Regulation in Alzheimer's Disease.
  8. HIV Increases the Inhibitory Impact of Morphine and Antiretrovirals on Autophagy in Primary Human Macrophages: Contributions to Neuropathogenesis.
  9. The multifaceted regulation of mitophagy by endogenous metabolites.
  10. Role of chaperone-mediated autophagy in the pathophysiology including pulmonary disorders.
  11. Substrate Reduction Therapy Reverses Mitochondrial, mTOR, and Autophagy Alterations in a Cell Model of Gaucher Disease.
  12. In vivo autophagy quantification: Measuring LC3 and P62 puncta in 3D image system from zebrafish larvae.
  13. Crosstalk between Autophagy and Inflammatory Processes in Cancer.
  14. mTOR Signaling: The Interface Linking Cellular Metabolism and Hepatitis B Virus Replication.
  15. Porin 1 Modulates Autophagy in Yeast.
  16. AMPK-mTOR Signaling and Cellular Adaptations in Hypoxia.
  17. A brief overview of BNIP3L/NIX receptor-mediated mitophagy.
  18. Persistent mTORC1 activation via Depdc5 deletion results in spontaneous hepatocellular carcinoma but does not exacerbate carcinogen- and high-fat diet-induced hepatic carcinogenesis in mice.
  19. Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells.
  20. Targeting Autophagy with Natural Products as a Potential Therapeutic Approach for Cancer.
  21. Activating Autophagy to Prevent Doxorubicin Cardiomyopathy: The Timing Matters.
  22. Alpha-Synuclein and the Endolysosomal System in Parkinson's Disease: Guilty by Association.
  23. Inhibition of autophagy by a small molecule through covalent modification of LC3.
  24. Storage of Mutant Human SOD1 in Non-Neural Cells from the Type-1 Amyotrophic Lateral Sclerosis ratG93A Model Correlated with the Lysosomes' Dysfunction.
  25. Autophagy Promotes the Survival of Adipose Mesenchymal Stem/Stromal Cells and Enhances Their Therapeutic Effects in Cisplatin-Induced Liver Injury via Modulating TGF-β1/Smad and PI3K/AKT Signaling Pathways.
  26. Cadmium affects autophagy in the human intestinal cells Caco-2 through ROS-mediated ERK activation.
  27. Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.
  28. TBK1 has a new Akt.
  29. A single copy transgenic mutant FUS strain reproduces age-dependent ALS phenotypes in C. elegans.
  30. Impaired Autophagy in Psoriasis and Atopic Dermatitis: A New Therapeutic Target?
  31. Circadian autophagy drives iTRF-mediated longevity.
  32. Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.
  33. Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.
  34. Autophagy-Associated IL-15 Production Is Involved in the Pathogenesis of Leprosy Type 1 Reaction.
  35. GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.
  36. Redefining the Scope of Targeted Protein Degradation: Translational Opportunities in Hijacking the Autophagy-Lysosome Pathway.
  37. IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease.
  38. Nonstructural Protein NS1 of Influenza Virus Disrupts Mitochondrial Dynamics and Enhances Mitophagy via ULK1 and BNIP3.
  39. Mechanistic Target of Rapamycin Complex 1 Signaling Links Hypoxia to Increased IGFBP-1 Phosphorylation in Primary Human Decidualized Endometrial Stromal Cells.
  40. Exacerbated Age-Related Hippocampal Alterations of Microglia Morphology, β-Amyloid and Lipofuscin Deposition and Presenilin Overexpression in Per1-/--Mice.
  41. Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche.
  42. Autophagy, Mesenchymal Stem Cell Differentiation, and Secretion.
  43. Autophagy as a Mechanism for Adaptive Prediction-Mediated Emergence of Drug Resistance.
  44. α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast.
  45. Perturbation-Based Modeling Unveils the Autophagic Modulation of Chemosensitivity and Immunogenicity in Breast Cancer Cells.
  46. GDF-15 Deficiency Reduces Autophagic Activity in Human Macrophages In Vitro and Decreases p62-Accumulation in Atherosclerotic Lesions in Mice.
  47. A human iPSC-derived inducible neuronal model of Niemann-Pick disease, type C1.
  1. Metabolites. 2021 Aug 31. pii: 588. [Epub ahead of print]11(9):
    Selective Autophagy as a Potential Therapeutic Target in Age-Associated Pathologies.
    Margarita-Elena Papandreou, Nektarios Tavernarakis.
      Progressive accumulation of damaged cellular constituents contributes to age-related diseases. Autophagy is the main catabolic process, which recycles cellular material in a multitude of tissues and organs. Autophagy is activated upon nutrient deprivation, and oncogenic, heat or oxidative stress-induced stimuli to selectively degrade cell constituents and compartments. Specificity and accuracy of the autophagic process is maintained via the precision of interaction of autophagy receptors or adaptors and substrates by the intricate, stepwise orchestration of specialized integrating stimuli. Polymorphisms in genes regulating selective autophagy have been linked to aging and age-associated disorders. The involvement of autophagy perturbations in aging and disease indicates that pharmacological agents balancing autophagic flux may be beneficial, in these contexts. Here, we introduce the modes and mechanisms of selective autophagy, and survey recent experimental evidence of dysfunctional autophagy triggering severe pathology. We further highlight identified pharmacological targets that hold potential for developing therapeutic interventions to alleviate cellular autophagic cargo burden and associated pathologies.
    Keywords:  age-related disease; aggrephagy; aging; mitophagy; neurodegeneration; nucleophagy; pexophagy; rapamycin; selective autophagy
    DOI:  https://doi.org/10.3390/metabo11090588
  2. Elife. 2021 Sep 29. pii: e72328. [Epub ahead of print]10
    Quantitative proteomics reveals the selectivity of ubiquitin-binding autophagy receptors in the turnover of damaged lysosomes by lysophagy.
    Vinay V Eapen, Sharan Swarup, Melissa J Hoyer, Joao A Paulo, Wade Harper.
      Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding Galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their inter-relationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and Galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor OPTN can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.
    Keywords:  biochemistry; cell biology; chemical biology; human
    DOI:  https://doi.org/10.7554/eLife.72328
  3. Cells. 2021 Sep 06. pii: 2328. [Epub ahead of print]10(9):
    Advances in ER-Phagy and Its Diseases Relevance.
    Lingang He, Xuehong Qian, Yixian Cui.
      As an important form of selective autophagy in cells, ER-phagy (endoplasmic reticulum-selective autophagy), the autophagic degradation of endoplasmic reticulum (ER), degrades ER membranes and proteins to maintain cellular homeostasis. The relationship between ER-phagy and human diseases, including neurodegenerative disorders, cancer, and other metabolic diseases has been unveiled by extensive research in recent years. Starting with the catabolic process of ER-phagy and key mediators in this pathway, this paper reviews the advances in the mechanism of ER-phagy and its diseases relevance. We hope to provide some enlightenment for further study on ER-phagy and the development of novel therapeutic strategies for related diseases.
    Keywords:  ER-phagy; cancer; metabolic diseases; neurodegenerative diseases; receptor
    DOI:  https://doi.org/10.3390/cells10092328
  4. Cells. 2021 Aug 30. pii: 2247. [Epub ahead of print]10(9):
    Aberrant Stress Granule Dynamics and Aggrephagy in ALS Pathogenesis.
    Yi Zhang, Jiayu Gu, Qiming Sun.
      Stress granules are conserved cytosolic ribonucleoprotein (RNP) compartments that undergo dynamic assembly and disassembly by phase separation in response to stressful conditions. Gene mutations may lead to aberrant phase separation of stress granules eliciting irreversible protein aggregations. A selective autophagy pathway called aggrephagy may partially alleviate the cytotoxicity mediated by these protein aggregates. Cells must perceive when and where the stress granules are transformed into toxic protein aggregates to initiate autophagosomal engulfment for subsequent autolysosomal degradation, therefore, maintaining cellular homeostasis. Indeed, defective aggrephagy has been causally linked to various neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). In this review, we discuss stress granules at the intersection of autophagy and ALS pathogenesis.
    Keywords:  aggrephagy; amyotrophic lateral sclerosis; neurodegenerative disease; phase separation; stress granule
    DOI:  https://doi.org/10.3390/cells10092247
  5. Cell Rep. 2021 Sep 28. pii: S2211-1247(21)01216-X. [Epub ahead of print]36(13): 109762
    ULK1 phosphorylation of striatin activates protein phosphatase 2A and autophagy.
    Zehan Hu, Devanarayanan Siva Sankar, Bich Vu, Alexandre Leytens, Christine Vionnet, Wenxian Wu, Michael Stumpe, Esther Martínez-Martínez, Björn Stork, Jörn Dengjel.
      The evolutionarily conserved ULK1 kinase complex acts as gatekeeper of canonical autophagy and regulates induction of autophagosome biogenesis. To better understand control of ULK1 and analyze whether ULK1 has broader functions that are also linked to the later steps of autophagy, we perform comprehensive phosphoproteomic analyses. Combining in vivo with in vitro data, we identify numerous direct ULK1 target sites within autophagy-relevant proteins that are critical for autophagosome maturation and turnover. In addition, we highlight an intimate crosstalk between ULK1 and several phosphatase complexes. ULK1 is not only a PP2A target but also directly phosphorylates the regulatory PP2A subunit striatin, activating PP2A and serving as positive feedback to promote autophagy-dependent protein turnover. Thus, ULK1 and phosphatase activities are tightly coordinated to robustly regulate protein degradation by autophagy.
    Keywords:  PP2A; STRIPAK; STRN; autophagy; in vitro kinase assay; kinase; mass spectrometry; phosphatase; phosphoproteomics; signaling
    DOI:  https://doi.org/10.1016/j.celrep.2021.109762
  6. Ageing Res Rev. 2021 Sep 23. pii: S1568-1637(21)00215-4. [Epub ahead of print] 101468
    Autophagy and the Hallmarks of Aging.
    Susmita Kaushik, Inmaculada Tasset, Esperanza Arias, Olatz Pampliega, Esther Wong, Marta Martinez-Vicente, Ana Maria Cuervo.
      Autophagy, an essential cellular process that mediates degradation of proteins and organelles in lysosomes, has been tightly linked to cellular quality control for its role as part of the proteostasis network. The current interest in identifying the cellular and molecular determinants of aging, has highlighted the important contribution of malfunctioning of autophagy with age to the loss of proteostasis that characterizes all old organisms. However, the diversity of cellular functions of the different types of autophagy and the often reciprocal interactions of autophagy with other determinants of aging, is placing autophagy at the center of the aging process. In this work, we summarize evidence for the contribution of autophagy to health- and lifespan and provide examples of the bidirectional interplay between autophagic pathways and several of the so-called hallmarks of aging. This central role of autophagy in aging, and the dependence on autophagy of many geroprotective interventions, has motivated a search for direct modulators of autophagy that could be used to slow aging and extend healthspan. Here, we review some of those ongoing therapeutic efforts and comment on the potential of targeting autophagy in aging.
    Keywords:  aging; chaperones; lysosomes; organelle turnover; proteolysis; proteostasis
    DOI:  https://doi.org/10.1016/j.arr.2021.101468
  7. J Alzheimers Dis. 2021 Sep 22.
    A Review of APOE Genotype-Dependent Autophagic Flux Regulation in Alzheimer's Disease.
    Huiyi Chen, Feng Chen, Miaoping Zhang, Yanting Chen, Lili Cui, Chunmei Liang.
      Autophagy is a basic physiological process maintaining cell renewal, the degradation of dysfunctional organelles, and the clearance of abnormal proteins and has recently been identified as a main mechanism underlying the onset and progression of Alzheimer's disease (AD). The APOE ɛ4 genotype is the strongest genetic determinant of AD pathogenesis and initiates autophagic flux at different times. This review synthesizes the current knowledge about the potential pathogenic effects of ApoE4 on autophagy and describes its associations with the biological hallmarks of autophagy and AD from a novel perspective. Via a remarkable variety of widely accepted signaling pathway markers, such as mTOR, TFEB, SIRT1, LC3, p62, LAMP1, LAMP2, CTSD, Rabs, and V-ATPase, ApoE isoforms differentially modulate autophagy initiation; membrane expansion, recruitment, and enclosure; autophagosome and lysosome fusion; and lysosomal degradation. Although the precise pathogenic mechanism varies for different genes and proteins, the dysregulation of autophagic flux is a key mechanism on which multiple pathogenic processes converge.
    Keywords:  Alzheimer’s disease; apolipoproteins E; autophagy; neurodegenerative diseases
    DOI:  https://doi.org/10.3233/JAD-210602
  8. Cells. 2021 Aug 24. pii: 2183. [Epub ahead of print]10(9):
    HIV Increases the Inhibitory Impact of Morphine and Antiretrovirals on Autophagy in Primary Human Macrophages: Contributions to Neuropathogenesis.
    John M Barbaro, Ana Maria Cuervo, Joan W Berman.
      HIV enters the CNS early after peripheral infection, establishing reservoirs in perivascular macrophages that contribute to development of HIV-associated neurocognitive disorders (HAND) in 15-40% of people with HIV (PWH) despite effective antiretroviral therapy (ART). Opioid use may contribute to dysregulated macrophage functions resulting in more severe neurocognitive symptoms in PWH taking opioids. Macroautophagy helps maintain quality control in long-lived cell types, such as macrophages, and has been shown to regulate, in part, some macrophage functions in the CNS that contribute to HAND. Using Western blotting and confocal immunofluorescence in primary human macrophages, we demonstrated that morphine and a commonly prescribed ART regimen induce bulk autophagy. Morphine and ART also inhibited completion of autophagy. HIV infection increased these inhibitory effects. We also examined two types of selective autophagy that degrade aggregated proteins (aggrephagy) and dysfunctional mitochondria (mitophagy). Morphine and ART inhibited selective autophagy mediated by p62 regardless of HIV infection, and morphine inhibited mitophagic flux in HIV-infected cells demonstrating potential mitotoxicity. These results indicate that inhibition of autophagy, both in bulk and selective, in CNS macrophages may mediate neurocognitive dysfunction in PWH using opioids. Increasing autophagic activity in the context of HIV may represent a novel therapeutic strategy for reducing HAND in these individuals.
    Keywords:  HIV-associated neurocognitive disorders; LC3; antiretroviral therapy; autophagy; mitophagy; myeloid cells; opioids; p62/SQSTM1; selective autophagy
    DOI:  https://doi.org/10.3390/cells10092183
  9. Autophagy. 2021 Sep 29. 1-24
    The multifaceted regulation of mitophagy by endogenous metabolites.
    Ting Zhang, Qian Liu, Weihua Gao, Sheikh Arslan Sehgal, Hao Wu.
      Owing to the dominant functions of mitochondria in multiple cellular metabolisms and distinct types of regulated cell death, maintaining a functional mitochondrial network is fundamental for the cellular homeostasis and body fitness in response to physiological adaptations and stressed conditions. The process of mitophagy, in which the dysfunctional or superfluous mitochondria are selectively engulfed by autophagosome and subsequently degraded in lysosome, has been well formulated as one of the major mechanisms for mitochondrial quality control. To date, the PINK1-PRKN-dependent and receptors (including proteins and lipids)-dependent pathways have been characterized to determine the mitophagy in mammalian cells. The mitophagy is highly responsive to the dynamics of endogenous metabolites, including iron-, calcium-, glycolysis-TCA-, NAD+-, amino acids-, fatty acids-, and cAMP-associated metabolites. Herein, we summarize the recent advances toward the molecular details of mitophagy regulation in mammalian cells. We also highlight the key regulations of mammalian mitophagy by endogenous metabolites, shed new light on the bidirectional interplay between mitophagy and cellular metabolisms, with attempting to provide a perspective insight into the nutritional intervention of metabolic disorders with mitophagy deficit.Abbreviations: acetyl-CoA: acetyl-coenzyme A; ACO1: aconitase 1; ADCYs: adenylate cyclases; AMPK: AMP-activated protein kinase; ATM: ATM serine/threonine kinase; BCL2L1: BCL2 like 1; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; Ca2+: calcium ion; CALCOCO2: calcium binding and coiled-coil domain 2; CANX: calnexin; CO: carbon monoxide; CYCS: cytochrome c, somatic; DFP: deferiprone; DNM1L: dynamin 1 like; ER: endoplasmic reticulum; FKBP8: FKBP prolyl isomerase 8; FOXO3: forkhead box O3; FTMT: ferritin mitochondrial; FUNDC1: FUN14 domain containing 1; GABA: γ-aminobutyric acid; GSH: glutathione; HIF1A: hypoxia inducible factor 1 subunit alpha; IMMT: inner membrane mitochondrial protein; IRP1: iron regulatory protein 1; ISC: iron-sulfur cluster; ITPR2: inositol 1,4,5-trisphosphate type 2 receptor; KMO: kynurenine 3-monooxygenase; LIR: LC3 interacting region; MAM: mitochondria-associated membrane; MAP1LC3: microtubule associated protein 1 light chain 3; MFNs: mitofusins; mitophagy: mitochondrial autophagy; mPTP: mitochondrial permeability transition pore; MTOR: mechanistic target of rapamycin kinase; NAD+: nicotinamide adenine dinucleotide; NAM: nicotinamide; NMN: nicotinamide mononucleotide; NO: nitric oxide; NPA: Niemann-Pick type A; NR: nicotinamide riboside; NR4A1: nuclear receptor subfamily 4 group A member 1; NRF1: nuclear respiratory factor 1; OPA1: OPA1 mitochondrial dynamin like GTPase; OPTN: optineurin; PARL: presenilin associated rhomboid like; PARPs: poly(ADP-ribose) polymerases; PC: phosphatidylcholine; PHB2: prohibitin 2; PINK1: PTEN induced kinase 1; PPARG: peroxisome proliferator activated receptor gamma; PPARGC1A: PPARG coactivator 1 alpha; PRKA: protein kinase AMP-activated; PRKDC: protein kinase, DNA-activated, catalytic subunit; PRKN: parkin RBR E3 ubiquitin protein ligase; RHOT: ras homolog family member T; ROS: reactive oxygen species; SIRTs: sirtuins; STK11: serine/threonine kinase 11; TCA: tricarboxylic acid; TP53: tumor protein p53; ULK1: unc-51 like autophagy activating kinase 1; VDAC1: voltage dependent anion channel 1.
    Keywords:  Cell metabolism; metabolite; mitochondria; mitophagy; mitophagy receptor
    DOI:  https://doi.org/10.1080/15548627.2021.1975914
  10. Inflamm Regen. 2021 Oct 01. 41(1): 29
    Role of chaperone-mediated autophagy in the pathophysiology including pulmonary disorders.
    Yusuke Hosaka, Jun Araya, Yu Fujita, Kazuyoshi Kuwano.
      Autophagy is a highly conserved mechanism of delivering cytoplasmic components for lysosomal degradation. Among the three major autophagic pathways, chaperone-mediated autophagy (CMA) is primarily characterized by its selective nature of protein degradation, which is mediated by heat shock cognate 71 kDa protein (HSC70: also known as HSPA8) recognition of the KFERQ peptide motif in target proteins. Lysosome-associated membrane protein type 2A (LAMP2A) is responsible for substrate binding and internalization to lysosomes, and thus, the lysosomal expression level of LAMP2A is a rate-limiting factor for CMA. Recent advances have uncovered not only physiological but also pathological role of CMA in multiple organs, including neurodegenerative disorders, kidney diseases, liver diseases, heart diseases, and cancers through the accumulation of unwanted proteins or increased degradation of target proteins with concomitant metabolic alterations resulting from CMA malfunction. With respect to pulmonary disorders, the involvement of CMA has been demonstrated in lung cancer and chronic obstructive pulmonary disease (COPD) pathogenesis through regulating apoptosis. Further understanding of CMA machinery may shed light on the molecular mechanisms of refractory disorders and lead to novel treatment modalities through CMA modulation.
    Keywords:  Autophagy; Chaperone-mediated autophagy; Chronic obstructive pulmonary disease; Lung cancer
    DOI:  https://doi.org/10.1186/s41232-021-00180-9
  11. Cells. 2021 Sep 02. pii: 2286. [Epub ahead of print]10(9):
    Substrate Reduction Therapy Reverses Mitochondrial, mTOR, and Autophagy Alterations in a Cell Model of Gaucher Disease.
    Yanyan Peng, Benjamin Liou, Yi Lin, Venette Fannin, Wujuan Zhang, Ricardo A Feldman, Kenneth D R Setchell, Gregory A Grabowski, Ying Sun.
      Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout (Gba-/-) mouse model. Gba-/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba-/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba-/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba-/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy-lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.
    Keywords:  cell biology; glucosylceramide; glucosylsphingosine; lysosomal storage disorders; neurons
    DOI:  https://doi.org/10.3390/cells10092286
  12. J Cell Biochem. 2021 Oct;122(10): 1435-1444
    In vivo autophagy quantification: Measuring LC3 and P62 puncta in 3D image system from zebrafish larvae.
    Abdalla Elbialy.
      Autophagy is a central pathway in maintaining cellular homeostasis through the recycling of damaged proteins and organelles. Detection of LC3 protein levels by immunofluorescence or western blot analysis is one of the most common ways to measure autophagy. For quantitative autophagy analysis, LC3 western blot analysis is commonly used, whereas immunostaining is used for qualitative autophagy analysis. However, zebrafish larvae have a lot of proteases that rapidly degrade LC3 protein in samples. P62 is another autophagy marker that bind to damaged proteins and can reflects autophagic status. This study demonstrates a fast and accurate way to quantify autophagy from LC3 and/or P62 immunostaining images. We used a three-dimensional analysis of whole-mount LC3 immunostaining images of zebrafish larvae. Counting LC3 and P62 punctate by two dimensions can be used as a qualitative method for the analysis of autophagy. However, here we demonstrate that 3D image analysis can be used as a quantitative, rapid tool for monitoring autophagy in zebrafish larvae and avoiding drawbacks of LC3 western blot analysis.
    Keywords:  3D image; LC3; P62; autophagy; quantification
    DOI:  https://doi.org/10.1002/jcb.30021
  13. Life (Basel). 2021 Aug 30. pii: 903. [Epub ahead of print]11(9):
    Crosstalk between Autophagy and Inflammatory Processes in Cancer.
    Eun-Ji Lee, Hyun-Jeong Kim, Min Sik Choi, Ji-Eun Chang.
      Inflammation is an adaptive response to tissue injury, which is a critical process in order to restore tissue functionality and homeostasis. The association between inflammation and cancer has been a topic of interest for many years, not only inflammatory cells themselves but also the chemokines and cytokines they produce, which affect cancer development. Autophagy is an intracellular self-degradative process providing elimination of damaged or dysfunctional organelles under stressful conditions such as nutrient deficiency, hypoxia, or chemotherapy. Interestingly, the signaling pathways that are involved in cancer-associated inflammation may regulate autophagy as well. These are (1) the toll-like receptor (TLR) signaling cascade, (2) the reactive oxygen species (ROS) signaling pathway, (3) the inflammatory cytokine signaling pathway, and (4) the IκB kinase (IKK)/Nuclear factor-κB (NF-κB) signaling axis. Moreover, the studies on the context-specific functions of autophagy during inflammatory responses in cancer will be discussed here. On that basis, we focus on autophagy inhibitors and activators regulating inflammatory process in cancer as useful candidates for enhancing anticancer effects. This review summarizes how the autophagic process regulates these key inflammatory processes and vice versa in various cancers.
    Keywords:  IκB kinase/nuclear factor-κB; autophagy; autophagy activators; autophagy inhibitors; cancer; inflammation; inflammatory cytokine; reactive oxygen species; toll-like receptor
    DOI:  https://doi.org/10.3390/life11090903
  14. Virol Sin. 2021 Sep 28.
    mTOR Signaling: The Interface Linking Cellular Metabolism and Hepatitis B Virus Replication.
    Xueyu Wang, Zhiqiang Wei, Yongfang Jiang, Zhongji Meng, Mengji Lu.
      Mammalian target of rapamycin (mTOR) is a conserved Ser/Thr kinase that includes mTOR complex (mTORC) 1 and mTORC2. The mTOR pathway is activated in viral hepatitis, including hepatitis B virus (HBV) infection-induced hepatitis. Currently, chronic HBV infection remains one of the most serious public health issues worldwide. The unavailability of effective therapeutic strategies for HBV suggests that clarification of the pathogenesis of HBV infection is urgently required. Increasing evidence has shown that HBV infection can activate the mTOR pathway, indicating that HBV utilizes or hijacks the mTOR pathway to benefit its own replication. Therefore, the mTOR signaling pathway might be a crucial target for controlling HBV infection. Here, we summarize and discuss the latest findings from model biology research regarding the interaction between the mTOR signaling pathway and HBV replication.
    Keywords:  Autophagy; Hepatitis B virus (HBV); Mammalian target of rapamycin (mTOR); Metabolism
    DOI:  https://doi.org/10.1007/s12250-021-00450-3
  15. Cells. 2021 Sep 14. pii: 2416. [Epub ahead of print]10(9):
    Porin 1 Modulates Autophagy in Yeast.
    Filomena Broeskamp, Elizabeth S M Edrich, Oskar Knittelfelder, Lisa Neuhaus, Thorsten Meyer, Jonas Heyden, Lukas Habernig, Florian Kreppel, Campbell W Gourlay, Patrick Rockenfeller.
      Autophagy is a cellular recycling program which efficiently reduces the cellular burden of ageing. Autophagy is characterised by nucleation of isolation membranes, which grow in size and further expand to form autophagosomes, engulfing cellular material to be degraded by fusion with lysosomes (vacuole in yeast). Autophagosomal membranes do not bud from a single cell organelle, but are generated de novo. Several lipid sources for autophagosomal membranes have been identified, but the whole process of their generation is complex and not entirely understood. In this study, we investigated how the mitochondrial outer membrane protein porin 1 (Por1), the yeast orthologue of mammalian voltage-dependent anion channel (VDAC), affects autophagy in yeast. We show that POR1 deficiency reduces the autophagic capacity and leads to changes in vacuole and lipid homeostasis. We further investigated whether limited phosphatidylethanolamine (PE) availability in por1∆ was causative for reduced autophagy by overexpression of the PE-generating phosphatidylserine decarboxylase 1 (Psd1). Altogether, our results show that POR1 deficiency is associated with reduced autophagy, which can be circumvented by additional PSD1 overexpression. This suggests a role for Por1 in Psd1-mediated autophagy regulation.
    Keywords:  autophagy; phosphatidylethanolamine; phosphatidylserine decarboxylase; voltage dependent anion channel (VDAC)
    DOI:  https://doi.org/10.3390/cells10092416
  16. Int J Mol Sci. 2021 Sep 09. pii: 9765. [Epub ahead of print]22(18):
    AMPK-mTOR Signaling and Cellular Adaptations in Hypoxia.
    Yoomi Chun, Joungmok Kim.
      Cellular energy is primarily provided by the oxidative degradation of nutrients coupled with mitochondrial respiration, in which oxygen participates in the mitochondrial electron transport chain to enable electron flow through the chain complex (I-IV), leading to ATP production. Therefore, oxygen supply is an indispensable chapter in intracellular bioenergetics. In mammals, oxygen is delivered by the bloodstream. Accordingly, the decrease in cellular oxygen level (hypoxia) is accompanied by nutrient starvation, thereby integrating hypoxic signaling and nutrient signaling at the cellular level. Importantly, hypoxia profoundly affects cellular metabolism and many relevant physiological reactions induce cellular adaptations of hypoxia-inducible gene expression, metabolism, reactive oxygen species, and autophagy. Here, we introduce the current knowledge of hypoxia signaling with two-well known cellular energy and nutrient sensing pathways, AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin complex 1 (mTORC1). Additionally, the molecular crosstalk between hypoxic signaling and AMPK/mTOR pathways in various hypoxic cellular adaptions is discussed.
    Keywords:  AMPK; hypoxia; hypoxia-inducible factor (HIF); hypoxic cellular adaptations; mTORC1
    DOI:  https://doi.org/10.3390/ijms22189765
  17. FEBS Open Bio. 2021 Oct 01.
    A brief overview of BNIP3L/NIX receptor-mediated mitophagy.
    Mija Marinković, Ivana Novak.
      Mitophagy is a form of autophagy specialized to selectively remove mitochondria. Although the PINK1/Parkin pathway is the best described mitophagy of damaged mitochondria, receptor/mediated mitophagy seems to have a pivotal role in cellular development and specialization. The most studied mitophagy receptor BNIP3L/NIX is shown to be important for the programmed removal of healthy mitochondria during terminal differentiation of erythrocytes, but its role has been proven in various cell types. Despite recent advances in our understanding of its regulation by phosphorylation and dimerization, there remain numerous questions on how BNIP3L/NIX tightly balances between cellular life and death decisions. This brief review intends to summarize ongoing dilemmas related to BNIP3L/NIX.
    Keywords:  BNIP3L/NIX; mitochondria; mitophagy; reticulocytes
    DOI:  https://doi.org/10.1002/2211-5463.13307
  18. Biochem Biophys Res Commun. 2021 Sep 20. pii: S0006-291X(21)01329-2. [Epub ahead of print]578 142-149
    Persistent mTORC1 activation via Depdc5 deletion results in spontaneous hepatocellular carcinoma but does not exacerbate carcinogen- and high-fat diet-induced hepatic carcinogenesis in mice.
    Lin Xu, Chenyan Yang, Jing Wang, Zun Li, Rong Huang, Honghui Ma, Jie Ma, Qingzhi Wang, Xiwen Xiong.
      The mechanistic target of rapamycin complex 1 (mTORC1) acts as a central regulator of metabolic pathways that drive cellular growth. Abnormal activation of mTORC1 occurs at high frequency in human and mouse hepatocellular carcinoma (HCC). DEP domain-containing protein 5 (DEPDC5), a component of GATOR1 complex, is a repressor of amino acid-sensing branch of the mTORC1 pathway. In the current study, we found that persistent activation of hepatic mTORC1 signaling caused by Depdc5 ablation was sufficient to induce a pathological program of liver damage, inflammation and fibrosis that triggers spontaneous HCC development. Take advantage of the combinatory treatment with a single dose of diethylnitrosamine (DEN) and chronic feeding with high-fat diet (HFD), we demonstrated that hepatic depdc5 deletion did not aggravate DEN&HFD induced liver tumorigenesis, probably due to its protective effects on diet-induced liver steatosis. In addition, we further showed that chronic rapamycin treatment did not have any apparent tumor-suppressing effects on DEN&HFD treated control mice, whereas it dramatically reduced the tumor burden in mice with hepatic Depdc5 ablation. This study provides the novel in vivo evidence for Depdc5 deletion mediated mTORC1 hyperactivation in liver tumorigenesis caused by aging or DEN&HFD treatment. Moreover, our findings also propose that pharmacological inhibition of mTORC1 signaling maybe a promising strategy to treat HCC patients with mutations in DEPDC5 gene.
    Keywords:  DEPDC5; Diethylnitrosamine (DEN); Hepatocellular carcinoma (HCC); High-fat diet (HFD); Liver; mTORC1
    DOI:  https://doi.org/10.1016/j.bbrc.2021.09.036
  19. Biomolecules. 2021 Sep 06. pii: 1314. [Epub ahead of print]11(9):
    Insights into the Interaction of Lysosomal Amino Acid Transporters SLC38A9 and SLC36A1 Involved in mTORC1 Signaling in C2C12 Cells.
    Dan Wang, Xuebin Wan, Xiaoli Du, Zhuxia Zhong, Jian Peng, Qi Xiong, Jin Chai, Siwen Jiang.
      Amino acids are critical for mammalian target of rapamycin complex 1 (mTORC1) activation on the lysosomal surface. Amino acid transporters SLC38A9 and SLC36A1 are the members of the lysosomal amino acid sensing machinery that activates mTORC1. The current study aims to clarify the interaction of SLC38A9 and SLC36A1. Here, we discovered that leucine increased expressions of SLC38A9 and SLC36A1, leading to mTORC1 activation. SLC38A9 interacted with SLC36A1 and they enhanced each other's expression levels and locations on the lysosomal surface. Additionally, the interacting proteins of SLC38A9 in C2C12 cells were identified to participate in amino acid sensing mechanism, mTORC1 signaling pathway, and protein synthesis, which provided a resource for future investigations of skeletal muscle mass.
    Keywords:  SLC36A1; SLC38A9; amino acid transporter; leucine; lysosome; mTORC1 signaling pathway
    DOI:  https://doi.org/10.3390/biom11091314
  20. Int J Mol Sci. 2021 Sep 10. pii: 9807. [Epub ahead of print]22(18):
    Targeting Autophagy with Natural Products as a Potential Therapeutic Approach for Cancer.
    Md Abdul Alim Al-Bari, Yuko Ito, Samrein Ahmed, Nada Radwan, Hend S Ahmed, Nabil Eid.
      Macro-autophagy (autophagy) is a highly conserved eukaryotic intracellular process of self-digestion caused by lysosomes on demand, which is upregulated as a survival strategy upon exposure to various stressors, such as metabolic insults, cytotoxic drugs, and alcohol abuse. Paradoxically, autophagy dysfunction also contributes to cancer and aging. It is well known that regulating autophagy by targeting specific regulatory molecules in its machinery can modulate multiple disease processes. Therefore, autophagy represents a significant pharmacological target for drug development and therapeutic interventions in various diseases, including cancers. According to the framework of autophagy, the suppression or induction of autophagy can exert therapeutic properties through the promotion of cell death or cell survival, which are the two main events targeted by cancer therapies. Remarkably, natural products have attracted attention in the anticancer drug discovery field, because they are biologically friendly and have potential therapeutic effects. In this review, we summarize the up-to-date knowledge regarding natural products that can modulate autophagy in various cancers. These findings will provide a new position to exploit more natural compounds as potential novel anticancer drugs and will lead to a better understanding of molecular pathways by targeting the various autophagy stages of upcoming cancer therapeutics.
    Keywords:  anticancer drugs; autophagy; autophagy modulators; mTOR signaling; natural products; resveratrol; ω-3 PUFAs
    DOI:  https://doi.org/10.3390/ijms22189807
  21. Circ Res. 2021 Oct;129(8): 801-803
    Activating Autophagy to Prevent Doxorubicin Cardiomyopathy: The Timing Matters.
    Aarti Asnani.
      
    Keywords:  Editorials; autophagy; cardiomyopathies; doxorubicin
    DOI:  https://doi.org/10.1161/CIRCRESAHA.121.320063
  22. Biomolecules. 2021 Sep 09. pii: 1333. [Epub ahead of print]11(9):
    Alpha-Synuclein and the Endolysosomal System in Parkinson's Disease: Guilty by Association.
    Maxime Teixeira, Razan Sheta, Walid Idi, Abid Oueslati.
      Abnormal accumulation of the protein α- synuclein (α-syn) into proteinaceous inclusions called Lewy bodies (LB) is the neuropathological hallmark of Parkinson's disease (PD) and related disorders. Interestingly, a growing body of evidence suggests that LB are also composed of other cellular components such as cellular membrane fragments and vesicular structures, suggesting that dysfunction of the endolysosomal system might also play a role in LB formation and neuronal degeneration. Yet the link between α-syn aggregation and the endolysosomal system disruption is not fully elucidated. In this review, we discuss the potential interaction between α-syn and the endolysosomal system and its impact on PD pathogenesis. We propose that the accumulation of monomeric and aggregated α-syn disrupt vesicles trafficking, docking, and recycling, leading to the impairment of the endolysosomal system, notably the autophagy-lysosomal degradation pathway. Reciprocally, PD-linked mutations in key endosomal/lysosomal machinery genes (LRRK2, GBA, ATP13A2) also contribute to increasing α-syn aggregation and LB formation. Altogether, these observations suggest a potential synergistic role of α-syn and the endolysosomal system in PD pathogenesis and represent a viable target for the development of disease-modifying treatment for PD and related disorders.
    Keywords:  Parkinson’s disease; aggregation; alpha-synuclein; endolysosomal system; trafficking; vesicles
    DOI:  https://doi.org/10.3390/biom11091333
  23. Angew Chem Int Ed Engl. 2021 Sep 29.
    Inhibition of autophagy by a small molecule through covalent modification of LC3.
    Cheng Luo, Shijie Fan, Liyan Yue, Wei Wan, Yuanyuan Zhang, Bidong Zhang, Chinatsu Otomo, Quanfu Li, Tingting Lin, Junchi Hu, Pan Xu, Mingrui Zhu, Hongru Tao, Zhifeng Chen, Lianchun Li, Hong Ding, Zhiyi Yao, Junyan Lu, Yi Wen, Naixia Zhang, Minjia Tan, Kaixian Chen, Yuli Xie, Takanori Otomo, Bing Zhou, Hualiang Jiang, Yongjun Dang.
      The autophagic ubiquitin-like protein LC3 functions through interactions with LC3-interaction regions (LIRs) of other autophagy proteins including autophagy receptors, which stands out as a promising protein-protein interaction (PPI) target for the intervention of autophagy. Post-translational modifications like acetylation of Lys49 on the LIR-interacting surface could disrupt the interaction, offering an opportunity to design covalent small molecules interfering the interface. Through screening covalent compounds, we discover a small molecule modulator of LC3A/B that covalently modifies LC3A/B protein at Lys49. Activity-based protein profiling (ABPP) based evaluations reveal that a derivative molecule DC-LC3in-D5 exhibits a potent covalent reactivity and selectivity to LC3A/B in HeLa cells. DC-LC3in-D5 compromises LC3B lipidation in vitro and in HeLa cells, leading to deficiency in the formation of autophagic structures and autophagic substrate degradation. DC-LC3in-D5 could serve as a powerful tool for autophagy research as well as for therapeutic interventions.
    Keywords:  LC3, covalent modification, post-translational modification, autophagy
    DOI:  https://doi.org/10.1002/anie.202109464
  24. Biomedicines. 2021 Aug 24. pii: 1080. [Epub ahead of print]9(9):
    Storage of Mutant Human SOD1 in Non-Neural Cells from the Type-1 Amyotrophic Lateral Sclerosis ratG93A Model Correlated with the Lysosomes' Dysfunction.
    Ilaria Bicchi, Francesco Morena, Chiara Argentati, Laura Rota Nodari, Carla Emiliani, Maurizio Gelati, Angelo L Vescovi, Sabata Martino.
      Herein, we explored the impact of the lysosome dysfunction during the progression of Amyotrophic Lateral Sclerosis type-1 (ALS1). We conducted the study in non-neural cells, primary fibroblasts (rFFFs), and bone marrow-mesenchymal stem cells (rBM-MSCs), isolated from the animal model ratG93A for ALS1 at two stages of the disease: Pre-symptomatic-stage (ALS1-PreS) and Terminal-stage (ALS1-EndS). We documented the storage of human mutant Superoxide Dismutase 1, SOD1G93A (SOD1*) in the lysosomes of ALS1-rFFFs and ALS1-rBM-MSCs and demonstrated the hallmarks of the disease in non-neural cells as in ratG93A-ALS1-tissues. We showed that the SOD1* storage is associated with the altered glycohydrolases and proteases levels in tissues and both cell types from ALS1-PreS to ALS1-EndS. Only in ALS1-rFFFs, the lysosomes lost homeostasis, enlarge drastically, and contribute to the cell metabolic damage. Contrariwise, in ALS1-rBM-MSCs, we found a negligible metabolic dysfunction, which makes these cells' status similar to WT. We addressed this phenomenon to a safety mechanism perhaps associated with an enhanced lysosomal autophagic activity in ALS1-rBM-MSCs compared to ALS1-rFFFs, in which the lysosomal level of LC3-II/LC3I was comparable to that of WT-rFFFs. We suggested that the autophagic machinery could balance the storage of SOD1* aggregates and the lysosomal enzyme dysfunction even in ALS1-EndS-stem cells.
    Keywords:  ALS; GALC; Hexosaminidase; LC3; autophagy; bone marrow-mesenchymal stem cells; lysosomal storage disorders; mutant SOD1 lysosomal storage
    DOI:  https://doi.org/10.3390/biomedicines9091080
  25. Cells. 2021 Sep 18. pii: 2475. [Epub ahead of print]10(9):
    Autophagy Promotes the Survival of Adipose Mesenchymal Stem/Stromal Cells and Enhances Their Therapeutic Effects in Cisplatin-Induced Liver Injury via Modulating TGF-β1/Smad and PI3K/AKT Signaling Pathways.
    Eman Mohamad El Nashar, Mansour Abdullah Alghamdi, Wardah Abdullah Alasmari, Mohamed M A Hussein, Eman Hamza, Reham Ismail Taha, Mona M Ahmed, Khulood Mohammed Al-Khater, Ahmed Abdelfattah-Hassan.
      Autophagy is a key metabolic process where cells can recycle its proteins and organelles to regenerate its own cellular building blocks. Chemotherapy is indispensable for cancer treatment but associated with various side-effects, including organ damage. Stem cell-based therapy is a promising approach for reducing chemotherapeutic side effects, however, one of its main culprits is the poor survival of transplanted stem cells in damaged tissues. Here, we aimed to test the effects of activating autophagy in adipose-derived mesenchymal stem/stromal cells (ADSCs) on the survival of ADSCs, and their therapeutic value in cisplatin-induced liver injury model. Autophagy was activated in ADSCs by rapamycin (50 nM/L) for two hours before transplantation and were compared to non-preconditioned ADSCs. Rapamycin preconditioning resulted in activated autophagy and improved survival of ADSCs achieved by increased autophagosomes, upregulated autophagy-specific LC3-II gene, decreased protein degradation/ubiquitination by downregulated p62 gene, downregulated mTOR gene, and finally, upregulated antiapoptotic BCL-2 gene. In addition, autophagic ADSCs transplantation in the cisplatin liver injury model, liver biochemical parameters (AST, ALT and albumin), lipid peroxidation (MDA), antioxidant profile (SOD and GPX) and histopathological picture were improved, approaching near-normal conditions. These promising autophagic ADSCs effects were achieved by modulation of components in TGF-β1/Smad and PI3K-AKT signaling pathways, besides reducing NF-κB gene expression (marker for inflammation), reducing TGF-β1 levels (marker for fibrosis) and increasing SDF-1 levels (liver regeneration marker) in liver. Therefore, current results highlight the importance of autophagy in augmenting the therapeutic potential of stem cell therapy in alleviating cisplatin-associated liver damage and opens the path for improved cell-based therapies, in general, and with chemotherapeutics, in particular.
    Keywords:  ADSCs; apoptosis; cell-based therapy; hepatoprotective pathways; liver damage; rapamycin
    DOI:  https://doi.org/10.3390/cells10092475
  26. Cell Biol Toxicol. 2021 Sep 28.
    Cadmium affects autophagy in the human intestinal cells Caco-2 through ROS-mediated ERK activation.
    Myriam Mireault, Yong Xiao, Benoît Barbeau, Catherine Jumarie.
      Cadmium is a toxic metal that enters the food chain. Following oral ingestion, the intestinal epithelium has the capacity to accumulate high levels of this metal. We have previously shown that Cd induces ERK1/2 activation in differentiated but not proliferative human enterocytic-like Caco-2 cells. As autophagy is a dynamic process that plays a critical role in intestinal mucosa, we aimed the present study 1) to investigate the role of p-ERK1/2 in constitutive autophagy in proliferative Caco-2 cells and 2) to investigate whether Cd-induced activation of ERK1/2 modifies autophagic activity in postconfluent Caco-2 cell monolayers. Western blot analyses of ERK1/2 and autophagic markers (LC3, SQSTM1), and cellular staining with acridine orange showed that ERK1/2 and autophagic activities both decreased with time in culture. GFP-LC3 fluorescence was also associated with proliferative cells and the presence of a constitutive ERK1/2-dependent autophagic flux was demonstrated in proliferative but not in postconfluent cells. In the latter condition, serum and glucose deprivation triggered autophagy via a transient phosphorylation of ERK1/2, whereas Cd-modified autophagy via a ROS-dependent sustained activation of ERK1/2. Basal autophagy flux in proliferative cells and Cd-induced increases in autophagic markers in postconfluent cells both involved p-ERK1/2. Whether Cd blocks autophagic flux in older cell cultures remains to be clarified but our data suggest dual effects. Our results prompt further studies investigating the consequences that Cd-induced ERK1/2 activation and the related effect on autophagy may have on the intestinal cells, which may accumulate and trap high levels of Cd under some nutritional conditions.
    Keywords:  Autophagy; Cadmium; ERK signaling; Intestinal cells; Reactive oxygen species
    DOI:  https://doi.org/10.1007/s10565-021-09655-4
  27. Mol Carcinog. 2021 Oct 01.
    Rapalogs induce non-apoptotic, autophagy-dependent cell death in HPV-negative TP53 mutant head and neck squamous cell carcinoma.
    Md Maksudul Alam, Janmaris Marin Fermin, Patrick T Spiller, Chaning Burnett, Xiaohua Rong, Tara Moore-Medlin, Caden O Maxwell, Alok R Khandelwal, Cherie-Ann O Nathan.
      TP53 is the most frequently mutated gene in head and neck squamous cell carcinoma (HNSCC). Patients with HPV-negative TP53 mutant HNSCC have the worst prognosis, necessitating additional agents for treatment. Since mutant p53 causes sustained activation of the PI3K/AKT/mTOR signaling pathway, we investigated the effect of rapalogs RAD001 and CCI-779 on HPV-negative mutTP53 HNSCC cell lines and xenografts. Rapalogs significantly reduced cell viability and colony formation. Interestingly, rapalogs-induced autophagy with no effect on apoptosis. Pretreatment with autophagy inhibitors, 3-methyladenine (3-MA) and ULK-101 rescued the cell viability by inhibiting rapalog-induced autophagy, suggesting that both RAD001 and CCI-779 induce non-apoptotic autophagy-dependent cell death (ADCD). Moreover, rapalogs upregulated the levels of ULK1 and pULK1 S555 with concomitant downregulation of the mTORC1 pathway. However, pretreatment of cells with rapalogs prevented the ULK-101-mediated inhibition of ULK1 to sustained autophagy, suggesting that rapalogs induce ADCD through the activation of ULK1. To further translate our in vitro studies, we investigated the effect of RAD001 in HPV-negative mutTP53 (HN31 and FaDu) tumor cell xenograft model in nude mice. Mice treated with RAD001 exhibited a significant tumor volume reduction without induction of apoptosis, and with a concomitant increase in autophagy. Further, treatment with RAD001 was associated with a considerable increase in pULK1 S555 and ULK1 levels through the inhibition of mTORC1. 3-MA reversed the effect of RAD001 on FaDu tumor growth suggesting that RAD001 promotes ACDC in HPV-negative mutTP53 xenograft. This is the first report demonstrating that rapalogs promote non-apoptotic ADCD in HPV-negative mutTP53 HNSCC via the ULK1 pathway. Further studies are required to establish the promising role of rapalogs in preventing the regrowth of HPV-negative mutTP53 HNSCC.
    Keywords:  HNSCC; TP53; autophagy; mTOR; rapalog
    DOI:  https://doi.org/10.1002/mc.23357
  28. J Biol Chem. 2021 Sep 23. pii: S0021-9258(21)01047-4. [Epub ahead of print] 101244
    TBK1 has a new Akt.
    Leslie M Shaw.
      TANK-binding kinase 1 (TBK1) is a non-canonical IκB kinase that plays an essential role in the innate immune response to foreign pathogens. Recent studies have highlighted additional roles for TBK1 in the regulation of metabolism, although the mechanisms of this regulation have not been well characterized. In a recent issue, Tooley et al demonstrated that TBK1-dependent activation of downstream kinase Akt is mediated via mTOR complex 2 (mTORC2). This novel action of TBK1 reveals a key role for this kinase in the regulation of cellular metabolism and growth by diverse environmental inputs.
    Keywords:  AKT; TBK1; mTORC2
    DOI:  https://doi.org/10.1016/j.jbc.2021.101244
  29. MicroPubl Biol. 2021 ;2021
    A single copy transgenic mutant FUS strain reproduces age-dependent ALS phenotypes in C. elegans.
    Audrey Labarre, Gilles Tossing, Claudia Maios, James J Doyle, J Alex Parker.
      Mutations in the human DNA/RNA binding protein FUS are associated with amyotrophic lateral sclerosis and frontotemporal lobar degeneration, including some aggressive and juvenile onset forms. Cytoplasmic inclusions of human FUS proteins are observed in various neurodegenerative disorders, such as Huntington's disease or spinocerebellar ataxia, suggesting that FUS proteinopathy may be a key player in neurodegeneration. To better understand the pathogenic mechanisms of FUS, we created single copy transgenic Caenorhabditis elegans strains expressing full-length, untagged human FUS in the worm's GABAergic neurons. These transgenic worms expressing human mutant FUS (mFUS) display the same ALS-associated phenotypes than our previous multiple copy transgenic model, including adult-onset age-dependent loss of motility, progressive paralysis and GABAergic neurodegeneration. These phenotypes are distinct from the transgenic worms expressing human wild-type FUS (wtFUS). We introduce here our C. elegans single copy transgenic for human mutant FUS motor neuron toxicity that may be used for rapid genetic and pharmacological suppressor screening.
    DOI:  https://doi.org/10.17912/micropub.biology.000473
  30. J Invest Dermatol. 2021 Sep 23. pii: S0022-202X(21)01409-3. [Epub ahead of print]
    Impaired Autophagy in Psoriasis and Atopic Dermatitis: A New Therapeutic Target?
    Stephan Hailfinger, Klaus Schulze-Osthoff.
      Dysfunctional autophagy is linked to various diseases, including psoriasis and atopic dermatitis. Recent evidence suggests that exposure of keratinocytes to TNF-α results in impaired autophagy and lysosomal function. The skin of patients with psoriasis and atopic dermatitis reveals a decreased expression of lysosomal cathepsins. Impaired autophagy is presumably involved in inflammation and disturbed keratinocyte differentiation, whereas stimulating autophagy might be a treatment option in inflammatory skin disease.
    DOI:  https://doi.org/10.1016/j.jid.2021.06.006
  31. Nature. 2021 Sep 29.
    Circadian autophagy drives iTRF-mediated longevity.
    Matt Ulgherait, Adil M Midoun, Scarlet J Park, Jared A Gatto, Samantha J Tener, Julia Siewert, Naomi Klickstein, Julie C Canman, William W Ja, Mimi Shirasu-Hiza.
      Time-restricted feeding (TRF) has recently gained interest as a potential anti-ageing treatment for organisms from Drosophila to humans1-5. TRF restricts food intake to specific hours of the day. Because TRF controls the timing of feeding, rather than nutrient or caloric content, TRF has been hypothesized to depend on circadian-regulated functions; the underlying molecular mechanisms of its effects remain unclear. Here, to exploit the genetic tools and well-characterized ageing markers of Drosophila, we developed an intermittent TRF (iTRF) dietary regimen that robustly extended fly lifespan and delayed the onset of ageing markers in the muscles and gut. We found that iTRF enhanced circadian-regulated transcription and that iTRF-mediated lifespan extension required both circadian regulation and autophagy, a conserved longevity pathway. Night-specific induction of autophagy was both necessary and sufficient to extend lifespan on an ad libitum diet and also prevented further iTRF-mediated lifespan extension. By contrast, day-specific induction of autophagy did not extend lifespan. Thus, these results identify circadian-regulated autophagy as a critical contributor to iTRF-mediated health benefits in Drosophila. Because both circadian regulation and autophagy are highly conserved processes in human ageing, this work highlights the possibility that behavioural or pharmaceutical interventions that stimulate circadian-regulated autophagy might provide people with similar health benefits, such as delayed ageing and lifespan extension.
    DOI:  https://doi.org/10.1038/s41586-021-03934-0
  32. Cells. 2021 Sep 17. pii: 2450. [Epub ahead of print]10(9):
    Mammalian Cell-Free System Recapitulates the Early Events of Post-Fertilization Sperm Mitophagy.
    Won-Hee Song, Dalen Zuidema, Young-Joo Yi, Michal Zigo, Zhibing Zhang, Miriam Sutovsky, Peter Sutovsky.
      Propagation of paternal sperm-contributed mitochondrial genes, resulting in heteroplasmy, is seldom observed in mammals due to post-fertilization degradation of sperm mitochondria, referred to as sperm mitophagy. Whole organelle sperm mitochondrion degradation is thought to be mediated by the interplay between the ubiquitin-proteasome system (UPS) and the autophagic pathway (Song et al., Proc. Natl. Acad. Sci. USA, 2016). Both porcine and primate post-fertilization sperm mitophagy rely on the ubiquitin-binding autophagy receptor, sequestosome 1 (SQSTM1), and the proteasome-interacting ubiquitinated protein dislocase, valosin-containing protein (VCP). Consequently, we anticipated that sperm mitophagy could be reconstituted in a cell-free system consisting of permeabilized mammalian spermatozoa co-incubated with porcine oocyte extracts. We found that SQSTM1 was detected in the midpiece/mitochondrial sheath of the sperm tail after, but not before, co-incubation with oocyte extracts. VCP was prominent in the sperm mitochondrial sheath both before and after the extract co-incubation and was also detected in the acrosome and postacrosomal sheath and the subacrosomal layer of the spermatozoa co-incubated with extraction buffer as control. Such patterns are consistent with our previous observation of SQSTM1 and VCP associating with sperm mitochondria inside the porcine zygote. In addition, it was observed that sperm head expansion mimicked the early stages of paternal pronucleus development in a zygote during prolonged sperm-oocyte extract co-incubation. Treatment with anti-SQSTM1 antibody during extract co-incubation prevented ooplasmic SQSTM1 binding to sperm mitochondria. Even in an interspecific cellular environment encompassing bull spermatozoa and porcine oocyte extract, ooplasmic SQSTM1 was recruited to heterospecific sperm mitochondria. Complementary with the binding of SQSTM1 and VCP to sperm mitochondria, two sperm-borne pro-mitophagy proteins, parkin co-regulated gene product (PACRG) and spermatogenesis associated 18 (SPATA18), underwent localization changes after extract coincubation, which were consistent with their degradation observed inside fertilized porcine oocytes. These results demonstrate that the early developmental events of post-fertilization sperm mitophagy observed in porcine zygote can be reconstituted in a cell-free system, which could become a useful tool for identifying additional molecules that regulate mitochondrial inheritance in mammals.
    Keywords:  PACRG; SPATA18; SQSTM1; VCP; autophagy; cell-free system; mitochondria; mtDNA; ubiquitin-proteasome system
    DOI:  https://doi.org/10.3390/cells10092450
  33. Nutrients. 2021 Aug 29. pii: 3026. [Epub ahead of print]13(9):
    Metabolome Characteristics of Liver Autophagy Deficiency under Starvation Conditions in Infancy.
    Kazuhito Sekiguchi, Hiroaki Miyahara, Masanori Inoue, Kyoko Kiyota, Kumiko Sakai, Toshikatsu Hanada, Kenji Ihara.
      The liver function is essential for metabolism, detoxification, and bile synthesis, even in the neonatal period. Autophagy plays significance roles in THE adult liver, whereas the role of liver autophagy in the early neonatal period largely remains unclear. To clarify the importance of liver autophagy in the neonatal starvation period, we generated liver-specific autophagy-deficient (Atg5flox/flox; Albumin-Cre) mice and investigated under starvation conditions comparing with control (Atg5flox/+; Albumin-Cre) mice, focusing on serum metabolites and liver histopathology. As a result, autophagy in the liver was found to unessential for the survival under postnatal starvation. A metabolomics analysis of serum metabolites by gas chromatography-tandem mass spectrometry showed a significant difference between the groups, especially after 12-h starvation, suggesting the synergistical adaption of metabolic pathways, such as the "malate-aspartate shuttle", "aspartate metabolism", "urea cycle", and "glycine and serine metabolism". Liver-specific autophagy-deficiency under postnatal starvation conditions can cause a characteristic metabolic alteration suggesting a change of the mitochondrial function. Neonates seemed to maintain ketone production under starvation conditions, even in the autophagy-deficient liver, through a change in the mitochondrial function, which may be an adaptive mechanism for avoiding fatal starvation.
    Keywords:  autophagy; infant; liver; metabolome; starvation
    DOI:  https://doi.org/10.3390/nu13093026
  34. Cells. 2021 Aug 27. pii: 2215. [Epub ahead of print]10(9):
    Autophagy-Associated IL-15 Production Is Involved in the Pathogenesis of Leprosy Type 1 Reaction.
    Bruno Jorge de Andrade Silva, Tamiris Lameira Bittencourt, Thyago Leal-Calvo, Mayara Abud Mendes, Rhana Berto da Silva Prata, Mayara Garcia de Mattos Barbosa, Priscila Ribeiro Andrade, Suzana Côrte-Real, Gilberto Marcelo Sperandio da Silva, Milton Ozório Moraes, Euzenir Nunes Sarno, Roberta Olmo Pinheiro.
      Leprosy reactional episodes are acute inflammatory events that may occur during the clinical course of the disease. Type 1 reaction (T1R) is associated with an increase in neural damage, and the understanding of the molecular pathways related to T1R onset is pivotal for the development of strategies that may effectively control the reaction. Interferon-gamma (IFN-γ) is a key cytokine associated with T1R onset and is also associated with autophagy induction. Here, we evaluated the modulation of the autophagy pathway in Mycobacterium leprae-stimulated cells in the presence or absence of IFN-γ. We observed that IFN-γ treatment promoted autophagy activation and increased the expression of genes related to the formation of phagosomes, autophagy regulation and function, or lysosomal pathways in M. leprae-stimulated cells. IFN-γ increased interleukin (IL)-15 secretion in M. leprae-stimulated THP-1 cells in a process associated with autophagy activation. We also observed higher IL15 gene expression in multibacillary (MB) patients who later developed T1R during clinical follow-up when compared to MB patients who did not develop the episode. By overlapping gene expression patterns, we observed 13 common elements shared between T1R skin lesion cells and THP-1 cells stimulated with both M. leprae and IFN-γ. Among these genes, the autophagy regulator Translocated Promoter Region, Nuclear Basket Protein (TPR) was significantly increased in T1R cells when compared with non-reactional MB cells. Overall, our results indicate that IFN-γ may induce a TPR-mediated autophagy transcriptional program in M. leprae-stimulated cells similar to that observed in skin cells during T1R by a pathway that involves IL-15 production, suggesting the involvement of this cytokine in the pathogenesis of T1R.
    Keywords:  IL-15; Mycobacterium leprae; autophagy; leprosy or T1R; lysosomes; macrophage or THP-1; phagosome
    DOI:  https://doi.org/10.3390/cells10092215
  35. Sci Adv. 2021 Oct;7(40): eabj2485
    GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis.
    Jonathan M Goodwin, Ward G Walkup, Kirsty Hooper, Taoyingnan Li, Chieko Kishi-Itakura, Aylwin Ng, Timothy Lehmberg, Archana Jha, Sravya Kommineni, Katherine Fletcher, Jorge Garcia-Fortanet, Yaya Fan, Qing Tang, Menghao Wei, Asmita Agrawal, Sagar R Budhe, Sreekanth R Rouduri, Dan Baird, Jeff Saunders, Janna Kiselar, Mark R Chance, Andrea Ballabio, Brent A Appleton, John H Brumell, Oliver Florey, Leon O Murphy.
      [Figure: see text].
    DOI:  https://doi.org/10.1126/sciadv.abj2485
  36. Biochemistry. 2021 Sep 27.
    Redefining the Scope of Targeted Protein Degradation: Translational Opportunities in Hijacking the Autophagy-Lysosome Pathway.
    Katelyn Cassidy, Heng Zhao.
      The advent of multi-specific targeted protein degradation (TPD) therapies has made it possible to drug targets that have long been considered to be inaccessible. For this reason, the foremost TPD modalities - molecular glues and proteolysis targeting chimeras (PROTACs) -have been widely adopted and developed in therapeutic programs across the pharmaceutical and biotechnology industries. While there are many clear advantages to these two approaches, there are also blind spots. Specifically, PROTACs and molecular glues are inherently mechanistically analogous in that targets of both are degraded via the 26s proteasome; however, not all disease-relevant targets are suitable for ubiquitin proteasome system (UPS)-mediated degradation. The alternative mammalian protein degradation pathway, the autophagy-lysosome system (or ALS), is capable of degrading targets that elude the UPS such as long-lived proteins, insoluble protein aggregates, and even abnormal organelles. Emerging TPD strategies- such as ATTEC, AUTAC, and LYTAC- take advantage of the substrate diversity of the ALS to greatly expand the clinical utility of TPD. In this Perspective, we will discuss the array of current TPD modalities, with a focus on critical evaluation of these novel ALS-mediated degradation techniques.
    DOI:  https://doi.org/10.1021/acs.biochem.1c00330
  37. Proc Natl Acad Sci U S A. 2021 Oct 05. pii: e2110629118. [Epub ahead of print]118(40):
    IP3R-driven increases in mitochondrial Ca2+ promote neuronal death in NPC disease.
    Scott A Tiscione, Maria Casas, Jonathan D Horvath, Vincent Lam, Keiko Hino, Daniel S Ory, L Fernando Santana, Sergi Simó, Rose E Dixon, Eamonn J Dickson.
      Ca2+ is the most ubiquitous second messenger in neurons whose spatial and temporal elevations are tightly controlled to initiate and orchestrate diverse intracellular signaling cascades. Numerous neuropathologies result from mutations or alterations in Ca2+ handling proteins; thus, elucidating molecular pathways that shape Ca2+ signaling is imperative. Here, we report that loss-of-function, knockout, or neurodegenerative disease-causing mutations in the lysosomal cholesterol transporter, Niemann-Pick Type C1 (NPC1), initiate a damaging signaling cascade that alters the expression and nanoscale distribution of IP3R type 1 (IP3R1) in endoplasmic reticulum membranes. These alterations detrimentally increase Gq-protein coupled receptor-stimulated Ca2+ release and spontaneous IP3R1 Ca2+ activity, leading to mitochondrial Ca2+ cytotoxicity. Mechanistically, we find that SREBP-dependent increases in Presenilin 1 (PS1) underlie functional and expressional changes in IP3R1. Accordingly, expression of PS1 mutants recapitulate, while PS1 knockout abrogates Ca2+ phenotypes. These data present a signaling axis that links the NPC1 lysosomal cholesterol transporter to the damaging redistribution and activity of IP3R1 that precipitates cell death in NPC1 disease and suggests that NPC1 is a nanostructural disease.
    Keywords:  GPCR; IP3R; NPC1; calcium; neurodegeneration
    DOI:  https://doi.org/10.1073/pnas.2110629118
  38. Viruses. 2021 Sep 15. pii: 1845. [Epub ahead of print]13(9):
    Nonstructural Protein NS1 of Influenza Virus Disrupts Mitochondrial Dynamics and Enhances Mitophagy via ULK1 and BNIP3.
    Jae-Hwan Lee, Soo-Jin Oh, Jeanho Yun, Ok Sarah Shin.
      Nonstructural protein 1 (NS1) of influenza virus (IFV) is essential for evading interferon (IFN)-mediated antiviral responses, thereby contributing to the pathogenesis of influenza. Mitophagy is a type of autophagy that selectively removes damaged mitochondria. The role of NS1 in IFV-mediated mitophagy is currently unknown. Herein, we showed that overexpression of NS1 protein led to enhancement of mitophagy. Mitophagy induction via carbonyl cyanide 3-chlorophenylhydrazone treatment in IFV-infected A549 cells led to increased viral replication efficiency, whereas the knockdown of PTEN-induced kinase 1 (PINK1) led to the opposite effect on viral replication. Overexpression of NS1 protein led to changes in mitochondrial dynamics, including depolarization of mitochondrial membrane potential. In contrast, infection with NS1-deficient virus resulted in impaired mitochondrial fragmentation, subsequent mitolysosomal formation, and mitophagy induction, suggesting an important role of NS1 in mitophagy. Meanwhile, NS1 protein increased the phosphorylation of Unc-51-like autophagy activating kinase 1 (ULK1) and the mitochondrial expression of BCL2- interacting protein 3 (BNIP3), both of which were found to be important for IFV-mediated mitophagy. Overall, these data highlight the importance of IFV NS1, ULK1, and BNIP3 during mitophagy activation.
    Keywords:  BNIP3; NS1; antiviral immune responses; influenza a virus; mitophagy
    DOI:  https://doi.org/10.3390/v13091845
  39. Biomolecules. 2021 Sep 18. pii: 1382. [Epub ahead of print]11(9):
    Mechanistic Target of Rapamycin Complex 1 Signaling Links Hypoxia to Increased IGFBP-1 Phosphorylation in Primary Human Decidualized Endometrial Stromal Cells.
    Pinki Nandi, Chloe E Jang, Kyle Biggar, Chidambra D Halari, Thomas Jansson, Madhulika B Gupta.
      Insulin-like growth factor-1 (IGF-1) bioavailability in pregnancy is governed by IGF binding protein (IGFBP-1) and its phosphorylation, which enhances the affinity of IGFBP-1 for the growth factor. The decidua is the predominant source of maternal IGFBP-1; however, the mechanisms regulating decidual IGFBP-1 secretion/phosphorylation are poorly understood. Using decidualized primary human endometrial stromal cells (HESCs) from first-trimester placenta, we tested the hypothesis that mTORC1 signaling mechanistically links hypoxia to decidual IGFBP-1 secretion/phosphorylation. Hypoxia inhibited mechanistic target of rapamycin (mTORC1) (p-P70-S6K/Thr389, -47%, p = 0.038; p-4E-BP1/Thr70, -55%, p = 0.012) and increased IGFBP-1 (total, +35%, p = 0.005; phosphorylated, Ser101/+82%, p = 0.018; Ser119/+88%, p = 0.039; Ser 169/+157%, p = 0.019). Targeted parallel reaction monitoring-mass spectrometry (PRM-MS) additionally demonstrated markedly increased dual IGFBP-1 phosphorylation (pSer98+Ser101; pSer169+Ser174) in hypoxia. IGFBP-1 hyperphosphorylation inhibited IGF-1 receptor autophosphorylation/ Tyr1135 (-29%, p = 0.002). Furthermore, silencing of tuberous sclerosis complex 2 (TSC2) activated mTORC1 (p-P70-S6K/Thr389, +68%, p = 0.038; p-4E-BP1/Thr70, +30%, p = 0.002) and reduced total/site-specific IGFBP-1 phosphorylation. Importantly, TSC2 siRNA prevented inhibition of mTORC1 and the increase in secretion/site-specific IGFBP-1 phosphorylation in hypoxia. PRM-MS indicated concomitant changes in protein kinase autophosphorylation (CK2/Tyr182; PKC/Thr497; PKC/Ser657). Overall, mTORC1 signaling mechanistically links hypoxia to IGFBP-1 secretion/phosphorylation in primary HESC, implicating decidual mTORC1 inhibition as a novel mechanism linking uteroplacental hypoxia to fetal growth restriction.
    Keywords:  IGF Type 1; fetal development; humans; insulin-like growth factor I; placental insufficiency; receptor
    DOI:  https://doi.org/10.3390/biom11091382
  40. Antioxidants (Basel). 2021 Aug 24. pii: 1330. [Epub ahead of print]10(9):
    Exacerbated Age-Related Hippocampal Alterations of Microglia Morphology, β-Amyloid and Lipofuscin Deposition and Presenilin Overexpression in Per1-/--Mice.
    Jan Hendrik Börner, Oliver Rawashdeh, Abdelhaq Rami.
      In humans, alterations of circadian rhythms and autophagy are linked to metabolic, cardiovascular and neurological dysfunction. Autophagy constitutes a specific form of cell recycling in many eukaryotic cells. Aging is the principal risk factor for the development of neurodegenerative diseases. Thus, we assume that both the circadian clock and autophagy are indispensable to counteract aging. We have previously shown that the hippocampus of Per1-/--mice exhibits a reduced autophagy and higher neuronal susceptibility to ischemic insults compared to wild type (WT). Therefore, we chose to study the link between aging and loss of clock gene Per1-/--mice. Young and aged C3H- and Per1-/--mice were used as models to analyze the hippocampal distribution of Aβ42, lipofuscin, presenilin, microglia, synaptophysin and doublecortin. We detected several changes in the hippocampus of aged Per1-/--mice compared to their wild type littermates. Our results show significant alterations of microglia morphology, an increase in Aβ42 deposition, overexpression of presenilin, decrease in synaptophysin levels and massive accumulation of lipofuscin in the hippocampus of 24-month-old Per1-/--mice, without alteration of adult neurogenesis. We suggest that the marked lipofuscin accumulation, Aβ42 deposition, and overexpression of presenilin-2 observed in our experiments may be some of the consequences of the slowed autophagy in the hippocampus of aged Per1-/--mice. This may lead during aging to excessive accumulation of misfolded proteins which may, consequently, result in higher neuronal vulnerability.
    Keywords:  Per1−/−-mice; aging; autophagy; hippocampus; lipofuscin; microglia; oxidative stress; ß-amyloid
    DOI:  https://doi.org/10.3390/antiox10091330
  41. Proc Natl Acad Sci U S A. 2021 Oct 05. pii: e2105367118. [Epub ahead of print]118(40):
    Matrix stiffness drives stromal autophagy and promotes formation of a protumorigenic niche.
    Anna Hupfer, Anna Brichkina, Anke Koeniger, Corinna Keber, Carsten Denkert, Petra Pfefferle, Frederik Helmprobst, Axel Pagenstecher, Alexander Visekruna, Matthias Lauth.
      Increased stiffness of solid tissues has long been recognized as a diagnostic feature of several pathologies, most notably malignant diseases. In fact, it is now well established that elevated tissue rigidity enhances disease progression and aggressiveness and is associated with a poor prognosis in patients as documented, for instance, for lung fibrosis or the highly desmoplastic cancer of the pancreas. The underlying mechanisms of the interplay between physical properties and cellular behavior are, however, not very well understood. Here, we have found that switching culture conditions from soft to stiff substrates is sufficient to evoke (macro) autophagy in various fibroblast types. Mechanistically, this is brought about by stiffness-sensing through an Integrin αV-focal adhesion kinase module resulting in sequestration and posttranslational stabilization of the metabolic master regulator AMPKα at focal adhesions, leading to the subsequent induction of autophagy. Importantly, stiffness-induced autophagy in stromal cells such as fibroblasts and stellate cells critically supports growth of adjacent cancer cells in vitro and in vivo. This process is Integrin αV dependent, opening possibilities for targeting tumor-stroma crosstalk. Our data thus reveal that the mere change in mechanical tissue properties is sufficient to metabolically reprogram stromal cell populations, generating a tumor-supportive metabolic niche.
    Keywords:  AMPK; ITGAV; autophagy; pancreatic stellate cells; tumor stroma
    DOI:  https://doi.org/10.1073/pnas.2105367118
  42. Biomedicines. 2021 Sep 07. pii: 1178. [Epub ahead of print]9(9):
    Autophagy, Mesenchymal Stem Cell Differentiation, and Secretion.
    Mikhail Menshikov, Ekaterina Zubkova, Iuri Stafeev, Yelena Parfyonova.
      Mesenchymal stem cells (MSC) are multipotent cells capable to differentiate into adipogenic, osteogenic, and chondrogenic directions, possessing immunomodulatory activity and a capability to stimulate angiogenesis. A scope of these features and capabilities makes MSC a significant factor of tissue homeostasis and repair. Among factors determining the fate of MSC, a prominent place belongs to autophagy, which is activated under different conditions including cell starvation, inflammation, oxidative stress, and some others. In addition to supporting cell homeostasis by elimination of protein aggregates, and non-functional and damaged proteins, autophagy is a necessary factor of change in cell phenotype on the process of cell differentiation. In present review, some mechanisms providing participation of autophagy in cell differentiation are discussed.
    Keywords:  autophagy; differentiation; immunomodulation; mesenchymal stem cells; signal transduction
    DOI:  https://doi.org/10.3390/biomedicines9091178
  43. Front Microbiol. 2021 ;12 712631
    Autophagy as a Mechanism for Adaptive Prediction-Mediated Emergence of Drug Resistance.
    Nivedita Nivedita, John D Aitchison, Nitin S Baliga.
      Drug resistance is a major problem in treatment of microbial infections and cancers. There is growing evidence that a transient drug tolerant state may precede and potentiate the emergence of drug resistance. Therefore, understanding the mechanisms leading to tolerance is critical for combating drug resistance and for the development of effective therapeutic strategy. Through laboratory evolution of yeast, we recently demonstrated that adaptive prediction (AP), a strategy employed by organisms to anticipate and prepare for a future stressful environment, can emerge within 100 generations by linking the response triggered by a neutral cue (caffeine) to a mechanism of protection against a lethal agent (5-fluoroorotic acid, 5-FOA). Here, we demonstrate that mutations selected across multiple laboratory-evolved lines had linked the neutral cue response to core genes of autophagy. Across these evolved lines, conditional activation of autophagy through AP conferred tolerance, and potentiated subsequent selection of mutations in genes specific to overcoming the toxicity of 5-FOA. These results offer a new perspective on how extensive genome-wide genetic interactions of autophagy could have facilitated the emergence of AP over short evolutionary timescales to potentiate selection of 5-FOA resistance-conferring mutations.
    Keywords:  adaptive prediction; autophagy; conditional fitness; drug resistance; structured environments
    DOI:  https://doi.org/10.3389/fmicb.2021.712631
  44. Cells. 2021 Aug 28. pii: 2229. [Epub ahead of print]10(9):
    α-Synuclein Decreases the Abundance of Proteasome Subunits and Alters Ubiquitin Conjugates in Yeast.
    Blagovesta Popova, Dajana Galka, Nicola Häffner, Dan Wang, Kerstin Schmitt, Oliver Valerius, Michael Knop, Gerhard H Braus.
      Parkinson's disease (PD) is the most prevalent movement disorder characterized with loss of dopaminergic neurons in the brain. One of the pathological hallmarks of the disease is accumulation of aggregated α-synuclein (αSyn) in cytoplasmic Lewy body inclusions that indicates significant dysfunction of protein homeostasis in PD. Accumulation is accompanied with highly elevated S129 phosphorylation, suggesting that this posttranslational modification is linked to pathogenicity and altered αSyn inclusion dynamics. To address the role of S129 phosphorylation on protein dynamics further we investigated the wild type and S129A variants using yeast and a tandem fluorescent timer protein reporter approach to monitor protein turnover and stability. Overexpression of both variants leads to inhibited yeast growth. Soluble S129A is more stable and additional Y133F substitution permits αSyn degradation in a phosphorylation-independent manner. Quantitative cellular proteomics revealed significant αSyn-dependent disturbances of the cellular protein homeostasis, which are increased upon S129 phosphorylation. Disturbances are characterized by decreased abundance of the ubiquitin-dependent protein degradation machinery. Biotin proximity labelling revealed that αSyn interacts with the Rpt2 base subunit. Proteasome subunit depletion by reducing the expression of the corresponding genes enhances αSyn toxicity. Our studies demonstrate that turnover of αSyn and depletion of the proteasome pool correlate in a complex relationship between altered proteasome composition and increased αSyn toxicity.
    Keywords:  Parkinson disease; alpha-synuclein; posttranslational modifications; protein aggregation; protein degradation; protein homeostasis; ubiquitin–proteasome system; yeast
    DOI:  https://doi.org/10.3390/cells10092229
  45. Metabolites. 2021 Sep 18. pii: 637. [Epub ahead of print]11(9):
    Perturbation-Based Modeling Unveils the Autophagic Modulation of Chemosensitivity and Immunogenicity in Breast Cancer Cells.
    Isaac Quiros-Fernandez, Lucía Figueroa-Protti, Jorge L Arias-Arias, Norman Brenes-Cordero, Francisco Siles, Javier Mora, Rodrigo Antonio Mora-Rodríguez.
      In the absence of new therapeutic strategies, chemotherapeutic drugs are the most widely used strategy against metastatic breast cancer, in spite of eliciting multiple adverse effects and having low responses with an average 5-year patient survival rate. Among the new therapeutic targets that are currently in clinical trials, here, we addressed the association between the regulation of the metabolic process of autophagy and the exposure of damage-associated molecular patterns associated (DAMPs) to immunogenic cell death (ICD), which has not been previously studied. After validating an mCHR-GFP tandem LC3 sensor capacity to report dynamic changes of the autophagic metabolic flux in response to external stimuli and demonstrating that both basal autophagy levels and response to diverse autophagy regulators fluctuate among different cell lines, we explored the interaction between autophagy modulators and chemotherapeutic agents in regards of cytotoxicity and ICD using three different breast cancer cell lines. Since these interactions are very complex and variable throughout different cell lines, we designed a perturbation-based model in which we propose specific modes of action of chemotherapeutic agents on the autophagic flux and the corresponding strategies of modulation to enhance the response to chemotherapy. Our results point towards a promising therapeutic potential of the metabolic regulation of autophagy to overcome chemotherapy resistance by eliciting ICD.
    Keywords:  autophagy; chemotherapy; immunogenic cell death; perturbation-based modeling
    DOI:  https://doi.org/10.3390/metabo11090637
  46. Cells. 2021 Sep 07. pii: 2346. [Epub ahead of print]10(9):
    GDF-15 Deficiency Reduces Autophagic Activity in Human Macrophages In Vitro and Decreases p62-Accumulation in Atherosclerotic Lesions in Mice.
    Aline Heduschke, Kathrin Ackermann, Beate Wilhelm, Lilli Mey, Gabriel Alejandro Bonaterra, Ralf Kinscherf, Anja Schwarz.
      (1) Background: Growth differentiation factor-15 (GDF-15) is associated with cardiovascular diseases and autophagy in human macrophages (MΦ). Thus, we are interested in investigating autophagic mechanisms with special respect to the role of GDF-15. (2) Methods: Recombinant (r)GDF-15 and siRNA GDF-15 were used to investigate the effects of GDF-15 on autophagic and lysosomal activity, as well as autophagosome formation by transmission electron microscopy (TEM) in MΦ. To ascertain the effects of GDF-15-/- on the progression of atherosclerotic lesions, we used GDF-15-/-/ApoE-/- and ApoE-/- mice under a cholesterol-enriched diet (CED). Body weight, body mass index (BMI), blood lipid levels and lumen stenosis in the brachiocephalic trunk (BT) were analyzed. Identification of different cell types and localization of autophagy-relevant proteins in atherosclerotic plaques were performed by immunofluorescence. (3) Results: siGDF-15 reduced and, conversely, rGDF-15 increased the autophagic activity in MΦ, whereas lysosomal activity was unaffected. Autophagic degradation after starvation and rGDF-15 treatment was observed by TEM. GDF-15-/-/ApoE-/- mice, after CED, showed reduced lumen stenosis in the BT, while body weight, BMI and triglycerides were increased compared with ApoE-/- mice. GDF-15-/- decreased p62-accumulation in atherosclerotic lesions, especially in endothelial cells (ECs). (4) Conclusion: GDF-15 seems to be an important factor in the regulation of autophagy, especially in ECs of atherosclerotic lesions, indicating its crucial pathophysiological function during atherosclerosis development.
    Keywords:  atherosclerosis; autophagy; growth-differentiation factor-15; p62
    DOI:  https://doi.org/10.3390/cells10092346
  47. BMC Biol. 2021 Oct 01. 19(1): 218
    A human iPSC-derived inducible neuronal model of Niemann-Pick disease, type C1.
    Anika V Prabhu, Insung Kang, Raffaella De Pace, Christopher A Wassif, Hideji Fujiwara, Pamela Kell, Xuntian Jiang, Daniel S Ory, Juan S Bonifacino, Michael E Ward, Forbes D Porter.
       BACKGROUND: Niemann-Pick disease, type C (NPC) is a childhood-onset, lethal, neurodegenerative disorder caused by autosomal recessive mutations in the genes NPC1 or NPC2 and characterized by impaired cholesterol homeostasis, a lipid essential for cellular function. Cellular cholesterol levels are tightly regulated, and mutations in either NPC1 or NPC2 lead to deficient transport and accumulation of unesterified cholesterol in the late endosome/lysosome compartment, and progressive neurodegeneration in affected individuals. Previous cell-based studies to understand the NPC cellular pathophysiology and screen for therapeutic agents have mainly used patient fibroblasts. However, these do not allow modeling the neurodegenerative aspect of NPC disease, highlighting the need for an in vitro system that permits understanding the cellular mechanisms underlying neuronal loss and identifying appropriate therapies. This study reports the development of a novel human iPSC-derived, inducible neuronal model of Niemann-Pick disease, type C1 (NPC1).
    RESULTS: We generated a null i3Neuron (inducible × integrated × isogenic) (NPC1-/- i3Neuron) iPSC-derived neuron model of NPC1. The NPC1-/- and the corresponding isogenic NPC1+/+ i3Neuron cell lines were used to efficiently generate homogenous, synchronized neurons that can be used in high-throughput screens. NPC1-/- i3Neurons recapitulate cardinal cellular NPC1 pathological features including perinuclear endolysosomal storage of unesterified cholesterol, accumulation of GM2 and GM3 gangliosides, mitochondrial dysfunction, and impaired axonal lysosomal transport. Cholesterol storage, mitochondrial dysfunction, and axonal trafficking defects can be ameliorated by treatment with 2-hydroxypropyl-β-cyclodextrin, a drug that has shown efficacy in NPC1 preclinical models and in a phase 1/2a trial.
    CONCLUSION: Our data demonstrate the utility of this new cell line in high-throughput drug/chemical screens to identify potential therapeutic agents. The NPC1-/- i3Neuron line will also be a valuable tool for the NPC1 research community to explore the pathological mechanisms contributing to neuronal degeneration.
    Keywords:  Human induced pluripotent stem cells; Human neurons; Lysosomal disease; NPC1; Neurodegeneration; Niemann-Pick disease, type C1
    DOI:  https://doi.org/10.1186/s12915-021-01133-x
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