bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023–10–08
eighty papers selected by
Viktor Korolchuk, Newcastle University
  1. Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease.
  2. Autophagy: Regulator of cell death.
  3. Iron blocks autophagic flux and induces autophagosomes accumulation in microglia.
  4. Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy.
  5. Metabolic control from the endolysosome: lysosome-resident amino acid transporters open novel therapeutic possibilities.
  6. Mitophagy for cardioprotection.
  7. USP13 deubiquitinates p62/SQSTM1 to induce autophagy and Nrf2 release for activating antioxidant response genes.
  8. S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.
  9. A historical perspective of macroautophagy regulation by biochemical and biomechanical stimuli.
  10. Atg5 knockout induces alternative autophagy via the downregulation of Akt expression.
  11. Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae.
  12. Microorganism-regulated autophagy in gastrointestinal cancer.
  13. Non-canonical functions of a mutant TSC2 protein in mitotic division.
  14. ApoE4-dependent lysosomal cholesterol accumulation impairs mitochondrial homeostasis and oxidative phosphorylation in human astrocytes.
  15. Blazing a trail for the clinical use of rapamycin as a geroprotecTOR.
  16. The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps.
  17. Spatiotemporal regulation of de novo and salvage purine synthesis during brain development.
  18. Chaperone-mediated autophagy protects against hyperglycemic stress.
  19. L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.
  20. Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells.
  21. Perturbation of autophagy pathways in murine alveolar macrophage by 2D TMDCs is chalcogen-dependent.
  22. TLR7 promotes skin inflammation via activating NFκB-mTORC1 axis in rosacea.
  23. VPS35, the core component of the retromer complex, and Parkinson's disease.
  24. PACS-2 deficiency aggravates tubular injury in diabetic kidney disease by inhibiting ER-phagy.
  25. Unraveling the interplay between RAS/RAF/MEK/ERK signaling pathway and autophagy in cancer: From molecular mechanisms to targeted therapy.
  26. Assessment of Autophagy Markers Suggests Increased Activity Following LVAD Therapy.
  27. CoCl2 -triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299.
  28. Liquid-liquid phase separation regulates alpha-synuclein aggregate and mitophagy in Parkinson's disease.
  29. Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy.
  30. Improving lysosomal ferroptosis with NMN administration protects against heart failure.
  31. The role of autophagy protein Atg5 in multiple sclerosis.
  32. HIF-1α Contributes to Hypoxia-induced VSMC Proliferation and Migration by Regulating Autophagy in Type A Aortic Dissection.
  33. Epstein-Barr Virus Encoded BCL2, BHRF1, Downregulates Autophagy by Noncanonical Binding of BECN1.
  34. Phosphoribosyl modification of poly-ubiquitin chains at the Legionella-containing vacuole prohibiting autophagy adaptor recognition.
  35. FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation.
  36. Targeted protein degradation in drug development: Recent advances and future challenges.
  37. Autophagy in the LVAD-Supported Heart: A Sign of Hope or a Marker of Unloading.
  38. The Prognosis of Cancer Depends on the Interplay of Autophagy, Apoptosis, and Anoikis within the Tumor Microenvironment.
  39. Molecular mechanism by which RRM2-inhibitor (cholagogue osalmid) plus bafilomycin A1 cause autophagic cell death in multiple myeloma.
  40. Deficient chaperone-mediated autophagy facilitates LPS-induced microglial activation via regulation of the p300/NF-κB/NLRP3 pathway.
  41. NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma.
  42. Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice.
  43. Calcium storage in multivesicular endo-lysosome.
  44. Divergence of TORC1-mediated Stress Response Leads to Novel Acquired Stress Resistance in a Pathogenic Yeast.
  45. miR-22a targets p62/SQSTM1 to negatively affect autophagy and disease resistance of grass carp (Ctenopharyngodon idella).
  46. Sortilin inhibition treats multiple neurodegenerative lysosomal storage disorders.
  47. Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.
  48. STING channels its proton power.
  49. The Role of Prohibitin-2 in Diseases.
  50. ARL8B mediates lipid droplet contact and delivery to lysosomes for lipid remobilization.
  51. Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells.
  52. Leflunomide alleviates obesity via activation of the TAK1-AMPK pathway and induction of lipophagy.
  53. Amino acid transporter SLC38A5 is a tumor promoter and a novel therapeutic target for pancreatic cancer.
  54. The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.
  55. Metformin activates AMPK and mTOR to Inhibit RANKL-stimulated osteoclast formation.
  56. Mechanistic target of rapamycin (mTOR): a potential new therapeutic target for rheumatoid arthritis.
  57. CENPN suppresses autophagy and increases paclitaxel resistance in nasopharyngeal carcinoma cells by inhibiting the CREB-VAMP8 signaling axis.
  58. Mitochondrial dysfunction: A fatal blow in depression.
  59. mTOR inhibits starvation-induced nuclear export of the proteasome.
  60. Modulation of TRPM8 alters the phagocytic activity of microglia and induces changes in sub-cellular organelle functions.
  61. Identification of autophagy and angiogenesis modulators in colorectal cancer based on bioinformatics analysis.
  62. Engeletin alleviates depression-like phenotype by increasing synaptic plasticity via the BDNF-TrkB-mTORC1 signalling pathway.
  63. Syntaxin 17 Protects Against Heart Failure Through Recruitment of CDK1 to Promote DRP1-Dependent Mitophagy.
  64. Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.
  65. Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole.
  66. Melatonin Potentiates Sensitivity to 5-Fluorouracil in Gastric Cancer Cells by Upregulating Autophagy and Downregulating Myosin Light-Chain Kinase.
  67. Herp regulates intracellular survival of Mycobacterium tuberculosis H37Ra in macrophages by regulating reactive oxygen species-mediated autophagy.
  68. Trehalose consumption ameliorates pathogenesis in an inducible mouse model of the Fragile X-associated tremor/ataxia syndrome.
  69. ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.
  70. Hotspots and future trends of autophagy in Traditional Chinese Medicine: A Bibliometric analysis.
  71. Pleural Fibrosis: Now That's What mTORC(ing) About.
  72. High Autophagy Patterns in Swelling Platelets During Apheresis Platelet Storage.
  73. P2Y6 receptor-dependent microglial phagocytosis of synapses during development regulates synapse density and memory.
  74. Cathepsin D overexpression in the nervous system rescues lethality and Aβ42 accumulation of cathepsin D systemic knockout in vivo.
  75. Protein restriction slows the development and progression of Alzheimer's disease in mice.
  76. Curcumin in vitro Neuroprotective Effects Are Mediated by p62/keap-1/Nrf2 and PI3K/AKT Signaling Pathway and Autophagy Inhibition.
  77. YTHDF2 facilitates aggresome formation via UPF1 in an m6A-independent manner.
  78. TSC2 S1365A mutation potently regulates CD8+T cell function and differentiation improving adoptive cellular cancer therapy.
  79. ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis.
  80. Invariant BECN1 CXXC motifs bind Zn2+ and regulate structure and function of the BECN1 intrinsically disordered region.
  1. Signal Transduct Target Ther. 2023 10 02. 8(1): 375
    Multifaceted role of mTOR (mammalian target of rapamycin) signaling pathway in human health and disease.
    Vivek Panwar, Aishwarya Singh, Manini Bhatt, Rajiv K Tonk, Shavkatjon Azizov, Agha Saquib Raza, Shinjinee Sengupta, Deepak Kumar, Manoj Garg.
      The mammalian target of rapamycin (mTOR) is a protein kinase that controls cellular metabolism, catabolism, immune responses, autophagy, survival, proliferation, and migration, to maintain cellular homeostasis. The mTOR signaling cascade consists of two distinct multi-subunit complexes named mTOR complex 1/2 (mTORC1/2). mTOR catalyzes the phosphorylation of several critical proteins like AKT, protein kinase C, insulin growth factor receptor (IGF-1R), 4E binding protein 1 (4E-BP1), ribosomal protein S6 kinase (S6K), transcription factor EB (TFEB), sterol-responsive element-binding proteins (SREBPs), Lipin-1, and Unc-51-like autophagy-activating kinases. mTOR signaling plays a central role in regulating translation, lipid synthesis, nucleotide synthesis, biogenesis of lysosomes, nutrient sensing, and growth factor signaling. The emerging pieces of evidence have revealed that the constitutive activation of the mTOR pathway due to mutations/amplification/deletion in either mTOR and its complexes (mTORC1 and mTORC2) or upstream targets is responsible for aging, neurological diseases, and human malignancies. Here, we provide the detailed structure of mTOR, its complexes, and the comprehensive role of upstream regulators, as well as downstream effectors of mTOR signaling cascades in the metabolism, biogenesis of biomolecules, immune responses, and autophagy. Additionally, we summarize the potential of long noncoding RNAs (lncRNAs) as an important modulator of mTOR signaling. Importantly, we have highlighted the potential of mTOR signaling in aging, neurological disorders, human cancers, cancer stem cells, and drug resistance. Here, we discuss the developments for the therapeutic targeting of mTOR signaling with improved anticancer efficacy for the benefit of cancer patients in clinics.
    DOI:  https://doi.org/10.1038/s41392-023-01608-z
  2. Cell Death Dis. 2023 Oct 04. 14(10): 648
    Autophagy: Regulator of cell death.
    ShiZuo Liu, ShuaiJie Yao, Huan Yang, ShuaiJie Liu, YanJiao Wang.
      Autophagy is the process by which cells degrade and recycle proteins and organelles to maintain intracellular homeostasis. Generally, autophagy plays a protective role in cells, but disruption of autophagy mechanisms or excessive autophagic flux usually leads to cell death. Despite recent progress in the study of the regulation and underlying molecular mechanisms of autophagy, numerous questions remain to be answered. How does autophagy regulate cell death? What are the fine-tuned regulatory mechanisms underlying autophagy-dependent cell death (ADCD) and autophagy-mediated cell death (AMCD)? In this article, we highlight the different roles of autophagy in cell death and discuss six of the main autophagy-related cell death modalities, with a focus on the metabolic changes caused by excessive endoplasmic reticulum-phagy (ER-phagy)-induced cell death and the role of mitophagy in autophagy-mediated ferroptosis. Finally, we discuss autophagy enhancement in the treatment of diseases and offer a new perspective based on the use of autophagy for different functional conversions (including the conversion of autophagy and that of different autophagy-mediated cell death modalities) for the clinical treatment of tumors.
    DOI:  https://doi.org/10.1038/s41419-023-06154-8
  3. Food Chem Toxicol. 2023 Sep 28. pii: S0278-6915(23)00456-8. [Epub ahead of print] 114054
    Iron blocks autophagic flux and induces autophagosomes accumulation in microglia.
    Deqiang Fu, Xingyue Liang, Yuxuan Jiang, Jieping Liu, Xiaosi Lin, Quan Yang, Xue Chen, Ping Huang, Wei Wang, Wenlin Wu.
      Iron is an essential dietary micronutrient for maintaining physiological homeostasis. However, disruption of cerebral iron regulation with the accumulation of iron in different brain structures appears to have a role in the pathogenesis of various neurodegenerative disorders. Studies have reported that autophagy induction could potentially mitigate progression in neurodegenerative diseases with iron deposition, but the relationship between autophagy and iron remains poorly understood. Meanwhile, abnormal autophagy in microglia is closely related to the occurrence of neurodegenerative diseases. Therefore, the effect of iron on microglia autophagy needs to be elaborated. In the present study, we found that iron induces autophagosome accumulation but inhibits its initiation in an Akt-mTOR pathway independent manner. Meanwhile, it caused autophagy flux defects and dysfunction of lysosomes. We also found that iron overload reduced the expression of Rab7, which is an essential protein for the fusion of autophagosomes and lysosomes. These results suggest that iron induces the accumulation of autophagosome in microglia and disrupts the autophagic flux in late stage of autophagy. Therefore, our work provides new insights into the molecular mechanisms of iron neurotoxicity.
    Keywords:  Autophagic flux; Autophagy; Iron; Microglia
    DOI:  https://doi.org/10.1016/j.fct.2023.114054
  4. J Cell Biol. 2023 Dec 04. pii: e202303078. [Epub ahead of print]222(12):
    Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy.
    Jake Cross, Joanne Durgan, David G McEwan, Matthew Tayler, Kevin M Ryan, Oliver Florey.
      Cells harness multiple pathways to maintain lysosome integrity, a central homeostatic process. Damaged lysosomes can be repaired or targeted for degradation by lysophagy, a selective autophagy process involving ATG8/LC3. Here, we describe a parallel ATG8/LC3 response to lysosome damage, mechanistically distinct from lysophagy. Using a comprehensive series of biochemical, pharmacological, and genetic approaches, we show that lysosome damage induces non-canonical autophagy and Conjugation of ATG8s to Single Membranes (CASM). Following damage, ATG8s are rapidly and directly conjugated onto lysosome membranes, independently of ATG13/WIPI2, lipidating to PS (and PE), a molecular hallmark of CASM. Lysosome damage drives V-ATPase V0-V1 association, direct recruitment of ATG16L1 via its WD40-domain/K490A, and is sensitive to Salmonella SopF. Lysosome damage-induced CASM is associated with formation of dynamic, LC3A-positive tubules, and promotes robust LC3A engagement with ATG2, a lipid transfer protein central to lysosome repair. Together, our data identify direct ATG8 conjugation as a rapid response to lysosome damage, with important links to lipid transfer and dynamics.
    DOI:  https://doi.org/10.1083/jcb.202303078
  5. Front Immunol. 2023 ;14 1243104
    Metabolic control from the endolysosome: lysosome-resident amino acid transporters open novel therapeutic possibilities.
    Toshihiko Kobayashi, Noriko Toyama-Sorimachi.
      Amino acid transporters are generally recognized as machinery that transport amino acids from the extracellular environment into the cytoplasm. Although their primary function is the uptake of amino acids to supply the cell with nutrients and energy, endolysosome-resident amino acid (EL-aa) transporters possess several unique functions in accordance with their localization in intracellular vesicular membranes. They play pivotal roles in the maintenance of metabolic homeostasis via direct involvement in the amino acid sensing pathway, which regulates the activity of mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of cellular metabolism. Additionally, some EL-aa transporters contribute to the maintenance of dynamic homeostasis of endolysosomes, including the regulation of endolysosomal acidity, by carrying amino acids out of endolysosomes. In addition, EL-aa transporters act as a scaffold to gather signaling molecules and multiple enzymes to control cellular metabolism on the endolysosomal membrane. Among EL-aa transporters, solute carrier family 15 member 4 (SLC15A4) is preferentially expressed in immune cells, including macrophages, dendritic cells, and B cells, and plays a key role in the integration of metabolic and inflammatory signals. In this review, we summarize our recent findings on EL-aa transporter contributions to inflammatory and metabolic signaling in the endolysosomes of immune cells by focusing on the SLC15 family, including SLC15A4 and SLC15A3, and discuss their uniqueness and universality. We also discuss the potential of targeting these EL-aa transporters in immune cells for the development of novel therapeutic strategies for inflammatory diseases. Because these transporters are highly expressed in immune cells and significantly alter the functions of immune cells, targeting them would provide a great advantage in ensuring a wide safety margin.
    Keywords:  amino acid transporter; endolysosome; immune cells; inflammation; mTORC1; metabolism; solute carrier family 15; therapeutic target
    DOI:  https://doi.org/10.3389/fimmu.2023.1243104
  6. Basic Res Cardiol. 2023 Oct 05. 118(1): 42
    Mitophagy for cardioprotection.
    Allen Sam Titus, Eun-Ah Sung, Daniela Zablocki, Junichi Sadoshima.
      Mitochondrial function is maintained by several strictly coordinated mechanisms, collectively termed mitochondrial quality control mechanisms, including fusion and fission, degradation, and biogenesis. As the primary source of energy in cardiomyocytes, mitochondria are the central organelle for maintaining cardiac function. Since adult cardiomyocytes in humans rarely divide, the number of dysfunctional mitochondria cannot easily be diluted through cell division. Thus, efficient degradation of dysfunctional mitochondria is crucial to maintaining cellular function. Mitophagy, a mitochondria specific form of autophagy, is a major mechanism by which damaged or unnecessary mitochondria are targeted and eliminated. Mitophagy is active in cardiomyocytes at baseline and in response to stress, and plays an essential role in maintaining the quality of mitochondria in cardiomyocytes. Mitophagy is mediated through multiple mechanisms in the heart, and each of these mechanisms can partially compensate for the loss of another mechanism. However, insufficient levels of mitophagy eventually lead to mitochondrial dysfunction and the development of heart failure. In this review, we discuss the molecular mechanisms of mitophagy in the heart and the role of mitophagy in cardiac pathophysiology, with the focus on recent findings in the field.
    Keywords:  Alternative mitophagy; Drp1; Mitochondrial quality control; Mitophagy
    DOI:  https://doi.org/10.1007/s00395-023-01009-x
  7. Free Radic Biol Med. 2023 Sep 28. pii: S0891-5849(23)00648-2. [Epub ahead of print]
    USP13 deubiquitinates p62/SQSTM1 to induce autophagy and Nrf2 release for activating antioxidant response genes.
    Bin Lee, Young Hun Kim, Woori Lee, Hee Youn Choi, Jisun Lee, Jiwon Kim, Dương Ngọc Mai, Su Ful Jung, Man Sup Kwak, Jeon-Soo Shin.
      SQSTM1/p62 (sequestosome 1) is a multifunctional protein that serves as a receptor for selective autophagy and scaffold. In selective autophagy, p62 functions as a bridge between polyubiquitinated proteins and autophagosomes. Further, p62 acts as a signaling hub for many cellular pathways including mTORC1, NF-κB, and Keap1-Nrf2. Post-translational modifications of p62, such as ubiquitination and phosphorylation, are known to determine its binding partners and regulate their intracellular functions. However, the mechanism of p62 deubiquitination remains unclear. In this study, we found that ubiquitin-specific protease 13 (USP13), a member of the USP family, directly binds p62 and removes ubiquitin at Lys7 (K7) of the PB1 domain. USP13-mediated p62 deubiquitination enhances p62 protein stability and facilitates p62 oligomerization, resulting in increased autophagy and degradation of Keap1, which is a negative regulator of the antioxidant response that promotes Nrf2 activation. Thus, USP13 can be considered a therapeutic target as a deubiquitination enzyme of p62 in autophagy-related diseases.
    Keywords:  Antioxidant response; Autophagic flux; Deubiquitinase; Keap1-Nrf2 pathway; Ubiquitin-specific protease (USP) 13; p62/sequestosome 1
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.09.024
  8. Mol Cell. 2023 Oct 05. pii: S1097-2765(23)00699-8. [Epub ahead of print]83(19): 3485-3501.e11
    S-acylation of p62 promotes p62 droplet recruitment into autophagosomes in mammalian autophagy.
    Xue Huang, Jia Yao, Lu Liu, Jing Chen, Ligang Mei, Jingjing Huangfu, Dong Luo, Xinyi Wang, Changhai Lin, Xiaorong Chen, Yi Yang, Sheng Ouyang, Fujing Wei, Zhuolin Wang, Shaolin Zhang, Tingxiu Xiang, Dante Neculai, Qiming Sun, Eryan Kong, Edward W Tate, Aimin Yang.
      p62 is a well-characterized autophagy receptor that recognizes and sequesters specific cargoes into autophagosomes for degradation. p62 promotes the assembly and removal of ubiquitinated proteins by forming p62-liquid droplets. However, it remains unclear how autophagosomes efficiently sequester p62 droplets. Herein, we report that p62 undergoes reversible S-acylation in multiple human-, rat-, and mouse-derived cell lines, catalyzed by zinc-finger Asp-His-His-Cys S-acyltransferase 19 (ZDHHC19) and deacylated by acyl protein thioesterase 1 (APT1). S-acylation of p62 enhances the affinity of p62 for microtubule-associated protein 1 light chain 3 (LC3)-positive membranes and promotes autophagic membrane localization of p62 droplets, thereby leading to the production of small LC3-positive p62 droplets and efficient autophagic degradation of p62-cargo complexes. Specifically, increasing p62 acylation by upregulating ZDHHC19 or by genetic knockout of APT1 accelerates p62 degradation and p62-mediated autophagic clearance of ubiquitinated proteins. Thus, the protein S-acylation-deacylation cycle regulates p62 droplet recruitment to the autophagic membrane and selective autophagic flux, thereby contributing to the control of selective autophagic clearance of ubiquitinated proteins.
    Keywords:  APT1; S-acylation; ZDHHC19; autophagy; autophagy receptor; liquid-liquid phase separation; p62 droplet; p62 protein; protein posttranslational modification; selective autophagy
    DOI:  https://doi.org/10.1016/j.molcel.2023.09.004
  9. FEBS Lett. 2023 Sep 30.
    A historical perspective of macroautophagy regulation by biochemical and biomechanical stimuli.
    Nicolas Dupont, Aurore Claude-Taupin, Patrice Codogno.
      Macroautophagy is a lysosomal degradative pathway for intracellular macromolecules, protein aggregates and organelles. The formation of the autophagosome, a double membrane-bound structure that sequesters cargoes before their delivery to the lysosome, is regulated by several stimuli in multicellular organisms. Pioneering studies in rat liver showed the importance of amino acids, insulin and glucagon in controlling macroautophagy. Thereafter, many studies have deciphered the signaling pathways downstream of these biochemical stimuli to control autophagosome formation. Two signaling hubs have emerged: the kinase mTOR, in a complex at the surface of lysosomes which is sensitive to nutrients and hormones; and AMPK, which is sensitive to the cellular energetic status. Besides nutritional, hormonal and energetic fluctuations, many organs have to respond to mechanical forces (compression, stretching and shear stress). Recent studies have shown the importance of mechanotransduction in controlling macroautophagy. This regulation engages cell surface sensors, such as the primary cilium, in order to translate mechanical stimuli into biological responses.
    Keywords:  autophagy; growth factors; mechanotransduction; nutrients; primary cilium; shear stress; signaling pathway
    DOI:  https://doi.org/10.1002/1873-3468.14744
  10. Toxicol Res. 2023 Oct;39(4): 637-647
    Atg5 knockout induces alternative autophagy via the downregulation of Akt expression.
    Hye-Gyo Kim, Myeong-Han Ro, Michael Lee.
      Autophagy play contradictory roles in cellular transformation. We previously found that the knockout (KO) of autophagy-related 5 (Atg5), which is essential for autophagy, leads to the malignant transformation of NIH 3T3 cells. In this study, we explored the mechanism by which autophagy contributes to this malignant transformation using two transformed cell lines, Atg5 KO and Ras-NIH 3T3. Monomeric red fluorescent protein-green fluorescent protein-light chain 3 reporter and Cyto-ID staining revealed that Ras-NIH 3T3 cells exhibited higher basal autophagy activity than NIH 3T3 cells. Additionally, transformed cells, regardless of their Atg5 KO status, were more sensitive to autophagy inhibitors (SBI-0206965, chloroquine, and obatoclax) than the untransformed NIH 3T3 cells, suggesting that the transformed cells are more autophagy-dependent than the normal cells. Loss of Atg5 improved the cell viability and mobility, especially in Ras-NIH 3T3 cells. Furthermore, we discovered that autophagy was alternatively induced in a Rab9-dependent manner in Ras-NIH 3T3 and NIH 3T3/Atg5 KO cells. In particular, Atg5 KO cells showed reduced mTOR-mediated phosphorylation of Akt (pAkt S473), indicating the mTOR-independent occurrence of alternative autophagy in Atg5 KO cells. Therefore, our study provides evidence that alternative autophagy may contribute to tumorigenesis in cells with an impaired Atg5-dependent autophagy pathway.
    Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00191-3.
    Keywords:  Atg5; Autophagy; Cell transformation; Knockout; Tumorigenesis
    DOI:  https://doi.org/10.1007/s43188-023-00191-3
  11. Cell Rep. 2023 Oct 03. pii: S2211-1247(23)01217-2. [Epub ahead of print]42(10): 113205
    Metabolism of glucose activates TORC1 through multiple mechanisms in Saccharomyces cerevisiae.
    Mohammad Alfatah, Liang Cui, Corinna Jie Hui Goh, Trishia Yi Ning Cheng, Yizhong Zhang, Arshia Naaz, Jin Huei Wong, Jacqueline Lewis, Wei Jie Poh, Prakash Arumugam.
      Target of Rapamycin Complex 1 (TORC1) is a conserved eukaryotic protein complex that links the presence of nutrients with cell growth. In Saccharomyces cerevisiae, TORC1 activity is positively regulated by the presence of amino acids and glucose in the medium. However, the mechanisms underlying nutrient-induced TORC1 activation remain poorly understood. By utilizing an in vivo TORC1 activation assay, we demonstrate that differential metabolism of glucose activates TORC1 through three distinct pathways in yeast. The first "canonical Rag guanosine triphosphatase (GTPase)-dependent pathway" requires conversion of glucose to fructose 1,6-bisphosphate, which activates TORC1 via the Rag GTPase heterodimer Gtr1GTP-Gtr2GDP. The second "non-canonical Rag GTPase-dependent pathway" requires conversion of glucose to glucose 6-phosphate, which activates TORC1 via a process that involves Gtr1GTP-Gtr2GTP and mitochondrial function. The third "Rag GTPase-independent pathway" requires complete glycolysis and vacuolar ATPase reassembly for TORC1 activation. We have established a roadmap to deconstruct the link between glucose metabolism and TORC1 activation.
    Keywords:  CP: Cell biology; CP: Metabolism; glucose signaling; glycolysis; mitochondrial function; target of rapamycin complex 1; vacuolar ATPase reassembly
    DOI:  https://doi.org/10.1016/j.celrep.2023.113205
  12. PeerJ. 2023 ;11 e16130
    Microorganism-regulated autophagy in gastrointestinal cancer.
    Jun-Yu Xu, Jiao-Xiu Fan, Min Hu, Jun Zeng.
      Gastrointestinal cancer has always been one of the most urgent problems to be solved, and it has become a major global health issue. Microorganisms in the gastrointestinal tract regulate normal physiological and pathological processes. Accumulating evidence reveals the role of the imbalance in the microbial community during tumorigenesis. Autophagy is an important intracellular homeostatic process, where defective proteins and organelles are degraded and recycled under stress. Autophagy plays a dual role in tumors as both tumor suppressor and tumor promoter. Many studies have shown that autophagy plays an important role in response to microbial infection. Here, we provide an overview on the regulation of the autophagy signaling pathway by microorganisms in gastrointestinal cancer.
    Keywords:  Autophagy; Gastrointestinal cancer; Microorganisms
    DOI:  https://doi.org/10.7717/peerj.16130
  13. PLoS One. 2023 ;18(10): e0292086
    Non-canonical functions of a mutant TSC2 protein in mitotic division.
    Mary-Bronwen L Chalkley, Rachel B Mersfelder, Maria Sundberg, Laura C Armstrong, Mustafa Sahin, Rebecca A Ihrie, Kevin C Ess.
      Tuberous Sclerosis Complex (TSC) is a debilitating developmental disorder characterized by a variety of clinical manifestations. TSC is caused by mutations in the TSC1 or TSC2 genes, which encode the hamartin/tuberin proteins respectively. These proteins function as a heterodimer that negatively regulates the mechanistic Target of Rapamycin Complex 1 (mTORC1). TSC research has focused on the effects of mTORC1, a critical signaling hub, on regulation of diverse cell processes including metabolism, cell growth, translation, and neurogenesis. However, non-canonical functions of TSC2 are not well studied, and the potential disease-relevant biological mechanisms of mutations affecting these functions are not well understood. We observed aberrant multipolar mitotic division, a novel phenotype, in TSC2 mutant iPSCs. The multipolar phenotype is not meaningfully affected by treatment with the inhibitor rapamycin. We further observed dominant negative activity of the mutant form of TSC2 in producing the multipolar division phenotype. These data expand the knowledge of TSC2 function and pathophysiology which will be highly relevant to future treatments for patients with TSC.
    DOI:  https://doi.org/10.1371/journal.pone.0292086
  14. Cell Rep. 2023 Sep 29. pii: S2211-1247(23)01195-6. [Epub ahead of print]42(10): 113183
    ApoE4-dependent lysosomal cholesterol accumulation impairs mitochondrial homeostasis and oxidative phosphorylation in human astrocytes.
    Hyein Lee, Sukhee Cho, Mi-Jin Kim, Yeo Jin Park, Eunji Cho, Yeon Suk Jo, Yong-Seok Kim, Jung Yi Lee, Themis Thoudam, Seung-Hwa Woo, Se-In Lee, Juyeong Jeon, Young-Sam Lee, Byung-Chang Suh, Jong Hyuk Yoon, Younghoon Go, In-Kyu Lee, Jinsoo Seo.
      Recent developments in genome sequencing have expanded the knowledge of genetic factors associated with late-onset Alzheimer's disease (AD). Among them, genetic variant ε4 of the APOE gene (APOE4) confers the greatest disease risk. Dysregulated glucose metabolism is an early pathological feature of AD. Using isogenic ApoE3 and ApoE4 astrocytes derived from human induced pluripotent stem cells, we find that ApoE4 increases glycolytic activity but impairs mitochondrial respiration in astrocytes. Ultrastructural and autophagy flux analyses show that ApoE4-induced cholesterol accumulation impairs lysosome-dependent removal of damaged mitochondria. Acute treatment with cholesterol-depleting agents restores autophagic activity, mitochondrial dynamics, and associated proteomes, and extended treatment rescues mitochondrial respiration in ApoE4 astrocytes. Taken together, our study provides a direct link between ApoE4-induced lysosomal cholesterol accumulation and abnormal oxidative phosphorylation.
    Keywords:  ApoE4; CP: Cell biology; CP: Metabolism; glucose metabolism; human astrocytes; lysosomal cholesterol accumulation; mitophagy
    DOI:  https://doi.org/10.1016/j.celrep.2023.113183
  15. Geroscience. 2023 Oct 06.
    Blazing a trail for the clinical use of rapamycin as a geroprotecTOR.
    RAP PAC Investigators
      Treatment with rapamycin, an inhibitor of the mechanistic Target Of Rapamycin Complex One (mTORC1) protein kinase, has been repeatedly demonstrated to extend lifespan and prevent or delay age-related diseases in diverse model systems. Concerns over the risk of potentially serious side effects in humans, including immunosuppression and metabolic disruptions, have cautiously limited the translation of rapamycin and its analogs as a treatment for aging associated conditions. During the last decade, we and others have developed a working model that suggests that while inhibition of mTORC1 promotes healthy aging, many of the negative side effects of rapamycin are associated with "off-target" inhibition of a second mTOR complex, mTORC2. Differences in the kinetics and molecular mechanisms by which rapamycin inhibits mTORC1 and mTORC2 suggest that a therapeutic window for rapamycin could be exploited using intermittent dosing schedules or alternative rapalogs that may enable more selective inhibition of mTORC1. However, the optimal dosing schedules and the long-term efficacy of such interventions in humans are unknown. Here, we highlight ongoing or upcoming clinical trials that will address outstanding questions regarding the safety, pharmacokinetics, pharmacodynamics, and efficacy of rapamycin and rapalogs on several clinically oriented outcomes. Results from these early phase studies will help guide the design of phase 3 clinical trials to determine whether rapamycin can be used safely to inhibit mTORC1 for the treatment and prevention of age-related diseases in humans.
    Keywords:  Aging; Everolimus; Metabolism; Muscle; Sirolimus; mTOR
    DOI:  https://doi.org/10.1007/s11357-023-00935-x
  16. J Cell Biol. 2023 Dec 04. pii: e202204048. [Epub ahead of print]222(12):
    The TMEM192-mKeima probe specifically assays lysophagy and reveals its initial steps.
    Takayuki Shima, Monami Ogura, Ruriko Matsuda, Shuhei Nakamura, Natsuko Jin, Tamotsu Yoshimori, Akiko Kuma.
      Membrane rupture of lysosomes results in leakage of their contents, which is harmful to cells. Recent studies have reported that several systems contribute to the repair or elimination of damaged lysosomes. Lysophagy is a type of selective autophagy that plays a crucial role in the lysosomal damage response. Because multiple pathways are involved in this response, an assay that specifically evaluates lysophagy is needed. Here, we developed the TMEM192-mKeima probe to evaluate lysophagy. By comparing the use of this probe with the conventional galectin-3 assay, we showed that this probe is more specific to lysophagy. Using TMEM192-mKeima, we showed that TFEB and p62 are important for the lysosomal damage response but not for lysophagy, although they have previously been considered to be involved in lysophagy. We further investigated the initial steps in lysophagy and identified UBE2L3, UBE2N, TRIM10, 16, and 27 as factors involved in it. Our results demonstrate that the TMEM192-mKeima probe is a useful tool for investigating lysophagy.
    DOI:  https://doi.org/10.1083/jcb.202204048
  17. eNeuro. 2023 Sep 28. pii: ENEURO.0159-23.2023. [Epub ahead of print]
    Spatiotemporal regulation of de novo and salvage purine synthesis during brain development.
    Tomoya Mizukoshi, Seiya Yamada, Shin-Ichi Sakakibara.
      The levels of purines, essential molecules to sustain eukaryotic cell homeostasis, are regulated by the coordination of the de novo and salvage synthesis pathways. In the embryonic central nervous system (CNS), the de novo pathway is considered crucial to meet the requirements for the active proliferation of neural stem/progenitor cells (NSPCs). However, how these two pathways are balanced or separately utilized during CNS development remains poorly understood. In this study, we showed a dynamic shift in pathway utilization, with greater reliance on the de novo pathway during embryonic stages and on the salvage pathway in postnatal-adult mouse brain. The pharmacological effects of various purine synthesis inhibitors in vitro and the expression profile of purine synthesis enzymes indicated that NSPCs in the embryonic cerebrum mainly utilize the de novo pathway. Simultaneously, NSPCs in the cerebellum require both the de novo and the salvage pathways. In vivo administration of de novo inhibitors resulted in severe hypoplasia of the forebrain cortical region, indicating a gradient of purine demand along the anteroposterior axis of the embryonic brain, with cortical areas of the dorsal forebrain having higher purine requirements than ventral or posterior areas such as the striatum and thalamus. This histological defect of the neocortex was accompanied by strong downregulation of the mechanistic target of rapamycin complex 1 (mTORC1)/ribosomal protein S6 kinase (S6K)/S6 signaling cascade, a crucial pathway for cell metabolism, growth, and survival. These findings indicate the importance of the spatiotemporal regulation of both purine pathways for mTORC1 signaling and proper brain development.Significance StatementBrain development requires a balance of de novo and salvage purine synthetic pathways. However, the utilization of these pathways during brain development remains poorly understood. This study provides evidence that the spatiotemporal regulation of these two purine synthesis pathways is essential for normal brain development. We revealed that inhibition of de novo purine synthesis results in the downregulation of mammalian/mechanistic target of rapamycin (mTOR) signaling, leading to malformations in specific embryonic brain regions such as the cerebral neocortex. These results suggest a temporal and spatial gradient of purine demand during embryonic brain development. These findings could improve our understanding of neurological diseases caused by defects in purine metabolism.
    Keywords:  NSPCs; cortical development; de novo pathway; mTOR; purine metabolism; salvage pathway
    DOI:  https://doi.org/10.1523/ENEURO.0159-23.2023
  18. Autophagy. 2023 Oct 05.
    Chaperone-mediated autophagy protects against hyperglycemic stress.
    Emilio J Vélez, Simon Schnebert, Maxime Goguet, Sara Balbuena-Pecino, Karine Dias, Linda Beauclair, Stéphanie Fontagné-Dicharry, Vincent Véron, Alexandra Depincé, Florian Beaumatin, Amaury Herpin, Iban Seiliez.
      Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis critical for cellular homeostasis and metabolism, and whose defects have been associated with several human pathologies. While CMA has been well described in mammals, functional evidence has only recently been documented in fish, opening up new perspectives to tackle this function under a novel angle. Now we propose to explore CMA functions in the rainbow trout (RT, Oncorhynchus mykiss), a fish species recognized as a model organism of glucose intolerance and characterized by the presence of two paralogs of the CMA-limiting factor Lamp2A (lysosomal associated membrane protein 2A). To this end, we validated a fluorescent reporter (KFERQ-PA-mCherry1) previously used to track functional CMA in mammalian cells, in an RT hepatoma-derived cell line (RTH-149). We found that incubation of cells with high-glucose levels (HG, 25 mM) induced translocation of the CMA reporter to lysosomes and/or late endosomes in a KFERQ- and Lamp2A-dependent manner, as well as reduced its half-life compared to the control (5 mM), thus demonstrating increased CMA flux. Furthermore, we observed that activation of CMA upon HG exposure was mediated by generation of mitochondrial reactive oxygen species, and involving the antioxidant transcription factor Nfe2l2/Nrf2 (nfe2 like bZIP transcription factor 2). Finally, we demonstrated that CMA plays an important protective role against HG-induced stress, primarily mediated by one of the two RT Lamp2As. Together, our results provide unequivocal evidence for CMA activity existence in RT and highlight both the role and regulation of CMA during glucose-related metabolic disorders.
    Keywords:  CMA; Lamp2A; NFE2L2/NRF2; ROS; RTH-149; fish; glucose; glucose intolerance; metabolism; trout
    DOI:  https://doi.org/10.1080/15548627.2023.2267415
  19. Biochim Biophys Acta Mol Cell Res. 2023 Sep 29. pii: S0167-4889(23)00175-1. [Epub ahead of print] 119602
    L-Dopa decarboxylase modulates autophagy in hepatocytes and is implicated in dengue virus-caused inhibition of autophagy completion.
    Vassilina Tsopela, Evangelos Korakidis, Despoina Lagou, Katerina I Kalliampakou, Raphaela S Milona, Eirini Kyriakopoulou, George Mpekoulis, Ioanna Gemenetzi, Elli-Anna Stylianaki, Constantinos D Sideris, Aggelina Sioli, Dionysis Kefallinos, Diamantis C Sideris, Vassilis Aidinis, Aristides G Eliopoulos, Konstantinos Kambas, Dido Vassilacopoulou, Niki Vassilaki.
      The enzyme L-Dopa Decarboxylase (DDC) synthesizes the catecholamine dopamine and the indolamine serotonin. Apart from its role in the brain as a neurotransmitter biosynthetic enzyme, DDC has been detected also in the liver and other peripheral organs, where it is implicated in cell proliferation, apoptosis, and host-virus interactions. Dengue virus (DENV) suppresses DDC expression at the later stages of infection, during which DENV also inhibits autophagosome-lysosome fusion. As dopamine affects autophagy in neuronal cells, we investigated the possible association of DDC with autophagy in human hepatocytes and examined whether DDC mediates the relationship between DENV infection and autophagy. We performed DDC silencing/overexpression and evaluated autophagic markers upon induction of autophagy, or suppression of autophagosome-lysosome fusion. Our results showed that DDC expression favored the autophagic process, at least in part, through its biosynthetic function, while knockdown of DDC or inhibition of its enzymatic activity prevented autophagy completion. In turn, autophagy induction upregulated DDC, while autophagy reduction by chemical or genetic (ATG14L KO) ways caused the opposite effect. This study also implicated DDC with the cellular energetic status, as its silencing reduced the oxidative phosphorylation activity of the cell. We also report that upon DDC silencing, the repressive effect of DENV on the completion of autophagy was enhanced, and the inhibition of autolysosome formation did not exert an additive effect on viral proliferation. These data unravel a novel role of DDC in the autophagic process and suggest that DENV downregulates DDC expression to inhibit the completion of autophagy, reinforcing the importance of this protein in viral infections.
    Keywords:  Autolysosomes; Autophagic flux; Autophagy; Catecholamines; DENV; Dengue virus; L-Dopa decarboxylase (DDC); Serotonin; Viral proliferation
    DOI:  https://doi.org/10.1016/j.bbamcr.2023.119602
  20. J Vis Exp. 2023 09 15.
    Complementary Approaches to Interrogate Mitophagy Flux in Pancreatic β-Cells.
    Elena Levi-D'Ancona, Vaibhav Sidarala, Scott A Soleimanpour.
      Mitophagy is a quality control mechanism necessary to maintain optimal mitochondrial function. Dysfunctional β-cell mitophagy results in insufficient insulin release. Advanced quantitative assessments of mitophagy often require the use of genetic reporters. The mt-Keima mouse model, which expresses a mitochondria-targeted pH-sensitive dual-excitation ratiometric probe for quantifying mitophagy via flow cytometry, has been optimized in β-cells. The ratio of acidic-to-neutral mt-Keima wavelength emissions can be used to robustly quantify mitophagy. However, using genetic mitophagy reporters can be challenging when working with complex genetic mouse models or difficult-to-transfect cells, such as primary human islets. This protocol describes a novel complementary dye-based method to quantify β-cell mitophagy in primary islets using MtPhagy. MtPhagy is a pH-sensitive, cell-permeable dye that accumulates in the mitochondria and increases its fluorescence intensity when mitochondria are in low pH environments, such as lysosomes during mitophagy. By combining the MtPhagy dye with Fluozin-3-AM, a Zn2+ indicator that selects for β-cells, and Tetramethylrhodamine, ethyl ester (TMRE) to assess mitochondrial membrane potential, mitophagy flux can be quantified specifically in β-cells via flow cytometry. These two approaches are highly complementary, allowing for flexibility and precision in assessing mitochondrial quality control in numerous β-cell models.
    DOI:  https://doi.org/10.3791/65789
  21. J Environ Sci (China). 2024 Jan;pii: S1001-0742(22)00644-1. [Epub ahead of print]135 97-107
    Perturbation of autophagy pathways in murine alveolar macrophage by 2D TMDCs is chalcogen-dependent.
    Xiaofei Zhou, Weitao Jin, Rui Zhang, Xuan Mao, Jianbo Jia, Hongyu Zhou.
      Increasing risks of incidental and occupational exposures to two-dimensional transition metal dichalcogenides (2D TMDCs) due to their broad application in various areas raised their public health concerns. While the composition-dependent cytotoxicity of 2D TMDCs has been well-recognized, how the outer chalcogenide atoms and inner transition metal atoms differentially contribute to their perturbation on cell homeostasis at non-lethal doses remains to be identified. In the present work, we compared the autophagy induction and related mechanisms in response to WS2, NbS2, WSe2 and NbSe2 nanosheets exposures in MH-S murine alveolar macrophages. All these 2D TMDCs had comparable physicochemical properties, overall cytotoxicity and capability in triggering autophagy in MH-S cells, but showed outer chalcogen-dependent subcellular localization and activation of autophagy pathways. Specifically, WS2 and NbS2 nanosheets adhered on the cell surface and internalized in the lysosomes, and triggered mTOR-dependent activation of autophagy. Meanwhile, WSe2 and NbSe2 nanosheets had extensive distribution in cytoplasm of MH-S cells and induced autophagy in an mTOR-independent manner. Furthermore, the 2D TMDCs-induced perturbation on autophagy aggravated the cytotoxicity of respirable benzo[a]pyrene. These findings provide a deeper insight into the potential health risk of environmental 2D TMDCs from the perspective of homeostasis perturbation.
    Keywords:  Autophagy pathway; Cell function; Chalcogen group; Endosomal escape; Inhalation toxicity
    DOI:  https://doi.org/10.1016/j.jes.2022.12.029
  22. PeerJ. 2023 ;11 e15976
    TLR7 promotes skin inflammation via activating NFκB-mTORC1 axis in rosacea.
    Yaqun Huang, Da Liu, Mengting Chen, San Xu, Qinqin Peng, Yan Zhu, Juan Long, Tangxiele Liu, Zhili Deng, Hongfu Xie, Ji Li, Fangfen Liu, Wenqin Xiao.
      Rosacea is a chronic inflammatory skin disease originated from damaged skin barrier and innate/adaptive immune dysregulation. Toll-like receptors (TLRs) sense injured skin and initiate downstream inflammatory and immune responses, whose role in rosacea is not fully understood. Here, via RNA-sequencing analysis, we found that the TLR signaling pathway is the top-ranked signaling pathway enriched in rosacea skin lesions, in which TLR7 is highlighted and positively correlated with the inflammation severity of disease. In LL37-induced rosacea-like mouse models, silencing TLR7 prevented the development of rosacea-like skin inflammation. Specifically, we demonstrated that overexpressing TLR7 in keratinocytes stimulates rapamycin-sensitive mTOR complex 1 (mTORC1) pathway via NFκB signaling. Ultimately, TLR7/NFκ B/mTORC1 axis promotes the production of cytokines and chemokines, leading to the migration of CD4+T cells, which are infiltrated in the lesional skin of rosacea. Our report reveals the crucial role of TLR7 in rosacea pathogenesis and indicatesa promising candidate for rosacea treatments.
    Keywords:  MTOR; NFκB; Rosacea; Skin immunology; Skin inflammation; TLR7
    DOI:  https://doi.org/10.7717/peerj.15976
  23. Ibrain. 2021 ;7(4): 318-324
    VPS35, the core component of the retromer complex, and Parkinson's disease.
    Ai-Di Luo, Zu-Cai Xu, Shu-Sheng Liao.
      Parkinson's disease (PD) is a neurodegenerative disease that is common in middle-aged and elderly people, and its onset is related to multiple factors, such as heredity, environment, and age. The vesicle protein sorting 35 (VPS35) gene was found to be a late-onset autosomal dominant familial PD (PARK17) causative gene. The protein encoded by this gene is located in the endosome and aggregates with other membrane proteins to form a retromer complex, which participates in the membrane protein cycle between the endosome and the Golgi network. Increasing evidence shows that VPS35 may participate in the pathogenesis of PD by affecting autophagy, mitochondria, neurosynaptic transmission, dopamine signaling pathways, and so forth, and it can interact with other disease-causing genes of familial PD. This article aimed to review the functions of VPS35 and the mechanism of its mutations in PD that have been discovered in recent years.
    Keywords:  Parkinson's disease (PD); mechanism; retromer complex; vesicle protein sorting 35 (VPS35)
    DOI:  https://doi.org/10.1002/ibra.12004
  24. Cell Death Dis. 2023 Oct 04. 14(10): 649
    PACS-2 deficiency aggravates tubular injury in diabetic kidney disease by inhibiting ER-phagy.
    Jinfei Yang, Li Li, Chenrui Li, Wei Chen, Yan Liu, Shilu Luo, Chanyue Zhao, Yachun Han, Ming Yang, Hao Zhao, Na Jiang, Yiyun Xi, Chengyuan Tang, Juan Cai, Li Xiao, Huafeng Liu, Lin Sun.
      Autophagy of endoplasmic reticulum (ER-phagy) selectively removes damaged ER through autophagy-lysosome pathway, acting as an adaptive mechanism to alleviate ER stress and restore ER homeostasis. However, the role and precise mechanism of ER-phagy in tubular injury of diabetic kidney disease (DKD) remain obscure. In the present study, we demonstrated that ER-phagy of renal tubular cells was severely impaired in streptozocin (STZ)-induced diabetic mice, with a decreased expression of phosphofurin acidic cluster sorting protein 2 (PACS-2), a membrane trafficking protein which was involved in autophagy, and a reduction of family with sequence similarity 134 member B (FAM134B), one ER-phagy receptor. These changes were further aggravated in mice with proximal tubule specific knockout of Pacs-2 gene. In vitro, transfection of HK-2 cells with PACS-2 overexpression plasmid partially improved the impairment of ER-phagy and the reduction of FAM134B, both of which were induced in high glucose ambience; while the effect was blocked by FAM134B siRNA. Mechanistically, PACS-2 interacted with and promoted the nuclear translocation of transcription factor EB (TFEB), which was reported to activate the expression of FAM134B. Collectively, these data unveiled that PACS-2 deficiency aggravates renal tubular injury in DKD via inhibiting ER-phagy through TFEB/FAM134B pathway.
    DOI:  https://doi.org/10.1038/s41419-023-06175-3
  25. Biochem Pharmacol. 2023 Oct 04. pii: S0006-2952(23)00433-1. [Epub ahead of print] 115842
    Unraveling the interplay between RAS/RAF/MEK/ERK signaling pathway and autophagy in cancer: From molecular mechanisms to targeted therapy.
    Yunli Huang, Yongqi Zhen, Yanmei Chen, Shaoguang Sui, Lan Zhang.
      RAS/RAF/MEK/ERK signaling pathway is one of the most important pathways of Mitogen-activated protein kinases (MAPK), which widely participate in regulating cell proliferation, differentiation, apoptosis and signaling transduction. Autophagy is an essential mechanism that maintains cellular homeostasis by degrading aged and damaged organelles. Recently, some studies revealed RAS/RAF/MEK/ERK signaling pathway is closely related to autophagy regulation and has a dual effect in tumor cells. However, the specific mechanism by which RAS/RAF/MEK/ERK signaling pathway participates in autophagy regulation is not fully understood. This article provides a comprehensive review of the research progress with regard to the RAS/RAF/MEK/ERK signaling pathway and autophagy, as well as their interplay in cancer therapy. The impact of small molecule inhibitors that target the RAS/RAF/MEK/ERK signaling pathway on autophagy is discussed in this study. The advantages and limitations of the clinical combination of these small molecule inhibitors with autophagy inhibitors are also explored. The findings from this study may provide additional perspectives for future cancer treatment strategies.
    Keywords:  Autophagy; Cancer; Interplay; RAS/RAF/MEK/ERK signaling pathway; Targeted therapy
    DOI:  https://doi.org/10.1016/j.bcp.2023.115842
  26. JACC Basic Transl Sci. 2023 Sep;8(9): 1043-1056
    Assessment of Autophagy Markers Suggests Increased Activity Following LVAD Therapy.
    Thomas G Martin, Miranda A Juarros, Joseph C Cleveland, Michael R Bristow, Amrut V Ambardekar, Peter M Buttrick, Leslie A Leinwand.
      Left ventricular reverse remodeling in heart failure is associated with improved clinical outcomes. However, the molecular features that drive this process are poorly defined. Left ventricular assist devices (LVADs) are the therapy associated with the greatest reverse remodeling and lead to partial myocardial recovery in most patients. In this study, we examined whether autophagy may be implicated in post-LVAD reverse remodeling. We found expression of key autophagy factors increased post-LVAD, while autophagic substrates decreased. Autolysosome numbers increased post-LVAD, further indicating increased autophagy. These findings support the conclusion that mechanical unloading activates autophagy, which may underly the reverse remodeling observed.
    Keywords:  LVAD; autolysosome; autophagy; reverse remodeling
    DOI:  https://doi.org/10.1016/j.jacbts.2023.05.015
  27. FEBS Open Bio. 2023 Oct 06.
    CoCl2 -triggered pseudohypoxic stress induces proteasomal degradation of SIRT4 via polyubiquitination of lysines K78 and K299.
    Nils Hampel, Jacqueline Georgy, Mehrnaz Mehrabipour, Alexander Lang, Isabell Lehmkuhl, Jürgen Scheller, Mohammad R Ahmadian, Doreen M Floss, Roland P Piekorz.
      SIRT4, together with SIRT3 and SIRT5, comprises the mitochondrially-localized subgroup of sirtuins. SIRT4 regulates mitochondrial bioenergetics, dynamics (mitochondrial fusion), and quality control (mitophagy) via its NAD+ -dependent enzymatic activities. Here, we address the regulation of SIRT4 itself by characterizing its protein stability and degradation upon CoCl2 -induced pseudohypoxic stress that typically triggers mitophagy. Interestingly, we observed that of the mitochondrial sirtuins, only the protein levels of SIRT4 or ectopically expressed SIRT4-eGFP decrease upon CoCl2 treatment of HEK293 cells. Co-treatment with BafA1, an inhibitor of autophagosome-lysosome fusion required for autophagy/mitophagy, or the use of the proteasome inhibitor MG132, prevented CoCl2 -induced SIRT4 downregulation. Consistent with the proteasomal degradation of SIRT4, the lysine mutants SIRT4(K78R) and SIRT4(K299R) showed significantly reduced polyubiquitination upon CoCl2 treatment and were more resistant to pseudohypoxia-induced degradation as compared to SIRT4. Moreover, SIRT4(K78R) and SIRT4(K299R) displayed increased basal protein stability as compared to wild-type SIRT4 when subjected to MG132 treatment or cycloheximide (CHX) chase assays. Thus, our data indicate that stress-induced protein degradation of SIRT4 occurs through two mechanisms: (i) via mitochondrial autophagy/mitophagy, and (ii) as a separate process via proteasomal degradation within the cytoplasm.
    Keywords:  SIRT4; Sirtuin; autophagy; proteasome; pseudohypoxia; ubiquitination
    DOI:  https://doi.org/10.1002/2211-5463.13715
  28. Front Neurosci. 2023 ;17 1250532
    Liquid-liquid phase separation regulates alpha-synuclein aggregate and mitophagy in Parkinson's disease.
    Kaiying Hou, Tingting Liu, Jingwen Li, Meiyan Xian, Lin Sun, Jianshe Wei.
      Parkinson's disease (PD) is the second most common neurodegenerative disease in the world, and alpha-synuclein (α-syn) abnormal aggregate and mitochondrial dysfunction play a crucial role in its pathological development. Recent studies have revealed that proteins can form condensates through liquid-liquid phase separation (LLPS), and LLPS has been found to be widely present in α-syn aberrant aggregate and mitophagy-related protein physiological processes. This review summarizes the occurrence of α-syn LLPS and its influencing factors, introduces the production and transformation of the related protein LLPS during PINK1-Parkin-mediated mitophagy, hoping to provide new ideas and methods for the study of PD pathology.
    Keywords:  PINK1-Parkin; Parkinson’s disease; alpha-synuclein; liquid-liquid phase separation; mitophagy
    DOI:  https://doi.org/10.3389/fnins.2023.1250532
  29. Nat Commun. 2023 Oct 06. 14(1): 6230
    Rab41-mediated ESCRT machinery repairs membrane rupture by a bacterial toxin in xenophagy.
    Takashi Nozawa, Hirotaka Toh, Junpei Iibushi, Kohei Kogai, Atsuko Minowa-Nozawa, Junko Satoh, Shinji Ito, Kazunori Murase, Ichiro Nakagawa.
      Xenophagy, a type of selective autophagy, is a bactericidal membrane trafficking that targets cytosolic bacterial pathogens, but the membrane homeostatic system to cope with bacterial infection in xenophagy is not known. Here, we show that the endosomal sorting complexes required for transport (ESCRT) machinery is needed to maintain homeostasis of xenophagolysosomes damaged by a bacterial toxin, which is regulated through the TOM1L2-Rab41 pathway that recruits AAA-ATPase VPS4. We screened Rab GTPases and identified Rab41 as critical for maintaining the acidification of xenophagolysosomes. Confocal microscopy revealed that ESCRT components were recruited to the entire xenophagolysosome, and this recruitment was inhibited by intrabody expression against bacterial cytolysin, indicating that ESCRT targets xenophagolysosomes in response to a bacterial toxin. Rab41 translocates to damaged autophagic membranes via adaptor protein TOM1L2 and recruits VPS4 to complete ESCRT-mediated membrane repair in a unique GTPase-independent manner. Finally, we demonstrate that the TOM1L2-Rab41 pathway-mediated ESCRT is critical for the efficient clearance of bacteria through xenophagy.
    DOI:  https://doi.org/10.1038/s41467-023-42039-2
  30. Life Sci Alliance. 2023 Dec;pii: e202302116. [Epub ahead of print]6(12):
    Improving lysosomal ferroptosis with NMN administration protects against heart failure.
    Mikako Yagi, Yura Do, Haruka Hirai, Kenji Miki, Takahiro Toshima, Yukina Fukahori, Daiki Setoyama, Chiaki Abe, Yo-Ichi Nabeshima, Dongchon Kang, Takeshi Uchiumi.
      Myocardial mitochondria are primary sites of myocardial energy metabolism. Mitochondrial disorders are associated with various cardiac diseases. We previously showed that mice with cardiomyocyte-specific knockout of the mitochondrial translation factor p32 developed heart failure from dilated cardiomyopathy. Mitochondrial translation defects cause not only mitochondrial dysfunction but also decreased nicotinamide adenine dinucleotide (NAD+) levels, leading to impaired lysosomal acidification and autophagy. In this study, we investigated whether nicotinamide mononucleotide (NMN) administration, which compensates for decreased NAD+ levels, improves heart failure because of mitochondrial dysfunction. NMN administration reduced damaged lysosomes and improved autophagy, thereby reducing heart failure and extending the lifespan in p32cKO mice. We found that lysosomal damage due to mitochondrial dysfunction induced ferroptosis, involving the accumulation of iron in lysosomes and lipid peroxide. The ameliorative effects of NMN supplementation were found to strongly affect lysosomal function rather than mitochondrial function, particularly lysosome-mediated ferroptosis. NMN supplementation can improve lysosomal, rather than mitochondrial, function and prevent chronic heart failure.
    DOI:  https://doi.org/10.26508/lsa.202302116
  31. Mult Scler Relat Disord. 2023 Sep 27. pii: S2211-0348(23)00530-8. [Epub ahead of print]79 105029
    The role of autophagy protein Atg5 in multiple sclerosis.
    Maria-Elizabeth Baeva, Carlos Camara-Lemarroy.
      Multiple sclerosis (MS) is a neurological disease which has a strong autoimmune component to its pathology. Although there are currently many approved immunomodulatory treatments that reduce the rate of relapse and slow down the progression of the disease, the cure is still elusive. This may be due to the underlying etiology still being unknown. Autophagy is the potential link between neurodegeneration and autoimmunity. Specifically, this review will focus on the autophagy protein Atg5 and examine the in vitro cell culture, animal and human studies that have examined its expression and effects in the context of MS. The findings of these investigations are summarized, and a model is proposed in which elevated Atg5 levels leads to dysfunctional autophagy, neurodegeneration, inflammation, and eventually clinical disability. While there are currently no drugs that specifically target Atg5, our review recommends that further investigations into the role that Atg5 plays in MS pathophysiology may eventually lead to the development of autophagy-specific treatments of MS.
    Keywords:  Atg5; Autophagy; Experimental autoimmune encephalomyelitis; Multiple sclerosis; Neurodegeneration; Relapsing-remitting multiple sclerosis
    DOI:  https://doi.org/10.1016/j.msard.2023.105029
  32. Adv Biol (Weinh). 2023 Oct 02. e2300292
    HIF-1α Contributes to Hypoxia-induced VSMC Proliferation and Migration by Regulating Autophagy in Type A Aortic Dissection.
    Ben Huang, Nan Chen, Zhenhang Chen, Jinqiang Shen, Hao Zhang, Chunsheng Wang, Yongxin Sun.
      Type A aortic dissection (AD) is a catastrophic cardiovascular disease. Hypoxia-inducible factor-1α (HIF-1α) and autophagy are reported to be upregulated in the AD specimens. However, the interaction between HIF-1α and autophagy in the pathogenesis of AD remains to be explored. HIF-1α and LC3 levels are evaluated in 10 AD and 10 normal aortic specimens. MDC staining, autophagic vacuoles, and autophagic flux are detected in human aortic smooth muscle cells (HASMCs) under hypoxia treatment. CCK-8, transwell, and wound healing assay are used to identify proliferation and migration under hypoxia treatment. Furthermore, 3-MA is used to inhibit autophagy in hypoxia-treated HASMCs. This study reveals that AD tissues highly express HIF-1α and the LC3. Autophagy is induced under hypoxia in a time-dependent manner, and autophagy is positively related to HIF-1α in HASMCs. Moreover, the proliferation and migration of HASMCs are enhanced by hypoxia, whereas the knockdown of HIF-1α attenuates this effect. Additionally, inhibiting autophagy with 3-MA ameliorates hypoxia-induced proliferation and migration of HASMCs. In summary, the above results indicate that HIF-1α facilitates HASMC proliferation and migration by upregulating autophagy in a hypoxic microenvironment. Thus, inhibition of autophagy may be a novel therapeutic target for the prevention and treatment of AD.
    Keywords:  HASM; HIF-1α; aortic dissection; autophagy; hypoxia
    DOI:  https://doi.org/10.1002/adbi.202300292
  33. Biochemistry. 2023 Sep 30.
    Epstein-Barr Virus Encoded BCL2, BHRF1, Downregulates Autophagy by Noncanonical Binding of BECN1.
    Samuel Wyatt, Karen Glover, Srinivasulu Dasanna, Monica Lewison, Maribel González-García, Christopher L Colbert, Sangita C Sinha.
      γ-herpesviruses (γHVs) encode BCL2 homologues (vBCL2) that bind the Bcl-2 homology 3 domains (BH3Ds) of diverse proteins, inhibiting apoptosis and promoting host cell and virus survival. vBCLs encoded by Kaposi sarcoma-associated HV (KSHV) and γHV68 downregulate autophagy, a degradative cellular process crucial for homeostasis and innate immune responses to pathogens, by binding to a BH3D in BECN1, a key autophagy protein. Epstein-Barr virus (EBV) encodes a vBCL2 called BHRF1. Here we show that unlike the KSHV and γHV68 vBCL2s, BHRF1 does not bind the isolated BECN1 BH3D. We use yeast two-hybrid assays to identify the minimal region of BECN1 required and sufficient for binding BHRF1. We confirm that this is a direct, albeit weak, interaction via affinity pull-down assays and isothermal titration calorimetry. To understand the structural bases of BHRF1 specificity, we determined the 2.6 Å crystal structure of BHRF1 bound to the BID BH3D, which binds ∼400-times tighter to BHRF1 than does BECN1, and performed a detailed structural comparison with complexes of diverse BH3Ds bound to BHRF1 and to other antiapoptotic BCL2s. Lastly, we used mammalian cell autophagy assays to demonstrate that BHRF1 downregulates autophagy and that a cell-permeable peptide derived from the BID BH3D inhibits BHRF1-mediated downregulation of autophagy. In summary, our results suggest that BHRF1 downregulates autophagy by noncanonical binding of a flexible region of BECN1 that includes but is not limited to the BH3D and that BH3D-derived peptides that bind better to BHRF1 can block downregulation of autophagy by BHRF1.
    DOI:  https://doi.org/10.1021/acs.biochem.3c00225
  34. Res Sq. 2023 Sep 14. pii: rs.3.rs-3266941. [Epub ahead of print]
    Phosphoribosyl modification of poly-ubiquitin chains at the Legionella-containing vacuole prohibiting autophagy adaptor recognition.
    Yuxin Mao, Min Wan, Marena Minelli, Qiuye Zhao, Shannon Marshall, Haiyuan Yu, Marcus Smolka.
      Ubiquitination is a crucial posttranslational modification in eukaryotes that plays a significant role in the infection of intracellular microbial pathogens, such as Legionella pneumophila , the bacterium responsible for Legionnaires' disease. While the Legionella -containing vacuole (LCV) is coated with ubiquitin (Ub), it avoids recognition by autophagy adaptors. In this study, we report that the Sdc and Sde families of effectors work together to build ubiquitinated species around the LCV. The Sdc effectors catalyze canonical polyubiquitination directly on host targets or on the phosphoribosyl-Ub (PR-Ub) conjugated to host targets by Sde. Remarkably, the Ub moieties within the poly-Ub chains are either modified with a phosphoribosyl group by Sde and other PDE domain-containing effectors or covalently attached to other host substrates via Sde-mediated PR-ubiquitination. Furthermore, these modifications prevent the recognition by Ub adaptors, such as p62, and therefore exclude host autophagy adaptors from the LCV. Our findings shed light on the nature of the poly-ubiquitinated species present at the surface of the LCV and provide a molecular mechanism for the avoidance of autophagy adaptors by the Ub-decorated LCV.
    DOI:  https://doi.org/10.21203/rs.3.rs-3266941/v1
  35. J Reprod Dev. 2023 Oct 02.
    FABP4 mediates endoplasmic reticulum stress and autophagy to regulate endometrial epithelial cell function during early sheep gestation.
    Meijun Song, Kexing Hao, Fenghua Qi, Wenjuan Zhao, Zhengrong Wang, Jing Wang, Guangdong Hu.
      Dynamic changes in the endometrium are crucial for establishing early pregnancy in ruminants. Blastocyst elongation and implantation require hormones and nutrients to be secreted from the maternal endometrium. The fatty acid-binding protein FABP4 is a widely expressed fatty acid transport protein that promotes cell proliferation, migration, and invasion and is involved in conceptus implantation. However, the mechanism underlying the functional regulation of endometrial epithelial cells (EECs) by FABP4 during ovine peri-implantation remains unclear. We simulated hormonal changes in vitro in sheep EECs (SEECs) during the peri-implantation period and found that it elevated FABP4 expression. FABP4 inhibition significantly reduced cell migration, endoplasmic reticulum stress, and autophagy, suggesting that FABP4 regulates endometrial function in sheep. Moreover, the FABP4 inhibitor BMS309403 counteracted hormone-mediated functional changes in SEECs, and an endoplasmic reticulum stress activator and autophagy inhibitor reversed the abnormal secretion of prostaglandins induced by FABP4 inhibition. These results suggest that FABP4 affects ovine endometrial function during early gestation by regulating endoplasmic reticulum stress and autophagy in SEECs.
    Keywords:  Autophagy; Endometrial epithelial cells; Endoplasmic reticulum stress; Fatty acid-binding protein; Implantation
    DOI:  https://doi.org/10.1262/jrd.2023-015
  36. Eur J Med Chem. 2023 Sep 27. pii: S0223-5234(23)00806-1. [Epub ahead of print]261 115839
    Targeted protein degradation in drug development: Recent advances and future challenges.
    Jian Song, Mingzheng Hu, Jun Zhou, Songbo Xie, Tianliang Li, Yan Li.
      Targeted protein degradation (TPD) has emerged as a promising therapeutic approach with potential advantages over traditional occupancy-based inhibitors in terms of dosing, side effects and targeting "undruggable" proteins. Targeted degraders can theoretically bind any nook or cranny of targeted proteins to drive degradation. This offers convenience versus the small-molecule inhibitors that must function in a well-defined pocket. The degradation process depends mainly on two cell self-destruction mechanisms, namely the ubiquitin-proteasome system and the lysosomal degradation pathway. Various TPD strategies (e.g., proteolytic-targeting chimeras, molecular glues, lysosome-targeting chimeras, and autophagy-targeting chimeras) have been developed. These approaches hold great potential for targeting dysregulated proteins, potentially offering therapeutic benefits. In this article, we systematically review the mechanisms of various TPD strategies, potential applications to drug discovery, and recent advances. We also discuss the benefits and challenges associated with these TPD strategies, aiming to provide insight into the targeting of dysregulated proteins and facilitate their clinical applications.
    Keywords:  Drug discovery; Dysregulated protein; Lysosome-targeting chimera; Proteolytic-targeting chimera; Targeted protein degradation
    DOI:  https://doi.org/10.1016/j.ejmech.2023.115839
  37. JACC Basic Transl Sci. 2023 Sep;8(9): 1057-1059
    Autophagy in the LVAD-Supported Heart: A Sign of Hope or a Marker of Unloading.
    David R Rawnsley, Abhinav Diwan.
      
    Keywords:  acyl-CoA binding protein; autophagy; diazepam binding inhibitor; heart failure; left ventricular assist device; lysosome; sodium-glucose cotransporter-2 inhibitors
    DOI:  https://doi.org/10.1016/j.jacbts.2023.07.015
  38. Cell Biochem Biophys. 2023 Oct 03.
    The Prognosis of Cancer Depends on the Interplay of Autophagy, Apoptosis, and Anoikis within the Tumor Microenvironment.
    Shweta Gulia, Prakash Chandra, Asmita Das.
      Within the tumor microenvironment, the fight between the immune system and cancer influences tumor transformation. Metastasis formation is an important stage in the progression of cancer. This process is aided by cellular detachment and resistance to anoikis, which are achieved by altering intercellular signaling. Autophagy, specifically pro-survival autophagy, aids cancer cells in developing treatment resistance. Numerous studies have shown that autophagy promotes tumor growth and resistance to anoikis. To regulate protective autophagy, cancer-related genes phosphorylate both pro- and anti-apoptotic proteins. Apoptosis, a type of controlled cell death, eliminates damaged or unwanted cells. Anoikis is a type of programmed cell death in which cells lose contact with the extracellular matrix. The dysregulation of these cellular pathways promotes tumor growth and spread. Apoptosis, anoikis, and autophagy interact meticulously and differently depending on the cellular circumstances. For instance, autophagy can protect cancer cells from apoptosis by removing cellular components that are damaged and might otherwise trigger apoptotic pathways. Similarly, anoikis dysregulation can trigger autophagy by causing cellular harm and metabolic stress. In order to prevent or treat metastatic disease, specifically, targeting these cellular mechanisms may present a promising prospect for cancer therapy. This review discourses the state of our understanding of the molecular and cellular mechanisms underlying tumor transformation and the establishment of metastatic tumors. To enhance the prognosis for cancer, we highlight and discuss potential therapeutic approaches that target these processes and genes involved in them.
    Keywords:  Anoikis; Apoptosis; Autophagy; Metastasis; Tumor progression
    DOI:  https://doi.org/10.1007/s12013-023-01179-4
  39. Arch Biochem Biophys. 2023 Sep 28. pii: S0003-9861(23)00270-9. [Epub ahead of print]747 109771
    Molecular mechanism by which RRM2-inhibitor (cholagogue osalmid) plus bafilomycin A1 cause autophagic cell death in multiple myeloma.
    Shushan Guo, Zhijian Xu, Qilin Feng, Hui Zhang, Dandan Yu, Bo Li, Ke Hu, Xuejie Gao, Qikai Zhang, Hongfei Yi, Xiaosong Wu, Dongliang Song, Huabin Zhu, Haiyan Cai, Yu Peng, Weiliang Zhu, Jumei Shi.
      Despite significant improvement in the prognosis of multiple myeloma (MM), the disease remains incurable; thus, more effective therapies are required. Ribonucleoside-diphosphate reductase subunit M2 (RRM2) is significantly associated with drug resistance, rapid relapse, and poor prognosis. Previously, we found that 4-hydroxysalicylanilide (osalmid), a specific inhibitor of RRM2, exhibits anti-MM activity in vitro, in vivo, and in human patients; however, the mechanism remains unclear. Osalmid inhibits the translocation of RRM2 to the nucleus and stimulates autophagosome synthesis but inhibits subsequent autophagosome-lysosome fusion. We confirm that RRM2 binds to receptor-interacting protein kinase 3 (RIPK3) and reduces RIPK3, inhibiting autophagosome-lysosome fusion. Interestingly, the combination of osalmid and bafilomycin A1 (an autophagy inhibitor) depletes RIPK3 and aggravates p62 and autophagosome accumulation, leading to autophagic cell death. Combination therapy demonstrates synergistic cytotoxicity both in vitro and in vivo. Therefore, we propose that combining osalmid and bafilomycin A1(BafA1) may have clinical benefits against MM.
    Keywords:  Autophagy; Bafilomycin A1; Cholagogue osalmid; RIPK3; RRM2; p62
    DOI:  https://doi.org/10.1016/j.abb.2023.109771
  40. Sci Adv. 2023 Oct 06. 9(40): eadi8343
    Deficient chaperone-mediated autophagy facilitates LPS-induced microglial activation via regulation of the p300/NF-κB/NLRP3 pathway.
    Jin Wu, Yingying Han, Hao Xu, Hongyang Sun, Rui Wang, Haigang Ren, Guanghui Wang.
      Neuroinflammation is a pathological change that is involved in the progression of Parkinson's disease. Dysfunction of chaperone-mediated autophagy (CMA) has proinflammatory effects. However, the mechanism by which CMA mediates inflammation and whether CMA affects microglia and microglia-mediated neuronal damage remain to be elucidated. In the present study, we found that LAMP2A, a limiting protein for CMA, was decreased in lipopolysaccharide (LPS)-treated primary microglia. Activation of CMA by the activator CA significantly repressed LPS-induced microglial activation, whereas CMA dysfunction exacerbated microglial activation. We further identified that the protein p300 was a substrate of CMA. Degradation of p300 by CMA reduced p65 acetylation, thereby inhibiting the transcription of proinflammatory factors and the activation of the NLRP3 inflammasome. Furthermore, CA pretreatment inhibited microglia-mediated inflammation and, in turn, attenuated neuronal death in vitro and in vivo. Our findings suggest repressive effects of CMA on microglial activation through the p300-associated NF-κB signaling pathway, thus uncovering a mechanistic link between CMA and neuroinflammation.
    DOI:  https://doi.org/10.1126/sciadv.adi8343
  41. bioRxiv. 2023 Sep 22. pii: 2023.09.19.558537. [Epub ahead of print]
    NSD1 supports cell growth and regulates autophagy in HPV-negative head and neck squamous cell carcinoma.
    Iuliia Topchu, Igor Bychkov, Demirkan Gursel, Petr Makhov, Yanis Boumber.
      Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Despite advances in therapeutic management and immunotherapy, the five-year survival rate for head and neck cancer remains at ∼66% of all diagnosed cases. A better definition of drivers of HPV-negative HNSCC that are targetable points of tumor vulnerability could lead to significant clinical advances. NSD1 is a histone methyltransferase which catalyzes histone H3 lysine 36 di-methylation (H3K36me 2 ); mutations inactivating NSD1 have been linked to improved outcomes in HNSCC. In this study, we show that NSD1 induces H3K36me 2 levels in HNSCC, and that the depletion of NSD1 reduces HNSCC of cell growth in vitro and in vivo . We also find that NSD1 strongly promotes activation of the Akt/mTORC1 signaling pathway. NSD1 depletion in HNSCC induces an autophagic gene program activation, causes accumulation of the p62 and LC3B-II proteins, and decreases the autophagic signaling protein ULK1 at both protein and mRNA levels. Reflecting these signaling defects, knockdown of NSD1 disrupts autophagic flux in HNSCC cells. Taken together, these data identify positive regulation of Akt/mTORC1 signaling and autophagy as novel NSD1 functions in HNSCC, suggesting that NSD1 may be of value as a therapeutic target in this cancer.
    DOI:  https://doi.org/10.1101/2023.09.19.558537
  42. Ecotoxicol Environ Saf. 2023 Sep 28. pii: S0147-6513(23)01038-2. [Epub ahead of print]265 115534
    Activation of the p62-Keap1-Nrf2 pathway protects against oxidative stress and excessive autophagy in ovarian granulosa cells to attenuate DEHP-induced ovarian impairment in mice.
    Bo Xu, Tiantian He, Hong Yang, Wenjie Dai, Ling Liu, Xiaoqian Ma, Jiaxue Ma, Guoqin Yang, Rui Si, Xing Du, Xufeng Fu, Xiuying Pei.
      Di-(2-ethylhexyl) phthalate (DEHP) is widely used in various plastics but has been demonstrated to cause female reproductive toxicity. However, the exact mechanism underlying the ovarian damage induced by DEHP remains unclear. In this study, DEHP was administered orally to 5-week-old female mice for 30 days at doses of 0, 250, 500, and 1000 mg/kg/day. The findings demonstrated that DEHP exposure disrupted ovarian function and follicular development as well as induced oxidative stress and autophagy in ovarian granulosa cells (GCs). Further, 200 µM mono-(2-ethylhexyl) phthalate (MEHP), the primary metabolite of DEHP in vivo, induced autophagy in both human ovarian granulosa cells line (KGN) and mouse primary GCs within 24 h in vitro. However, it did not affect the p62-dependent autophagy flux. Furthermore, MEHP-induced autophagy was inhibited by the autophagy inhibitor 3-MA and exacerbated by the autophagy activator rapamycin, indicating that MEHP induces excessive autophagy in GCs. Subsequently, we found that MEHP-induced autophagic cell death was primarily attributed to oxidative damage from elevated intracellular ROS levels. Meanwhile, MEHP exposure induced nuclear translocation of erythroid-derived factor 2-related factor (Nrf2), a key regulator of antioxidant activity resulting in activating antioxidant effects. Interestingly, we also found that MEHP-induced increase in p62 competitively binds Keap1, thereby facilitating nuclear translocation of Nrf2 and establishing a positive feedback loop in antioxidant regulation. Therefore, this study demonstrated that inhibition of Nrf2 could aggravate oxidative damage and enhance excessive autophagy caused by MEHP, while activation of Nrf2 could reverse the trend. These findings have also been reinforced in studies of cultured ovaries in vitro. Our study suggests that the p62-Keap1-Nrf2 pathway may serve as a potential protective mechanism against DEHP-induced oxidative stress and excessive autophagy in mouse GCs.
    Keywords:  Di-(2-ethylhexyl) phthalate; Excessive autophagy; Female reproductive toxicity; Ovarian granulosa cells; Oxidative stress; P62-Keap1-Nrf2 pathway
    DOI:  https://doi.org/10.1016/j.ecoenv.2023.115534
  43. Phys Biol. 2023 Sep 28.
    Calcium storage in multivesicular endo-lysosome.
    Cameron C Scott, Vaibhav Wasnik, Paula Nunes-Hasler, Nicolas Demaurex, Karsten Kruse, Jean Gruenberg.
      It is now established that endo-lysosomes, also referred to as late endosomes, serve as intracellular calcium store, in addition to the endoplasmic reticulum. While abundant calcium-binding proteins provide the latter compartment with its calcium storage capacity, essentially nothing is known about the mechanism responsible for calcium storage in endo-lysosomes. In this paper, we propose that the structural organization of endo-lysosomal membranes drives the calcium storage capacity of the compartment. Indeed, endo-lysosomes exhibit a characteristic multivesicular ultrastructure, with intralumenal membranes providing a large amount of additional bilayer surface. We used a theoretical approach to investigate the calcium storage capacity of endosomes, using known calcium binding affinities for bilayers and morphological data on endo-lysosome membrane organization. Finally, we tested our predictions experimentally after SNX3 depletion to decrease the intralumenal membrane content. We conclude that the major negatively-charge lipids and proteins of endo-lysosomes serve as calcium-binding molecules in the acidic calcium stores of mammalian cells, while the large surface area of intralumenal membranes provide the necessary storage capacity.
    Keywords:  Calcium; acidic calcium stores; endosomes; intralumenal; lysobisphosphatidic acid; lysosomes; multivesicular
    DOI:  https://doi.org/10.1088/1478-3975/acfe6a
  44. bioRxiv. 2023 Sep 19. pii: 2023.06.20.545716. [Epub ahead of print]
    Divergence of TORC1-mediated Stress Response Leads to Novel Acquired Stress Resistance in a Pathogenic Yeast.
    Jinye Liang, Hanxi Tang, Lindsey F Snyder, Christopher E Youngstrom, Bin Z He.
      Acquired stress resistance (ASR) enables organisms to prepare for environmental changes that occur after an initial stressor. However, the genetic basis for ASR and how the underlying network evolved remain poorly understood. In this study, we discovered that a short phosphate starvation induces oxidative stress response (OSR) genes in the pathogenic yeast C. glabrata and protects it against a severe H 2 O 2 stress; the same treatment, however, provides little benefit in the low pathogenic-potential relative, S. cerevisiae . This ASR involves the same transcription factors (TFs) as the OSR, but with different combinatorial logics. We show that Target-of- Rapamycin Complex 1 (TORC1) is differentially inhibited by phosphate starvation in the two species and contributes to the ASR via its proximal effector, Sch9. Therefore, evolution of the phosphate starvation-induced ASR involves the rewiring of TORC1's response to phosphate limitation and the repurposing of TF-target gene networks for the OSR using new regulatory logics.
    DOI:  https://doi.org/10.1101/2023.06.20.545716
  45. Fish Shellfish Immunol. 2023 Sep 28. pii: S1050-4648(23)00610-1. [Epub ahead of print] 109124
    miR-22a targets p62/SQSTM1 to negatively affect autophagy and disease resistance of grass carp (Ctenopharyngodon idella).
    Hongyan Yu, Lingli Xie, Zheyan Chen, Huiqin Niu, Xuewen Jia, Biao Du, Yubang Shen, Lang Gui, Xiaoyan Xu, Jiale Li.
      MicroRNAs (miRNAs) are integral to many biological functions, including autophagy, a process recently proven to be closely linked to innate immunity. In this study, we present findings on miR-22a, a teleost homolog of mammalian miR-22, illustrating its capacity to target the autophagy adaptor p62, subsequently inducing downregulation at both mRNA and protein levels. Utilizing Western blot analyses, we demonstrated that miR-22a inhibits the autophagy flux of CIK cells, correlated with an elevated presence of LC3 II. Additionally, the overexpression of miR-22a resulted in the suppression of NF-κB signaling, leading to reduced cellar antimicrobial abilities and increased apoptosis. These findings provide novel insights into the role of miR-22a as an autophagy-related miRNA and its immune mechanisms against pathogens via p62 in teleost, enriching our understanding of the interplay between autophagy and innate immunity.
    Keywords:  Autophagy; Grass carp; Immune; miR-22a
    DOI:  https://doi.org/10.1016/j.fsi.2023.109124
  46. bioRxiv. 2023 Sep 22. pii: 2023.09.22.559064. [Epub ahead of print]
    Sortilin inhibition treats multiple neurodegenerative lysosomal storage disorders.
    Hannah G Leppert, Joelle T Anderson, Kaylie J Timm, Cristina Davoli, Melissa A Pratt, Clarissa D Booth, Katherine A White, Mitchell J Rechtzigel, Brandon L Meyerink, Tyler B Johnson, Jon J Brudvig, Jill M Weimer.
      Lysosomal storage disorders (LSDs) are a genetically and clinically diverse group of diseases characterized by lysosomal dysfunction. Batten disease is a family of severe LSDs primarily impacting the central nervous system. Here we show that AF38469, a small molecule inhibitor of sortilin, improves lysosomal and glial pathology across multiple LSD models. Live-cell imaging and comparative transcriptomics demonstrates that the transcription factor EB (TFEB), an upstream regulator of lysosomal biogenesis, is activated upon treatment with AF38469. Utilizing CLN2 and CLN3 Batten disease mouse models, we performed a short-term efficacy study and show that treatment with AF38469 prevents the accumulation of lysosomal storage material and the development of neuroinflammation, key disease associated pathologies. Tremor phenotypes, an early behavioral phenotype in the CLN2 disease model, were also completely rescued. These findings reveal sortilin inhibition as a novel and highly efficacious therapeutic modality for the treatment of multiple forms of Batten disease.
    DOI:  https://doi.org/10.1101/2023.09.22.559064
  47. Redox Biol. 2023 Sep 21. pii: S2213-2317(23)00296-3. [Epub ahead of print]67 102895
    Increasing glutathione levels by a novel posttranslational mechanism inhibits neuronal hyperexcitability.
    Ashwini Sri Hari, Rajeswari Banerji, Li-Ping Liang, Ruth E Fulton, Christopher Quoc Huynh, Timothy Fabisiak, Pallavi Bhuyan McElroy, James R Roede, Manisha Patel.
      Glutathione (GSH) depletion, and impaired redox homeostasis have been observed in experimental animal models and patients with epilepsy. Pleiotropic strategies that elevate GSH levels via transcriptional regulation have been shown to significantly decrease oxidative stress and seizure frequency, increase seizure threshold, and rescue certain cognitive deficits. Whether elevation of GSH per se alters neuronal hyperexcitability remains unanswered. We previously showed that thiols such as dimercaprol (DMP) elevate GSH via post-translational activation of glutamate cysteine ligase (GCL), the rate limiting GSH biosynthetic enzyme. Here, we asked if elevation of cellular GSH by DMP altered neuronal hyperexcitability in-vitro and in-vivo. Treatment of primary neuronal-glial cerebrocortical cultures with DMP elevated GSH and inhibited a voltage-gated potassium channel blocker (4-aminopyridine, 4AP) induced neuronal hyperexcitability. DMP increased GSH in wildtype (WT) zebrafish larvae and significantly attenuated convulsant pentylenetetrazol (PTZ)-induced acute 'seizure-like' swim behavior. DMP treatment increased GSH and inhibited convulsive, spontaneous 'seizure-like' swim behavior in the Dravet Syndrome (DS) zebrafish larvae (scn1Lab). Furthermore, DMP treatment significantly decreased spontaneous electrographic seizures and associated seizure parameters in scn1Lab zebrafish larvae. We investigated the role of the redox-sensitive mammalian target of rapamycin (mTOR) pathway due to the presence of several cysteine-rich proteins and their involvement in regulating neuronal excitability. Treatment of primary neuronal-glial cerebrocortical cultures with 4AP or l-buthionine-(S,R)-sulfoximine (BSO), an irreversible inhibitor of GSH biosynthesis, significantly increased mTOR complex I (mTORC1) activity which was rescued by pre-treatment with DMP. Furthermore, BSO-mediated GSH depletion oxidatively modified the tuberous sclerosis protein complex (TSC) consisting of hamartin (TSC1), tuberin (TSC2), and TBC1 domain family member 7 (TBC1D7) which are critical negative regulators of mTORC1. In summary, our results suggest that DMP-mediated GSH elevation by a novel post-translational mechanism can inhibit neuronal hyperexcitability both in-vitro and in-vivo and a plausible link is the redox sensitive mTORC1 pathway.
    Keywords:  Glutamate cysteine ligase; Glutathione; Neuronal hyperexcitability; Oxidative stress; mTORC1
    DOI:  https://doi.org/10.1016/j.redox.2023.102895
  48. Mol Cell. 2023 Oct 05. pii: S1097-2765(23)00737-2. [Epub ahead of print]83(19): 3402-3403
    STING channels its proton power.
    Mingqi Dong, Katherine A Fitzgerald.
      Induction of type I interferon by the STING pathway is a cornerstone of innate immunity. STING also turns on non-canonical autophagy and inflammasome activation although the underlying mechanisms remain ill defined. Liu et al.1 discovered that STING forms a channel that directs proton efflux from the Golgi to drive these responses.
    DOI:  https://doi.org/10.1016/j.molcel.2023.09.014
  49. Front Biosci (Landmark Ed). 2023 Sep 22. 28(9): 211
    The Role of Prohibitin-2 in Diseases.
    Fang Liu, You Zhang, Zhiyong Guo, An-Jing Ren.
      Prohibitin-2 (PHB2) is a conserved protein in mitochondria that regulates various biological processes, including cell cycle, proliferation, apoptosis, transcription, signal transduction, and mitochondrial ridge morphogenesis. Recently, there has been growing interest in the biological function of PHB2. This article primarily discusses the recent advances in the role of PHB2 in diseases.
    Keywords:  autophagy; diseases; mitochondria; prohibitin-2
    DOI:  https://doi.org/10.31083/j.fbl2809211
  50. Cell Rep. 2023 Sep 30. pii: S2211-1247(23)01215-9. [Epub ahead of print]42(10): 113203
    ARL8B mediates lipid droplet contact and delivery to lysosomes for lipid remobilization.
    Dilip Menon, Apoorva Bhapkar, Bhoomika Manchandia, Gitanjali Charak, Surabhi Rathore, Rakesh Mohan Jha, Arpita Nahak, Moumita Mondal, Mohyeddine Omrane, Akash Kumar Bhaskar, Lipi Thukral, Abdou Rachid Thiam, Sheetal Gandotra.
      Lipid droplets (LDs) play a crucial role in maintaining cellular lipid balance by storing and delivering lipids as needed. However, the intricate lipolytic pathways involved in LD turnover remain poorly described, hindering our comprehension of lipid catabolism and related disorders. Here, we show a function of the small GTPase ARL8B in mediating LD turnover in lysosomes. ARL8B-GDP localizes to LDs, while ARL8-GTP predominantly favors lysosomes. GDP binding induces a conformation with an exposed N-terminal amphipathic helix, enabling ARL8B to bind to LDs. By associating with LDs and lysosomes, and with its property to form a heterotypic complex, ARL8B mediates LD-lysosome contacts and efficient lipid transfer between these organelles. In human macrophages, this ARL8B-dependent LD turnover mechanism appears as the major lipolytic pathway. Our finding opens exciting possibilities for understanding the molecular mechanisms underlying LD degradation and its potential implications for inflammatory disorders.
    Keywords:  ARL8B; CP: Cell biology; CP: Immunology; lipid droplet; lysosome; macrophage; microlipophagy; small GTPase
    DOI:  https://doi.org/10.1016/j.celrep.2023.113203
  51. Front Immunol. 2023 ;14 1203645
    Zika virus modulates mitochondrial dynamics, mitophagy, and mitochondria-derived vesicles to facilitate viral replication in trophoblast cells.
    Jae Kyung Lee, Ok Sarah Shin.
      Zika virus (ZIKV) remains a global public health threat with the potential risk of a future outbreak. Since viral infections are known to exploit mitochondria-mediated cellular processes, we investigated the effects of ZIKV infection in trophoblast cells in terms of the different mitochondrial quality control pathways that govern mitochondrial integrity and function. Here we demonstrate that ZIKV (PRVABC59) infection of JEG-3 trophoblast cells manipulates mitochondrial dynamics, mitophagy, and formation of mitochondria-derived vesicles (MDVs). Specifically, ZIKV nonstructural protein 4A (NS4A) translocates to the mitochondria, triggers mitochondrial fission and mitophagy, and suppresses mitochondrial associated antiviral protein (MAVS)-mediated type I interferon (IFN) response. Furthermore, proteomics profiling of small extracellular vesicles (sEVs) revealed an enrichment of mitochondrial proteins in sEVs secreted by ZIKV-infected JEG-3 cells, suggesting that MDV formation may also be another mitochondrial quality control mechanism manipulated during placental ZIKV infection. Altogether, our findings highlight the different mitochondrial quality control mechanisms manipulated by ZIKV during infection of placental cells as host immune evasion mechanisms utilized by ZIKV at the placenta to suppress the host antiviral response and facilitate viral infection.
    Keywords:  mitochondria-derived vesicles (MDVs); mitochondrial quality control; mitophagy; nonstructural protein 4A (NS4A); zika virus (ZIKV)
    DOI:  https://doi.org/10.3389/fimmu.2023.1203645
  52. FASEB J. 2023 Nov;37(11): e23227
    Leflunomide alleviates obesity via activation of the TAK1-AMPK pathway and induction of lipophagy.
    Xiaoyue Ji, Junhong Chen, Chaoying You, Jing Sun, Xiulong Xu.
      Lipophagy is a subset of selective autophagy that specifically degrades lipid droplets and plays an important role in obesity. Leflunomide treatment in rheumatoid arthritis (RA) patients has been associated with weight loss and decreased blood glucose levels, which cannot be attributed to its known side effects. Our prior studies showed that A77 1726, the active metabolite of leflunomide, acts as an inhibitor of S6K1 to sensitize the insulin receptor and control hyperglycemia. Whether the anti-obesity effect of leflunomide is mediated by targeting S6K1 and its underlying mechanisms remain unclear. Here, we report that A77 1726 induced LC3 lipidation and increased the formation of autophagosomes and lipoautolysosomes in 3T3-L1 adipocytes by activating TGF-β-activated kinase 1 (TAK1), AMP-activated kinase (AMPK), and Unc-51 like autophagy-activated kinase 1 (ULK1). A77 1726 reduced the content of lipid droplets in 3T3-L1 adipocytes, which was blocked by bafilomycin or by beclin-1 knockdown. Similar observations were made in murine adipocytes differentiated from S6K1-/- embryonic fibroblasts (MEFs). Leflunomide treatment restricted bodyweight gains in ob/ob mice and reduced the visceral fat deposit and the size of adipocytes. Leflunomide treatment induced autophagy in adipose and liver tissues and reduced hepatic lipid contents. Consistently, S6K1 knockout increased the levels of LC3 lipidation in the liver, muscle, and fat of S6K-/- mice. Leflunomide treatment and S6K1 deficiency both induced TAK1, AMPK, and ULK1 phosphorylation in these tissues. These observations collectively suggest that leflunomide controls obesity in part by activating AMPK and inducing lipophagy. Our study provides insights into the mechanisms of leflunomide-mediated anti-obesity activity.
    Keywords:  A77 1726; autophagy; leflunomide; lipophagy; obesity; p70 S6 kinase 1
    DOI:  https://doi.org/10.1096/fj.202301162R
  53. Sci Rep. 2023 Oct 06. 13(1): 16863
    Amino acid transporter SLC38A5 is a tumor promoter and a novel therapeutic target for pancreatic cancer.
    Tyler Sniegowski, Devaraja Rajasekaran, Souad R Sennoune, Sukumaran Sunitha, Fang Chen, Mohamed Fokar, Sudhir Kshirsagar, P Hemachandra Reddy, Ksenija Korac, Mosharaf Mahmud Syed, Tanima Sharker, Vadivel Ganapathy, Yangzom D Bhutia.
      Pancreatic ductal adenocarcinoma (PDAC) cells have a great demand for nutrients in the form of sugars, amino acids, and lipids. Particularly, amino acids are critical for cancer growth and, as intermediates, connect glucose, lipid and nucleotide metabolism. PDAC cells meet these requirements by upregulating selective amino acid transporters. Here we show that SLC38A5 (SN2/SNAT5), a neutral amino acid transporter is highly upregulated and functional in PDAC cells. Using CRISPR/Cas9-mediated knockout of SLC38A5, we show its tumor promoting role in an in vitro cell line model as well as in a subcutaneous xenograft mouse model. Using metabolomics and RNA sequencing, we show significant reduction in many amino acid substrates of SLC38A5 as well as OXPHOS inactivation in response to SLC38A5 deletion. Experimental validation demonstrates inhibition of mTORC1, glycolysis and mitochondrial respiration in KO cells, suggesting a serious metabolic crisis associated with SLC38A5 deletion. Since many SLC38A5 substrates are activators of mTORC1 as well as TCA cycle intermediates/precursors, we speculate amino acid insufficiency as a possible link between SLC38A5 deletion and inactivation of mTORC1, glycolysis and mitochondrial respiration, and the underlying mechanism for PDAC attenuation. Overall, we show that SLC38A5 promotes PDAC, thereby identifying a novel, hitherto unknown, therapeutic target for PDAC.
    DOI:  https://doi.org/10.1038/s41598-023-43983-1
  54. Cell Commun Signal. 2023 Oct 05. 21(1): 275
    The Golgi stacking protein GRASP55 is targeted by the natural compound prodigiosin.
    Lena Berning, Thomas Lenz, Ann Kathrin Bergmann, Gereon Poschmann, Hannah U C Brass, David Schlütermann, Annabelle Friedrich, María José Mendiburo, Céline David, Seda Akgün, Jörg Pietruszka, Kai Stühler, Björn Stork.
       BACKGROUND: The bacterial secondary metabolite prodigiosin has been shown to exert anticancer, antimalarial, antibacterial and immunomodulatory properties. With regard to cancer, it has been reported to affect cancer cells but not non-malignant cells, rendering prodigiosin a promising lead compound for anticancer drug discovery. However, a direct protein target has not yet been experimentally identified.
    METHODS: We used mass spectrometry-based thermal proteome profiling in order to identify target proteins of prodigiosin. For target validation, we employed a genetic knockout approach and electron microscopy.
    RESULTS: We identified the Golgi stacking protein GRASP55 as target protein of prodigiosin. We show that prodigiosin treatment severely affects Golgi morphology and functionality, and that prodigiosin-dependent cytotoxicity is partially reduced in GRASP55 knockout cells. We also found that prodigiosin treatment results in decreased cathepsin activity and overall blocks autophagic flux, whereas co-localization of the autophagosomal marker LC3 and the lysosomal marker LAMP1 is clearly promoted. Finally, we observed that autophagosomes accumulate at GRASP55-positive structures, pointing towards an involvement of an altered Golgi function in the autophagy-inhibitory effect of this natural compound.
    CONCLUSION: Taken together, we propose that prodigiosin affects autophagy and Golgi apparatus integrity in an interlinked mode of action involving the regulation of organelle alkalization and the Golgi stacking protein GRASP55. Video Abstract.
    Keywords:  Autophagy; Golgi apparatus; Natural compound; Prodigiosin; Target identification
    DOI:  https://doi.org/10.1186/s12964-023-01275-1
  55. Eur Rev Med Pharmacol Sci. 2023 Sep;pii: 33801. [Epub ahead of print]27(18): 8795-8811
    Metformin activates AMPK and mTOR to Inhibit RANKL-stimulated osteoclast formation.
    Y-S Kim, B-S Park, H-S Baek, H-M Kang, J-M Oh, I-R Kim.
       OBJECTIVE: Metformin is a medication used to treat type 2 diabetes by inhibiting hepatic glucose production through adenosine monophosphate-activated protein kinase (AMPK) activation. Autophagy is closely related to the homeostasis and stress mechanisms of the body. In recent years, much research has arisen on therapeutic methods utilizing autophagy mechanisms to treat diagnoses such as metabolic diseases, tumors, and Alzheimer's disease. This study thus aimed to investigate the effects of metformin treatment on the differentiation of osteoclasts and changes in AMPK mechanisms, which play an important role in regulating energy homeostasis, and mTOR, a highly conserved kinase that regulates autophagy.
    MATERIALS AND METHODS: Experimentation, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, tartrate-resistant acid phosphate (TRAP) staining, pit formation assay, immunofluorescence, western blotting, and real-time polymerase chain reaction (PCR) was performed to investigate the effects of metformin on osteoclast differentiation. Additionally, to investigate its association with AMPK and pathways, the AMPK inhibitor compound C and mammalian targets of rapamycin (mTOR) activator leucine were used to examine the expression of osteoclast- or autophagy-related proteins, genes, and TRAP-positive cells.
    RESULTS: Metformin showed no cytotoxic effects on mouse osteoblastic cell lines (MC3T3-E1) and murine macrophage cell lines (RAW264.7) but did inhibit osteoclast differentiation. Furthermore, metformin was found to inhibit osteoclast differentiation through AMPK activation and mTOR inhibition. In turn, AMPK inhibition using compound C promoted osteoclast differentiation, and mTOR activation using leucine inhibited autophagy and osteoclast differentiation.
    CONCLUSIONS: Metformin activates the AMPK pathway while functioning as an activator of mTOR, thereby leading to the inhibition of autophagy and osteoclast differentiation.
    DOI:  https://doi.org/10.26355/eurrev_202309_33801
  56. Arthritis Res Ther. 2023 Oct 02. 25(1): 187
    Mechanistic target of rapamycin (mTOR): a potential new therapeutic target for rheumatoid arthritis.
    Fen Zhang, Ting Cheng, Sheng-Xiao Zhang.
      Rheumatoid arthritis (RA) is an autoimmune disease characterized by systemic synovitis and bone destruction. Proinflammatory cytokines activate pathways of immune-mediated inflammation, which aggravates RA. The mechanistic target of rapamycin (mTOR) signaling pathway associated with RA connects immune and metabolic signals, which regulates immune cell proliferation and differentiation, macrophage polarization and migration, antigen presentation, and synovial cell activation. Therefore, therapy strategies targeting mTOR have become an important direction of current RA treatment research. In the current review, we summarize the biological functions of mTOR, its regulatory effects on inflammation, and the curative effects of mTOR inhibitors in RA, thus providing references for the development of RA therapeutic targets and new drugs.
    Keywords:  Rheumatoid arthritis; Treatment; mTOR
    DOI:  https://doi.org/10.1186/s13075-023-03181-w
  57. Autophagy. 2023 Sep 30. 1-20
    CENPN suppresses autophagy and increases paclitaxel resistance in nasopharyngeal carcinoma cells by inhibiting the CREB-VAMP8 signaling axis.
    Bin-Ru Wang, Ji-Bo Han, Yang Jiang, Shan Xu, Rui Yang, Yong-Gang Kong, Ze-Zhang Tao, Qing-Quan Hua, You Zou, Shi-Ming Chen.
      Chemotherapeutic resistance is one of the most common reasons for poor prognosis of patients with nasopharyngeal carcinoma (NPC). We found that CENPN can promote the growth, proliferation and apoptosis resistance of NPC cells, but its relationship with chemotherapeutic resistance in NPC is unclear. Here we verified that the CENPN expression level in NPC patients was positively correlated with the degree of paclitaxel (PTX) resistance and a poor prognosis through analysis of clinical cases. VAMP8 expression was significantly increased after knockdown of CENPN by transcriptome sequencing. We found in cell experiments that CENPN inhibited macroautophagy/autophagy and VAMP8 expression and significantly increased PTX resistance. Overexpression of CENPN reduced the inhibitory effects of PTX on survival, cell proliferation, cell cycle progression and apoptosis resistance in NPC cells by inhibiting autophagy. In turn, knockdown of CENPN can affect the phenotype of NPC cells by increasing autophagy to achieve PTX sensitization. Sequential knockdown of CENPN and VAMP8 reversed the PTX-sensitizing effect of CENPN knockdown alone. Experiments in nude mice confirmed that knockdown of CENPN can increase VAMP8 expression, enhance autophagy and increase the sensitivity of NPC cells to PTX. Mechanistic studies showed that CENPN inhibited the translocation of p-CREB into the nucleus of NPC cells, resulting in the decreased binding of p-CREB to the VAMP8 promoter, thereby inhibiting the transcription of VAMP8. These results demonstrate that CENPN may be a marker for predicting chemotherapeutic efficacy and a potential target for inducing chemosensitization to agents such as PTX.Abbreviations: 3-MA: 3-methyladenine; ATG5: autophagy related 5; CENPN: centromere protein N; CQ: chloroquine; CREB: cAMP responsive element binding protein; ChIP: chromatin immunoprecipitation assay; IC50: half-maximal inhibitory concentration; LAMP2A: lysosomal associated membrane protein 2A; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NPC: nasopharyngeal carcinoma; NPG: nasopharyngitis; oeCENPN: overexpressed CENPN; PTX: paclitaxel; RAPA: rapamycin; RNA-seq: transcriptome sequencing; shCENPN: small hairpin RNA expression vector targeting the human CENPN gene; shCENPN-shVAMP8: sequential knockdown targeting the human CENPN gene and VAMP8 gene; shVAMP8: small hairpin RNA expression vector targeting the human VAMP8 gene; TEM: transmission electron microscopy; TIR: tumor inhibitory rate; VAMP8: vesicle associated membrane protein 8.
    Keywords:  Autophagy; CENPN; chemotherapeutic resistance; nasopharyngeal carcinoma; paclitaxel
    DOI:  https://doi.org/10.1080/15548627.2023.2258052
  58. Biomed Pharmacother. 2023 Oct 04. pii: S0753-3322(23)01450-6. [Epub ahead of print]167 115652
    Mitochondrial dysfunction: A fatal blow in depression.
    Yu Song, Huan Cao, Chengchao Zuo, Zhongya Gu, Yaqi Huang, Jinfeng Miao, Yufeng Fu, Yu Guo, Yongsheng Jiang, Furong Wang.
      Mitochondria maintain the normal physiological function of nerve cells by producing sufficient cellular energy and performing crucial roles in maintaining the metabolic balance through intracellular Ca2+ homeostasis, oxidative stress, and axonal development. Depression is a prevalent psychiatric disorder with an unclear pathophysiology. Damage to the hippocampal neurons is a key component of the plasticity regulation of synapses and plays a critical role in the mechanism of depression. There is evidence suggesting that mitochondrial dysfunction is associated with synaptic impairment. The maintenance of mitochondrial homeostasis includes quantitative maintenance and quality control of mitochondria. Mitochondrial biogenesis produces new and healthy mitochondria, and mitochondrial dynamics cooperates with mitophagy to remove damaged mitochondria. These processes maintain mitochondrial population stability and exert neuroprotective effects against early depression. In contrast, mitochondrial dysfunction is observed in various brain regions of patients with major depressive disorders. The accumulation of defective mitochondria accelerates cellular nerve dysfunction. In addition, impaired mitochondria aggravate alterations in the brain microenvironment, promoting neuroinflammation and energy depletion, thereby exacerbating the development of depression. This review summarizes the influence of mitochondrial dysfunction and the underlying molecular pathways on the pathogenesis of depression. Additionally, we discuss the maintenance of mitochondrial homeostasis as a potential therapeutic strategy for depression.
    Keywords:  Major depressive disorder; Mitochondrial dysfunction; Mitochondrial quality control; Neuroinflammation
    DOI:  https://doi.org/10.1016/j.biopha.2023.115652
  59. Nat Rev Mol Cell Biol. 2023 Oct 06.
    mTOR inhibits starvation-induced nuclear export of the proteasome.
    Kim Baumann.
      
    DOI:  https://doi.org/10.1038/s41580-023-00678-9
  60. Biochem Biophys Res Commun. 2023 Sep 26. pii: S0006-291X(23)01120-8. [Epub ahead of print]682 56-63
    Modulation of TRPM8 alters the phagocytic activity of microglia and induces changes in sub-cellular organelle functions.
    Deep Shikha, Chandan Mahish, Raima Sing, Subhasis Chattopadhyay, Chandan Goswami.
      In this work, we investigated the presence and function of TRPM8, a non-selective and cold-sensitive Ca2+-permeable ion channel in the primary microglia cell as well as in microglia cell line BV2. We demonstrate that primary microglia as well as BV2 express TRPM8 endogenously. Both pharmacological activation or inhibition of TRPM8 causes enhanced uptake of bacterial particles at early time points of infection. In BV2, TRPM8 activation and/or LPS-signaling alters its surface expression and cytosolic ROS production. TRPM8 modulation in the absence and presence of LPS causes differential regulation of cytosolic pH and lysosomal pH. Notably, TRPM8 modulation also alters the correlation between lysosomal pH and cytosolic pH depending on TRPM8 modulation and the presence or absence of LPS. Collectively our data suggest that TRPM8 is involved in the regulation of subcellular organelle, i.e. mitochondrial and lysosomal functions. Data also suggest that primarily TRPM8 activation, but often deviation from endogenous TRPM8 function is linked with better innate immune function mediated by microglial cells. We suggest that TRPM8-mediated regulations of sub-cellular organelle functions are more context-dependent manner. Such understanding is relevant in the context of microglial cell functions and innate immunity.
    Keywords:  Ca(2+)-signaling; Lysosome; Mitochondria; NOS; Phagocytosis; ROS; Surface expression; TRPM8
    DOI:  https://doi.org/10.1016/j.bbrc.2023.09.078
  61. Nucleosides Nucleotides Nucleic Acids. 2023 Oct 04. 1-16
    Identification of autophagy and angiogenesis modulators in colorectal cancer based on bioinformatics analysis.
    Fariba Shakeri, Parisa Mohamadynejad, Mehdi Moghanibashi.
      Colorectal cancer (CRC) is the third most common cancer and the fourth leading cause of cancer-related death worldwide. The purpose of this study was to discover novel molecular pathways and potential prognosis biomarkers. To achieve this, we acquired five microarray datasets from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes between CRC and adjacent normal tissue samples and further validated them using The Cancer Genome Atlas (TCGA) database. Using various analytical approaches, including the construction of a competing endogenous RNA (ceRNA) network, Gene Ontology term and Kyoto Encyclopedia of Genes and Genomes pathway analyses, as well as survival analysis, we identified key genes and pathways associated with the diagnosis and prognosis of CRC. We obtained a total of 185 differentially expressed genes, comprising 17 lncRNAs, 30 miRNAs, and 138 mRNAs. The ceRNA network consisted of 17 lncRNAs, 25 miRNAs, and 7 mRNAs. Among the 7 mRNAs involved in the ceRNA network, SLC7A5 and KRT80 were found to be upregulated, while ADIPOQ, CCBE1, KCNB1, CADM2, and CHRDL1 were downregulated in CRC. Further analysis revealed that ADIPOQ and SLC7A5 are involved in the AMPK and mTOR signaling pathway, respectively. In addition, survival analysis demonstrated a statistically significant relationship between ADIPOQ, SLC7A5, and overall survival rates in CRC patients. In conclusion, our findings suggest that downregulation of ADIPOQ and upregulation of SLC7A5 in tumor cells lead to increased mTORC1 activity, reduced autophagy, enhanced angiogenesis, and ultimately contribute to cancer progression and decreased survival in CRC patients.
    Keywords:  ADIPOQ; Colorectal cancer; SLC7A5; angiogenesis; autophagy; bioinformatics
    DOI:  https://doi.org/10.1080/15257770.2023.2259431
  62. J Cell Mol Med. 2023 Oct 06.
    Engeletin alleviates depression-like phenotype by increasing synaptic plasticity via the BDNF-TrkB-mTORC1 signalling pathway.
    Yangyang Xu, Jie Zhang, Linyao Yu, Wei Zhang, Yingtian Zhang, Yaoqin Shi, Shuping Zhang, Chunmei Li, Jingwei Tian.
      Major depressive disorder (MDD) is a severe mental disorder associated with high rates of morbidity and mortality. Current first-line pharmacotherapies for MDD are based on enhancement of monoaminergic neurotransmission, but these antidepressants are still insufficient and produce significant side-effects. Consequently, the development of novel antidepressants and therapeutic targets is desired. Engeletin, a natural Smilax glabra rhizomilax derivative, is a compound with proven efficacy in treating ischemic stroke, yet its therapeutic effects and mechanisms for depression remain unexplored. The effects of engeletin were assessed in the forced swimming test (FST) and tail suspension test (TST) in mice. Engeletin was also investigated in the chronic restraint stress (CRS) mouse model of depression with fluoxetine (FLX) as the positive control. Changes in prefrontal cortex (PFC) spine density, synaptic plasticity-linked protein expressions and the brain-derived neurotrophic factor (BDNF)-tyrosine kinase B (TrkB)- mammalian target of rapamycin complex 1 (mTORC1) signalling pathway after chronic stress and engeletin treatment were then investigated. The TrkB and mTORC1 selective inhibitors, ANA-12 and rapamycin, respectively, were utilized to assess the engeletin's antidepressive mechanisms. Our data shows that engeletin exhibited antidepressant-like activity in the FST and TST in mice without affecting locomotor activity. Furthermore, it exhibited efficiency against the depression of CRS model. Moreover, it enhanced the BDNF-TrkB-mTORC1 pathway in the PFC during CRS and altered the reduction in dendritic spine density and levels of synaptic plasticity-linked protein induced by CRS. In conclusion, engeletin has antidepressant activity via activation of the BDNF-TrkB-mTORC1 signalling pathway and upregulation of PFC synaptic plasticity.
    Keywords:  BDNF-TrkB-mTORC1 signalling pathway; chronic restraint stress; engeletin; major depressive disorder; prefrontal cortex; synaptic plasticity
    DOI:  https://doi.org/10.1111/jcmm.17975
  63. JACC Basic Transl Sci. 2023 Sep;8(9): 1215-1239
    Syntaxin 17 Protects Against Heart Failure Through Recruitment of CDK1 to Promote DRP1-Dependent Mitophagy.
    Haixia Xu, Xiang Wang, Wenjun Yu, Shiqun Sun, Ne N Wu, Junbo Ge, Jun Ren, Yingmei Zhang.
      Mitochondrial dysfunction is suggested to be a major contributor for the progression of heart failure (HF). Here we examined the role of syntaxin 17 (STX17) in the progression of HF. Cardiac-specific Stx17 knockout manifested cardiac dysfunction and mitochondrial damage, associated with reduced levels of p(S616)-dynamin-related protein 1 (DRP1) in mitochondria-associated endoplasmic reticulum membranes and dampened mitophagy. Cardiac STX17 overexpression promoted DRP1-dependent mitophagy and attenuated transverse aortic constriction-induced contractile and mitochondrial damage. Furthermore, STX17 recruited cyclin-dependent kinase-1 through its SNARE domain onto mitochondria-associated endoplasmic reticulum membranes, to phosphorylate DRP1 at Ser616 and promote DRP1-mediated mitophagy upon transverse aortic constriction stress. These findings indicate the potential therapeutic benefit of targeting STX17 in the mitigation of HF.
    Keywords:  CDK1; DRP1; MAMs; STX17; mitophagy; pressure overload–induced heart failure
    DOI:  https://doi.org/10.1016/j.jacbts.2023.04.006
  64. Cell Rep. 2023 Sep 25. pii: S2211-1247(23)01172-5. [Epub ahead of print] 113160
    Analysis of proteome-wide degradation dynamics in ALS SOD1 iPSC-derived patient neurons reveals disrupted VCP homeostasis.
    Konstantinos Tsioras, Kevin C Smith, Seby L Edassery, Mehraveh Garjani, Yichen Li, Chloe Williams, Elizabeth D McKenna, Wenxuan Guo, Anika P Wilen, Timothy J Hark, Stefan L Marklund, Lyle W Ostrow, Jonathan D Gilthorpe, Justin K Ichida, Robert G Kalb, Jeffrey N Savas, Evangelos Kiskinis.
      Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS) through gain-of-function effects, yet the mechanisms by which misfolded mutant SOD1 (mutSOD1) protein impairs human motor neurons (MNs) remain unclear. Here, we use induced-pluripotent-stem-cell-derived MNs coupled to metabolic stable isotope labeling and mass spectrometry to investigate proteome-wide degradation dynamics. We find several proteins, including the ALS-causal valosin-containing protein (VCP), which predominantly acts in proteasome degradation and autophagy, that degrade slower in mutSOD1 relative to isogenic control MNs. The interactome of VCP is altered in mutSOD1 MNs in vitro, while VCP selectively accumulates in the affected motor cortex of ALS-SOD1 patients. Overexpression of VCP rescues mutSOD1 toxicity in MNs in vitro and in a C. elegans model in vivo, in part due to its ability to modulate the degradation of insoluble mutSOD1. Our results demonstrate that VCP contributes to mutSOD1-dependent degeneration, link two distinct ALS-causal genes, and highlight selective protein degradation impairment in ALS pathophysiology.
    Keywords:  ALS; CP: Neuroscience; CP: Stem cell research; SILAC-based mass spectrometry; SOD1; VCP/p97; amyotrophic lateral sclerosis; iPSCs; motor neurons; protein degradation; ubiquitin
    DOI:  https://doi.org/10.1016/j.celrep.2023.113160
  65. Res Sq. 2023 Sep 18. pii: rs.3.rs-3269310. [Epub ahead of print]
    Sde Proteins Coordinate Ubiquitin Utilization and Phosphoribosylation to Promote Establishment and Maintenance of the Legionella Replication Vacuole.
    Ralph Isberg, Kristin Kotewicz, Mengyun Zheng, Seongok Kim, Albert Tai.
      The Legionella pneumophila Sde family of translocated proteins promote host tubular endoplasmic reticulum (ER) rearrangements that are tightly linked to phosphoribosyl-ubiquitin (pR-Ub) modification of Reticulon 4 (Rtn4). Sde proteins have two additional activities of unclear relevance to the infection process: K63 linkage-specific deubiquitination and phosphoribosyl modification of polyubiquitin (pR-Ub). We show here that the deubiquitination activity (DUB) stimulates ER rearrangements while pR-Ub protects the replication vacuole from cytosolic surveillance by autophagy. Loss of DUB activity was tightly linked to lowered pR-Ub modification of Rtn4, consistent with the DUB activity fueling the production of pR-Ub-Rtn4. In parallel, phosphoribosyl modification of polyUb, in a region of the protein known as the isoleucine patch, caused an absolute block in binding by the autophagy adapter p62. An inability of Sde mutants to modify polyUb resulted in immediate p62 association, a critical precursor to autophagic attack. The ability of Sde WT to block p62 association decayed quickly after bacterial infection, as predicted by the presence of previously characterized L. pneumophila effectors that inactivate Sde and remove polyUb. In sum, these results show that the accessory Sde activities act to stimulate ER rearrangements and protect from host innate immune sensing in a temporal fashion.
    DOI:  https://doi.org/10.21203/rs.3.rs-3269310/v1
  66. J Cancer. 2023 ;14(14): 2608-2618
    Melatonin Potentiates Sensitivity to 5-Fluorouracil in Gastric Cancer Cells by Upregulating Autophagy and Downregulating Myosin Light-Chain Kinase.
    Xiaorui Shi, Hongxia Li, Zhangyong Dan, Chuanlin Shu, Rumeng Zhu, Qingling Yang, Yi Wang, Huaqing Zhu.
      5-Fluorouracil is an effective chemotherapeutic drug for gastric cancer. However, the acquisition of chemotherapeutic resistance remains a challenge in treatment. Melatonin can enhance the therapeutic effect of 5-fluorouracil; however, the underlying mechanisms are not well understood. We investigated the effects of combinations of melatonin and 5-fluorouracil on the proliferation, migration and invasion of gastric cancer cells. Melatonin significantly potentiated the 5-fluorouracil-mediated inhibition of proliferation, migration and invasion in gastric cancer cells, which potentiates sensitivity to 5-FU by promoting the activation of Beclin-1-dependent autophagy and targeting the myosin light-chain kinase (MLCK) signaling pathway. Previous studies have shown that autophagy might be associated with the MLCK signaling pathway. The autophagy inhibitor, 3-methyladenine, effectively rescued the migratory and invasive capabilities of gastric cancer cells, while also reducing expression level of MLCK and the phosphorylation level of MLC. This indicates that autophagy is involved in tumor metastasis, which may be related to inhibition of the MLCK signaling pathway. Our findings indicate that melatonin can improve the effectiveness of 5-fluorouracil in gastric cancer and could be used as a supplemental agent in the treatment of gastric cancer with 5-fluorouracil.
    Keywords:  5-Fluorouracil; MLCK; autophagy; gastric cancer; melatonin
    DOI:  https://doi.org/10.7150/jca.85353
  67. mBio. 2023 Oct 06. e0153523
    Herp regulates intracellular survival of Mycobacterium tuberculosis H37Ra in macrophages by regulating reactive oxygen species-mediated autophagy.
    Sang-Hun Son, Junghwan Lee, Soo-Na Cho, Ji-Ae Choi, Jaewhan Kim, Tam Doan Nguyen, Seong-Ahn Lee, Doyi Son, Chang-Hwa Song.
      Novel treatment strategies for tuberculosis (TB), such as host-directed therapeutics, may offer therapeutic options for patients with drug-resistant TB. Endoplasmic reticulum (ER) stress-mediated apoptosis is one of the host defense mechanisms used to remove mycobacteria. It is reported that homocysteine-inducible ER protein with ubiquitin-like domain 1 (Herp) inhibits apoptosis by preventing the loss of ER Ca2+ and mitochondrial potential during ER stress. However, the roles of Herp in ER stress and apoptosis during H37Ra infection are largely unknown. Here, we show that Herp is induced in H37Ra-infected macrophages through an activating transcription factor 6 (ATF6)-dependent ER stress response. Suppressing Herp by genetic approaches decreased production of HRD1, conserved branch of mammalian ER-associated degradation (ERAD) machinery, and increased the production levels of ER stress-associated molecules such as p-IRE1ɑ and BiP after H37Ra infection. Suppressing Herp also increased both the NADPH oxidase 2 and inositol triphosphate receptor, which sequentially led to increased reactive oxygen species (ROS) production during H37Ra infection. Interestingly, the Herp depletion-mediated ROS increment led to autophagy induction, which led to decreased intracellular survival of mycobacteria in H37Ra-infected macrophages. The role of Herp was further confirmed by the fact that blocking this molecule in vitro and in vivo significantly reduced mycobacterial survival. These findings indicate that Herp mediates crosstalk between ER stress and ROS-mediated autophagy during H37Ra infection, suggesting the potential of Herp manipulation as a therapeutic strategy for Mycobacterium tuberculosis (Mtb) infection.IMPORTANCESeveral studies have suggested that endoplasmic reticulum (ER) stress is important in the pathogenesis of infectious diseases; however, the precise function of ER stress regulation and the role of Herp as a regulator in Mtb H37Ra-induced ER stress remain elusive. Therefore, our study investigated ER stress and autophagy associated with Herp expression in Mycobacterium tuberculosis-infected macrophages to determine the role of Herp in the pathogenesis of tuberculosis.
    Keywords:  ER stress; ER-associated degradation; Mycobacterium tuberculosis; autophagy; homocysteine-inducible ER protein with ubiquitin-like domain 1; reactive oxygen species
    DOI:  https://doi.org/10.1128/mbio.01535-23
  68. Nutr Neurosci. 2023 Sep 30. 1-10
    Trehalose consumption ameliorates pathogenesis in an inducible mouse model of the Fragile X-associated tremor/ataxia syndrome.
    Emre Kul, Oliver Stork.
      Trehalose is a naturally occurring sugar found in various food and pharmaceutical preparations with the ability to enhance cellular proteostasis and reduce the formation of toxic intracellular protein aggregates, making it a promising therapeutic candidate for various neurodegenerative disorders.
    OBJECTIVES: Here, we explored the effectiveness of nutritional trehalose supplementation in ameliorating symptoms in a mouse model of Fragile X-associated tremor/ataxia syndrome (FXTAS), an incurable late onset manifestation of moderately expanded trinucleotide CGG repeat expansion mutations in the 5' untranslated region of the fragile X messenger ribonucleoprotein 1 gene (FMR1).
    METHODS: An inducible mouse model of FXTAS expressing 90 CGG repeats in the brain had been previously developed, which faithfully captures hallmarks of the disorder, the formation of intracellular inclusions, and the disturbance of motor function. Taking advantage of the inducible nature of the model, we investigated the therapeutic potential of orally administered trehalose under two regimens, modelling disease prevention and disease treatment.
    RESULTS AND DISCUSSION: Trehalose's effectiveness in combating protein aggregation is frequently attributed to its ability to induce autophagy. Accordingly, trehalose supplementation under the prevention regimen ameliorated the formation of intranuclear inclusions and improved the motor deficiencies resulting from the induced expression of 90 CGG repeats, but it failed to reverse the existing nuclear pathology as a treatment strategy. Given the favorable safety profile of trehalose, it is promising to further explore the potential of this agent for early stage FXTAS.
    Keywords:  FXTAS; autophagy; cerebellum; motor behavior; neurodegeneration; repeat expansion; transgenic mouse; trehalose
    DOI:  https://doi.org/10.1080/1028415X.2023.2261682
  69. Mol Cell. 2023 Sep 22. pii: S1097-2765(23)00698-6. [Epub ahead of print]
    ATR promotes clearance of damaged DNA and damaged cells by rupturing micronuclei.
    Yoon Ki Joo, Elizabeth M Black, Isabelle Trier, Wisse Haakma, Lee Zou, Lilian Kabeche.
      The human ataxia telangiectasia mutated and Rad3-related (ATR) kinase functions in the nucleus to protect genomic integrity. Micronuclei (MN) arise from genomic and chromosomal instability and cause aneuploidy and chromothripsis, but how MN are removed is poorly understood. Here, we show that ATR is active in MN and promotes their rupture in S phase by phosphorylating Lamin A/C at Ser395, which primes Ser392 for CDK1 phosphorylation and destabilizes the MN envelope. In cells harboring MN, ATR or CDK1 inhibition reduces MN rupture. Consequently, ATR inhibitor (ATRi) diminishes activation of the cytoplasmic DNA sensor cGAS and compromises cGAS-dependent autophagosome accumulation in MN and clearance of micronuclear DNA. Furthermore, ATRi reduces cGAS-mediated senescence and killing of MN-bearing cancer cells by natural killer cells. Thus, in addition to the canonical ATR signaling pathway, an ATR-CDK1-Lamin A/C axis promotes MN rupture to clear damaged DNA and cells, protecting the genome in cell populations through unexpected cell-autonomous and cell-non-autonomous mechanisms.
    Keywords:  ATR; CDK1; DNA damage; STING; TREX1; autophagosome; autophagy; cGAS; cell-autonomous; cell-non-autonomous; clearance; damaged DNA; micronuclear DNA; micronuclei; micronucleus; natural killer cells; nuclear envelope; rupture
    DOI:  https://doi.org/10.1016/j.molcel.2023.09.003
  70. Heliyon. 2023 Oct;9(10): e20142
    Hotspots and future trends of autophagy in Traditional Chinese Medicine: A Bibliometric analysis.
    Jun Zhao, Guang-Wei Liu, Cheng Tao.
       Objective: To discuss the hotspots and future trends of autophagy in traditional Chinese medicine (TCM) and provide a reference for researchers in this field.
    Method: Using visual analysis tools, metrological statistics and visual research on the pertinent literature in the area of autophagy use in TCM were undertaken in the core collection database of the Web of Science. By examining the authors, keywords, research circumstances, research hotspots, and trends of linked research, the use of autophagy in TCM was investigated.
    Results and Conclusions: A total of 916 studies were included, among which Beijing University Chinese Medicine was the largest number of advantageous research institutions, followed by Shanghai University Traditional Chinese Medicine and Guangzhou University Chinese Medicine.The keywords of literature research primarily comprise apoptosis, activation, inhibition, pathway, mechanism, oxidative stress, proliferation, NF-κB, cancer, mtor, etc. At present, the research on autophagy in the field of TCM is increasing on a year-to-year basis. The research has focused on the role played by TCM in malignant tumors, atherosclerosis, Alzheimer's disease through autophagy, and the regulation of autophagy signaling pathways (e.g., PI3K/AKT/mTOR signaling pathway, TLR4 signaling pathway,nrf2 signaling pathway and NF-κB signaling pathway). In the future, the therapeutic effect of TCM on chemotherapy-resistant tumor cells through autophagy pathway, the role of TCM mediating mitophagy and activating autophagy function, and the therapeutic effect of TCM components represented by luteolin on tumors, asthma, myocardial injury and other diseases through autophagy mechanism will be the research hotspots in the future.
    Keywords:  Autophagy; Bibliometrics analysis; CiteSpace; Traditional Chinese Medicine (TCM); Visualized Analysis
    DOI:  https://doi.org/10.1016/j.heliyon.2023.e20142
  71. Am J Respir Cell Mol Biol. 2023 Oct 03.
    Pleural Fibrosis: Now That's What mTORC(ing) About.
    Steven E Mutsaers, Vera P Krymskaya.
      
    Keywords:  mTOR; pleural fibrosis; signalling pathway inhibition; signalling pathways
    DOI:  https://doi.org/10.1165/rcmb.2023-0327ED
  72. Indian J Hematol Blood Transfus. 2023 Oct;39(4): 670-678
    High Autophagy Patterns in Swelling Platelets During Apheresis Platelet Storage.
    Lu Yu, Shifang Yu, Yunlei He, Gang Deng, Qiang Li.
      Platelets undergo remarkable morphological changes during storage. Platelets change into different sizes and densities and differ in their biochemistry and functions. However, the correlation between structural heterogeneity and platelet autophagy is largely unknown. The aim of this study was to investigate the autophagy process in vitro, such as routine storage of platelets, and explore the role of reactive oxygen species (ROS) involved in the regulation of platelet autophagy. The ROS and autophagy levels of platelet concentrates from apheresis platelets were evaluated through flow cytometry. The expression levels of autophagy-associated proteins (LC3I, LC3II, Beclin1, Parkin, and PINK1) were measured via Western blot. All biomarkers were dynamically monitored for seven days. Moreover, the morphological characteristics of platelet morphology during storage were analyzed through transmission electron microscopy (TEM). Flow cytometry showed that the levels of total cell ROS and mitochondria ROS increased in the stored platelets. Together with the increase in mitochondrial ROS, the autophagy signal LC3 in the platelets was strongly amplified. The number of swollen platelets (large platelets) considerably increased, and that of autophagy signal LC3 was remarkably higher than that of the normal platelets. Western blot revealed that the expression levels of Beclin1 and LC3 II/LC3 I ratio were enhanced, whereas those of Parkin and PINK1 almost did not change during the seven days of storage. The existence of autophagosomes or autophagolysosomes in the platelets at the middle stage of platelet storage was observed via TEM. Our data demonstrated that the subpopulation of large (swollen) platelets exhibited different autophagy patterns. Furthermore, increased platelet autophagy was associated with mitochondrial ROS. These preliminary results suggest that swelling platelets have a higher autophagy pattern than normal platelets during storage.
    Keywords:  Autophagy; LC3; Platelet; ROS
    DOI:  https://doi.org/10.1007/s12288-023-01638-1
  73. J Neurosci. 2023 Sep 27. pii: JN-RM-1089-23. [Epub ahead of print]
    P2Y6 receptor-dependent microglial phagocytosis of synapses during development regulates synapse density and memory.
    Jacob M Dundee, Mar Puigdellívol, Richard Butler, Guy C Brown.
      During brain development, excess synapses are pruned (i.e. removed), in part by microglial phagocytosis, and dysregulated synaptic pruning can lead to behavioral deficits. The P2Y6 receptor (P2Y6R) is known to regulate microglial phagocytosis of neurons, and to regulate microglial phagocytosis of synapses in cell culture and in vivo during aging. However, currently it is unknown whether P2Y6R regulates synaptic pruning during development. Here, we show that P2Y6R knockout mice of both sexes had strongly reduced microglial internalization of synaptic material, measured as Vglut1 within CD68-staining lysosomes of microglia at postnatal day 30 (P30), suggesting reduced microglial phagocytosis of synapses. Consistent with this, we found an increased density of synapses in the somatosensory cortex and the CA3 region and dentate gyrus of the hippocampus at P30. We also show that adult P2Y6R knockout mice have impaired short- and long-term spatial memory and impaired short- and long-term recognition memory compared to wild-type mice, as measured by novel location recognition, novel object recognition, and Y-maze memory tests. Overall, this indicates P2Y6R regulates microglial phagocytosis of synapses during development, and this contributes to memory capacity.Significance StatementThe P2Y6 receptor (P2Y6R) is activated by UDP released by neurons, inducing microglial phagocytosis of such neurons or synapses. We tested whether P2Y6R regulates developmental synaptic pruning in mice and found that P2Y6R knockout mice have reduced synaptic material within microglial lysosomes, and increased synaptic density in the brains of postnatal day 30 mice, consistent with reduced synaptic pruning during development. We also found that adult P2Y6R knockout mice had reduced memory, consistent with persistent deficits in brain function, resulting from impaired synaptic pruning. Overall, the results suggest that P2Y6R mediates microglial phagocytosis of synapses during development, and the absence of this results in memory deficits in the adult.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1089-23.2023
  74. Acta Pharm Sin B. 2023 Oct;13(10): 4172-4184
    Cathepsin D overexpression in the nervous system rescues lethality and Aβ42 accumulation of cathepsin D systemic knockout in vivo.
    Xiaosen Ouyang, Willayat Y Wani, Gloria A Benavides, Matthew J Redmann, Hai Vo, Thomas van Groen, Victor M Darley-Usmar, Jianhua Zhang.
      The lysosome is responsible for protein and organelle degradation and homeostasis and the cathepsins play a key role in maintaining protein quality control. Cathepsin D (CTSD), is one such lysosomal protease, which when deficient in humans lead to neurolipofuscinosis (NCL) and is important in removing toxic protein aggregates. Prior studies demonstrated that CTSD germ-line knockout-CtsdKO (CDKO) resulted in accumulation of protein aggregates, decreased proteasomal activities, and postnatal lethality on Day 26 ± 1. Overexpression of wildtype CTSD, but not cathepsin B, L or mutant CTSD, decreased α-synuclein toxicity in worms and mammalian cells. In this study we generated a mouse line expressing human CTSD with a floxed STOP cassette between the ubiquitous CAG promoter and the cDNA. After crossing with Nestin-cre, the STOP cassette is deleted in NESTIN + cells to allow CTSD overexpression-CTSDtg (CDtg). The CDtg mice exhibited normal behavior and similar sensitivity to sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced neurodegeneration. By breeding CDtg mice with CDKO mice, we found that over-expression of CTSD extended the lifespan of the CDKO mice, partially rescued proteasomal deficits and the accumulation of Aβ42 in the CDKO. This new transgenic mouse provides supports for the key role of CTSD in protecting against proteotoxicity and offers a new model to study the role of CTSD enhancement in vivo.
    Keywords:  Apoptosis; Autophagy; Behavior; Cathepsin D knockout mice; Cathepsin D transgenic mouse; Dopamine; Life expectancy; Lysosome; Mitochondrial bioenergetics; Neuronal ceroid lipofuscinosis
    DOI:  https://doi.org/10.1016/j.apsb.2023.07.015
  75. Res Sq. 2023 Sep 12. pii: rs.3.rs-3342413. [Epub ahead of print]
    Protein restriction slows the development and progression of Alzheimer's disease in mice.
    Reji Babygirija, Michelle M Sonsalla, Jessica H Han, Isabella James, Cara L Green, Mariah F Calubag, Jericha Mill, Gina Wade, Anna Tobon, John Michael, Michaela M Trautman, Ryan Matoska, Chung-Yang Yeh, Isaac Grunow, Heidi H Pak, Michael J Rigby, John M Denu, Luigi Puglielli, Judith Simcox, Dudley W Lamming.
      Over the last decade, it has become evident that dietary protein is a critical regulator of metabolic health and aging. Low protein diets are associated with healthy aging in humans, and we and others have shown that dietary protein restriction (PR) extends the lifespan and healthspan of mice. Here, we examined the effect of PR on metabolic health and the development and progression of Alzheimer's disease (AD) in the 3xTg mouse model of AD. We found that PR has metabolic benefits for 3xTg mice and non-transgenic controls of both sexes, promoting leanness and glycemic control in 3xTg mice. We found that PR induces sex-specific alterations in circulating metabolites and in the brain lipidome, downregulating sphingolipid subclasses including ceramides, glucosylceramides, and sphingomyelins in 3xTg females. Consumption of a PR diet starting at 6 months of age reduced AD pathology in conjunction with reduced mTORC1 activity, increased autophagy, and had cognitive benefits for 3xTg mice. Finally, PR improved the survival of 3xTg mice. Our results demonstrate that PR slows the progression of AD at molecular and pathological levels, preserves cognition in this mouse model of AD, and suggests that PR or pharmaceutical interventions that mimic the effects of this diet may hold promise as a treatment for AD.
    DOI:  https://doi.org/10.21203/rs.3.rs-3342413/v1
  76. Physiol Res. 2023 Aug 31. 72(4): 497-510
    Curcumin in vitro Neuroprotective Effects Are Mediated by p62/keap-1/Nrf2 and PI3K/AKT Signaling Pathway and Autophagy Inhibition.
    X Li, P Sung, D Zhang, L Yan.
      Oxidative stress and autophagy are potential mechanisms associated with cerebral ischemia/reperfusion injury (IRI) and is usually linked to inflammatory responses and apoptosis. Curcumin has recently been demonstrated to exhibit anti-inflammatory, anti-oxidant, anti-apoptotic and autophagy regulation properties. However, mechanism of curcumin on IRI-induced oxidative stress and autophagy remains not well understood. We evaluated the protective effects and potential mechanisms of curcumin on cerebral microvascular endothelial cells (bEnd.3) and neuronal cells (HT22) against oxygen glucose deprivation/reoxygenation (OGD/R) in vitro models that mimic in vivo cerebral IRI. The cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) activity assays revealed that curcumin attenuated the OGD/R-induced injury in a dose-specific manner. OGD/R induced elevated levels of inflammatory cytokines TNF-alpha, IL-6 as well as IL-1beta, and these effects were notably reduced by curcumin. OGD/R-mediated apoptosis was suppressed by curcumin via upregulating B-cell lymphoma-2 (Bcl-2) and downregulating Bcl-associated X (Bax), cleaved-caspase3 and TUNEL apoptosis marker. Additionally, curcumin increased superoxide dismutase (SOD) and glutathione (GSH), but suppressed malondialdehyde (MDA) and reactive oxygen species (ROS) content. Curcumin inhibited the levels of autophagic biomarkers such as LC3 II/LC3 I and Beclin1. Particularly, curcumin induced p62 accumulation and its interactions with keap1 and promoted NF-E2-related factor 2 (Nrf2) translocation to nucleus, accompanied by increased NADPH quinone dehydrogenase (Nqo1) and heme oxygenase 1 (HO-1). Treatment of curcumin increased phosphorylation-phosphatidylinositol 3 kinase (p-PI3K) and p-protein kinase B (p-AKT). The autophagy inhibitor 3-methyladenine (3-MA) activated the keap-1/Nrf2 and PI3K/AKT pathways. This study highlights the neuroprotective effects of curcumin on cerebral IRI.
  77. Nat Commun. 2023 Oct 06. 14(1): 6248
    YTHDF2 facilitates aggresome formation via UPF1 in an m6A-independent manner.
    Hyun Jung Hwang, Tae Lim Park, Hyeong-In Kim, Yeonkyoung Park, Geunhee Kim, Chiyeol Song, Won-Ki Cho, Yoon Ki Kim.
      YTHDF2 has been extensively studied and typified as an RNA-binding protein that specifically recognizes and destabilizes RNAs harboring N6-methyladenosine (m6A), the most prevalent internal modification found in eukaryotic RNAs. In this study, we unravel the m6A-independent role of YTHDF2 in the formation of an aggresome, where cytoplasmic protein aggregates are selectively sequestered upon failure of protein homeostasis mediated by the ubiquitin-proteasome system. Downregulation of YTHDF2 in HeLa cells reduces the circularity of aggresomes and the rate of movement of misfolded polypeptides, inhibits aggresome formation, and thereby promotes cellular apoptosis. Mechanistically, YTHDF2 is recruited to a misfolded polypeptide-associated complex composed of UPF1, CTIF, eEF1A1, and DCTN1 through its interaction with UPF1. Subsequently, YTHDF2 increases the interaction between the dynein motor protein and the misfolded polypeptide-associated complex, facilitating the diffusion dynamics of the movement of misfolded polypeptides toward aggresomes. Therefore, our data reveal that YTHDF2 is a cellular factor involved in protein quality control.
    DOI:  https://doi.org/10.1038/s41467-023-42015-w
  78. JCI Insight. 2023 Oct 03. pii: e167829. [Epub ahead of print]
    TSC2 S1365A mutation potently regulates CD8+T cell function and differentiation improving adoptive cellular cancer therapy.
    Chirag H Patel, Yi Dong, Navid Koleini, Xiaoxu Wang, Brittany L Dunkerly-Eyring, Jiayu Wen, Mark J Ranek, Laura M Bartle, Daniel B Henderson, Jason G Sagert, David A Kass, Jonathan D Powell.
      MTORC1 integrates signaling from the immune microenvironment to regulate T cell activation, differentiation, and function. TSC2 in the tuberous sclerosis complex tightly regulates mTORC1 activation. CD8+ T cells lacking TSC2 have constitutively enhanced mTORC1 activity and generate robust effector T cells; however sustained mTORC1 activation prevents generation of long-lived memory CD8+ T cells. Here we show manipulating TSC2 at Ser1365 potently regulates activated but not basal mTORC1 signaling in CD8+ T cells. Unlike non-stimulated TSC2 knockout cells, CD8+ T cells expressing a phospho-silencing mutant TSC2-S1365A (SA) retain normal basal mTORC1 activity. PKC and T-cell Receptor (TCR) stimulation induces TSC2 S1365 phosphorylation and preventing this with the SA mutation markedly increases mTORC1 activation and T-cell effector function. Consequently, SA CD8+ T cells display greater effector responses while retaining their capacity to become long-lived memory T cells. SA CD8+ T cells also display enhanced effector function under hypoxic and acidic conditions. In murine and human solid-tumor models, CD8+ SA T cells used as adoptive cell therapy display greater anti-tumor immunity than WT CD8+ T cells. These findings reveal an upstream mechanism to regulate mTORC1 activity in T cells. The TSC2-SA mutation enhances both T cell effector function and long-term persistence/memory formation, supporting an approach to engineer better CAR-T cells for treating cancer.
    Keywords:  Adaptive immunity; Cancer immunotherapy; Cell Biology; Immunology; T cells
    DOI:  https://doi.org/10.1172/jci.insight.167829
  79. Commun Biol. 2023 Oct 05. 6(1): 1012
    ClpP/ClpX deficiency impairs mitochondrial functions and mTORC1 signaling during spermatogenesis.
    Chenxi Guo, Yuan Xiao, Jingkai Gu, Peikun Zhao, Zhe Hu, Jiahuan Zheng, Renwu Hua, Zhuo Hai, Jiaping Su, Jian V Zhang, William S B Yeung, Tianren Wang.
      Caseinolytic protease proteolytic subunit (ClpP) and caseinolytic protease X (ClpX) are mitochondrial matrix peptidases that activate mitochondrial unfolded protein response to maintain protein homeostasis in the mitochondria. However, the role of ClpP and ClpX in spermatogenesis remains largely unknown. In this study, we demonstrated the importance of ClpP/ClpX for meiosis and spermatogenesis with two conditional knockout (cKO) mouse models. We found that ClpP/ClpX deficiency reduced mitochondrial functions and quantity in spermatocytes, affected energy supply during meiosis and attenuated zygotene-pachytene transformation of the male germ cells. The dysregulated spermatocytes finally underwent apoptosis resulting in decreased testicular size and vacuolar structures within the seminiferous tubules. We found mTORC1 pathway was over-activated after deletion of ClpP/ClpX in spermatocytes. Long-term inhibition of the mTORC1 signaling via rapamycin treatment in vivo partially rescue spermatogenesis. The data reveal the critical roles of ClpP and ClpX in regulating meiosis and spermatogenesis.
    DOI:  https://doi.org/10.1038/s42003-023-05372-2
  80. Autophagy. 2023 Oct 04. 1-17
    Invariant BECN1 CXXC motifs bind Zn2+ and regulate structure and function of the BECN1 intrinsically disordered region.
    Shreya Mukhopadhyay, Subeksha Subedi, Jesse B Hopkins, Angel Ugrinov, Srinivas Chakravarthy, Christopher L Colbert, Sangita C Sinha.
       ABBREVIATIONS: AFM: aromatic finger mutant; BH3D: BCL2 homology 3 domain; CCD: coiled-coil domain; CD: circular dichroism spectroscopy; [CysDM1]: C18S and C21S double mutant; [CysDM2]: C137S, and C140S double mutant; [CysTM], C18S, C21S, C137S, and C140S tetrad mutant; Dmax: maximum particle diameter; dRI, differential refractive index; EFA: evolving factor analysis; FHD: flexible helical domain; FL: full length; GFP: green fluorescent protein; HDX-MS: hydrogen/deuterium exchange mass spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; IDR: intrinsically disordered region; ITC, isothermal titration calorimetry; MALS, multi angle light scattering; MBP: maltose-binding protein; MoRFs: molecular recognition features; P(r): pairwise-distance distribution; PtdIns3K: class III phosphatidylinositol 3-kinase; Rg: radius of gyration; SASBDB: small angle scattering biological data bank; SEC: size-exclusion chromatography; SEC-SAXS: size-exclusion chromatography in tandem with small angle X-ray scattering; TEV: tobacco-etch virus; TFE: 2,2,2-trifluoroethanol; TPEN: N,N,N,N-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine; Vc: volume of correlation; WT: wild-type.
    Keywords:  Autophagy; BECN1 (Beclin 1); Zn2+-binding; conformational transitions; intrinsically disordered region (IDR); invariant CxxC motifs
    DOI:  https://doi.org/10.1080/15548627.2023.2259707
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