bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023–04–23
sixty papers selected by
Viktor Korolchuk, Newcastle University
  1. NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.
  2. Ubiquitin-binding autophagic receptors in yeast: Cue5 and beyond.
  3. Phagophore-lysosome/vacuole fusion in mutant yeast and mammalian cells.
  4. Role of FLCN Phosphorylation in Insulin-Mediated mTORC1 Activation and Tumorigenesis.
  5. Endophilin-A/SH3GL2 calcium switch for synaptic autophagy induction is impaired by a Parkinson's risk variant.
  6. The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.
  7. An Update on Autophagy as a Target in the Treatment of Alzheimer's Disease.
  8. FRIENDLY is required for efficient dark-induced mitophagy and controlled senescence in Arabidopsis.
  9. New insights into the autophagy-NAD axis in brain disease.
  10. Inhibition of STX17-SNAP29-VAMP8 complex formation by costunolide sensitizes ovarian cancer cells to cisplatin via the AMPK/mTOR signaling pathway.
  11. Endoplasmic reticulum chaperone GRP78 participates in fluoride-induced autophagy in LS8 cells by regulating the IRE1-TRAF2-JNK pathway.
  12. Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.
  13. mTOR/α-ketoglutarate-mediated signaling pathways in the context of brain neurodegeneration and neuroprotection.
  14. Impact of context-dependent autophagy states on tumor progression.
  15. Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.
  16. A TRAF-like E3 ubiquitin ligase TrafE coordinates ESCRT and autophagy in endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.
  17. The role of autophagy in cardiovascular disease: Cross-interference of signaling pathways and underlying therapeutic targets.
  18. The crosslinks between ferroptosis and autophagy in asthma.
  19. Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy.
  20. Goblet cell stress management.
  21. Glucose-Induced Activation of mTORC1 is Associated with Hexokinase2 Binding to Sestrins in HEK293T Cells.
  22. Fountain of youth-Targeting autophagy in aging.
  23. Driving the degradation of oncofusion proteins for targeted cancer therapy.
  24. Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice.
  25. Synaptojanin1 modifies endolysosomal parameters in cultured ventral midbrain neurons.
  26. Nutrient-sensing AgRP neurons relay control of liver autophagy during energy deprivation.
  27. Vesicle tethering and fusion promoted by LC3/GABARAP proteins is modulated by the ATG12-ATG5-ATG16L1 complex.
  28. Dentin defects caused by a Dspp-1 frameshift mutation are associated with the activation of autophagy.
  29. Control of circadian rhythm on cortical excitability and synaptic plasticity.
  30. Doxorubicin downregulates autophagy to promote apoptosis-induced dilated cardiomyopathy via regulating the AMPK/mTOR pathway.
  31. Alzheimer's disease-associated mutant ubiquitin (UBB+1) is secreted through an autophagosome-like vesicle-mediated unconventional pathway.
  32. The relationship between HMGB1 and autophagy in the pathogenesis of diabetes and its complications.
  33. The TRAF2-p62 axis promotes proliferation and survival of liver cancer by activating mTORC1 pathway.
  34. A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila.
  35. SARS-CoV-2 ORF3a expression in brain disrupts the autophagy-lysosomal pathway, impairs sphingolipid homeostasis, and drives neuropathogenesis.
  36. Upregulation of the ESCRT pathway and multivesicular bodies accelerates degradation of proteins associated with neurodegeneration.
  37. Knockout of the neonatal Fc receptor alters immune complex trafficking and lysosomal function in cultured podocytes.
  38. Lyso-globotriaosylsphingosine induces endothelial dysfunction via autophagy-dependent regulation of necroptosis.
  39. The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts.
  40. Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast.
  41. Autophagy in protists and their hosts: When, how and why?
  42. Non-coding RNAs: The recently accentuated molecules in the regulation of cell autophagy for ovarian cancer pathogenesis and therapeutic response.
  43. Lysosomal lipid peroxidation mediates immunogenic cell death.
  44. Cotton Leaf Curl Multan virus C4 protein suppresses autophagy to facilitate viral infection.
  45. Interaction between dual specificity phosphatase-1 and cullin-1 attenuates alcohol-related liver disease by restoring p62-mediated mitophagy.
  46. Pathogenic LRRK2 compromises the subcellular distribution of lysosomes in a Rab12-RILPL1-dependent manner.
  47. Cellular battle against endoplasmic reticulum stress and its adverse effect on health.
  48. Parkinson's disease-associated ATP13A2/PARK9 functions as a lysosomal H+,K+-ATPase.
  49. SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration.
  50. TRIM21 enhances bortezomib sensitivity in multiple myeloma by halting prosurvival autophagy.
  51. Pretreatment of 3-MA prevents doxorubicin-induced cardiotoxicity through inhibition of autophagy initiation.
  52. A ketogenic diet alters mTOR activity, systemic metabolism and potentially prevents collagen degradation associated with chronic alcohol consumption in mice.
  53. An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.
  54. HRD1 Promotes Non-small Cell Lung Carcinoma Metastasis by Blocking Autophagy-mediated MIEN1 Degradation.
  55. Defective iron homeostasis and hematological abnormalities in Niemann-Pick disease type C1.
  56. MicroRNA-593-5p contributes to cell death following exposure to 1-methyl-4-phenylpyridinium (MPP+) by targeting PTEN-induced putative kinase 1 (PINK1).
  57. Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1.
  58. Autophagy-Activated Self-reporting Photosensitizer Promoting Cell Mortality in Cancer Starvation Therapy.
  59. Targeting autophagy and lipid metabolism in cancer stem cells.
  60. Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration.
  1. Cell Rep. 2023 Apr 20. pii: S2211-1247(23)00383-2. [Epub ahead of print]42(5): 112372
    NAD depletion mediates cytotoxicity in human neurons with autophagy deficiency.
    Congxin Sun, Elena Seranova, Malkiel A Cohen, Miruna Chipara, Jennie Roberts, Dewi Astuti, Adina M Palhegyi, Animesh Acharjee, Lucia Sedlackova, Tetsushi Kataura, Elsje G Otten, Prashanta K Panda, Samuel Lara-Reyna, Miriam E Korsgen, Kevin J Kauffman, Alejandro Huerta-Uribe, Malgorzata Zatyka, Luiz F S E Silva, Jorge Torresi, Shupei Zhang, Georgina W Hughes, Carl Ward, Erich R Kuechler, David Cartwright, Sergey Trushin, Eugenia Trushina, Gaurav Sahay, Yosef Buganim, Gareth G Lavery, Joerg Gsponer, Daniel G Anderson, Eva-Maria Frickel, Tatiana R Rosenstock, Timothy Barrett, Oliver D K Maddocks, Daniel A Tennant, Haoyi Wang, Rudolf Jaenisch, Viktor I Korolchuk, Sovan Sarkar.
      Autophagy is a homeostatic process critical for cellular survival, and its malfunction is implicated in human diseases including neurodegeneration. Loss of autophagy contributes to cytotoxicity and tissue degeneration, but the mechanistic understanding of this phenomenon remains elusive. Here, we generated autophagy-deficient (ATG5-/-) human embryonic stem cells (hESCs), from which we established a human neuronal platform to investigate how loss of autophagy affects neuronal survival. ATG5-/- neurons exhibit basal cytotoxicity accompanied by metabolic defects. Depletion of nicotinamide adenine dinucleotide (NAD) due to hyperactivation of NAD-consuming enzymes is found to trigger cell death via mitochondrial depolarization in ATG5-/- neurons. Boosting intracellular NAD levels improves cell viability by restoring mitochondrial bioenergetics and proteostasis in ATG5-/- neurons. Our findings elucidate a mechanistic link between autophagy deficiency and neuronal cell death that can be targeted for therapeutic interventions in neurodegenerative and lysosomal storage diseases associated with autophagic defect.
    Keywords:  CP: Cell biology; CP: Metabolism; NAD; NADases; NAM; NMN; NR; autophagy; cell death; cell survival, human embryonic stem cell-derived neurons; mitochondria; nicotinamide; nicotinamide adenine dinucleotide; nicotinamide mononucleotide; nicotinamide riboside
    DOI:  https://doi.org/10.1016/j.celrep.2023.112372
  2. Autophagy. 2023 Apr 16. 1-5
    Ubiquitin-binding autophagic receptors in yeast: Cue5 and beyond.
    Theresah Nana Ama Mensah, Ankit Shroff, Taras Y Nazarko.
      The selectivity in selective macroautophagy/autophagy pathways is achieved via selective autophagy receptors (SARs) - proteins that bind a ligand on the substrate to be degraded and an Atg8-family protein on the growing autophagic membrane, phagophore, effectively bridging them. In mammals, the most common ligand of SARs is ubiquitin, a small protein modifier that tags substrates for their preferential degradation by autophagy. Consequently, most common SARs are ubiquitin-binding SARs, such as SQSTM1/p62 (sequestosome 1). Surprisingly, there is only one SAR of this type in yeast - Cue5, which acts as the receptor for aggrephagy and proteaphagy - pathways that remove ubiquitinated protein aggregates and proteasomes, respectively. However, recent studies described ubiquitin-dependent autophagic pathways that do not require Cue5, e.g. the stationary phase lipophagy for lipid droplets or nitrogen starvation-induced mitophagy for mitochondria. What is the role of ubiquitin in these pathways? Here, we propose that ubiquitinated lipid droplets and mitochondria are recognized by alternative ubiquitin-binding SARs. Our analysis identifies proteins that could potentially fulfill this role in yeast. We think that matching of ubiquitin-dependent (but Cue5-independent) autophagic pathways with ubiquitin- and Atg8-binding proteins enlisted here might uncover novel ubiquitin-binding SARs in yeast.Abbreviations: AIM: Atg8-family interacting motif; CUE: coupling of ubiquitin conjugation to ER degradation; ERMES: endoplasmic reticulum-mitochondria encounter structure; HECT: homologous to the E6-AP carboxyl terminus; LD: lipid droplet; SAR: selective autophagy receptor; SGD: Saccharomyces Genome Database; UBA: ubiquitin-associated; UBX: ubiquitin regulatory X; UIM: ubiquitin-interacting motif.
    Keywords:  Cue5; autophagic receptor; selective autophagy; selective autophagy receptor; ubiquitin-binding protein; ubiquitin-binding receptor
    DOI:  https://doi.org/10.1080/15548627.2023.2196878
  3. Autophagy. 2023 Apr 21.
    Phagophore-lysosome/vacuole fusion in mutant yeast and mammalian cells.
    Yongheng Liang.
      Macroautophagy/autophagy is a process through which the phagophores engulf non-essential or damaged cellular materials, forming double-membrane autophagosomes (APs) and fusing with lysosomes/vacuoles, after which the materials are degraded for recycling purposes. Autophagy is associated with increased cell survival under different stress conditions. AP-lysosome/vacuole fusion is a critical step in autophagy. Some mutant cells can accumulate phagophores under autophagy-induction conditions. Autophagy is interrupted when accumulated phagophores cannot fuse with lysosomes/vacuoles, resulting in a significant decrease in cell survivability. However, phagophore-lysosome/vacuole fusion has been reported in related mammalian cells and yeast mutant cells. This observation indicates that it is possible to restore a partial autophagy process after interruption. Furthermore, these findings indicate that phagophore closure is not a prerequisite for its fusion with the lysosome/vacuole in the mutant cells. The phagophore-lysosome/vacuole fusion strategy can significantly rescue defective autophagy due to failed phagophore closure. This commentary discusses the fusion of phagophores and lysosomes/vacuoles and implications of such fusion events.
    Keywords:  autolysosome; autophagic body; autophagosome; inner membrane of phagophore or autophagosome; lysosome; outer membrane of phagophore or autophagosome; phagophore; phagophore-lysosome/vacuole fusion; sporulation; vacuole
    DOI:  https://doi.org/10.1080/15548627.2023.2205272
  4. Adv Sci (Weinh). 2023 Apr 21. e2206826
    Role of FLCN Phosphorylation in Insulin-Mediated mTORC1 Activation and Tumorigenesis.
    Guoyan Wang, Lei Chen, Xinjian Lei, Senlin Qin, Huijun Geng, Yining Zheng, Chao Xia, Junhu Yao, Tong Meng, Lu Deng.
      The amino acid-stimulated Rag GTPase pathway is one of the main pathways that regulate mechanistic target of rapamycin complex 1 (mTORC1) activation and function, but little is known about the effects of growth factors on Rag GTPase-mediated mTORC1 activation. Here, a highly conserved insulin-responsive phosphorylation site on folliculin (FLCN), Ser62, that is phosphorylates by AKT1 is identified and characterized. mTORC2-AKT1 is localized on lysosomes, and RagD-specific recruitment of mTORC2-AKT1 on lysosomes is identified as an essential step in insulin-AKT1-mediated FLCN phosphorylation. Additionally, FLCN phosphorylation inhibits the activity of RagC GTPase and is essential for insulin-induced mTORC1 activation. Functionally, phosphorylated FLCN promotes cell viability and induces autophagy, and also regulates in vivo tumor growth in an mTORC1-dependent manner. Its expression is also positively correlated with mTORC1 activity in colon cancer, clear cell renal cell carcinoma, and chordoma. These results indicate that FLCN is an important intermediate for cross-talk between the amino acid and growth factor pathways. Further, FLCN phosphorylation may be a promising therapeutic target for diseases characterized by mTORC1 dysregulation.
    Keywords:  FLCN phosphorylation; insulin; mTORC1; tumorigenesis
    DOI:  https://doi.org/10.1002/advs.202206826
  5. Autophagy. 2023 Apr 17. 1-3
    Endophilin-A/SH3GL2 calcium switch for synaptic autophagy induction is impaired by a Parkinson's risk variant.
    Marianna Decet, Sandra-Fausia Soukup.
      At the synapse, proteins are reused several times during neuronal activity, causing a decline in protein function over time. Although emerging evidence supports a role of autophagy in synaptic function, the precise molecular mechanisms linking neuronal activity, autophagy and synaptic dysfunction are vastly unknown. We show how extracellular calcium influx in the pre-synaptic terminal constitutes the initial stimulus for autophagosome formation in response to neuronal activity. This mechanism likely acts to rapidly support synaptic homeostasis and protein quality control when intense neuronal activity challenges the synaptic proteome. We identified a residue in the flexible region of EndoA (Endophilin A) that dictates calcium-dependent EndoA mobility from the plasma membrane to the cytosol, where this protein interacts with autophagic membranes to promote autophagosome formation. We discovered that a novel Parkinson's disease-risk mutation in SH3GL2 (SH3 domain containing GRB2 like 2, endophilin A1) disrupts the calcium sensing of SH3GL2, leading to an immobile protein that cannot respond to calcium influx and therefore disrupting autophagy induction at synapses. Our work shows how neuronal activity is connected with autophagy to maintain synaptic homeostasis and survival.
    Keywords:  Neuronal activity; Parkinson disease; SH3GL2/ Endophilin-A; autophagy; calcium; neurodegeneration
    DOI:  https://doi.org/10.1080/15548627.2023.2200627
  6. J Biol Chem. 2023 Apr 13. pii: S0021-9258(23)01740-4. [Epub ahead of print] 104712
    The dynamin Vps1 mediates Atg9 transport to the sites of autophagosome formation.
    Henning Arlt, Babu Raman, Yasmina Filali-Mouncef, Yan Hu, Alexandre Leytens, Ralph Hardenberg, Rodrigo Guimarães, Franziska Kriegenburg, Muriel Mari, Iwona I Smaczynska-de Rooij, Kathryn R Ayscough, Jörn Dengjel, Christian Ungermann, Fulvio Reggiori.
      Autophagy is a key process in eukaryotes to maintain cellular homeostasis by delivering cellular components to lysosomes/vacuoles for degradation and reuse of the resulting metabolites. Membrane rearrangements and trafficking events are mediated by the core machinery of autophagy-related (Atg) proteins, which carry out a variety of functions. How Atg9, a lipid scramblase and the only conserved transmembrane protein within this core Atg machinery, is trafficked during autophagy remained largely unclear. Here, we addressed this question in yeast Saccharomyces cerevisiae and found that retromer complex and dynamin Vps1 mutants alter Atg9 subcellular distribution and severely impair the autophagic flux by affecting two separate autophagy steps. We provide evidence that Vps1 interacts with Atg9 at Atg9 reservoirs. In the absence of Vps1, Atg9 fails to reach the sites of autophagosome formation, and this results in an autophagy defect. The function of Vps1 in autophagy requires its GTPase activity. Moreover, Vps1 point mutants associated with human diseases such as microcytic anemia and Charcot-Marie-Tooth are unable to sustain autophagy and affect Atg9 trafficking. Together, our data provide novel insights on the role of dynamins in Atg9 trafficking and suggest that a defect in this autophagy step could contribute to severe human pathologies.
    Keywords:  Atg9; Autophagy; PAS; SNX; Vps1; autophagosome; dynamin; phagophore; retromer; sorting nexin
    DOI:  https://doi.org/10.1016/j.jbc.2023.104712
  7. Curr Drug Targets. 2023 Apr 17.
    An Update on Autophagy as a Target in the Treatment of Alzheimer's Disease.
    Parnika M Sose, Gaurav Mahesh Doshi, Pravin P Kale.
      Proteostasis is crucial for the maintenance and proper operation of cells. Under typical circumstances, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway are used to clean out undesired, damaged, misfolded, or aggregated proteins. Any dysregulation in the above-mentioned pathways leads to neurodegeneration. One of the most renowned neurodegenerative disorders is AD. This condition is more prevalent in senior people and is frequently linked to dementia, progressive memory loss, and cognitive function decline, which further contributes to cholinergic neuron degradation and synaptic plasticity loss. Extracellular accumulation of amyloid beta plaques and the intraneuronal deposition of misfolded neurofibrillary tangles are two prime pathological reasons for AD. At present, there is no treatment for AD. All that remains available is the symptomatic treatment of this disease. Autophagy is the major mechanism by which the cells degrade the protein aggregates. Deposited immature autophagic vacuoles (AVs) in AD brains suggest interruption of a person's normal autophagy process. This review has briefly covered various forms and mechanisms of autophagy. Furthermore, the discussion in the article is supported by different ways and mechanisms via which autophagy can be stimulated in a beneficial way and can emerge as a novel target in the treatment of various metabolic CNS related disorders. In the current review article, the mTOR-dependent ones are PI3K/Akt/TSC/mTOR, AMPK/TSC/mTOR, and Rag/mTOR pathways and mTOR-independent ones which include Ca2+/calpain, inositol-dependent, cAMP/EPAC/PLC, and JNK1/Beclin-1/PI3K pathways have been discussed in details. The article sheds light on drugs which are validated with details in tabular form from recent updates in clinical trials.
    Keywords:  AD; Aging and Alzheimer’s; Autophagy; beta plaques; mTORC1 inhibitors.; neurofibrillary tangles
    DOI:  https://doi.org/10.2174/1389450124666230417104325
  8. Free Radic Biol Med. 2023 Apr 19. pii: S0891-5849(23)00377-5. [Epub ahead of print]
    FRIENDLY is required for efficient dark-induced mitophagy and controlled senescence in Arabidopsis.
    Sylwia M Kacprzak, Olivier Van Aken.
      Mitochondria play essential roles in plant metabolism, supporting both development and stress responses. To maintain a healthy pool of mitochondria, several quality control systems are in place. Selected degradation of mitochondria by autophagy -mitophagy- has been extensively studied in yeast and animals, but information on mitophagy components in plants is limited. The 'Friendly Mitochondria' (FRIENDLY; FMT) protein, homologous to 'clustered mitochondria protein homolog' CLU in animals, was recently suggested to mediate mitophagy of depolarized mitochondria. In this study, we evaluated the role of FMT in carbon starvation- and dark senescence-induced mitophagy in Arabidopsis. Using mitophagy flux assays, we show that loss of FMT results in decreased mitophagy during dark-induced senescence. Mitophagy induced by inhibition of Target of Rapamycin (TOR) signalling is also partially impaired in fmt mutants. The FMT protein is mostly localised in the cytosol, but we show that during darkness FMT is redistributed into speckles, some of which associate with mitochondria. Fmt mutants were initially identified for their abnormal mitochondrial morphology, with mitochondria often forming clusters. We found that dark senescence strongly increases the number and size of mitochondrial clusters in fmt mutants. In agreement with a role for FMT in mitophagy, we show that fmt mutants show accelerated senescence phenotypes comparable to autophagy 11 (atg11) mutants, but less prominent than in atg5 mutants. Furthermore, FMT prevents excessive dark-induced cell death and hydrogen peroxide production, and supports mitophagy and greening in etiolated seedlings. Our findings thus indicate that FMT contributes to mitophagy and provide evidence that mitophagy is required for controlled senescence and prevention of accelerated cell death. We propose that FMT mediates efficient mitophagy by preventing mitochondrial clustering, thereby allowing mitochondria to be captured more effectively by autophagosomes, rather than by acting as a direct mitophagy receptor.
    Keywords:  Arabidopsis; Autophagy; Clustered mitochondria; Mitochondria oxidative stress; Mitophagy; Senescence
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.04.007
  9. Cell Rep. 2023 Apr 20. pii: S2211-1247(23)00431-X. [Epub ahead of print]42(5): 112420
    New insights into the autophagy-NAD axis in brain disease.
    Maria Jose Perez, Michela Deleidi.
      Sun et al. demonstrate that defects in autophagy cause nicotinamide adenine dinucleotide (NAD) depletion and neurotoxicity.1 Restoring NAD levels rescues cytotoxicity in autophagy-deficient neurons, providing a potential therapy for neurodegenerative and lysosomal storage diseases associated with autophagy defects.
    DOI:  https://doi.org/10.1016/j.celrep.2023.112420
  10. Biochem Pharmacol. 2023 Apr 13. pii: S0006-2952(23)00140-5. [Epub ahead of print]212 115549
    Inhibition of STX17-SNAP29-VAMP8 complex formation by costunolide sensitizes ovarian cancer cells to cisplatin via the AMPK/mTOR signaling pathway.
    Xiao Liang, Chunlei Yu, Yunhong Tian, Xiaocong Xiang, Yuexi Luo.
      Ovarian cancer (OC) is the most common gynecological malignancy. Chemotherapy failure is a major challenge in OC treatment. Targeting autophagy is a promising strategy to enhance the cytotoxicity of chemotherapeutic agents. In this study, we found that costunolide (CTD) inhibits autophagic flux and exhibits high therapeutic efficacy for OC treatment in an in vitro model. Mechanistically, CTD inactivates AMPK/mTOR signaling to inhibit autophagy initiation at the early stage and blocks mTORC1-dependent autophagosome-lysosome fusion at the late stage during autophagy by disrupting SNARE complex (STX17-SNAP29-VAMP8) formation, resulting in lethal autophagy arrest in OC cells. Furthermore, CTD sensitizes OC cells to cisplatin (CDDP) by blocking CDDP-induced autophagy both in vitro and in vivo. Together, our data provide novel mechanistic insights into CTD-induced autophagy arrest and suggest a new autophagy inhibitor for effective treatment of OC.
    Keywords:  Autophagy; Cisplatin; Costunolide; Lysosome; Ovarian cancer
    DOI:  https://doi.org/10.1016/j.bcp.2023.115549
  11. Environ Toxicol. 2023 Apr 18.
    Endoplasmic reticulum chaperone GRP78 participates in fluoride-induced autophagy in LS8 cells by regulating the IRE1-TRAF2-JNK pathway.
    Lin Zhao, Liyuan Wang, Han Wang, Tao Xi, Sijia Liu, Yang Li, Jianping Ruan, Yongqing Huang.
      Fluoride induces endoplasmic reticulum (ER) stress in ameloblasts, which is responsible for enamel mineralization disorder. Fluoride induces autophagy in ameloblasts, but the molecular mechanisms through which ameloblasts respond to fluoride-induced cellular stress and autophagy remain unclear. This study investigated ER stress-induced autophagy and the regulatory role of the ER molecular chaperone GRP78 in fluoride-induced autophagy in ameloblast LS8 cells. To explore the relationship between fluoride-induced ER stress and autophagy, we assessed changes in fluoride-induced autophagy in LS8 cells following overexpression and/or silencing of the ER stress molecular chaperone GRP78. We found that autophagy induced by fluoride was further increased after GRP78 overexpression in LS8 cells. Fluoride-induced autophagy was reduced in GRP78-silenced LS8 cells. Furthermore, we found that ER stress can regulate autophagy in fluoride-treated ameloblasts (LS8 cells) and that the GRP78/IRE1/TRAF2/JNK pathway is involved in the underlying regulation. Our study suggests that ER stress plays a role in fluoride-induced damage by inducing ameloblast autophagy.
    Keywords:  GRP78; LS8; NaF; autophagy; endoplasmic reticulum stress
    DOI:  https://doi.org/10.1002/tox.23805
  12. STAR Protoc. 2023 Apr 18. pii: S2666-1667(23)00194-6. [Epub ahead of print]4(2): 102236
    Monitoring and assessment of lysosomal membrane damage in cultured cells using the high-content imager.
    Keisuke Tabata, Marika Saeki, Tamotsu Yoshimori, Maho Hamasaki.
      Autophagy is an intracellular self-degradation process in which part of the cytoplasm, aggregates, or damaged organelles are degraded in lysosomes. Lysophagy is a specific form of selective autophagy responsible for clearing damaged lysosomes. Here, we present a protocol for inducing lysosomal damage in cultured cells and assessing lysosomal damage using a high-content imager and software program. We describe steps for induction of lysosomal damage, image acquisition with spinning disk confocal microscopy, and image analysis using Pathfinder. We then detail data analysis of the clearance of damaged lysosomes. For complete details on the use and execution of this protocol, please refer to Teranishi et al. (2022).1.
    Keywords:  Cell Biology; Cell Culture; High-throughput Screening; Microscopy
    DOI:  https://doi.org/10.1016/j.xpro.2023.102236
  13. BBA Adv. 2022 ;2 100066
    mTOR/α-ketoglutarate-mediated signaling pathways in the context of brain neurodegeneration and neuroprotection.
    Olha Kostiuchenko, Iryna Lushnikova, Magdalena Kowalczyk, Galyna Skibo.
      Cerebral disorders are largely associated with impaired cellular metabolism, despite the regulatory mechanisms designed to ensure cell viability and adequate brain function. Mechanistic target of rapamycin (mTOR) signaling is one of the most crucial factors in the regulation of energy homeostasis and its imbalance is linked with a variety of neurodegenerative diseases. Recent advances in the metabolic pathways' modulation indicate the role of α-ketoglutarate (AKG) as a major signaling hub, additionally highlighting its anti-aging and neuroprotective properties, but the mechanisms of its action are not entirely clear. In this review, we analyzed the physiological and pathophysiological aspects of mTOR in the brain. We also discussed AKG's multifunctional properties, as well as mTOR/AKG-mediated functional communications in cellular metabolism. Thus, this article provides a broad overview of the mTOR/AKG-mediated signaling pathways, in the context of neurodegeneration and endogenous neuroprotection, with the aim to find novel therapeutic strategies.
    Keywords:  Aging; Autophagy; Metabolism; Neurodegeneration; Neuroprotection; mTOR; α-ketoglutarate (AKG)
    DOI:  https://doi.org/10.1016/j.bbadva.2022.100066
  14. Nat Cancer. 2023 Apr 17.
    Impact of context-dependent autophagy states on tumor progression.
    Mohamad Assi, Alec C Kimmelman.
      Macroautophagy is a cellular quality-control process that degrades proteins, protein aggregates and damaged organelles. Autophagy plays a fundamental role in cancer where, in the presence of stressors (for example, nutrient starvation, hypoxia, mechanical pressure), tumor cells activate it to degrade intracellular substrates and provide energy. Cell-autonomous autophagy in tumor cells and cell-nonautonomous autophagy in the tumor microenvironment and in the host converge on mechanisms that modulate metabolic fitness, DNA integrity and immune escape and, consequently, support tumor growth. In this Review, we will discuss insights into the tumor-modulating roles of autophagy in different contexts and reflect on how future studies using physiological culture systems may help to understand the complexity and open new therapeutic avenues.
    DOI:  https://doi.org/10.1038/s43018-023-00546-7
  15. Sci Adv. 2023 04 21. 9(16): eadf9284
    Autophagy supports mitochondrial metabolism through the regulation of iron homeostasis in pancreatic cancer.
    Subhadip Mukhopadhyay, Joel Encarnación-Rosado, Elaine Y Lin, Albert S W Sohn, Huan Zhang, Joseph D Mancias, Alec C Kimmelman.
      Pancreatic ductal adenocarcinoma (PDAC) cells maintain a high level of autophagy, allowing them to thrive in an austere microenvironment. However, the processes through which autophagy promotes PDAC growth and survival are still not fully understood. Here, we show that autophagy inhibition in PDAC alters mitochondrial function by losing succinate dehydrogenase complex iron sulfur subunit B expression by limiting the availability of the labile iron pool. PDAC uses autophagy to maintain iron homeostasis, while other tumor types assessed require macropinocytosis, with autophagy being dispensable. We observed that cancer-associated fibroblasts can provide bioavailable iron to PDAC cells, promoting resistance to autophagy ablation. To overcome this cross-talk, we used a low-iron diet and demonstrated that this augmented the response to autophagy inhibition therapy in PDAC-bearing mice. Our work highlights a critical link between autophagy, iron metabolism, and mitochondrial function that may have implications for PDAC progression.
    DOI:  https://doi.org/10.1126/sciadv.adf9284
  16. Elife. 2023 Apr 18. pii: e85727. [Epub ahead of print]12
    A TRAF-like E3 ubiquitin ligase TrafE coordinates ESCRT and autophagy in endolysosomal damage response and cell-autonomous immunity to Mycobacterium marinum.
    Lyudmil Raykov, Manon Mottet, Jahn Nitschke, Thierry Soldati.
      Cells are perpetually challenged by pathogens, protein aggregates or chemicals, that induce plasma membrane or endolysosomal compartments damage. This severe stress is recognised and controlled by the endosomal sorting complex required for transport (ESCRT) and the autophagy machineries, which are recruited to damaged membranes to either repair or to remove membrane remnants. Yet, insight is limited about how damage is sensed and which effectors lead to extensive tagging of the damaged organelles with signals, such as K63-polyubiquitin, required for the recruitment of membrane repair or removal machineries. To explore the key factors responsible for detection and marking of damaged compartments, we use the professional phagocyte Dictyostelium discoideum. We found an evolutionary conserved E3-ligase, TrafE, that is robustly recruited to intracellular compartments disrupted after infection with Mycobacterium marinum or after sterile damage caused by chemical compounds. TrafE acts at the intersection of ESCRT and autophagy pathways and plays a key role in functional recruitment of the ESCRT subunits ALIX, Vps32 and Vps4 to damage sites. Importantly, we show that the absence of TrafE severely compromises the xenophagy restriction of mycobacteria as well as ESCRT-mediated and autophagy-mediated endolysosomal membrane damage repair, resulting in early cell death.
    Keywords:  cell biology; dictyostelium; infectious disease; microbiology
    DOI:  https://doi.org/10.7554/eLife.85727
  17. Front Cardiovasc Med. 2023 ;10 1088575
    The role of autophagy in cardiovascular disease: Cross-interference of signaling pathways and underlying therapeutic targets.
    Bing Jiang, Xuan Zhou, Tao Yang, Linlin Wang, Longfei Feng, Zheng Wang, Jin Xu, Weiyao Jing, Tao Wang, Haixiang Su, GuoWei Yang, Zheng Zhang.
      Autophagy is a conserved lysosomal pathway for the degradation of cytoplasmic proteins and organelles, which realizes the metabolic needs of cells and the renewal of organelles. Autophagy-related genes (ATGs) are the main molecular mechanisms controlling autophagy, and their functions can coordinate the whole autophagic process. Autophagy can also play a role in cardiovascular disease through several key signaling pathways, including PI3K/Akt/mTOR, IGF/EGF, AMPK/mTOR, MAPKs, p53, Nrf2/p62, Wnt/β-catenin and NF-κB pathways. In this paper, we reviewed the signaling pathway of cross-interference between autophagy and cardiovascular diseases, and analyzed the development status of novel cardiovascular disease treatment by targeting the core molecular mechanism of autophagy as well as the critical signaling pathway. Induction or inhibition of autophagy through molecular mechanisms and signaling pathways can provide therapeutic benefits for patients. Meanwhile, we hope to provide a unique insight into cardiovascular treatment strategies by understanding the molecular mechanism and signaling pathway of crosstalk between autophagy and cardiovascular diseases.
    Keywords:  autophagy; autophagy-related gene; cardiovascular disease; crosstalk; potential target; signaling pathway
    DOI:  https://doi.org/10.3389/fcvm.2023.1088575
  18. Front Immunol. 2023 ;14 1140791
    The crosslinks between ferroptosis and autophagy in asthma.
    Xiaodi Lv, Weifeng Tang, Jingjing Qin, Wenqian Wang, Jingcheng Dong, Ying Wei.
      Autophagy is an evolutionarily conserved cellular process capable of degrading various biological molecules and organelles via the lysosomal pathway. Ferroptosis is a type of oxidative stress-dependent regulated cell death associated with the iron accumulation and lipid peroxidation. The crosslinks between ferroptosis and autophagy have been focused on since the dependence of ferroptosis on autophagy was discovered. Although the research and theories on the relationship between autophagy and ferroptosis remain scattered and fragmented, the crosslinks between these two forms of regulated cell death are closely related to the treatment of various diseases. Thereof, asthma as a chronic inflammatory disease has a tight connection with the occurrence of ferroptosis and autophagy since the crosslinked signal pathways may be the crucial regulators or exactly regulated by cells and secretion in the immune system. In addition, non-immune cells associated with asthma are also closely related to autophagy and ferroptosis. Further studies of cross-linking asthma inflammation with crosslinked signaling pathways may provide us with several key molecules that regulate asthma through specific regulators. The crosslinks between autophagy and ferroptosis provide us with a new perspective to interpret and understand the manifestations of asthma, potential drug discovery targets, and new therapeutic options to effectively intervene in the imbalance caused by abnormal inflammation in asthma. Herein, we introduce the main molecular mechanisms of ferroptosis, autophagy, and asthma, describe the role of crosslinks between ferroptosis and autophagy in asthma based on their common regulatory cells or molecules, and discuss potential drug discovery targets and therapeutic applications in the context of immunomodulatory and symptom alleviation.
    Keywords:  asthma; autophagy; crosslinks; ferroptosis; targets
    DOI:  https://doi.org/10.3389/fimmu.2023.1140791
  19. J Vis Exp. 2023 Mar 31.
    Ultrastructural Localization of Endogenous LC3 by On-Section Correlative Light-Electron Microscopy.
    Jan van der Beek, Tineke Veenendaal, Cecilia de Heus, Suzanne van Dijk, Corlinda Ten Brink, Nalan Liv, Judith Klumperman.
      The visualization of autophagic organelles at the ultrastructural level by electron microscopy (EM) is essential to establish their identity and reveal details that are important for understanding the autophagic process. However, EM methods often lack molecular information, obstructing the correlation of ultrastructural information obtained by EM to fluorescence microscopy-based localization of specific autophagy proteins. Furthermore, the rarity of autophagosomes in unaltered cellular conditions hampers investigation by EM, which requires high magnification, and hence provides a limited field of view. In answer to both challenges, an on-section correlative light-electron microscopy (CLEM) method based on fluorescent labeling was applied to correlate a common autophagosomal marker, LC3, to EM ultrastructure. The method was used to rapidly screen cells in fluorescence microscopy for LC3 labeling in combination with other relevant markers. Subsequently, the underlying ultrastructural features of selected LC3-labeled spots were identified by CLEM. The method was applied to starved cells without adding inhibitors of lysosomal acidification. In these conditions, LC3 was found predominantly on autophagosomes and rarely in autolysosomes, in which LC3 is rapidly degraded. These data show both the feasibility and sensitivity of this approach, demonstrating that CLEM can be used to provide ultrastructural insights on LC3-mediated autophagy in native conditions-without drug treatments or genetic alterations. Overall, this method presents a valuable tool for ultrastructural localization studies of autophagy proteins and other scarce antigens by bridging light microscopy to EM data.
    DOI:  https://doi.org/10.3791/65067
  20. Sci Signal. 2023 Apr 18. 16(781): eadi2176
    Goblet cell stress management.
    Annalisa M VanHook.
      Autophagy augments the mucus-secreting capacity of goblet cells by reducing ER stress.
    DOI:  https://doi.org/10.1126/scisignal.adi2176
  21. J Nutr. 2023 Apr;pii: S0022-3166(22)13243-8. [Epub ahead of print]153(4): 988-998
    Glucose-Induced Activation of mTORC1 is Associated with Hexokinase2 Binding to Sestrins in HEK293T Cells.
    Paul A Roberson, Gregory N Kincheloe, Jaclyn E Welles, Dandan Xu, Mahalia Sam-Clarke, Paul S MacLean, Charles H Lang, Leonard S Jefferson, Scot R Kimball.
       BACKGROUND: Sestrins (SESN1-3) act as proximal sensors in leucine-induced activation of the protein kinase mechanistic target of rapamycin (mTOR) in complex 1 (mTORC1), a key regulator of cell growth and metabolism.
    OBJECTIVE: In the present study, the hypothesis that SESNs also mediate glucose-induced activation of mTORC1 was tested.
    METHODS: Rats underwent overnight fasting, and in the morning, either saline or a glucose solution (4 g⋅kg-1 BW/10 mL⋅kg-1) was administered by oral gavage; mTORC1 activation in the tibialis anterior muscle was assessed. To further assess the mechanism through which glucose promotes mTORC1 activation, wild-type (WT) HEK293T and HEK293T cells lacking either all 3 SESNs (SESNTKO) or hexokinase 2 (HK2KO) were deprived of glucose, followed by glucose addback, and mTORC1 activation was assessed. In addition, glucose-induced changes in the association of the SESNs with components of the GAP activity toward the Rags (GATOR2) complex and with hexokinase 2 (HK2) were assessed by co-immunoprecipitation. One- and two-way ANOVA with Tukey post hoc comparisons were used.
    RESULTS: Glucose administration to fasted rats promoted mTORC1 activation. Similarly, glucose readdition (GluAB) to the medium of glucose-deprived WT cells also promoted mTORC1 activation. By contrast, SESNTKO cells demonstrated attenuated mTORC1 activation following GluAB compared with WT cells. Interestingly, HK2 associated with all 3 SESNs in a glucose-dependent manner, i.e., HK2 abundance in SESN immunoprecipitates was high in cells deprived of glucose and decreased in response to GluAB. Moreover, similar to SESNTKO cells, the sensitivity of mTORC1 to GluAB was attenuated in HK2KO cells compared with WT cells.
    CONCLUSIONS: The results of this study demonstrate that the SESNs and HK2 play important roles in glucose-induced mTORC1 activation in HEK293T cells. However, unlike leucine-induced mTORC1 activation, the effect was independent of the changes in SESN-GATOR2 interaction, and instead, it was associated with alterations in the association of SESNs with HK2.
    Keywords:  glucose; mTORC1; metabolism; nutrient signaling; skeletal muscle
    DOI:  https://doi.org/10.1016/j.tjnut.2022.11.021
  22. Front Aging Neurosci. 2023 ;15 1125739
    Fountain of youth-Targeting autophagy in aging.
    Lea Danics, Anna Anoir Abbas, Balázs Kis, Karolina Pircs.
      As our society ages inexorably, geroscience and research focusing on healthy aging is becoming increasingly urgent. Macroautophagy (referred to as autophagy), a highly conserved process of cellular clearance and rejuvenation has attracted much attention due to its universal role in organismal life and death. Growing evidence points to autophagy process as being one of the key players in the determination of lifespan and health. Autophagy inducing interventions show significant improvement in organismal lifespan demonstrated in several experimental models. In line with this, preclinical models of age-related neurodegenerative diseases demonstrate pathology modulating effect of autophagy induction, implicating its potential to treat such disorders. In humans this specific process seems to be more complex. Recent clinical trials of drugs targeting autophagy point out some beneficial effects for clinical use, although with limited effectiveness, while others fail to show any significant improvement. We propose that using more human-relevant preclinical models for testing drug efficacy would significantly improve clinical trial outcomes. Lastly, the review discusses the available cellular reprogramming techniques used to model neuronal autophagy and neurodegeneration while exploring the existing evidence of autophagy's role in aging and pathogenesis in human-derived in vitro models such as embryonic stem cells (ESCs), induced pluripotent stem cell derived neurons (iPSC-neurons) or induced neurons (iNs).
    Keywords:  aging; autophagy; autophagy-modifying drugs; clinical trial; direct reprogramming; disease modeling; neurodegenerative diseases; rejuvenation
    DOI:  https://doi.org/10.3389/fnagi.2023.1125739
  23. Drug Discov Today. 2023 Apr 13. pii: S1359-6446(23)00100-9. [Epub ahead of print] 103584
    Driving the degradation of oncofusion proteins for targeted cancer therapy.
    Xingya Zhang, Yingqian Chen, Bo Yang, Xuejing Shao, Meidan Ying.
      Oncofusion proteins drive the development of about 16.5% of human cancers, functioning as the unique pathogenic factor in some cancers. The targeting of oncofusion proteins is an attractive strategy to treat malignant tumors. Recently, triggering the degradation of oncofusion proteins has been shown to hold great promise as a therapeutic strategy. Here, we review the recent findings on the mechanisms that maintain the high stability of oncofusion proteins. Then, we summarize strategies to target the degradation of oncofusion proteins through the ubiquitin-proteasome pathway, the autophagy-lysosomal pathway, and the caspase-dependent pathway. By examining oncofusion protein degradation in cancer, we not only gain better insight into the carcinogenic mechanisms that involve oncofusion proteins, but also raise the possibility of treating oncofusion-driven cancer.
    Keywords:  autophagy-lysosomal pathway; caspase-dependent pathway; oncofusion protein; protein degradation; protein stability; ubiquitin-proteasome pathway
    DOI:  https://doi.org/10.1016/j.drudis.2023.103584
  24. Nat Microbiol. 2023 Apr 20.
    Modulation of virus-induced neuroinflammation by the autophagy receptor SHISA9 in mice.
    Yanyan Zheng, Liqiu Wang, Qingxiang Liu, Huifang Xian, Chenqiu Zhang, Sihui Cai, Shuai Yang, Shouheng Jin, Jun Cui.
      Microglia and astrocytes are subgroups of brain glia cells that support and protect neurons within the central nervous system (CNS). At early stages of viral infection in the CNS, they are predominant responding cells and lead to recruitment of peripheral immune cells for viral clearance. Inhibitor of nuclear factor κB kinase subunit epsilon (IKKi) is critical for type I interferon signalling and inflammation, which modulate heterogenic immune responses during CNS infection. Balanced autophagy is vital to maintain brain integrity, yet regulation of autophagy and immune activity within brain glia cells is poorly understood. Here we identify SHISA9 as an autophagy cargo receptor that mediates the autophagy-dependent degradation of IKKi during herpes simplex virus type 1 infection. IKKi is recognized by SHISA9 through unanchored K48-linked poly-ubiquitin chains and bridged to autophagosome membrane components GABARAPL1. Single-cell RNA sequencing analysis shows that SHISA9 has temporal characteristics while modulating both antiviral and inflammatory responses in microglia and astrocytes at different stages during viral infection. We found that Shisa9-/- mice are highly susceptible to herpes simplex virus encephalitis, have pathogenic astrocytes and display more severe neuroinflammation compared with wild-type mice. Taken together, our study unravels a critical role of selective autophagy by orchestrating immune heterogeneity of different CNS resident cells through the SHISA9-IKKi axis.
    DOI:  https://doi.org/10.1038/s41564-023-01357-3
  25. eNeuro. 2023 Apr 17. pii: ENEURO.0426-22.2023. [Epub ahead of print]
    Synaptojanin1 modifies endolysosomal parameters in cultured ventral midbrain neurons.
    Xinyu Zhu, Sanjana Surya Prakash, Geoffrey McAuliffe, Ping-Yue Pan.
      The accumulation of α-syn enriched protein aggregates is thought to arise from dysfunction in degradation systems within the brain. Recently, missense mutations of SYNJ1 encoding the SAC1 and 5'-phosphatase domains have been found in families with hereditary early-onset Parkinsonism. Previous studies showed that Synj1 haploinsufficiency (Synj1+/-) leads to accumulation of the autophagy substrate p62 and pathological α-syn proteins in the midbrain (MB) and striatum of aged mice. In this study, we aim to investigate the neuronal degradation pathway using the Synj1+/- MB culture from mouse pups of mixed sex as a model. Our data show that GFP-LC3 puncta formation and cumulative mKeima puncta formation are unaltered at baseline in Synj1+/- MB neurons. However, GFP-LAMP1 puncta is reduced with a similar decrease in endogenous proteins, including LAMP1, LAMP2, and LAMP2A. The LAMP1 vesicles are hyperacidified with enhanced enzymatic activity in Synj1+/- MB neurons. Using a combination of light and electron microscopy, we show that endolysosomal changes are primarily associated with a lack of SAC1 activity. Consistently, expressing the SYNJ1 R258Q mutant in N2a cells reduces the lysosome number. Interestingly, the endolysosomal defects in Synj1+/- neurons does not impact the clearance of exogenously expressed wild-type α-syn; however, the clearance of α-syn A53T was impaired in the axons of Synj1+/- MB neurons. Taken together, our results suggest axonal vulnerability to endolysosomal defects in Synj1 deficient MB neurons.Significance StatementIn the study, Zhu et al. discovered a previously uncharacterized role of Synj1 in regulating endolysosomal number, protein, and acidity in ventral midbrain neurons. These alterations are associated with a specific impairment in the clearance of α-syn A53T, but not WT α-syn in axons, suggesting an essential role of Synj1 in axonal degradative capacity under pathological stress. This work in cultured mammalian neurons complements recent research efforts in Drosophila, C. elegans and zebra fish, and provides a novel insight for the role Synj1 in neuronal degradation.
    Keywords:  alpha-synuclein; autophagy; lysosome; synaptojanin1
    DOI:  https://doi.org/10.1523/ENEURO.0426-22.2023
  26. Cell Metab. 2023 Apr 12. pii: S1550-4131(23)00124-9. [Epub ahead of print]
    Nutrient-sensing AgRP neurons relay control of liver autophagy during energy deprivation.
    Weiyi Chen, Oliver Mehlkop, Alexandra Scharn, Hendrik Nolte, Paul Klemm, Sinika Henschke, Lukas Steuernagel, Tamara Sotelo-Hitschfeld, Ecem Kaya, Claudia Maria Wunderlich, Thomas Langer, Natalia L Kononenko, Patrick Giavalisco, Jens Claus Brüning.
      Autophagy represents a key regulator of aging and metabolism in sensing energy deprivation. We find that fasting in mice activates autophagy in the liver paralleled by activation of hypothalamic AgRP neurons. Optogenetic and chemogenetic activation of AgRP neurons induces autophagy, alters phosphorylation of autophagy regulators, and promotes ketogenesis. AgRP neuron-dependent induction of liver autophagy relies on NPY release in the paraventricular nucleus of the hypothalamus (PVH) via presynaptic inhibition of NPY1R-expressing neurons to activate PVHCRH neurons. Conversely, inhibiting AgRP neurons during energy deprivation abrogates induction of hepatic autophagy and rewiring of metabolism. AgRP neuron activation increases circulating corticosterone concentrations, and reduction of hepatic glucocorticoid receptor expression attenuates AgRP neuron-dependent activation of hepatic autophagy. Collectively, our study reveals a fundamental regulatory principle of liver autophagy in control of metabolic adaptation during nutrient deprivation.
    Keywords:  AgRP neurons; CRH neurons; HPA axis; NPY1R; autophagy; corticosterone; hypothalamus; liver metabolism; non-cell autonomous; short-term fasting
    DOI:  https://doi.org/10.1016/j.cmet.2023.03.019
  27. Autophagy. 2023 Apr 18. 1-3
    Vesicle tethering and fusion promoted by LC3/GABARAP proteins is modulated by the ATG12-ATG5-ATG16L1 complex.
    Marina N Iriondo, Alicia Alonso.
      Recently, we have examined the membrane anchoring and subsequent lipidation of six members of the LC3/GABARAP protein family, together with their ability to promote membrane tethering and fusion. GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggested the existence of a lipidation threshold as a requisite for tethering and inter-vesicular lipid mixing. The presence of ATG12-ATG5-ATG16L1 (E3 in short) increased and accelerated LC3/GABARAP lipidation and subsequent vesicle tethering. However, E3 hampered LC3/GABARAP capacity to induce inter-vesicular lipid mixing and/or fusion. Our results suggest a model in which, together with the recently described inter-membrane lipid transfer mechanism, LC3/GABARAP could help in the phagophore expansion process through their ability to tether and fuse vesicles. The growing regions would be areas where the LC3/GABARAP proteins could be lipidated in the absence of E3, or else an independent regulatory mechanism would allow lipid/vesicle incorporation and phagophore growth when E3 was present.
    Keywords:  ATG12–ATG5-ATG16L1; ATG8; Autophagy proteins; E3 complex; LC3/GABARAP; autophagosome expansion
    DOI:  https://doi.org/10.1080/15548627.2023.2202557
  28. Sci Rep. 2023 Apr 19. 13(1): 6393
    Dentin defects caused by a Dspp-1 frameshift mutation are associated with the activation of autophagy.
    Tian Liang, Charles E Smith, Yuanyuan Hu, Hong Zhang, Chuhua Zhang, Qian Xu, Yongbo Lu, Ling Qi, Jan C-C Hu, James P Simmer.
      Dentin sialophosphoprotein (DSPP) is primarily expressed by differentiated odontoblasts (dentin-forming cells), and transiently expressed by presecretory ameloblasts (enamel-forming cells). Disease-causing DSPP mutations predominantly fall into two categories: 5' mutations affecting targeting and trafficking, and 3' - 1 frameshift mutations converting the repetitive, hydrophilic, acidic C-terminal domain into a hydrophobic one. We characterized the dental phenotypes and investigated the pathological mechanisms of DsppP19L and Dspp-1fs mice that replicate the two categories of human DSPP mutations. In DsppP19L mice, dentin is less mineralized but contains dentinal tubules. Enamel mineral density is reduced. Intracellular accumulation and ER retention of DSPP is observed in odontoblasts and ameloblasts. In Dspp-1fs mice, a thin layer of reparative dentin lacking dentinal tubules is deposited. Odontoblasts show severe pathosis, including intracellular accumulation and ER retention of DSPP, strong ubiquitin and autophagy activity, ER-phagy, and sporadic apoptosis. Ultrastructurally, odontoblasts show extensive autophagic vacuoles, some of which contain fragmented ER. Enamel formation is comparable to wild type. These findings distinguish molecular mechanisms underlying the dental phenotypes of DsppP19L and Dspp-1fs mice and support the recently revised Shields classification of dentinogenesis imperfecta caused by DSPP mutations in humans. The Dspp-1fs mice may be valuable for the study of autophagy and ER-phagy.
    DOI:  https://doi.org/10.1038/s41598-023-33362-1
  29. Front Neural Circuits. 2023 ;17 1099598
    Control of circadian rhythm on cortical excitability and synaptic plasticity.
    Claudia Lodovichi, Gian Michele Ratto.
      Living organisms navigate through a cyclic world: activity, feeding, social interactions are all organized along the periodic succession of night and day. At the cellular level, periodic activity is controlled by the molecular machinery driving the circadian regulation of cellular homeostasis. This mechanism adapts cell function to the external environment and its crucial importance is underlined by its robustness and redundancy. The cell autonomous clock regulates cell function by the circadian modulation of mTOR, a master controller of protein synthesis. Importantly, mTOR integrates the circadian modulation with synaptic activity and extracellular signals through a complex signaling network that includes the RAS-ERK pathway. The relationship between mTOR and the circadian clock is bidirectional, since mTOR can feedback on the cellular clock to shift the cycle to maintain the alignment with the environmental conditions. The mTOR and ERK pathways are crucial determinants of synaptic plasticity and function and thus it is not surprising that alterations of the circadian clock cause defective responses to environmental challenges, as witnessed by the bi-directional relationship between brain disorders and impaired circadian regulation. In physiological conditions, the feedback between the intrinsic clock and the mTOR pathway suggests that also synaptic plasticity should undergo circadian regulation.
    Keywords:  LTP; chloride homeostasis; circadian rhythm; mTOR; memory and learning; neuronal excitability
    DOI:  https://doi.org/10.3389/fncir.2023.1099598
  30. Biomed Pharmacother. 2023 Apr 13. pii: S0753-3322(23)00479-1. [Epub ahead of print]162 114691
    Doxorubicin downregulates autophagy to promote apoptosis-induced dilated cardiomyopathy via regulating the AMPK/mTOR pathway.
    Sheng Zhang, Xueping Wei, Haijin Zhang, Youping Wu, Junsong Jing, Rongrong Huang, Ting Zhou, Jingjin Hu, Yueguo Wu, Yuanyuan Li, Zhenqiang You.
      The broad-spectrum antineoplastic drug doxorubicin (DOX) has one of the most serious chronic side effects on the heart, dilated cardiomyopathy, but the precise molecular mechanisms underlying disease progression subsequent to long latency periods remain puzzling. Here, we established a model of DOX-induced dilated cardiomyopathy. In a cardiac cytology exploration, we found that differentially expressed genes in the KEGG signaling pathway enrichment provided a novel complex network of mTOR bridging autophagy and oxidative stress. Validation results showed that DOX caused intracellular reactive oxygen species accumulation in cardiomyocytes, disrupted mitochondria, led to imbalanced intracellular energy metabolism, and triggered cardiomyocyte apoptosis. Apoptosis showed a negative correlation with DOX-regulated cardiomyocyte autophagy. To evaluate whether the inhibition of mTOR could upregulate autophagy to protect cardiomyocytes, we used rapamycin to restore autophagy depressed by DOX. Rapamycin increased cardiomyocyte survival by easing the autophagic flux blocked by DOX. In addition, rapamycin reduced oxidative stress, prevented mitochondrial damage, and restored energy metabolic homeostasis in DOX-treated cardiomyocytes. In vivo, we used metformin (Met) which is an AMPK activator to protect cardiac tissue to alleviate DOX-induced dilated cardiomyopathy. In this study, Met significantly attenuated the oxidative stress response of myocardial tissue caused by DOX and activated cardiomyocyte autophagy to maintain cardiomyocyte energy metabolism and reduce cardiomyocyte apoptosis by downregulating mTOR activity. Overall, our study revealed the role of autophagy and apoptosis in DOX-induced dilated cardiomyopathy and demonstrated the potential role of regulation of the AMPK/mTOR axis in the treatment of DOX-induced dilated cardiomyopathy.
    Keywords:  AMPK; Apoptosis; Autophagy; Dilated cardiomyopathy; Doxorubicin; MTOR
    DOI:  https://doi.org/10.1016/j.biopha.2023.114691
  31. Biochim Biophys Acta Gene Regul Mech. 2023 Apr 17. pii: S1874-9399(23)00031-7. [Epub ahead of print] 194936
    Alzheimer's disease-associated mutant ubiquitin (UBB+1) is secreted through an autophagosome-like vesicle-mediated unconventional pathway.
    Ajay R Wagh, Prasad Sulakshane, Michael H Glickman.
      Misfolded protein aggregation at both intracellular and extracellular milieus is thought to be the major etiology of Alzheimer's disease (AD). UBB+1, a frameshift variant of the ubiquitin B gene (UBB) results in a folded ubiquitin domain fused to a flexible unstructured extension. Accumulation of UBB+1 in extracellular plaques in the brains of AD patients undoubtedly suggests a role of the ubiquitin-proteasome system in AD. However, the exact mechanism of extracellular secretion of UBB+1 remains unknown. In an attempt to understand the molecular mechanism of UBB+1 secretion, we performed a survey of secretory pathways and identified the involvement of unconventional autophagosome-mediated UBB+1 secretion. Expression of UBB+1 was sufficient to stimulate LC3B/Atg8 conversion from LC3B-I to LC3B-II, which indicates initiation of the autophagy pathway. Furthermore, deficiency of ATG5 - a key player in autophagosome formation - inhibited UBB+1 secretion. Based on immunofluorescence 3D structured illumination (SIM) microscopy and co-immunoprecipitation, we provide evidence that UBB+1 is associated with the secretory autophagosome marker, SEC22B, while HSP90 possibly acts as a carrier. Using LC-MS/MS and mutagenesis we found that in cells, UBB+1 is ubiquitinated on lysine 11, 29, and 48, however, this ubiquitination does not contribute to its secretion. By contrast, proteasome or lysosome inhibition slightly enhanced secretion. Taken together, this study suggests that by ridding cells of UBB+1, secretory autophagosomes may alleviate the cellular stress associated with UBB+1, yet simultaneously mediate the spreading of a mutant specie with disordered characteristics to the extracellular milieu.
    Keywords:  Autophagosome mediated secretion; Autophagy; HSP90; Lysosome; Mutant ubiquitin; Proteasome; SEC22B; UBB(+1); Ubiquitination
    DOI:  https://doi.org/10.1016/j.bbagrm.2023.194936
  32. Front Endocrinol (Lausanne). 2023 ;14 1141516
    The relationship between HMGB1 and autophagy in the pathogenesis of diabetes and its complications.
    Kun Yang, Feng Cao, Weili Wang, Zhenyu Tian, Lu Yang.
      Diabetes mellitus is a chronic metabolic disorder characterized by elevated blood glucose levels and has become the third leading threat to human health after cancer and cardiovascular disease. Recent studies have shown that autophagy is closely associated with diabetes. Under normal physiological conditions, autophagy promotes cellular homeostasis, reduces damage to healthy tissues and has bidirectional effects on regulating diabetes. However, under pathological conditions, unregulated autophagy activation leads to cell death and may contribute to the progression of diabetes. Therefore, restoring normal autophagy may be a key strategy to treat diabetes. High-mobility group box 1 protein (HMGB1) is a chromatin protein that is mainly present in the nucleus and can be actively secreted or passively released from necrotic, apoptotic, and inflammatory cells. HMGB1 can induce autophagy by activating various pathways. Studies have shown that HMGB1 plays an important role in insulin resistance and diabetes. In this review, we will introduce the biological and structural characteristics of HMGB1 and summarize the existing knowledge on the relationship between HMGB1, autophagy, diabetes, and diabetic complications. We will also summarize potential therapeutic strategies that may be useful for the prevention and treatment of diabetes and its complications.
    Keywords:  HMGB1; TCM; autophagy; diabetes; diabetic complication
    DOI:  https://doi.org/10.3389/fendo.2023.1141516
  33. Cell Death Differ. 2023 Apr 20.
    The TRAF2-p62 axis promotes proliferation and survival of liver cancer by activating mTORC1 pathway.
    Xue Liang, Jiping Yao, Danrui Cui, Weiyang Zheng, Yanning Liu, Guohua Lou, Bingjue Ye, Liyan Shui, Yi Sun, Yongchao Zhao, Min Zheng.
      TRAF2 (Tumor necrosis factor receptor-associated factor 2) is a dual function protein, acting as an adaptor protein and a ubiquitin E3 ligase, which plays an essential role in mediating the TNFα-NFκB signal pathway. Dysregulated expression of TRAF2 has been reported in a variety of human cancers. Whether and how TRAF2 regulates the growth of liver cancer cells remains elusive. The goal of this study is to investigate potential dysregulation of TRAF2 and its biological function in liver cancer, and to elucidate the underlying mechanism, leading to validation of TRAF2 as an attractive liver cancer target. Here, we reported TRAF2 is up-regulated in human liver cancer cell lines and tissues, and high TRAF2 expression is associated with a poor prognosis of HCC patients. Proteomics profiling along with Co-immunoprecipitation analysis revealed that p62 is a new substrate of TRAF2, which is subjected to TRAF2-induced polyubiquitination via the K63 linkage at the K420 residue. A strong negative correlation was found between the protein levels of p62 and TRAF2 in human HCC samples. TRAF2 depletion inhibited growth and survival of liver cancer cells both in vitro and in vivo by causing p62 accumulation, which is partially rescued by simultaneous p62 knockdown. Mechanistically, TRAF2-mediated p62 polyubiquitylation activates the mTORC1 by forming the p62-mTORC1-Rag complex, which facilitates the lysosome localization of mTORC1. TRAF2 depletion inhibited mTORC1 activity through the disruption of interaction between p62 and the mTORC1 complex. In conclusion, our study provides the proof-of-concept evidence that TRAF2 is a valid target for liver cancer.
    DOI:  https://doi.org/10.1038/s41418-023-01164-7
  34. Cell Mol Life Sci. 2023 Apr 22. 80(5): 129
    A neuroprotective role of Ufmylation through Atg9 in the aging brain of Drosophila.
    Huifang Li, Zhenghong Yu, Zikang Niu, Yun Cheng, Zhenhao Wei, Yafei Cai, Fei Ma, Lanxin Hu, Jiejie Zhu, Wei Zhang.
      Ufmylation is a recently identified small ubiquitin-like modification, whose biological function and relevant cellular targets are poorly understood. Here we present evidence of a neuroprotective role for Ufmylation involving Autophagy-related gene 9 (Atg9) during Drosophila aging. The Ufm1 system ensures the health of aged neurons via Atg9 by coordinating autophagy and mTORC1, and maintaining mitochondrial homeostasis and JNK (c-Jun N-terminal kinase) activity. Neuron-specific expression of Atg9 suppresses the age-associated movement defect and lethality caused by loss of Ufmylation. Furthermore, Atg9 is identified as a conserved target of Ufm1 conjugation mediated by Ddrgk1, a critical regulator of Ufmylation. Mammalian Ddrgk1 was shown to be indispensable for the stability of endogenous Atg9A protein in mouse embryonic fibroblast (MEF) cells. Taken together, our findings might have important implications for neurodegenerative diseases in mammals.
    Keywords:  Life span; Oxidative stress; Patj; Puc; Uba5; Ufl1
    DOI:  https://doi.org/10.1007/s00018-023-04778-9
  35. FASEB J. 2023 05;37(5): e22919
    SARS-CoV-2 ORF3a expression in brain disrupts the autophagy-lysosomal pathway, impairs sphingolipid homeostasis, and drives neuropathogenesis.
    Hongling Zhu, Colleen Byrnes, Y Terry Lee, Galina Tuymetova, Hannah B D Duffy, Jenna Y Bakir, Sydney N Pettit, Jabili Angina, Danielle A Springer, Maria L Allende, Mari Kono, Richard L Proia.
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes injury to multiple organ systems, including the brain. SARS-CoV-2's neuropathological mechanisms may include systemic inflammation and hypoxia, as well as direct cell damage resulting from viral infections of neurons and glia. How the virus directly causes injury to brain cells, acutely and over the long term, is not well understood. In order to gain insight into this process, we studied the neuropathological effects of open reading frame 3a (ORF3a), a SARS-CoV-2 accessory protein that is a key pathological factor of the virus. Forced ORF3a brain expression in mice caused the rapid onset of neurological impairment, neurodegeneration, and neuroinflammation-key neuropathological features found in coronavirus disease (COVID-19, which is caused by SARS-CoV-2 infection). Furthermore, ORF3a expression blocked autophagy progression in the brain and caused the neuronal accumulation of α-synuclein and glycosphingolipids, all of which are linked to neurodegenerative disease. Studies with ORF3-expressing HeLa cells confirmed that ORF3a disrupted the autophagy-lysosomal pathway and blocked glycosphingolipid degradation, resulting in their accumulation. These findings indicate that, in the event of neuroinvasion by SARS-CoV-2, ORF3a expression in brain cells may drive neuropathogenesis and be an important mediator of both short- and long-term neurological manifestations of COVID-19.
    DOI:  https://doi.org/10.1096/fj.202300149R
  36. Autophagy Rep. 2023 ;pii: 2166722. [Epub ahead of print]2(1):
    Upregulation of the ESCRT pathway and multivesicular bodies accelerates degradation of proteins associated with neurodegeneration.
    Ron Benyair, Sai Srinivas Panapakkam Giridharan, Pilar Rivero-Ríos, Junya Hasegawa, Emily Bristow, Eeva-Liisa Eskelinen, Merav D Shmueli, Vered Fishbain-Yoskovitz, Yifat Merbl, Lisa M Sharkey, Henry L Paulson, Phyllis I Hanson, Samarjit Patnaik, Ismael Al-Ramahi, Juan Botas, Juan Marugan, Lois S Weisman.
      Many neurodegenerative diseases, including Huntington's disease (HD) and Alzheimer's disease (AD), occur due to an accumulation of aggregation-prone proteins, which results in neuronal death. Studies in animal and cell models show that reducing the levels of these proteins mitigates disease phenotypes. We previously reported a small molecule, NCT-504, which reduces cellular levels of mutant huntingtin (mHTT) in patient fibroblasts as well as mouse striatal and cortical neurons from an HdhQ111 mutant mouse. Here, we show that NCT-504 has a broader potential, and in addition reduces levels of Tau, a protein associated with Alzheimer's disease, as well as other tauopathies. We find that in untreated cells, Tau and mHTT are degraded via autophagy. Notably, treatment with NCT-504 diverts these proteins to multivesicular bodies (MVB) and the ESCRT pathway. Specifically, NCT-504 causes a proliferation of endolysosomal organelles including MVB, and an enhanced association of mHTT and Tau with endosomes and MVB. Importantly, depletion of proteins that act late in the ESCRT pathway blocked NCT-504 dependent degradation of Tau. Moreover, NCT-504-mediated degradation of Tau occurred in cells where Atg7 is depleted, which indicates that this pathway is independent of canonical autophagy. Together, these studies reveal that upregulation of traffic through an ESCRT-dependent MVB pathway may provide a therapeutic approach for neurodegenerative diseases.
    Keywords:  ESCRT; Huntington; NCT-504; Neurodegeneration; Tau; huntingtin; multivesicular bodies (MVB)
    DOI:  https://doi.org/10.1080/27694127.2023.2166722
  37. PLoS One. 2023 ;18(4): e0284636
    Knockout of the neonatal Fc receptor alters immune complex trafficking and lysosomal function in cultured podocytes.
    George Haddad, James Dylewski, River Evans, Linda Lewis, Judith Blaine.
      Podocytes are key to preventing the filtration of serum proteins into the urine. Recent evidence also suggests that in immune mediated kidney diseases, podocytes are the targets of immune complexes (ICs). The mechanisms whereby podocytes handle and respond to ICs remain unknown. The neonatal Fc receptor (FcRn) is involved in IgG handling in podocytes and is also required in dendritic cells to traffic ICs to the lysosome for proteolytic degradation of antigen and presentation on MHC II. Here we examine the role of FcRn in handling ICs in podocytes. We show that knockout of FcRn in podocytes results in decreased trafficking of ICs to the lysosome and increases IC trafficking to recycling endosomes. FcRn KO also alters lysosomal distribution, decreases lysosomal surface area and decreases cathepsin B expression and activity. We demonstrate that signaling pathways in cultured podocytes differ after treatment with IgG alone versus ICs and that podocyte proliferation in both WT and KO podocytes is suppressed by IC treatment. Our findings suggest that podocytes respond differentially to IgG versus ICs and that FcRn modifies the lysosomal response to ICs. Elucidating the mechanisms underlying podocyte handling of ICs may provide novel pathways to modulate immune mediated kidney disease progression.
    DOI:  https://doi.org/10.1371/journal.pone.0284636
  38. Korean J Physiol Pharmacol. 2023 May 01. 27(3): 231-240
    Lyso-globotriaosylsphingosine induces endothelial dysfunction via autophagy-dependent regulation of necroptosis.
    Ae-Rang Hwang, Seonghee Park, Chang-Hoon Woo.
      Fabry disease is a lysosomal storage disorder characterized by the lysosomal accumulations of glycosphingolipids in a variety of cytotypes, which include endothelial cells. The disease is inherited and originates from an error in glycosphingolipid catabolism caused by insufficient α-galactosidase A activity, which causes uncontrolled progressive storage of intracellular globotriaosylceramide (Gb3) in the vasculature and extracellular accumulation of lyso-Gb3 (a deacetylated soluble form of Gb3). Necrosis can lead to inflammation, which exacerbates necrosis and creates a positive feedback loop that triggers necroinflammation. However, the role played by necroptosis, a form of programmed necrotic cell death, in the cell-to-cell inflammatory reaction between epithelial and endothelial cells is unclear. Thus, the present study was undertaken to determine whether lyso-Gb3 induces necroptosis and whether necroptosis inhibition protects endothelial dysfunction against lyso-Gb3 inflamed retinal pigment epithelial cells. We found lyso-Gb3 induced necroptosis of a retinal pigment epithelial cell line (ARPE-19) in an autophagy-dependent manner and that conditioned media (CM) from ARPE-19 cells treated with lyso-Gb3 induced the necroptosis, inflammation, and senescence of human umbilical vein endothelial cells. In addition, a pharmacological study showed CM from lyso-Gb3 treated ARPE-19 cells induced endothelial necroptosis, inflammation, and senescence were significantly inhibited by an autophagy inhibitor (3-MA) and by two necroptosis inhibitors (necrostatin and GSK-872), respectively. These results demonstrate lyso-Gb3 induces necroptosis via autophagy and suggest that lyso-Gb3 inflamed retinal pigment epithelial cells trigger endothelial dysfunction via the autophagy-dependent necroptosis pathway. This study suggests the involvement of a novel autophagy-dependent necroptosis pathway in the regulation of endothelial dysfunction in Fabry disease.
    Keywords:  Autophagy; Cellular senescence; Glycosphingolipids; Inflammation; Necroptosis
    DOI:  https://doi.org/10.4196/kjpp.2023.27.3.231
  39. Elife. 2023 Apr 18. pii: e86030. [Epub ahead of print]12
    The autophagy receptor NBR1 directs the clearance of photodamaged chloroplasts.
    Han Nim Lee, Jenu Chacko, Ariadna Gonzalez Solís, Kuo-En Chen, Jessica A S Barros, Santiago Signorelli, A Harvey Millar, Richard David Vierstra, Kevin W Eliceiri, Marisa S Otegui.
      The ubiquitin-binding NBR1 autophagy receptor plays a prominent role in recognizing ubiquitylated protein aggregates for vacuolar degradation by macroautophagy. Here, we show that upon exposing Arabidopsis plants to intense light, NBR1 associates with photodamaged chloroplasts independently of ATG7, a core component of the canonical autophagy machinery. NBR1 coats both the surface and interior of chloroplasts, which is then followed by direct engulfment of the organelles into the central vacuole via a microautophagy-type process. The relocalization of NBR1 into chloroplasts does not require the chloroplast translocon complexes embedded in the envelope but is instead greatly enhanced by removing the self-oligomerization mPB1 domain of NBR1. The delivery of NBR1-decorated chloroplasts into vacuoles depends on the ubiquitin-binding UBA2 domain of NBR1 but is independent of the ubiquitin E3 ligases SP1 and PUB4, known to direct the ubiquitylation of chloroplast surface proteins. Compared to wild-type plants, nbr1 mutants have altered levels of a subset of chloroplast proteins and display abnormal chloroplast density and sizes upon high light exposure. We postulate that, as photodamaged chloroplasts lose envelope integrity, cytosolic ligases reach the chloroplast interior to ubiquitylate thylakoid and stroma proteins which are then recognized by NBR1 for autophagic clearance. This study uncovers a new function of NBR1 in the degradation of damaged chloroplasts by microautophagy.
    Keywords:  A. thaliana; cell biology; plant biology
    DOI:  https://doi.org/10.7554/eLife.86030
  40. Front Microbiol. 2023 ;14 1152249
    Severe acute respiratory syndrome coronavirus-2 accessory proteins ORF3a and ORF7a modulate autophagic flux and Ca2+ homeostasis in yeast.
    José Luis Garrido-Huarte, Josep Fita-Torró, Rosa Viana, Amparo Pascual-Ahuir, Markus Proft.
      Virus infection involves the manipulation of key host cell functions by specialized virulence proteins. The Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) small accessory proteins ORF3a and ORF7a have been implicated in favoring virus replication and spreading by inhibiting the autophagic flux within the host cell. Here, we apply yeast models to gain insights into the physiological functions of both SARS-CoV-2 small open reading frames (ORFs). ORF3a and ORF7a can be stably overexpressed in yeast cells, producing a decrease in cellular fitness. Both proteins show a distinguishable intracellular localization. ORF3a localizes to the vacuolar membrane, whereas ORF7a targets the endoplasmic reticulum. Overexpression of ORF3a and ORF7a leads to the accumulation of Atg8 specific autophagosomes. However, the underlying mechanism is different for each viral protein as assessed by the quantification of the autophagic degradation of Atg8-GFP fusion proteins, which is inhibited by ORF3a and stimulated by ORF7a. Overexpression of both SARS-CoV-2 ORFs decreases cellular fitness upon starvation conditions, where autophagic processes become essential. These data confirm previous findings on SARS-CoV-2 ORF3a and ORF7a manipulating autophagic flux in mammalian cell models and are in agreement with a model where both small ORFs have synergistic functions in stimulating intracellular autophagosome accumulation, ORF3a by inhibiting autophagosome processing at the vacuole and ORF7a by promoting autophagosome formation at the ER. ORF3a has an additional function in Ca2+ homeostasis. The overexpression of ORF3a confers calcineurin-dependent Ca2+ tolerance and activates a Ca2+ sensitive FKS2-luciferase reporter, suggesting a possible ORF3a-mediated Ca2+ efflux from the vacuole. Taken together, we show that viral accessory proteins can be functionally investigated in yeast cells and that SARS-CoV-2 ORF3a and ORF7a proteins interfere with autophagosome formation and processing as well as with Ca2+ homeostasis from distinct cellular targets.
    Keywords:  Ca2+ homeostasis; ORF3a; ORF7a; SARS-CoV-2; Saccharomyces cerevisiae; autophagy
    DOI:  https://doi.org/10.3389/fmicb.2023.1152249
  41. Autophagy Rep. 2023 ;pii: 2149211. [Epub ahead of print]2(1):
    Autophagy in protists and their hosts: When, how and why?
    Patricia Silvia Romano, Takahiko Akematsu, Sébastien Besteiro, Annina Bindschedler, Vern B Carruthers, Zeinab Chahine, Isabelle Coppens, Albert Descoteaux, Thabata Lopes Alberto Duque, Cynthia Y He, Volker Heussler, Karine G Le Roch, Feng-Jun Li, Juliana Perrone Bezerra de Menezes, Rubem Figueiredo Sadok Menna-Barreto, Jeremy C Mottram, Jacqueline Schmuckli-Maurer, Boris Turk, Patricia Sampaio Tavares Veras, Betiana Nebai Salassa, María Cristina Vanrell.
      Pathogenic protists are a group of organisms responsible for causing a variety of human diseases including malaria, sleeping sickness, Chagas disease, leishmaniasis, and toxoplasmosis, among others. These diseases, which affect more than one billion people globally, mainly the poorest populations, are characterized by severe chronic stages and the lack of effective antiparasitic treatment. Parasitic protists display complex life-cycles and go through different cellular transformations in order to adapt to the different hosts they live in. Autophagy, a highly conserved cellular degradation process, has emerged as a key mechanism required for these differentiation processes, as well as other functions that are crucial to parasite fitness. In contrast to yeasts and mammals, protist autophagy is characterized by a modest number of conserved autophagy-related proteins (ATGs) that, even though, can drive the autophagosome formation and degradation. In addition, during their intracellular cycle, the interaction of these pathogens with the host autophagy system plays a crucial role resulting in a beneficial or harmful effect that is important for the outcome of the infection. In this review, we summarize the current state of knowledge on autophagy and other related mechanisms in pathogenic protists and their hosts. We sought to emphasize when, how, and why this process takes place, and the effects it may have on the parasitic cycle. A better understanding of the significance of autophagy for the protist life-cycle will potentially be helpful to design novel anti-parasitic strategies.
    Keywords:  Autophagy; Biological cycle; Leishmania sp.; Plasmodium sp.; Protists; Toxoplasma gondii; Trypanosoma brucei; Trypanosoma cruzi
    DOI:  https://doi.org/10.1080/27694127.2022.2149211
  42. Front Pharmacol. 2023 ;14 1162045
    Non-coding RNAs: The recently accentuated molecules in the regulation of cell autophagy for ovarian cancer pathogenesis and therapeutic response.
    Bi Peng, Jing Li, Yuanliang Yan, Yuanhong Liu, Qiuju Liang, Wei Liu, Abhimanyu Thakur, Kui Zhang, Zhijie Xu, Jian Wang, Fan Zhang.
      Autophagy is a self-recycling and conserved process, in which the senescent cytoplasmic components are degraded in cells and then recycled to maintain homeostatic balance. Emerging evidence has suggested the involvement of autophagy in oncogenesis and progression of various cancers, such as ovarian cancer (OC). Meanwhile, the non-coding RNAs (ncRNAs) frequently regulate the mRNA transcription and other functional signaling pathways in cell autophagy, displaying promising roles in human cancer pathogenesis and therapeutic response. This article mainly reviews the cutting-edge research advances about the interactions between ncRNAs and autophagy in OC. This review not only summarizes the underlying mechanisms of dynamic ncRNA-autophagy association in OC, but also discusses their prognostic implications and therapeutic biomarkers. The aim of this review was to provide a more in-depth knowledge framework exploring the ncRNA-autophagy crosstalk and highlight the promising treatment strategies for OC patients.
    Keywords:  cancer pathogenesis; cell autophagy; non-coding RNAs; ovarian cancer; prognosis
    DOI:  https://doi.org/10.3389/fphar.2023.1162045
  43. J Clin Invest. 2023 Apr 17. pii: e169240. [Epub ahead of print]133(8):
    Lysosomal lipid peroxidation mediates immunogenic cell death.
    Pravin Phadatare, Jayanta Debnath.
      Cancer cells rely on lysosome-dependent degradation to recycle nutrients that serve their energetic and biosynthetic needs. Despite great interest in repurposing the antimalarial hydroxychloroquine as a lysosomal inhibitor in clinical oncology trials, the mechanisms by which hydroxychloroquine and other lysosomal inhibitors induce tumor-cell cytotoxicity remain unclear. In this issue of the JCI, Bhardwaj et al. demonstrate that DC661, a dimeric form of chloroquine that inhibits palmitoyl-protein thioesterase 1 (PPT1), promoted lysosomal lipid peroxidation, resulting in lysosomal membrane permeabilization and tumor cell death. Remarkably, this lysosomal cell death pathway elicited cell-intrinsic immunogenicity and promoted T lymphocyte-mediated tumor cell clearance. The findings provide the mechanistic foundation for the potential combined use of immunotherapy and lysosomal inhibition in clinical trials.
    DOI:  https://doi.org/10.1172/JCI169240
  44. Plant Physiol. 2023 Apr 19. pii: kiad235. [Epub ahead of print]
    Cotton Leaf Curl Multan virus C4 protein suppresses autophagy to facilitate viral infection.
    Meng Yang, Asigul Ismayil, Teng Gao, Zihan Ye, Ning Yue, Jie Wu, Xiyin Zheng, Yiqing Li, Yan Wang, Yiguo Hong, Yule Liu.
      Autophagy plays an important role in plant antiviral defense. Several plant viruses are reported to encode viral suppressor of autophagy (VSA) to prevent autophagy for effective virus infection. However, whether and how other viruses, in particular DNA viruses, also encode VSAs to affect viral infection in plants is unknown. Here, we report that the C4 protein encoded by Cotton leaf curl Multan geminivirus (CLCuMuV) inhibits autophagy by binding to the autophagy negative regulator eukaryotic translation initiation factor 4A (eIF4A) to enhance the eIF4A - Autophagy-related protein 5 (ATG5) interaction. By contrast, the R54A or R54K mutation in C4 abolishes its capacity to interact with eIF4A, and neither C4R54A nor C4R54K can suppress autophagy. However, the R54 residue is not essential for C4 to interfere with transcriptional gene silencing or post-transcriptional gene silencing. Moreover, plants infected with mutated CLCuMuV-C4R54K develop less severe symptoms with decreased levels of viral DNA. These findings reveal a molecular mechanism underlying how the DNA virus CLCuMuV deploys a VSA to subdue host cellular antiviral autophagy defense and uphold viral infection in plants.
    DOI:  https://doi.org/10.1093/plphys/kiad235
  45. Int J Biol Sci. 2023 ;19(6): 1831-1845
    Interaction between dual specificity phosphatase-1 and cullin-1 attenuates alcohol-related liver disease by restoring p62-mediated mitophagy.
    Ruibing Li, Zhe Dai, Xiaoman Liu, Chunling Wang, Jia Huang, Ting Xin, Ying Tong, Yijin Wang.
      Besides abstinence, no effective treatment exists for alcohol-related liver disease (ALD), a dreaded consequence of alcohol abuse. In this study, we assessed the roles on ALD of dual specificity phosphatase-1 (DUSP1), an hepatoprotective enzyme, and Cullin-1 (CUL1), a member of the E3 ubiquitin ligase complex that exerts also transcriptional suppression of mitochondrial genes. Alcohol treatment downregulated hepatic DUSP1 expression in wild-type mice. Notably, DUSP1 transgenic (Dusp1Tg ) mice showed resistance to alcohol-mediated hepatic dysfunction, as evidenced by decreased AST/ALT activity, improved alcohol metabolism, and suppressed liver fibrosis, inflammation, and oxidative stress. Functional experiments demonstrated that DUSP1 overexpression prevents alcohol-mediated mitochondrial damage in hepatocytes through restoring mitophagy. Accordingly, pharmacological blockade of mitophagy abolished the hepatoprotective actions of DUSP1. Molecular assays showed that DUSP1 binds cytosolic CUL1 and prevents its translocation to the nucleus. Importantly, CUL1 silencing restored the transcription of p62 and Parkin, resulting in mitophagy activation, and sustained mitochondrial integrity and hepatocyte function upon alcohol stress. These results indicate that alcohol-mediated DUSP1 downregulation interrupts DUSP1/CUL1 interaction, leading to CUL1 nuclear translocation and mitophagy inhibition via transcriptional repression of p62 and Parkin. Thus, targeting the DUSP1/CUL1/p62 axis will be a key approach to restore hepatic mitophagy as well as alleviate symptoms of ALD.
    Keywords:  Alcohol-related liver disease; CUL1; DUSP1; mitophagy
    DOI:  https://doi.org/10.7150/ijbs.81447
  46. FASEB J. 2023 May;37(5): e22930
    Pathogenic LRRK2 compromises the subcellular distribution of lysosomes in a Rab12-RILPL1-dependent manner.
    Kyohei Ito, Miho Araki, Yuta Katai, Yuki Nishimura, Sota Imotani, Haruki Inoue, Genta Ito, Taisuke Tomita.
      Mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial Parkinson's disease (PD). Recent studies have shown that LRRK2 physiologically phosphorylates several Rab family proteins including Rab12 and that this phosphorylation is accelerated by the pathogenic mutations in LRRK2, although the significance in the PD pathogenesis remains unknown. Here we examined the effect of the overexpression of LRRK2 on the distribution of organelles in cultured cells and found that lysosomes become clustered in a perinuclear region upon the overexpression of pathogenic mutant LRRK2 in a manner dependent on its kinase activity. The perinuclear clustering of lysosomes was abolished by knocking out RAB12 as well as its effector protein RILPL1. Re-expression of Rab12 in RAB12 knockout cells suggested that the phosphorylation at Ser106 of Rab12 is required for the perinuclear clustering of lysosomes. Moreover, phosphorylated Rab12 was also accumulated on the clustered lysosomes, and the phosphorylation of Rab12 increased its interaction with RILPL1, leading us to conclude that the increase in the phosphorylation of Rab12 by pathogenic LRRK2 compromised intracellular lysosomal transport via the enhanced interaction of Rab12 with RILPL1. These data suggest the involvement of abnormal regulation of lysosomal transport in the LRRK2-mediated pathogenesis of PD.
    Keywords:  Rab-interacting lysosomal protein-like 1; Rab12; leucine-rich repeat kinase 2; lysosome
    DOI:  https://doi.org/10.1096/fj.202200780RR
  47. Life Sci. 2023 Apr 17. pii: S0024-3205(23)00339-9. [Epub ahead of print]323 121705
    Cellular battle against endoplasmic reticulum stress and its adverse effect on health.
    Subramaniyan Divya, Palaniyandi Ravanan.
      The endoplasmic reticulum (ER) is a dynamic organelle and a reliable performer for precisely folded proteins. To maintain its function and integrity, arrays of sensory and quality control systems enhance protein folding fidelity and resolve the highest error-prone areas. Yet numerous internal and external factors disrupt its homeostasis and trigger ER stress responses. Cells try to reduce the number of misfolded proteins via the UPR mechanism, and ER-related garbage disposals systems like ER-associated degradation (ERAD), ER-lysosome-associated degradation (ERLAD), ER-Associated RNA Silencing (ERAS), extracellular chaperoning, and autophagy systems, which activates and increase the cell survival rate by degrading misfolded proteins, prevent the aggregated proteins and remove the dysfunctional organelles. Throughout life, organisms must confront environmental stress to survive and develop. Communication between the ER & other organelles, signaling events mediated by calcium, reactive oxygen species, and inflammation are linked to diverse stress signaling pathways and regulate cell survival or cell death mechanisms. Unresolved cellular damages can cross the threshold limit of their survival, resulting in cell death or driving for various diseases. The multifaceted ability of unfolded protein response facilitates the therapeutic target and a biomarker for various diseases, helping with early diagnosis and detecting the severity of diseases.
    Keywords:  Biomarkers; ER-associated degradation; ER-phagy; ER-to-lysosome-associated degradation; ERAS; Terminal UPR
    DOI:  https://doi.org/10.1016/j.lfs.2023.121705
  48. Nat Commun. 2023 Apr 20. 14(1): 2174
    Parkinson's disease-associated ATP13A2/PARK9 functions as a lysosomal H+,K+-ATPase.
    Takuto Fujii, Shushi Nagamori, Pattama Wiriyasermkul, Shizhou Zheng, Asaka Yago, Takahiro Shimizu, Yoshiaki Tabuchi, Tomoyuki Okumura, Tsutomu Fujii, Hiroshi Takeshima, Hideki Sakai.
      Mutations in the human ATP13A2 (PARK9), a lysosomal ATPase, cause Kufor-Rakeb Syndrome, an early-onset form of Parkinson's disease (PD). Here, we demonstrate that ATP13A2 functions as a lysosomal H+,K+-ATPase. The K+-dependent ATPase activity and the lysosomal K+-transport activity of ATP13A2 are inhibited by an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase, thapsigargin, and K+-competitive inhibitors of gastric H+,K+-ATPase, such as vonoprazan and SCH28080. Interestingly, these H+,K+-ATPase inhibitors cause lysosomal alkalinization and α-synuclein accumulation, which are pathological hallmarks of PD. Furthermore, PD-associated mutants of ATP13A2 show abnormal expression and function. Our results suggest that the H+/K+-transporting function of ATP13A2 contributes to acidification and α-synuclein degradation in lysosomes.
    DOI:  https://doi.org/10.1038/s41467-023-37815-z
  49. bioRxiv. 2023 Apr 03. pii: 2023.04.03.534570. [Epub ahead of print]
    SKA2 regulated hyperactive secretory autophagy drives neuroinflammation-induced neurodegeneration.
    Jakob Hartmann, Thomas Bajaj, Joy Otten, Claudia Klengel, Anne-Kathrin Gellner, Ellen Junglas, Kathrin Hafner, Elmira A Anderzhanova, Fiona Tang, Galen Missig, Lindsay Rexrode, Katelyn Li, Max L Pöhlmann, Daniel E Heinz, Roy Lardenoije, Nina Dedic, Kenneth M McCullough, Tomasz Próchnicki, Thomas Rhomberg, Silvia Martinelli, Antony Payton, Andrew C Robinson, Valentin Stein, Eicke Latz, William A Carlezon, Mathias V Schmidt, Chris Murgatroyd, Sabina Berretta, Torsten Klengel, Harry Pantazopoulos, Kerry J Ressler, Nils C Gassen.
      High levels of proinflammatory cytokines induce neurotoxicity and catalyze inflammation-driven neurodegeneration, but the specific release mechanisms from microglia remain elusive. We demonstrate that secretory autophagy (SA), a non-lytic modality of autophagy for secretion of vesicular cargo, regulates neuroinflammation-mediated neurodegeneration via SKA2 and FKBP5 signaling. SKA2 inhibits SA-dependent IL-1β release by counteracting FKBP5 function. Hippocampal Ska2 knockdown in mice hyperactivates SA resulting in neuroinflammation, subsequent neurodegeneration and complete hippocampal atrophy within six weeks. The hyperactivation of SA increases IL-1β release, initiating an inflammatory feed-forward vicious cycle including NLRP3-inflammasome activation and Gasdermin D (GSDMD)-mediated neurotoxicity, which ultimately drives neurodegeneration. Results from protein expression and co-immunoprecipitation analyses of postmortem brains demonstrate that SA is hyperactivated in Alzheimer's disease. Overall, our findings suggest that SKA2-regulated, hyperactive SA facilitates neuroinflammation and is linked to Alzheimer's disease, providing new mechanistic insight into the biology of neuroinflammation.
    DOI:  https://doi.org/10.1101/2023.04.03.534570
  50. Blood Adv. 2023 Apr 21. pii: bloodadvances.2022008241. [Epub ahead of print]
    TRIM21 enhances bortezomib sensitivity in multiple myeloma by halting prosurvival autophagy.
    Jing Chen, Wen Cao, Xi Huang, Qingxiao Chen, Shuting Ye, Jianwei Qu, Yang Liu, Xing Guo, Shunnan Yao, Enfan Zhang, Jingsong He, Anqi Li, Li Yang, Zhen Cai.
      Bortezomib (bort) is an effective therapeutic agent for multiple myeloma (MM) patients, however, the majority of patients develop drug resistance. Autophagy, a highly conserved process that recycles cytosol or entire organelles via lysosomal activity, is essential for the survival, homeostasis and drug resistance in MM. Growing evidence has highlighted the E3 ligase tripartite motif-containing protein 21 (TRIM21) not only interacts with multiple autophagy regulators but also participates in drug resistance in various cancers. However, to date, the direct substrates and additional roles of TRIM21 in MM remain unknown. In this study, we demonstrated that low TRIM21 expression was a factor for relapse in MM. TRIM21 knockdown (KD) made MM cells more resistant to bort, while TRIM21 overexpression (OE) resulted in increased MM sensitivity to bort. Proteomic and phospho-proteomic studies of TRIM21 KD MM cells showed bort resistance was associated with increased oxidative stress and elevated pro-survival autophagy. Our results provided that TRIM21 KD MM cell lines induced pro-survival autophagy after bort treatment, and suppressing autophagy by 3-methyladenine treatment or by the short hairpin RNA of ATG5 restored bort sensitivity. Indeed, autophagy-related gene 5 (ATG5) expression was increased and decreased by TRIM21 KD and OE, respectively. TRIM21 affected autophagy by ubiquitinating ATG5 through K48 for proteasomal degradation. Importantly, we confirmed that TRIM21 could potentiate the anti-myeloma effect of bort through in vitro and in vivo experiments. Overall, our findings define the key role for TRIM21 in MM bort resistance and provide the basis for a novel targeted therapeutic approach.
    DOI:  https://doi.org/10.1182/bloodadvances.2022008241
  51. Toxicology. 2023 Apr 14. pii: S0300-483X(23)00098-7. [Epub ahead of print] 153512
    Pretreatment of 3-MA prevents doxorubicin-induced cardiotoxicity through inhibition of autophagy initiation.
    Xiaofan Sun, Heng Meng, Jia Xiao, Fangshu Liu, Juan Du, Hui Zeng.
      Anthracycline antineoplastics are effective in the treatment of hematological malignancies and solid tumors. However, the anthracycline-induced cardiotoxicity (AIC) limits their use as chemotherapeutic agents. Autophagy-based therapies have been explored to prevent AIC. Yet, whether inhibition of autophagy during its early stage could alleviate AIC remains unclear. In this study, we firstly observed the activation of autophagy during AIC in both cardiomyocyte cell lines AC16 and H9c2. Moreover, knockdown of Atg7, a key regulatory factor in early autophagy, could ameliorate the effects of DOX-induced AIC. Importantly, the use of early autophagy inhibitor 3-MA protected cardiomyocyte cells from DOX-induced cardiotoxicity in vitro and in a chronic AIC mouse model. Our findings demonstrate that inhibiting early stage of autophagy may be an effective preventative therapeutic strategy to protect cardiac function from AIC.
    Keywords:  3-MA; Atg7; Autophagy; Cardiotoxicity; Doxorubicin
    DOI:  https://doi.org/10.1016/j.tox.2023.153512
  52. Metabolomics. 2023 Apr 19. 19(5): 43
    A ketogenic diet alters mTOR activity, systemic metabolism and potentially prevents collagen degradation associated with chronic alcohol consumption in mice.
    Luciano Willemse, Karin Terburgh, Roan Louw.
       INTRODUCTION: A ketogenic diet (KD), which is a high fat, low carbohydrate diet has been shown to inhibit the mammalian target of rapamycin (mTOR) pathway and alter the redox state. Inhibition of the mTOR complex has been associated with the attenuation and alleviation of various metabolic and- inflammatory diseases such as neurodegeneration, diabetes, and metabolic syndrome. Various metabolic pathways and signalling mechanisms have been explored to assess the therapeutic potential of mTOR inhibition. However, chronic alcohol consumption has also been reported to alter mTOR activity, the cellular redox- and inflammatory state. Thus, a relevant question that remains is what effect chronic alcohol consumption would have on mTOR activity and overall metabolism during a KD-based intervention.
    OBJECTIVES: The aim of this study was to evaluate the effect of alcohol and a KD on the phosphorylation of the mTORC1 target p70S6K, systemic metabolism as well as the redox- and inflammatory state in a mouse model.
    METHODS: Mice were fed either a control diet with/without alcohol or a KD with/without alcohol for three weeks. After the dietary intervention, samples were collected and subjected towards western blot analysis, multi-platform metabolomics analysis and flow cytometry.
    RESULTS: Mice fed a KD exhibited significant mTOR inhibition and reduction in growth rate. Alcohol consumption alone did not markedly alter mTOR activity or growth rate but moderately increased mTOR inhibition in mice fed a KD. In addition, metabolic profiling showed alteration of several metabolic pathways as well as the redox state following consumption of a KD and alcohol. A KD was also observed to potentially prevent bone loss and collagen degradation associated with chronic alcohol consumption, as indicated by hydroxyproline metabolism.
    CONCLUSION: This study sheds light on the influence that a KD alongside alcohol intake can exert on not just mTOR, but also their effect on metabolic reprogramming and the redox state.
    Keywords:  Ketogenic diet; Metabolic reprogramming; Metabolomics; Mtor; Redox; Western blot
    DOI:  https://doi.org/10.1007/s11306-023-02006-w
  53. Sci Adv. 2023 04 21. 9(16): eadf8966
    An SPNS1-dependent lysosomal lipid transport pathway that enables cell survival under choline limitation.
    Samantha G Scharenberg, Wentao Dong, Ali Ghoochani, Kwamina Nyame, Roni Levin-Konigsberg, Aswini R Krishnan, Eshaan S Rawat, Kaitlyn Spees, Michael C Bassik, Monther Abu-Remaileh.
      Lysosomes degrade macromolecules and recycle their nutrient content to support cell function and survival. However, the machineries involved in lysosomal recycling of many nutrients remain to be discovered, with a notable example being choline, an essential metabolite liberated via lipid degradation. Here, we engineered metabolic dependency on lysosome-derived choline in pancreatic cancer cells to perform an endolysosome-focused CRISPR-Cas9 screen for genes mediating lysosomal choline recycling. We identified the orphan lysosomal transmembrane protein SPNS1 as critical for cell survival under choline limitation. SPNS1 loss leads to intralysosomal accumulation of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE). Mechanistically, we reveal that SPNS1 is a proton gradient-dependent transporter of LPC species from the lysosome for their re-esterification into phosphatidylcholine in the cytosol. Last, we establish that LPC efflux by SPNS1 is required for cell survival under choline limitation. Collectively, our work defines a lysosomal phospholipid salvage pathway that is essential under nutrient limitation and, more broadly, provides a robust platform to deorphan lysosomal gene function.
    DOI:  https://doi.org/10.1126/sciadv.adf8966
  54. J Biol Chem. 2023 Apr 17. pii: S0021-9258(23)01751-9. [Epub ahead of print] 104723
    HRD1 Promotes Non-small Cell Lung Carcinoma Metastasis by Blocking Autophagy-mediated MIEN1 Degradation.
    Cheng Zeng, Jing Guo, Jiajia Wu, Tiantian Che, Xiaoping Huang, Huawen Liu, Zhenghong Lin.
      Dysregulation of autophagy has been implicated in the development of many diseases, including cancer. Here, we revealed a novel function of the E3 ubiquitin ligase HRD1 in non-small cell lung carcinoma (NSCLC) metastasis by regulating autophagy. Mechanistically, HRD1 inhibits autophagy by promoting ATG3 ubiquitination and degradation. Additionally, a pro-migratory and invasive factor, MIEN1 (migration and invasion enhancer 1), was found to be autophagically degraded upon HRD1 deficiency. Importantly, both HRD1 and MIEN1 expression are upregulated and positively correlated in lung tumors. Based on these results, we proposed a novel mechanism of HRD1 function that the degradation of ATG3 protein by HRD1 leads to autophagy inhibition and MIEN1 release, thus promoting NSCLC metastasis. Therefore, our findings provided new insights into the role of HRD1 in NSCLC metastasis and new therapeutic targets for lung cancer treatment.
    Keywords:  ATG3; HRD1; MIEN1; NSCLC metastasis; autophagy
    DOI:  https://doi.org/10.1016/j.jbc.2023.104723
  55. Wellcome Open Res. 2022 ;7 267
    Defective iron homeostasis and hematological abnormalities in Niemann-Pick disease type C1.
    Oscar C W Chen, Stephan Siebel, Alexandria Colaco, Elena-Raluca Nicoli, Nick Platt, Dawn Shepherd, Stephanie Newman, Andrew E Armitage, Nicole Y Farhat, George Seligmann, Claire Smith, David A Smith, Alaa Abdul-Sada, Mylvaganam Jeyakumar, Hal Drakesmith, Forbes D Porter, Frances M Platt.
      Background: Niemann-Pick disease type C1 (NPC1) is a neurodegenerative lysosomal storage disorder characterized by the accumulation of multiple lipids in the late endosome/lysosomal system and reduced acidic store calcium. The lysosomal system regulates key aspects of iron homeostasis, which prompted us to investigate whether there are hematological abnormalities and iron metabolism defects in NPC1. Methods: Iron-related hematological parameters, systemic and tissue metal ion and relevant hormonal and proteins levels, expression of specific pro-inflammatory mediators and erythrophagocytosis were evaluated in an authentic mouse model and in a large cohort of NPC patients. Results: Significant changes in mean corpuscular volume and corpuscular hemoglobin were detected in Npc1-/- mice from an early age. Hematocrit, red cell distribution width and hemoglobin changes were observed in late-stage disease animals. Systemic iron deficiency, increased circulating hepcidin, decreased ferritin and abnormal pro-inflammatory cytokine levels were also found. Furthermore, there is evidence of defective erythrophagocytosis in Npc1 -/- mice and in an in vitro NPC1 cellular model. Comparable hematological changes, including low normal serum iron and transferrin saturation and low cerebrospinal fluid ferritin were confirmed in NPC1 patients. Conclusions: These data suggest loss of iron homeostasis and hematological abnormalities in NPC1 may contribute to the pathophysiology of this disease.
    Keywords:  Niemann-Pick disease type C; haematology; iron; lysosomal storage diseases; lysosome
    DOI:  https://doi.org/10.12688/wellcomeopenres.17261.1
  56. J Biol Chem. 2023 Apr 13. pii: S0021-9258(23)01737-4. [Epub ahead of print] 104709
    MicroRNA-593-5p contributes to cell death following exposure to 1-methyl-4-phenylpyridinium (MPP+) by targeting PTEN-induced putative kinase 1 (PINK1).
    Myungsik Yoo, Doo Chul Choi, Aleta Murphy, Atiq M Ahsan, Eunsung Junn.
      Neurodegenerative diseases are characterized by a decline in neuronal function and structure, leading to neuronal death. Understanding the molecular mechanisms of neuronal death is crucial for developing therapeutics. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression by degrading target mRNAs or inhibiting translation. MiR dysregulation has been linked to many neurodegenerative diseases, but the underlying mechanisms are not well understood. As mitochondrial dysfunction is one of the common molecular mechanisms leading to neuronal death in many neurodegenerative diseases, here we studied miRs that modulate neuronal death caused by 1-methyl-4-phenylpyridinium (MPP+), an inhibitor of complex I in mitochondria. We identified miR-593-5p, levels of which were increased in SH-SY5Y human neuronal cells, after exposure to MPP+. We found that intracellular Ca2+, but not of reactive oxygen species (ROS), mediated this miR-593-5p increase. Furthermore, we found the increase in miR-593-5p was due to enhanced stability, not increased transcription or miR processing. Importantly, we show the increase in miR-593-5p contributed to MPP+-induced cell death. Our data revealed that miR-593-5p inhibits a signaling pathway involving PTEN-induced putative kinase 1 (PINK1) and Parkin, two proteins responsible for the removal of damaged mitochondria from cells, by targeting the coding sequence of PINK1 mRNA. Our findings suggest that miR-593-5p contributes to neuronal death resulting from MPP+ toxicity, in part, by impeding the PINK1/Parkin-mediated pathway that facilitates the clearance of damaged mitochondria. Taken together, our observations highlight the potential significance of inhibiting miR-593-5p as a therapeutic approach for neurodegenerative diseases.
    Keywords:  PTEN‐induced putative kinase 1; cell death; microRNA; molecular cell biology; neurodegeneration
    DOI:  https://doi.org/10.1016/j.jbc.2023.104709
  57. Science. 2023 Apr 21. 380(6642): eabj5559
    Induction of lysosomal and mitochondrial biogenesis by AMPK phosphorylation of FNIP1.
    Nazma Malik, Bibiana I Ferreira, Pablo E Hollstein, Stephanie D Curtis, Elijah Trefts, Sammy Weiser Novak, Jingting Yu, Rebecca Gilson, Kristina Hellberg, Lingjing Fang, Arlo Sheridan, Nasun Hah, Gerald S Shadel, Uri Manor, Reuben J Shaw.
      Cells respond to mitochondrial poisons with rapid activation of the adenosine monophosphate-activated protein kinase (AMPK), causing acute metabolic changes through phosphorylation and prolonged adaptation of metabolism through transcriptional effects. Transcription factor EB (TFEB) is a major effector of AMPK that increases expression of lysosome genes in response to energetic stress, but how AMPK activates TFEB remains unresolved. We demonstrate that AMPK directly phosphorylates five conserved serine residues in folliculin-interacting protein 1 (FNIP1), suppressing the function of the folliculin (FLCN)-FNIP1 complex. FNIP1 phosphorylation is required for AMPK to induce nuclear translocation of TFEB and TFEB-dependent increases of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) and estrogen-related receptor alpha (ERRα) messenger RNAs. Thus, mitochondrial damage triggers AMPK-FNIP1-dependent nuclear translocation of TFEB, inducing sequential waves of lysosomal and mitochondrial biogenesis.
    DOI:  https://doi.org/10.1126/science.abj5559
  58. Adv Sci (Weinh). 2023 Apr 21. e2301295
    Autophagy-Activated Self-reporting Photosensitizer Promoting Cell Mortality in Cancer Starvation Therapy.
    Ruoyao Zhang, Chen Zhang, Chao Chen, Minggang Tian, Joe H C Chau, Zhao Li, Yuanzhan Yang, Xiaoqiong Li, Ben Zhong Tang.
      Cancer starvation therapy have received continuous attention as an efficient method to fight against wide-spectrum cancer. However, during cancer starvation therapy, the protective autophagy promotes cancer cells survival, compromising the therapeutic effect. Herein, a novel strategy by combination of autophagy-activated fluorescent photosensitizers (PSs) and cancer starvation therapy to realize the controllable and efficient ablation of tumor is conceived. Two dual-emissive self-reporting aggregation-induced emission luminogens (AIEgens), TPAQ and TPAP, with autophagy-activated reactive oxygen species (ROS) generation are prepared to fight against the protective autophagy in cancer starvation therapy. When protective autophagy occurs, a portion of TPAQ and TPAP will translocate from lipid droplets to acidic lysosomes with significant redshift in fluorescence emission and enhanced ROS generation ability. The accumulation of ROS induced by TPAQ-H and TPAP-H causes lysosomal membrane permeabilization (LMP), which further results in cell apoptosis and promotes cell death. In addition, TPAQ and TPAP can enable the real-time self-reporting to cell autophagy and cell death process by observing the change of red-emissive fluorescence signals. Particularly, the efficient ablation of tumor via the combination of cancer starvation therapy and photodynamic therapy (PDT) induced by TPAQ has been successfully confirmed in 3D tumor spheroid chip, suggesting the validation of this strategy.
    Keywords:  3D tumor spheroid chip; autophagy-activated photosensitizer; cancer starvation therapy; dual-emissive self-reporting AIEgen; photodynamic therapy
    DOI:  https://doi.org/10.1002/advs.202301295
  59. Biochem Pharmacol. 2023 Apr 13. pii: S0006-2952(23)00141-7. [Epub ahead of print]212 115550
    Targeting autophagy and lipid metabolism in cancer stem cells.
    Bandana Chakravarti, Jawed Akhtar Siddiqui, Rohit Anthony Sinha, Sana Raza.
      Cancer stem cells (CSCs) are a subset of cancer cells with self-renewal ability and tumor initiating properties. Unlike the other non-stem cancer cells, CSCs resist traditional therapy and remain a major cause of disease relapse. With the recent advances in metabolomics, various studies have demonstrated that CSCs have distinct metabolic properties. Metabolic reprogramming in CSCs contributes to self-renewal and maintenance of stemness. Accumulating evidence suggests that rewiring of energy metabolism is a key player that enables to meet energy demands, maintains stemness, and sustains cancer growth and invasion. CSCs use various mechanisms such as increased glycolysis, redox signaling, and autophagy modulation to overcome nutritional deficiency and sustain cell survival. The alterations in lipid metabolism acquired by the CSCs support biomass production through increased dependence on fatty acid synthesis and β-oxidation, and contribute to oncogenic signaling pathways. This review summarizes our current understanding of lipid metabolism in CSCs and how pharmacological regulation of autophagy and lipid metabolism influences CSC phenotype. Increased dependence on lipid metabolism appears as an attractive strategy to eliminate CSCs using therapeutic agents that specifically target CSCs based on their modulation of lipid metabolism.
    Keywords:  Autophagy; Cancer stem cells (CSCs); Heterogeneity; Lipid metabolism; Therapy
    DOI:  https://doi.org/10.1016/j.bcp.2023.115550
  60. Sci Adv. 2023 Apr 21. 9(16): eabq0651
    Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration.
    Poulomi Banerjee, Arpan R Mehta, Raja S Nirujogi, James Cooper, Owen G James, Jyoti Nanda, James Longden, Karen Burr, Karina McDade, Andrea Salzinger, Evdokia Paza, Judith Newton, David Story, Suvankar Pal, Colin Smith, Dario R Alessi, Bhuvaneish T Selvaraj, Josef Priller, Siddharthan Chandran.
      Although microglial activation is widely found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the underlying mechanism(s) are poorly understood. Here, using human-induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harboring the most common ALS/FTD mutation (C9orf72, mC9-MG), gene-corrected isogenic controls (isoC9-MG), and C9orf72 knockout hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated immune response upon stimulation with lipopolysaccharide. Analysis of the C9ORF72 interactome revealed that C9ORF72 interacts with regulators of autophagy and functional studies showed impaired initiation of autophagy in mC9-MG and C9KO-MG. Coculture studies with motor neurons (MNs) demonstrated that the autophagy deficit in mC9-MG drives increased vulnerability of mC9-MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated both cell-autonomous functional deficits in hiPSC-MG and MN death in MG-MN coculture. Together, these findings reveal an important role for C9ORF72 in regulating immune homeostasis and identify dysregulation in myeloid cells as a contributor to neurodegeneration in ALS/FTD.
    DOI:  https://doi.org/10.1126/sciadv.abq0651
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