bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023‒07‒09
sixty-four papers selected by
Viktor Korolchuk, Newcastle University
  1. High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.
  2. Imaging ATG9A, a Multi-Spanning Membrane Protein.
  3. PTEN is both an activator and a substrate of chaperone-mediated autophagy.
  4. Autophagy is required for stem-cell-mediated endometrial programming and the establishment of pregnancy.
  5. Tracking the Transition from an ATG9A Vesicle to an Autophagosome.
  6. Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.
  7. Metamorphosis by ATG13 and ATG101 in human autophagy initiation.
  8. Ratiometric and discriminative visualization of autophagic processes with a novel dual-responded lysosome-specific fluorescent probe.
  9. Autophagy dictates sensitivity to PRMT5 inhibitor in breast cancer.
  10. ARL-17477 is a dual inhibitor of NOS1 and the autophagic-lysosomal system that prevents tumor growth in vitro and in vivo.
  11. LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans.
  12. Aging inverts the effects of p75NTR -modulated mTOR manipulation on hippocampal neuron synaptic plasticity in male mice.
  13. Emerging roles of the MiT/TFE factors in cancer.
  14. CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.
  15. The role of redox-mediated lysosomal dysfunction and therapeutic strategies.
  16. HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates.
  17. Autophagy inhibitors for cancer therapy: Small molecules and nanomedicines.
  18. Curcumin as a Perspective Protection for Retinal Pigment Epithelium during Autophagy Inhibition in the Course of Retinal Degeneration.
  19. TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.
  20. The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs.
  21. Canagliflozin shares common mTOR and MAPK signaling mechanisms with other lifespan extension treatments.
  22. Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.
  23. Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence.
  24. mTORC1-mediated acquisition of reward-related representations by hippocampal somatostatin interneurons.
  25. A Positive Feedback Regulation of SnRK1 Signaling by Autophagy in Plants.
  26. Constitutively active autophagy in macrophages dampens inflammation through metabolic and post-transcriptional regulation of cytokine production.
  27. Progranulin regulation of autophagy contributes to its chondroprotective effect in osteoarthritis.
  28. Autophagy regulation by inorganic, organic, and organic/inorganic hybrid nanoparticles: Organelle damage, regulation factors, and potential pathways.
  29. Autophagy up-regulation upon FeHV-1 infection on permissive cells.
  30. Mitochondrial quality control: prime targets for antiviral therapy?
  31. Recent insights into autophagy and metals/nanoparticles exposure.
  32. Leaderless secretory proteins of the neurodegenerative diseases via TNTs: a structure-function perspective.
  33. mTORC2 acts as a gatekeeper for mTORC1 deficiency-mediated impairments in ILC3 development.
  34. TRAPPIII requires Drs2 binding to transport Atg9 vesicles at cold temperatures.
  35. TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.
  36. Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis.
  37. A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer.
  38. In vivo measurement of autophagic flux by fluorescence correlation spectroscopy.
  39. Survival in sporadic ALS is associated with lower p62 burden in the spinal cord.
  40. Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin.
  41. Foot-and-mouth disease virus structural protein VP3 interacts with HDAC8 and promotes its autophagic degradation to facilitate viral replication.
  42. TBK1 Knockdown Alleviates Axonal Transport Deficits in Retinal Ganglion Cells Via mTORC1 Activation in a Retinal Damage Mouse Model.
  43. Glaucoma-associated Optineurin mutations increase transmitophagy in a vertebrate optic nerve.
  44. FUNDC1: a key mediator of adenosine A2BR activation-induced inhibition of cardiac mitophagy under ischemia/reperfusion conditions.
  45. Modulation of the immunity and inflammation by autophagy.
  46. Autophagy in intestinal fibrosis: relevance in inflammatory bowel disease.
  47. Hierarchical phosphorylation of HOXB13 by mTOR dictates its activity and oncogenic function in prostate cancer.
  48. Realgar-Induced Neurotoxicity: Crosstalk Between the Autophagic Flux and the p62-NRF2 Feedback Loop Mediates p62 Accumulation to Promote Apoptosis.
  49. Lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.
  50. Compensatory cross-talk between autophagy and glycolysis regulates senescence and stemness in heterogeneous glioblastoma tumor subpopulations.
  51. Inhibiting autophagy by miR-19a-3p/PTEN regulation protected retinal pigment epithelial cells from hyperglycemic damage.
  52. The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8).
  53. The regulatory role of lipophagy in central nervous system diseases.
  54. DMT1 differentially regulates mitochondrial complex activities to reduce glutathione loss and mitigate ferroptosis.
  55. Mitochondrial homeostasis: a potential target for delaying renal aging.
  56. Lower dose of metformin combined with artesunate induced autophagy-dependent apoptosis of glioblastoma by activating ROS-AMPK-mTOR axis.
  57. Impaired mitochondrial dynamics and removal of the damaged mitochondria in diabetic retinopathy.
  58. Targeting autophagy with tamoxifen in breast cancer: From molecular mechanisms to targeted therapy.
  59. Transcription factor EB (TFEB) promotes autophagy in early brain injury after subarachnoid hemorrhage in rats.
  60. SIRT3 regulates mitophagy in liver fibrosis through deacetylation of PINK1/NIPSNAP1.
  61. IFITM3 restricts RABV infection through inhibiting viral entry and mTORC1- dependent autophagy.
  62. Intracellular Calcium links Milk Stasis to Lysosome Dependent Cell Death by Activating a TGFβ3/TFEB/STAT3 Pathway Early during Mammary Gland Involution.
  63. Emerging trends and research foci in autophagy of pancreatic cancer: a bibliometric and visualized study.
  64. Rictor induces AKT signaling to regulate lymphatic valve formation.
  1. Autophagy. 2023 Jul 05. 1-20
    High-resolution visualization and assessment of basal and OXPHOS-induced mitophagy in H9c2 cardiomyoblasts.
    Gustav Godtliebsen, Kenneth Bowitz Larsen, Zambarlal Bhujabal, Ida S Opstad, Mireia Nager, Abhinanda R Punnakkal, Trine B Kalstad, Randi Olsen, Trine Lund, Dilip K Prasad, Krishna Agarwal, Truls Myrmel, Asa Birna Birgisdottir.
      Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations: ATG: autophagy related; ATG7: autophagy related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OXPHOS: oxidative phosphorylation; PepA: pepstatin A; PLA: proximity ligation assay; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; RAB9A: RAB9A, member RAS oncogene family; ROS: reactive oxygen species; SIM: structured illumination microscopy; siRNA: short interfering RNA; SYNJ2BP: synaptojanin 2 binding protein; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.
    Keywords:  CLEM; SIM; deep learning; lysosomes; mitochondria; quality control
    DOI:  https://doi.org/10.1080/15548627.2023.2230837
  2. J Vis Exp. 2023 06 16.
    Imaging ATG9A, a Multi-Spanning Membrane Protein.
    Alexander R van Vliet, Stefano De Tito, Eugenia Almacellas, Sharon A Tooze.
      Autophagy is a highly conserved pathway that the cell uses to maintain homeostasis, degrade damaged organelles, combat invading pathogens, and survive pathological conditions. A set of proteins, called ATG proteins, comprise the core autophagy machinery and work together in a defined hierarchy. Studies in recent years have improved our knowledge of the autophagy pathway. Most recently, it has been proposed that ATG9A vesicles are at the heart of autophagy, as they control the rapid de novo synthesis of an organelle called the phagophore. The study of ATG9A has proven challenging, since ATG9A is a transmembrane protein, and it is present in different membrane compartments. As such, understanding its trafficking is an important element for understanding autophagy. Here, detailed methods are presented that can be used to study ATG9A and, in particular, its localization using immunofluorescence techniques, which can be assessed and quantified. The pitfalls of transient overexpression are also addressed. The correct characterization of ATG9A function and the standardization of techniques to analyze its trafficking are crucial to further characterize the events governing autophagy initiation.
    DOI:  https://doi.org/10.3791/65349
  3. J Cell Biol. 2023 Sep 04. pii: e202208150. [Epub ahead of print]222(9):
    PTEN is both an activator and a substrate of chaperone-mediated autophagy.
    Katherine K Zhang, Calvin M Burns, Mary E Skinner, David B Lombard, Richard A Miller, S Joseph Endicott.
      PTEN is a crucial negative regulator of the INS/PI3K/AKT pathway and is one of the most commonly mutated tumor suppressors in cancer. Global overexpression (OE) of PTEN in mice shifts metabolism to favor oxidative phosphorylation over glycolysis, reduces fat mass, and extends the lifespan of both sexes. We demonstrate that PTEN regulates chaperone-mediated autophagy (CMA). Using cultured cells and mouse models, we show that PTEN OE enhances CMA, dependent upon PTEN's lipid phosphatase activity and AKT inactivation. Reciprocally, PTEN knockdown reduces CMA, which can be rescued by inhibiting class I PI3K or AKT. Both PTEN and CMA are negative regulators of glycolysis and lipid droplet formation. We show that suppression of glycolysis and lipid droplet formation downstream of PTEN OE depends on CMA activity. Finally, we show that PTEN protein levels are sensitive to CMA and that PTEN accumulates in lysosomes with elevated CMA. Collectively, these data suggest that CMA is both an effector and a regulator of PTEN.
    DOI:  https://doi.org/10.1083/jcb.202208150
  4. Autophagy. 2023 Jul 05. 1-3
    Autophagy is required for stem-cell-mediated endometrial programming and the establishment of pregnancy.
    Pooja Popli, Ramakrishna Kommagani.
      Autophagy plays an important role in the normal growth and morphogenesis of a variety of tissues. Its role in uterine maturation, however, is not fully characterized. Recently, we reported that BECN1 (Beclin1)-dependent autophagy, but not apoptosis, is crucial for stem cell-mediated endometrial programming and the establishment of pregnancy in mice. Upon genetic and pharmacological inhibition of BECN1-mediated autophagy, female mice displayed severe endometrial structural and functional defects leading to infertility. Specifically, conditional loss of Becn1 in the uterus induces apoptosis and results in the gradual loss of endometrial progenitor stem cells. Importantly, the restoration of BECN1-driven autophagy, but not apoptosis in Becn1 conditionally ablated mice promoted normal uterine adenogenesis and morphogenesis. Overall, our findings emphasize the critical role of intrinsic autophagy in endometrial homeostasis and on the molecular underpinnings of uterine differentiation.
    Keywords:  Autophagy; apoptosis; beclin-1; endocytosis; endometrium; morphogenesis; stem cells
    DOI:  https://doi.org/10.1080/15548627.2023.2231270
  5. Autophagy. 2023 Jul 05.
    Tracking the Transition from an ATG9A Vesicle to an Autophagosome.
    David Broadbent, Carlo Barnaba, Jens C Schmidt.
      Live-cell imaging is a well-established tool to gain insights into the dynamics of autophagosome formation. We have recently generated a panel of human cancer cell lines that express HaloTagged autophagy-related (ATG) proteins from their endogenous loci, providing a highly sensitive tool to study autophagosome biogenesis. Using these cell lines in combination with automated autophagic foci tracking we dissected the molecular mechanisms of phagophore initiation and expansion. Our work supports a model in which phosphorylations by the Unc-51-Like activating Kinases (ULK1/2) and phosphoinositide 3-kinases (PI3Ks) complexes trigger the recruitment of autophagy factors to mobile ATG9A-positive vesicles transforming them into phagophores. Only a small fraction, approximately 10-20%, of these phagophores expand into autophagosomes, suggesting that autophagosome biogenesis is inefficient. Finally, we demonstrate that ATG2A recruitment commits mobile phagophores to autophagosome formation via tethering to donor membranes and promoting the conjugation of human ATG8 homologues.
    Keywords:  ATG9A vesicles; Autophagy; Genome editing; Quantitative imaging; Single-particle tracking
    DOI:  https://doi.org/10.1080/15548627.2023.2232641
  6. Nat Commun. 2023 07 03. 14(1): 3911
    Loss of the batten disease protein CLN3 leads to mis-trafficking of M6PR and defective autophagic-lysosomal reformation.
    Alessia Calcagni', Leopoldo Staiano, Nicolina Zampelli, Nadia Minopoli, Niculin J Herz, Giuseppe Di Tullio, Tuong Huynh, Jlenia Monfregola, Alessandra Esposito, Carmine Cirillo, Aleksandar Bajic, Mahla Zahabiyon, Rachel Curnock, Elena Polishchuk, Luke Parkitny, Diego Luis Medina, Nunzia Pastore, Peter J Cullen, Giancarlo Parenti, Maria Antonietta De Matteis, Paolo Grumati, Andrea Ballabio.
      Batten disease, one of the most devastating types of neurodegenerative lysosomal storage disorders, is caused by mutations in CLN3. Here, we show that CLN3 is a vesicular trafficking hub connecting the Golgi and lysosome compartments. Proteomic analysis reveals that CLN3 interacts with several endo-lysosomal trafficking proteins, including the cation-independent mannose 6 phosphate receptor (CI-M6PR), which coordinates the targeting of lysosomal enzymes to lysosomes. CLN3 depletion results in mis-trafficking of CI-M6PR, mis-sorting of lysosomal enzymes, and defective autophagic lysosomal reformation. Conversely, CLN3 overexpression promotes the formation of multiple lysosomal tubules, which are autophagy and CI-M6PR-dependent, generating newly formed proto-lysosomes. Together, our findings reveal that CLN3 functions as a link between the M6P-dependent trafficking of lysosomal enzymes and lysosomal reformation pathway, explaining the global impairment of lysosomal function in Batten disease.
    DOI:  https://doi.org/10.1038/s41467-023-39643-7
  7. Autophagy. 2023 Jul 02. 1-2
    Metamorphosis by ATG13 and ATG101 in human autophagy initiation.
    Anoshi Patel, Alex C Faesen.
      ABBREVIATIONS: ATG, Autophagy-related, HORMA, protein domain named after HOP1-MAD2-REV7; RB1CC1, RB1 inducible coiled-coil 1; ULK, Unc-51-like kinase.
    Keywords:  ATG2A; ATG9A; lipid transfer; membrane contact site; metamorphosis; super-complex
    DOI:  https://doi.org/10.1080/15548627.2023.2230054
  8. Biomater Res. 2023 Jul 06. 27(1): 66
    Ratiometric and discriminative visualization of autophagic processes with a novel dual-responded lysosome-specific fluorescent probe.
    Fan Zheng, Yeshuo Ma, Jipeng Ding, Shuai Huang, Shengwang Zhang, Xueyan Huang, Bin Feng, Hongliang Zeng, Fei Chen, Wenbin Zeng.
      BACKGROUND: Autophagy is a critical self-eating pathway involved in numerous physiological and pathological processes. Lysosomal degradation of dysfunctional organelles and invading microorganisms is central to the autophagy mechanism and essential for combating disease-related conditions. Therefore, monitoring fluctuations in the lysosomal microenvironment is vital for tracking the dynamic process of autophagy. Although much effort has been put into designing probes for measuring lysosomal viscosity or pH separately, there is a need to validate the concurrent imaging of the two elements to enhance the understanding of the dynamic progression of autophagy.METHODS: Probe HFI was synthesized in three steps and was developed to visualize changes in viscosity and pH within lysosomes for real-time autophagy tracking. Then, the spectrometric determination was carried out. Next, the probe was applied to image autophagy in cells under nutrient-deprivation or external stress. Additionally, the performance of HFI to monitor autophagy was employed to evaluate acetaminophen-induced liver injury.
    RESULTS: We constructed a ratiometric dual-responsive probe, HFI, with a large Stokes shift over 200 nm, dual-wavelength emission, and small background interference. The ratiometric fluorescent signal (R = I 610/I 460) of HFI had an excellent correlation with both viscosity and pH. More importantly, high viscosity and low pH had a synergistic promotion effect on the emission intensity of HFI, which enabled it to specially lit lysosomes without disturbing the inherent microenvironment. We then successfully used HFI to monitor intracellular autophagy induced by starvation or drugs in real-time. Interestingly, HFI also enabled us to visualize the occurrence of autophagy in the liver tissue of a DILI model, as well as the reversible effect of hepatoprotective drugs on this event.
    CONCLUSIONS: In this study, we developed the first ratiometric dual-responsive fluorescent probe, HFI, for real-time revealing autophagic details. It could image lysosomes with minimal perturbation to their inherent pH, allowing us to track changes in lysosomal viscosity and pH in living cells. Ultimately, HFI has great potential to serve as a useful indicator for autophagic changes in viscosity and pH in complex biological samples and can also be used to assess drug safety.
    Keywords:  Acetaminophen-induced liver injury; Autophagy visualization; Dual-responsive; Fluorescent probe; Lysosome-specific; Ratiometric imaging; Viscosity; pH
    DOI:  https://doi.org/10.1186/s40824-023-00409-3
  9. Sci Rep. 2023 07 03. 13(1): 10752
    Autophagy dictates sensitivity to PRMT5 inhibitor in breast cancer.
    Charles Brobbey, Shasha Yin, Liu Liu, Lauren E Ball, Philip H Howe, Joe R Delaney, Wenjian Gan.
      Protein arginine methyltransferase 5 (PRMT5) catalyzes mono-methylation and symmetric di-methylation on arginine residues and has emerged as a potential antitumor target with inhibitors being tested in clinical trials. However, it remains unknown how the efficacy of PRMT5 inhibitors is regulated. Here we report that autophagy blockage enhances cellular sensitivity to PRMT5 inhibitor in triple negative breast cancer cells. Genetic ablation or pharmacological inhibition of PRMT5 triggers cytoprotective autophagy. Mechanistically, PRMT5 catalyzes monomethylation of ULK1 at R532 to suppress ULK1 activation, leading to attenuation of autophagy. As a result, ULK1 inhibition blocks PRMT5 deficiency-induced autophagy and sensitizes cells to PRMT5 inhibitor. Our study not only identifies autophagy as an inducible factor that dictates cellular sensitivity to PRMT5 inhibitor, but also unearths a critical molecular mechanism by which PRMT5 regulates autophagy through methylating ULK1, providing a rationale for the combination of PRMT5 and autophagy inhibitors in cancer therapy.
    DOI:  https://doi.org/10.1038/s41598-023-37706-9
  10. Sci Rep. 2023 07 04. 13(1): 10757
    ARL-17477 is a dual inhibitor of NOS1 and the autophagic-lysosomal system that prevents tumor growth in vitro and in vivo.
    Teiko Komori Nomura, Satoshi Endo, Takuma Kuwano, Kazuya Fukasawa, Shigeo Takashima, Tomoki Todo, Kyoji Furuta, Takuhei Yamamoto, Eiichi Hinoi, Hiroko Koyama, Ryo Honda.
      ARL-17477 is a selective neuronal nitric oxide synthase (NOS1) inhibitor that has been used in many preclinical studies since its initial discovery in the 1990s. In the present study, we demonstrate that ARL-17477 exhibits a NOS1-independent pharmacological activity that involves inhibition of the autophagy-lysosomal system and prevents cancer growth in vitro and in vivo. Initially, we screened a chemical compound library for potential anticancer agents, and identified ARL-17477 with micromolar anticancer activity against a wide spectrum of cancers, preferentially affecting cancer stem-like cells and KRAS-mutant cancer cells. Interestingly, ARL-17477 also affected NOS1-knockout cells, suggesting the existence of a NOS1-independent anticancer mechanism. Analysis of cell signals and death markers revealed that LC3B-II, p62, and GABARAP-II protein levels were significantly increased by ARL-17477. Furthermore, ARL-17477 had a chemical structure similar to that of chloroquine, suggesting the inhibition of autophagic flux at the level of lysosomal fusion as an underlying anticancer mechanism. Consistently, ARL-17477 induced lysosomal membrane permeabilization, impaired protein aggregate clearance, and activated transcription factor EB and lysosomal biogenesis. Furthermore, in vivo ARL-17477 inhibited the tumor growth of KRAS-mutant cancer. Thus, ARL-17477 is a dual inhibitor of NOS1 and the autophagy-lysosomal system that could potentially be used as a cancer therapeutic.
    DOI:  https://doi.org/10.1038/s41598-023-37797-4
  11. Elife. 2023 Jul 03. pii: e85748. [Epub ahead of print]12
    LGG-1/GABARAP lipidation is not required for autophagy and development in Caenorhabditis elegans.
    Romane Leboutet, Céline Largeau, Leonie Müller, Magali Prigent, Grégoire Quinet, Manuel S Rodriguez, Marie-Hélène Cuif, Thorsten Hoppe, Emmanuel Culetto, Christophe Lefebvre, Renaud Legouis.
      The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.
    Keywords:  C. elegans; S. cerevisiae; cell biology
    DOI:  https://doi.org/10.7554/eLife.85748
  12. FASEB J. 2023 08;37(8): e23067
    Aging inverts the effects of p75NTR -modulated mTOR manipulation on hippocampal neuron synaptic plasticity in male mice.
    Zijun Wang, Brian K Kennedy, Lik-Wei Wong, Sreedharan Sajikumar.
      Age-induced impairments in learning and memory are in part caused by changes to hippocampal synaptic plasticity during aging. The p75 neurotrophin receptor (p75NTR ) and mechanistic target of rapamycin (mTOR) are implicated in synaptic plasticity processes. mTOR is also well known for its involvement in aging. Recently, p75NTR and mTOR were shown to be mechanistically linked, and that p75NTR mediates age-induced impairment of hippocampal synaptic plasticity. Yet the consequences of p75NTR -mTOR interaction on hippocampal synaptic plasticity, and the role of mTOR in age-induced cognitive decline, are unclear. In this study, we utilize field electrophysiology to study the effects of mTOR inhibition and activation on long-term potentiation (LTP) in male young and aged wild-type (WT) mice. We then repeated the experiments on p75NTR knockout mice. The results demonstrate that mTOR inhibition blocks late-LTP in young WT mice but rescues age-related late-LTP impairment in aged WT mice. mTOR activation suppresses late-LTP in aged WT mice while lacking observable effects on young WT mice. These effects were not observed in p75NTR knockout mice. These results demonstrate that the role of mTOR in hippocampal synaptic plasticity is distinct between young and aged mice. Such effects could be explained by differing sensitivity of young and aged hippocampal neurons to changes in protein synthesis or autophagic activity levels. Additionally, elevated mTOR in the aged hippocampus could cause excessive mTOR signaling, which is worsened by activation and alleviated by inhibition. Further research on mTOR and p75NTR may prove useful for advancing understanding and, ultimately, mitigation of age-induced cognitive decline.
    Keywords:  TOR serine-threonine kinases; aging; long-term potentiation; neuronal plasticity; receptor, nerve growth factor
    DOI:  https://doi.org/10.1096/fj.202201640RRR
  13. Trends Cancer. 2023 Jul 01. pii: S2405-8033(23)00106-1. [Epub ahead of print]
    Emerging roles of the MiT/TFE factors in cancer.
    Roberto Zoncu, Rushika M Perera.
      The microphthalmia/transcription factor E (MiT/TFE) transcription factors (TFs; TFEB, TFE3, MITF, and TFEC) play a central role in cellular catabolism and quality control and are subject to extensive layers of regulation that influence their localization, stability, and activity. Recent studies have highlighted a broader role for these TFs in driving diverse stress-adaptation pathways, which manifest in a context- and tissue-dependent manner. Several human cancers upregulate the MiT/TFE factors to survive extreme fluctuations in nutrients, energy, and pharmacological challenges. Emerging data suggest that reduced activity of the MiT/TFE factors can also promote tumorigenesis. Here, we outline recent findings relating to novel mechanisms of regulation and activity of MiT/TFE proteins across some of the most aggressive human cancers.
    Keywords:  TFE3; TFEB; autophagy; cancer; lysosome; mTORC1; metabolism
    DOI:  https://doi.org/10.1016/j.trecan.2023.06.005
  14. bioRxiv. 2023 Jun 26. pii: 2023.06.16.545386. [Epub ahead of print]
    CRISPR screens in iPSC-derived neurons reveal principles of tau proteostasis.
    Avi J Samelson, Nabeela Ariqat, Justin McKetney, Gita Rohanitazangi, Celeste Parra Bravo, Darrin Goodness, Ruilin Tian, Parker Grosjean, Romany Abskharon, David Eisenberg, Nicholas M Kanaan, Li Gan, Carlo Condello, Danielle L Swaney, Martin Kampmann.
      A hallmark of age-associated neurodegenerative diseases is the aggregation of proteins. Aggregation of the protein tau defines tauopathies, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to the accumulation of tau aggregates, and subsequent dysfunction and death. The mechanisms underlying cell type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi-based modifier screen in iPSC-derived neurons. The screen uncovered expected pathways, including autophagy, but also unexpected pathways including UFMylation and GPI anchor synthesis, that control tau oligomer levels. We identify the E3 ubiquitin ligase CUL5 as a tau interactor and potent modifier of tau levels. In addition, disruption of mitochondrial function increases tau oligomer levels and promotes proteasomal misprocessing of tau. These results reveal new principles of tau proteostasis in human neurons and pinpoint potential therapeutic targets for tauopathies.
    DOI:  https://doi.org/10.1101/2023.06.16.545386
  15. Biomed Pharmacother. 2023 Jul 05. pii: S0753-3322(23)00912-5. [Epub ahead of print]165 115121
    The role of redox-mediated lysosomal dysfunction and therapeutic strategies.
    Xiaomeng He, Xuening Li, Wei Tian, Chenyu Li, Pengfei Li, Jingyuan Zhao, Shilei Yang, Shuai Li.
      Redox homeostasis refers to the dynamic equilibrium between oxidant and reducing agent in the body which plays a crucial role in maintaining normal physiological activities of the body. The imbalance of redox homeostasis can lead to the development of various human diseases. Lysosomes regulate the degradation of cellular proteins and play an important role in influencing cell function and fate, and lysosomal dysfunction is closely associated with the development of various diseases. In addition, several studies have shown that redox homeostasis plays a direct or indirect role in regulating lysosomes. Therefore, this paper systematically reviews the role and mechanisms of redox homeostasis in the regulation of lysosomal function. Therapeutic strategies based on the regulation of redox exerted to disrupt or restore lysosomal function are further discussed. Uncovering the role of redox in the regulation of lysosomes helps to point new directions for the treatment of many human diseases.
    Keywords:  Autophagy; LMP; Lysosomal; Redox homeostasis; Targeting ROS
    DOI:  https://doi.org/10.1016/j.biopha.2023.115121
  16. bioRxiv. 2023 May 30. pii: 2023.05.30.542866. [Epub ahead of print]
    HUWE1 Amplifies Ubiquitin Modifications to Broadly Stimulate Clearance of Proteins and Aggregates.
    Mengying Zhou, Rui Fang, Louis Colson, Katherine A Donovan, Moritz Hunkeler, Yuyu Song, Can Zhang, Siyi Chen, Dong-Hoon Lee, Gary A Bradshaw, Robyn Eisert, Yihong Ye, Marian Kalocsay, Alfred Goldberg, Eric S Fischer, Ying Lu.
      Selective breakdown of proteins and aggregates is crucial for maintaining normal cellular activities and is involved in the pathogenesis of diverse diseases. How the cell recognizes and tags these targets in different structural states for degradation by the proteasome and autophagy pathways has not been well understood. Here, we discovered that a HECT-family ubiquitin ligase HUWE1 is broadly required for the efficient degradation of soluble factors and for the clearance of protein aggregates/condensates. Underlying this capacity of HUWE1 is a novel Ubiquitin-Directed ubiquitin Ligase (UDL) activity which recognizes both soluble substrates and aggregates that carry a high density of ubiquitin chains and rapidly expand the ubiquitin modifications on these targets. Ubiquitin signal amplification by HUWE1 recruits the ubiquitin-dependent segregase p97/VCP to process these targets for subsequent degradation or clearance. HUWE1 controls the cytotoxicity of protein aggregates, mediates Targeted Protein Degradation and regulates cell-cycle transitions with its UDL activity.
    DOI:  https://doi.org/10.1101/2023.05.30.542866
  17. Pharmacol Ther. 2023 Jul 03. pii: S0163-7258(23)00149-3. [Epub ahead of print] 108485
    Autophagy inhibitors for cancer therapy: Small molecules and nanomedicines.
    Jian-Li Chen, Xuan Wu, Dan Yin, Xiao-Hui Jia, Xu Chen, Ze-Yun Gu, Xiao-Ming Zhu.
      Autophagy is a conserved process in which the cytosolic materials are degraded and eventually recycled for cellular metabolism to maintain homeostasis. The dichotomous role of autophagy in pathogenesis is complicated. Accumulating reports have suggested that cytoprotective autophagy is responsible for tumor growth and progression. Autophagy inhibitors, such as chloroquine (CQ) and hydroxychloroquine (HCQ), are promising for treating malignancies or overcoming drug resistance in chemotherapy. With the rapid development of nanotechnology, nanomaterials also show autophagy-inhibitory effects or are reported as the carriers delivering autophagy inhibitors. In this review, we summarize the small-molecule compounds and nanomaterials inhibiting autophagic flux as well as the mechanisms involved. The nanocarrier-based drug delivery systems for autophagy inhibitors and their distinct advantages are also described. The progress of autophagy inhibitors for clinical applications is finally introduced, and their future perspectives are discussed.
    Keywords:  Autophagy inhibitors; Cancer; Chemosensitization; Chemotherapy; Nanomaterials; Nanomedicines
    DOI:  https://doi.org/10.1016/j.pharmthera.2023.108485
  18. Curr Neuropharmacol. 2023 Jul 05.
    Curcumin as a Perspective Protection for Retinal Pigment Epithelium during Autophagy Inhibition in the Course of Retinal Degeneration.
    Roberto Pinelli, Michela Ferrucci, Francesca Biagioni, Violet Bumah, Elena Scaffidi, Stefano Puglisi-Allegra, Francesco Fornai.
      Defective autophagy in the retinal pigment epithelium (RPE) is involved in retinal degeneration, mostly in the course of age-related macular degeneration (AMD), which is an increasingly prevalent retinal disorder, eventually leading to blindness. However, most autophagy activators own serious adverse effects when administered systemically. Curcumin is a phytochemical, which induces autophagy with a wide dose-response curve, which brings minimal side effects. Recent studies indicating defective autophagy in AMD were analyzed. Accordingly, in this perspective, we discuss and provide some evidence about the protective effects of curcumin in preventing RPE cell damage induced by the autophagy inhibitor 3-methyladenine (3-MA). Cells from human RPE were administered the autophagy inhibitor 3-MA. The cell damage induced by 3-MA was assessed at light microscopy by hematoxylin eosin, Fluoro Jade-B, and ZO1 immunohistochemistry along with electron microscopy. The autophagy inhibitor 3-MA produces cell loss and cell degeneration of RPE cells. These effects are counteracted dose-dependently by curcumin. In line with the hypothesis that the autophagy machinery is key in sustaining the integrity of the RPE, here we provide evidence that the powerful autophagy inhibitor 3-MA produces dose-dependently cell loss and cell degeneration in cultured RPE cells, while inhibiting autophagy as shown by LC3-II/LC3-I ratio and gold-standard assessment of autophagy through LC3-positive autophagy vacuoles. These effects are prevented dose-dependently by curcumin, which activates autophagy. These data shed the perspective of validating the role of phytochemicals as safe autophagy activators to treat AMD.
    Keywords:  3-methyladenine; Phytochemicals; age-related macular degeneration.; autophagy; neurodegeneration; retinal degeneration; retinal pigment epithelium
    DOI:  https://doi.org/10.2174/1570159X21666230705103839
  19. EMBO J. 2023 Jul 06. e113105
    TECPR1 is activated by damage-induced sphingomyelin exposure to mediate noncanonical autophagy.
    Namrita Kaur, Laura Rodriguez de la Ballina, Håvard Styrkestad Haukaas, Maria Lyngaas Torgersen, Maja Radulovic, Michael J Munson, Alan Sabirsh, Harald Stenmark, Anne Simonsen, Sven R Carlsson, Alf Håkon Lystad.
      Cells use noncanonical autophagy, also called conjugation of ATG8 to single membranes (CASM), to label damaged intracellular compartments with ubiquitin-like ATG8 family proteins in order to signal danger caused by pathogens or toxic compounds. CASM relies on E3 complexes to sense membrane damage, but so far, only the mechanism to activate ATG16L1-containing E3 complexes, associated with proton gradient loss, has been described. Here, we show that TECPR1-containing E3 complexes are key mediators of CASM in cells treated with a variety of pharmacological drugs, including clinically relevant nanoparticles, transfection reagents, antihistamines, lysosomotropic compounds, and detergents. Interestingly, TECPR1 retains E3 activity when ATG16L1 CASM activity is obstructed by the Salmonella Typhimurium pathogenicity factor SopF. Mechanistically, TECPR1 is recruited by damage-induced sphingomyelin (SM) exposure using two DysF domains, resulting in its activation and ATG8 lipidation. In vitro assays using purified human TECPR1-ATG5-ATG12 complex show direct activation of its E3 activity by SM, whereas SM has no effect on ATG16L1-ATG5-ATG12. We conclude that TECPR1 is a key activator of CASM downstream of SM exposure.
    Keywords:  CASM; DysF; membrane damage; noncanonical autophagy; sphingomyelin
    DOI:  https://doi.org/10.15252/embj.2022113105
  20. Autophagy. 2023 Jul 06.
    The potent BECN2-ATG14 coiled-coil interaction is selectively critical for endolysosomal degradation of GPRASP1/GASP1-associated GPCRs.
    Xianxiu Qiu, Na Li, Qifan Yang, Shuai Wu, Xiaohua Li, Xuehua Pan, Soh Yamamoto, Xiaozhe Zhang, Jincheng Zeng, Jiahao Liao, Congcong He, Renxiao Wang, Yanxiang Zhao.
      BECN2 is a mammal-specific homolog of BECN1. Both proteins serve as a scaffolding molecule in the class III phosphatidylinositol 3-kinase complex (PtdIns3K) to promote macroautophagy/autophagy and endolysosomal trafficking. Our previous studies have shown that the BECN1 coiled-coil domain forms a metastable homodimer and readily self-dissociate to form the BECN1-ATG14 or BECN1-UVRAG coiled-coil complex as part of the scaffolding "arm" of the PtdIns3K complex. Here we report the crystal structure of the BECN2 coiled-coil domain, which forms a metastable homodimer similar to BECN1 but shows reduced stability and large deviation from the ideal coiled-coil geometry due to extra "imperfect" residues. We also report the crystal structure of BECN2-ATG14 coiled-coil complex, which is structurally similar to the BECN1-UVRAG coiled-coil complex we reported previously but functionally distinct. The potent BECN2-ATG14 interaction is selectively critical for endolysosomal degradation of the GPRASP1-associated DRD2/D2R but shows limited effect on EGFR, a cargo that depends on the BECN1-UVRAG interaction. We designed stapled peptides that selectively interacted with the BECN2 coiled-coil domain to enhance the BECN2-ATG14 or BECN1-UVRAG interaction. One such peptide specifically promoted BECN2-dependent processes including autophagy and endolysosomal degradation of DRD2/D2R but did not affect BECN1-dependent EGFR degradation. Our findings suggest that, despite high sequence identity to BECN1, BECN2 coiled-coil domain has unique structural features and the BECN2-ATG14 interaction selectively promotes autophagic or endolysosomal degradation of BECN2-specific cargos including GPRASP1-associated GPCRs.
    Keywords:  ATG14; BECN2; PtdIns3K; autophagy; coiled-coil
    DOI:  https://doi.org/10.1080/15548627.2023.2233872
  21. Life Sci. 2023 Jul 03. pii: S0024-3205(23)00539-8. [Epub ahead of print] 121904
    Canagliflozin shares common mTOR and MAPK signaling mechanisms with other lifespan extension treatments.
    Eric Jiang, Arjun Dinesh, Sohan Jadhav, Richard A Miller, Gonzalo G Garcia.
      Long-lived mouse models and treatments that extend lifespan, such as Rapamycin, acarbose and 17α- -estradiol, lead to reduction in mTORC1 activity, declines in cap-dependent translation and increases in cap-independent translation. In addition, these treatments reduce the MEK-ERK-MNK (ERK1-2) signaling cascade, leading to reduction in eIF4E phosphorylation, which also regulates mRNA translation. Here, we report that Canagliflozin, a drug that extends lifespan only in male mice reduces mTORC1 and ERK1-2 signaling in male mice only. The data suggest reduction in mTORC1 and ERK pathways are common mechanisms shared by both genetic and pharmacological models of slowed aging in mice. Our data also reveal a significant sexual dimorphism in the ERK1-2 signaling pathway which might help to explain why some drugs can extend lifespan in males but have no effects in female mice.
    Keywords:  Aging; ERK; MAPK; Signal transduction; mTOR
    DOI:  https://doi.org/10.1016/j.lfs.2023.121904
  22. bioRxiv. 2023 Jun 01. pii: 2023.06.01.541136. [Epub ahead of print]
    Expression of ALS-PFN1 impairs vesicular degradation in iPSC-derived microglia.
    Salome Funes, Del Hayden Gadd, Michelle Mosqueda, Jianjun Zhong, Jonathan Jung, Shankaracharya, Matthew Unger, Debra Cameron, Pepper Dawes, Pamela J Keagle, Justin A McDonough, Sivakumar Boopathy, Miguel Sena-Esteves, Cathleen Lutz, William C Skarnes, Elaine T Lim, Dorothy P Schafer, Francesca Massi, John E Landers, Daryl A Bosco.
      Microglia play a pivotal role in neurodegenerative disease pathogenesis, but the mechanisms underlying microglia dysfunction and toxicity remain to be fully elucidated. To investigate the effect of neurodegenerative disease-linked genes on the intrinsic properties of microglia, we studied microglia-like cells derived from human induced pluripotent stem cells (iPSCs), termed iMGs, harboring mutations in profilin-1 (PFN1) that are causative for amyotrophic lateral sclerosis (ALS). ALS-PFN1 iMGs exhibited lipid dysmetabolism and deficits in phagocytosis, a critical microglia function. Our cumulative data implicate an effect of ALS-linked PFN1 on the autophagy pathway, including enhanced binding of mutant PFN1 to the autophagy signaling molecule PI3P, as an underlying cause of defective phagocytosis in ALS-PFN1 iMGs. Indeed, phagocytic processing was restored in ALS-PFN1 iMGs with Rapamycin, an inducer of autophagic flux. These outcomes demonstrate the utility of iMGs for neurodegenerative disease research and highlight microglia vesicular degradation pathways as potential therapeutic targets for these disorders.
    DOI:  https://doi.org/10.1101/2023.06.01.541136
  23. Mol Biol Cell. 2023 Jul 05. mbcE22060200
    Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence.
    Sumaiya A Soha, Araniy Santhireswaran, Saaimatul Huq, Jayde Casimir-Powell, Nicala Jenkins, Gregory K Hodgson, Michael Sugiyama, Costin N Antonescu, Stefania Impellizzeri, Roberto J Botelho.
      The dynamics of living cells can be studied by live-cell fluorescence microscopy. However, this requires the use of excessive light energy to obtain good signal-to-noise ratio, which can then photobleach fluorochromes, and more worrisomely, lead to photo-toxicity. Upon light excitation, noble metal nanoparticles such as silver nanoparticles (AgNP) generate plasmons, which can then amplify excitation in direct proximity of the nanoparticle's surface and couple to the oscillating dipole of nearby radiating fluorophores, modifying their rate of emission and thus, enhancing their fluorescence. Here, we show that AgNP fed to cells to accumulate within lysosomes enhanced the fluorescence of lysosome-targeted Alexa488-conjugated dextran, BODIPY-cholesterol, and DQ-BSA. Moreover, AgNP increased the fluorescence of GFP fused to the cytosolic tail of LAMP1, showing that metal enhanced fluorescence can occur across the lysosomal membrane. The inclusion of AgNPs in lysosomes did not disturb lysosomal properties such as lysosomal pH, degradative capacity, autophagy and autophagic flux, and membrane integrity, though AgNP seemed to increase basal lysosome tubulation. Importantly, by using AgNP, we could track lysosome motility with reduced laser power without damaging and altering lysosome dynamics. Overall, AgNP-enhanced fluorescence may be a useful tool to study the dynamics of the endo-lysosomal pathway while minimizing photo-toxicity. [Media: see text] [Media: see text] [Media: see text] [Media: see text].
    DOI:  https://doi.org/10.1091/mbc.E22-06-0200
  24. Mol Brain. 2023 07 03. 16(1): 55
    mTORC1-mediated acquisition of reward-related representations by hippocampal somatostatin interneurons.
    François-Xavier Michon, Isabel Laplante, Anthony Bosson, Richard Robitaille, Jean-Claude Lacaille.
      Plasticity of principal cells and inhibitory interneurons underlies hippocampal memory. Bidirectional modulation of somatostatin cell mTORC1 activity, a crucial translational control mechanism in synaptic plasticity, causes parallel changes in hippocampal CA1 somatostatin interneuron (SOM-IN) long-term potentiation and hippocampus-dependent memory, indicating a key role in learning. However, SOM-IN activity changes and behavioral correlates during learning, and the role of mTORC1 in these processes, remain ill-defined. To address these questions, we used two-photon Ca2+ imaging from SOM-INs during a virtual reality goal-directed spatial memory task in head-fixed control mice (SOM-IRES-Cre mice) or in mice with conditional knockout of Rptor (SOM-Rptor-KO mice) to block mTORC1 activity in SOM-INs. We found that control mice learn the task, but SOM-Raptor-KO mice exhibit a deficit. Also, SOM-IN Ca2+ activity became increasingly related to reward during learning in control mice but not in SOM-Rptor-KO mice. Four types of SOM-IN activity patterns related to reward location were observed, "reward off sustained", "reward off transient", "reward on sustained" and "reward on transient", and these responses showed reorganization after reward relocation in control but not SOM-Rptor-KO mice. Thus, SOM-INs develop mTORC1-dependent reward- related activity during learning. This coding may bi-directionally interact with pyramidal cells and other structures to represent and consolidate reward location.
    Keywords:  Goal-directed task; Hippocampus; In vivo 2-photon Ca2+ imaging; Mechanistic target of rapamycin complex 1 (mTORC1); Reward-related activity; Somatostatin interneuron; Spatial memory task; Virtual reality
    DOI:  https://doi.org/10.1186/s13041-023-01042-w
  25. Mol Plant. 2023 Jul 04. pii: S1674-2052(23)00177-6. [Epub ahead of print]
    A Positive Feedback Regulation of SnRK1 Signaling by Autophagy in Plants.
    Chao Yang, Xibao Li, Lianming Yang, Shunquan Chen, Jun Liao, Kailin Li, Jun Zhou, Wenjin Shen, Xiaohong Zhuang, Mingyi Bai, Diane C Bassham, Caiji Gao.
      SnRK1, an evolutionarily conserved heterotrimeric kinase complex that acts as a key metabolic sensor in maintaining energy homeostasis in plants, is an important upstream activator of autophagy, which serves as a cellular degradation mechanism for the healthy growth of plants. However, whether and how the autophagy pathway is involved in regulating SnRK1 activity remains unknown. Here, we identified a clade of plant-specific and mitochondria-localized FCS-like zinc finger (FLZ) proteins as currently unknown ATG8-interacting partners that actively inhibit SnRK1 signaling by repressing the T-loop phosphorylation of the catalytic α subunits of SnRK1, thereby negatively modulating autophagy and plant tolerance to energy deprivation caused by long-term carbon starvation. Interestingly, these AtFLZs are transcriptionally repressed by low energy stress, and AtFLZ proteins undergo a selective autophagy-dependent pathway to be delivered to the vacuole for degradation, thereby constituting a positive feedback regulation to relieve their repression of SnRK1 signaling. Bioinformatic analyses show that the ATG8-FLZ-SnRK1 regulatory axis first appears in gymnosperms and seems to be highly conserved during the evolution of seed plants. Consistent with this, depletion of ATG8-interacting ZmFLZ14 confers enhanced tolerance, whereas overexpression of ZmFLZ14 leads to reduced tolerance to energy deprivation in maize. Thus, our study reveals a previously unknown mechanism by which autophagy contributes to the positive feedback regulation of SnRK1 signaling, thereby conferring better adaptation of plants to stressful environments.
    Keywords:  ATG8; FLZ; SnRK1; autophagy; carbon starvation
    DOI:  https://doi.org/10.1016/j.molp.2023.07.001
  26. Cell Rep. 2023 Jun 30. pii: S2211-1247(23)00719-2. [Epub ahead of print]42(7): 112708
    Constitutively active autophagy in macrophages dampens inflammation through metabolic and post-transcriptional regulation of cytokine production.
    Jinjin Xu, Lingjia Kong, Blayne A Oliver, Bihua Li, Elizabeth A Creasey, Gaelen Guzman, Monica Schenone, Kimberly L Carey, Steven A Carr, Daniel B Graham, Jacques Deguine, Ramnik J Xavier.
      Autophagy is an essential cellular process that is deeply integrated with innate immune signaling; however, studies that examine the impact of autophagic modulation in the context of inflammatory conditions are lacking. Here, using mice with a constitutively active variant of the autophagy gene Beclin1, we show that increased autophagy dampens cytokine production during a model of macrophage activation syndrome and in adherent-invasive Escherichia coli (AIEC) infection. Moreover, loss of functional autophagy through conditional deletion of Beclin1 in myeloid cells significantly enhances innate immunity in these contexts. We further analyzed primary macrophages from these animals with a combination of transcriptomics and proteomics to identify mechanistic targets downstream of autophagy. Our study reveals glutamine/glutathione metabolism and the RNF128/TBK1 axis as independent regulators of inflammation. Altogether, our work highlights increased autophagic flux as a potential approach to reduce inflammation and defines independent mechanistic cascades involved in this control.
    Keywords:  CP: Immunology; RNF128; TBK1 signaling; autophagy; glutamine metabolism; glutathione metabolism; infection; inflammation; macrophages; proteogenomics
    DOI:  https://doi.org/10.1016/j.celrep.2023.112708
  27. Genes Dis. 2023 Jul;10(4): 1582-1595
    Progranulin regulation of autophagy contributes to its chondroprotective effect in osteoarthritis.
    Yiming Pan, Yuyou Yang, Mengtian Fan, Cheng Chen, Rong Jiang, Li Liang, Menglin Xian, Biao Kuang, Nana Geng, Naibo Feng, Lin Deng, Wei Zheng, Fengmei Zhang, Xiaoli Li, Fengjin Guo.
      Progranulin (PGRN) is a multifunctional growth factor involved in many physiological processes and disease states. The apparent protective role of PGRN and the importance of chondrocyte autophagic function in the progression of osteoarthritis (OA) led us to investigate the role of PGRN in the regulation of chondrocyte autophagy. PGRN knockout chondrocytes exhibited a deficient autophagic response with limited induction following rapamycin, serum starvation, and IL-1β-induced autophagy. PGRN-mediated anabolism and suppression of IL-1β-induced catabolism were largely abrogated in the presence of the BafA1 autophagy inhibitor. Mechanistically, during the process of OA, PGRN and the ATG5-ATG12 conjugate form a protein complex; PGRN regulates autophagy in chondrocytes and OA through, at least partially, the interactions between PGRN and the ATG5-ATG12 conjugate. Furthermore, the ATG5-ATG12 conjugate is critical for cell proliferation and apoptosis. Knockdown or knockout of ATG5 reduces the expression of ATG5-ATG12 conjugate and inhibits the chondroprotective effect of PGRN on anabolism and catabolism. Overexpression of PGRN partially reversed this effect. In brief, the PGRN-mediated regulation of chondrocyte autophagy plays a key role in the chondroprotective role of PGRN in OA. Such studies provide new insights into the pathogenesis of OA and PGRN-associated autophagy in chondrocyte homeostasis.
    Keywords:  ATG12; ATG5; Anabolism; Autophagy; Catabolism; Osteoarthritis; PGRN
    DOI:  https://doi.org/10.1016/j.gendis.2022.05.031
  28. J Biochem Mol Toxicol. 2023 Jul 06. e23429
    Autophagy regulation by inorganic, organic, and organic/inorganic hybrid nanoparticles: Organelle damage, regulation factors, and potential pathways.
    Dong Qiao, Ting Zhang, Meng Tang.
      The rapid development of nanotechnology requires a more thorough understanding of the potential health effects caused by nanoparticles (NPs). As a programmed cell death, autophagy is one of the biological effects induced by NPs, which maintain intracellular homeostasis by degrading damaged organelles and removing aggregates of defective proteins through lysosomes. Currently, autophagy has been shown to be associated with the development of several diseases. A significant number of research have demonstrated that most NPs can regulate autophagy, and their regulation of autophagy is divided into induction and blockade. Studying the autophagy regulation by NPs will facilitate a more comprehensive understanding of the toxicity of NPs. In this review, we will illustrate the effects of different types of NPs on autophagy, including inorganic NPs, organic NPs, and organic/inorganic hybrid NPs. The potential mechanisms by which NPs regulate autophagy are highlighted, including organelle damage, oxidative stress, inducible factors, and multiple signaling pathways. In addition, we list the factors influencing NPs-regulated autophagy. This review may provide basic information for the safety assessment of NPs.
    Keywords:  autophagy; nanoparticles; organelle damage; oxidative stress; toxicity
    DOI:  https://doi.org/10.1002/jbt.23429
  29. Front Vet Sci. 2023 ;10 1174681
    Autophagy up-regulation upon FeHV-1 infection on permissive cells.
    Gianmarco Ferrara, Mariafrancesca Sgadari, Consiglia Longobardi, Giuseppe Iovane, Ugo Pagnini, Serena Montagnaro.
      FeHV-1 is a member of the Herpesviridae family that is distributed worldwide and causes feline viral rhinotracheitis (FVR). Since its relationship with the autophagic process has not yet been elucidated, the aim of this work was to evaluate the autophagy mediated by FeHV-1 and to determine its proviral or antiviral role. Our data showed that autophagy is induced by FeHV-1 in a viral dose and time-dependent manner. Phenotypic changes in LC3/p62 axis (increase of LC3-II and degradation of p62) were detected from 12 h post infection using western blot and immuno-fluorescence assays. In a second step, by using late autophagy inhibitors and inducers, the possible proviral role of autophagy during FeHV-1 infection was investigating by assessing the effects of each chemical in terms of viral yield, cytotoxic effects, and expression of viral glycoproteins. Our findings suggest that late-stage autophagy inhibitors (bafilomycin and chloroquine) have a negative impact on viral replication. Interestingly, we observed an accumulation of gB, a viral protein, when cells were pretreated with bafilomycin, whereas the opposite effect was observed when an autophagy inducer was used. The importance of autophagy during FeHV-1 infection was further supported by the results obtained with ATG5 siRNA. In summary, this study demonstrates FeHV-1-mediated autophagy induction, its proviral role, and the negative impact of late autophagy inhibitors on viral replication.
    Keywords:  ATG5; LC3-II; autophagy; bafilomycin A1; chloroquine; feline herpesvirus; late-stage autophagy inhibitors; rapamycin
    DOI:  https://doi.org/10.3389/fvets.2023.1174681
  30. Trends Pharmacol Sci. 2023 Jul 04. pii: S0165-6147(23)00133-5. [Epub ahead of print]
    Mitochondrial quality control: prime targets for antiviral therapy?
    Yijin Wang, Peicong Ji, Qiuwei Pan.
      Mitochondrial quality control (MQC) plays a crucial role in maintaining mitochondrial health. Mitochondrial dynamics and mitophagy are two intricate processes of the MQC machinery acting at the organelle level to orchestrate mitochondrial homeostasis. Here, we discuss how viruses perturb these two processes to facilitate their infections and emphasize the rationale and challenges of therapeutically targeting MQC for treating viral diseases.
    DOI:  https://doi.org/10.1016/j.tips.2023.06.006
  31. Toxicol Res. 2023 Jul;39(3): 355-372
    Recent insights into autophagy and metals/nanoparticles exposure.
    Qiong Li, Yajing Feng, Ruike Wang, Rundong Liu, Yue Ba, Hui Huang.
      Some anthropogenic pollutants, such as heavy metals and nanoparticles (NPs), are widely distributed and a major threat to environmental safety and public health. In particular, lead (Pb), cadmium (Cd), chromium (Cr), arsenic (As), and mercury (Hg) have systemic toxicity even at extremely low concentrations, so they are listed as priority metals in relation to their significant public health burden. Aluminum (Al) is also toxic to multiple organs and is linked to Alzheimer's disease. As the utilization of many metal nanoparticles (MNPs) gradually gain traction in industrial and medical applications, they are increasingly being investigated to address potential toxicity by impairing certain biological barriers. The dominant toxic mechanism of these metals and MNPs is the induction of oxidative stress, which subsequently triggers lipid peroxidation, protein modification, and DNA damage. Notably, a growing body of research has revealed the linkage between dysregulated autophagy and some diseases, including neurodegenerative diseases and cancers. Among them, some metals or metal mixtures can act as environmental stimuli and disturb basal autophagic activity, which has an underlying adverse health effect. Some studies also revealed that specific autophagy inhibitors or activators could modify the abnormal autophagic flux attributed to continuous exposure to metals. In this review, we have gathered recent data about the contribution of the autophagy/mitophagy mediated toxic effects and focused on the involvement of some key regulatory factors of autophagic signaling during exposure to selected metals, metal mixtures, as well as MNPs in the real world. Besides this, we summarized the potential significance of interactions between autophagy and excessive reactive oxygen species (ROS)-mediated oxidative damage in the regulation of cell survival response to metals/NPs. A critical view is given on the application of autophagy activators/inhibitors to modulate the systematic toxicity of various metals/MNPs.
    Keywords:  Autophagy; Metals; Nanoparticles; Toxicity
    DOI:  https://doi.org/10.1007/s43188-023-00184-2
  32. Front Mol Neurosci. 2023 ;16 983108
    Leaderless secretory proteins of the neurodegenerative diseases via TNTs: a structure-function perspective.
    Sreedevi Padmanabhan, Ravi Manjithaya.
      Neurodegenerative disease-causing proteins such as alpha-synuclein, tau, and huntingtin are known to traverse across cells via exosomes, extracellular vesicles and tunneling nanotubes (TNTs). There seems to be good synergy between exosomes and TNTs in intercellular communication. Interestingly, many of the known major neurodegenerative proteins/proteolytic products are leaderless and are also reported to be secreted out of the cell via unconventional protein secretion. Such classes contain intrinsically disordered proteins and regions (IDRs) within them. The dynamic behavior of these proteins is due to their heterogenic conformations that is exhibited owing to various factors that occur inside the cells. The amino acid sequence along with the chemical modifications has implications on the functional roles of IDRs inside the cells. Proteins that form aggregates resulting in neurodegeneration become resistant to degradation by the processes of autophagy and proteasome system thus leading to Tunneling nanotubes, TNT formation. The proteins that traverse across TNTs may or may not be dependent on the autophagy machinery. It is not yet clear whether the conformation of the protein plays a crucial role in its transport from one cell to another without getting degraded. Although there is some experimental data, there are many grey areas which need to be revisited. This review provides a different perspective on the structural and functional aspects of these leaderless proteins that get secreted outside the cell. In this review, attention has been focused on the characteristic features that lead to aggregation of leaderless secretory proteins (from structural-functional aspect) with special emphasis on TNTs.
    Keywords:  autophagy; intrinsic disordered regions; leaderless proteins; neurodegenerative disease; tunneling nanotubes; unconventional protein secretion
    DOI:  https://doi.org/10.3389/fnmol.2023.983108
  33. Acta Pharmacol Sin. 2023 Jul 05.
    mTORC2 acts as a gatekeeper for mTORC1 deficiency-mediated impairments in ILC3 development.
    Ya-Fei Deng, Shu-Ting Wu, Hong-Yan Peng, Lei Tian, Ya-Na Li, Yao Yang, Meng Meng, Lan-Lan Huang, Pei-Wen Xiong, Song-Yang Li, Qing-Lan Yang, Li-Li Wang, Xiao-Yao Li, Li-Ping Li, Xiu-Lan Lu, Xiao-Hui Li, Yan-Ling Wei, Zheng-Hui Xiao, Jian-Hua Yu, You-Cai Deng.
      Group 3 innate lymphoid cells (ILC3s) are mediators of intestinal immunity and barrier function. Recent studies have investigated the role of the mammalian target of rapamycin complex (mTOR) in ILC3s, whereas the mTORC1-related mechanisms and crosstalk between mTORC1 and mTORC2 involved in regulating ILC3 homeostasis remain unknown. In this study, we found that mTORC1 but not mTORC2 was critical in ILC3 development, IL-22 production, and ILC3-mediated intestinal homeostasis. Single-cell RNA sequencing revealed that mTORC1 deficiency led to disruption of ILC3 heterogeneity, showing an increase in differentiation into ILC1-like phenotypes. Mechanistically, mTORC1 deficiency decreased the expression of NFIL3, which is a critical transcription factor responsible for ILC3 development. The activities of both mTORC1 and mTORC2 were increased in wild-type ILC3s after activation by IL-23, whereas inhibition of mTORC1 by Raptor deletion or rapamycin treatment resulted in increased mTORC2 activity. Previous studies have demonstrated that S6K, the main downstream target of mTORC1, can directly phosphorylate Rictor to dampen mTORC2 activity. Our data found that inhibition of mTORC1 activity by rapamycin reduced Rictor phosphorylation in ILC3s. Reversing the increased mTORC2 activity via heterozygous or homozygous knockout of Rictor in Raptor-deleted ILC3s resulted in severe ILC3 loss and complete susceptibility to intestinal infection in mice with mTORC1 deficiency (100% mortality). Thus, mTORC1 acts as a rheostat of ILC3 heterogeneity, and mTORC2 protects ILC3s from severe loss of cells and immune activity against intestinal infection when mTORC1 activity is diminished.
    Keywords:  Group 3 innate lymphoid cells; NFIL3; mTORC1; mTORC2; single-cell RNA sequencing
    DOI:  https://doi.org/10.1038/s41401-023-01120-8
  34. Autophagy. 2023 Jul 06. 1-2
    TRAPPIII requires Drs2 binding to transport Atg9 vesicles at cold temperatures.
    Marta Puig-Tintó, Irene Pazos, Laura Betancur, Altair C Hernández, Oriol Gallego.
      ABBREVIATIONS: Autophagy-related 9 (Atg9); cytoplasm-to-vacuole targeting (Cvt); Golgi-associated retrograde protein (GARP); multisubunit tethering complexes (MTCs); phagophore assembly site (PAS); phosphatidylserine (PS); Protein interactions from Imaging Complexes after Translocation (PICT); transport protein particle III (TRAPPIII); type IV P-type ATPases (P4-ATPases).
    Keywords:  Atg9; Drs2; P4-ATPases; TRAPPIII; cytoplasm-to-vacuole targeting; multisubunit tethering complex
    DOI:  https://doi.org/10.1080/15548627.2023.2233365
  35. EMBO J. 2023 Jul 06. e113012
    TECPR1 conjugates LC3 to damaged endomembranes upon detection of sphingomyelin exposure.
    Keith B Boyle, Cara J Ellison, Paul R Elliott, Martina Schuschnig, Krista Grimes, Marc S Dionne, Chihiro Sasakawa, Sean Munro, Sascha Martens, Felix Randow.
      Invasive bacteria enter the cytosol of host cells through initial uptake into bacteria-containing vacuoles (BCVs) and subsequent rupture of the BCV membrane, thereby exposing to the cytosol intraluminal, otherwise shielded danger signals such as glycans and sphingomyelin. The detection of glycans by galectin-8 triggers anti-bacterial autophagy, but how cells sense and respond to cytosolically exposed sphingomyelin remains unknown. Here, we identify TECPR1 (tectonin beta-propeller repeat containing 1) as a receptor for cytosolically exposed sphingomyelin, which recruits ATG5 into an E3 ligase complex that mediates lipid conjugation of LC3 independently of ATG16L1. TECPR1 binds sphingomyelin through its N-terminal DysF domain (N'DysF), a feature not shared by other mammalian DysF domains. Solving the crystal structure of N'DysF, we identified key residues required for the interaction, including a solvent-exposed tryptophan (W154) essential for binding to sphingomyelin-positive membranes and the conjugation of LC3 to lipids. Specificity of the ATG5/ATG12-E3 ligase responsible for the conjugation of LC3 is therefore conferred by interchangeable receptor subunits, that is, the canonical ATG16L1 and the sphingomyelin-specific TECPR1, in an arrangement reminiscent of certain multi-subunit ubiquitin E3 ligases.
    Keywords:  ATG5-ATG12 E3 ligase; DysF; autophagy; membrane damage; sphingomyelin
    DOI:  https://doi.org/10.15252/embj.2022113012
  36. Nat Cell Biol. 2023 Jul 06.
    Class 3 PI3K coactivates the circadian clock to promote rhythmic de novo purine synthesis.
    Chantal Alkhoury, Nathaniel F Henneman, Volodymyr Petrenko, Yui Shibayama, Arianna Segaloni, Alexis Gadault, Ivan Nemazanyy, Edouard Le Guillou, Amare Desalegn Wolide, Konstantina Antoniadou, Xin Tong, Teruya Tamaru, Takeaki Ozawa, Muriel Girard, Karim Hnia, Dominik Lutter, Charna Dibner, Ganna Panasyuk.
      Metabolic demands fluctuate rhythmically and rely on coordination between the circadian clock and nutrient-sensing signalling pathways, yet mechanisms of their interaction remain not fully understood. Surprisingly, we find that class 3 phosphatidylinositol-3-kinase (PI3K), known best for its essential role as a lipid kinase in endocytosis and lysosomal degradation by autophagy, has an overlooked nuclear function in gene transcription as a coactivator of the heterodimeric transcription factor and circadian driver Bmal1-Clock. Canonical pro-catabolic functions of class 3 PI3K in trafficking rely on the indispensable complex between the lipid kinase Vps34 and regulatory subunit Vps15. We demonstrate that although both subunits of class 3 PI3K interact with RNA polymerase II and co-localize with active transcription sites, exclusive loss of Vps15 in cells blunts the transcriptional activity of Bmal1-Clock. Thus, we establish non-redundancy between nuclear Vps34 and Vps15, reflected by the persistent nuclear pool of Vps15 in Vps34-depleted cells and the ability of Vps15 to coactivate Bmal1-Clock independently of its complex with Vps34. In physiology we find that Vps15 is required for metabolic rhythmicity in liver and, unexpectedly, it promotes pro-anabolic de novo purine nucleotide synthesis. We show that Vps15 activates the transcription of Ppat, a key enzyme for the production of inosine monophosphate, a central metabolic intermediate for purine synthesis. Finally, we demonstrate that in fasting, which represses clock transcriptional activity, Vps15 levels are decreased on the promoters of Bmal1 targets, Nr1d1 and Ppat. Our findings open avenues for establishing the complexity for nuclear class 3 PI3K signalling for temporal regulation of energy homeostasis.
    DOI:  https://doi.org/10.1038/s41556-023-01171-3
  37. bioRxiv. 2023 May 31. pii: 2023.05.30.542934. [Epub ahead of print]
    A new vulnerability to BET inhibition due to enhanced autophagy in BRCA2 deficient pancreatic cancer.
    EunJung Lee, Suyakarn Archasappawat, Keely Ji, Jocelyn Pena, Virneliz Fernandez-Vega, Ritika Gangaraju, Nitin Sai Beesabathuni, Martin Jean Kim, Qi Tian, Priya Shah, Louis Scampavia, Timothy Spicer, Chang-Il Hwang.
      Pancreatic cancer is one of the deadliest diseases in human malignancies. Among total pancreatic cancer patients, ∼10% of patients are categorized as familial pancreatic cancer (FPC) patients, carrying germline mutations of the genes involved in DNA repair pathways ( e.g., BRCA2 ). Personalized medicine approaches tailored toward patients' mutations would improve patients' outcome. To identify novel vulnerabilities of BRCA2 -deficient pancreatic cancer, we generated isogenic Brca2 -deficient murine pancreatic cancer cell lines and performed high-throughput drug screens. High-throughput drug screening revealed that Brca2 -deficient cells are sensitive to Bromodomain and Extraterminal Motif (BET) inhibitors, suggesting that BET inhibition might be a potential therapeutic approach. We found that BRCA2 deficiency increased autophagic flux, which was further enhanced by BET inhibition in Brca2 -deficient pancreatic cancer cells, resulting in autophagy-dependent cell death. Our data suggests that BET inhibition can be a novel therapeutic strategy for BRCA2 -deficient pancreatic cancer.
    DOI:  https://doi.org/10.1101/2023.05.30.542934
  38. Analyst. 2023 Jul 06.
    In vivo measurement of autophagic flux by fluorescence correlation spectroscopy.
    Haohan Song, Chaoqing Dong, Jicun Ren.
      Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays a very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence imaging techniques have been used to assess autophagic flux, we remain less able to in vivo monitor autophagic flux in a highly sensitive, robust, and well-quantified manner. Here, we reported a new method for real-time and quantitatively monitoring autophagosomes and assessing autophagic flux in living cells based on fluorescence correlation spectroscopy (FCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with an enhanced green fluorescent protein (EGFP-LC3B) was used as a biomarker to label autophagosomes in living cells, and FCS was used to monitor EGFP-LC3B labeled autophagosomes by using the characteristic diffusion time (τD) value and brightness per particle (BPP) value. By analyzing the distribution frequency of the τD values in living cells stably expressing EGFP-LC3B, mutant EGFP-LC3B (EGFP-LC3BΔG) and enhanced green fluorescent protein (EGFP), we found that the τD value greater than 10 ms was attributed to the signal of EGFP-LC3B labeled autophagosomes. So, we proposed a parameter PAP as an indicator to assess the basal autophagic activity and induced autophagic flux. This new method was able to evaluate autophagy inducers, early-stage autophagy inhibitors, and late-stage autophagy inhibitors. Compared with current methods, our method shows high spatiotemporal resolution and very high sensitivity for autophagosomes in low EGFP-LC3B expressing cells and will become an attractive and alternative method for biological and medical studies, some drug screening, and disease treatment.
    DOI:  https://doi.org/10.1039/d3an00651d
  39. J Neuropathol Exp Neurol. 2023 Jul 06. pii: nlad051. [Epub ahead of print]
    Survival in sporadic ALS is associated with lower p62 burden in the spinal cord.
    Monica Pinkerton, Guinevere Lourenco, Maria Torres Pacheco, Glenda M Halliday, Matthew C Kiernan, Rachel H Tan.
      The autophagy marker p62 appears as a consistent component of pathological aggregates in amyotrophic lateral sclerosis (ALS) and its modulation to facilitate protein degradation has been proposed as a potential therapeutic target. Importantly, recent studies have implicated diffuse phosphorylated TDP-43 inclusions that are immuno-negative for p62 in more rapid disease, highlighting the need for better understanding of p62 involvement in ALS pathogenesis. The present study set out to assess p62 pathology in the motor neurons of 31 patients with sporadic ALS that had either a short (<2 years) or longer (4-7 years) disease duration to determine its association with pTDP-43 pathology, motor neuron loss, and survival in sporadic disease. Our results identified significantly more cytoplasmic p62 aggregates in the spinal cord of patients with a shorter survival. Disease duration demonstrated a negative association with p62 burden and density of remaining motor neurons in the spinal cord, suggesting that survival in sporadic ALS is associated with the successful clearance of lower motor neurons with p62 aggregates. These findings implicate the autophagy pathway in ALS survival and provide support for further study of p62 as a potential prognostic biomarker in ALS.
    Keywords:  Amyotrophic lateral sclerosis; Motor neurons; p62 pathology; pTDP-43
    DOI:  https://doi.org/10.1093/jnen/nlad051
  40. Mol Ther. 2023 Jul 01. pii: S1525-0016(23)00375-1. [Epub ahead of print]
    Increased expression or activation of TRPML1 reduces hepatic storage of toxic Z alpha-1 antitrypsin.
    Nunzia Pastore, Francesco Annunziata, Rita Colonna, Veronica Maffia, Teresa Giuliano, Bruno Maria Custode, Bernadette Lombardi, Elena Polishchuk, Vincenzo Cacace, Lucia De Stefano, Edoardo Nusco, Nicolina Cristina Sorrentino, Pasquale Piccolo, Nicola Brunetti-Pierri.
      Mutant Z-alpha-1 antitrypsin (ATZ) accumulates in globules in the liver and is the prototype of proteotoxic hepatic disease. Therapeutic strategies aiming at clearance of polymeric ATZ are needed. Transient receptor potential mucolipin 1 (TRPML1) is a lysosomal Ca2+ channel that maintains lysosomal homeostasis. In this study, we show that by increasing lysosomal exocytosis, TRPML1 gene transfer or small molecule-mediated activation of TRPML1 reduce hepatic ATZ globules and fibrosis in PiZ transgenic mice that express the human ATZ. ATZ globule clearance induced by TRPML1 occurred without increase in autophagy or nuclear translocation of TFEB. Our results show that targeting TRPML1 and lysosomal exocytosis is a novel approach for treatment of the liver disease due to ATZ and potentially other diseases due to proteotoxic liver storage.
    DOI:  https://doi.org/10.1016/j.ymthe.2023.06.018
  41. Autophagy. 2023 Jul 05.
    Foot-and-mouth disease virus structural protein VP3 interacts with HDAC8 and promotes its autophagic degradation to facilitate viral replication.
    Huijun Zhang, Xiangwei Wang, Min Qu, Zhiyong Li, Xiangping Yin, Lijie Tang, Xiangtao Liu, Yuefeng Sun.
      Macroautophagy/autophagy has been utilized by many viruses, including foot-and-mouth disease virus (FMDV), to facilitate replication, while the underlying mechanism of the interplay between autophagy and innate immune responses is still elusive. This study showed that HDAC8 (histone deacetylase 8) inhibits FMDV replication by regulating innate immune signal transduction and antiviral response. To counteract the HDAC8 effect, FMDV utilizes autophagy to promote HDAC8 degradation. Further data showed that FMDV structural protein VP3 promotes autophagy during virus infection and interacts with and degrades HDAC8 in an AKT-MTOR-ATG5-dependent autophagy pathway. Our data demonstrated that FMDV evolved a strategy to counteract host antiviral activity by autophagic degradation of a protein that regulates innate immune response during virus infection.
    Keywords:  Autophagy; FMDV; HDAC8, innate immune response; virus replication
    DOI:  https://doi.org/10.1080/15548627.2023.2233847
  42. Invest Ophthalmol Vis Sci. 2023 07 03. 64(10): 1
    TBK1 Knockdown Alleviates Axonal Transport Deficits in Retinal Ganglion Cells Via mTORC1 Activation in a Retinal Damage Mouse Model.
    Meng Ye, Yuanyuan Hu, Bowen Zhao, Qianxue Mou, Yueqi Ni, Jing Luo, Lu Li, Hong Zhang, Yin Zhao.
      Purpose: Glaucoma is the leading cause of irreversible blindness worldwide and is characterized by progressive retinal ganglion cell (RGC) death and optic nerve degeneration. Axonal transport deficits are the earliest crucial pathophysiological changes in glaucoma. Genetic variation in the TANK-binding kinase 1 gene (TBK1) plays a role in the pathogenesis of glaucoma. This study was designed to investigate intrinsic factors underlying RGCs' damage and to explore the molecular mechanism of TBK1 involvement in glaucomatous pathogenesis.Methods: We established a mouse model of acute ocular hypertension and used TBK1 conditional knockdown mice to investigate the role of TBK1 in glaucoma. CTB-Alexa 555 was utilized to evaluate axonal transport in mice. To observe the efficiency of gene knockdown, we performed immunofluorescence staining. Immunoblotting and immunoprecipitation assays were performed to examine protein‒protein colocalization. RT‒qPCR was performed to measure the mRNA levels of Tbk1.
    Results: In this study, we found that conditional TBK1 knockdown in RGCs resulted in increased axonal transport and protection against axonal degeneration. Through mechanistic studies, we found that TBK1 inhibited mTORC1 pathway activation by phosphorylating RAPTOR at Ser1189. Phosphorylation of RAPTOR at Ser1189 abrogated the interaction of RAPTOR with the deubiquitinase USP9X, leading to an increase in RAPTOR ubiquitination and a subsequent decline in protein stabilization.
    Conclusions: Our study identified a novel mechanism involving an interaction between the glaucoma risk gene TBK1 and the pivotal mTORC1 pathway, which may provide new therapeutic targets in glaucoma and other neurodegenerative diseases.
    DOI:  https://doi.org/10.1167/iovs.64.10.1
  43. bioRxiv. 2023 May 30. pii: 2023.05.26.542507. [Epub ahead of print]
    Glaucoma-associated Optineurin mutations increase transmitophagy in a vertebrate optic nerve.
    Yaeram Jeong, Chung-Ha O Davis, Aaron M Muscarella, Viraj Deshpande, Keun-Young Kim, Mark H Ellisman, Nicholas Marsh-Armstrong.
      We previously described a process referred to as transmitophagy where mitochondria shed by retinal ganglion cell (RGC) axons are transferred to and degraded by surrounding astrocytes in the optic nerve head of mice. Since the mitophagy receptor Optineurin (OPTN) is one of few large- effect glaucoma genes and axonal damage occurs at the optic nerve head in glaucoma, here we explored whether OPTN mutations perturb transmitophagy. Live-imaging of Xenopus laevis optic nerves revealed that diverse human mutant but not wildtype OPTN increase stationary mitochondria and mitophagy machinery and their colocalization within, and in the case of the glaucoma-associated OPTN mutations also outside of, RGC axons. These extra-axonal mitochondria are degraded by astrocytes. Our studies support the view that in RGC axons under baseline conditions there are low levels of mitophagy, but that glaucoma-associated perturbations in OPTN result in increased axonal mitophagy involving the shedding and astrocytic degradation of the mitochondria.
    DOI:  https://doi.org/10.1101/2023.05.26.542507
  44. Cardiovasc Diagn Ther. 2023 Jun 30. 13(3): 509-522
    FUNDC1: a key mediator of adenosine A2BR activation-induced inhibition of cardiac mitophagy under ischemia/reperfusion conditions.
    Yanyi Tian, Haize Ge, Xiyun Bian, Yao Wang, Yuezhao Lai, Yunqi Wang, Yanwen Bai, Xi Zhang, Jingman Xu, Wei Tian.
      Background: Mitophagy is an essential factor in mitochondrial quality control and myocardial ischaemia/reperfusion (I/R) injury protection. Because adenosine A2B receptor (A2BR) activation exerts a major role in reducing myocardial I/R injury, the effects of adenosine A2BR activation on cardiac mitophagy under reperfusion conditions were investigated.Methods: 110 adult Wistar rats (7-10 w), weighing 250-350 grams, were cultured in specific-pathogen-free (SPF) conditions before experiments. All hearts were removed and reperfused by Langendorff device. Six hearts with coronary flow (CF) values >28 or <10 mL/min were excluded. Others were arbitrarily divided into the following groups: sham operation group, I/R group, BAY60-6583 (BAY) (1-1,000 nM) + I/R group, PP2 + BAY + I/R group. After ischemia in rats, reperfusion was performed. H9c2 cells were placed in an imitated ischemic environment followed by Tyrode's solution to stimulate hypoxia/reoxygenation (H/R) injury. The mitochondrial fluorescence indicator MitoTracker Green and lysosomal fluorescence indicator LysoTracker Red were used to examine mitochondria and lysosomes, respectively. Colocalization of mitochondrial and autophagy marker proteins was determined by immunofluorescence. Autophagic flow currents were tested by Ad-mCherry-GFP-LC3B. Protein-protein interactions were predicted using a database and analyzed by co-immunoprecipitation. Autophagy marker protein, mitophagy marker protein, and mitophagy protein FUNDC1 were detected by immunoblotting.
    Results: Compared with those in the I/R group, myocardial autophagy and mitophagy were suppressed by the selective adenosine A2BR agonist BAY, and this effect was inhibited by the selective Src tyrosine kinase inhibitor PP2, indicating that adenosine A2BR activation could inhibit myocardial autophagy and mitophagy by activating Src tyrosine kinase. In support, in H9c2 cells, the selective Src tyrosine kinase inhibitor PP2 inhibited the effect of BAY on TOM20 with LC3 or mitochondria with lysosomes colocalization and autophagy flow. Here, we showed that mitochondrial FUNDC1 co-precipitated with Src tyrosine kinase after BAY was added. Consistently, the immunofluorescence and western blotting results demonstrated that compared to that in the H/R group, the expression of mitochondrial FUNDC1 was reduced by BAY, but this effect was reversed by PP2.
    Conclusions: Adenosine A2BR activation may inhibit myocardial mitophagy by downregulating expression of the mitochondrial FUNDC1 by activating Src tyrosine kinase under I/R conditions and could increase the interaction between Src tyrosine kinase and FUNDC1.
    Keywords:  Adenosine A2B receptor; FUNDC1; Src tyrosine kinase
    DOI:  https://doi.org/10.21037/cdt-22-468
  45. MedComm (2020). 2023 Aug;4(4): e311
    Modulation of the immunity and inflammation by autophagy.
    Ting Gan, Shu Qu, Hong Zhang, Xu-Jie Zhou.
      Autophagy, a highly conserved cellular self-degradation pathway, has emerged with novel roles in the realms of immunity and inflammation. Genome-wide association studies have unveiled a correlation between genetic variations in autophagy-related genes and heightened susceptibility to autoimmune and inflammatory diseases. Subsequently, substantial progress has been made in unraveling the intricate involvement of autophagy in immunity and inflammation through functional studies. The autophagy pathway plays a crucial role in both innate and adaptive immunity, encompassing various key functions such as pathogen clearance, antigen processing and presentation, cytokine production, and lymphocyte differentiation and survival. Recent research has identified novel approaches in which the autophagy pathway and its associated proteins modulate the immune response, including noncanonical autophagy. This review provides an overview of the latest advancements in understanding the regulation of immunity and inflammation through autophagy. It summarizes the genetic associations between variants in autophagy-related genes and a range of autoimmune and inflammatory diseases, while also examining studies utilizing transgenic animal models to uncover the in vivo functions of autophagy. Furthermore, the review delves into the mechanisms by which autophagy dysregulation contributes to the development of three common autoimmune and inflammatory diseases and highlights the potential for autophagy-targeted therapies.
    Keywords:  autoimmune diseases; autophagy; immunity; inflammation; pathogenesis
    DOI:  https://doi.org/10.1002/mco2.311
  46. Front Pharmacol. 2023 ;14 1170436
    Autophagy in intestinal fibrosis: relevance in inflammatory bowel disease.
    Dulce C Macias-Ceja, María D Barrachina, Dolores Ortiz-Masià.
      Chronic inflammation is often associated with fibrotic disorders in which an excessive deposition of extracellular matrix is a hallmark. Long-term fibrosis starts with tissue hypofunction and finally ends in organ failure. Intestinal fibrosis is not an exception, and it is a frequent complication of inflammatory bowel disease (IBD). Several studies have confirmed the link between deregulated autophagy and fibrosis and the presence of common prognostic markers; indeed, both up- and downregulation of autophagy are presumed to be implicated in the progression of fibrosis. A better knowledge of the role of autophagy in fibrosis may lead to it becoming a potential target of antifibrotic therapy. In this review we explore novel advances in the field that highlight the relevance of autophagy in fibrosis, and give special focus to fibrosis in IBD patients.
    Keywords:  Crohn’s disease; autophagy; colitis; fibrosis; inflammatory bowel disease; intestinal fibrosis
    DOI:  https://doi.org/10.3389/fphar.2023.1170436
  47. Mol Cancer Res. 2023 Jul 06. pii: MCR-23-0086. [Epub ahead of print]
    Hierarchical phosphorylation of HOXB13 by mTOR dictates its activity and oncogenic function in prostate cancer.
    Yonghong Chen, Catherine R Dufour, Lingwei Han, Ting Li, Hui Xia, Vincent Giguere.
      Dysregulation of mTOR signaling plays a critical role in promoting prostate cancer growth. HOXB13, a homeodomain transcription factor, is known to influence the androgen response and prostate cancer development. Recently, HOXB13 was found to complex with mTOR on chromatin. However, the functional crosstalk between HOXB13 and mTOR remains elusive. We now report that mTOR directly interacts with and hierarchically phosphorylates HOXB13 at threonine 8 and 41 then serine 31 to promote its interaction with the E3 ligase SKP2 while enhancing its oncogenic properties. Expression of HOXB13 harboring phosphomimetic mutations at the mTOR-targeted sites stimulates prostate cancer cellular growth both in vitro and in murine xenografts. Transcriptional profiling studies revealed a phospho-HOXB13-dependent gene signature capable of robustly discriminating between normal prostate tissues, primary and metastatic prostate cancer samples. This work uncovers a previously unanticipated molecular cascade by which mTOR directly phosphorylates HOXB13 to dictate a specific gene program with oncogenic implications in prostate cancer. Implications: Control of HOXB13 transcriptional activity via its direct phosphorylation by the mTOR kinase is a potential therapeutic avenue for the management of advanced prostate cancer.
    DOI:  https://doi.org/10.1158/1541-7786.MCR-23-0086
  48. Mol Neurobiol. 2023 Jul 03.
    Realgar-Induced Neurotoxicity: Crosstalk Between the Autophagic Flux and the p62-NRF2 Feedback Loop Mediates p62 Accumulation to Promote Apoptosis.
    Rui Feng, Jieyu Liu, Zhao Yang, Tiantian Yao, Ping Ye, Xiuhan Li, Jiaxin Zhang, Hong Jiang.
      Realgar is a traditional Chinese medicine that contains arsenic. It has been reported that the abuse of medicine-containing realgar has potential central nervous system (CNS) toxicity, but the toxicity mechanism has not been elucidated. In this study, we established an in vivo realgar exposure model and selected the end product of realgar metabolism, DMA, to treat SH-SY5Y cells in vitro. Many assays, including behavioral, analytical chemistry, and molecular biology, were used to elucidate the roles of the autophagic flux and the p62-NRF2 feedback loop in realgar-induced neurotoxicity. The results showed that arsenic could accumulate in the brain, causing cognitive impairment and anxiety-like behavior. Realgar impairs the ultrastructure of neurons, promotes apoptosis, perturbs autophagic flux homeostasis, amplifies the p62-NRF2 feedback loop, and leads to p62 accumulation. Further analysis showed that realgar promotes the formation of the Beclin1-Vps34 complex by activating JNK/c-Jun to induce autophagy and recruit p62. Meanwhile, realgar inhibits the activities of CTSB and CTSD and changes the acidity of lysosomes, leading to the inhibition of p62 degradation and p62 accumulation. Moreover, the amplified p62-NRF2 feedback loop is involved in the accumulation of p62. Its accumulation promotes neuronal apoptosis by upregulating the expression levels of Bax and cleaved caspase-9, resulting in neurotoxicity. Taken together, these data suggest that realgar can perturb the crosstalk between the autophagic flux and the p62-NRF2 feedback loop to mediate p62 accumulation, promote apoptosis, and induce neurotoxicity. Realgar promotes p62 accumulation to produce neurotoxicity by perturbing the autophagic flux and p62-NRF2 feedback loop crosstalk.
    Keywords:  Arsenic; Autophagic flux; Neurotoxicity; Realgar; p62-NRF2 feedback loop
    DOI:  https://doi.org/10.1007/s12035-023-03452-2
  49. Oncogene. 2023 Jul 01.
    Lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.
    Jianchun Wu, Kaitrin Kramer, David L Crowe.
      The International Agency for Research on Cancer determined that obesity is the primary preventable cause of breast cancer. The nuclear receptor peroxisome proliferator activated receptor γ (PPARγ) binds inflammatory mediators in obesity and its expression is reduced in human breast cancer. We created a new model to better understand how the obese microenvironment alters nuclear receptor function in breast cancer. The obesity related cancer phenotype was PPARγ dependent; deletion of PPARγ in mammary epithelium which is a tumor suppressor in lean mice unexpectedly increased tumor latency, reduced the luminal progenitor (LP) tumor cell fraction, and increased autophagic and senescent cells. Loss of PPARγ expression in mammary epithelium of obese mice increased expression of 2-aminoadipate semialdehyde synthase (AASS) which regulates lysine catabolism to acetoacetate. PPARγ-associated co-repressors and activators regulated AASS expression via a canonical response element. AASS expression was significantly reduced in human breast cancer, and AASS overexpression or acetoacetate treatment inhibited proliferation and induced autophagy and senescence in human breast cancer cell lines. Genetic or pharmacologic HDAC inhibition promoted autophagy and senescence in mammary tumor cells in vitro and in vivo. We concluded that lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.
    DOI:  https://doi.org/10.1038/s41388-023-02766-8
  50. Acta Neuropathol Commun. 2023 Jul 07. 11(1): 110
    Compensatory cross-talk between autophagy and glycolysis regulates senescence and stemness in heterogeneous glioblastoma tumor subpopulations.
    Emma Martell, Helgi Kuzmychova, Harshal Senthil, Esha Kaul, Chirayu R Chokshi, Chitra Venugopal, Christopher M Anderson, Sheila K Singh, Tanveer Sharif.
      Despite tremendous research efforts, successful targeting of aberrant tumor metabolism in clinical practice has remained elusive. Tumor heterogeneity and plasticity may play a role in the clinical failure of metabolism-targeting interventions for treating cancer patients. Moreover, compensatory growth-related processes and adaptive responses exhibited by heterogeneous tumor subpopulations to metabolic inhibitors are poorly understood. Here, by using clinically-relevant patient-derived glioblastoma (GBM) cell models, we explore the cross-talk between glycolysis, autophagy, and senescence in maintaining tumor stemness. We found that stem cell-like GBM tumor subpopulations possessed higher basal levels of glycolytic activity and increased expression of several glycolysis-related enzymes including, GLUT1/SLC2A1, PFKP, ALDOA, GAPDH, ENO1, PKM2, and LDH, compared to their non-stem-like counterparts. Importantly, bioinformatics analysis also revealed that the mRNA expression of glycolytic enzymes positively correlates with stemness markers (CD133/PROM1 and SOX2) in patient GBM tumors. While treatment with glycolysis inhibitors induced senescence in stem cell-like GBM tumor subpopulations, as evidenced by increased β-galactosidase staining and upregulation of the cell cycle regulators p21Waf1/Cip1/CDKN1A and p16INK4A/CDKN2A, these cells maintained their aggressive stemness features and failed to undergo apoptotic cell death. Using various techniques including autophagy flux and EGFP-MAP1LC3B+ puncta formation analysis, we determined that inhibition of glycolysis led to the induction of autophagy in stem cell-like GBM tumor subpopulations, but not in their non-stem-like counterparts. Similarly, blocking autophagy in stem cell-like GBM tumor subpopulations induced senescence-associated growth arrest without hampering stemness capacity or inducing apoptosis while reciprocally upregulating glycolytic activity. Combinatorial treatment of stem cell-like GBM tumor subpopulations with autophagy and glycolysis inhibitors blocked the induction of senescence while drastically impairing their stemness capacity which drove cells towards apoptotic cell death. These findings identify a novel and complex compensatory interplay between glycolysis, autophagy, and senescence that helps maintain stemness in heterogeneous GBM tumor subpopulations and provides a survival advantage during metabolic stress.
    Keywords:  Autophagy; Cancer stem cell-like cells; Glioblastoma; Glycolysis; Metabolism; Senescence; Tumor heterogeneity
    DOI:  https://doi.org/10.1186/s40478-023-01604-y
  51. Biochim Biophys Acta Mol Cell Res. 2023 Jun 29. pii: S0167-4889(23)00102-7. [Epub ahead of print]1870(7): 119530
    Inhibiting autophagy by miR-19a-3p/PTEN regulation protected retinal pigment epithelial cells from hyperglycemic damage.
    Qiaoyun Gong, Dawei Luo, Haiyan Wang, Xun Xu, Ying Fan, Zhi Zheng, Tianwei Qian.
      OBJECTIVE: The catabolic process of autophagy is arousing the attention of researchers studying diabetic retinopathy (DR), but the role and molecular mechanism of autophagy in DR are still unclear.METHODS: An in vivo diabetic rat model and in vitro hyperglycemic-exposed retinal pigment epithelium (RPE) cell cultures were established to mimic early DR. Transmission electron microscopy and mRFP-GFP-LC3 adenovirus transfection were applied for autophagic flux analysis. MicroRNA (miR)-19a-3p, members of the phosphate and tensin homolog (PTEN)/Akt/mammalian target of rapamycin (mTOR) pathway, and the autophagy-related proteins light chain (LC)3II/I and p62 were detected. Annexin V, transwell, Cell Counting Kit-8, fluorescein isothiocyanate-dextran monolayer permeability assay, and transepithelial electrical resistance were performed to evaluate the effects of regulating autophagy on RPE cells under the DR condition.
    RESULTS: Autophagy was aberrantly activated in DR as evidenced by autophagosome accumulation. Further mechanistic experiments revealed that DR induced PTEN expression, thus inhibiting Akt/mTOR phosphorylation and stimulating aberrant autophagy and apoptosis. Notably, these events could be reversed by miR-19a-3p directly targeting PTEN. Downregulation of autophagy by miR-19a-3p overexpression, PTEN knockdown, or 3-methyladenine (3-MA) treatment inhibited autophagosome formation and thus effectively ameliorated hyperglycemia-induced RPE cell apoptosis, increased migration, inhibited viability, and enhanced monolayer permeability under the DR condition.
    CONCLUSIONS: Our findings suggest that upregulation of miR-19a-3p inhibits aberrant autophagy by directly targeting PTEN, thus protecting RPE cells against DR damage. miR-19a-3p may represent a novel therapeutic target for inducing protective autophagy in early DR.
    Keywords:  Autophagy; Hyperglycemia; PTEN; Retinal pigment epithelium cell; microRNA
    DOI:  https://doi.org/10.1016/j.bbamcr.2023.119530
  52. Autophagy. 2023 Jul 07. 1-19
    The autophagic protein FYCO1 controls TNFRSF10/TRAIL receptor induced apoptosis and is inactivated by CASP8 (caspase 8).
    Valeria Coppola, Ilaria Marino, Uwe Warnken, Mario Falchi, Luca Pasquini, Mauro Biffoni, Ruggero De Maria, Tobias Longin Haas.
      Apoptosis is a tightly controlled cell death program executed by proteases, the so-called caspases. It plays an important role in tissue homeostasis and is often dysregulated in cancer. Here, we identified FYCO1, a protein that promotes microtubule plus end-directed transport of autophagic and endosomal vesicles as a molecular interaction partner of activated CASP8 (caspase 8). The absence of FYCO1 sensitized cells to basal and TNFSF10/TRAIL-induced apoptosis by receptor accumulation and stabilization of the Death Inducing Signaling Complex (DISC). Loss of FYCO1 resulted in impaired transport of TNFRSF10B/TRAIL-R2/DR5 (TNF receptor superfamily member 10b) to the lysosomes in TNFSF10/TRAIL-stimulated cells. More in detail, we show that FYCO1 interacted via its C-terminal GOLD domain with the CCZ1-MON1A complex, which is necessary for RAB7A activation and for the fusion of autophagosomal/endosomal vesicles with lysosomes. We demonstrated that FYCO1 is a novel and specific CASP8 substrate. The cleavage at aspartate 1306 resulted in the release of the C-terminal GOLD domain, inactivating FYCO1 function, and allowing for the progression of apoptosis. Furthermore, the lack of FYCO1 resulted in a stronger and prolonged formation of the TNFRSF1A/TNF-R1 signaling complex. Thus, FYCO1 limits the ligand-induced and steady-state signaling of TNFR-superfamily members, providing a control mechanism that fine-tunes both apoptotic and inflammatory answers.Abbreviations: AP: affinity purification; CHX: cycloheximide; co-IP: co-immunoprecipitation; CRISPR: clustered regularly interspaced short palindromic repeats; DISC: death-inducing signaling complex; DR: death receptors; doxy: doxycycline; GEF: guanine nucleotide exchange factor; ind: inducible; KD: knockdown; KO: knockout; MS: mass spectrometry; shRNA: short hairpin RNA; siRNA: small interfering RNA; TIP: two-step co-immunoprecipitation; WB: western blot.
    Keywords:  FYCO1; TRAIL; apoptosis; autophagy; caspase 8; lysosomal degradation
    DOI:  https://doi.org/10.1080/15548627.2023.2229656
  53. Cell Death Discov. 2023 Jul 06. 9(1): 229
    The regulatory role of lipophagy in central nervous system diseases.
    Zhuo-Qing Lan, Zi-Yi Ge, Shu-Kai Lv, Bing Zhao, Cai-Xia Li.
      Lipid droplets (LDs) are the organelles for storing neutral lipids, which are broken down when energy is insufficient. It has been suggested that excessive accumulation of LDs can affect cellular function, which is important to coordinate homeostasis of lipids in vivo. Lysosomes play an important role in the degradation of lipids, and the process of selective autophagy of LDs through lysosomes is known as lipophagy. Dysregulation of lipid metabolism has recently been associated with a variety of central nervous system (CNS) diseases, but the specific regulatory mechanisms of lipophagy in these diseases remain to be elucidated. This review summarizes various forms of lipophagy and discusses the role that lipophagy plays in the development of CNS diseases in order to reveal the related mechanisms and potential therapeutic targets for these diseases.
    DOI:  https://doi.org/10.1038/s41420-023-01504-z
  54. Free Radic Biol Med. 2023 Jul 05. pii: S0891-5849(23)00511-7. [Epub ahead of print]
    DMT1 differentially regulates mitochondrial complex activities to reduce glutathione loss and mitigate ferroptosis.
    Qing Tan, Xiaoqian Zhang, Shuxiang Li, Wenbin Liu, Jiaqi Yan, Siqi Wang, Feng Cui, Dan Li, Jun Li.
      Mitochondria are vital for energy production and Redox homeostasis, yet knowledge of relevant mechanisms remains limited. Here, through a genome-wide CRISPR-Cas9 knockout screening, we have identified DMT1 as a major regulator of mitochondria membrane potential. Our findings demonstrate that DMT1 deficiency increased the activity of mitochondrial complex I and reduced that of complex III. Enhanced complex I activity leads to increased NAD+ production, which activates IDH2 by promoting its deacetylation via SIRT3, This results in higher levels of NADPH and GSH, which improve antioxidant capacity during Erastin-induced ferroptosis. Meanwhile, loss of complex III activity impairs mitochondrial biogenesis and promotes mitophagy, contributing to suppression of ferroptosis. Thus, DMT1 differentially regulates activities of mitochondrial complex I and III to cooperatly suppress Erastin-induced ferroptosis. Furthermore, NMN, an alternative method of increasing mitochondrial NAD+, exhibits similar protective effects against ferroptosis by boosting GSH in a manner similar to DMT1 deficiency, shedding a light on potential therapeutic strategy for ferroptosis-related pathologies.
    Keywords:  DMT1; Ferroptosis; Mitochondria NAD(+); REDOX homeostasis
    DOI:  https://doi.org/10.1016/j.freeradbiomed.2023.06.023
  55. Front Pharmacol. 2023 ;14 1191517
    Mitochondrial homeostasis: a potential target for delaying renal aging.
    Ming Yang, Chongbin Liu, Na Jiang, Yan Liu, Shilu Luo, Chenrui Li, Hao Zhao, Yachun Han, Wei Chen, Li Li, Li Xiao, Lin Sun.
      Mitochondria, which are the energy factories of the cell, participate in many life activities, and the kidney is a high metabolic organ that contains abundant mitochondria. Renal aging is a degenerative process associated with the accumulation of harmful processes. Increasing attention has been given to the role of abnormal mitochondrial homeostasis in renal aging. However, the role of mitochondrial homeostasis in renal aging has not been reviewed in detail. Here, we summarize the current biochemical markers associated with aging and review the changes in renal structure and function during aging. Moreover, we also review in detail the role of mitochondrial homeostasis abnormalities, including mitochondrial function, mitophagy and mitochondria-mediated oxidative stress and inflammation, in renal aging. Finally, we describe some of the current antiaging compounds that target mitochondria and note that maintaining mitochondrial homeostasis is a potential strategy against renal aging.
    Keywords:  inflammation; mitochondria; mitophagy; oxidative stress; renal aging
    DOI:  https://doi.org/10.3389/fphar.2023.1191517
  56. Exp Cell Res. 2023 Jul 01. pii: S0014-4827(23)00239-2. [Epub ahead of print] 113691
    Lower dose of metformin combined with artesunate induced autophagy-dependent apoptosis of glioblastoma by activating ROS-AMPK-mTOR axis.
    Wencong Ding, Lingxiao Liao, Jia Liu, Jiaxing Zhao, Qiongyan Tang, Yongshi Liao.
      Glioblastoma multiform (GBM), one of the most common, aggressive primary brain tumours, demonstrates resistance to radiotherapy and chemotherapy after surgical resection and treatment failure. Metformin (MET) has been shown to suppress the proliferative capacity and invasion ability of GBM cells by activating AMPK and inhibiting mTOR, but the effective dose exceeded the maximum tolerated dose. Artesunate (ART) can exert certain anti-tumour effects by activating the AMPK-mTOR axis and inducing autophagy in tumour cells. Therefore, this study investigated the effects of MET combined with ART combination therapy on autophagy and apoptosis in GBM cells. MET combined with ART treatment effectively suppressed the viability, mono-cloning ability, migration and invasion capacities, as well as metastatic ability of GBM cells. The underlying mechanism involved modulation of the ROS-AMPK-mTOR axis, which was confirmed using 3-methyladenine and rapamycin to inhibit or promote the effects of MET combined with ART, respectively. The study findings suggest that MET used in combination with ART can induce autophagy-dependent apoptosis in GBM cells by activating the ROS-AMPK-mTOR pathway, providing a potential new treatment for GBM.
    Keywords:  Artesunate; Autophagy; Autophagy-dependent apoptosis; Glioblastoma; Metformin
    DOI:  https://doi.org/10.1016/j.yexcr.2023.113691
  57. Front Endocrinol (Lausanne). 2023 ;14 1160155
    Impaired mitochondrial dynamics and removal of the damaged mitochondria in diabetic retinopathy.
    Kumari Alka, Jay Kumar, Renu A Kowluru.
      Introduction: Mitochondrial dynamic plays a major role in their quality control, and the damaged mitochondrial components are removed by autophagy. In diabetic retinopathy, mitochondrial fusion enzyme, mitofusin 2 (Mfn2), is downregulated and mitochondrial dynamic is disturbed resulting in depolarized and dysfunctional mitochondria. Our aim was to investigate the mechanism of inhibition of Mfn2, and its role in the removal of the damaged mitochondria, in diabetic retinopathy.Methods: Using human retinal endothelial cells, effect of high glucose (20mM) on the GTPase activity of Mfn2 and its acetylation were determined. Role of Mfn2 in the removal of the damaged mitochondria was confirmed by regulating its acetylation, or by Mfn2 overexpression, on autophagosomes- autolysosomes formation and the mitophagy flux.
    Results: High glucose inhibited GTPase activity and increased acetylation of Mfn2. Inhibition of acetylation, or Mfn2 overexpression, attenuated decrease in GTPase activity and mitochondrial fragmentation, and increased the removal of the damaged mitochondria. Similar phenomenon was observed in diabetic mice; overexpression of sirtuin 1 (a deacetylase) ameliorated diabetes-induced inhibition of retinal Mfn2 and facilitated the removal of the damaged mitochondria.
    Conclusions: Acetylation of Mfn2 has dual roles in mitochondrial homeostasis in diabetic retinopathy, it inhibits GTPase activity of Mfn2 and increases mitochondrial fragmentation, and also impairs removal of the damaged mitochondria. Thus, protecting Mfn2 activity should maintain mitochondrial homeostasis and inhibit the development/progression of diabetic retinopathy.
    Keywords:  diabetic retinopathy; mitochondria; mitochondrial dynamics; mitofusin; mitophagy; retina
    DOI:  https://doi.org/10.3389/fendo.2023.1160155
  58. Fundam Clin Pharmacol. 2023 Jul 04.
    Targeting autophagy with tamoxifen in breast cancer: From molecular mechanisms to targeted therapy.
    Mohammad Yasin Zamanian, Maryam Golmohammadi, Amir Nili-Ahmadabadi, Ameer A Alameri, Mohammed Al-Hassan, Shadia Hamoud Alshahrani, Mohammed Sami Hasan, Andrés Alexis Ramírez-Coronel, Qutaiba A Qasim, Mahsa Heidari, Amita Verma.
      BACKGROUND: Tamoxifen (TAM) is often recommended as a first-line treatment for estrogen receptor-positive breast cancer (BC). However, TAM resistance continues to be a medical challenge for BC with hormone receptor positivity. The function of macro-autophagy and autophagy has recently been identified to be altered in BC, which suggests a potential mechanism for TAM resistance. Autophagy is a cellular stress-induced response to preserve cellular homeostasis. Also, therapy-induced autophagy, which is typically cytoprotective and activated in tumor cells, could sometimes be non-protective, cytostatic, or cytotoxic depending on how it is regulated.OBJECTIVE: This review explored the literature on the connections between hormonal therapies and autophagy. We investigated how autophagy could develop drug resistance in BC cells.
    METHODS: Scopus, Science Direct, PubMed, and Google Scholar were used to search articles for this study.
    RESULTS: The results demonstrated that protein kinases such as pAMPK, BAX, and p-p70S6K could be a sign of autophagy in developing TAM resistance. According to the study's findings, autophagy plays an important role in BC patients' TAM resistance.
    CONCLUSION: Therefore, by overcoming endocrine resistance in estrogen receptor-positive breast tumors, autophagy inhibition may improve the therapeutic efficacy of TAM.
    Keywords:  anticancer properties; autophagy; breast cancer; tamoxifen
    DOI:  https://doi.org/10.1111/fcp.12936
  59. Neurochirurgie. 2023 Jul 01. pii: S0028-3770(23)00062-0. [Epub ahead of print] 101465
    Transcription factor EB (TFEB) promotes autophagy in early brain injury after subarachnoid hemorrhage in rats.
    Wenqi Lu, Haichao Chu, Xiaoxu Li.
      BACKGROUND: Early brain injury (EBI) is responsible for devastating outcomes for patients with subarachnoid hemorrhage (SAH). Autophagy and apoptosis regulate the process of cell death. The transcription factor EB (TFEB) can increase autophagic flux by promoting autophagosome formation and autophagosome-lysosome fusion, and dysregulation of TFEB activity might induce the development of several diseases. However, the biological functions of TFEB in EBI post-SAH remain unknown.METHODS: We established an animal model of SAH by modified endovascular perforation and a cellular model by treating primary cortical neurons with oxyhemoglobin (oxyHb). A recombinant adenovirus containing a shRNA targeting TFEB (adv-sh-TFEB) was used to knock down TFEB expression. Protein levels of TFEB, Bax, Bcl-2, cleaved caspase-3, LC3, and Beclin-1 were measured by western blotting and immunofluorescence staining. Neuronal apoptosis was assessed by flow cytometry and TUNEL staining. Short-term neurobehavioral functions were examined by modified Garcia and beam balance scores. Brain edema was determined through assessment of brain water content.
    RESULTS: TFEB was increased at the protein level in cellular and animal models of SAH. TFEB depletion attenuated the oxyHb-induced apoptosis of primary cortical neurons and autophagy. TFEB downregulation improved the short-term neurobehavioral functions and reduced brain edema after SAH and inhibited neuronal apoptosis in SAH rats.
    CONCLUSION: TFEB depletion improves short-term neurologic performance and reduces brain edema by preventing autophagy and apoptosis after SAH.
    Keywords:  TFEB; apoptosis; autophagy; neurobehavioral functions; subarachnoid hemorrhage
    DOI:  https://doi.org/10.1016/j.neuchi.2023.101465
  60. J Cell Physiol. 2023 Jul 07.
    SIRT3 regulates mitophagy in liver fibrosis through deacetylation of PINK1/NIPSNAP1.
    Ruixi Li, Zhecheng Wang, Yue Wang, Ruimin Sun, Boyang Zou, Xinyao Tian, Deshun Liu, Xuzi Zhao, Junjun Zhou, Yan Zhao, Jihong Yao.
      Damaged mitochondria, a key factor in liver fibrosis, can be removed by the mitophagy pathway to maintain homeostasis of the intracellular environment to alleviate the development of fibrosis. PINK1 (PTEN-induced kinase 1) and NIPSNAP1 (nonneuronal SNAP25-like protein 1), which cooperatively regulate mitophagy, have been predicted to include the sites of lysine acetylation related to SIRT3 (mitochondrial deacetylase sirtuin 3). Our study aimed to discuss whether SIRT3 deacetylates PINK1 and NIPSNAP1 to regulate mitophagy in liver fibrosis. Carbon tetrachloride (CCl4 )-induced liver fibrosis as an in vivo model and LX-2 cells as activated cells were used to simulate liver fibrosis. SIRT3 expression was significantly decreased in mice in response to CCl4 , and SIRT3 knockout in vivo significantly deepened the severity of liver fibrosis, as indicated by increased α-SMA and Col1a1 levels both in vivo and in vitro. SIRT3 overexpression decreased α-SMA and Col1a1 levels. Furthermore, SIRT3 significantly regulated mitophagy in liver fibrosis, as demonstrated by LC3-Ⅱ/Ⅰ and p62 expression and colocalization between TOM20 and LAMP1. Importantly, PINK1 and NIPSNAP1 expression was also decreased in liver fibrosis, and PINK1 and NIPSNAP1 overexpression significantly improved mitophagy and attenuated ECM production. Furthermore, after simultaneously interfering with PINK1 or NIPSNAP1 and overexpressing SIRT3, the effect of SIRT3 on improving mitophagy and alleviating liver fibrosis was disrupted. Mechanistically, we show that SIRT3, as a mitochondrial deacetylase, specifically regulates the acetylation of PINK1 and NIPSNAP1 to mediate the mitophagy pathway in liver fibrosis. SIRT3-mediated PINK1 and NIPSNAP1 deacetylation is a novel molecular mechanism in liver fibrosis.
    Keywords:  NIPSNAP1; PINK1; SIRT3; liver fibrosis; mitophagy
    DOI:  https://doi.org/10.1002/jcp.31069
  61. Vet Microbiol. 2023 Jun 28. pii: S0378-1135(23)00175-X. [Epub ahead of print]284 109823
    IFITM3 restricts RABV infection through inhibiting viral entry and mTORC1- dependent autophagy.
    Jiaqi Ma, Zhongzhong Tu, Shouwen Du, Xinying Zhang, Jie Wang, Jianxiong Guo, Ye Feng, Hongbin He, Hongmei Wang, Chang Li, Changchun Tu, Yan Liu.
      Rabies, which caused by rabies virus (RABV), is a zoonotic and life-threatening disease with 100% mortality, and there is no effective treatment thus far due to the unclear pathogenesis and less of treatment targets. Interferon-induced transmembrane protein 3 (IFITM3) has recently been identified as an important anti-viral host effector induced by type I interferon. However, the role of IFITM3 in RABV infection has not been elucidated. In this study, we demonstrated that IFITM3 is a crucial restriction factor for RABV, the viral-induced IFITM3 significantly inhibited RABV replication, while knockdown of IFITM3 had the opposite effect. We then identified that IFNβ induces the upregulation of IFITM3 in the absence or presence of RABV infection, meanwhile, IFITM3 positively regulates RABV-triggered production of IFNβ in a feedback manner. In-depth research we found that IFITM3 not only inhibits the virus absorb and entry, but also inhibits viral replication through mTORC1-dependent autophagy. All these findings broaden our understanding of IFITM3 function and uncover a novel mechanism against RABV infection.
    Keywords:  Anti-viral effect; Autophagy; IFITM3; IFNβ; RABV
    DOI:  https://doi.org/10.1016/j.vetmic.2023.109823
  62. Res Sq. 2023 Jun 16. pii: rs.3.rs-3030763. [Epub ahead of print]
    Intracellular Calcium links Milk Stasis to Lysosome Dependent Cell Death by Activating a TGFβ3/TFEB/STAT3 Pathway Early during Mammary Gland Involution.
    Jaekwang Jeong, Jongwon Lee, Gabriel Talaia, Wonnam Kim, Junho Song, Juhyeon Hong, Kwangmin Yoo, David Gonzalez, Diana Athonvarangkul, Jaehun Shin, Pamela Dann, Ann Haberman, Lark Kyun Kim, Shawn Ferguson, Jungmin Choi, John Wysolmerski.
      Involution of the mammary gland after lactation is a dramatic example of coordinated cell death. Weaning causes distension of the alveolar structures due to the accumulation of milk, which, in turn, activates STAT3 and initiates a caspase-independent but lysosome-dependent cell death (LDCD) pathway. Although the importance of STAT3 and LDCD in early mammary involution is well established, it has not been entirely clear how milk stasis activates STAT3. In this report, we demonstrate that protein levels of the PMCA2 calcium pump are significantly downregulated within 2-4 hours of experimental milk stasis. Reductions in PMCA2 expression correlate with an increase in cytoplasmic calcium in vivo as measured by multiphoton intravital imaging of GCaMP6f fluorescence. These events occur concomitant with the appearance of nuclear pSTAT3 expression but prior to significant activation of LDCD or its previously implicated mediators such as LIF, IL6 and TGFβ3, all of which appear to be upregulated by increased intracellular calcium. We also observed that milk stasis, loss of PMCA2 expression and increased intracellular calcium levels activate TFEB, an important regulator of lysosome biogenesis. This is the result of increased TGFβ signaling and inhibition of cell cycle progression. Finally, we demonstrate that increased intracellular calcium activates STAT3 by inducing degradation of its negative regulator, SOCS3, a process which also appears to be mediated by TGFβ signaling. In summary, these data suggest that intracellular calcium serves as an important proximal biochemical signal linking milk stasis to STAT3 activation, increased lysosomal biogenesis, and lysosome-mediated cell death.
    DOI:  https://doi.org/10.21203/rs.3.rs-3030763/v1
  63. Front Oncol. 2023 ;13 1220435
    Emerging trends and research foci in autophagy of pancreatic cancer: a bibliometric and visualized study.
    Linlin Fan, Zhiyong Wei, Lili Liu, Xiaojie Qi, Hong Yu.
      Objective: The purpose of this study was to analyze the trends by year, country, institution, journal, reference and keyword in publications on the autophagy of pancreatic cancer (PC) and to predict future research hotspots.Methods: The Web of Science Core Collection was used to search for publications. The contributions of various countries/regions, institutes, authors, identified research hotspots, and promising future trends were analyzed using the VOSviewer1.6.16 and CiteSpace6.6.R2 programs. We also summarized autophagy relevant clinical trials of PC.
    Results: A total of 1293 papers on the autophagy of PC published between 2013 and 2023 were included in the study. The average number of citations per article was 33.76. The China had the most publications, followed by USA, and a total of 50 influential articles were identified through co-citation analysis. Clustering analysis revealed clusters of keywords: metabolic reprogramming and ER stress, mTOR-mediated apoptosis, extracellular trap as the most concerned clusters. The co-occurrence cluster analysis showed pancreatic stellate cell, autophagy-dependent ferroptosis, autophagy-related pathway, metabolic rewiring, on-coding RNA as the highly concerned research topics in recently.
    Conclusion: The number of publications and research interest have generally increased over the past few years. The China and USA have made prominent contributions to the study of the autophagy of PC. The current research hotspots mainly focus not only on the related modulation, metabolic reprogramming, ferroptosis of tumor cells themselves, but also on tumor microenvironments such as autophagy associated pancreatic stellate cells and new treatments targeting autophagy.
    Keywords:  autophagy; bibliometric study; ferroptosis; pancreatic cancer; pancreatic stellate cells; tumor microenvironment
    DOI:  https://doi.org/10.3389/fonc.2023.1220435
  64. bioRxiv. 2023 Jun 13. pii: 2023.06.12.544698. [Epub ahead of print]
    Rictor induces AKT signaling to regulate lymphatic valve formation.
    Richa Banerjee, Luz A Knauer, Drishya Iyer, Sara E Barlow, Joshua P Scallan, Ying Yang.
      Lymphatic valves are specialized structures of the collecting lymphatic vessels and are crucial for preventing retrograde lymph flow. Mutations in valve-forming genes have been clinically implicated in the pathology of congenital lymphedema. Lymphatic valves form when oscillatory shear stress (OSS) from lymph flow signals through the PI3K/AKT pathway to promote the transcription of valve-forming genes that trigger the growth and maintenance of lymphatic valves throughout life. Conventionally, in other tissue types, AKT activation requires dual kinase activity and the mammalian target of rapamycin complex 2 (mTORC2) commands this process by phosphorylating AKT at Ser473. Here we showed that embryonic and postnatal lymphatic deletion of Rictor , a critical component of mTORC2, led to a significant decrease in lymphatic valves and prevented the maturation of collecting lymphatic vessels. RICTOR knockdown in human lymphatic endothelial cells (hdLECs) not only significantly reduced the level of activated AKT and the expression of valve-forming genes under no-flow conditions, but also abolished the upregulation of AKT activity and valve-forming genes in response to flow. We further showed that the AKT target, FOXO1, a repressor of lymphatic valve formation, had increased nuclear activity in Rictor knockout mesenteric LECs, in vivo . Deletion of Foxo1 in Rictor knockout mice restored the number of valves to control levels in both mesenteric and ear lymphatics. Our work revealed a novel role of RICTOR signaling in the mechanotransduction signaling pathway, wherein it activates AKT and prevents the nuclear accumulation of the valve repressor, FOXO1, which ultimately allows the formation and maintenance of a normal lymphatic valve.
    DOI:  https://doi.org/10.1101/2023.06.12.544698
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