bims-auttor 2021-05-02 papers

bims-auttor

Biomed News

on Autophagy and mTOR

Issue of 2021–05–02
35 papers selected by
Viktor Korolchuk, Newcastle University

How autophagy controls the intestinal epithelial barrier.
Common Principles and Specific Mechanisms of Mitophagy from Yeast to Humans.
TFG binds LC3C to regulate ULK1 localization and autophagosome formation.
Current Approaches and Future Directions for the Treatment of mTORopathies.
Intersection between Redox Homeostasis and Autophagy: Valuable Insights into Neurodegeneration.
Platycodin D inhibits autophagy and increases glioblastoma cell death via LDLR upregulation.
Coordinate regulation of the senescent state by selective autophagy.
[New structures of mTORC1: Focus on Rag GTPases].
Autophagic Organelles in DNA Damage Response.
Leucine-sensing mechanism of leucyl-tRNA synthetase 1 for mTORC1 activation.
Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.
Convergent and Divergent Mechanisms of Epileptogenesis in mTORopathies.
PINK1-mediated mitophagy maintains pluripotency through optineurin.
Murine Models of Lysosomal Storage Diseases Exhibit Differences in Brain Protein Aggregation and Neuroinflammation.
Membrane supply and remodeling during autophagosome biogenesis.
Autophagic Cell Death During Development - Ancient and Mysterious.
Changes in Drp1 Function and Mitochondrial Morphology Are Associated with the α-Synuclein Pathology in a Transgenic Mouse Model of Parkinson's Disease.
Autophagosome biogenesis comes out of the black box.
S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux.
Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine.
Cell scientist to watch - Liang Ge.
Moments in autophagy and disease: Past and present.
Marliolide Derivative Induces Melanosome Degradation via Nrf2/p62-Mediated Autophagy.
Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons.
4E-BP2-dependent translation in cerebellar Purkinje cells controls spatial memory but not autism-like behaviors.
Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan.
Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.
The autophagy protein ATG16L1 cooperates with IFT20 and INPP5E to regulate the turnover of phosphoinositides at the primary cilium.
The influence of circular RNAs on autophagy and disease progression.
An Experimentally Induced Mutation in the UBA Domain of p62 Changes the Sensitivity of Cisplatin by Up-Regulating HK2 Localisation on the Mitochondria and Increasing Mitophagy in A2780 Ovarian Cancer Cells.
Antiretroviral Drugs Impact Autophagy with Toxic Outcomes.
The Characterization of a Subependymal Giant Astrocytoma-Like Cell Line from Murine Astrocyte with mTORC1 Hyperactivation.
Integrated Action of Autophagy and Adipose Tissue Triglyceride Lipase Ameliorates Diet-Induced Hepatic Steatosis in Liver-Specific PLIN2 Knockout Mice.
Pharmacological Modulators of Autophagy as a Potential Strategy for the Treatment of COVID-19.
ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells.

Autophagy. 2021 Apr 27. 1-18

How autophagy controls the intestinal epithelial barrier.

Elisabeth G Foerster, Tapas Mukherjee, Liliane Cabral-Fernandes, Juliana D B Rocha, Stephen E Girardin, Dana J Philpott.

  Macroautophagy/autophagy is a cellular catabolic process that results in lysosome-mediated recycling of organelles and protein aggregates, as well as the destruction of intracellular pathogens. Its role in the maintenance of the intestinal epithelium is of particular interest, as several autophagy-related genes have been associated with intestinal disease. Autophagy and its regulatory mechanisms are involved in both homeostasis and repair of the intestine, supporting intestinal barrier function in response to cellular stress through tight junction regulation and protection from cell death. Furthermore, a clear role has emerged for autophagy not only in secretory cells but also in intestinal stem cells, where it affects their metabolism, as well as their proliferative and regenerative capacity. Here, we review the physiological role of autophagy in the context of intestinal epithelial maintenance and how genetic mutations affecting autophagy contribute to the development of intestinal disease.Abbreviations: AKT1S1: AKT1 substrate 1; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; APC: APC regulator of WNT signaling pathway; ATF6: activating transcription factor 6; ATG: autophagy related; atg16l1[ΔIEC] mice: mice with a specific deletion of Atg16l1 in intestinal epithelial cells; ATP: adenosine triphosphate; BECN1: beclin 1; bsk/Jnk: basket; CADPR: cyclic ADP ribose; CALCOCO2: calcium binding and coiled-coil domain 2; CASP3: caspase 3; CD: Crohn disease; CDH1/E-cadherin: cadherin 1; CF: cystic fibrosis; CFTR: CF transmembrane conductance regulator; CGAS: cyclic GMP-AMP synthase; CLDN2: claudin 2; CoPEC: colibactin-producing E. coli; CRC: colorectal cancer; CYP1A1: cytochrome P450 family 1 subfamily A member 1; DC: dendritic cell; DDIT3: DNA damage inducible transcript 3; DEPTOR: DEP domain containing MTOR interacting protein; DSS: dextran sulfate sodium; EGF: epidermal growth factor; EGFR: epidermal growth factor receptor; EIF2A: eukaryotic translation initiation factor 2A; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2AK4/GCN2: eukaryotic translation initiation factor 2 alpha kinase 4; ER: endoplasmic reticulum; ERN1: endoplasmic reticulum to nucleus signaling 1; GABARAP: GABA type A receptor-associated protein; HMGB1: high mobility group box 1; HSPA5/GRP78: heat shock protein family A (Hsp70) member 5; IBD: inflammatory bowel disease; IEC: intestinal epithelial cell; IFN: interferon; IFNG/IFNγ:interferon gamma; IL: interleukin; IRGM: immunity related GTPase M; ISC: intestinal stem cell; LGR5: leucine rich repeat containing G protein-coupled receptor 5; LRRK2: leucine rich repeat kinase 2; MAP1LC3A/LC3: microtubule associated protein 1 light chain 3 alpha; MAPK/JNK: mitogen-activated protein kinase; MAPK14/p38 MAPK: mitogen-activated protein kinase 14; MAPKAP1: MAPK associated protein 1; MAVS: mitochondrial antiviral signaling protein; miRNA: microRNA; MLKL: mixed lineage kinase domain like pseudokinase; MLST8: MTOR associated protein, LST8 homolog; MNV: murine norovirus; MTOR: mechanistic target of rapamycin kinase; NBR1: NBR1 autophagy cargo receptor; NLRP: NLR family pyrin domain containing; NOD: nucleotide binding oligomerization domain containing; NRBF2: nuclear receptor binding factor 2; OPTN: optineurin; OXPHOS: oxidative phosphorylation; P: phosphorylation; Patj: PATJ crumbs cell polarity complex component; PE: phosphatidyl-ethanolamine; PI3K: phosphoinositide 3-kinase; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PPARG: peroxisome proliferator activated receptor gamma; PRR5: proline rich 5; PRR5L: proline rich 5 like; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; RB1CC1/FIP200: RB1 inducible coiled-coil 1; RER: rough endoplasmic reticulum; RHEB: Ras homolog, MTORC1 binding; RICTOR: RPTOR independent companion of MTOR complex 2; RIPK1: receptor interacting serine/threonine kinase 1; ROS: reactive oxygen species; RPTOR: regulatory associated protein of MTOR complex 1; RPS6KB1: ribosomal protein S6 kinase B1; SH3GLB1: SH3 domain containing GRB2 like, endophilin B1; SNP: single-nucleotide polymorphism; SQSTM1: sequestosome 1; STAT3: signal transducer and activator of transcription 3; STING1: stimulator of interferon response cGAMP interactor 1; TA: transit-amplifying; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TGM2: transglutaminase 2; TJ: tight junction; TJP1/ZO1: tight junction protein 1; TNBS: 2,4,6-trinitrobenzene sulfonic acid; TNF/TNFα: tumor necrosis factor; Tor: target of rapamycin; TRAF: TNF receptor associated factor; TRIM11: tripartite motif containing 11; TRP53: transformation related protein 53; TSC: TSC complex subunit; Ub: ubiquitin; UC: ulcerative colitis; ULK1: unc-51 like autophagy activating kinase 1; USO1/p115: USO1 vesicle transport factor; UVRAG: UV radiation resistance associated; WIPI: WD repeat domain, phosphoinositide interacting; WNT: WNT family member; XBP1: X-box binding protein 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

Keywords:  Autophagy; Crohn disease; IBD; MTOR; intestinal epithelium; intestinal stem cells

DOI:  https://doi.org/10.1080/15548627.2021.1909406
Int J Mol Sci. 2021 Apr 22. pii: 4363. [Epub ahead of print]22(9):

Common Principles and Specific Mechanisms of Mitophagy from Yeast to Humans.

Rajesh Kumar, Andreas S Reichert.

  Mitochondria are double membrane-bound organelles in eukaryotic cells essential to a variety of cellular functions including energy conversion and ATP production, iron-sulfur biogenesis, lipid and amino acid metabolism, and regulating apoptosis and stress responses. Mitochondrial dysfunction is mechanistically linked to several neurodegenerative diseases, cancer, and ageing. Excessive and dysfunctional/damaged mitochondria are degraded by selective autophagic pathways known as mitophagy. Both budding yeast and mammals use the well-conserved machinery of core autophagy-related genes (ATGs) to execute and regulate mitophagy. In mammalian cells, the PINK1-PARKIN mitophagy pathway is a well-studied pathway that senses dysfunctional mitochondria and marks them for degradation in the lysosome. PINK1-PARKIN mediated mitophagy relies on ubiquitin-binding mitophagy adaptors that are non-ATG proteins. Loss-of-function mutations in PINK1 and PARKIN are linked to Parkinson´s disease (PD) in humans, and defective mitophagy is proposed to be a main pathomechanism. Despite the common view that yeast cells lack PINK1- and PARKIN-homologs and that mitophagy in yeast is solely regulated by receptor-mediated mitophagy, some studies suggest that a ubiquitination-dependent mitophagy pathway also exists. Here, we will discuss shared mechanisms between mammals and yeast, how mitophagy in the latter is regulated in a ubiquitin-dependent and -independent manner, and why these pathways are essential for yeast cell survival and fitness under various physiological stress conditions.

Keywords:  PARKIN; PINK1; autophagy; cancer; mitophagy; quality control; ubiquitin

DOI:  https://doi.org/10.3390/ijms22094363
EMBO J. 2021 May 01. e103563

TFG binds LC3C to regulate ULK1 localization and autophagosome formation.

Marianna Carinci, Beatrice Testa, Matteo Bordi, Giacomo Milletti, Massimo Bonora, Laura Antonucci, Caterina Ferraina, Marta Carro, Mukesh Kumar, Donatella Ceglie, Franziska Eck, Roberta Nardacci, Francois le Guerroué, Stefania Petrini, Maria E Soriano, Ignazio Caruana, Valentina Doria, Maria Manifava, Camille Peron, Matteo Lambrughi, Valeria Tiranti, Christian Behrends, Elena Papaleo, Paolo Pinton, Carlotta Giorgi, Nicholas T Ktistakis, Franco Locatelli, Francesca Nazio, Francesco Cecconi.

  The early secretory pathway and autophagy are two essential and evolutionarily conserved endomembrane processes that are finely interlinked. Although growing evidence suggests that intracellular trafficking is important for autophagosome biogenesis, the molecular regulatory network involved is still not fully defined. In this study, we demonstrate a crucial effect of the COPII vesicle-related protein TFG (Trk-fused gene) on ULK1 puncta number and localization during autophagy induction. This, in turn, affects formation of the isolation membrane, as well as the correct dynamics of association between LC3B and early ATG proteins, leading to the proper formation of both omegasomes and autophagosomes. Consistently, fibroblasts derived from a hereditary spastic paraparesis (HSP) patient carrying mutated TFG (R106C) show defects in both autophagy and ULK1 puncta accumulation. In addition, we demonstrate that TFG activity in autophagy depends on its interaction with the ATG8 protein LC3C through a canonical LIR motif, thereby favouring LC3C-ULK1 binding. Altogether, our results uncover a link between TFG and autophagy and identify TFG as a molecular scaffold linking the early secretion pathway to autophagy.

Keywords:  ERGIC; LC3C; TFG; autophagy

DOI:  https://doi.org/10.15252/embj.2019103563
Dev Neurosci. 2021 Apr 28. 1-16

Current Approaches and Future Directions for the Treatment of mTORopathies.

Vasiliki Karalis, Helen S Bateup.

  The mechanistic target of rapamycin (mTOR) is a kinase at the center of an evolutionarily conserved signaling pathway that orchestrates cell growth and metabolism. mTOR responds to an array of intra- and extracellular stimuli and in turn controls multiple cellular anabolic and catabolic processes. Aberrant mTOR activity is associated with numerous diseases, with particularly profound impact on the nervous system. mTOR is found in two protein complexes, mTOR complex 1 (mTORC1) and 2 (mTORC2), which are governed by different upstream regulators and have distinct cellular actions. Mutations in genes encoding for mTOR regulators result in a collection of neurodevelopmental disorders known as mTORopathies. While these disorders can affect multiple organs, neuropsychiatric conditions such as epilepsy, intellectual disability, and autism spectrum disorder have a major impact on quality of life. The neuropsychiatric aspects of mTORopathies have been particularly challenging to treat in a clinical setting. Current therapeutic approaches center on rapamycin and its analogs, drugs that are administered systemically to inhibit mTOR activity. While these drugs show some clinical efficacy, adverse side effects, incomplete suppression of mTOR targets, and lack of specificity for mTORC1 or mTORC2 may limit their utility. An increased understanding of the neurobiology of mTOR and the underlying molecular, cellular, and circuit mechanisms of mTOR-related disorders will facilitate the development of improved therapeutics. Animal models of mTORopathies have helped unravel the consequences of mTOR pathway mutations in specific brain cell types and developmental stages, revealing an array of disease-related phenotypes. In this review, we discuss current progress and potential future directions for the therapeutic treatment of mTORopathies with a focus on findings from genetic mouse models.

Keywords:  Epilepsy; Neurodevelopmental disorders; PTEN; Rapamycin; Raptor; Rictor; Tuberous sclerosis complex; mTORC1; mTORC2; mTORopathy

DOI:  https://doi.org/10.1159/000515672
Antioxidants (Basel). 2021 Apr 28. pii: 694. [Epub ahead of print]10(5):

Intersection between Redox Homeostasis and Autophagy: Valuable Insights into Neurodegeneration.

Hyungsun Park, Jongyoon Kim, Chihoon Shin, Seongju Lee.

  Autophagy, a main degradation pathway for maintaining cellular homeostasis, and redox homeostasis have recently been considered to play protective roles in neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. Increased levels of reactive oxygen species (ROS) in neurons can induce mitochondrial damage and protein aggregation, thereby resulting in neurodegeneration. Oxidative stress is one of the major activation signals for the induction of autophagy. Upon activation, autophagy can remove ROS, damaged mitochondria, and aggregated proteins from the cells. Thus, autophagy can be an effective strategy to maintain redox homeostasis in the brain. However, the interaction between redox homeostasis and autophagy is not clearly elucidated. In this review, we discuss recent studies on the relationship between redox homeostasis and autophagy associated with neurodegenerative diseases and propose that autophagy induction through pharmacological intervention or genetic activation might be a promising strategy to treat these disorders.

Keywords:  ROS; autophagy; neurodegenerative diseases; redox homeostasis

DOI:  https://doi.org/10.3390/antiox10050694
Mol Oncol. 2021 May 01.

Platycodin D inhibits autophagy and increases glioblastoma cell death via LDLR upregulation.

Sol Ji Lee, Yu-Jeong Choi, Hyo In Kim, Hyo Eun Moon, Sun Ha Paek, Tai Young Kim, Seong-Gyu Ko.

  Targeting autophagy is a promising therapeutic approach in cancer therapy. Here, we screened 30 traditional herbal medicines to identify novel autophagy regulators, and found that Platycodon grandiflorus (PG) and platycodin D (PD), a triterpenoid saponin from PG, inhibited autophagy in glioblastoma multiforme (GBM) cells. Mechanistically, PD prevented lysosomal degradation and the fusion between autophagosomes and lysosomes by inducing sequestration of free cholesterol in lysosomes. The autophagy inhibitory effect of PD was mimicked by both genetic and pharmacological inhibition of Niemann-Pick C1 (NPC1), which exports low density lipoprotein (LDL)-derived cholesterol from lysosomes. Moreover, PD promoted the uptake of exogenous LDL cholesterol via upregulation of LDL receptor (LDLR), leading to further accumulation of cholesterol within lysosomes and GBM cell death. Importantly, these phenomena were more pronounced in LDLR-overexpressing GBM cells than in normal astrocytes. Finally, blockade of cholesterol uptake by LDLR knockdown reversed the PD-induced inhibition of autophagy and GBM cell growth. Our study proposes that PD could be a potent anti-GBM drug by disrupting cholesterol trafficking and autophagy.

Keywords:  GBM; LDLR; Platycodin D; autophagy; cholesterol; lysosome

DOI:  https://doi.org/10.1002/1878-0261.12966
Dev Cell. 2021 Apr 27. pii: S1534-5807(21)00317-8. [Epub ahead of print]

Coordinate regulation of the senescent state by selective autophagy.

Yeonghyeon Lee, Jaejin Kim, Mi-Sung Kim, Yoojin Kwon, Sanghee Shin, Hyerim Yi, Hyeonkyeong Kim, Moon Jong Chang, Chong Bum Chang, Seung-Baik Kang, V Narry Kim, Jin-Hong Kim, Jong-Seo Kim, Stephen J Elledge, Chanhee Kang.

  Cellular senescence is a complex stress response implicated in aging. Autophagy can suppress senescence but is counterintuitively necessary for full senescence. Although its anti-senescence role is well described, to what extent autophagy contributes to senescence establishment and the underlying mechanisms is poorly understood. Here, we show that selective autophagy of multiple regulatory components coordinates the homeostatic state of senescence. We combined a proteomic analysis of autophagy components with protein stability profiling, identifying autophagy substrate proteins involved in several senescence-related processes. Selective autophagy of KEAP1 promoted redox homeostasis during senescence. Furthermore, selective autophagy limited translational machinery components to ameliorate senescence-associated proteotoxic stress. Lastly, selective autophagy of TNIP1 enhanced senescence-associated inflammation. These selective autophagy networks appear to operate in vivo senescence during human osteoarthritis. Our data highlight a caretaker role of autophagy in the stress support network of senescence through regulated protein stability and unravel the intertwined relationship between two important age-related processes.

Keywords:  aging; autophagy; cellular senescence; inflammation; oxidative stress; proteostasis; regulated protein stability; selective autophagy; stress response

DOI:  https://doi.org/10.1016/j.devcel.2021.04.008
Med Sci (Paris). 2021 Apr;37(4): 372-378

[New structures of mTORC1: Focus on Rag GTPases].

Agata Nawrotek, Jacqueline Cherfils.

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane.

DOI: https://doi.org/10.1051/medsci/2021033
Front Cell Dev Biol. 2021 ;9 668735

Autophagic Organelles in DNA Damage Response.

Jeongha Kim, Sungmin Lee, Hyunwoo Kim, Haksoo Lee, Ki Moon Seong, HyeSook Youn, BuHyun Youn.

  Autophagy is an important subcellular event engaged in the maintenance of cellular homeostasis via the degradation of cargo proteins and malfunctioning organelles. In response to cellular stresses, like nutrient deprivation, infection, and DNA damaging agents, autophagy is activated to reduce the damage and restore cellular homeostasis. One of the responses to cellular stresses is the DNA damage response (DDR), the intracellular pathway that senses and repairs damaged DNA. Proper regulation of these pathways is crucial for preventing diseases. The involvement of autophagy in the repair and elimination of DNA aberrations is essential for cell survival and recovery to normal conditions, highlighting the importance of autophagy in the resolution of cell fate. In this review, we summarized the latest information about autophagic recycling of mitochondria, endoplasmic reticulum (ER), and ribosomes (called mitophagy, ER-phagy, and ribophagy, respectively) in response to DNA damage. In addition, we have described the key events necessary for a comprehensive understanding of autophagy signaling networks. Finally, we have highlighted the importance of the autophagy activated by DDR and appropriate regulation of autophagic organelles, suggesting insights for future studies. Especially, DDR from DNA damaging agents including ionizing radiation (IR) or anti-cancer drugs, induces damage to subcellular organelles and autophagy is the key mechanism for removing impaired organelles.

Keywords:  DNA damage response; ER-phagy; mitophagy; ribophagy; therapeutic approach

DOI:  https://doi.org/10.3389/fcell.2021.668735
Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00345-4. [Epub ahead of print]35(4): 109031

Leucine-sensing mechanism of leucyl-tRNA synthetase 1 for mTORC1 activation.

Sulhee Kim, Ina Yoon, Jonghyeon Son, Junga Park, Kibum Kim, Ji-Ho Lee, Sam-Yong Park, Beom Sik Kang, Jung Min Han, Kwang Yeon Hwang, Sunghoon Kim.

  Leucyl-tRNA synthetase 1 (LARS1) mediates activation of leucine-dependent mechanistic target of rapamycin complex 1 (mTORC1) as well as ligation of leucine to its cognate tRNAs, yet its mechanism of leucine sensing is poorly understood. Here we describe leucine binding-induced conformational changes of LARS1. We determine different crystal structures of LARS1 complexed with leucine, ATP, and a reaction intermediate analog, leucyl-sulfamoyl-adenylate (Leu-AMS), and find two distinct functional states of LARS1 for mTORC1 activation. Upon leucine binding to the synthetic site, H251 and R517 in the connective polypeptide and 50FPYPY54 in the catalytic domain change the hydrogen bond network, leading to conformational change in the C-terminal domain, correlating with RagD association. Leucine binding to LARS1 is increased in the presence of ATP, further augmenting leucine-dependent interaction of LARS1 and RagD. Thus, this work unveils the structural basis for leucine-dependent long-range communication between the catalytic and RagD-binding domains of LARS1 for mTORC1 activation.

Keywords:  X-ray crystallography; conformational change; leucine sensing; leucyl-tRNA synthetase 1; mechanistic target of rapamycin complex 1

DOI:  https://doi.org/10.1016/j.celrep.2021.109031
Int J Mol Sci. 2021 Apr 19. pii: 4220. [Epub ahead of print]22(8):

Genistein Activates Transcription Factor EB and Corrects Niemann-Pick C Phenotype.

Graciela Argüello, Elisa Balboa, Pablo J Tapia, Juan Castro, María José Yañez, Pamela Mattar, Rodrigo Pulgar, Silvana Zanlungo.

  Niemann-Pick type C disease (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.

Keywords:  Niemann–Pick C; TFEB; cholesterol; genistein; lysosomal storage diseases; lysosome clearance

DOI:  https://doi.org/10.3390/ijms22084220
Front Neuroanat. 2021 ;15 664695

Convergent and Divergent Mechanisms of Epileptogenesis in mTORopathies.

Lena H Nguyen, Angélique Bordey.

  Hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1) due to mutations in genes along the PI3K-mTOR pathway and the GATOR1 complex causes a spectrum of neurodevelopmental disorders (termed mTORopathies) associated with malformation of cortical development and intractable epilepsy. Despite these gene variants' converging impact on mTORC1 activity, emerging findings suggest that these variants contribute to epilepsy through both mTORC1-dependent and -independent mechanisms. Here, we review the literature on in utero electroporation-based animal models of mTORopathies, which recapitulate the brain mosaic pattern of mTORC1 hyperactivity, and compare the effects of distinct PI3K-mTOR pathway and GATOR1 complex gene variants on cortical development and epilepsy. We report the outcomes on cortical pyramidal neuronal placement, morphology, and electrophysiological phenotypes, and discuss some of the converging and diverging mechanisms responsible for these alterations and their contribution to epileptogenesis. We also discuss potential therapeutic strategies for epilepsy, beyond mTORC1 inhibition with rapamycin or everolimus, that could offer personalized medicine based on the gene variant.

Keywords:  GATOR1 complex; cortical development; epilepsy; focal cortical dysplasia; in utero electroporation; mTOR; neuron migration; tuberous sclerosis complex

DOI:  https://doi.org/10.3389/fnana.2021.664695
Cell Prolif. 2021 May 01. e13034

PINK1-mediated mitophagy maintains pluripotency through optineurin.

Chaoqun Wang, Kun Liu, Jiani Cao, Liang Wang, Qian Zhao, Zheng Li, Honghai Zhang, Quan Chen, Tongbiao Zhao.

   OBJECTIVES: Dysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self-renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.
MATERIALS AND METHODS: Parkin+/+ and parkin-/- ESCs were established from E3.5 blastocysts of parkin+/- x parkin+/- mating mice. The pink1-/- , optn-/- and ndp52-/- ESCs were generated by CRISPR-Cas9. shRNAs were used for function loss assay of target genes. Mito-Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q-PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.
RESULTS: PINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1-/- ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub-S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub-binding dead OPTN mutant (D477N).
CONCLUSIONS: The mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1-activated OPTN binding of PINK1-phosphorylated Ubiquitin.

Keywords:  OPTN; PINK1; embryonic stem cells; mitochondria; mitophagy; reprogramming

DOI:  https://doi.org/10.1111/cpr.13034
Biomedicines. 2021 Apr 21. pii: 446. [Epub ahead of print]9(5):

Murine Models of Lysosomal Storage Diseases Exhibit Differences in Brain Protein Aggregation and Neuroinflammation.

Jennifer Clarke, Can Kayatekin, Catherine Viel, Lamya Shihabuddin, Sergio Pablo Sardi.

  Genetic, epidemiological and experimental evidence implicate lysosomal dysfunction in Parkinson's disease (PD) and related synucleinopathies. Investigate several mouse models of lysosomal storage diseases (LSDs) and evaluate pathologies reminiscent of synucleinopathies. We obtained brain tissue from symptomatic mouse models of Gaucher, Fabry, Sandhoff, Niemann-Pick A (NPA), Hurler, Pompe and Niemann-Pick C (NPC) diseases and assessed for the presence of Lewy body-like pathology (proteinase K-resistant α-synuclein and tau aggregates) and neuroinflammation (microglial Iba1 and astrocytic GFAP) by immunofluorescence. All seven LSD models exhibited evidence of proteinopathy and/or inflammation in the central nervous system (CNS). However, these phenotypes were divergent. Gaucher and Fabry mouse models displayed proteinase K-resistant α-synuclein and tau aggregates but no neuroinflammation; whereas Sandhoff, NPA and NPC showed marked neuroinflammation and no overt proteinopathy. Pompe disease animals uniquely displayed widespread distribution of tau aggregates accompanied by moderate microglial activation. Hurler mice also demonstrated proteinopathy and microglial activation. The present study demonstrated additional links between LSDs and pathogenic phenotypes that are hallmarks of synucleinopathies. The data suggest that lysosomal dysregulation can contribute to brain region-specific protein aggregation and induce widespread neuroinflammation in the brain. However, only a few LSD models examined exhibited phenotypes consistent with synucleinopathies. While no model can recapitulate the complexity of PD, they can enable the study of specific pathways and mechanisms contributing to disease pathophysiology. The present study provides evidence that there are existing, previously unutilized mouse models that can be employed to study pathogenic mechanisms and gain insights into potential PD subtypes, helping to determine if they are amenable to pathway-specific therapeutic interventions.

Keywords:  lysosomal diseases; mouse models of disease; neuroinflammation; synuclein; tau

DOI:  https://doi.org/10.3390/biomedicines9050446
Curr Opin Cell Biol. 2021 Apr 27. pii: S0955-0674(21)00022-3. [Epub ahead of print]71 112-119

Membrane supply and remodeling during autophagosome biogenesis.

Rubén Gómez-Sánchez, Sharon A Tooze, Fulvio Reggiori.

  The de novo generation of double-membrane autophagosomes is the hallmark of autophagy. The initial membranous precursor cisterna, the phagophore, is very likely generated by the fusion of vesicles and acts as a membrane seed for the subsequent expansion into an autophagosome. This latter step requires a massive convoy of lipids into the phagophore. In this review, we present recent advances in our understanding of the intracellular membrane sources and lipid delivery mechanisms, which principally rely on vesicular transport and membrane contact sites that contribute to autophagosome biogenesis. In this context, we discuss lipid biosynthesis and lipid remodeling events that play a crucial role in both phagophore nucleation and expansion.

Keywords:  ATG proteins; Autophagy; Isolation membrane; Lipid source; Lipids; Membrane contact site; Membrane origin; Omegasome; Phagophore; Vesicular transport

DOI:  https://doi.org/10.1016/j.ceb.2021.02.001
Front Cell Dev Biol. 2021 ;9 656370

Autophagic Cell Death During Development - Ancient and Mysterious.

Lawrence M Schwartz.

  While cell death is a normal and essential component of development and homeostasis, dysregulation of this process underlies most human diseases, including cancer, autoimmunity and neurodegeneration. The best characterized mechanism for cell death is apoptosis, although some cells die by a distinct process known as autophagy-dependent cell death (ADCD). Autophagy is mediated by the formation of double membrane vesicles that contain protein aggregates, damaged organelles like mitochondria, and bulk cytoplasm, which then fuse with lysosomes to degrade and recycle their contents. Autophagy is typically viewed as an adaptive process that allows cells to survive stresses like nutrient deprivation, although increasing evidence suggests that it may also mediate cell death during development and pathogenesis. An aggressive form of autophagy termed autosis has been described in cells following either ischemia/reperfusion injury or in response to autophagy-inducing proteins like Tat-Beclin 1. Despite an extensive literature on autophagic cell death in a variety of contexts, there are still fundamental gaps in our understanding of this process. As examples: Does autophagy directly kill cells and if so how? Is ADCD activated concurrently when cells are triggered to die via apoptosis? And is ADCD essentially a more protracted version of autosis or a distinct pathway? The goal of this mini-review is to summarize the field and to identify some of the major gaps in our knowledge. Understanding the molecular mechanisms that mediate ADCD will not only provide new insights into development, they may facilitate the creation of better tools for both the diagnostics and treatment of disease.

Keywords:  Tat-Beclin 1; apoptosis; autophagy; autosis; lysosome; necrosis; programmed cell death

DOI:  https://doi.org/10.3389/fcell.2021.656370
Cells. 2021 Apr 13. pii: 885. [Epub ahead of print]10(4):

Changes in Drp1 Function and Mitochondrial Morphology Are Associated with the α-Synuclein Pathology in a Transgenic Mouse Model of Parkinson's Disease.

Philipp Portz, Michael K Lee.

  Alterations in mitochondrial function and morphology are associated with many human diseases, including cancer and neurodegenerative diseases. Mitochondrial impairment is linked to Parkinson's disease (PD) pathogenesis, and alterations in mitochondrial dynamics are seen in PD models. In particular, α-synuclein (αS) abnormalities are often associated with pathological changes to mitochondria. However, the relationship between αS pathology and mitochondrial dynamics remains poorly defined. Herein, we examined a mouse model of α-synucleinopathy for αS pathology-linked alterations in mitochondrial dynamics in vivo. We show that α-synucleinopathy in a transgenic (Tg) mouse model expressing familial PD-linked mutant A53T human αS (TgA53T) is associated with a decrease in Drp1 localization and activity in the mitochondria. In addition, we show that the loss of Drp1 function in the mitochondria is associated with two distinct phenotypes of enlarged neuronal mitochondria. Mitochondrial enlargement was only present in diseased animals and, apart from Drp1, other proteins involved in mitochondrial dynamics are unlikely to cause these changes, as their levels remained mostly unchanged. Further, the levels of Mfn1, a protein that facilitates mitochondrial fusion, was decreased nonspecifically with transgene expression. These results support the view that altered mitochondrial dynamics are a significant neuropathological factor in α-synucleinopathies.

Keywords:  Drp1; Parkinson; alpha-synuclein; fission; fusion; mitochondria; mitochondrial dynamics; mitophagy

DOI:  https://doi.org/10.3390/cells10040885
Nat Cell Biol. 2021 Apr 26.

Autophagosome biogenesis comes out of the black box.

Chunmei Chang, Liv E Jensen, James H Hurley.

Macroautophagic clearance of cytosolic materials entails the initiation, growth and closure of autophagosomes. Cargo triggers the assembly of a web of cargo receptors and core machinery. Autophagy-related protein 9 (ATG9) vesicles seed the growing autophagosomal membrane, which is supplied by de novo phospholipid synthesis, phospholipid transport via ATG2 proteins and lipid flipping by ATG9. Autophagosomes close via ESCRT complexes. Here, we review recent discoveries that illuminate the molecular mechanisms of autophagosome formation and discuss emerging questions in this rapidly developing field.

DOI: https://doi.org/10.1038/s41556-021-00669-y
Cells. 2021 Apr 17. pii: 929. [Epub ahead of print]10(4):

S6K1 Is Indispensible for Stress-Induced Microtubule Acetylation and Autophagic Flux.

Aleksandra Hać, Karolina Pierzynowska, Anna Herman-Antosiewicz.

  Autophagy is a specific macromolecule and organelle degradation process. The target macromolecule or organelle is first enclosed in an autophagosome, and then delivered along acetylated microtubules to the lysosome. Autophagy is triggered by stress and largely contributes to cell survival. We have previously shown that S6K1 kinase is essential for autophagic flux under stress conditions. Here, we aimed to elucidate the underlying mechanism of S6K1 involvement in autophagy. We stimulated autophagy in S6K1/2 double-knockout mouse embryonic fibroblasts by exposing them to different stress conditions. Transient gene overexpression or silencing, immunoblotting, immunofluorescence, flow cytometry, and ratiometric fluorescence analyses revealed that the perturbation of autophagic flux in S6K1-deficient cells did not stem from impaired lysosomal function. Instead, the absence of S6K1 abolished stress-induced tubulin acetylation and disrupted the acetylated microtubule network, in turn impairing the autophagosome-lysosome fusion. S6K1 overexpression restored tubulin acetylation and autophagic flux in stressed S6K1/2-deficient cells. Similar effect of S6K1 status was observed in prostate cancer cells. Furthermore, overexpression of an acetylation-mimicking, but not acetylation-resistant, tubulin variant effectively restored autophagic flux in stressed S6K1/2-deficient cells. Collectively, S6K1 controls tubulin acetylation, hence contributing to the autophagic flux induced by different stress conditions and in different cells.

Keywords:  S6 kinase 1 (S6K1); autophagic flux; autophagosome-lysosome fusion; lysosome; serum deprivation; sulforaphane; tubulin acetylation

DOI:  https://doi.org/10.3390/cells10040929
Mol Cell. 2021 Apr 16. pii: S1097-2765(21)00215-X. [Epub ahead of print]

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine.

Joanne Durgan, Alf H Lystad, Katherine Sloan, Sven R Carlsson, Michael I Wilson, Elena Marcassa, Rachel Ulferts, Judith Webster, Andrea F Lopez-Clavijo, Michael J Wakelam, Rupert Beale, Anne Simonsen, David Oxley, Oliver Florey.

  Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.

Keywords:  ATG4; ATG8; LC3-associated phagocytosis; non-canonical autophagy; phosphatidylserine

DOI:  https://doi.org/10.1016/j.molcel.2021.03.020
J Cell Sci. 2021 May 01. pii: jcs258670. [Epub ahead of print]134(9):

Cell scientist to watch - Liang Ge.

Liang Ge pursued his PhD in the lab of Dr Bao-Liang Song at the Institutes of Biochemistry and Cell Biology, Chinese Academy of Sciences, where he studied the molecular mechanisms of cholesterol absorption. In 2011 he moved to California for a postdoc and later a research specialist position with Randy Schekman at the University of California, Berkeley. There, he discovered key roles for LC3 lipidation and the ER-Golgi intermediate compartment in autophagosome biogenesis. Liang established his group in the School of Life Sciences at Tsinghua University at the end of 2017, where he combines cell biology and biochemistry techniques, mouse models and computational biology to study the mechanisms of autophagy and unconventional protein secretion.

DOI: https://doi.org/10.1242/jcs.258670
Mol Aspects Med. 2021 Apr 27. pii: S0098-2997(21)00026-1. [Epub ahead of print] 100966

Moments in autophagy and disease: Past and present.

Xin Wen, Ying Yang, Daniel J Klionsky.

  Over the past several decades, research on autophagy, a highly conserved lysosomal degradation pathway, has been advanced by studies in different model organisms, especially in the field of its molecular mechanism and regulation. The malfunction of autophagy is linked to various diseases, among which cancer and neurodegenerative diseases are the major focus. In this review, we cover some other important diseases, including cardiovascular diseases, infectious and inflammatory diseases, and metabolic disorders, as well as rare diseases, with a hope of providing a more complete understanding of the spectrum of autophagy's role in human health.

Keywords:  Autophagy; COVID-19; Cardiovascular diseases; Metabolic disorders; Rare diseases

DOI:  https://doi.org/10.1016/j.mam.2021.100966
Int J Mol Sci. 2021 Apr 13. pii: 3995. [Epub ahead of print]22(8):

Marliolide Derivative Induces Melanosome Degradation via Nrf2/p62-Mediated Autophagy.

Cheong-Yong Yun, Nahyun Choi, Jae Un Lee, Eun Jung Lee, Ji Young Kim, Won Jun Choi, Sang Ho Oh, Jong-Hyuk Sung.

  Nuclear factor erythroid 2-related factor 2 (Nrf2), which is linked to autophagy regulation and melanogenesis regulation, is activated by marliolide. In this study, we investigated the effect of a marliolide derivative on melanosome degradation through the autophagy pathway. The effect of the marliolide derivative on melanosome degradation was investigated in α-melanocyte stimulating hormone (α-MSH)-treated melanocytes, melanosome-incorporated keratinocyte, and ultraviolet (UV)B-exposed HRM-2 mice (melanin-possessing hairless mice). The marliolide derivative, 5-methyl-3-tetradecylidene-dihydro-furan-2-one (DMF02), decreased melanin pigmentation by melanosome degradation in α-MSH-treated melanocytes and melanosome-incorporated keratinocytes, evidenced by premelanosome protein (PMEL) expression, but did not affect melanogenesis-associated proteins. The UVB-induced hyperpigmentation in HRM-2 mice was also reduced by a topical application of DMF02. DMF02 activated Nrf2 and induced autophagy in vivo, evidenced by decreased PMEL in microtubule-associated proteins 1A/1B light chain 3B (LC3)-II-expressed areas. DMF02 also induced melanosome degradation via autophagy in vitro, and DMF02-induced melanosome degradation was recovered by chloroquine (CQ), which is a lysosomal inhibitor. In addition, Nrf2 silencing by siRNA attenuated the DMF02-induced melanosome degradation via the suppression of p62. DMF02 induced melanosome degradation in melanocytes and keratinocytes by regulating autophagy via Nrf2-p62 activation. Therefore, Nrf2 activator could be a promising therapeutic agent for reducing hyperpigmentation.

Keywords:  Nrf2; autophagy; melanosome degradation; p62

DOI:  https://doi.org/10.3390/ijms22083995
Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00350-8. [Epub ahead of print]35(4): 109034

Post-Golgi carriers, not lysosomes, confer lysosomal properties to pre-degradative organelles in normal and dystrophic axons.

Pearl P Y Lie, Dun-Sheng Yang, Philip Stavrides, Chris N Goulbourne, Ping Zheng, Panaiyur S Mohan, Anne M Cataldo, Ralph A Nixon.

  Lysosomal trafficking and maturation in neurons remain poorly understood and are unstudied in vivo despite high disease relevance. We generated neuron-specific transgenic mice to track vesicular CTSD acquisition, acidification, and traffic within the autophagic-lysosomal pathway in vivo, revealing that mature lysosomes are restricted from axons. Moreover, TGN-derived transport carriers (TCs), not lysosomes, supply lysosomal components to axonal organelles. Ultrastructurally distinctive TCs containing TGN and lysosomal markers enter axons, engaging autophagic vacuoles and late endosomes. This process is markedly upregulated in dystrophic axons of Alzheimer models. In cultured neurons, most axonal LAMP1 vesicles are weakly acidic TCs that shuttle lysosomal components bidirectionally, conferring limited degradative capability to retrograde organelles before they mature fully to lysosomes within perikarya. The minor LAMP1 subpopulation attaining robust acidification are retrograde Rab7+ endosomes/amphisomes, not lysosomes. Restricted lysosome entry into axons explains the unique lysosome distribution in neurons and their vulnerability toward neuritic dystrophy in disease.

Keywords:  LAMP1; acidification; autophagic vacuole; axonal transport; late endosome; lysosome; post-Golgi traffic; transport carrier

DOI:  https://doi.org/10.1016/j.celrep.2021.109034
Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00352-1. [Epub ahead of print]35(4): 109036

4E-BP2-dependent translation in cerebellar Purkinje cells controls spatial memory but not autism-like behaviors.

Mehdi Hooshmandi, Vinh Tai Truong, Eviatar Fields, Riya Elizabeth Thomas, Calvin Wong, Vijendra Sharma, Ilse Gantois, Patricia Soriano Roque, Kleanthi Chalkiadaki, Neil Wu, Anindyo Chakraborty, Soroush Tahmasebi, Masha Prager-Khoutorsky, Nahum Sonenberg, Aparna Suvrathan, Alanna J Watt, Christos G Gkogkas, Arkady Khoutorsky.

  Recent studies have demonstrated that selective activation of mammalian target of rapamycin complex 1 (mTORC1) in the cerebellum by deletion of the mTORC1 upstream repressors TSC1 or phosphatase and tensin homolog (PTEN) in Purkinje cells (PCs) causes autism-like features and cognitive deficits. However, the molecular mechanisms by which overactivated mTORC1 in the cerebellum engenders these behaviors remain unknown. The eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2) is a central translational repressor downstream of mTORC1. Here, we show that mice with selective ablation of 4E-BP2 in PCs display a reduced number of PCs, increased regularity of PC action potential firing, and deficits in motor learning. Surprisingly, although spatial memory is impaired in these mice, they exhibit normal social interaction and show no deficits in repetitive behavior. Our data suggest that, downstream of mTORC1/4E-BP2, there are distinct cerebellar mechanisms independently controlling social behavior and memory formation.

Keywords:  4E-BP2; Purkinje cells; autism spectrum disorders; motor learning; spatial memory

DOI:  https://doi.org/10.1016/j.celrep.2021.109036
Aging (Albany NY). 2021 Apr 26. 13

Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan.

Tamara Jiménez-Saucedo, Juan José Berlanga, Miguel Rodríguez-Gabriel.

  Although the stress response in eukaryotes depends on early events triggered in cells by environmental insults, long-term processes such as aging are also affected. The loss of cellular proteostasis greatly impacts aging, which is regulated by the balancing of protein synthesis and degradation systems. As translation is the input event in proteostasis, we decided to study the role of translational activity on cell lifespan. Our hypothesis was that a reduction on translational activity or specific changes in translation may increase cellular longevity. Using mutant strains of Schizosaccharomyces pombe and various stress conditions, we showed that translational reduction caused by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) during the exponential growth phase enhances chronological lifespan (CLS). Furthermore, through next-generation sequence analysis, we found eIF2α phosphorylation-dependent translational activation of some specific genes, especially those involved in autophagy. This fact, together with the observed regulation of autophagy, points to a conserved mechanism involving general and specific control of translation and autophagy as mediators of the role of eIF2α phosphorylation in aging.

Keywords:  autophagy; eIF2 factor; gene expression; longevity; translational control

DOI:  https://doi.org/10.18632/aging.203018
Cancers (Basel). 2021 Apr 21. pii: 2004. [Epub ahead of print]13(9):

Quinacrine-Induced Autophagy in Ovarian Cancer Triggers Cathepsin-L Mediated Lysosomal/Mitochondrial Membrane Permeabilization and Cell Death.

Prabhu Thirusangu, Christopher L Pathoulas, Upasana Ray, Yinan Xiao, Julie Staub, Ling Jin, Ashwani Khurana, Viji Shridhar.

  We previously reported that the antimalarial compound quinacrine (QC) induces autophagy in ovarian cancer cells. In the current study, we uncovered that QC significantly upregulates cathepsin L (CTSL) but not cathepsin B and D levels, implicating the specific role of CTSL in promoting QC-induced autophagic flux and apoptotic cell death in OC cells. Using a Magic Red® cathepsin L activity assay and LysoTracker red, we discerned that QC-induced CTSL activation promotes lysosomal membrane permeability (LMP) resulting in the release of active CTSL into the cytosol to promote apoptotic cell death. We found that QC-induced LMP and CTSL activation promotes Bid cleavage, mitochondrial outer membrane permeabilization (MOMP), and mitochondrial cytochrome-c release. Genetic (shRNA) and pharmacological (Z-FY(tBU)-DMK) inhibition of CTSL markedly reduces QC-induced autophagy, LMP, MOMP, apoptosis, and cell death; whereas induced overexpression of CTSL in ovarian cancer cell lines has an opposite effect. Using recombinant CTSL, we identified p62/SQSTM1 as a novel substrate of CTSL, suggesting that CTSL promotes QC-induced autophagic flux. CTSL activation is specific to QC-induced autophagy since no CTSL activation is seen in ATG5 knockout cells or with the anti-malarial autophagy-inhibiting drug chloroquine. Importantly, we showed that upregulation of CTSL in QC-treated HeyA8MDR xenografts corresponds with attenuation of p62, upregulation of LC3BII, cytochrome-c, tBid, cleaved PARP, and caspase3. Taken together, the data suggest that QC-induced autophagy and CTSL upregulation promote a positive feedback loop leading to excessive autophagic flux, LMP, and MOMP to promote QC-induced cell death in ovarian cancer cells.

Keywords:  CTSL; LMP; MOMP; autophagy; ovarian cancer; quinacrine

DOI:  https://doi.org/10.3390/cancers13092004
Cell Rep. 2021 Apr 27. pii: S2211-1247(21)00361-2. [Epub ahead of print]35(4): 109045

The autophagy protein ATG16L1 cooperates with IFT20 and INPP5E to regulate the turnover of phosphoinositides at the primary cilium.

Asma Boukhalfa, Federica Roccio, Nicolas Dupont, Patrice Codogno, Etienne Morel.

  The primary cilium (PC) regulates signalization linked to external stress sensing. Previous works established a functional interplay between the PC and the autophagic machinery. When ciliogenesis is promoted by serum deprivation, the autophagy protein ATG16L1 and the ciliary protein IFT20 are co-transported to the PC. Here, we demonstrate that IFT20 and ATG16L1 are part of the same complex requiring the WD40 domain of ATG16L1 and a Y-E-F-I motif in IFT20. We show that ATG16L1-deficient cells exhibit aberrant ciliary structures, which accumulate PI4,5P2, whereas PI4P, a lipid normally concentrated in the PC, is absent. Finally, we demonstrate that INPP5E, a phosphoinositide-associated phosphatase responsible for PI4P generation, interacts with ATG16L1 and that a perturbation of the ATG16L1/IFT20 complex alters its trafficking to the PC. Altogether, our results reveal a function of ATG16L1 in ciliary lipid and protein trafficking, thus directly contributing to proper PC dynamics and functions.

Keywords:  ATG; IFT; INPP5E; PI4P; macroautophagy; phosphoinositides; primary cilium; trafficking

DOI:  https://doi.org/10.1016/j.celrep.2021.109045
Autophagy. 2021 Apr 27. 1-14

The influence of circular RNAs on autophagy and disease progression.

Yian Wang, Yongzhen Mo, Miao Peng, Shanshan Zhang, Zhaojian Gong, Qijia Yan, Yanyan Tang, Yi He, Qianjin Liao, Xiayu Li, Xu Wu, Bo Xiang, Ming Zhou, Yong Li, Guiyuan Li, Xiaoling Li, Zhaoyang Zeng, Can Guo, Wei Xiong.

  Circular RNAs (circRNAs) are non-coding RNAs that have attracted considerable attention in recent years. Owing to their distinct circular structure, circRNAs are stable in cells. Autophagy is a catabolic process that helps in the degradation and recycling of harmful or inessential biological macromolecules in cells and enables cells to adapt to stress and changes in the internal and external environments. Evidence has shown that circRNAs influence the course of a disease by regulating autophagy, which indicates that autophagy is involved in the onset and development of various diseases and can affect drug resistance (for example, it affects cisplatin resistance in tumors). In this review, we summarized the role of circRNAs in autophagy and their influence on disease onset and progression as well as drug resistance. The review will expand our understanding of tumors as well as cardiovascular and neurological diseases and also suggest novel therapeutic strategies.Abbreviations: ACR: autophagy-related circRNA; ADSCs: adipogenic mesenchymal stem cells; AMPK: AMP-activated protein kinase; ATG: autophagy related; BCL2: BCL2 apoptosis regulator; BECN1: beclin 1; ceRNA: competing endogenous RNA; circRNA: circular RNA; CMA: chaperone-mediated autophagy; EPCs: endothelial progenitor cells; LE/MVBs: late endosomes/multivesicular bodies; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSCLC: non-small cell lung cancer; PDLSCs: periodontal ligament stem cells; PE: phosphatidylethanolamine; PtdIns: phosphatidylinositol; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate 1,2-dipalmitoyl; PTEN: phosphatase and tensin homolog; RBPs: RNA-binding proteins; SiO2: silicon dioxide; TFEB: transcription factor EB; ULK: unc-51 like autophagy activating kinase 1.

Keywords:  Autophagy; cancer; cardiovascular disease; circRNAs; neurological disease

DOI:  https://doi.org/10.1080/15548627.2021.1917131
Int J Mol Sci. 2021 Apr 13. pii: 3983. [Epub ahead of print]22(8):

An Experimentally Induced Mutation in the UBA Domain of p62 Changes the Sensitivity of Cisplatin by Up-Regulating HK2 Localisation on the Mitochondria and Increasing Mitophagy in A2780 Ovarian Cancer Cells.

Sihang Yu, Xiaoyu Yan, Rui Tian, Long Xu, Yuanxin Zhao, Liankun Sun, Jing Su.

  The study of cisplatin sensitivity is the key to the development of ovarian cancer treatment strategies. Mitochondria are one of the main targets of cisplatin, its self-clearing ability plays an important role in determining the fate of ovarian cancer cells. First, we proved that the sensitivity of ovarian cancer cells to cisplatin depends on mitophagy, and p62 acts as a broad autophagy receptor to regulate this process. However, p62's regulation of mitophagy does not depend on its location on the mitochondria. Our research shows that the mutation of the UBA domain of p62 increases the localisation of HK2 on the mitochondria, thereby increasing the phosphorylated ubiquitin form of parkin, then stabilising the process of mitophagy and ultimately cell survival. Collectively, our results showed that a mutation in the UBA domain of p62 regulates the level of apoptosis stimulated by cisplatin in ovarian cancer.

Keywords:  UBA; apoptosis; cisplatin; mitophagy; ovarian cancer; p62

DOI:  https://doi.org/10.3390/ijms22083983
Cells. 2021 Apr 15. pii: 909. [Epub ahead of print]10(4):

Antiretroviral Drugs Impact Autophagy with Toxic Outcomes.

Laura Cheney, John M Barbaro, Joan W Berman.

  Antiretroviral drugs have dramatically improved the morbidity and mortality of people living with HIV (PLWH). While current antiretroviral therapy (ART) regimens are generally well-tolerated, risks for side effects and toxicity remain as PLWH must take life-long medications. Antiretroviral drugs impact autophagy, an intracellular proteolytic process that eliminates debris and foreign material, provides nutrients for metabolism, and performs quality control to maintain cell homeostasis. Toxicity and adverse events associated with antiretrovirals may be due, in part, to their impacts on autophagy. A more complete understanding of the effects on autophagy is essential for developing antiretroviral drugs with decreased off target effects, meaning those unrelated to viral suppression, to minimize toxicity for PLWH. This review summarizes the findings and highlights the gaps in our knowledge of the impacts of antiretroviral drugs on autophagy.

Keywords:  ER stress; HIV; antiretroviral drugs; autophagy; mitochondria; mitophagy; side effects; toxicity

DOI:  https://doi.org/10.3390/cells10040909
Int J Mol Sci. 2021 Apr 16. pii: 4116. [Epub ahead of print]22(8):

The Characterization of a Subependymal Giant Astrocytoma-Like Cell Line from Murine Astrocyte with mTORC1 Hyperactivation.

Xin Tang, Gabrielle Angst, Michael Haas, Fuchun Yang, Chenran Wang.

  Tuberous sclerosis complex (TSC) is a genetic disorder caused by inactivating mutations in TSC1 (hamartin) or TSC2 (tuberin), crucial negative regulators of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. TSC affects multiple organs including the brain. The neurologic manifestation is characterized by cortical tubers, subependymal nodules (SEN), and subependymal giant cell astrocytoma (SEGA) in brain. SEGAs may result in hydrocephalus in TSC patients and mTORC1 inhibitors are the current recommended therapy for SEGA. Nevertheless, a major limitation in the research for SEGA is the lack of cell lines or animal models for mechanistic investigations and development of novel therapy. In this study, we generated TSC1-deficient neural cells from spontaneously immortalized mouse astrocytes in an attempt to mimic human SEGA. The TSC1-deficient cells exhibit mTORC1 hyperactivation and characteristics of transition from astrocytes to neural stem/progenitor cell phenotypes. Rapamycin efficiently decreased mTORC1 activity of these TSC1-deficient cells in vitro. In vivo, TSC1-deficient cells could form SEGA-like tumors and Rapamycin treatment decreased tumor growth. Collectively, our study generates a novel SEGA-like cell line that is invaluable for studying mTORC1-driven molecular and pathological alterations in neurologic tissue. These SEGA-like cells also provide opportunities for the development of novel therapeutic strategy for TSC patients with SEGA.

Keywords:  SEGA-like tumorigenesis; TSC1; astrocyte; mTORC1; rapamycin

DOI:  https://doi.org/10.3390/ijms22084116
Cells. 2021 Apr 25. pii: 1016. [Epub ahead of print]10(5):

Integrated Action of Autophagy and Adipose Tissue Triglyceride Lipase Ameliorates Diet-Induced Hepatic Steatosis in Liver-Specific PLIN2 Knockout Mice.

John D Griffin, Eloy Bejarano, Xiang-Dong Wang, Andrew S Greenberg.

  An imbalance in the storage and breakdown of hepatic lipid droplet (LD) triglyceride (TAG) leads to hepatic steatosis, a defining feature of non-alcoholic fatty liver disease (NAFLD). The two primary cellular pathways regulating hepatic TAG catabolism are lipolysis, initiated by adipose triglyceride lipase (ATGL), and lipophagy. Each of these processes requires access to the LD surface to initiate LD TAG catabolism. Ablation of perilipin 2 (PLIN2), the most abundant lipid droplet-associated protein in steatotic liver, protects mice from diet-induced NAFLD. However, the mechanisms underlaying this protection are unclear. We tested the contributions of ATGL and lipophagy mediated lipolysis to reduced hepatic TAG in mice with liver-specific PLIN2 deficiency (PLIN2LKO) fed a Western-type diet for 12 weeks. We observed enhanced autophagy in the absence of PLIN2, as determined by ex vivo p62 flux, as well as increased p62- and LC3-positive autophagic vesicles in PLIN2LKO livers and isolated primary hepatocytes. Increased levels of autophagy correlated with significant increases in cellular fatty acid (FA) oxidation in PLIN2LKO hepatocytes. We observed that inhibition of either autophagy or ATGL blunted the increased FA oxidation in PLIN2LKO hepatocytes. Additionally, combined inhibition of ATGL and autophagy reduced FA oxidation to the same extent as treatment with either inhibitor alone. In sum, these studies show that protection against NAFLD in the absence of hepatic PLIN2 is driven by the integrated actions of both ATGL and lipophagy.

Keywords:  adipose tissue triglyceride lipase; autophagy; lipid droplet; non-alcoholic fatty liver disease; perilipin

DOI:  https://doi.org/10.3390/cells10051016
Int J Mol Sci. 2021 Apr 15. pii: 4067. [Epub ahead of print]22(8):

Pharmacological Modulators of Autophagy as a Potential Strategy for the Treatment of COVID-19.

Gustavo José da Silva Pereira, Anderson Henrique França Figueredo Leão, Adolfo Garcia Erustes, Ingrid Beatriz de Melo Morais, Talita Aparecida de Moraes Vrechi, Lucas Dos Santos Zamarioli, Cássia Arruda Souza Pereira, Laís de Oliveira Marchioro, Letícia Paulino Sperandio, Ísis Valeska Freire Lins, Mauro Piacentini, Gian Maria Fimia, Patrícia Reckziegel, Soraya Soubhi Smaili, Claudia Bincoletto.

  The family of coronaviruses (CoVs) uses the autophagy machinery of host cells to promote their growth and replication; thus, this process stands out as a potential target to combat COVID-19. Considering the different roles of autophagy during viral infection, including SARS-CoV-2 infection, in this review, we discuss several clinically used drugs that have effects at different stages of autophagy. Among them, we mention (1) lysosomotropic agents, which can prevent CoVs infection by alkalinizing the acid pH in the endolysosomal system, such as chloroquine and hydroxychloroquine, azithromycin, artemisinins, two-pore channel modulators and imatinib; (2) protease inhibitors that can inhibit the proteolytic cleavage of the spike CoVs protein, which is necessary for viral entry into host cells, such as camostat mesylate, lopinavir, umifenovir and teicoplanin and (3) modulators of PI3K/AKT/mTOR signaling pathways, such as rapamycin, heparin, glucocorticoids, angiotensin-converting enzyme inhibitors (IECAs) and cannabidiol. Thus, this review aims to highlight and discuss autophagy-related drugs for COVID-19, from in vitro to in vivo studies. We identified specific compounds that may modulate autophagy and exhibit antiviral properties. We hope that research initiatives and efforts will identify novel or "off-label" drugs that can be used to effectively treat patients infected with SARS-CoV-2, reducing the risk of mortality.

Keywords:  COVID-19; autophagy; pharmacology

DOI:  https://doi.org/10.3390/ijms22084067
Sci Rep. 2021 Apr 27. 11(1): 9011

ALK inhibition activates LC3B-independent, protective autophagy in EML4-ALK positive lung cancer cells.

Anna M Schläfli, Igor Tokarchuk, Sarah Parejo, Susanne Jutzi, Sabina Berezowska, Nikolai Engedal, Mario P Tschan.

ALK inhibitors effectively target EML4-ALK positive non-small cell lung cancer, but their effects are hampered by treatment resistance. In the present study, we asked whether ALK inhibition affects autophagy, and whether this may influence treatment response. Whereas the impact of targeted therapies on autophagic activity previously have been assessed by surrogate marker proteins such as LC3B, we here thoroughly examined effects on functional autophagic activity, i.e. on the sequestration and degradation of autophagic cargo, in addition to autophagic markers. Interestingly, the ALK inhibitor Ceritinib decreased mTOR activity and increased GFP-WIPI1 dot formation in H3122 and H2228 EML4-ALK+ lung cancer cells, suggesting autophagy activation. Moreover, an mCherry-EGFP-LC3B based assay indicated elevated LC3B carrier flux upon ALK inhibition. In accordance, autophagic cargo sequestration and long-lived protein degradation significantly increased upon ALK inhibition. Intriguingly, autophagic cargo flux was dependent on VPS34 and ULK1, but not LC3B. Co-treating H3122 cells with Ceritinib and a VPS34 inhibitor or Bafilomycin A1 resulted in reduced cell numbers. Moreover, VPS34 inhibition reduced clonogenic recovery of Ceritinib-treated cells. In summary, our results indicate that ALK inhibition triggers LC3B-independent macroautophagic flux in EML4-ALK+ cells to support cancer cell survival and clonogenic growth.

DOI: https://doi.org/10.1038/s41598-021-87966-6