bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2024‒01‒14
sixty-four papers selected by
Viktor Korolchuk, Newcastle University
  1. PRKAA2, MTOR, and TFEB in the regulation of lysosomal damage response and autophagy.
  2. Unexpected roles for AMPK in suppression of autophagy and reactivation of mTORC1 signaling during prolonged amino acid deprivation.
  3. COPI Vesicle Disruption Inhibits Mineralization via mTORC1-Mediated Autophagy.
  4. Disruption of lysosomal nutrient sensing scaffold hyperactivates mTORC1 contributing to pathogenesis of a fatal neurodegenerative disease.
  5. Neuronal Autophagy: Regulations and Implications in Health and Disease.
  6. Upregulated pexophagy limits the capacity of selective autophagy.
  7. TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex.
  8. Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.
  9. Autophagy-mediated nanomaterials for tumor therapy.
  10. Contrasting views on the role of AMPK in autophagy.
  11. Autophagy-from yeast to humans: Thirty years of molecular autophagy.
  12. Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.
  13. Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.
  14. Molecular Mechanism of Calycosin Inhibited Vascular Calcification.
  15. Varicella zoster virus glycoprotein E facilitates PINK1/Parkin-mediated mitophagy to evade STING and MAVS-mediated antiviral innate immunity.
  16. Herpesvirus lytic infection-induced mitophagy via viral interferon regulatory factor 1.
  17. Antioxidants and azd0156 Rescue Inflammatory Response in Autophagy-Impaired Macrophages.
  18. A new GUV-based assay to reconstitute membrane tethering in vitro demonstrates the vesicle tethering ability of the selective autophagy scaffold Atg11.
  19. Rescue of ApoE4-related lysosomal autophagic failure in Alzheimer's disease by targeted small molecules.
  20. TRPM2-dependent autophagy inhibition exacerbates oxidative stress-induced CXCL16 secretion by keratinocytes in vitiligo.
  21. Mechanisms and therapeutic targets of mitophagy after intracerebral hemorrhage.
  22. Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function.
  23. Neuroactive steroid effects on autophagy in a human embryonic kidney 293 (HEK) cell model.
  24. Berberine Induces Mitophagy through Adenosine Monophosphate-Activated Protein Kinase and Ameliorates Mitochondrial Dysfunction in PINK1 Knockout Mouse Embryonic Fibroblasts.
  25. The aging mouse CNS is protected by an autophagy-dependent microglia population promoted by IL-34.
  26. White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells.
  27. Rapamycin protects mouse skin from ultraviolet B-induced photodamage by modulating Hspb2-mediated autophagy and apoptosis.
  28. AMPK-mTORC1 pathway mediates hepatic IGFBP-1 phosphorylation in glucose deprivation.
  29. Cell-type specific role of autophagy in the liver and its implications in non-alcoholic fatty liver disease.
  30. The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.
  31. Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy.
  32. Spartin-mediated lipid transfer facilitates lipid droplet turnover.
  33. CK1δ/ε inhibition induces ULK1-mediated autophagy in tumorigenesis.
  34. The role of TSC1 and TSC2 proteins in neuronal axons.
  35. Tangshen formula protects against podocyte apoptosis via enhancing the TFEB-mediated autophagy-lysosome pathway in diabetic nephropathy.
  36. Atg8 family proteins, LIR/AIM motifs and other interaction modes.
  37. EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.
  38. M2 Muscarinic Receptor Stimulation Induces Autophagy in Human Glioblastoma Cancer Stem Cells via mTOR Complex-1 Inhibition.
  39. Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance.
  40. Interconnection of cellular autophagy and endosomal vesicle trafficking and its role in hepatitis B virus replication and release.
  41. Autophagic blockage by metformin-loaded PLGA nanoparticles causes cell cycle arrest of HepG2 cells.
  42. Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.
  43. miR-214-3p promotes the pathogenesis of Parkinson's disease by inhibiting autophagy.
  44. Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.
  45. Identification and Potential Participation of Lipases in Autophagic Body Degradation in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.
  46. Intracellular calcium links milk stasis to lysosome-dependent cell death during early mammary gland involution.
  47. The cell cycle, autophagy, and cell wall integrity pathway jointly governed by MoSwe1 in Magnaporthe oryzae.
  48. Olanzapine, risperidone and ziprasidone differently affect lysosomal function and autophagy, reflecting their different metabolic risk in patients.
  49. MET Oncogene Enhances Pro-Migratory Functions by Counteracting NMDAR2B Cleavage.
  50. Should proteins be interrogated when they conspire?
  51. Nanoreceptors promote mutant p53 protein degradation by mimicking selective autophagy receptors.
  52. Cytochrome c levels affect the TOR pathway to regulate growth and metabolism under energy-deficient conditions.
  53. Acute ingestion of a ketone monoester, whey protein, or their co-ingestion in the overnight postabsorptive state elicit a similar stimulation of myofibrillar protein synthesis rates in young males: a double-blind randomized trial.
  54. Role of Autophagy and Pyroptosis in Intervertebral Disc Degeneration.
  55. MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury.
  56. Response to Bruton's tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy.
  57. Endurance Exercise Training Mitigates Diastolic Dysfunction in Diabetic Mice Independent of Phosphorylation of Ulk1 at S555.
  58. Arrestin-3 binds parkin and enhances parkin-dependent mitophagy.
  59. Serine to proline mutation at position 341 of MYOC impairs trabecular meshwork function by causing autophagy deregulation.
  60. Curcumin inhibits propofol-induced autophagy of MN9D cells via Akt/mTOR/p70S6K signaling pathway.
  61. ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia.
  62. Disruption of lysosomal proteolysis in astrocytes facilitates midbrain organoid proteostasis failure in an early-onset Parkinson's disease model.
  63. mTOR Pathway Substrates Present High Activation in Vascular Malformations and Significantly Decrease with Age.
  64. Autophagy is essential for human myelopoiesis.
  1. J Mol Med (Berl). 2024 Jan 06.
    PRKAA2, MTOR, and TFEB in the regulation of lysosomal damage response and autophagy.
    Mohd Shariq, Mohammad Firoz Khan, Reshmi Raj, Nuzhat Ahsan, Pramod Kumar.
      Lysosomes function as critical signaling hubs that govern essential enzyme complexes. LGALS proteins (LGALS3, LGALS8, and LGALS9) are integral to the endomembrane damage response. If ESCRT fails to rectify damage, LGALS-mediated ubiquitination occurs, recruiting autophagy receptors (CALCOCO2, TRIM16, and SQSTM1) and VCP/p97 complex containing UBXN6, PLAA, and YOD1, initiating selective autophagy. Lysosome replenishment through biogenesis is regulated by TFEB. LGALS3 interacts with TFRC and TRIM16, aiding ESCRT-mediated repair and autophagy-mediated removal of damaged lysosomes. LGALS8 inhibits MTOR and activates TFEB for ATG and lysosomal gene transcription. LGALS9 inhibits USP9X, activates PRKAA2, MAP3K7, ubiquitination, and autophagy. Conjugation of ATG8 to single membranes (CASM) initiates damage repair mediated by ATP6V1A, ATG16L1, ATG12, ATG5, ATG3, and TECPR1. ATG8ylation or CASM activates the MERIT system (ESCRT-mediated repair, autophagy-mediated clearance, MCOLN1 activation, Ca2+ release, RRAG-GTPase regulation, MTOR modulation, TFEB activation, and activation of GTPase IRGM). Annexins ANAX1 and ANAX2 aid damage repair. Stress granules stabilize damaged membranes, recruiting FLCN-FNIP1/2, G3BP1, and NUFIP1 to inhibit MTOR and activate TFEB. Lysosomes coordinate the synergistic response to endomembrane damage and are vital for innate and adaptive immunity. Future research should unveil the collaborative actions of ATG proteins, LGALSs, TRIMs, autophagy receptors, and lysosomal proteins in lysosomal damage response.
    Keywords:  Autophagy receptors; LGALS; Lysosomal damage response; MTOR; PRKAA2; TFEB
    DOI:  https://doi.org/10.1007/s00109-023-02411-7
  2. bioRxiv. 2023 Dec 21. pii: 2023.12.20.572593. [Epub ahead of print]
    Unexpected roles for AMPK in suppression of autophagy and reactivation of mTORC1 signaling during prolonged amino acid deprivation.
    Dubek Kazyken, Sydney G Dame, Claudia Wang, Maxwell Wadley, Diane C Fingar.
      Amino acid withdrawal suppresses mTORC1 signaling rapidly, which initiates macroautophagy (herein, autophagy). Prolonged amino acid deprivation, however, leads to partial reactivation of mTORC1 due to the liberation of free amino acids by autophagic proteolysis. We observed impaired reactivation of mTORC1 signaling and increased apoptotic cell death upon prolonged amino acid withdrawal in cells lacking the AMPKα1/α2 catalytic subunits. These findings align well with the role of AMPK in promoting cell survival during energetic stress but oppose the well-documented inhibitory action of AMPK toward mTORC1. AMPK-mediated reactivation of mTORC1 during prolonged amino acid deprivation could be explained, however, if AMPK were required for autophagy. Indeed, a prevailing view posits that activation of AMPK by glucose withdrawal promotes autophagy and mitophagy through multisite phosphorylation of ULK1. When we examined the role of AMPK in autophagy induced by amino acid deprivation, however, we found unexpectedly that autophagy remained unimpaired in cells lacking AMPK α1/α2, as monitored by several autophagic readouts in several cell lines. Moreover, the absence of AMPK increased ULK1 signaling, LC3b lipidation, and lysosomal acidity, and the phosphorylation of ULK1 S555 (an AMPK site proposed to induce autophagy) decreased upon amino acid withdrawal or pharmacological mTORC1 inhibition. In addition, activation of AMPK with compound 991, glucose deprivation, or AICAR blunted basal and amino acid withdrawal-induced autophagy. Together our results demonstrate that AMPK suppresses rather than promotes autophagy and supports mTORC1 signaling during prolonged amino acid deprivation, revealing unexpected roles for AMPK in control of these processes.
    DOI:  https://doi.org/10.1101/2023.12.20.572593
  3. Int J Mol Sci. 2023 Dec 26. pii: 339. [Epub ahead of print]25(1):
    COPI Vesicle Disruption Inhibits Mineralization via mTORC1-Mediated Autophagy.
    Jiaming Nie, Shaoyang Ma, Yuchen Zhang, Shuchen Yu, Jiajia Yang, Ang Li, Dandan Pei.
      Bone mineralization is a sophisticated regulated process composed of crystalline calcium phosphate and collagen fibril. Autophagy, an evolutionarily conserved degradation system, whereby double-membrane vesicles deliver intracellular macromolecules and organelles to lysosomes for degradation, has recently been shown to play an essential role in mineralization. However, the formation of autophagosomes in mineralization remains to be determined. Here, we show that Coat Protein Complex I (COPI), responsible for Golgi-to-ER transport, plays a pivotal role in autophagosome formation in mineralization. COPI vesicles were increased after osteoinduction, and COPI vesicle disruption impaired osteogenesis. Mechanistically, COPI regulates autophagy activity via the mTOR complex 1 (mTORC1) pathway, a key regulator of autophagy. Inhibition of mTOR1 rescues the impaired osteogenesis by activating autophagy. Collectively, our study highlights the functional importance of COPI in mineralization and identifies COPI as a potential therapeutic target for treating bone-related diseases.
    Keywords:  COPI vesicle; autophagy; mineralization
    DOI:  https://doi.org/10.3390/ijms25010339
  4. J Biol Chem. 2024 Jan 09. pii: S0021-9258(24)00017-6. [Epub ahead of print] 105641
    Disruption of lysosomal nutrient sensing scaffold hyperactivates mTORC1 contributing to pathogenesis of a fatal neurodegenerative disease.
    Maria B Bagh, Abhilash P Appu, Tamal Sadhukhan, Avisek Mondal, Nisha Plavelil, Mahadevan Raghavankutty, Ajayan M Supran, Sriparna Sadhukhan, Aiyi Liu, Anil B Mukherjee.
      The CLN1-disease, formerly called infantile neuronal ceroid lipofuscinosis (INCL), is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1-disease has remained elusive. Here we report that in the brain of Cln1-/- mice, which mimic CLN1-disease, the mechanistic target of rapamycin complex-1 (mTORC1)-kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1-deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1-activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1-activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.
    Keywords:  CLN1-disease; IGF1-signaling; Lysosomal storage disease; Neurodegeneration; S-palmitoylation; mTORC1 activation
    DOI:  https://doi.org/10.1016/j.jbc.2024.105641
  5. Cells. 2024 Jan 04. pii: 103. [Epub ahead of print]13(1):
    Neuronal Autophagy: Regulations and Implications in Health and Disease.
    Caroline Liénard, Alexandre Pintart, Pascale Bomont.
      Autophagy is a major degradative pathway that plays a key role in sustaining cell homeostasis, integrity, and physiological functions. Macroautophagy, which ensures the clearance of cytoplasmic components engulfed in a double-membrane autophagosome that fuses with lysosomes, is orchestrated by a complex cascade of events. Autophagy has a particularly strong impact on the nervous system, and mutations in core components cause numerous neurological diseases. We first review the regulation of autophagy, from autophagosome biogenesis to lysosomal degradation and associated neurodevelopmental/neurodegenerative disorders. We then describe how this process is specifically regulated in the axon and in the somatodendritic compartment and how it is altered in diseases. In particular, we present the neuronal specificities of autophagy, with the spatial control of autophagosome biogenesis, the close relationship of maturation with axonal transport, and the regulation by synaptic activity. Finally, we discuss the physiological functions of autophagy in the nervous system, during development and in adulthood.
    Keywords:  autophagy; compartmentalisation; neurodegeneration; neurodevelopment; regulations
    DOI:  https://doi.org/10.3390/cells13010103
  6. Nat Commun. 2024 Jan 09. 15(1): 375
    Upregulated pexophagy limits the capacity of selective autophagy.
    Kyla Germain, Raphaella W L So, Laura F DiGiovanni, Joel C Watts, Robert H J Bandsma, Peter K Kim.
      Selective autophagy is an essential process to maintain cellular homeostasis through the constant recycling of damaged or superfluous components. Over a dozen selective autophagy pathways mediate the degradation of diverse cellular substrates, but whether these pathways can influence one another remains unknown. We address this question using pexophagy, the autophagic degradation of peroxisomes, as a model. We show in cells that upregulated pexophagy impairs the selective autophagy of both mitochondria and protein aggregates by exhausting the autophagy initiation factor, ULK1. We confirm this finding in cell models of the pexophagy-mediated form of Zellweger Spectrum Disorder, a disease characterized by peroxisome dysfunction. Further, we extend the generalizability of limited selective autophagy by determining that increased protein aggregate degradation reciprocally reduces pexophagy using cell models of Parkinson's Disease and Huntington's Disease. Our findings suggest that the degradative capacity of selective autophagy can become limited by an increase in one substrate.
    DOI:  https://doi.org/10.1038/s41467-023-44005-4
  7. Nat Commun. 2024 Jan 09. 15(1): 406
    TFEB drives mTORC1 hyperactivation and kidney disease in Tuberous Sclerosis Complex.
    Nicola Alesi, Damir Khabibullin, Dean M Rosenthal, Elie W Akl, Pieter M Cory, Michel Alchoueiry, Samer Salem, Melissa Daou, William F Gibbons, Jennifer A Chen, Long Zhang, Harilaos Filippakis, Laura Graciotti, Caterina Miceli, Jlenia Monfregola, Claudia Vilardo, Manrico Morroni, Chiara Di Malta, Gennaro Napolitano, Andrea Ballabio, Elizabeth P Henske.
      Tuberous Sclerosis Complex (TSC) is caused by TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and lesions  in multiple organs including lung (lymphangioleiomyomatosis) and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in TSC. Here, we generated two mouse models of TSC in which kidney pathology is the primary phenotype. Knockout of TFEB rescues kidney pathology and overall survival, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, increased mTORC1 activity in the TSC2 knockout kidneys is normalized by TFEB knockout. In TSC2-deficient cells, Rheb knockdown or Rapamycin treatment paradoxically increases TFEB phosphorylation at the mTORC1-sites and relocalizes TFEB from nucleus to cytoplasm. In mice, Rapamycin treatment normalizes lysosomal gene expression, similar to TFEB knockout, suggesting that Rapamycin's benefit in TSC is TFEB-dependent. These results change the view of the mechanisms of mTORC1 hyperactivation in TSC and may lead to therapeutic avenues.
    DOI:  https://doi.org/10.1038/s41467-023-44229-4
  8. Curr Protoc. 2024 Jan;4(1): e950
    Histodenz Separation of Lysosomal Subpopulations for Analysis of Chaperone-mediated Autophagy.
    Calvin M Burns, Richard A Miller, S Joseph Endicott.
      Chaperone-mediated autophagy (CMA) is the most selective form of lysosomal proteolysis, in which proteins are individually selected for lysosomal degradation. CMA degradation targets bear a pentapeptide consensus motif that is recognized by the cytosolic chaperone HSPA8 (Hsc70), which participates in the trafficking of the target to the lysosomal surface. From there, it is translocated into the lysosomal lumen, independent of vesicle fusion, in a process dependent upon the lysosomal transmembrane protein LAMP2A. There are limited tools for studying CMA in whole cells and tissues, and many of the best techniques for studying CMA rely on the preparation of lysosome enriched fractions. Such experiments include (1) the in vitro evaluation of CMA substrate uptake activity, (2) the characterization of changes to lysosomal resident and CMA regulatory proteins, and (3) lysosomal targetomics, i.e., the use of quantitative proteomics to characterize lysosomal degradation targets. Previous studies using discontinuous metrizamide gradients have shown that a subpopulation of liver lysosomes is responsible for the majority of CMA activity ("CMA+ "). These CMA+ lysosomes are low density and have higher levels of MTORC2 relative to the "CMA- " lysosomes, which are high density and have higher levels of MTORC1. Because of safety concerns surrounding metrizamide, however, this compound is difficult to obtain, and it is impractically expensive. Here, we have provided protocols for isolation of lysosomal subpopulations for CMA-related analyses from mouse liver using Histodenz, a safe and affordable alternative to metrizamide. Supplementary protocols show how to perform CMA activity assays with appropriate statistical analysis, and how to analyze for lysosomal breakage/membrane integrity. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation of lysosomal subpopulations from mouse liver using discontinuous Histodenz gradients Alternate Protocol: Isolation of lysosomes from cultured cells using discontinuous Histodenz gradients Support Protocol 1: Verifying enrichment of lysosomal markers in lysosome-enriched fractions Support Protocol 2: Measuring in vitro uptake of CMA substrates Support Protocol 3: Measuring lysosomal membrane integrity by hexosaminidase assay.
    Keywords:  autophagy; density purification; lysosomes; organelle isolation
    DOI:  https://doi.org/10.1002/cpz1.950
  9. Front Oncol. 2023 ;13 1194524
    Autophagy-mediated nanomaterials for tumor therapy.
    Zijian Liao, Xiyu Liu, Dianfa Fan, Xingjun Sun, Zhikun Zhang, Pan Wu.
      Autophagy is a lysosomal self-degradation pathway that plays an important protective role in maintaining intracellular environment. Deregulation of autophagy is related to several diseases, including cancer, infection, neurodegeneration, aging, and heart disease. In this review, we will summarize recent advances in autophagy-mediated nanomaterials for tumor therapy. Firstly, the autophagy signaling pathway for tumor therapy will be reviewed, including oxidative stress, mammalian target of rapamycin (mTOR) signaling and autophagy-associated genes pathway. Based on that, many autophagy-mediated nanomaterials have been developed and applied in tumor therapy. According to the different structure of nanomaterials, we will review and evaluate these autophagy-mediated nanomaterials' therapeutic efficacy and potential clinical application.
    Keywords:  apoptosis; autophagy; nanomaterials; signaling pathway; tumor therapy
    DOI:  https://doi.org/10.3389/fonc.2023.1194524
  10. Bioessays. 2024 Jan 12. e2300211
    Contrasting views on the role of AMPK in autophagy.
    Do-Hyung Kim.
      Efficient management of low energy states is vital for cells to maintain basic functions and metabolism and avoid cell death. While autophagy has long been considered a critical mechanism for ensuring survival during energy depletion, recent research has presented conflicting evidence, challenging the long-standing concept. This recent development suggests that cells prioritize preserving essential cellular components while restraining autophagy induction when cellular energy is limited. This essay explores the conceptual discourse on autophagy regulation during energy stress, navigating through the studies that established the current paradigm and the recent research that has challenged its validity while proposing an alternative model. This exploration highlights the far-reaching implications of the alternative model, which represents a conceptual departure from the established paradigm, offering new perspectives on how cells respond to energy stress.
    Keywords:  AMPK; autophagy; energy stress response; mTOR
    DOI:  https://doi.org/10.1002/bies.202300211
  11. FEBS Lett. 2024 Jan;598(1): 3-6
    Autophagy-from yeast to humans: Thirty years of molecular autophagy.
    Tassula Proikas-Cezanne, Michael Thumm.
      
    DOI:  https://doi.org/10.1002/1873-3468.14796
  12. Nutrients. 2023 Dec 26. pii: 84. [Epub ahead of print]16(1):
    Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.
    Kotaro Tamura, Hidefumi Kitazawa, Satoshi Sugita, Kohjiro Hashizume, Masazumi Iwashita, Takaaki Ishigami, Yoshihiko Minegishi, Akira Shimotoyodome, Noriyasu Ota.
      Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.
    Keywords:  Sestrin1/2; amino acids; mTOR complex 1; muscle cells
    DOI:  https://doi.org/10.3390/nu16010084
  13. Vet Microbiol. 2024 Jan 03. pii: S0378-1135(23)00326-7. [Epub ahead of print]290 109972
    Bovine parainfluenza type 3 virus induces incomplete autophagy to promote viral replication by activated beclin1 in vitro.
    Yu Han, Chongyang Wang, Kejia Lu, Xiaoyu Dong, Zhengwu Chang, Riteng Zhang, Qili Hou, Xinglong Wang, Sa Xiao, Haijin Liu, Zengqi Yang.
      Bovine Parainfluenza virus Type 3 (BPIV3) is one of the most important pathogens in cattle, capable of causing severe respiratory symptoms. Numerous studies have shown that autophagy plays a diverse role in the infection process of various pathogens. The influence of autophagy machinery on BPIV3 infection has not yet been confirmed. In the present study, we initially demonstrated that the expression of LC3 was significantly increased and exhibited a notable increase in double or single-membrane vesicles under a transmission electron microscope during BPIV3 infection. These observations unequivocally establish the induction of steady-state autophagy in vitro consequent to BPIV3 infection. Furthermore, quantification of autophagic flux substantiates the induction of an incomplete autophagic process during BPIV3 infection. Additionally, through targeted interventions, we demonstrate the regulatory impact of pharmacological agents influencing autophagy and RNA interference targeting an autophagy-associated protein on viral replication. Intriguingly, our data revealed that BPIV3 infection enhanced the phosphorylation of rapamycin kinase (mTOR). This result demonstrated that mTOR does not operate as a counteractive regulator of BPIV3-induced autophagy. Instead, we discern an augmentation in the expression of Beclin1, a key autophagy initiator, which complexes with Vps34, constituting a Class III phosphatidylinositol 3-kinase. This phenomenon serves as a hallmark in the inaugural phase of autophagy initiation during BPIV3 infection. Collectively, these discernments underscore that BPIV3 infection actively stimulates autophagy, thereby enhancing viral replication through the activation of Beclin1, independently of the mTOR signaling pathway. This nuanced comprehension significantly contributes to unraveling the intricate molecular mechanisms governing BPIV3-induced autophagy.
    Keywords:  Autophagy; Beclin1; Bovine parainfluenza virus; Viral replication
    DOI:  https://doi.org/10.1016/j.vetmic.2023.109972
  14. Nutrients. 2023 Dec 27. pii: 99. [Epub ahead of print]16(1):
    Molecular Mechanism of Calycosin Inhibited Vascular Calcification.
    Zekun Zhou, Yi Li, Wei Jiang, Zengli Wang.
      Vascular calcification (VC) is a pathological condition frequently observed in cardiovascular diseases. Primary factors contributing to VC are osteogenic differentiation of vascular smooth muscle and hydroxyapatite deposition. Targeted autophagy (a lysosome-mediated mechanism for degradation/recycling of unnecessary cellular components) is a useful approach for inhibiting VC and promoting vascular cell health. Calycosin has been shown to alleviate atherosclerosis by enhancing macrophage autophagy, but its therapeutic effect on VC has not been demonstrated. Using an in vitro model (rat thoracic aortic smooth muscle cell line A7r5), we demonstrated effective inhibition of VC using calycosin (the primary flavonoid component of astragalus), based on the enhancement of autophagic flux. Calycosin treatment activated AMPK/mTOR signaling to induce initiation of autophagy and restored mTORC1-dependent autophagosome-lysosome fusion in late-stage autophagy by promoting soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation, thereby preventing stoppage of autophagy in calcified cells. Calycosin substantially reduced degrees of both osteogenic differentiation and calcium deposition in our VC cell model by enhancing autophagy. The present findings clarify the mechanism whereby calycosin mitigates autophagy stoppage in calcified smooth muscle cells and provide a basis for effective VC treatment via autophagy enhancement.
    Keywords:  SNARE complex; autophagy; calycosin; lysosome; vascular calcification
    DOI:  https://doi.org/10.3390/nu16010099
  15. Cell Death Dis. 2024 Jan 06. 15(1): 16
    Varicella zoster virus glycoprotein E facilitates PINK1/Parkin-mediated mitophagy to evade STING and MAVS-mediated antiviral innate immunity.
    Soo-Jin Oh, Je-Wook Yu, Jin-Hyun Ahn, Seok Tae Choi, Hosun Park, Jeanho Yun, Ok Sarah Shin.
      Viruses have evolved to control mitochondrial quality and content to facilitate viral replication. Mitophagy is a selective autophagy, in which the damaged or unnecessary mitochondria are removed, and thus considered an essential mechanism for mitochondrial quality control. Although mitophagy manipulation by several RNA viruses has recently been reported, the effect of mitophagy regulation by varicella zoster virus (VZV) remains to be fully determined. In this study, we showed that dynamin-related protein-1 (DRP1)-mediated mitochondrial fission and subsequent PINK1/Parkin-dependent mitophagy were triggered during VZV infection, facilitating VZV replication. In addition, VZV glycoprotein E (gE) promoted PINK1/Parkin-mediated mitophagy by interacting with LC3 and upregulating mitochondrial reactive oxygen species. Importantly, VZV gE inhibited MAVS oligomerization and STING translocation to disrupt MAVS- and STING-mediated interferon (IFN) responses, and PINK1/Parkin-mediated mitophagy was required for VZV gE-mediated inhibition of IFN production. Similarly, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-mediated mitophagy induction led to increased VZV replication but attenuated IFN production in a three-dimensional human skin organ culture model. Our results provide new insights into the immune evasion mechanism of VZV gE via PINK1/Parkin-dependent mitophagy.
    DOI:  https://doi.org/10.1038/s41419-023-06400-z
  16. Autophagy Rep. 2023 ;pii: 2281135. [Epub ahead of print]2(1):
    Herpesvirus lytic infection-induced mitophagy via viral interferon regulatory factor 1.
    Mai Tram Vo, Young Bong Choi.
      Viral control of mitochondria via mitophagy has a dampening effect on mitochondrion-mediated innate immune responses. We previously found that human herpesvirus 8 (HHV-8) could activate mitophagy via its lytic gene product vIRF-1 (viral interferon regulatory factor 1). Mechanistically, we previously demonstrated that vIRF-1 interacts with the mitophagic proteins BNIP3L (BCL2 interacting protein 3 like) and TUFM (Tu translation elongation factor, mitochondrial). Despite these significant findings, however, the precise molecular mechanisms underlying vIRF-1-activated mitophagy, particularly with core components of the autophagy machinery, remained to be fully elucidated. We recently reported that vIRF-1 binds preferentially and directly to GABARAPL1 (GABA type A receptor associated protein like 1) in a noncanonical manner, and this interaction is essential for virus-productive replication. Furthermore, we found that BNIP3L is a crucial factor that promotes vIRF-1 oligomerization and associated mitophagy activation, including GABARAPL1 interaction with vIRF-1 and TUFM dimerization. Together, our findings deepen our understanding of lytic infection-induced mitophagy and provide the key protein-protein interactions involved in vIRF-1-mediated mitophagy.
    Keywords:  BNIP3L; GABARAPL1; Human herpesvirus 8; Mitochondria; Mitophagy; Nix; TUFM; vIRF-1
    DOI:  https://doi.org/10.1080/27694127.2023.2281135
  17. Int J Mol Sci. 2023 Dec 21. pii: 169. [Epub ahead of print]25(1):
    Antioxidants and azd0156 Rescue Inflammatory Response in Autophagy-Impaired Macrophages.
    Abdalla Elbialy, Mai Kitauchi, Dai Yamanouchi.
      Autophagy is a lysosomal degradation system that eliminates and recycles damaged intracellular organelles and proteins. Inflammatory macrophages play a critical role in the development of various age-related inflammatory illnesses such as abdominal aortic aneurysm, atherosclerosis, and rheumatoid arthritis; therefore, identifying the mechanisms that cause macrophage inflammation is crucial for a better understanding of and developing therapeutics for inflammatory diseases. Previous research has linked autophagy to macrophage inflammation; Atg16L1-deficient macrophages increase IL-1 and IL-18 production via inflammasome activation. In this study, however, we show an alternative pathway of macrophage inflammation in an autophagy-deficient environment. We found that inhibiting autophagy in THP1 macrophages progressively increased the expression of p65-mediated inflammatory genes. This effect was reversed by treatment with antioxidants or azd0156, an ataxia telangiectasia mutated (ATM) inhibitor. In addition, our results showed that M1 macrophages inhibit autophagy and induce DNA damage, whereas M2 macrophages activate autophagy and reduce DNA damage. Importantly, the chemical activation of autophagy or ATM inhibition during M1 polarization reduced the M1 phenotype and inflammation, whereas inhibiting autophagy during M2 polarization also reduced the M2 phenotype. Thus, our findings highlight the importance of the autophagy-ATM pathway in driving macrophage inflammation.
    Keywords:  NF-kB; abdominal aortic aneurysm; ataxia telangiectasia mutated; autophagy; macrophage
    DOI:  https://doi.org/10.3390/ijms25010169
  18. bioRxiv. 2023 Dec 19. pii: 2023.12.19.572332. [Epub ahead of print]
    A new GUV-based assay to reconstitute membrane tethering in vitro demonstrates the vesicle tethering ability of the selective autophagy scaffold Atg11.
    Devika Andhare, Michael J Ragusa.
      Membrane tethering is essential for the generation of organelle contact sites and to anchor incoming vesicles to their target membranes before vesicle fusion. During autophagy in yeast, tethering of 30 nm vesicles to cargo is one of the first steps in the generation of the isolation membrane that engulfs the cargo to be degraded. While membrane tethering is critical for cellular function, many of the current biochemical techniques to assay for membrane tethering rely on indirect readouts and are limited in their ability to monitor protein localization at sites of tethering. As such, we developed a fluorescence-microscopy-based G UV liposome tethering assay (GLT) to directly visualize membrane tethering and monitor protein localization at tethering sites simultaneously. We initially used GLT with engineered membrane tethers to demonstrate the ease of use, versatility and sensitivity of the assay. We also demonstrated that the selective autophagy scaffolding protein Atg11 can bind, and tether negatively charged membranes but is unable to bind GUVs mimicking the charge of the outer mitochondrial membrane. Atg11 instead requires the selective autophagy receptor Atg32 to be recruited to mitochondrial membranes. Lastly, we demonstrate that Atg11 bound to Atg32 on GUVs can tether negatively charged vesicles to GUVs. Collectively, our results reconstitute one of the first steps during the initiation of mitochondrial autophagy and highlight the versatility of GLT to study a range of membrane tethering events biochemically.
    DOI:  https://doi.org/10.1101/2023.12.19.572332
  19. Commun Biol. 2024 Jan 08. 7(1): 60
    Rescue of ApoE4-related lysosomal autophagic failure in Alzheimer's disease by targeted small molecules.
    Meenakshisundaram Balasubramaniam, Jagadeesh Narasimhappagari, Ling Liu, Akshatha Ganne, Srinivas Ayyadevara, Ramani Atluri, Haarika Ayyadevara, Guy Caldwell, Robert J Shmookler Reis, Steven W Barger, W Sue T Griffin.
      Homozygosity for the ε4 allele of APOE increases the odds of developing Alzheimer's by 12 to 15 times relative to the most common ε3;ε3 genotype, and its association with higher plaque loads comports with evidence that APOEε4 compromises autophagy. The ApoE4 protein specifically binds a cis element ("CLEAR") in the promoters of several autophagy genes to block their transcription. We used a multifaceted approach to identify a druggable site in ApoE4, and virtual screening of lead-like compounds identified small molecules that specifically bind to this site to impede ApoE4::DNA binding. We validated these molecules both in vitro and in vivo with models expressing ApoE4, including ApoE4 targeted-replacement mice. One compound was able to significantly restore transcription of several autophagy genes and protected against amyloid-like aggregation in a C. elegans AD model. Together, these findings provide proof-of-principle evidence for pharmacological remediation of lysosomal autophagy by ApoE4 via ApoE4-targeted lead molecules that represent a novel tack on neurodegenerative disorders.
    DOI:  https://doi.org/10.1038/s42003-024-05767-9
  20. J Pathol. 2024 Jan 08.
    TRPM2-dependent autophagy inhibition exacerbates oxidative stress-induced CXCL16 secretion by keratinocytes in vitiligo.
    Pan Kang, Yinghan Wang, Jianru Chen, Yuqian Chang, Weigang Zhang, Tingting Cui, Xiuli Yi, Shuli Li, Chunying Li.
      Vitiligo is a depigmented skin disease due to the destruction of melanocytes. Under oxidative stress, keratinocyte-derived chemokine C-X-C motif ligand 16 (CXCL16) plays a critical role in recruiting CD8+ T cells, which kill melanocytes. Autophagy serves as a protective cell survival mechanism and impairment of autophagy has been linked to increased secretion of the proinflammatory cytokines. However, the role of autophagy in the secretion of CXCL16 under oxidative stress has not been investigated. Herein, we initially found that autophagy was suppressed in both keratinocytes of vitiligo lesions and keratinocytes exposed to oxidative stress in vitro. Autophagy inhibition also promoted CXCL16 secretion. Furthermore, upregulated transient receptor potential cation channel subfamily M member 2 (TRPM2) functioned as an upstream oxidative stress sensor to inhibit autophagy. Moreover, TRPM2-mediated Ca2+ influx activated calpain to shear autophagy related 5 (Atg5) and Atg12-Atg5 conjugate formation was blocked to inhibit autophagy under oxidative stress. More importantly, Atg5 downregulation enhanced the binding of interferon regulatory factor 3 (IRF3) to the CXCL16 promoter region by activating Tank-binding kinase 1 (TBK1), thus promoting CXCL16 secretion. These findings suggested that TRPM2-restrained autophagy promotes CXCL16 secretion via the Atg5-TBK1-IRF3 signaling pathway under oxidative stress. Inhibition of TRPM2 may serve as a potential target for the treatment of vitiligo. © 2024 The Pathological Society of Great Britain and Ireland.
    Keywords:  CXCL16; TRPM2; autophagy; keratinocyte; oxidative stress; vitiligo
    DOI:  https://doi.org/10.1002/path.6247
  21. Heliyon. 2024 Jan 15. 10(1): e23941
    Mechanisms and therapeutic targets of mitophagy after intracerebral hemorrhage.
    Qinghua Huang, Xiaoqin Yu, Peijie Fu, Moxin Wu, Xiaoping Yin, Zhiying Chen, Manqing Zhang.
      Mitochondria are dynamic organelles responsible for cellular energy production. In addition to regulating energy homeostasis, mitochondria are responsible for calcium homeostasis, clearance of damaged organelles, signaling, and cell survival in the context of injury and pathology. In stroke, the mechanisms underlying brain injury secondary to intracerebral hemorrhage are complex and involve cellular hypoxia, oxidative stress, inflammatory responses, and apoptosis. Recent studies have shown that mitochondrial damage and autophagy are essential for neuronal metabolism and functional recovery after intracerebral hemorrhage, and are closely related to inflammatory responses, oxidative stress, apoptosis, and other pathological processes. Because hypoxia and inflammatory responses can cause secondary damage after intracerebral hemorrhage, the restoration of mitochondrial function and timely clearance of damaged mitochondria have neuroprotective effects. Based on studies on mitochondrial autophagy (mitophagy), cellular inflammation, apoptosis, ferroptosis, the BNIP3 autophagy gene, pharmacological and other regulatory approaches, and normobaric oxygen (NBO) therapy, this article further explores the neuroprotective role of mitophagy after intracerebral hemorrhage.
    Keywords:  Autophagy; BNIP3; Hypoxia; Inflammation and oxidative stress; Intracerebral hemorrhage; Mitophagy
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e23941
  22. EMBO J. 2024 Jan 11.
    Control of NAD+ homeostasis by autophagic flux modulates mitochondrial and cardiac function.
    Quanjiang Zhang, Zhonggang Li, Qiuxia Li, Samuel Aj Trammell, Mark S Schmidt, Karla Maria Pires, Jinjin Cai, Yuan Zhang, Helena Kenny, Sihem Boudina, Charles Brenner, E Dale Abel.
      Impaired autophagy is known to cause mitochondrial dysfunction and heart failure, in part due to altered mitophagy and protein quality control. However, whether additional mechanisms are involved in the development of mitochondrial dysfunction and heart failure in the setting of deficient autophagic flux remains poorly explored. Here, we show that impaired autophagic flux reduces nicotinamide adenine dinucleotide (NAD+) availability in cardiomyocytes. NAD+ deficiency upon autophagic impairment is attributable to the induction of nicotinamide N-methyltransferase (NNMT), which methylates the NAD+ precursor nicotinamide (NAM) to generate N-methyl-nicotinamide (MeNAM). The administration of nicotinamide mononucleotide (NMN) or inhibition of NNMT activity in autophagy-deficient hearts and cardiomyocytes restores NAD+ levels and ameliorates cardiac and mitochondrial dysfunction. Mechanistically, autophagic inhibition causes the accumulation of SQSTM1, which activates NF-κB signaling and promotes NNMT transcription. In summary, we describe a novel mechanism illustrating how autophagic flux maintains mitochondrial and cardiac function by mediating SQSTM1-NF-κB-NNMT signaling and controlling the cellular levels of NAD+.
    Keywords:  Autophagic Flux; Heart Dysfunction; Mitochondrial Homeostasis; NAD+ Metabolism
    DOI:  https://doi.org/10.1038/s44318-023-00009-w
  23. Sci Rep. 2024 01 10. 14(1): 1042
    Neuroactive steroid effects on autophagy in a human embryonic kidney 293 (HEK) cell model.
    Sofia V Salvatore, Ma Xenia G Ilagan, Hongjin Shu, Peter M Lambert, Ann Benz, Mingxing Qian, Douglas F Covey, Charles F Zorumski, Steven Mennerick.
      Neuropsychiatric and neurodegenerative disorders are correlated with cellular stress. Macroautophagy (autophagy) may represent an important protective pathway to maintain cellular homeostasis and functionality, as it targets cytoplasmic components to lysosomes for degradation and recycling. Given recent evidence that some novel psychiatric treatments, such as the neuroactive steroid (NAS) allopregnanolone (AlloP, brexanolone), may induce autophagy, we stably transfected human embryonic kidney 293 (HEK) cells with a ratiometric fluorescent probe to assay NAS effects on autophagy. We hypothesized that NAS may modulate autophagy in part by the ability of uncharged NAS to readily permeate membranes. Microscopy revealed a weak effect of AlloP on autophagic flux compared with the positive control treatment of Torin1. In high-throughput microplate experiments, we found that autophagy induction was more robust in early passages of HEK cells. Despite limiting studies to early passages for maximum sensitivity, a range of NAS structures failed to reliably induce autophagy or interact with Torin1 or starvation effects. To probe NAS in a system where AlloP effects have been shown previously, we surveyed astrocytes and again saw minimal autophagy induction by AlloP. Combined with other published results, our results suggest that NAS may modulate autophagy in a cell-specific or context-specific manner. Although there is merit to cell lines as a screening tool, future studies may require assaying NAS in cells from brain regions involved in neuropsychiatric disorders.
    DOI:  https://doi.org/10.1038/s41598-024-51582-x
  24. Int J Mol Sci. 2023 Dec 22. pii: 219. [Epub ahead of print]25(1):
    Berberine Induces Mitophagy through Adenosine Monophosphate-Activated Protein Kinase and Ameliorates Mitochondrial Dysfunction in PINK1 Knockout Mouse Embryonic Fibroblasts.
    Jee-Hyun Um, Kang-Min Lee, Young-Yeon Kim, Da-Ye Lee, Eunmi Kim, Dong-Hyun Kim, Jeanho Yun.
      Mitophagy stimulation has been shown to have a therapeutic effect on various neurodegenerative diseases. However, nontoxic mitophagy inducers are still very limited. In this study, we found that the natural alkaloid berberine exhibited mitophagy stimulation activity in various human cells. Berberine did not interfere with mitochondrial function, unlike the well-known mitophagy inducer carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and subsequently induced mitochondrial biogenesis. Berberine treatment induced the activation of adenosine monophosphate-activated protein kinase (AMPK), and the AMPK inhibitor compound C abolished berberine-induced mitophagy, suggesting that AMPK activation is essential for berberine-induced mitophagy. Notably, berberine treatment reversed mitochondrial dysfunction in PINK1 knockout mouse embryonic fibroblasts. Our results suggest that berberine is a mitophagy-specific inducer and can be used as a therapeutic treatment for neurodegenerative diseases, including Parkinson's disease, and that natural alkaloids are potential sources of mitophagy inducers.
    Keywords:  Parkinson’s disease; berberine; mitochondrial dysfunction; mitophagy
    DOI:  https://doi.org/10.3390/ijms25010219
  25. Nat Commun. 2024 Jan 09. 15(1): 383
    The aging mouse CNS is protected by an autophagy-dependent microglia population promoted by IL-34.
    Rasmus Berglund, Yufei Cheng, Eliane Piket, Milena Z Adzemovic, Manuel Zeitelhofer, Tomas Olsson, Andre Ortlieb Guerreiro-Cacais, Maja Jagodic.
      Microglia harness an unutilized health-promoting potential in age-related neurodegenerative and neuroinflammatory diseases, conditions like progressive multiple sclerosis (MS). Our research unveils an microglia population emerging in the cortical brain regions of aging mice, marked by ERK1/2, Akt, and AMPK phosphorylation patterns and a transcriptome indicative of activated autophagy - a process critical for cellular adaptability. By deleting the core autophagy gene Ulk1 in microglia, we reduce this population in the central nervous system of aged mice. Notably, this population is found dependent on IL-34, rather than CSF1, although both are ligands for CSF1R. When aging mice are exposed to autoimmune neuroinflammation, the loss of autophagy-dependent microglia leads to neural and glial cell death and increased mortality. Conversely, microglial expansion mediated by IL-34 exhibits a protective effect. These findings shed light on an autophagy-dependent neuroprotective microglia population as a potential target for treating age-related neuroinflammatory conditions, including progressive MS.
    DOI:  https://doi.org/10.1038/s41467-023-44556-6
  26. bioRxiv. 2024 Jan 02. pii: 2023.12.18.566359. [Epub ahead of print]
    White matter hyperintensity genetic risk factor TRIM47 regulates autophagy in brain endothelial cells.
    Sunny Hoi-Sang Yeung, Ralph Hon-Sun Lee, Gerald Wai-Yeung Cheng, Iris Wai-Ting Ma, Julia Kofler, Candice Kent, Fulin Ma, Karl Herrup, Myriam Fornage, Ken Arai, Kai-Hei Tse.
      White matter hyperintensity (WMH) is strongly correlated with age-related dementia and hypertension, but its pathogenesis remains obscure. GWAS identified TRIM47 at 17q25 locus as a top genetic risk factor for WMH formation. TRIM family is a class of E3 ubiquitin ligase with pivotal functions in autophagy, which is critical for brain endothelial cell (ECs) remodeling during hypertension. We hypothesize that TRIM47 regulates autophagy and its loss-of-function disturbs cerebrovasculature. Based on transcriptomics and immunohistochemistry, TRIM47 is found selectively expressed by brain ECs in human and mouse, and its transcription is upregulated by artificially-induced autophagy while downregulated in hypertension-like conditions. Using in silico simulation, immunocytochemistry and super-resolution microscopy, we identified the highly conserved binding site between TRIM47 and the LIR (LC3-interacting region) motif of LC3B. Importantly, pharmacological autophagy induction increased Trim47 expression on mouse ECs (b.End3) culture, while silencing Trim47 significantly increased autophagy with ULK1 phosphorylation induction, transcription and vacuole formation. Together, we confirm that TRIM47 is an endogenous inhibitor of autophagy in brain ECs, and such TRIM47-mediated regulation connects genetic and physiological risk factors for WMH formation but warrants further investigation.SUMMARY STATEMENT: TRIM47, top genetic risk factor for white matter hyperintensity formation, is a negative regulator of autophagy in brain endothelial cells and implicates a novel cellular mechanism for age-related cerebrovascular changes.
    DOI:  https://doi.org/10.1101/2023.12.18.566359
  27. Mol Biol Rep. 2024 Jan 06. 51(1): 80
    Rapamycin protects mouse skin from ultraviolet B-induced photodamage by modulating Hspb2-mediated autophagy and apoptosis.
    Ang Li, Ai-Jun Chen, Jing Xu, Zhu-Yuan Wen, Gen-Long Bai, Zi-Yue Wang, Yu-Xin Jiang, Ping Wang.
      BACKGROUND: Continuous exposure to UVB is the main extrinsic cause of skin photodamage, which is associated with oxidative stress, DNA damage, apoptosis and degradation of collagen. Rapamycin, a mechanistic target inhibitor of rapamycin complex 1 (mTORC1), has been shown to play a crucial role anti-tumor and aging retardation, but its mechanism of action in UVB-induced photodamage still remains unknown. In this study, we investigated the role of rapamycin and Hspb2 (also known as Hsp27) in UVB-induced photodamage in mice.METHODS AND RESULTS: We constructed skin acute photodamage models on the ears of WT and Hspb2 KO mice, respectively, and administered rapamycin treatment. Histological results showed that knockout of the hspb2 exacerbated the skin damage, as evidenced by thickening of the epidermis, breakage and disruption of collagen fibers and reduction in their number, which is reversed by rapamycin treatment. In addition, hspb2 knockout promoted UVB-induced apoptosis and reduced autophagy levels, with a significant increase in p53 levels and Bax/Bcl-2 ratio, a reduction in LC3II/I ratio and an increase in p62 levels in the KO mice compared to those in WT mice after the same dose of UVB irradiation. Rapamycin was also found to inhibit collagen degradation induced by hspb2 knockdown through activation of the TGF-β/Smad signaling pathway.
    CONCLUSIONS: Rapamycin can alleviate skin photodamage from Hspb2 knockout to some extent. It may be a potential therapeutic drug for skin photodamage. In this study, we investigated the role of rapamycin and Hspb2 in UVB-induced photodamage in mice. Histological results showed that knockout of the hspb2 exacerbated the skin damage, as evidenced by thickening of the epidermis, breakage and disruption of collagen fibers and reduction in their number, which is reversed by rapamycin treatment. In addition, hspb2 knockout promoted UVB-induced apoptosis and reduced autophagy levels. Rapamycin was also found to inhibit collagen degradation induced by hspb2 knockdown through activation of the TGF-β/Smad signaling pathway. We conclude that rapamycin and Hspb2 exert a synergistic protective effect in skin photodamage.
    Keywords:  Apoptosis; Autophagy; Photodamage; Rapamycin; UVB
    DOI:  https://doi.org/10.1007/s11033-023-08954-9
  28. J Mol Endocrinol. 2024 Jan 01. pii: JME-23-0137. [Epub ahead of print]
    AMPK-mTORC1 pathway mediates hepatic IGFBP-1 phosphorylation in glucose deprivation.
    Jenica H Kakadia, Muhammad U Khalid, Ilka U Heinemann, Victor Han.
      Mechanisms underlying limitations in glucose supply that restrict fetal growth are not well established. IGF-1 is an important regulator of fetal growth and IGF-1 bioavailability is markedly inhibited by IGFBP-1 especially when the binding protein is hyperphosphorylated. We hypothesized that the AMPK-mTORC1 pathway increases IGFBP-1 phosphorylation in response to glucose deprivation. Glucose deprivation in HepG2 cells activated AMPK and TSC2, inhibited mTORC1 and increased IGFBP-1 secretion and site-specific phosphorylation. Glucose deprivation also decreased IGF-1 bioavailability and IGF-dependent activation of IGF-1R. AICAR (an AMPK-activator) activated TSC2, inhibited mTORC1 and increased IGFBP-1 secretion/ phosphorylation. Further, siRNA silencing of either AMPK or TSC2 prevented mTORC1 inhibition and IGFBP-1 secretion and phosphorylation in glucose deprivation. Our data suggest that the increase in IGFBP-1 phosphorylation in response to glucose deprivation is mediated by the activation of AMPK/TSC2 and inhibition of mTORC1, providing a possible mechanistic link between glucose deprivation and restricted fetal growth.
    DOI:  https://doi.org/10.1530/JME-23-0137
  29. World J Hepatol. 2023 Dec 27. 15(12): 1272-1283
    Cell-type specific role of autophagy in the liver and its implications in non-alcoholic fatty liver disease.
    Sana Raza, Sangam Rajak, Rajani Singh, Jin Zhou, Rohit A Sinha, Amit Goel.
      Autophagy, a cellular degradative process, has emerged as a key regulator of cellular energy production and stress mitigation. Dysregulated autophagy is a common phenomenon observed in several human diseases, and its restoration offers curative advantage. Non-alcoholic fatty liver disease (NAFLD), more recently renamed metabolic dysfunction-associated steatotic liver disease, is a major metabolic liver disease affecting almost 30% of the world population. Unfortunately, NAFLD has no pharmacological therapies available to date. Autophagy regulates several hepatic processes including lipid metabolism, inflammation, cellular integrity and cellular plasticity in both parenchymal (hepatocytes) and non-parenchymal cells (Kupffer cells, hepatic stellate cells and sinusoidal endothelial cells) with a profound impact on NAFLD progression. Understanding cell type-specific autophagy in the liver is essential in order to develop targeted treatments for liver diseases such as NAFLD. Modulating autophagy in specific cell types can have varying effects on liver function and pathology, making it a promising area of research for liver-related disorders. This review aims to summarize our present understanding of cell-type specific effects of autophagy and their implications in developing autophagy centric therapies for NAFLD.
    Keywords:  Autophagy; Hepatic stellate cells; Hepatocytes; Macrophages; Non-alcoholic fatty liver disease
    DOI:  https://doi.org/10.4254/wjh.v15.i12.1272
  30. Cell Rep. 2024 Jan 05. pii: S2211-1247(23)01642-X. [Epub ahead of print]43(1): 113631
    The paracaspase MALT1 controls cholesterol homeostasis in glioblastoma stem-like cells through lysosome proteome shaping.
    Clément Maghe, Kilian Trillet, Gwennan André-Grégoire, Mathilde Kerhervé, Laura Merlet, Kathryn A Jacobs, Kristine Schauer, Nicolas Bidère, Julie Gavard.
      Glioblastoma stem-like cells (GSCs) compose a tumor-initiating and -propagating population remarkably vulnerable to variation in the stability and integrity of the lysosomal compartment. Previous work has shown that the expression and activity of the paracaspase MALT1 control GSC viability via lysosome abundance. However, the underlying mechanisms remain elusive. By combining RNA sequencing (RNA-seq) with proteome-wide label-free quantification, we now report that MALT1 repression in patient-derived GSCs alters the homeostasis of cholesterol, which accumulates in late endosomes (LEs)-lysosomes. This failure in cholesterol supply culminates in cell death and autophagy defects, which can be partially reverted by providing exogenous membrane-permeable cholesterol to GSCs. From a molecular standpoint, a targeted lysosome proteome analysis unraveled that Niemann-Pick type C (NPC) lysosomal cholesterol transporters are diluted when MALT1 is impaired. Accordingly, we found that NPC1/2 inhibition and silencing partially mirror MALT1 loss-of-function phenotypes. This supports the notion that GSC fitness relies on lysosomal cholesterol homeostasis.
    Keywords:  CP: Cancer; CP: Cell biology; Niemann-Pick disease; SREBP2; autophagy; cholesterol; glioblastoma; glioma; late endosome; lysosome; proteome; transporter
    DOI:  https://doi.org/10.1016/j.celrep.2023.113631
  31. Bio Protoc. 2024 Jan 05. 14(1): e4917
    Real-Time Monitoring of ATG8 Lipidation in vitro Using Fluorescence Spectroscopy.
    Wenxin Zhang, Taki Nishimura, Sharon A Tooze.
      Autophagy is an essential catabolic pathway used to sequester and engulf cytosolic substrates via a unique double-membrane structure, called an autophagosome. The ubiquitin-like ATG8 proteins play an important role in mediating autophagosome membrane expansion. They are covalently conjugated to phosphatidylethanolamine (PE) on the autophagosomes via a ubiquitin-like conjugation system called ATG8 lipidation. In vitro reconstitution of ATG8 lipidation with synthetic liposomes has been previously established and used widely to characterise the function of the E1 ATG7, the E2 ATG3, and the E3 complex ATG12-ATG5-ATG16L1. However, there is still a lack of a tool to provide kinetic measurements of this enzymatic reaction. In this protocol, we describe a real-time lipidation assay using NBD-labelled ATG8. This real-time assay can distinguish the formation of ATG8 intermediates (ATG7~ATG8 and/or ATG3~ATG8) and, finally, ATG8-PE conjugation. It allows kinetic characterisation of the activity of ATG7, ATG3, and the E3 complex during ATG8 lipidation. Furthermore, this protocol can be adapted to characterise the upstream regulators that may affect protein activity in ATG8 lipidation reaction with a kinetic readout. Key features • Preparation of ATG7 E1 from insect cells (Sf9 cells). • Preparation of ATG3 E2 from bacteria (E. coli). • Preparation of LC3B S3C from bacteria (E. coli). • Preparation of liposomes to monitor the kinetics of ATG8 lipidation in a real-time manner.
    Keywords:  Autophagy; Fluorescence spectroscopy; In vitro ATG8 lipidation; Liposomes; NBD; Real-time ATG8 lipidation assay; Site-directed fluorescence; Ubiquitin-like conjugation
    DOI:  https://doi.org/10.21769/BioProtoc.4917
  32. Proc Natl Acad Sci U S A. 2024 Jan 16. 121(3): e2314093121
    Spartin-mediated lipid transfer facilitates lipid droplet turnover.
    Neng Wan, Zhouping Hong, Matthew A H Parson, Justin L Korfhage, John E Burke, Thomas J Melia, Karin M Reinisch.
      Lipid droplets (LDs) are organelles critical for energy storage and membrane lipid homeostasis, whose number and size are carefully regulated in response to cellular conditions. The molecular mechanisms underlying lipid droplet biogenesis and degradation, however, are not well understood. The Troyer syndrome protein spartin (SPG20) supports LD delivery to autophagosomes for turnover via lipophagy. Here, we characterize spartin as a lipid transfer protein whose transfer ability is required for LD degradation. Spartin copurifies with phospholipids and neutral lipids from cells and transfers phospholipids in vitro via its senescence domain. A senescence domain truncation that impairs lipid transfer in vitro also impairs LD turnover in cells while not affecting spartin association with either LDs or autophagosomes, supporting that spartin's lipid transfer ability is physiologically relevant. Our data indicate a role for spartin-mediated lipid transfer in LD turnover.
    Keywords:  lipid droplet turnover; lipid transport protein; membrane dynamics
    DOI:  https://doi.org/10.1073/pnas.2314093121
  33. Transl Oncol. 2024 Jan 06. pii: S1936-5233(23)00249-8. [Epub ahead of print]40 101863
    CK1δ/ε inhibition induces ULK1-mediated autophagy in tumorigenesis.
    Vivian Weiwen Xue, Shanshan Liu, Qi Sun, Jiong Ning, Huan Li, Weilan Wang, Sapna Sayed, Xibao Zhao, Li Fu, Desheng Lu.
      INTRODUCTION: Autophagy is an important mechanism of cell homeostasis maintenance. As essential serine/threonine-protein kinases, casein kinase I family members affect tumorigenesis by regulating a variety of cellular progression. However, the mechanism by which they regulate autophagy remains unclear.MATERIALS AND METHODS: We silenced CK1δ/ε in cancer cells and observed cell morphology, the expression of autophagy-related genes, and its impact on cancer cell growth and viability. By inhibiting CK1δ/ε-induced upregulation of autophagy genes, we profiled the regulatory mechanism of CK1δ/ε on autophagy and cancer cell growth. The impact of CK1δ/ε inhibition on tumor cell growth was also assessed in vivo.
    RESULTS: Here, we found that CK1δ/ε played an important role in ULK1-mediated autophagy regulation in both lung cancer and melanoma cells. Mechanically, silencing CK1δ/ε increased ULK1 expression with enhanced autophagic flux and suppressed cancer cell proliferation, while ULK1 knockdown blocked the activation of autophagy caused by CK1δ/ε inhibition. By silencing CK1δ/ε in syngeneic mouse model bearing LLC1 murine lung cancer cells in vivo, we observed tumor growth suppression mediated by CK1δ/ε inhibition.
    CONCLUSION: Our results provide evidence for the role of CK1δ/ε in the regulation of tumorigenesis via the ULK1-mediated autophagy, and also suggest the impact of CK1δ/ε inhibition on tumor growth and its significance as a potential therapeutic target.
    Keywords:  Autophagy; CK1δ/ε; Tumorigenesis; ULK1
    DOI:  https://doi.org/10.1016/j.tranon.2023.101863
  34. Mol Psychiatry. 2024 Jan 11.
    The role of TSC1 and TSC2 proteins in neuronal axons.
    Vasiliki Karalis, Delaney Wood, Nicole A Teaney, Mustafa Sahin.
      Tuberous Sclerosis Complex 1 and 2 proteins, TSC1 and TSC2 respectively, participate in a multiprotein complex with a crucial role for the proper development and function of the nervous system. This complex primarily acts as an inhibitor of the mechanistic target of rapamycin (mTOR) kinase, and mutations in either TSC1 or TSC2 cause a neurodevelopmental disorder called Tuberous Sclerosis Complex (TSC). Neurological manifestations of TSC include brain lesions, epilepsy, autism, and intellectual disability. On the cellular level, the TSC/mTOR signaling axis regulates multiple anabolic and catabolic processes, but it is not clear how these processes contribute to specific neurologic phenotypes. Hence, several studies have aimed to elucidate the role of this signaling pathway in neurons. Of particular interest are axons, as axonal defects are associated with severe neurocognitive impairments. Here, we review findings regarding the role of the TSC1/2 protein complex in axons. Specifically, we will discuss how TSC1/2 canonical and non-canonical functions contribute to the formation and integrity of axonal structure and function.
    DOI:  https://doi.org/10.1038/s41380-023-02402-7
  35. J Ethnopharmacol. 2024 Jan 08. pii: S0378-8741(24)00021-7. [Epub ahead of print] 117721
    Tangshen formula protects against podocyte apoptosis via enhancing the TFEB-mediated autophagy-lysosome pathway in diabetic nephropathy.
    Yuyang Wang, Liang Peng, Xiaoguang Lu, Haojun Zhang, Hailing Zhao, Tingting Zhao, Liping Yang, Huimin Mao, Fang Ma, Tongtong Liu, Ping Li, Yongli Zhan.
      ETHNOPHARMACOLOGICAL RELEVANCE: Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease and currently there are no specific and effective drugs for its treatment. Podocyte injury is a detrimental feature and the major cause of albuminuria in DN. We previously reported Tangshen Formula (TSF), a Chinese herbal medicine, has shown therapeutic effects on DN. However, the underlying mechanisms remain obscure.AIM OF THE STUDY: This study aimed to explore the protective effect of TSF on podocyte apoptosis in DN and elucidate the potential mechanism.
    MATERIALS AND METHODS: The effects of TSF were assessed in a murine model using male KKAy diabetic mice, as well as in advanced glycation end products-stimulated primary mice podocytes. Transcription factor EB (TFEB) knockdown primary podocytes were employed for mechanistic studies. In vivo and in vitro studies were performed and results assessed using transmission electron microscopy, immunofluorescence staining, and western blotting.
    RESULTS: TSF treatment alleviated podocyte apoptosis and structural impairment, decreased albuminuria, and mitigated renal dysfunction in KKAy mice. Notably, TSF extracted twice showed a more significant reduction in proteinuria than TSF extracted three times. Accumulation of autophagic biomarkers p62 and LC3, and aberrant autophagic flux in podocytes of DN mice were significantly altered by TSF therapy. Consistent with the in vivo results, TSF prevented the apoptosis of primary podocytes exposed to AGEs and activated autophagy. However, the anti-apoptosis capacity of TSF was countered by the autophagy-lysosome inhibitor chloroquine. We found that TSF increased the nuclear translocation of TFEB in diabetic podocytes, and thus upregulated transcription of its several autophagic target genes. Pharmacological activation of TFEB by TSF accelerated the conversion of autophagosome to autolysosome and lysosomal biogenesis, further augmented autophagic flux. Conversely, TFEB knockdown negated the favorable effects of TSF on autophagy in AGEs-stimulated primary podocytes.
    CONCLUSIONS: These findings indicate TSF appears to attenuate podocyte apoptosis and promote autophagy in DN via the TFEB-mediated autophagy-lysosome system. Thus, TSF may be a therapeutic candidate for DN.
    Keywords:  Autophagic flux; Diabetic nephropathy; Lysosome; Podocyte apoptosis; Tangshen formula; Transcription factor EB
    DOI:  https://doi.org/10.1016/j.jep.2024.117721
  36. Autophagy Rep. 2023 Dec 31. pii: 27694127.2023.2188523. [Epub ahead of print]2(1):
    Atg8 family proteins, LIR/AIM motifs and other interaction modes.
    Vladimir V Rogov, Ioannis P Nezis, Panagiotis Tsapras, Hong Zhang, Yasin Dagdas, Nobuo N Noda, Hitoshi Nakatogawa, Martina Wirth, Stephane Mouilleron, David G McEwan, Christian Behrends, Vojo Deretic, Zvulun Elazar, Sharon A Tooze, Ivan Dikic, Trond Lamark, Terje Johansen.
      The Atg8 family of ubiquitin-like proteins play pivotal roles in autophagy and other processes involving vesicle fusion and transport where the lysosome/vacuole is the end station. Nuclear roles of Atg8 proteins are also emerging. Here, we review the structural and functional features of Atg8 family proteins and their protein-protein interaction modes in model organisms such as yeast, Arabidopsis, C. elegans and Drosophila to humans. Although varying in number of homologs, from one in yeast to seven in humans, and more than ten in some plants, there is a strong evolutionary conservation of structural features and interaction modes. The most prominent interaction mode is between the LC3 interacting region (LIR), also called Atg8 interacting motif (AIM), binding to the LIR docking site (LDS) in Atg8 homologs. There are variants of these motifs like "half-LIRs" and helical LIRs. We discuss details of the binding modes and how selectivity is achieved as well as the role of multivalent LIR-LDS interactions in selective autophagy. A number of LIR-LDS interactions are known to be regulated by phosphorylation. New methods to predict LIR motifs in proteins have emerged that will aid in discovery and analyses. There are also other interaction surfaces than the LDS becoming known where we presently lack detailed structural information, like the N-terminal arm region and the UIM-docking site (UDS). More interaction modes are likely to be discovered in future studies.
    Keywords:  AIM; Atg8; Autophagy; LDS; LIR; UDS; phosphorylation; protein-protein interaction
    DOI:  https://doi.org/10.1080/27694127.2023.2188523
  37. Biochim Biophys Acta Mol Cell Res. 2024 Jan 10. pii: S0167-4889(24)00002-8. [Epub ahead of print] 119659
    EGCG-induced selective death of cancer cells through autophagy-dependent regulation of the p62-mediated antioxidant survival pathway.
    Ho Woon Lee, Jee-Hye Choi, Dongbeom Seo, Lkhagvasuren Gavaachimed, Jaesung Choi, Sehwan Park, Na Young Min, Dong Ho Lee, Hyo-Weon Bang, Seung Wook Ham, Jung-Woong Kim, Sung Chul Lee, Sangmyung Rhee, Sang-Beom Seo, Kwang-Ho Lee.
      The effects of EGCG on the selective death of cancer cells by modulating antioxidant pathways through autophagy were explored in various normal and cancer cells. EGCG positively regulated the p62-KEAP1-NRF2-HO-1 pathway in normal cells, while negatively regulating it in cancer cells, leading to selective apoptotic death of cancer cells. In EGCG-treated MRC5 cells (EGCG-MRC5), autophagic flux was blocked, which was accompanied by the formation of p62-positive aggregates. However, EGCG-treated HeLa cells (EGCG-HeLa) showed incomplete autophagic flux and no aggregate formation. The levels of P-ULK1 S556 and S758 increased in EGCG-MRC5 through AMPK-mTOR cooperative interaction. In contrast, EGCG treatment in HeLa cells led to AMPK-induced mTOR inactivation, resulting in abrogation of P-ULK1 S556 and S758 levels. AMPK knockout in EGCG-HeLa restored positive regulation of the p62-mediated pathway, which was accompanied by increased P-mTOR S2448 and P-ULK1 S758 levels. Knockdown of 67LR in EGCG-HeLa abolished AMPK activity but did not restore the p62-mediated pathway. Surprisingly, both AMPK knockout and 67LR knockdown in EGCG-HeLa markedly increased cell viability, despite differential regulation of the antioxidant enzyme HO-1. In conclusion, EGCG induces the selective death of cancer cells through the modulation of at least two autophagy-dependent and independent regulatory pathways: negative regulation involves the mTOR-ULK1 (S556 and S758)-p62-KEAP1-NRF2-HO-1 axis via AMPK activation, whereas positive regulation occurs through the 67LR-AMPK axis.
    Keywords:  (−)-Epigallocatechin-3-gallate (EGCG); AKT; AMPK; Autophagy; ROS; p62
    DOI:  https://doi.org/10.1016/j.bbamcr.2024.119659
  38. Cancers (Basel). 2023 Dec 20. pii: 25. [Epub ahead of print]16(1):
    M2 Muscarinic Receptor Stimulation Induces Autophagy in Human Glioblastoma Cancer Stem Cells via mTOR Complex-1 Inhibition.
    Claudia Guerriero, Marianna Manfredelli, Carlo Matera, Angela Iuzzolino, Luciano Conti, Clelia Dallanoce, Marco De Amici, Daniela Trisciuoglio, Ada Maria Tata.
      BACKGROUND: Although autophagy is a pro-survival process of tumor cells, it can stimulate cell death in particular conditions and when differently regulated by specific signals. We previously demonstrated that the selective stimulation of the M2 muscarinic receptor subtype (mAChR) negatively controls cell proliferation and survival and causes oxidative stress and cytotoxic and genotoxic effects in both GBM cell lines and GBM stem cells (GSCs). In this work, we have evaluated whether autophagy was induced as a downstream mechanism of the observed cytotoxic processes induced by M2 mAChR activation by the orthosteric agonist APE or the dualsteric agonist N8-Iper (N8).METHODS: To assess the activation of autophagy, we analyzed the expression of LC3B using Western blot analysis and in LC3B-EGFP transfected cell lines. Apoptosis was assessed by measuring the protein expression of Caspases 3 and 9.
    RESULTS: Our data indicate that activation of M2 mAChR by N8 promotes autophagy in both U251 and GB7 cell lines as suggested by the LC3B-II expression level and analysis of the transfected cells by fluorescence microscopy. Autophagy induction by M2 mAChRs is regulated by the decreased activity of the PI3K/AKT/mTORC1 pathway and upregulated by pAMPK expression. Downstream of autophagy activation, an increase in apoptosis was also observed in both cell lines after treatment with the two M2 agonists.
    CONCLUSIONS: N8 treatment causes autophagy via pAMPK upregulation, followed by apoptosis in both investigated cell lines. In contrast, the absence of autophagy in APE-treated GSC cells seems to indicate that cell death could be triggered by mechanisms alternative to those observed for N8.
    Keywords:  M2 muscarinic receptor; apoptosis; autophagy; cancer stem cells; glioblastoma; mTORC1; orthosteric and dualsteric muscarinic agonism
    DOI:  https://doi.org/10.3390/cancers16010025
  39. Int J Mol Sci. 2023 Dec 29. pii: 458. [Epub ahead of print]25(1):
    Tamoxifen Activates Transcription Factor EB and Triggers Protective Autophagy in Breast Cancer Cells by Inducing Lysosomal Calcium Release: A Gateway to the Onset of Endocrine Resistance.
    Cecilia Boretto, Chiara Actis, Pawan Faris, Francesca Cordero, Marco Beccuti, Giulio Ferrero, Giuliana Muzio, Francesco Moccia, Riccardo Autelli.
      Among the several mechanisms accounting for endocrine resistance in breast cancer, autophagy has emerged as an important player. Previous reports have evidenced that tamoxifen (Tam) induces autophagy and activates transcription factor EB (TFEB), which regulates the expression of genes controlling autophagy and lysosomal biogenesis. However, the mechanisms by which this occurs have not been elucidated as yet. This investigation aims at dissecting how TFEB is activated and contributes to Tam resistance in luminal A breast cancer cells. TFEB was overexpressed and prominently nuclear in Tam-resistant MCF7 cells (MCF7-TamR) compared with their parental counterpart, and this was not dependent on alterations of its nucleo-cytoplasmic shuttling. Tam promoted the release of lysosomal Ca2+ through the major transient receptor potential cation channel mucolipin subfamily member 1 (TRPML1) and two-pore channels (TPCs), which caused the nuclear translocation and activation of TFEB. Consistently, inhibiting lysosomal calcium release restored the susceptibility of MCF7-TamR cells to Tam. Our findings demonstrate that Tam drives the nuclear relocation and transcriptional activation of TFEB by triggering the release of Ca2+ from the acidic compartment, and they suggest that lysosomal Ca2+ channels may represent new druggable targets to counteract the onset of autophagy-mediated endocrine resistance in luminal A breast cancer cells.
    Keywords:  TFEB; TPCs; TRPML1; breast cancer; calcium signaling; endocrine resistance; lysosomal calcium channels; tamoxifen
    DOI:  https://doi.org/10.3390/ijms25010458
  40. Virol Sin. 2024 Jan 09. pii: S1995-820X(24)00001-4. [Epub ahead of print]
    Interconnection of cellular autophagy and endosomal vesicle trafficking and its role in hepatitis B virus replication and release.
    Jia Li, Yong Lin, Xueyu Wang, Mengji Lu.
      Hepatitis B virus (HBV) produces and releases various particle types, including complete virions, subviral particles with envelope proteins, and naked capsids. Recent studies demonstrate that HBV exploits distinct intracellular membrane trafficking pathways, including the endosomal vesicle trafficking and autophagy pathway, to assemble and release viral and subviral particles. Herein, we summarized the findings about the distinct roles of autophagy and endosomal membrane trafficking and the interaction of both pathways in HBV replication, assembly, and release.
    Keywords:  Amphisome; Autophagy; Endosomal vesicle; Hepatitis B virus (HBV)
    DOI:  https://doi.org/10.1016/j.virs.2024.01.001
  41. Nanomedicine (Lond). 2024 Jan 10.
    Autophagic blockage by metformin-loaded PLGA nanoparticles causes cell cycle arrest of HepG2 cells.
    Chen Chen, Li Yang, Ying Peng, Wen Jie Zhang, Xiao Xiao Yang, Wei Zhou.
      Aim: To fabricate and characterize metformin-loaded PLGA nanoparticles and investigate their inhibitory effect on HepG2 cells. Materials & methods: The nanoparticles were prepared using a double emulsification method, then characterized and subjected to a series of in vitro assays on HepG2 cells. Results: The nanoparticles were ~277.9 nm in size, and the entrapment efficiency and drug loading of metformin were 31.3 and 14.4%, respectively. In vitro studies suggested that the nanoparticles showed a higher inhibitory effect on HepG2 cells compared with metformin alone, mainly attributed to its blockage of autophagy, and ultimately result in cell cycle inhibition. Conclusion: The metformin-loaded PLGA nanoparticles could inhibit mTOR activity, increase p53 levels and decrease HIF1A levels, which ultimately caused HepG2 cell cycle arrest.
    Keywords:  HepG2; autophagy; cell cycle; metformin; nanoparticles; p53
    DOI:  https://doi.org/10.2217/nnm-2023-0160
  42. Int J Mol Sci. 2024 Jan 01. pii: 572. [Epub ahead of print]25(1):
    Investigating the Association between the Autophagy Markers LC3B, SQSTM1/p62, and DRAM and Autophagy-Related Genes in Glioma.
    Farheen Danish, Muhammad Asif Qureshi, Talat Mirza, Wajiha Amin, Sufiyan Sufiyan, Sana Naeem, Fatima Arshad, Nouman Mughal.
      High-grade gliomas are extremely fatal tumors, marked by severe hypoxia and therapeutic resistance. Autophagy is a cellular degradative process that can be activated by hypoxia, ultimately resulting in tumor advancement and chemo-resistance. Our study aimed to examine the link between autophagy markers' expression in low-grade gliomas (LGGs) and high-grade gliomas (HGGs). In 39 glioma cases, we assessed the protein expression of autophagy markers LC3B, SQSTM1/p62, and DRAM by immunohistochemistry (IHC) and the mRNA expression of the autophagy genes PTEN, PI3K, AKT, mTOR, ULK1, ULK2, UVRAG, Beclin 1, and VPS34 using RT-qPCR. LC3B, SQSTM1/p62, and DRAM expression were positive in 64.1%, 51.3%, and 28.2% of glioma cases, respectively. The expression of LC3B and SQSTM1/p62 was notably higher in HGGs compared to LGGs. VPS34 exhibited a significant differential expression, displaying increased fold change in HGGs compared to LGGs. Additionally, it exhibited robust positive associations with Beclin1 (rs = 0.768), UVRAG (rs = 0.802), and ULK2 (rs = 0.786) in HGGs. This underscores a potential association between autophagy and the progression of gliomas. We provide preliminary data for the functional analysis of autophagy using a cell culture model and to identify potential targets for therapeutic interventions.
    Keywords:  DRAM; LC3B; SQSTM1/p62; autophagy; autophagy-related genes (ATGs); high-grade gliomas (HGGs); low-grade gliomas (LGGs)
    DOI:  https://doi.org/10.3390/ijms25010572
  43. Biomed Pharmacother. 2024 Jan 09. pii: S0753-3322(24)00004-0. [Epub ahead of print]171 116123
    miR-214-3p promotes the pathogenesis of Parkinson's disease by inhibiting autophagy.
    Hui Dong, Jiahui Yan, Ping Huang, Xinyu Wang, Ru Zhang, Caiyun Zhang, Wenhui Wang, Wenxian Qian, Jin Zhou, Yunli Zhao, Jinghan Gao, Mengmeng Zhang, Xiuchang Ma, Zhizhong Wang, Changhua Yi, Jie Zhang, Wei Chen.
      Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by dopaminergic neuron death in the substantia nigra, leading to motor dysfunction. Autophagy dysregulation has been implicated in PD pathogenesis. This study explores the role of miR-214-3p in PD, focusing on its impact on autophagy and dopaminergic neuron viability. Using in vitro and in vivo models, we demonstrate that miR-214-3p inhibits autophagy and promotes dopaminergic neuron apoptosis. Behavioral assessments and molecular analyses reveal exacerbation of PD symptoms upon miR-214-3p overexpression. Furthermore, mechanistic investigations identify ATG3 as a target, shedding light on miR-214-3p's regulatory role in autophagy. These findings enhance our understanding of PD pathogenesis and propose miR-214-3p as a potential biomarker and therapeutic target for modulating autophagy and neuronal survival in PD.
    Keywords:  ATG3; Autophagy; Dopaminergic neurons; Parkinson's disease; miR-214–3p
    DOI:  https://doi.org/10.1016/j.biopha.2024.116123
  44. Mol Biol Cell. 2024 Jan 10. mbcE23080328
    Loss of the HOPS complex disrupts early-to-late endosome transition, impairs endosomal recycling and induces accumulation of amphisomes.
    Jan van der Beek, Cilia de Heus, Paolo Sanza, Nalan Liv, Judith Klumperman.
      The multisubunit HOPS tethering complex is a well-established regulator of lysosome fusion with late endosomes and autophagosomes. However, the role of the HOPS complex in other stages of endo-lysosomal trafficking is not well understood. To address this, we made HeLa cells knocked out for the HOPS-specific subunits Vps39 or Vps41, or the HOPS-CORVET-core subunits Vps18 or Vps11. In all four knockout cells we found that endocytosed cargos were trapped in enlarged endosomes that clustered in the perinuclear area. By correlative light-electron microscopy these endosomes showed a complex ultrastructure and hybrid molecular composition, displaying markers for early (Rab5, PtdIns3P, EEA1) as well as late (Rab7, CD63, LAMP1) endosomes. These 'HOPS bodies' were not acidified, contained enzymatically inactive cathepsins and accumulated endocytosed cargo and cation-independent mannose-6-phosphate receptor (CI-MPR). Consequently, CI-MPR was depleted from the TGN, and secretion of lysosomal enzymes to the extracellular space was enhanced. Strikingly, HOPS bodies also contained the autophagy proteins p62 and LC3, defining them as amphisomes. Together, these findings show that depletion of the lysosomal HOPS complex has a profound impact on the functional organization of the entire endosomal system and suggest the existence of a HOPS-independent mechanism for amphisome formation.
    DOI:  https://doi.org/10.1091/mbc.E23-08-0328
  45. Int J Mol Sci. 2023 Dec 20. pii: 90. [Epub ahead of print]25(1):
    Identification and Potential Participation of Lipases in Autophagic Body Degradation in Embryonic Axes of Lupin (Lupinus spp.) Germinating Seeds.
    Karolina Wleklik, Szymon Stefaniak, Katarzyna Nuc, Małgorzata Pietrowska-Borek, Sławomir Borek.
      Autophagy is a fundamental process for plants that plays a crucial role in maintaining cellular homeostasis and promoting survival in response to various environmental stresses. One of the lesser-known stages of plant autophagy is the degradation of autophagic bodies in vacuoles. To this day, no plant vacuolar enzyme has been confirmed to be involved in this process. On the other hand, several enzymes have been described in yeast (Saccharomyces cerevisiae), including Atg15, that possess lipolytic activity. In this preliminary study, which was conducted on isolated embryonic axes of the white lupin (Lupinus albus L.) and Andean lupin (Lupinus mutabilis Sweet), the potential involvement of plant vacuolar lipases in the degradation of autophagic bodies was investigated. We identified in transcriptomes (using next-generation sequencing (NGS)) of white and Andean lupin embryonic axes 38 lipases with predicted vacuolar localization, and for three of them, similarities in amino acid sequences with yeast Atg15 were found. A comparative transcriptome analysis of lupin isolated embryonic axes cultured in vitro under different sucrose and asparagine nutrition, evaluating the relations in the levels of the transcripts of lipase genes, was also carried out. A clear decrease in lipase gene transcript levels caused by asparagine, a key amino acid in lupin seed metabolism which retards the degradation of autophagic bodies during sugar-starvation-induced autophagy in lupin embryonic axes, was detected. Although the question of whether lipases are involved in the degradation of autophagic bodies during plant autophagy is still open, our findings strongly support such a hypothesis.
    Keywords:  Atg15; asparagine; autophagy; carbon starvation; lupin; seed embryo; sucrose; transcriptomics; vacuole; yeast
    DOI:  https://doi.org/10.3390/ijms25010090
  46. Cell Mol Life Sci. 2024 Jan 12. 81(1): 29
    Intracellular calcium links milk stasis to lysosome-dependent cell death during early mammary gland involution.
    Jaekwang Jeong, Jongwon Lee, Gabriel Talaia, Wonnam Kim, Junho Song, Juhyeon Hong, Kwangmin Yoo, David G Gonzalez, Diana Athonvarangkul, Jaehun Shin, Pamela Dann, Ann M Haberman, Lark Kyun Kim, Shawn M Ferguson, Jungmin Choi, John Wysolmerski.
      Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 h of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6, and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We further demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis through a process involving inhibition of CDK4/6 and cell cycle progression. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.
    Keywords:  Calcium; LDCD; PMCA2; STAT3; TFEB
    DOI:  https://doi.org/10.1007/s00018-023-05044-8
  47. Cell Commun Signal. 2024 Jan 09. 22(1): 19
    The cell cycle, autophagy, and cell wall integrity pathway jointly governed by MoSwe1 in Magnaporthe oryzae.
    Lin Li, Xue-Ming Zhu, Jian-Dong Bao, Jiao-Yu Wang, Xiao-Hong Liu, Fu-Cheng Lin.
      The cell cycle is pivotal to cellular differentiation in plant pathogenic fungi. Cell wall integrity (CWI) signaling plays an essential role in coping with cell wall stress. Autophagy is a degradation process in which cells decompose their components to recover macromolecules and provide energy under stress conditions. However, the specific association between cell cycle, autophagy and CWI pathway remains unclear in model pathogenic fungi Magnaporthe oryzae. Here, we have identified MoSwe1 as the conserved component of the cell cycle in the rice blast fungus. We have found that MoSwe1 targets MoMps1, a conserved critical MAP kinase of the CWI pathway, through protein phosphorylation that positively regulates CWI signaling. The CWI pathway is abnormal in the ΔMoswe1 mutant with cell cycle arrest. In addition, we provided evidence that MoSwe1 positively regulates autophagy by interacting with MoAtg17 and MoAtg18, the core autophagy proteins. Moreover, the S phase initiation was earlier, the morphology of conidia and appressoria was abnormal, and septum formation and glycogen degradation were impaired in the ΔMoswe1 mutant. Our research defines that MoSWE1 regulation of G1/S transition, CWI pathway, and autophagy supports its specific requirement for appressorium development and virulence in plant pathogenic fungi. Video Abstract.
    Keywords:  Appressorium formation; Autophagy; CWI pathway; Cell cycle; M. Oryzae; Virulence
    DOI:  https://doi.org/10.1186/s12964-023-01389-6
  48. Transl Psychiatry. 2024 Jan 08. 14(1): 13
    Olanzapine, risperidone and ziprasidone differently affect lysosomal function and autophagy, reflecting their different metabolic risk in patients.
    Marco Pozzi, Chiara Vantaggiato, Francesca Brivio, Genny Orso, Maria Teresa Bassi.
      The metabolic effects induced by antipsychotics in vitro depend on their action on the trafficking and biosynthesis of sterols and lipids. Previous research showed that antipsychotics with different adverse effects in patients cause similar alterations in vitro, suggesting the low clinical usefulness of cellular studies. Moreover, the inhibition of peripheral AMPK was suggested as potential aetiopathogenic mechanisms of olanzapine, and different effects on autophagy were reported for several antipsychotics. We thus assessed, in clinically-relevant culture conditions, the aetiopathogenic mechanisms of olanzapine, risperidone and ziprasidone, antipsychotics with respectively high, medium, low metabolic risk in patients, finding relevant differences among them. We highlighted that: olanzapine impairs lysosomal function affecting autophagy and autophagosome clearance, and increasing intracellular lipids and sterols; ziprasidone activates AMPK increasing the autophagic flux and reducing intracellular lipids; risperidone increases lipid accumulation, while it does not affect lysosomal function. These in vitro differences align with their different impact on patients. We also provided evidence that metformin add-on improved autophagy in olanzapine-treated cells and reduced lipid accumulation induced by both risperidone and olanzapine in an AMPK-dependent way; metformin also increased the production of bile acids to eliminate cholesterol accumulations caused by olanzapine. These results have different clinical implications. We demonstrated that antipsychotics with different metabolic impacts on patients actually have different mechanisms of action, thus supporting the possibility of a personalised antipsychotic treatment. Moreover, we found that metformin can fully revert the phenotype caused by risperidone but not the one caused by olanzapine, that still activates SREBP2.
    DOI:  https://doi.org/10.1038/s41398-023-02686-x
  49. Cells. 2023 Dec 21. pii: 28. [Epub ahead of print]13(1):
    MET Oncogene Enhances Pro-Migratory Functions by Counteracting NMDAR2B Cleavage.
    Simona Gallo, Annapia Vitacolonna, Paolo Maria Comoglio, Tiziana Crepaldi.
      The involvement of the N-methyl-D-aspartate receptor (NMDAR), a glutamate-gated ion channel, in promoting the invasive growth of cancer cells is an area of ongoing investigation. Our previous findings revealed a physical interaction between NMDAR and MET, the hepatocyte growth factor (HGF) receptor. However, the molecular mechanisms underlying this NMDAR/MET interaction remain unclear. In this study, we demonstrate that the NMDAR2B subunit undergoes proteolytic processing, resulting in a low-molecular-weight form of 100 kDa. Interestingly, when the NMDAR2B and MET constructs were co-transfected, the full-size high-molecular-weight NMDAR2B form of 160 kDa was predominantly observed. The protection of NMDAR2B from cleavage was dependent on the kinase activity of MET. We provide the following evidence that MET opposes the autophagic lysosomal proteolysis of NMDAR2B: (i) MET decreased the protein levels of lysosomal cathepsins; (ii) treatment with either an inhibitor of autophagosome formation or the fusion of the autophagosome and lysosome elevated the proportion of the NMDAR2B protein's uncleaved form; (iii) a specific mTOR inhibitor hindered the anti-autophagic effect of MET. Finally, we demonstrate that MET coopts NMDAR2B to augment cell migration. This implies that MET harnesses the functionality of NMDAR2B to enhance the ability of cancer cells to migrate.
    Keywords:  MET tyrosine kinase receptor; NMDAR; cancers; cell migration; glutamate receptor; proteolytic cleavage
    DOI:  https://doi.org/10.3390/cells13010028
  50. Autophagy. 2024 Jan 08.
    Should proteins be interrogated when they conspire?
    Daniel J Klionsky.
      As a follow up to that wildly popular11 At least in my own mind. Editor's Corner, "Look youse guys and gals, dat just ain't right" published in 2021, I have put together a follow-up guide to some common grammatical mistakes that I encounter in papers submitted to Autophagy. This guide is meant in particular to help non-native English speakers write more clearly but may also be of benefit to other authors who grew up at a time when learning grammar was replaced by autocorrect functions in Word and other programs, or the desire to simplify sentences so that they fit within a tweet, or to ease thumb strain when typing on a smartphone. The guide is also meant to help editors by reducing the number of changes needed to bring papers up to the high standards of clarity that we strive to maintain at Autophagy.
    Keywords:  Autophagy; grammar; it’s all about clarity; meaning what you write; writing what you mean
    DOI:  https://doi.org/10.1080/15548627.2024.2301800
  51. Nat Nanotechnol. 2024 Jan 12.
    Nanoreceptors promote mutant p53 protein degradation by mimicking selective autophagy receptors.
    Xiaowan Huang, Ziyang Cao, Jieying Qian, Tao Ding, Yanxia Wu, Hao Zhang, Suqin Zhong, Xiaoli Wang, Xiaoguang Ren, Wang Zhang, Youcui Xu, Guangyu Yao, Xingwu Wang, Xianzhu Yang, Longping Wen, Yunjiao Zhang.
      In some cancers mutant p53 promotes the occurrence, development, metastasis and drug resistance of tumours, with targeted protein degradation seen as an effective therapeutic strategy. However, a lack of specific autophagy receptors limits this. Here, we propose the synthesis of biomimetic nanoreceptors (NRs) that mimic selective autophagy receptors. The NRs have both a component for targeting the desired protein, mutant-p53-binding peptide, and a component for enhancing degradation, cationic lipid. The peptide can bind to mutant p53 while the cationic lipid simultaneously targets autophagosomes and elevates the levels of autophagosome formation, increasing mutant p53 degradation. The NRs are demonstrated in vitro and in a patient-derived xenograft ovarian cancer model in vivo. The work highlights a possible direction for treating diseases by protein degradation.
    DOI:  https://doi.org/10.1038/s41565-023-01562-5
  52. New Phytol. 2024 Jan 08.
    Cytochrome c levels affect the TOR pathway to regulate growth and metabolism under energy-deficient conditions.
    María Victoria Canal, Natanael Mansilla, Diana E Gras, Agustín Ibarra, Carlos M Figueroa, Daniel H Gonzalez, Elina Welchen.
      Mitochondrial function is essential for plant growth, but the mechanisms involved in adjusting growth and metabolism to changes in mitochondrial energy production are not fully understood. We studied plants with reduced expression of CYTC-1, one of two genes encoding the respiratory chain component cytochrome c (CYTc) in Arabidopsis, to understand how mitochondria communicate their status to coordinate metabolism and growth. Plants with CYTc deficiency show decreased mitochondrial membrane potential and lower ATP content, even when carbon sources are present. They also exhibit higher free amino acid content, induced autophagy, and increased resistance to nutritional stress caused by prolonged darkness, similar to plants with triggered starvation signals. CYTc deficiency affects target of rapamycin (TOR)-pathway activation, reducing S6 kinase (S6K) and RPS6A phosphorylation, as well as total S6K protein levels due to increased protein degradation via proteasome and autophagy. TOR overexpression restores growth and other parameters affected in cytc-1 mutants, even if mitochondrial membrane potential and ATP levels remain low. We propose that CYTc-deficient plants coordinate their metabolism and energy availability by reducing TOR-pathway activation as a preventive signal to adjust growth in anticipation of energy exhaustion, thus providing a mechanism by which changes in mitochondrial activity are transduced to the rest of the cell.
    Keywords:  Arabidopsis thaliana; cytochrome c; energy deficiency; mitochondria; starvation; target of rapamycin
    DOI:  https://doi.org/10.1111/nph.19506
  53. Am J Clin Nutr. 2024 Jan 10. pii: S0002-9165(24)00004-2. [Epub ahead of print]
    Acute ingestion of a ketone monoester, whey protein, or their co-ingestion in the overnight postabsorptive state elicit a similar stimulation of myofibrillar protein synthesis rates in young males: a double-blind randomized trial.
    Sarkis J Hannaian, Jamie Lov, Stephanie E Hawley, Manon Dargegen, Divine Malenda, Ari Gritsas, Gilles Gouspillou, José A Morais, Tyler A Churchward-Venne.
      BACKGROUND: Ketone bodies may have anabolic effects in skeletal muscle via their capacity to stimulate protein synthesis. Whether orally ingested exogenous ketones can stimulate postprandial myofibrillar protein synthesis (MyoPS) rates with and without dietary protein co-ingestion is unknown.OBJECTIVES: To evaluate the effects of ketone monoester intake and elevated blood β-hydroxybutyrate (β-OHB) concentration, with and without dietary protein co-ingestion, on postprandial MyoPS rates and mechanistic target of rapamycin complex 1 (mTORC1) pathway signalling.
    METHODS: In a randomized, double-blind, parallel group design, 36 recreationally active healthy young males (age: 24.2±4.1 y; body fat: 20.9±5.8 %; BMI: 23.4±2 kg/m2) received a primed continuous infusion of L-[ring-2H5]-phenylalanine and ingested either: 1) the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET+PRO). Blood and muscle biopsy samples were collected during basal and postprandial (300 min) conditions to assess β-OHB, glucose, insulin, and amino acid concentrations, MyoPS rates, and mTORC1 pathway signalling.
    RESULTS: Capillary blood β-OHB concentration increased similarly during postprandial conditions in KET and KET+PRO, with both being greater than PRO from 30-180 min (Treatment × Time Interaction: P<0.001). Postprandial plasma leucine and essential amino acid (EAA) incremental area under the curve (iAUC) over 300 min was greater (Treatment: both P<0.001) in KET+PRO vs. PRO and KET. KET, PRO, and KET+PRO stimulated postprandial MyoPS rates (0-300 min) above basal conditions (absolute change: 0.020%/h (95% CI: 0.013, 0.027%/h), 0.014%/h (95% CI: 0.009, 0.019%/h), 0.019%/h (95% CI: 0.014, 0.024%/h), respectively (Time: P<0.001), with no difference between treatments (Treatment: P=0.383) or treatment × time interaction (Interaction: P=0.245). mTORC1 pathway signalling responses did not differ between treatments (all P>0.05).
    CONCLUSION: Acute oral intake of a ketone monoester, 10 g whey protein, or their co-ingestion in the overnight postabsorptive state elicit a similar stimulation of postprandial MyoPS rates in healthy young males. This trial was registered at clinicaltrials.gov as NCT04565444. A direct link to the trial page is available here: https://clinicaltrials.gov/study/NCT04565444.
    Keywords:  amino acids; exogenous ketosis; ketone bodies; myofibrillar protein synthesis; skeletal muscle; whey protein; young males; β-hydroxybutyrate
    DOI:  https://doi.org/10.1016/j.ajcnut.2024.01.004
  54. J Inflamm Res. 2024 ;17 91-100
    Role of Autophagy and Pyroptosis in Intervertebral Disc Degeneration.
    Zhiqiang Wang, Xiaochun Li, Pengfei Yu, Yu Zhu, Feng Dai, Zhijia Ma, Xueqiang Shen, Hong Jiang, Jintao Liu.
      Intervertebral disc degeneration is a chronic degenerative disease caused by the interaction of genetic and environmental factors, mainly manifested as lower back pain. At present, the diagnosis of intervertebral disc degeneration mainly relies on imaging. However, early intervertebral disc degeneration is usually insidious, and there is currently a lack of relevant clinical biomarkers that can reliably reflect early disease progression. Pyroptosis is a regulatory form of cell death triggered by the activation of inflammatory bodies and caspase, which can induce the formation of plasma membrane pores and cell swelling or lysis. Previous studies have shown that during the progression of intervertebral disc degeneration, sustained activation of inflammasomes leads to nuclear cell pyroptosis, which can occur in the early stages of intervertebral disc degeneration. Moreover, intervertebral disc nucleus pulposus cells adapt to the external environment through autophagy and maintain cellular homeostasis and studying the mechanism of autophagy in IDD and intervening in its pathological and physiological processes can provide new ideas for the clinical treatment of IDD. This review analyzes the effects of pyroptosis and autophagy on IDD by reviewing relevant literature in recent years, in order to explore the relationship between pyroptosis, autophagy and IDD.
    Keywords:  autophagy; intervertebral disc degeneration; pyroptosis
    DOI:  https://doi.org/10.2147/JIR.S434896
  55. Am J Respir Cell Mol Biol. 2024 Jan 11.
    MLKL Protects Pulmonary Endothelial Cells in Acute Lung Injury.
    Ying Li, Yingxiang Liu, Xueya Yao, Ling Zhu, Liqun Yang, Qionghui Zhan.
      The role of autophagy in pulmonary microvascular endothelial cell (PMVEC) are controversial in lipopolysaccharide (LPS)-induced ALI. Mixed lineage kinase domain like pseudokinase (MLKL) has been recently reported to keep cell survival by facilitating autophagic flux in response to starvation, rather than its well-recognized role in necroptosis. Using mouse PMVEC and LPS-induced ALI model, we showed that in PMVEC, MLKL was phosphorylated (p-MLKL) and autophagic flux was accelerated at the early stage of LPS stimulation (1-3 h), manifested by increases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II), decreases in levels of SQSTM1/p62 (sequestosome 1), and fusion of autophagosome and lysosome by pHluorin-mKate2-human LC3 assay, which were all reversed by either MLKL inhibitor or siRNA MLKL. In mice, the inhibition of MLKL increased vascular permeability and aggravated mouse ALI upon 3-h LPS stimulation. The p-MLKL induced by short-term LPS formed multimers to facilitate the closure of phagophore by HaloTag-LC3 autophagosome completion assay. The charged multivesicular body protein 2A (CHMP2A) is essential in the process of phagophore closure into nascent autophagosome. Agreeing with the p-MLKL change, CHMP2A levels markedly increased during 1-3 h LPS stimulation. CHMP2A knockdown blocked autophagic flux upon LPS stimulation, whereas CHMP2A overexpression boosted autophagic flux and attenuated mouse ALI even in the presence of MLKL inhibitor. We propose that the activated MLKL induced by short-term LPS facilitates autophagic flux by accelerating the closure of phagophore via CHMP2A, thus protecting PMVEC and alleviating LPS-induced ALI.
    Keywords:  ALI; CHMP2A; MLKL; autophagic flux; pulmonary microvascular endothelial cell
    DOI:  https://doi.org/10.1165/rcmb.2023-0207OC
  56. Cancer Cell. 2024 Jan 06. pii: S1535-6108(23)00447-6. [Epub ahead of print]
    Response to Bruton's tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy.
    James D Phelan, Sebastian Scheich, Jaewoo Choi, George W Wright, Björn Häupl, Ryan M Young, Sara A Rieke, Martine Pape, Yanlong Ji, Henning Urlaub, Arnold Bolomsky, Carmen Doebele, Alena Zindel, Tanja Wotapek, Monica Kasbekar, Brett Collinge, Da Wei Huang, Zana A Coulibaly, Vivian M Morris, Xiaoxuan Zhuang, Julius C Enssle, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Zhuo Wang, Andy D Tran, Christopher J Shoemaker, Galina Shevchenko, Daniel J Hodson, Arthur L Shaffer, Louis M Staudt, Thomas Oellerich.
      Diffuse large B cell lymphoma (DLBCL) is an aggressive, profoundly heterogeneous cancer, presenting a challenge for precision medicine. Bruton's tyrosine kinase (BTK) inhibitors block B cell receptor (BCR) signaling and are particularly effective in certain molecular subtypes of DLBCL that rely on chronic active BCR signaling to promote oncogenic NF-κB. The MCD genetic subtype, which often acquires mutations in the BCR subunit, CD79B, and in the innate immune adapter, MYD88L265P, typically resists chemotherapy but responds exceptionally to BTK inhibitors. However, the underlying mechanisms of response to BTK inhibitors are poorly understood. Herein, we find a non-canonical form of chronic selective autophagy in MCD DLBCL that targets ubiquitinated MYD88L265P for degradation in a TBK1-dependent manner. MCD tumors acquire genetic and epigenetic alterations that attenuate this autophagic tumor suppressive pathway. In contrast, BTK inhibitors promote autophagic degradation of MYD88L265P, thus explaining their exceptional clinical benefit in MCD DLBCL.
    Keywords:  Bruton’s tyrosine kinase; DLBCL; autophagy; functional genomics; proteomics; targeted therapy
    DOI:  https://doi.org/10.1016/j.ccell.2023.12.019
  57. Int J Mol Sci. 2024 Jan 03. pii: 633. [Epub ahead of print]25(1):
    Endurance Exercise Training Mitigates Diastolic Dysfunction in Diabetic Mice Independent of Phosphorylation of Ulk1 at S555.
    Yuntian Guan, Mei Zhang, Christie Lacy, Soham Shah, Frederick H Epstein, Zhen Yan.
      Millions of diabetic patients suffer from cardiovascular complications. One of the earliest signs of diabetic complications in the heart is diastolic dysfunction. Regular exercise is a highly effective preventive/therapeutic intervention against diastolic dysfunction in diabetes, but the underlying mechanism(s) remain poorly understood. Studies have shown that the accumulation of damaged or dysfunctional mitochondria in the myocardium is at the center of this pathology. Here, we employed a mouse model of diabetes to test the hypothesis that endurance exercise training mitigates diastolic dysfunction by promoting cardiac mitophagy (the clearance of mitochondria via autophagy) via S555 phosphorylation of Ulk1. High-fat diet (HFD) feeding and streptozotocin (STZ) injection in mice led to reduced endurance capacity, impaired diastolic function, increased myocardial oxidative stress, and compromised mitochondrial structure and function, which were all ameliorated by 6 weeks of voluntary wheel running. Using CRISPR/Cas9-mediated gene editing, we generated non-phosphorylatable Ulk1 (S555A) mutant mice and showed the requirement of p-Ulk1at S555 for exercise-induced mitophagy in the myocardium. However, diabetic Ulk1 (S555A) mice retained the benefits of exercise intervention. We conclude that endurance exercise training mitigates diabetes-induced diastolic dysfunction independent of Ulk1 phosphorylation at S555.
    Keywords:  diabetes; diastolic dysfunction; echocardiography; exercise intervention; mitochondrial quality; mitophagy
    DOI:  https://doi.org/10.3390/ijms25010633
  58. J Neurochem. 2024 Jan 09.
    Arrestin-3 binds parkin and enhances parkin-dependent mitophagy.
    Chen Zheng, Kevin K Nguyen, Sergey A Vishnivetskiy, Vsevolod V Gurevich, Eugenia V Gurevich.
      Arrestins were discovered for their role in homologous desensitization of G-protein-coupled receptors (GPCRs). Later non-visual arrestins were shown to regulate several signaling pathways. Some of these pathways require arrestin binding to GPCRs, the regulation of others is receptor independent. Here, we demonstrate that arrestin-3 binds the E3 ubiquitin ligase parkin via multiple sites, preferentially interacting with its RING0 domain. Identification of the parkin domains involved suggests that arrestin-3 likely relieves parkin autoinhibition and/or stabilizes the enzymatically active "open" conformation of parkin. Arrestin-3 binding enhances ubiquitination by parkin of the mitochondrial protein mitofusin-1 and facilitates parkin-mediated mitophagy in HeLa cells. Furthermore, arrestin-3 and its mutant with enhanced parkin binding rescue mitofusin-1 ubiquitination and mitophagy in the presence of the Parkinson's disease-associated R275W parkin mutant, which is defective in both functions. Thus, modulation of parkin activity via arrestin-3 might be a novel strategy of anti-parkinsonian therapy.
    Keywords:  arrestin; mitochondria; mitophagy; parkin
    DOI:  https://doi.org/10.1111/jnc.16043
  59. Cell Death Discov. 2024 Jan 11. 10(1): 21
    Serine to proline mutation at position 341 of MYOC impairs trabecular meshwork function by causing autophagy deregulation.
    Xuejing Yan, Shen Wu, Qian Liu, Ying Cheng, Yufei Teng, Tianmin Ren, Jingxue Zhang, Ningli Wang.
      Glaucoma is a highly heritable disease, and myocilin was the first identified causal and most common pathogenic gene in glaucoma. Serine-to-proline mutation at position 341 of myocilin (MYOCS341P) is associated with severe glaucoma phenotypes in a five-generation primary open-angle glaucoma family. However, the underlying mechanisms are underexplored. Herein, we established the MYOCS341P transgenic mouse model and characterized the glaucoma phenotypes. Further, we systematically explored the functional differences between wild-type and MYOCS341P through immunoprecipitation, mass spectrometry, and RNA-seq analyses. We found that MYOCS341P transgenic mice exhibit glaucoma phenotypes, characterized by reduced aqueous humor outflow, elevated intraocular pressure, decreased trabecular meshwork (TM) cell number, narrowed Schlemm's canal, retinal ganglion cell loss, and visual impairment. Mechanistically, the secretion of dysfunctional MYOCS341P accumulated in the endoplasmic reticulum (ER), inducing ER stress and dysregulation of autophagy, thereby promoting TM cell death. We describe an effective transgenic model for mechanistic studies and the screening of therapeutic targets. Our data generated from high-throughput analyses help elucidate the mechanism underlying mutant MYOC-related glaucoma.
    DOI:  https://doi.org/10.1038/s41420-024-01801-1
  60. Cell Biol Int. 2024 Jan 09.
    Curcumin inhibits propofol-induced autophagy of MN9D cells via Akt/mTOR/p70S6K signaling pathway.
    Hongxia He, Yuping Han, Qiuyan Wan, Yao Yue, Shurong Li, Bingyin Su, Jun Li.
      The rapid rise in propofol dependency and abuse has highlighted limited resources for addressing substance abuse-related cognitive impairment, prompting the development of novel therapies. Dysregulated autophagy flow accelerates neuronal cell death, and interventions countering this dysregulation offer an appealing strategy for neuronal protection. Curcumin, a potent natural polyphenol derived from turmeric rhizomes, is renowned for its robust antineurotoxic properties and enhanced cognitive function. Utilizing CCK-8 and Ki67 fluorescent staining, our study revealed that curcumin treatment increased cell viability and proliferative potential in MN9D cells exposed to propofol-induced neurotoxicity. Furthermore, enzyme-linked immunosorbent assay and western blot analysis demonstrated the partial restoration of dopamine synthesis, secretion levels, and TH expression in damaged MN9D cells treated with curcumin. Scanning electrode microscope images displayed reduced autolysosomes and phagosomes in curcumin-treated cells compared to the propofol group. Immunoblotting revealed that curcumin mitigated the degradation of LC3I to LC3II and p62 induced by propofol stimulation, with green fluorescence expression of LC3 postcurcumin treatment resembling that following autophagy inhibitor HCQ treatment, indicating that modulating autophagy flow can alleviate propofol's toxic effects. Moreover, curcumin treatment upregulated the Akt/mTOR/p70S6K signaling pathway, suggesting that curcumin potentially curtails autophagy dysregulation in nerve cells by activating Akt/mTOR/p70S6K. In conclusion, our findings suggest that curcumin can ameliorate propofol abuse-induced neurotoxicity, partially through autophagy regulation and Akt/mTOR/p70S6K signaling activation.
    Keywords:  Akt/mTOR/p70S6K signaling pathway; autophagy; curcumin; propofol
    DOI:  https://doi.org/10.1002/cbin.12117
  61. Int J Mol Sci. 2024 Jan 04. pii: 646. [Epub ahead of print]25(1):
    ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia.
    Justine Lai, Claire Yang, Chuquan Shang, Will Chen, Michael P Chu, Joseph Brandwein, Raymond Lai, Peng Wang.
      We recently demonstrated that a small subset of cells in FLT3-mutated acute myeloid leukemia (AML) cell lines exhibit SORE6 reporter activity and cancer stem-like features including chemoresistance. To study why SORE6+ cells are more chemoresistant than SORE6- cells, we hypothesized that these cells carry higher autophagy, a mechanism linked to chemoresistance. We found that cytarabine (Ara-C) induced a substantially higher protein level of LC3B-II in SORE6+ compared to SORE6- cells. Similar observations were made using a fluorescence signal-based autophagy assay. Furthermore, chloroquine (an autophagy inhibitor) sensitized SORE6+ but not SORE6- cells to Ara-C. To decipher the molecular mechanisms underlying the high autophagic flux in SORE6+ cells, we employed an autophagy oligonucleotide array comparing gene expression between SORE6+ and SORE6- cells before and after Ara-C treatment. ULK2 was the most differentially expressed gene between the two cell subsets. To demonstrate the role of ULK2 in conferring higher chemoresistance in SORE6+ cells, we treated the two cell subsets with a ULK1/2 inhibitor, MRT68921. MRT68921 significantly sensitized SORE6+ but not SORE6- cells to Ara-C. Using our in vitro model for AML relapse, we found that regenerated AML cells contained higher ULK2 expression compared to pretreated cells. Importantly, inhibition of ULK2 using MRT68921 prevented in vitro AML relapse. Lastly, using pretreatment and relapsed AML patient bone marrow samples, we found that ULK2 expression was higher in relapsed AML. To conclude, our results supported the importance of autophagy in the relapse of FLT3-mutated AML and highlighted ULK2 in this context.
    Keywords:  acute myeloid leukemia; autophagy; cancer stemness; relapse
    DOI:  https://doi.org/10.3390/ijms25010646
  62. Nat Commun. 2024 Jan 10. 15(1): 447
    Disruption of lysosomal proteolysis in astrocytes facilitates midbrain organoid proteostasis failure in an early-onset Parkinson's disease model.
    Gustavo Morrone Parfitt, Elena Coccia, Camille Goldman, Kristen Whitney, Ricardo Reyes, Lily Sarrafha, Ki Hong Nam, Soha Sohail, Drew R Jones, John F Crary, Alban Ordureau, Joel Blanchard, Tim Ahfeldt.
      Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.
    DOI:  https://doi.org/10.1038/s41467-024-44732-2
  63. Diagnostics (Basel). 2023 Dec 25. pii: 38. [Epub ahead of print]14(1):
    mTOR Pathway Substrates Present High Activation in Vascular Malformations and Significantly Decrease with Age.
    Jakub Kopeć, Elżbieta Sałacińska-Łoś, Magdalena Orzechowska, Monika Sokolnicka, Aleksandra Gawłowska-Marciniak, Przewratil Przemysław.
      BACKGROUND: Vascular anomalies often result in aesthetic flaws, pain, and impair the quality of life. They require challenging treatments that frequently do not provide the desired results. The mammalian target of rapamycin (mTOR) is directly involved in the development of these malformations. However, the exact mechanism behind mTOR dysregulation has not been unambiguously defined. The purpose of this study is to investigate the activation of selected substrates of mTOR to partially assess its involvement in the disease process.METHODS: We analyzed tissue samples collected from patients with vascular anomalies treated in our department. We included patients with histopathological diagnoses of lymphatic, venous, capillary malformations, mixed lesions, and a control group of healthy skin samples. We stained the samples using H and E and immunohistochemistry. We used primary antibodies against p70 S6 Kinase, 4EBP1, and p-4EBP1. We graded their color reactions. The statistical analyses were performed using the FactoMineR and factoextra R v.4.1 packages. p-values < 0.05 were considered statistically significant.
    RESULTS: The analysis of 82 patients showed that healthy tissue vessels expressed lower levels of tested mTOR pathway substrates compared to high activation in vascular malformations. Elevated substrate expression in a comparison between sexes revealed higher P-4EBP1 expression in the female malformation group. We observed a decrease in mTOR substrate expression with age.
    CONCLUSION: The higher expression of mTOR substrates in vascular malformations compared to healthy tissue confirms their involvement in abnormal vascular development. Age-related changes in mTOR substrate expression highlight the need for timely intervention. Our study contributes to the understanding of the mTOR signaling pathway in vascular malformations and highlights its potential as a therapeutic target, contributing to personalized medicine.
    Keywords:  lymphatic malformation; mTOR; rapamycin; sirolimus; vascular anomalies; venus malformation
    DOI:  https://doi.org/10.3390/diagnostics14010038
  64. Stem Cell Reports. 2023 Dec 29. pii: S2213-6711(23)00498-8. [Epub ahead of print]
    Autophagy is essential for human myelopoiesis.
    Jiaming Gu, Yanling Zhu, Huaisong Lin, Yuhua Huang, Yanqi Zhang, Qi Xing, Baoqiang Kang, Zhishuai Zhang, Mingquan Wang, Tiancheng Zhou, Yuchan Mai, Qianyu Chen, Fei Li, Xing Hu, Shuoting Wang, Jiaojiao Peng, Xinrui Guo, Bing Long, Junwei Wang, Minghui Gao, Yongli Shan, Yazhou Cui, Guangjin Pan.
      Emergency myelopoiesis (EM) is essential in immune defense against pathogens for rapid replenishing of mature myeloid cells. During the EM process, a rapid cell-cycle switch from the quiescent hematopoietic stem cells (HSCs) to highly proliferative myeloid progenitors (MPs) is critical. How the rapid proliferation of MPs during EM is regulated remains poorly understood. Here, we reveal that ATG7, a critical autophagy factor, is essential for the rapid proliferation of MPs during human myelopoiesis. Peripheral blood (PB)-mobilized hematopoietic stem/progenitor cells (HSPCs) with ATG7 knockdown or HSPCs derived from ATG7-/- human embryonic stem cells (hESCs) exhibit severe defect in proliferation during fate transition from HSPCs to MPs. Mechanistically, we show that ATG7 deficiency reduces p53 localization in lysosome for a potential autophagy-mediated degradation. Together, we reveal a previously unrecognized role of autophagy to regulate p53 for a rapid proliferation of MPs in human myelopoiesis.
    DOI:  https://doi.org/10.1016/j.stemcr.2023.12.005
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