bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023‒06‒18
sixty-nine papers selected by
Viktor Korolchuk, Newcastle University
  1. PHAF1/MYTHO is a novel autophagy regulator that controls muscle integrity.
  2. Palbociclib-Induced Cellular Senescence Is Modulated by the mTOR Complex 1 and Autophagy.
  3. Autophagy in a Nutshell.
  4. Targeting the autophagy-lysosomal pathway in Huntington disease: a pharmacological perspective.
  5. Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.
  6. Emerging roles of p300/CBP in autophagy and autophagy-related human disorders.
  7. Assays of Monitoring and Measuring Autophagic Flux for iPSC-Derived Human Neurons and Other Brain Cell Types.
  8. SLIT2/ROBO1 signaling suppresses mTORC1 for organelle control and bacterial killing.
  9. Asteroid bodies and autophagy.
  10. mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.
  11. Extracellular Vesicles Released by Genetically Modified Macrophages Activate Autophagy and Produce Potent Neuroprotection in Mouse Model of Lysosomal Storage Disorder, Batten Disease.
  12. LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.
  13. The AMPK-dependent inhibition of autophagy plays a crucial role in protecting photoreceptor from photooxidative injury.
  14. Autophagy fuels mitochondrial function through regulation of iron metabolism in pancreatic cancer.
  15. ATG8 proteins are co-factors for human dopaminergic neuronal transcriptional control: implications for neuronal resilience in Parkinson disease.
  16. Restored autophagy is protective against PAK3-induced cardiac dysfunction.
  17. Is autophagy induction by PARP inhibitors a target for therapeutic benefit?
  18. Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning.
  19. Inner mitochondrial membrane fission protein, MTP18, serves as a mitophagy receptor to prevent apoptosis in oral cancer.
  20. Autophagy-mediated immune system regulation in reproductive system and pregnancy-associated complications.
  21. Ratiometric visualization of lysosomal pH fluctuations during autophagy by two-photon carbonized polymer dots-based probe.
  22. Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes.
  23. Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.
  24. Cinchonine exerts anti-tumor and immunotherapy sensitizing effects in lung cancer by impairing autophagic-lysosomal degradation.
  25. The Emerging Role of Autophagy-Associated lncRNAs in the Pathogenesis of Neurodegenerative Diseases.
  26. LAMP2A, and other chaperone-mediated autophagy related proteins, do not decline with age in genetically heterogeneous UM-HET3 mice.
  27. SNAP25 ameliorates postoperative cognitive dysfunction by facilitating PINK1-dependent mitophagy and impeding caspase-3/GSDME-dependent pyroptosis.
  28. Histone lysine demethylase 3B regulates autophagy via transcriptional regulation of GABARAPL1 in acute myeloid leukemia cells.
  29. Proteasomes of Autophagy-Deficient Cells Exhibit Alterations in Regulatory Proteins and a Marked Reduction in Activity.
  30. TMX4-driven LINC complex disassembly and asymmetric autophagy of the nuclear envelope upon acute ER stress.
  31. PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase.
  32. Role and clinical implication of autophagy in COVID-19.
  33. The RagA GTPase protects young egg chambers in Drosophila.
  34. TFEB is a central regulator of the aging process and age-related diseases.
  35. Advances in Understanding the Links between Metabolism and Autophagy in Acute Myeloid Leukemia: From Biology to Therapeutic Targeting.
  36. Mitophagy Activation Targeting PINK1 Is an Effective Treatment to Inhibit Zika Virus Replication.
  37. Interplay between septins and ubiquitin-mediated xenophagy during Shigella entrapment.
  38. Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.
  39. Reduction in Rubicon by cigarette smoke is associated with impaired phagocytosis and occurs through lysosomal degradation pathway.
  40. Dimethyl fumarate ameliorates Parkinsonian pathology by modulating autophagy and apoptosis via Nrf2-TIGAR-LAMP2/Cathepsin D axis.
  41. Nutrition and autophagy deficiency in critical illness.
  42. HSPA8 regulates anti-bacterial autophagy through liquid-liquid phase separation.
  43. Mechanism of NURP1 in temozolomide resistance in hypoxia-treated glioma cells via the KDM3A/TFEB axis.
  44. Reciprocal effects of alpha-synuclein aggregation and lysosomal homeostasis in synucleinopathy models.
  45. How does mitochondrial import machinery fine-tune mitophagy? Different paths and one destination.
  46. Genetic Targeting of dSAMTOR, A Negative dTORC1 Regulator, during Drosophila Aging: A Tissue-Specific Pathology.
  47. Fibroblast growth factor 21 (FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB (TFEB)-regulated lipophagy.
  48. The mitochondrial uncoupling effects of nitazoxanide enhances cellular autophagy and promotes the clearance of α-synuclein: Potential role of AMPK-JNK pathway.
  49. Editorial: The regulation of proteostasis in aging.
  50. Autophagy in homocysteine‑induced HUVEC senescence.
  51. TFEB: a double-edged sword for tumor metastasis.
  52. A Reactive Oxygen Species-Responsive Targeted Nanoscavenger to Promote Mitophagy for the Treatment of Alzheimer's Disease.
  53. Protein Accumulation and Impaired Autophagy Underlie Cognitive Dysfunction in Angelman Syndrome.
  54. Autophagy in maternal tissues contributes to Arabidopsis seed development.
  55. Epac1 participates in β1-adrenoreceptor autoantibody-mediated decreased autophagic flux in cardiomyocytes.
  56. C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.
  57. mTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.
  58. Dynamic Measurement of Endosome-Lysosome Fusion in Neurons Using High-Content Imaging.
  59. High levels of intracellular endotrophin in adipocytes mediate COPII vesicle supplies to autophagosome to impair autophagic flux and contribute to systemic insulin resistance in obesity.
  60. SPINK1 contributes to proliferation and clonal formation of HT29 cells through Beclin1 associated enhanced autophagy.
  61. Monitoring lysosomal acidity.
  62. mTOR Signaling Pathway in Bone Diseases Associated with Hyperglycemia.
  63. Activation of NLRP3 signaling contributes to cadmium-induced bone defects, associated with autophagic flux obstruction.
  64. Mammalian target of rapamycin (mTOR) inhibitors in dermatology.
  65. Chloroquine enhances the efficacy of chemotherapy drugs against acute myeloid leukemia by inactivating the autophagy pathway.
  66. Autophagy prevents microglial senescence.
  67. Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.
  68. Effects of leucine ingestion and contraction on the Sestrin/GATOR2 pathway and mTORC1 activation in rat fast-twitch muscle.
  69. Genetic Ablation of LAT1 Inhibits Growth of Liver Cancer Cells and Downregulates mTORC1 Signaling.
  1. Autophagy. 2023 Jun 12. 1-3
    PHAF1/MYTHO is a novel autophagy regulator that controls muscle integrity.
    Anais Franco-Romero, Jean Philippe Leduc-Gaudet, Sabah Na Hussain, Gilles Gouspillou, Marco Sandri.
      Skeletal muscles play key roles in movement, posture, thermogenesis, and whole-body metabolism. Autophagy plays essential roles in the regulation of muscle mass, function and integrity. However, the molecular machinery that regulates autophagy is still incompletely understood. In our recent study, we identified and characterized a novel Forkhead Box O (FoxO)-dependent gene, PHAF1/MYTHO (phagophore assembly factor 1/macro-autophagy and youth optimizer), as a novel autophagy regulator that controls muscle integrity. MYTHO/PHAF1 is upregulated in multiple conditions leading to muscle atrophy, and downregulation of its expression spares muscle atrophy triggered by fasting, denervation, cachexia and sepsis. Overexpression of PHAF1/MYTHO is sufficient to induce muscle atrophy. Prolonged downregulation of PHAF1/MYTHO causes a severe myopathic phenotype, which is characterized by impaired autophagy, muscle weakness, myofiber degeneration, mammalian target of rapamycin complex 1 (mTORC1) hyperactivation and extensive ultrastructural defects, such as accumulation of proteinaceous and membranous structures and tubular aggregates. This myopathic phenotype is attenuated upon administration of the mTORC1 inhibitor rapamycin. These findings position PHAF1/MYTHO as a novel regulator of skeletal muscle autophagy and tissue integrity.
    Keywords:  FoxO; Myopathy; autophagy; mTOR; muscle atrophy; myotonic dystrophy type 1
    DOI:  https://doi.org/10.1080/15548627.2023.2224206
  2. Int J Mol Sci. 2023 May 26. pii: 9284. [Epub ahead of print]24(11):
    Palbociclib-Induced Cellular Senescence Is Modulated by the mTOR Complex 1 and Autophagy.
    Angel Cayo, Whitney Venturini, Danitza Rebolledo-Mira, Rodrigo Moore-Carrasco, Andrés A Herrada, Estefanía Nova-Lamperti, Claudio Valenzuela, Nelson E Brown.
      Despite not dividing, senescent cells acquire the ability to synthesize and secrete a plethora of bioactive molecules, a feature known as the senescence-associated secretory phenotype (SASP). In addition, senescent cells often upregulate autophagy, a catalytic process that improves cell viability in stress-challenged cells. Notably, this "senescence-related autophagy" can provide free amino acids for the activation of mTORC1 and the synthesis of SASP components. However, little is known about the functional status of mTORC1 in models of senescence induced by CDK4/6 inhibitors (e.g., Palbociclib), or the effects that the inhibition of mTORC1 or the combined inhibition of mTORC1 and autophagy have on senescence and the SASP. Herein, we examined the effects of mTORC1 inhibition, with or without concomitant autophagy inhibition, on Palbociclib-driven senescent AGS and MCF-7 cells. We also assessed the pro-tumorigenic effects of conditioned media from Palbociclib-driven senescent cells with the inhibition of mTORC1, or with the combined inhibition of mTORC1 and autophagy. We found that Palbociclib-driven senescent cells display a partially reduced activity of mTORC1 accompanied by increased levels of autophagy. Interestingly, further mTORC1 inhibition exacerbated the senescent phenotype, a phenomenon that was reversed upon autophagy inhibition. Finally, the SASP varied upon inhibiting mTORC1, or upon the combined inhibition of mTORC1 and autophagy, generating diverse responses in cell proliferation, invasion, and migration of non-senescent tumorigenic cells. Overall, variations in the SASP of Palbociclib-driven senescent cells with the concomitant inhibition of mTORC1 seem to depend on autophagy.
    Keywords:  autophagy; cancer; mTORC1; palbociclib; senescence; senescence-associated secretory phenotype
    DOI:  https://doi.org/10.3390/ijms24119284
  3. FEBS Lett. 2023 Jun 16.
    Autophagy in a Nutshell.
    Oren Shatz, Zvulun Elazar.
      Autophagy is an intracellular catabolic process that eliminates cytoplasmic constituents selectively by tight engulfment in an isolation membrane or recycles bulk cytoplasm by nonselective sequestration. Completion of the isolation membrane forms a double membrane vesicle, termed autophagosome, that proceeds to fusion with the lysosome, where the inner membrane and its cytoplasmic content are degraded. Autophagosome biogenesis is unique in that the newly-formed membrane, termed phagophore, is elongated by direct lipid flow from a proximal ER-associated donor membrane. Recent years mark a tremendous advancement in delineating the direct regulation of this process by different lipid species and associated protein complexes. Here we schematically summarize the current view of autophagy and autophagosome biogenesis.
    Keywords:  PAS; autophagosome biogenesis; autophagy; lysosome; phagophore; vacuole
    DOI:  https://doi.org/10.1002/1873-3468.14679
  4. Front Aging Neurosci. 2023 ;15 1175598
    Targeting the autophagy-lysosomal pathway in Huntington disease: a pharmacological perspective.
    Junsheng Yang, Chaoyue Zhang.
      The autophagy-lysosomal pathway (ALP) is the major biological pathway responsible for clearing intracellular protein aggregates, therefore a promising target for treating diseases featuring the accumulation of aggregation-prone proteins, such as Huntington disease (HD). However, accumulating evidence indicated that targeting ALP to treat HD is pharmacologically challenging due to the complexity of autophagy and the autophagy defects in HD cells. Here in this mini-review, we summarized the current challenges in targeting ALP in HD and discussed a number of latest findings on aggrephagy and targeted protein degradation, which we believe will provide potential new targets and new strategies for treating HD via ALP.
    Keywords:  Huntington disease; aggrephagy; autophagy-lysosomal pathway; mHTT; targeted protein degradation
    DOI:  https://doi.org/10.3389/fnagi.2023.1175598
  5. Nat Chem Biol. 2023 Jun 15.
    Nanosensor-based monitoring of autophagy-associated lysosomal acidification in vivo.
    Mijin Kim, Chen Chen, Zvi Yaari, Rune Frederiksen, Ewelina Randall, Jaina Wollowitz, Christian Cupo, Xiaojian Wu, Janki Shah, Daniel Worroll, Rachel E Lagenbacher, Dana Goerzen, Yue-Ming Li, Heeseon An, YuHuang Wang, Daniel A Heller.
      Autophagy is a cellular process with important functions that drive neurodegenerative diseases and cancers. Lysosomal hyperacidification is a hallmark of autophagy. Lysosomal pH is currently measured by fluorescent probes in cell culture, but existing methods do not allow for quantitative, transient or in vivo measurements. In the present study, we developed near-infrared optical nanosensors using organic color centers (covalent sp3 defects on carbon nanotubes) to measure autophagy-mediated endolysosomal hyperacidification in live cells and in vivo. The nanosensors localize to the lysosomes, where the emission band shifts in response to local pH, enabling spatial, dynamic and quantitative mapping of subtle changes in lysosomal pH. Using the sensor, we observed cellular and intratumoral hyperacidification on administration of mTORC1 and V-ATPase modulators, revealing that lysosomal acidification mirrors the dynamics of S6K dephosphorylation and LC3B lipidation while diverging from p62 degradation. This sensor enables the transient and in vivo monitoring of the autophagy-lysosomal pathway.
    DOI:  https://doi.org/10.1038/s41589-023-01364-9
  6. J Cell Sci. 2023 06 15. pii: jcs261028. [Epub ahead of print]136(12):
    Emerging roles of p300/CBP in autophagy and autophagy-related human disorders.
    Yinfeng Xu, Wei Wan.
      As one of the major acetyltransferases in mammalian cells, p300 (also known as EP300) and its highly related protein CBP (also known as CREBBP), collectively termed p300/CBP, is characterized as a key regulator in gene transcription by modulating the acetylation of histones. In recent decades, proteomic analyses have revealed that p300 is also involved in the regulation of various cellular processes by acetylating many non-histone proteins. Among the identified substrates, some are key players involved in different autophagy steps, which together establish p300 as a master regulator of autophagy. Accumulating evidence has shown that p300 activity is controlled by many distinct cellular pathways to regulate autophagy in response to cellular or environmental stimuli. In addition, several small molecules have been shown to regulate autophagy by targeting p300, suggesting that manipulation of p300 activity is sufficient for controlling autophagy. Importantly, dysfunction of p300-regulated autophagy has been implicated in a number of human disorders, such as cancer, aging and neurodegeneration, highlighting p300 as a promising target for the drug development of autophagy-related human disorders. Here, we focus on the roles of p300-mediated protein acetylation in the regulation of autophagy and discuss implications for autophagy-related human disorders.
    Keywords:  Acetylation; Autophagy; Human disorders; p300/CBP
    DOI:  https://doi.org/10.1242/jcs.261028
  7. Methods Mol Biol. 2023 ;2683 221-233
    Assays of Monitoring and Measuring Autophagic Flux for iPSC-Derived Human Neurons and Other Brain Cell Types.
    Ryan O'Rourke, Guzide Ayse Erdemir, Yu-Wen Alvin Huang.
      Autophagy is a highly conserved, cytoprotective, catabolic process induced in response to conditions of cellular stress and nutrient deprivation. It is responsible for the degradation of large intracellular substrates such as misfolded or aggregated proteins and organelles. This self-degradative mechanism is crucial for proteostasis in post-mitotic neurons, requiring its careful regulation. Due to its homeostatic role and the implications, it has for certain disease pathologies, autophagy has become a growing area of research. We describe here two assays that can be used as part of a tool kit for measuring autophagy-lysosomal flux in human iPSC-derived neurons.One way to measure autophagic flux is through a western blotting assay, which can be used to analyze two important autophagy proteins: microtubule-associated protein 1 light chain 3 (LC3) and p62. In this chapter, we describe a western blotting assay for use in human iPSC neurons that can be used to quantify these two proteins of interest to measure autophagic flux.In addition to conventional western blotting techniques, more sophisticated tools have come available to readout autophagic flux in a sensitive and high-throughput manner. In the latter portion of this chapter, we describe a flow cytometry assay which utilizes a pH-sensitive fluorescent reporter which can also be used to measure autophagic flux.
    Keywords:  Aitophagic flux; Autophagy; Immunoblotting flow cytometry; Induced pluripotent stem cells (iPS cells); Microtubule associated protein 1 light chain 3 (LC3); Neurons; p62
    DOI:  https://doi.org/10.1007/978-1-0716-3287-1_18
  8. Life Sci Alliance. 2023 Aug;pii: e202301964. [Epub ahead of print]6(8):
    SLIT2/ROBO1 signaling suppresses mTORC1 for organelle control and bacterial killing.
    Vikrant K Bhosle, Joel Mj Tan, Taoyingnan Li, Rong Hua, Hyunwoo Kwon, Zhubing Li, Sajedabanu Patel, Marc Tessier-Lavigne, Lisa A Robinson, Peter K Kim, John H Brumell.
      SLIT/ROBO signaling impacts many aspects of tissue development and homeostasis, in part, through the regulation of cell growth and proliferation. Recent studies have also linked SLIT/ROBO signaling to the regulation of diverse phagocyte functions. However, the mechanisms by which SLIT/ROBO signaling acts at the nexus of cellular growth control and innate immunity remain enigmatic. Here, we show that SLIT2-mediated activation of ROBO1 leads to inhibition of mTORC1 kinase activity in macrophages, leading to dephosphorylation of its downstream targets, including transcription factor EB and ULK1. Consequently, SLIT2 augments lysosome biogenesis, potently induces autophagy, and robustly promotes the killing of bacteria within phagosomes. Concordant with these results, we demonstrate decreased lysosomal content and accumulated peroxisomes in the spinal cords of embryos from Robo1 -/- , Robo2 -/- double knockout mice. We also show that impediment of auto/paracrine SLIT-ROBO signaling axis in cancer cells leads to hyperactivation of mTORC1 and inhibition of autophagy. Together, these findings elucidate a central role of chemorepellent SLIT2 in the regulation of mTORC1 activity with important implications for innate immunity and cancer cell survival.
    DOI:  https://doi.org/10.26508/lsa.202301964
  9. J Cutan Pathol. 2023 Jun 14.
    Asteroid bodies and autophagy.
    Albert J H Suurmeijer.
      
    DOI:  https://doi.org/10.1111/cup.14483
  10. Int J Mol Sci. 2023 May 27. pii: 9364. [Epub ahead of print]24(11):
    mTOR Regulation of N-Myc Downstream Regulated 1 (NDRG1) Phosphorylation in Clear Cell Renal Cell Carcinoma.
    Anisha Valluri, Jessica Wellman, Chelsea L McCallister, Kathleen C Brown, Logan Lawrence, Rebecca Russell, James Jensen, James Denvir, Monica A Valentovic, Krista L Denning, Travis B Salisbury.
      The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.
    Keywords:  N-Myc Downstream Regulated 1; NDRG1; clear cell renal cell carcinoma; mTOR; mTORC1; mTORC2; proteomics
    DOI:  https://doi.org/10.3390/ijms24119364
  11. Cells. 2023 May 29. pii: 1497. [Epub ahead of print]12(11):
    Extracellular Vesicles Released by Genetically Modified Macrophages Activate Autophagy and Produce Potent Neuroprotection in Mouse Model of Lysosomal Storage Disorder, Batten Disease.
    Nazira El-Hage, Matthew J Haney, Yuling Zhao, Myosotys Rodriguez, Zhanhong Wu, Mori Liu, Carson J Swain, Hong Yuan, Elena V Batrakova.
      Over the recent decades, the use of extracellular vesicles (EVs) has attracted considerable attention. Herein, we report the development of a novel EV-based drug delivery system for the transport of the lysosomal enzyme tripeptidyl peptidase-1 (TPP1) to treat Batten disease (BD). Endogenous loading of macrophage-derived EVs was achieved through transfection of parent cells with TPP1-encoding pDNA. More than 20% ID/g was detected in the brain following a single intrathecal injection of EVs in a mouse model of BD, ceroid lipofuscinosis neuronal type 2 (CLN2) mice. Furthermore, the cumulative effect of EVs repetitive administrations in the brain was demonstrated. TPP1-loaded EVs (EV-TPP1) produced potent therapeutic effects, resulting in efficient elimination of lipofuscin aggregates in lysosomes, decreased inflammation, and improved neuronal survival in CLN2 mice. In terms of mechanism, EV-TPP1 treatments caused significant activation of the autophagy pathway, including altered expression of the autophagy-related proteins LC3 and P62, in the CLN2 mouse brain. We hypothesized that along with TPP1 delivery to the brain, EV-based formulations can enhance host cellular homeostasis, causing degradation of lipofuscin aggregates through the autophagy-lysosomal pathway. Overall, continued research into new and effective therapies for BD is crucial for improving the lives of those affected by this condition.
    Keywords:  Batten disease; autophagy; drug delivery; extracellular vesicles; lysosomal storage disorders; neuroprotection
    DOI:  https://doi.org/10.3390/cells12111497
  12. Dev Cell. 2023 Jun 08. pii: S1534-5807(23)00242-3. [Epub ahead of print]
    LC3B is lipidated to large lipid droplets during prolonged starvation for noncanonical autophagy.
    Mohyeddine Omrane, Kalthoum Ben M'Barek, Alexandre Santinho, Nathan Nguyen, Shanta Nag, Thomas J Melia, Abdou Rachid Thiam.
      Lipid droplets (LDs) store lipids that can be utilized during times of scarcity via autophagic and lysosomal pathways, but how LDs and autophagosomes interact remained unclear. Here, we discovered that the E2 autophagic enzyme, ATG3, localizes to the surface of certain ultra-large LDs in differentiated murine 3T3-L1 adipocytes or Huh7 human liver cells undergoing prolonged starvation. Subsequently, ATG3 lipidates microtubule-associated protein 1 light-chain 3B (LC3B) to these LDs. In vitro, ATG3 could bind alone to purified and artificial LDs to mediate this lipidation reaction. We observed that LC3B-lipidated LDs were consistently in close proximity to collections of LC3B-membranes and were lacking Plin1. This phenotype is distinct from macrolipophagy, but it required autophagy because it disappeared following ATG5 or Beclin1 knockout. Our data suggest that extended starvation triggers a noncanonical autophagy mechanism, similar to LC3B-associated phagocytosis, in which the surface of large LDs serves as an LC3B lipidation platform for autophagic processes.
    Keywords:  Atg3; LC3B; lipid droplets; noncanonical autophagy; organelle biogenesis; prolonged starvation
    DOI:  https://doi.org/10.1016/j.devcel.2023.05.009
  13. J Photochem Photobiol B. 2023 Jun 05. pii: S1011-1344(23)00089-1. [Epub ahead of print]245 112735
    The AMPK-dependent inhibition of autophagy plays a crucial role in protecting photoreceptor from photooxidative injury.
    Yu-Lin Li, Tian-Zi Zhang, Li-Kun Han, Chang He, Yi-Ran Pan, Bin Fan, Guang-Yu Li.
      Excessive light exposure can potentially cause irreversible damage to the various photoreceptor cells, and this aspect has been considered as an important factor leading to the progression of the different retinal diseases. AMP-activated protein kinase (AMPK) and the mammalian target of rapamycin (mTOR) are crucial intracellular signaling hubs involved in the regulation of cellular metabolism, energy homeostasis, cellular growth and autophagy. A number of previous studies have indicated that either AMPK activation or mTOR inhibition can promote autophagy in most cases. In the current study, we have established an in vitro as well as in vivo photooxidation-damaged photoreceptor model and investigated the possible influence of visible light exposure in the AMPK/mTOR/autophagy signaling pathway. We have also explored the potential regulatory effects of AMPK/mTOR on light-induced autophagy and protection achieved by suppressing autophagy in photooxidation-damaged photoreceptors. We observed that light exposure led to a significant activation of mTOR and autophagy in the photoreceptor cells. However, intriguingly, AMPK activation or mTOR inhibition significantly inhibited rather than promoting autophagy, which was termed as AMPK-dependent inhibition of autophagy. In addition, either indirectly suppressing autophagy by AMPK activation/ mTOR inhibition or directly blocking autophagy with an inhibitor exerted a significant protective effect on the photoreceptor cells against the photooxidative damage. Neuroprotective effects caused by the AMPK-dependent inhibition of autophagy were also verified with a retinal light injured mouse model in vivo. Overall, our findings demonstrated that AMPK / mTOR pathway could inhibit autophagy through AMPK-dependent inhibition of autophagy to significantly protect the photoreceptors from photooxidative injury, which may aid to further develop novel targeted retinal neuroprotective drugs.
    Keywords:  AMPK; Autophagy; Photooxidation; Photoreceptor; Retinal light injury; Retinal neuroprotection
    DOI:  https://doi.org/10.1016/j.jphotobiol.2023.112735
  14. Autophagy. 2023 Jun 13. 1-2
    Autophagy fuels mitochondrial function through regulation of iron metabolism in pancreatic cancer.
    Subhadip Mukhopadhyay, Joel Encarnacion-Rosado, Alec C Kimmelman.
      Pancreatic ductal adenocarcinoma (PDAC) has one of the lowest 5-year survival rates of any cancer in the United States. Our previous work has shown that autophagy can promote PDAC progression. We recently established the importance of autophagy in regulating bioavailable iron to control mitochondrial metabolism in PDAC. We found that inhibition of autophagy in PDAC leads to mitochondrial dysfunction due to abrogation of succinate dehydrogenase complex iron sulfur subunit B (SDHB) expression. Additionally, we observed that cancer-associated fibroblasts (CAFs) can provide iron to autophagy-inhibited PDAC tumor cells, thereby increasing their resistance to autophagy inhibition. To impede such metabolic compensation, we used a low iron diet together with autophagy inhibition and demonstrated a significant improvement of tumor response in syngeneic PDAC models.Abbreviations: PDAC: Pancreatic ductal adenocarcinoma; CAFs: cancer-associated fibroblasts; SDHB: succinate dehydrogenase complex iron sulfur subunit B; ISCA1: iron sulfur cluster assembly protein 1; FPN: ferroportin; LIP: labile iron pool; FAC: ferric ammonium chloride; OCR: oxygen consumption rate; OXPHOS: oxidative phosphorylation, IL6: interleukin 6; Fe-S: iron sulfur; ATP: adenosine triphosphate.
    Keywords:  Autophagy; cancer associated fibroblasts; iron metabolism; lysosome; mitochondria; pancreatic ductal adenocarcinoma
    DOI:  https://doi.org/10.1080/15548627.2023.2223473
  15. Autophagy. 2023 Jun 13.
    ATG8 proteins are co-factors for human dopaminergic neuronal transcriptional control: implications for neuronal resilience in Parkinson disease.
    Natalia Jiménez-Moreno, Jon D Lane.
      Parkinson disease (PD) is caused by the loss of ventral midbrain dopaminergic neurons (mDANs) in the substantia nigra pars compacta (SNpc). These cells are especially vulnerable to stress but can be protected by autophagy enhancement strategies in vitro and in vivo. In our recent study, we focused on the LIM (Lin11, Isl-1, and Mec-3)-domain homeobox transcription factors LMX1A (LIM homeobox transcription factor 1 alpha) and LMX1B (LIM homeobox transcription factor 1 beta), crucial drivers of mDAN differentiation with roles in autophagy gene expression for stress protection in the developed brain. Using human induced pluripotent stem cell (hiPSC)-derived mDANs and transformed human cell lines, we found that these autophagy gene transcription factors are themselves regulated by autophagy-mediated turnover. LMX1B possesses a non-canonical LC3-interacting region (LIR) in its C-terminus through which it interacts with ATG8 family members. The LMX1B LIR-like domain enables binding to ATG8 proteins in the nucleus, where ATG8 proteins act as co-factors for robust transcription of LMX1B target genes. Thus, we propose a novel role for ATG8 proteins as autophagy gene transcriptional co-factors for mDAN stress protection in PD.
    Keywords:  ATG8; Autophagy; Parkinson disease; dopaminergic neurons; stem cells; transcription factors
    DOI:  https://doi.org/10.1080/15548627.2023.2221958
  16. iScience. 2023 Jun 16. 26(6): 106970
    Restored autophagy is protective against PAK3-induced cardiac dysfunction.
    Andrea Ruiz-Velasco, Rida Raja, Xinyi Chen, Haresh Ganenthiran, Namrita Kaur, Nasser Hawimel O Alatawi, Jessica M Miller, Riham R E Abouleisa, Qinghui Ou, Xiangjun Zhao, Oveena Fonseka, Xin Wang, Susanne S Hille, Norbert Frey, Tao Wang, Tamer M A Mohamed, Oliver J Müller, Elizabeth J Cartwright, Wei Liu.
      Despite the development of clinical treatments, heart failure remains the leading cause of mortality. We observed that p21-activated kinase 3 (PAK3) was augmented in failing human and mouse hearts. Furthermore, mice with cardiac-specific PAK3 overexpression exhibited exacerbated pathological remodeling and deteriorated cardiac function. Myocardium with PAK3 overexpression displayed hypertrophic growth, excessive fibrosis, and aggravated apoptosis following isoprenaline stimulation as early as two days. Mechanistically, using cultured cardiomyocytes and human-relevant samples under distinct stimulations, we, for the first time, demonstrated that PAK3 acts as a suppressor of autophagy through hyper-activation of the mechanistic target of rapamycin complex 1 (mTORC1). Defective autophagy in the myocardium contributes to the progression of heart failure. More importantly, PAK3-provoked cardiac dysfunction was mitigated by administering an autophagic inducer. Our study illustrates a unique role of PAK3 in autophagy regulation and the therapeutic potential of targeting this axis for heart failure.
    Keywords:  Cell biology; Molecular biology; Omics
    DOI:  https://doi.org/10.1016/j.isci.2023.106970
  17. Oncol Res. 2022 ;30(1): 1-12
    Is autophagy induction by PARP inhibitors a target for therapeutic benefit?
    Ahmed M Elshazly, Tuong Vi V Nguyen, David A Gewirtz.
      PARP inhibitors have proven to be effective in conjunction with conventional therapeutics in the treatment of various solid as well as hematologic malignancies, particularly when the tumors are deficient in DNA repair pathways. However, as the case with other chemotherapeutic agents, their effectiveness is often compromised by the development of resistance. PARP inhibitors have consistently been reported to promote autophagy, a process that maintains cellular homeostasis and acts as an energy source by the degradation and reutilization of damaged subcellular organelles and proteins. Autophagy can exhibit different functional properties, the most prominent being cytoprotective. In addition, both cytotoxic and non-protective functions forms have also been identified. In this review, we explore the available literature regarding the different roles of autophagy in response to clinically-used PARP inhibitors, highlighting the possibility of targeting autophagy as an adjuvant therapy to potentially increase the effectiveness of PARP inhibition and to overcome the development of resistance.
    Keywords:  Autophagy; Cytoprotective; Cytotoxic; Niraparib; Non-protective; Olaparib; Rucaparib; Talazoparib
    DOI:  https://doi.org/10.32604/or.2022.026459
  18. Mol Metab. 2023 Jun 13. pii: S2212-8778(23)00087-X. [Epub ahead of print] 101753
    Salt-inducible kinase inhibition promotes the adipocyte thermogenic program and adipose tissue browning.
    Fubiao Shi, Flaviane de Fatima Silva, Dianxin Liu, Hari U Patel, Jonathan Xu, Wei Zhang, Clara Türk, Marcus Krüger, Sheila Collins.
      OBJECTIVE: Norepinephrine stimulates the adipose tissue thermogenic program through a β-adrenergic receptor (βAR) - cyclic adenosine monophosphate (cAMP) - protein kinase A (PKA) signaling cascade. We discovered that a noncanonical activation of the mechanistic target of rapamycin complex 1 (mTORC1) by PKA is required for the βAR-stimulation of adipose tissue browning. However, the downstream events triggered by PKA-phosphorylated mTORC1 activation that drive this thermogenic response are not well understood.METHODS: We used a proteomic approach of Stable Isotope Labeling by/with Amino acids in Cell culture (SILAC) to characterize the global protein phosphorylation profile in brown adipocytes treated with the βAR agonist. We identified salt-inducible kinase 3 (SIK3) as a candidate mTORC1 substrate and further tested the effect of SIK3 deficiency or SIK inhibition on the thermogenic gene expression program in brown adipocytes and in mouse adipose tissue.
    RESULTS: SIK3 interacts with RAPTOR, the defining component of the mTORC1 complex, and is phosphorylated at Ser884 in a rapamycin-sensitive manner. Pharmacological SIK inhibition by a pan-SIK inhibitor (HG-9-91-01) in brown adipocytes increases basal Ucp1 gene expression and restores its expression upon blockade of either mTORC1 or PKA. Short-hairpin RNA (shRNA) knockdown of Sik3 augments, while overexpression of SIK3 suppresses, Ucp1 gene expression in brown adipocytes. The regulatory PKA phosphorylation domain of SIK3 is essential for its inhibition. CRISPR-mediated Sik3 deletion in brown adipocytes increases type IIa histone deacetylase (HDAC) activity and enhances the expression of genes involved in thermogenesis such as Ucp1, Pgc1α, and mitochondrial OXPHOS complex protein. We further show that HDAC4 interacts with PGC1α after βAR stimulation and reduces lysine acetylation in PGC1α. Finally, a SIK inhibitor well-tolerated in vivo (YKL-05-099) can stimulate the expression of thermogenesis-related genes and browning of mouse subcutaneous adipose tissue.
    CONCLUSIONS: Taken together, our data reveal that SIK3, with the possible contribution of other SIKs, functions as a phosphorylation switch for β-adrenergic activation to drive the adipose tissue thermogenic program and indicates that more work to understand the role of the SIKs is warranted. Our findings also suggest that maneuvers targeting SIKs could be beneficial for obesity and related cardiometabolic disease.
    Keywords:  Adipocyte; SIK3; Salt-inducible kinase; Thermogenesis; UCP1; mTORC1
    DOI:  https://doi.org/10.1016/j.molmet.2023.101753
  19. J Cell Sci. 2023 Jun 14. pii: jcs.259986. [Epub ahead of print]
    Inner mitochondrial membrane fission protein, MTP18, serves as a mitophagy receptor to prevent apoptosis in oral cancer.
    Debasna P Panigrahi, Prakash P Praharaj, Bishnu P Behera, Srimanta Patra, Shankargouda Patil, Birija Sankar Patro, Sujit K Bhutia.
      MTP18, an inner mitochondrial membrane protein, plays a vital role in maintaining mitochondrial morphology. Furthermore, MTP18 induces mitochondrial fission with subsequent mitophagy, functioning as a mitophagy receptor that targets dysfunctional mitochondria into autophagosomes for elimination. Interestingly, MTP18 interacts with LC3 through its LC3 interacting region (LIR) to induce mitochondrial autophagy. Mutation in the LIR motif (mLIR) inhibits that interaction, thus suppressing mitophagy. Moreover, Parkin/PINK1 deficiency abrogates mitophagy in MTP18-overexpressing FaDu cells. Upon exposure to CCCP, MTP18[mLIR]-FaDu cells show decreased TOM20 expression without affecting COX IV expression. Conversely, loss of Parkin/PINK1 results in inhibition of TOM20 and COX IV degradation in MTP18[mLIR]-FaDu cells exposed to CCCP, establishing Parkin-mediated proteasomal degradation of outer mitochondrial membrane as essential for effective mitophagy. We found that MTP18 provides a survival advantage to oral cancer cells exposed to cellular stress and that inhibition of MTP18-dependent mitophagy induced cell death in oral cancer cells. The findings demonstrate that MTP18 is a novel mitophagy receptor and that MTP18-dependent mitophagy has pathophysiologic implications for oral cancer progression, indicating inhibition of MTP18-mitophagy could thus be a promising cancer therapy strategy.
    Keywords:  Apoptosis; MTP18; Mitochondrial fission; Mitophagy; Parkin
    DOI:  https://doi.org/10.1242/jcs.259986
  20. J Reprod Immunol. 2023 May 30. pii: S0165-0378(23)00179-1. [Epub ahead of print]158 103973
    Autophagy-mediated immune system regulation in reproductive system and pregnancy-associated complications.
    Erfan Komijani, Forough Parhizkar, Samaneh Abdolmohammadi-Vahid, Hamid Ahmadi, Narjes Nouri, Mehdi Yousefi, Leili Aghebati-Maleki.
      Autophagy lysosomal degradation is the main cell mechanism in cellular, tissue and organismal homeostasis and is controlled by autophagy-related genes (ATG). Autophagy has important effects in cellular physiology, including adaptation to metabolic stress, removal of dangerous cargo (such as protein aggregates, damaged organelles, and intracellular pathogens), regeneration during differentiation and development, and prevention of genomic damage in general. Also, it has been found that autophagy is essential for pre-implantation, development, and maintaining embryo survival in mammals. Under certain conditions, autophagy may be detrimental through pro-survival effects such as cancer progression or through possible cell death-promoting effects. Hormonal changes and environmental stress can initiate autophagy in reproductive physiology. The activity of autophagy can be upregulated under conditions like a lack of nutrients, inflammation, hypoxia, and infections. In this regard the dysregulation of autophagy involved in some pregnancy complications such as preeclampsia (PE) and pregnancy loss, and has a major impact on reproductive outcomes. Therefore, we aimed to discuss the relationship between autophagy and the female reproductive system, with a special focus on the immune system, and its role in fetal and maternal health.
    Keywords:  Autophagy; Immune system; Pregnancy
    DOI:  https://doi.org/10.1016/j.jri.2023.103973
  21. Anal Chim Acta. 2023 Aug 29. pii: S0003-2670(23)00669-4. [Epub ahead of print]1271 341448
    Ratiometric visualization of lysosomal pH fluctuations during autophagy by two-photon carbonized polymer dots-based probe.
    Genxiu Yao, Junyong Sun, Shan Miao, Yue Wang, Feng Gao.
      Monitoring the pH variation in lysosomes is very conducive to studying the autophagy process, and fluorescent ratiometric pH nanoprobes with inherent lysosome targeting ability are highly desirable. Here, a carbonized polymer dots-based pH probe (oAB-CPDs) was developed by self-condensation of o-aminobenzaldehyde and further carbonization at low temperature. The obtained oAB-CPDs display improved performance in pH sensing, including robust photostability, intrinsic lysosome-targeting ability, self-referenced ratiometric response, desirable two-photon-sensitized fluorescence property, and high selectivity. With the suitable pKa value of 5.89, the as-prepared nanoprobe was successfully applied to monitor the variation of lysosomal pH in HeLa cells. Moreover, the occurrence that lysosomal pH decreased during both starvation-induced and rapamycin-induced autophagy was observed by using oAB-CPDs as fluorescence probe. We believe that nanoprobe oAB-CPDs can work as a useful tool for visualizing autophagy in living cells.
    Keywords:  Autophagy; Carbonized polymer dots; Low-temperature synthesis; Ratiometric pH probe
    DOI:  https://doi.org/10.1016/j.aca.2023.341448
  22. J Vis Exp. 2023 May 19.
    Detection of Mitophagy in Caenorhabditis elegans and Mammalian Cells Using Organelle-Specific Dyes.
    Vijigisha Srivastava, Einav Gross.
      Mitochondria are essential for various biological functions, including energy production, lipid metabolism, calcium homeostasis, heme biosynthesis, regulated cell death, and the generation of reactive oxygen species (ROS). ROS are vital for key biological processes. However, when uncontrolled, they can lead to oxidative injury, including mitochondrial damage. Damaged mitochondria release more ROS, thereby intensifying cellular injury and the disease state. A homeostatic process named mitochondrial autophagy (mitophagy) selectively removes damaged mitochondria, which are then replaced by new ones. There are multiple mitophagy pathways, with the common endpoint being the breakdown of the damaged mitochondria in lysosomes. Several methodologies, including genetic sensors, antibody immunofluorescence, and electron microscopy, use this endpoint to quantify mitophagy. Each method for examining mitophagy has its advantages, such as specific tissue/cell targeting (with genetic sensors) and great detail (with electron microscopy). However, these methods often require expensive resources, trained personnel, and a lengthy preparation time before the actual experiment, such as for creating transgenic animals. Here, we present a cost-effective alternative for measuring mitophagy using commercially available fluorescent dyes targeting mitochondria and lysosomes. This method effectively measures mitophagy in the nematode Caenorhabditis elegans and human liver cells, which indicates its potential efficiency in other model systems.
    DOI:  https://doi.org/10.3791/65337
  23. Sci Adv. 2023 Jun 16. 9(24): eadd8910
    Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation.
    Dmitry Zenko, Jade Marsh, Andrew R Castle, Rahel Lewin, Roman Fischer, George K Tofaris.
      While defective α-synuclein homeostasis is central to Parkinson's pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of α-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.
    DOI:  https://doi.org/10.1126/sciadv.add8910
  24. Biomed Pharmacother. 2023 Jun 08. pii: S0753-3322(23)00770-9. [Epub ahead of print]164 114980
    Cinchonine exerts anti-tumor and immunotherapy sensitizing effects in lung cancer by impairing autophagic-lysosomal degradation.
    Huan Wang, Yuting Shi, Dannv Ma, Mengqing Cao, Yuchao Sun, Xinyuan Jiang, Zhiyong Xu, Yongfang Wang, Ying Yang, Yueli Shi, Kai Wang.
      Currently, there are several treatments approaches available for lung cancer; however, patients who develop drug resistance or have poor survival rates urgently require new therapeutic strategies for lung cancer. In autophagy, damaged proteins or organelles are enclosed within autophagic vesicles with a bilayer membrane structure and transported to the lysosomes for degradation and recirculation. Autophagy is a crucial pathway involved in the clearance of reactive oxygen species (ROS) and damaged mitochondria. Meanwhile, inhibiting autophagy is a promising strategy for cancer treatment. In this study, we found for the first time that Cinchonine (Cin) can act as an autophagy suppressor and exert anti-tumor effects. Cin significantly inhibited the proliferation, migration, and invasion of cancer cells in vitro and the tumor growth and metastasis in vivo, without obvious toxic effects. We found that Cin suppressed the autophagic process by blocking autophagosome degradation through the inhibition of the maturation of lysosomal hydrolases. Cin-mediated autophagy inhibition resulted in the elevated ROS level and the accumulation of damaged mitochondria, which in turn promoted apoptosis. N-acetylcysteine, a potential ROS scavenger, significantly suppressed Cin-induced apoptosis. Additionally, Cin upregulated programmed death-ligand 1 (PD-L1) expression in lung cancer cells by inhibiting autophagy. Compared with monotherapy and control group, the combined administration of anti-PD-L1 antibody and Cin significantly reduced tumor growth. These results suggest that Cin exerts anti-tumor effects by inhibiting autophagy, and that the combination of Cin and PD-L1 blockade has synergistic anti-tumor effects. The data demonstrates the significant clinical potential of Cin in lung cancer treatment.
    Keywords:  Autophagy; Cinchonine; Lung cancer; Lysosome; PD-L1
    DOI:  https://doi.org/10.1016/j.biopha.2023.114980
  25. Int J Mol Sci. 2023 Jun 02. pii: 9686. [Epub ahead of print]24(11):
    The Emerging Role of Autophagy-Associated lncRNAs in the Pathogenesis of Neurodegenerative Diseases.
    Yapei Jiang, Naihan Xu.
      Neurodegenerative diseases (NDDs) have become a significant global public health problem and a major societal burden. The World Health Organization predicts that NDDs will overtake cancer as the second most common cause of human mortality within 20 years. Thus, it is urgently important to identify pathogenic and diagnostic molecular markers related to neurodegenerative processes. Autophagy is a powerful process for removing aggregate-prone proteins in neurons; defects in autophagy are often associated with the pathogenesis of NDDs. Long non-coding RNAs (lncRNAs) have been suggested as key regulators in neurodevelopment; aberrant regulation of lncRNAs contributes to neurological disorders. In this review, we summarize the recent progress in the study of lncRNAs and autophagy in the context of neurodegenerative disorders, especially Alzheimer's disease (AD) and Parkinson's disease (PD). The information presented here should provide guidance for future in-depth investigations of neurodegenerative processes and related diagnostic molecular markers and treatment targets.
    Keywords:  Alzheimer’s disease; Parkinson’s disease; autophagy; lncRNA; neurodegenerative disease
    DOI:  https://doi.org/10.3390/ijms24119686
  26. Aging (Albany NY). 2023 Jun 13. 15
    LAMP2A, and other chaperone-mediated autophagy related proteins, do not decline with age in genetically heterogeneous UM-HET3 mice.
    Katherine K Zhang, Peichuan Zhang, Anagha Kodur, Ilkim Erturk, Calvin M Burns, Cynthia Kenyon, Richard A Miller, S Joseph Endicott.
      Chaperone-mediated autophagy (CMA) selectively degrades proteins that are crucial for glycolysis, fatty acid metabolism, and the progression of several age-associated diseases. Several previous studies, each of which evaluated males of a single inbred mouse or rat strain, have reported that CMA declines with age in many tissues, attributed to an age-related loss of LAMP2A, the primary and indispensable component of the CMA translocation complex. This has led to a paradigm in the field of CMA research, stating that the age-associated decline in LAMP2A in turn decreases CMA, contributing to the pathogenesis of late-life disease. We assessed LAMP2A levels and CMA substrate uptake in both sexes of the genetically heterogeneous UM-HET3 mouse stock, which is the current global standard for the evaluation of anti-aging interventions. We found no evidence for age-related changes in LAMP2A levels, CMA substrate uptake, or whole liver levels of CMA degradation targets, despite identifying sex differences in CMA.
    Keywords:  aging; autophagy; chaperone-mediated autophagy
    DOI:  https://doi.org/10.18632/aging.204796
  27. Exp Neurol. 2023 Jun 08. pii: S0014-4886(23)00148-6. [Epub ahead of print]367 114463
    SNAP25 ameliorates postoperative cognitive dysfunction by facilitating PINK1-dependent mitophagy and impeding caspase-3/GSDME-dependent pyroptosis.
    Wei Wang, Wenwei Gao, Lei Zhang, Zhongyuan Xia, Bo Zhao.
      Insufficient PTEN-induced kinase 1 (PINK1)-mediated mitophagy and activation of caspase-3/gasdermin E (GSDME)-dependent pyroptosis constitute the potential etiology of postoperative cognitive dysfunction (POCD), a severe neurological complication characterized by learning and memory deficits. Synaptosomal-Associated Protein 25 (SNAP25), a well-defined presynaptic protein that mediates the fusion between synaptic vesicles and plasma membrane, is crucial in autophagy and the trafficking of extracellular proteins to the mitochondria. We investigated whether SNAP25 regulates POCD via mitophagy and pyroptosis. SNAP25 downregulation was observed in the hippocampi of rats undergoing isoflurane anesthesia and laparotomy. SNAP25 silencing restrained PINK1-mediated mitophagy and promoted reactive oxygen species (ROS) production and caspase-3/GSDME-dependent pyroptosis in isoflurane (Iso) + lipopolysaccharide (LPS)-primed SH-SY5Y cells. SNAP25 depletion also destabilized PINK1 on the outer membrane of the mitochondria and blocked Parkin translocation to the mitochondria. In contrast, SNAP25 overexpression alleviated POCD and Iso + LPS-induced defective mitophagy and pyroptosis, which was reversed by PINK1 knockdown. These findings suggest that SNAP25 exerts neuroprotective effects against POCD by boosting PINK1-dependent mitophagy and hindering caspase-3/GSDME-dependent pyroptosis, providing a novel option for the management of POCD.
    Keywords:  Caspase-3; GSDME; Mitophagy; PINK1; Postoperative cognitive dysfunction; Pyroptosis; SNAP25
    DOI:  https://doi.org/10.1016/j.expneurol.2023.114463
  28. Int J Oncol. 2023 Jul;pii: 87. [Epub ahead of print]63(1):
    Histone lysine demethylase 3B regulates autophagy via transcriptional regulation of GABARAPL1 in acute myeloid leukemia cells.
    Ying Song, Jiaqi Zhang, Haihua Wang, Haiying Wang, Yong Liu, Zhenbo Hu.
      Macroautophagy (hereafter referred to as autophagy) is a highly conserved self‑digestion process that is critical for maintaining homeostasis in response to various stresses. The autophagy‑related protein family, including the GABA type A receptor‑associated protein (GABARAP) and microtubule‑associated protein 1 light chain 3 subfamilies, is crucial for autophagosome biogenesis. Although the regulatory machinery of autophagy in the cytoplasm has been widely studied, its transcriptional and epigenetic regulatory mechanisms still require more targeted investigations. The present study identified histone lysine demethylase 3B (KDM3B) as a crucial component of autophagy on a panel of leukemia cell lines, including K562, THP1 and U937, resulting in transcriptional activation of the autophagy‑related gene GABA type A receptor‑associated protein like 1 (GABARAPL1). KDM3B expression promoted autophagosome formation and affected the autophagic flux in leukemia cells under the induction of external stimuli. Notably, RNA‑sequencing and reverse transcription‑quantitative PCR analysis showed that KDM3B knockout inhibited the expression of GABARAPL1. Chromatin immunoprecipitation‑quantitative PCR and luciferase assay showed that KDM3B was associated with the GABARAPL1 gene promoter under stimulation and enhanced its transcription. The present findings demonstrated that KDM3B was critical for regulating the GABARAPL1 gene and influencing the process of autophagy in leukemia cells. These results provide a new insight for exploring the association between autophagy and KDM3B epigenetic regulation in leukemia.
    Keywords:  GABA type A receptor‑associated protein like 1; autophagy; histone lysine demethylase 3B; leukemia; transcriptional regulation
    DOI:  https://doi.org/10.3892/ijo.2023.5535
  29. Cells. 2023 May 30. pii: 1514. [Epub ahead of print]12(11):
    Proteasomes of Autophagy-Deficient Cells Exhibit Alterations in Regulatory Proteins and a Marked Reduction in Activity.
    Qiuhong Xiong, Rong Feng, Sarah Fischer, Malte Karow, Maria Stumpf, Susanne Meßling, Leonie Nitz, Stefan Müller, Christoph S Clemen, Ning Song, Ping Li, Changxin Wu, Ludwig Eichinger.
      Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is accumulating that there is extensive crosstalk between the two pathways, but the underlying mechanisms are still unclear. We previously found that autophagy 9 (ATG9) and 16 (ATG16) proteins are crucial for full proteasomal activity in the unicellular amoeba Dictyostelium discoideum. In comparison to AX2 wild-type cells, ATG9-and ATG16- cells displayed a 60%, and ATG9-/16- cells a 90%, decrease in proteasomal activity. Mutant cells also showed a significant increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates. Here, we focus on possible reasons for these results. Reanalysis of published tandem mass tag-based quantitative proteomic results of AX2, ATG9-, ATG16-, and ATG9-/16- cells revealed no change in the abundance of proteasomal subunits. To identify possible differences in proteasome-associated proteins, we generated AX2 wild-type and ATG16- cells expressing the 20S proteasomal subunit PSMA4 as GFP-tagged fusion protein, and performed co-immunoprecipitation experiments followed by mass spectrometric analysis. The results revealed no significant differences in the abundance of proteasomes between the two strains. However, we found enrichment as well as depletion of proteasomal regulators and differences in the ubiquitination of associated proteins for ATG16-, as compared to AX2 cells. Recently, proteaphagy has been described as a means to replace non-functional proteasomes. We propose that autophagy-deficient D. discoideum mutants suffer from inefficient proteaphagy, which results in the accumulation of modified, less-active, and also of inactive, proteasomes. As a consequence, these cells exhibit a dramatic decrease in proteasomal activity and deranged protein homeostasis.
    Keywords:  ATG16; ATG9; Dictyostelium; autophagy; ubiquitin proteasome system (UPS)
    DOI:  https://doi.org/10.3390/cells12111514
  30. Nat Commun. 2023 Jun 13. 14(1): 3497
    TMX4-driven LINC complex disassembly and asymmetric autophagy of the nuclear envelope upon acute ER stress.
    Marika K Kucińska, Juliette Fedry, Carmela Galli, Diego Morone, Andrea Raimondi, Tatiana Soldà, Friedrich Förster, Maurizio Molinari.
      The endoplasmic reticulum (ER) is an organelle of nucleated cells that produces proteins, lipids and oligosaccharides. ER volume and activity are increased upon induction of unfolded protein responses (UPR) and are reduced upon activation of ER-phagy programs. A specialized domain of the ER, the nuclear envelope (NE), protects the cell genome with two juxtaposed lipid bilayers, the inner and outer nuclear membranes (INM and ONM) separated by the perinuclear space (PNS). Here we report that expansion of the mammalian ER upon homeostatic perturbations results in TMX4 reductase-driven disassembly of the LINC complexes connecting INM and ONM and in ONM swelling. The physiologic distance between ONM and INM is restored, upon resolution of the ER stress, by asymmetric autophagy of the NE, which involves the LC3 lipidation machinery, the autophagy receptor SEC62 and the direct capture of ONM-derived vesicles by degradative LAMP1/RAB7-positive endolysosomes in a catabolic pathway mechanistically defined as micro-ONM-phagy.
    DOI:  https://doi.org/10.1038/s41467-023-39172-3
  31. Life Sci Alliance. 2023 Sep;pii: e202301920. [Epub ahead of print]6(9):
    PI3P-dependent regulation of cell size and autophagy by phosphatidylinositol 5-phosphate 4-kinase.
    Avishek Ghosh, Aishwarya Venugopal, Dhananjay Shinde, Sanjeev Sharma, Meera Krishnan, Swarna Mathre, Harini Krishnan, Sankhanil Saha, Padinjat Raghu.
      Phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 5-phosphate (PI5P) are low-abundance phosphoinositides crucial for key cellular events such as endosomal trafficking and autophagy. Phosphatidylinositol 5-phosphate 4-kinase (PIP4K) is an enzyme that regulates PI5P in vivo but can act on both PI5P and PI3P in vitro. In this study, we report a role for PIP4K in regulating PI3P levels in Drosophila Loss-of-function mutants of the only Drosophila PIP4K gene show reduced cell size in salivary glands. PI3P levels are elevated in dPIP4K 29 and reverting PI3P levels back towards WT, without changes in PI5P levels, can rescue the reduced cell size. dPIP4K 29 mutants also show up-regulation in autophagy and the reduced cell size can be reverted by depleting Atg8a that is required for autophagy. Lastly, increasing PI3P levels in WT can phenocopy the reduction in cell size and associated autophagy up-regulation seen in dPIP4K 29 Thus, our study reports a role for a PIP4K-regulated PI3P pool in the control of autophagy and cell size.
    DOI:  https://doi.org/10.26508/lsa.202301920
  32. Virol J. 2023 Jun 16. 20(1): 125
    Role and clinical implication of autophagy in COVID-19.
    Tianjiao Shan, Lan-Ya Li, Jin-Ming Yang, Yan Cheng.
      The ongoing coronavirus disease 2019 (COVID-19) pandemic constitutes a serious public health concern worldwide. Currently, more than 6 million deaths have occurred despite drastic containment measures, and this number is still increasing. Currently, no standard therapies for COVID-19 are available, which necessitates identifying effective preventive and therapeutic agents against COVID-19. However, developing new drugs and vaccines is a time-consuming process, and therefore, repurposing the existing drugs or redeveloping related targets seems to be the best strategy to develop effective therapeutics against COVID-19. Autophagy, a multistep lysosomal degradation pathway contributing to nutrient recycling and metabolic adaptation, is involved in the initiation and progression of numerous diseases as a part of an immune response. The key role of autophagy in antiviral immunity has been extensively studied. Moreover, autophagy can directly eliminate intracellular microorganisms by selective autophagy, that is, "xenophagy." However, viruses have acquired diverse strategies to exploit autophagy for their infection and replication. This review aims to trigger the interest in the field of autophagy as an antiviral target for viral pathogens (with an emphasis on COVID-19). We base this hypothesis on summarizing the classification and structure of coronaviruses as well as the process of SARS-CoV-2 infection and replication; providing the common understanding of autophagy; reviewing interactions between the mechanisms of viral entry/replication and the autophagy pathways; and discussing the current state of clinical trials of autophagy-modifying drugs in the treatment of SARS-CoV-2 infection. We anticipate that this review will contribute to the rapid development of therapeutics and vaccines against COVID-19.
    Keywords:  Autophagy; COVID-19; Clinical treatments; Coronavirus; Infectious diseases; SARS-CoV-2; Vaccines
    DOI:  https://doi.org/10.1186/s12985-023-02069-0
  33. Cell Rep. 2023 Jun 09. pii: S2211-1247(23)00642-3. [Epub ahead of print]42(6): 112631
    The RagA GTPase protects young egg chambers in Drosophila.
    Ying Zhou, Jianwen Guan, Guoqiang Meng, Weikang Fan, Churui Ge, Chunmei Niu, Yang Cheng, Yuanyuan Fu, Yingying Lu, Youheng Wei.
      The preservation of female fertility under unfavorable conditions is essential for animal reproduction. Inhibition of the target of rapamycin complex 1 (TORC1) is indispensable for Drosophila young egg chamber maintenance under nutrient starvation. Here, we show that knockdown of RagA results in young egg chamber death independent of TORC1 hyperactivity. RagA RNAi ovaries have autolysosomal acidification and degradation defects, which make the young egg chambers sensitive to autophagosome augmentation. Meanwhile, RagA RNAi ovaries have nuclear-localized Mitf, which promotes autophagic degradation and protects young egg chambers under stress. Interestingly, GDP-bound RagA rescues autolysosome defects, while GTP-bound RagA rescues Mitf nuclear localization in RagA RNAi young egg chambers. Moreover, Rag GTPase activity, rather than TORC1 activity, controls Mitf cellular localization in the Drosophila germ line. Our work suggests that RagA separately controls autolysosomal acidification and Mitf activity in the Drosophila young egg chambers.
    Keywords:  CP: Developmental biology; Drosophila; Mitf; Rag GTPases; apoptosis; autophagy; oogenesis
    DOI:  https://doi.org/10.1016/j.celrep.2023.112631
  34. Ageing Res Rev. 2023 Jun 13. pii: S1568-1637(23)00144-7. [Epub ahead of print] 101985
    TFEB is a central regulator of the aging process and age-related diseases.
    Samuel Abokyi, George Ghartey-Kwansah, Dennis Yan-Yin Tse.
      Old age is associated with a greater burden of disease, including neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, as well as other chronic diseases. Coincidentally, popular lifestyle interventions, such as caloric restriction, intermittent fasting, and regular exercise, in addition to pharmacological interventions intended to protect against age-related diseases, induce transcription factor EB (TFEB) and autophagy. In this review, we summarize emerging discoveries that point to TFEB activity affecting the hallmarks of aging, including inhibiting DNA damage and epigenetic modifications, inducing autophagy and cell clearance to promote proteostasis, regulating mitochondrial quality control, linking nutrient-sensing to energy metabolism, regulating pro- and anti-inflammatory pathways, inhibiting senescence and promoting cell regenerative capacity. Furthermore, the therapeutic impact of TFEB activation on normal aging and tissue-specific disease development is assessed in the contexts of neurodegeneration and neuroplasticity, stem cell differentiation, immune responses, muscle energy adaptation, adipose tissue browning, hepatic functions, bone remodeling, and cancer. Safe and effective strategies of activating TFEB hold promise as a therapeutic strategy for multiple age-associated diseases and for extending lifespan.
    Keywords:  caloric restriction; cell regenerative capacity; hallmarks of aging; lifespan extension; neuroplasticity; senescence
    DOI:  https://doi.org/10.1016/j.arr.2023.101985
  35. Cells. 2023 Jun 05. pii: 1553. [Epub ahead of print]12(11):
    Advances in Understanding the Links between Metabolism and Autophagy in Acute Myeloid Leukemia: From Biology to Therapeutic Targeting.
    Ernestina Saulle, Isabella Spinello, Maria Teresa Quaranta, Catherine Labbaye.
      Autophagy is a highly conserved cellular degradation process that regulates cellular metabolism and homeostasis under normal and pathophysiological conditions. Autophagy and metabolism are linked in the hematopoietic system, playing a fundamental role in the self-renewal, survival, and differentiation of hematopoietic stem and progenitor cells, and in cell death, particularly affecting the cellular fate of the hematopoietic stem cell pool. In leukemia, autophagy sustains leukemic cell growth, contributes to survival of leukemic stem cells and chemotherapy resistance. The high frequency of disease relapse caused by relapse-initiating leukemic cells resistant to therapy occurs in acute myeloid leukemia (AML), and depends on the AML subtypes and treatments used. Targeting autophagy may represent a promising strategy to overcome therapeutic resistance in AML, for which prognosis remains poor. In this review, we illustrate the role of autophagy and the impact of its deregulation on the metabolism of normal and leukemic hematopoietic cells. We report updates on the contribution of autophagy to AML development and relapse, and the latest evidence indicating autophagy-related genes as potential prognostic predictors and drivers of AML. We review the recent advances in autophagy manipulation, combined with various anti-leukemia therapies, for an effective autophagy-targeted therapy for AML.
    Keywords:  acute myeloid leukemia; autophagy; hematopoiesis; metabolism; therapy resistance
    DOI:  https://doi.org/10.3390/cells12111553
  36. ACS Infect Dis. 2023 Jun 10.
    Mitophagy Activation Targeting PINK1 Is an Effective Treatment to Inhibit Zika Virus Replication.
    Yike Huang, Qingyuan Li, Lan Kang, Bin Li, Haiyan Ye, Xiaoqiong Duan, He Xie, Man Jiang, Shilin Li, Ya Zhu, Qi Tan, Limin Chen.
      Mitophagy is a selective degradation mechanism that maintains mitochondrial homeostasis by eliminating damaged mitochondria. Many viruses manipulate mitophagy to promote their infection, but its role in Zika virus (ZIKV) is unclear. In this study, we investigated the effect of mitophagy activation on ZIKV replication by the mitochondrial uncoupling agent niclosamide. Our results demonstrate that niclosamide-induced mitophagy inhibits ZIKV replication by eliminating fragmented mitochondria, both in vitro and in a mouse model of ZIKV-induced necrosis. Niclosamide induces autophosphorylation of PTEN-induced putative kinase 1 (PINK1), leading to the recruitment of PRKN/Parkin to the outer mitochondrial membrane and subsequent phosphorylation of ubiquitin. Knockdown of PINK1 promotes ZIKV infection and rescues the anti-ZIKV effect of mitophagy activation, confirming the role of ubiquitin-dependent mitophagy in limiting ZIKV replication. These findings demonstrate the role of mitophagy in the host response in limiting ZIKV replication and identify PINK1 as a potential therapeutic target in ZIKV infection.
    Keywords:  PINK1; Zika virus; mitophagy; niclosamide; ubiquitin
    DOI:  https://doi.org/10.1021/acsinfecdis.3c00196
  37. Autophagy Rep. 2023 ;pii: 2213541. [Epub ahead of print]2(1):
    Interplay between septins and ubiquitin-mediated xenophagy during Shigella entrapment.
    Damián Lobato-Márquez, José Javier Conesa, Ana Teresa López-Jiménez, Michael E Divine, Jonathan N Pruneda, Serge Mostowy.
      Septins are cytoskeletal proteins implicated in numerous cellular processes including cytokinesis and morphogenesis. In the case of infection by Shigella flexneri, septins assemble into cage-like structures that entrap cytosolic bacteria targeted by autophagy. The interplay between septin cage entrapment and bacterial autophagy is poorly understood. We used a correlative light and cryo-soft X-ray tomography (cryo-SXT) pipeline to study septin cage entrapment of Shigella in its near-native state. Septin cages could be identified as X-ray dense structures, indicating they contain host cell proteins and lipids consistent with their autophagy links. Airyscan confocal microscopy of Shigella-septin cages showed that septins and lysine 63 (K63)-linked ubiquitin chains are present in separate bacterial microdomains, suggesting they are recruited separately. Finally, Cryo-SXT and live-cell imaging revealed an interaction between septins and microtubule-associated protein light chain 3B (LC3B)-positive membranes during autophagy of Shigella. Collectively our data present a new model for how septin-caged Shigella are targeted to autophagy.
    Keywords:  Shigella; autophagy; cryo-SXT; cytoskeleton; septins; ubiquitin
    DOI:  https://doi.org/10.1080/27694127.2023.2213541
  38. EMBO J. 2023 Jun 12. e113349
    Phosphorylation of phase-separated p62 bodies by ULK1 activates a redox-independent stress response.
    Ryo Ikeda, Daisuke Noshiro, Hideaki Morishita, Shuhei Takada, Shun Kageyama, Yuko Fujioka, Tomoko Funakoshi, Satoko Komatsu-Hirota, Ritsuko Arai, Elena Ryzhii, Manabu Abe, Tomoaki Koga, Hozumi Motohashi, Mitsuyoshi Nakao, Kenji Sakimura, Arata Horii, Satoshi Waguri, Yoshinobu Ichimura, Nobuo N Noda, Masaaki Komatsu.
      NRF2 is a transcription factor responsible for antioxidant stress responses that is usually regulated in a redox-dependent manner. p62 bodies formed by liquid-liquid phase separation contain Ser349-phosphorylated p62, which participates in the redox-independent activation of NRF2. However, the regulatory mechanism and physiological significance of p62 phosphorylation remain unclear. Here, we identify ULK1 as a kinase responsible for the phosphorylation of p62. ULK1 colocalizes with p62 bodies, directly interacting with p62. ULK1-dependent phosphorylation of p62 allows KEAP1 to be retained within p62 bodies, thus activating NRF2. p62S351E/+ mice are phosphomimetic knock-in mice in which Ser351, corresponding to human Ser349, is replaced by Glu. These mice, but not their phosphodefective p62S351A/S351A counterparts, exhibit NRF2 hyperactivation and growth retardation. This retardation is caused by malnutrition and dehydration due to obstruction of the esophagus and forestomach secondary to hyperkeratosis, a phenotype also observed in systemic Keap1-knockout mice. Our results expand our understanding of the physiological importance of the redox-independent NRF2 activation pathway and provide new insights into the role of phase separation in this process.
    Keywords:  KEAP1; NRF2/NFE2L2; ULK1; liquid-liquid phase separation; p62/SQSTM1
    DOI:  https://doi.org/10.15252/embj.2022113349
  39. Clin Exp Med. 2023 Jun 13.
    Reduction in Rubicon by cigarette smoke is associated with impaired phagocytosis and occurs through lysosomal degradation pathway.
    Patrick F Asare, Plinio R Hurtado, Hai B Tran, Griffith B Perkins, Eugene Roscioli, Sandra Hodge.
      BACKGROUND: A common feature of COPD is a defective lung macrophage phagocytic capacity that can contribute to chronic lung inflammation and infection. The precise mechanisms remain incompletely understood, although cigarette smoke is a known contributor. We previously showed deficiency of the LC3-associated phagocytosis (LAP) regulator, Rubicon, in macrophages from COPD subjects and in response to cigarette smoke. The current study investigated the molecular basis by which cigarette smoke extract (CSE) reduces Rubicon in THP-1, alveolar and blood monocyte-derived macrophages, and the relationship between Rubicon deficiency and CSE-impaired phagocytosis.METHODOLOGY: Phagocytic capacity of CSE-treated macrophages was measured by flow cytometry, Rubicon expression by Western blot and real time polymerase chain reaction, and autophagic-flux by LC3 and p62 levels. The effect of CSE on Rubicon degradation was determined using cycloheximide inhibition and Rubicon protein synthesis and half-life assessment.
    RESULTS: Phagocytosis was significantly impaired in CSE-exposed macrophages and strongly correlated with Rubicon expression. CSE-impaired autophagy, accelerated Rubicon degradation, and reduced its half-life. Lysosomal protease inhibitors, but not proteasome inhibitors, attenuated this effect. Autophagy induction did not significantly affect Rubicon expression.
    CONCLUSIONS: CSE decreases Rubicon through the lysosomal degradation pathway. Rubicon degradation and/or LAP impairment may contribute to dysregulated phagocytosis perpetuated by CSE.
    Keywords:  Autophagy; Inflammation; LC3-associated phagocytosis (LAP); Phagocytosis; Rubicon
    DOI:  https://doi.org/10.1007/s10238-023-01105-1
  40. Brain Res. 2023 Jun 12. pii: S0006-8993(23)00233-0. [Epub ahead of print] 148462
    Dimethyl fumarate ameliorates Parkinsonian pathology by modulating autophagy and apoptosis via Nrf2-TIGAR-LAMP2/Cathepsin D axis.
    Mayuri Khot, Anika Sood, Kamatham Pushpa Tryphena, Poojitha Pinjala, Saurabh Srivastava, Shashi Bala Singh, Dharmendra Kumar Khatri.
      Mounting evidence suggests a role for oxidative stress and accumulation of dysfunctional organelle and misfolded proteins in PD. Autophagosomes mediate the clearance of these cytoplasmic proteins via delivery to lysosomes to form autophagolysosomes, followed by degradation of the protein by lysosomal enzymes. In PD, autophagolysosome accumulation occurs initiating a plethora of events resulting in neuronal death by apoptosis. This study evaluated the effect of Dimethylfumarate (DMF), an Nrf2 activator in the rotenone-induced mouse PD model. In PD mice, there was decreased expression of LAMP2 and LC3, which resulted in inhibition of autophagic flux and increased expression of cathepsin D, which mediated apoptosis. The role of Nrf2 activation in alleviating oxidative stress is well known. Our study elucidated the novel mechanism underlying the neuroprotective effect of DMF. The loss of dopaminergic neurons induced by rotenone was lessened to a significant extent by pre-treatment with DMF. DMF promoted autophagosome formation and inhibited apoptosis by removing the inhibitory effect of p53 on TIGAR. TIGAR expression upregulated LAMP2 expression and downregulated Cathepsin D, promoting autophagy and inhibiting apoptosis. Thus, it was proved that DMF confers neuroprotection against rotenone-induced dopaminergic neurodegeneration and could be used as a potential therapeutic agent for PD and its progression.
    Keywords:  Dimethyl fumarate; Parkinson’s disease; apoptosis; autophagy; rotenone
    DOI:  https://doi.org/10.1016/j.brainres.2023.148462
  41. Curr Opin Crit Care. 2023 Jun 07.
    Nutrition and autophagy deficiency in critical illness.
    Ilse Vanhorebeek, Michaël Casaer, Jan Gunst.
      PURPOSE OF REVIEW: Critical illness imposes a severe insult on the body, with various stressors triggering pronounced cell damage. This compromises cellular function, leading to a high risk of multiple organ failure. Autophagy can remove damaged molecules and organelles but appears insufficiently activated during critical illness. This review discusses insight into the role of autophagy in critical illness and the involvement of artificial feeding in insufficient autophagy activation in critical illness.RECENT FINDINGS: Animal studies manipulating autophagy have shown its protective effects against kidney, lung, liver, and intestinal injury after several critical insults. Autophagy activation also protected peripheral, respiratory, and cardiac muscle function, despite aggravated muscle atrophy. Its role in acute brain injury is more equivocal. Animal and patient studies showed that artificial feeding suppressed autophagy activation in critical illness, particularly with high protein/amino acid doses. Feeding-suppressed autophagy may explain short and long-term harm by early enhanced calorie/protein feeding in large randomized controlled trials.
    SUMMARY: Insufficient autophagy during critical illness is at least partly explained by feeding-induced suppression. This may explain why early enhanced nutrition failed to benefit critically ill patients or even induced harm. Safe, specific activation of autophagy avoiding prolonged starvation opens perspectives for improving outcomes of critical illness.
    DOI:  https://doi.org/10.1097/MCC.0000000000001056
  42. Autophagy. 2023 Jun 13. 1-17
    HSPA8 regulates anti-bacterial autophagy through liquid-liquid phase separation.
    Chunhui Miao, Yajie Zhang, Mingyu Yu, Yuting Wei, Cheng Dong, Geng Pei, Yawen Xiao, Jianming Yang, Zhi Yao, Quan Wang.
      HSPA8 (heat shock protein family A (Hsp70) member 8) plays a significant role in the autophagic degradation of proteins, however, its effect on protein stabilization and anti-bacterial autophagy remains unknown. Here, it is discovered that HSPA8, as a binding partner of RHOB and BECN1, induce autophagy for intracellular bacteria clearance. Using its NBD and LID domains, HSPA8 physically binds to RHOB residues 1-42 and 89-118 as well as to BECN1 ECD domain, preventing RHOB and BECN1 degradation. Intriguingly, HSPA8 contains predicted intrinsically disordered regions (IDRs), and drives liquid-liquid phase separation (LLPS) to concentrate RHOB and BECN1 into HSPA8-formed liquid-phase droplets, resulting in improved RHOB and BECN1 interactions. Our study reveals a novel role and mechanism of HSPA8 in modulating anti-bacterial autophagy, and highlights the effect of LLPS-related HSPA8-RHOB-BECN1 complex on enhancing protein interaction and stabilization, which improves the understanding of autophagy-mediated defense against bacteria.
    Keywords:  Autophagy; HSPA8; LLPS; RHOB; bacteria
    DOI:  https://doi.org/10.1080/15548627.2023.2223468
  43. Oncol Res. 2023 ;31(3): 345-359
    Mechanism of NURP1 in temozolomide resistance in hypoxia-treated glioma cells via the KDM3A/TFEB axis.
    Tao Li, Xin Fu, Jie Wang, Wei Shang, Xiaotong Wang, Linyun Zhang, Jun Li.
      Temozolomide (TMZ) resistance is a major obstacle in glioma treatment. Nuclear protein-1 (NUPR1) is a regulator of glioma progression. This study investigated the mechanism of NUPR1 in TMZ resistance in hypoxia-treated glioma cells and its mechanism in modulating autophagy. We treated TMZ-resistant cells U251-TMZ and T98G-TMZ to normoxia or hypoxia and silenced NUPR1 in hypoxia-treated U251-TMZ and T98G-TMZ cells to assess cell viability, proliferation, apoptosis, LC3-II/LC3-I and p62 expressions, and autophagic flux under different concentrations of TMZ. We found that hypoxia upregulated NUPR1 expression and autophagy while NUPR1 silencing suppressed hypoxia-induced TMZ resistance and autophagy in glioma cells. We also investigated the interaction between NUPR1 and lysine demethylase 3A (KDM3A), as well as the enrichments of KDM3A and H3 lysine 9 dimethylation (H3K9me2) in the transcription factor EB (TFEB) promoter region. Our results suggest that hypoxia-induced NUPR1 promotes TFEB transcription by binding to KDM3A and reducing H3K9me2 levels, thereby augmenting glioma cell autophagy and TMZ resistance. Moreover, the overexpression of KDM3A or TFEB promoted glioma cell autophagy. In a xenograft tumor model, silencing NUPR1 suppressed TMZ resistance in glioma cells in vivo. Overall, our findings highlight a mechanism by which NUPR1 enhances glioma cell autophagy and TMZ resistance via the KDM3A/TFEB axis.
    Keywords:  Autophagy; Glioma; KDM3A; NUPR1; TMZ resistance
    DOI:  https://doi.org/10.32604/or.2023.028724
  44. Transl Neurodegener. 2023 Jun 13. 12(1): 31
    Reciprocal effects of alpha-synuclein aggregation and lysosomal homeostasis in synucleinopathy models.
    Alice Drobny, Fanni Annamária Boros, Denise Balta, Susy Prieto Huarcaya, Deniz Caylioglu, Niyeti Qazi, Julia Vandrey, Yanni Schneider, Jan Philipp Dobert, Caleb Pitcairn, Joseph Robert Mazzulli, Friederike Zunke.
      BACKGROUND: Lysosomal dysfunction has been implicated in a number of neurodegenerative diseases such as Parkinson's disease (PD). Various molecular, clinical and genetic studies have highlighted a central role of lysosomal pathways and proteins in the pathogenesis of PD. Within PD pathology the synaptic protein alpha-synuclein (αSyn) converts from a soluble monomer to oligomeric structures and insoluble amyloid fibrils. The aim of this study was to unravel the effect of αSyn aggregates on lysosomal turnover, particularly focusing on lysosomal homeostasis and cathepsins. Since these enzymes have been shown to be directly involved in the lysosomal degradation of αSyn, impairment of their enzymatic capacity has extensive consequences.METHODS: We used patient-derived induced pluripotent stem cells and a transgenic mouse model of PD to examine the effect of intracellular αSyn conformers on cell homeostasis and lysosomal function in dopaminergic (DA) neurons by biochemical analyses.
    RESULTS: We found impaired lysosomal trafficking of cathepsins in patient-derived DA neurons and mouse models with αSyn aggregation, resulting in reduced proteolytic activity of cathepsins in the lysosome. Using a farnesyltransferase inhibitor, which boosts hydrolase transport via activation of the SNARE protein ykt6, we enhanced the maturation and proteolytic activity of cathepsins and thereby decreased αSyn protein levels.
    CONCLUSIONS: Our findings demonstrate a strong interplay between αSyn aggregation pathways and function of lysosomal cathepsins. It appears that αSyn directly interferes with the enzymatic function of cathepsins, which might lead to a vicious cycle of impaired αSyn degradation. Lysosomal trafficking of cathepsin D (CTSD), CTSL and CTSB is disrupted when alpha-synuclein (αSyn) is aggregated. This results in a decreased proteolytic activity of cathepsins, which directly mediate αSyn clearance. Boosting the transport of the cathepsins to the lysosome increases their activity and thus contributes to efficient αSyn degradation.
    Keywords:  Dopaminergic neurons; Lysosome; Parkinson’s disease; Protein trafficking; Synucleinopathy; iPSC-derived models
    DOI:  https://doi.org/10.1186/s40035-023-00363-z
  45. Trends Endocrinol Metab. 2023 Jun 13. pii: S1043-2760(23)00107-8. [Epub ahead of print]
    How does mitochondrial import machinery fine-tune mitophagy? Different paths and one destination.
    Mohamed A Eldeeb, Andrea Soumbasis, Edward A Fon.
      Given their polyvalent roles, an intrinsic challenge that mitochondria face is the continuous exposure to various stressors including mitochondrial import defects, which leads to their dysfunction. Recent work has unveiled a presequence translocase-associated import motor (PAM) complex-dependent quality control pathway whereby misfolded proteins mitigate mitochondrial protein import and subsequently elicit mitophagy without the loss of mitochondrial membrane potential.
    Keywords:  PINK1; TOM complex; mitochondrial import; mitochondrial quality control; mitophagy; protein quality control
    DOI:  https://doi.org/10.1016/j.tem.2023.05.005
  46. Int J Mol Sci. 2023 Jun 02. pii: 9676. [Epub ahead of print]24(11):
    Genetic Targeting of dSAMTOR, A Negative dTORC1 Regulator, during Drosophila Aging: A Tissue-Specific Pathology.
    Stamatia A Katarachia, Sophia P Markaki, Athanassios D Velentzas, Dimitrios J Stravopodis.
      mTORC1 regulates mammalian cell metabolism and growth in response to diverse environmental stimuli. Nutrient signals control the localization of mTORC1 onto lysosome surface scaffolds that are critically implicated in its amino acid-dependent activation. Arginine, leucine and S-adenosyl-methionine (SAM) can serve as major mTORC1-signaling activators, with SAM binding to SAMTOR (SAM + TOR), a fundamental SAM sensor, preventing the protein's (SAMTOR's) inhibitory action(s) against mTORC1, thereby triggering its (mTORC1) kinase activity. Given the lack of knowledge regarding the role of SAMTOR in invertebrates, we have identified the Drosophila SAMTOR homologue (dSAMTOR) in silico and have, herein, genetically targeted it through the utilization of the GAL4/UAS transgenic tool. Survival profiles and negative geotaxis patterns were examined in both control and dSAMTOR-downregulated adult flies during aging. One of the two gene-targeted schemes resulted in lethal phenotypes, whereas the other one caused rather moderate pathologies in most tissues. The screening of head-specific kinase activities, via PamGene technology application, unveiled the significant upregulation of several kinases, including the dTORC1 characteristic substrate dp70S6K, in dSAMTOR-downregulated flies, thus strongly supporting the inhibitory dSAMTOR action(s) upon the dTORC1/dp70S6K signaling axis in Drosophila brain settings. Importantly, genetic targeting of the Drosophila BHMT bioinformatics counterpart (dBHMT), an enzyme that catabolizes betaine to produce methionine (the SAM precursor), led to severe compromises in terms of fly longevity, with glia-, motor neuron- and muscle-specific dBHMT downregulations exhibiting the strongest effects. Abnormalities in wing vein architectures were also detected in dBHMT-targeted flies, thereby justifying their notably reduced negative geotaxis capacities herein observed mainly in the brain-(mid)gut axis. In vivo adult fly exposure to clinically relevant doses of methionine revealed the mechanistic synergism of decreased dSAMTOR and increased methionine levels in pathogenic longevity, thus rendering (d)SAMTOR an important component in methionine-associated disorders, including homocystinuria(s).
    Keywords:  Drosophila; SAM; aging; betaine; dBHMT; dSAMTOR; dTORC1; kinase; methionine
    DOI:  https://doi.org/10.3390/ijms24119676
  47. J Zhejiang Univ Sci B. 2023 Jun 15. pii: 1673-1581(2023)06-0485-11. [Epub ahead of print]24(6): 485-495
    Fibroblast growth factor 21 (FGF21) attenuates tacrolimus-induced hepatic lipid accumulation through transcription factor EB (TFEB)-regulated lipophagy.
    Zhensheng Zhang, Li Xu, Xun Qiu, Xinyu Yang, Zhengxing Lian, Xuyong Wei, Di Lu, Xiao Xu.
      Tacrolimus (TAC), also called FK506, is one of the classical immunosuppressants to prevent allograft rejection after liver transplantation. However, it has been proved to be associated with post-transplant hyperlipemia. The mechanism behind this is unknown, and it is urgent to explore preventive strategies for hyperlipemia after transplantation. Therefore, we established a hyperlipemia mouse model to investigate the mechanism, by injecting TAC intraperitoneally for eight weeks. After TAC treatment, the mice developed hyperlipemia (manifested as elevated triglyceride (TG) and low-density lipoprotein cholesterol (LDL-c), as well as decreased high-density lipoprotein cholesterol (HDL-c)). Accumulation of lipid droplets was observed in the liver. In addition to lipid accumulation, TAC induced inhibition of the autophagy-lysosome pathway (microtubule-associated protein 1 light chain 3β (LC3B) II/I and LC3B II/actin ratios, transcription factor EB (TFEB), protein 62 (P62), and lysosomal-associated membrane protein 1 (LAMP1)) and downregulation of fibroblast growth factor 21 (FGF21) in vivo. Overexpression of FGF21 may reverse TAC-induced TG accumulation. In this mouse model, the recombinant FGF21 protein ameliorated hepatic lipid accumulation and hyperlipemia through repair of the autophagy-lysosome pathway. We conclude that TAC downregulates FGF21 and thus exacerbates lipid accumulation by impairing the autophagy-lysosome pathway. Recombinant FGF21 protein treatment could therefore reverse TAC-caused lipid accumulation and hypertriglyceridemia by enhancing autophagy.
    Keywords:  Autophagy; Fibroblast growth factor 21 (FGF21); Lipid; Lipophagy; Lysosome; Tacrolimus; Transcription factor EB (TFEB)
    DOI:  https://doi.org/10.1631/jzus.B2200562
  48. Cell Signal. 2023 Jun 12. pii: S0898-6568(23)00183-3. [Epub ahead of print] 110769
    The mitochondrial uncoupling effects of nitazoxanide enhances cellular autophagy and promotes the clearance of α-synuclein: Potential role of AMPK-JNK pathway.
    Niharika Amireddy, Vandana Dulam, Shweta Kaul, Rajeswari Pakkiri, Shasi V Kalivendi.
      Upregulation and aggregation of the pre-synaptic protein, α-synuclein plays a key role in Parkinson's disease (PD) and mitochondrial dysfunction was surmised to be an upstream event in the disease pathogenesis. Emerging reports identified the role of nitazoxanide (NTZ), an anti-helminth drug, in enhancing mitochondrial oxygen consumption rate (OCR) and autophagy. In the present study, we have examined the mitochondrial effects of NTZ in mediating cellular autophagy and subsequent clearance of both endogenous and pre-formed aggregates of α-synuclein in cellular model of PD. Our results demonstrate that the mitochondrial uncoupling effects of NTZ results in the activation of AMPK and JNK, which in-turn leads to the enhancement of cellular autophagy. Also,1-methyl-4-phenylpyridinium (MPP+) mediated decrease in autophagic flux with a concomitant increase in the α-synuclein levels were ameliorated in cells treated with NTZ. However, in cells lacking functional mitochondria (ρ0 cells), NTZ did not mitigate MPP+ mediated alterations in the autophagic clearance of α-synuclein, indicating that the mitochondrial effects of NTZ play a crucial role in the clearance of α-synuclein by autophagy. Also, the ability of AMPK inhibitor, compound C, in abrogating NTZ mediated enhancement in the autophagic flux and α-synuclein clearance highlight the pivotal role of AMPK in NTZ mediated autophagy. Further, NTZ per se enhanced the clearance of preformed α-synuclein aggregates that were exogenously added to the cells. Overall, the results of our present study suggest that NTZ activates macroautophagy in cells due to its uncoupling effects on mitochondrial respiration via activation of AMPK-JNK pathway resulting in the clearance of both endogenous and pre-formed α-synuclein aggregates. As NTZ happens to possess good bioavailability and safety profile, considering this drug for PD based on its mitochondrial uncoupling and autophagy enhancing properties for mitigating mitochondrial reactive oxygen species (ROS) and α-synuclein toxicity appears to be a promising therapeutic option.
    Keywords:  Autophagy; Mitochondria; Nitazoxanide; Protein aggregation; Uncoupler; α-Synuclein
    DOI:  https://doi.org/10.1016/j.cellsig.2023.110769
  49. Front Cell Dev Biol. 2023 ;11 1221510
    Editorial: The regulation of proteostasis in aging.
    Ji-Xin Tang, Fu-Hui Xiao.
      
    Keywords:  aging; autophagic flux; non-canonical autophagy; proteasome; protein translation; proteostasis
    DOI:  https://doi.org/10.3389/fcell.2023.1221510
  50. Exp Ther Med. 2023 Jul;26(1): 354
    Autophagy in homocysteine‑induced HUVEC senescence.
    Yexi Zhang, Juyan Ouyang, Liu Zhan, Yu Li, Shaoshan Li, Yi He, Hong Wang, Xiangyang Zhang.
      The senescence of vascular endothelial cells (VECs) drives the occurrence and development of cardiovascular disease (CVD). Homocysteine (HCY) is a general risk factor for age-associated CVDs. Autophagy, an evolutionarily conserved lysosomal protein degradation pathway, serves a part in VEC senescence. The purpose of this study was to investigate the role of autophagy in HCY-induced endothelial cell senescence and explore novel mechanisms and therapeutic approaches for related CVDs. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords of healthy pregnancies. Cell Counting Kit-8, flow cytometry and senescence-associated (SA) β-galactosidase (Gal) staining demonstrated that HCY induced HUVEC senescence by decreasing cell proliferation, arresting cell cycle and increasing the number of SA-β-Gal-positive cells. Stub-RFP-Sens-GFP-LC3 autophagy-related double fluorescence lentivirus revealed that HCY increased autophagic flux. Further, inhibition of autophagy using 3-methyladenine increased HCY-induced HUVEC senescence. By contrast, the induction of autophagy via rapamycin alleviated HCY-induced HUVEC senescence. Finally, the detection of reactive oxygen species (ROS) with ROS kit showed that HCY increased intracellular ROS, whereas induction of autophagy reduced intracellular ROS. In conclusion, HCY increased HUVEC senescence and upregulated autophagy; moderate autophagy could reverse HCY-induced cell senescence. Autophagy may alleviate HCY-induced cell senescence by decreasing intracellular ROS. This provides insight into the underlying mechanism of HCY-induced VEC senescence and potential treatments for age-associated CVDs.
    Keywords:  autophagy; cardiovascular disease; homocysteine; human umbilical vein endothelial cell; senescence
    DOI:  https://doi.org/10.3892/etm.2023.12053
  51. J Mol Med (Berl). 2023 Jun 17.
    TFEB: a double-edged sword for tumor metastasis.
    Jun-Hu Hu, Shou-Ye Li, Li-Hua Yu, Zhen-Rong Guan, Ya-Ping Jiang, Die Hu, Hao-Jie Wang, Li-Ping Zhao, Zhao-Huang Zhou, Ya-Xin Yan, Tian Xie, Zhi-Hui Huang, Jian-Shu Lou.
      Transcription factor EB, a member of the microphthalmia-associated transcription factor (MiTF/TFE) family, is a master regulator of autophagy, lysosome biogenesis, and TAMs. Metastasis is one of the main reasons for the failure of tumor therapy. Studies on the relationship between TFEB and tumor metastasis are contradictory. On the positive side, TFEB mainly affects tumor cell metastasis via five aspects, including autophagy, epithelial-mesenchymal transition (EMT), lysosomal biogenesis, lipid metabolism, and oncogenic signaling pathways; on the negative side, TFEB mainly affects tumor cell metastasis in two aspects, including tumor-associated macrophages (TAMs) and EMT. In this review, we described the detailed mechanism of TFEB-mediated regulation of metastasis. In addition, we also described the activation and inactivation of TFEB in several aspects, including the mTORC1 and Rag GTPase systems, ERK2, and AKT. However, the exact process by which TFEB regulates tumor metastasis remains unclear in some pathways, which requires further studies.
    Keywords:  Activation; Metastasis; Phosphorylation; TFEB; Tumor
    DOI:  https://doi.org/10.1007/s00109-023-02337-0
  52. Small. 2023 Jun 15. e2302284
    A Reactive Oxygen Species-Responsive Targeted Nanoscavenger to Promote Mitophagy for the Treatment of Alzheimer's Disease.
    Zhimin Yang, Haoyuan Shi, Guoen Cai, Sujun Jiang, Zhiyuan Hu, Zihua Wang.
      Mitophagy modulators are proposed as potential therapeutic intervention that enhance neuronal health and brain homeostasis in Alzheimer's disease (AD). Nevertheless, the lack of specific mitophagy inducers, low efficacies, and the severe side effects of nonselective autophagy during AD treatment have hindered their application. In this study, the P@NB nanoscavenger is designed with a reactive-oxygen-species-responsive (ROS-responsive) poly(l-lactide-co-glycolide) core and a surface modified with the Beclin1 and angiopoietin-2 peptides. Notably, nicotinamide adenine dinucleotide (NAD+ ) and Beclin1, which act as mitophagy promoters, are quickly released from P@NB in the presence of high ROS levels in lesions to restore mitochondrial homeostasis and induce microglia polarization toward the M2-type, thereby enabling it to phagocytose amyloid-peptide (Aβ). These studies demonstrate that P@NB accelerates Aβ degradation and alleviates excessive inflammatory responses by restoring autophagic flux, which ameliorates cognitive impairment in AD mice. This multitarget strategy induces autophagy/mitophagy through synergy, thereby normalizing mitochondrial dysfunction. Therefore, the developed method provides a promising AD-therapy strategy.
    Keywords:  Alzheimer's disease; anti-inflammatory; microglia polarization; mitophagy; nicotinamide adenine dinucleotide (NAD+)
    DOI:  https://doi.org/10.1002/smll.202302284
  53. Biol Psychiatry. 2023 07 01. pii: S0006-3223(23)01231-3. [Epub ahead of print]94(1): e1-e3
    Protein Accumulation and Impaired Autophagy Underlie Cognitive Dysfunction in Angelman Syndrome.
    Kirstie A Cummings.
      
    DOI:  https://doi.org/10.1016/j.biopsych.2023.04.015
  54. Plant Physiol. 2023 Jun 14. pii: kiad350. [Epub ahead of print]
    Autophagy in maternal tissues contributes to Arabidopsis seed development.
    Ori Avraham Erlichman, Shahar Weiss, Maria Abu-Arkia, Moria Ankary-Khaner, Yoram Soroka, Weronika Jasinska, Leah Rosental, Yariv Brotman, Tamar Avin-Wittenberg.
      Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development, including autophagy, which facilitates cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy affects nutrient remobilization from mother plants and functions in the embryo. However, it is impossible to distinguish between the contribution of autophagy in the source (i.e., the mother plant) and the sink tissue (i.e., the embryo) when using autophagy-knockout (atg mutant) plants. To address this, we employed an approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between WT and atg mutant Arabidopsis (Arabidopsis thaliana) plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from altered seed coat development. Our study emphasizes the importance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It also sheds light on the tissue-specific functions of autophagy, offering potential for research into the underlying mechanisms governing seed development and crop yield.
    Keywords:   Arabidopsis thaliana ; Autophagy; Maternal tissue; Seed; Source-sink relationship; Storage compounds
    DOI:  https://doi.org/10.1093/plphys/kiad350
  55. Biochim Biophys Acta Mol Cell Res. 2023 Jun 12. pii: S0167-4889(23)00084-8. [Epub ahead of print] 119512
    Epac1 participates in β1-adrenoreceptor autoantibody-mediated decreased autophagic flux in cardiomyocytes.
    Yang Li, Yuan Tian, Shu Shi, Xiaohong Hou, Haihu Hao, Mingxia Ma, Na Ning, Yuan Yuan, Xiaohui Wang, Huirong Liu, Li Wang.
      Decreased autophagic flux in cardiomyocytes is an important mechanism by which the β1-adrenoreceptor (β1-AR) autoantibody (β1-AA) induces heart failure. A previous study found that β1-AA imparts its biological effects via the β1-AR/Gs/AC/cAMP/PKA canonical signaling pathway, but PKA inhibition does not completely reverse β1-AA-induced reduction in autophagy in myocardial tissues, suggesting that other signaling molecules participate in this process. This study confirmed that Epac1 upregulation is indeed involved β1-AA-induced decreased cardiomyocyte autophagy through CE3F4 pretreatment, Epac1 siRNA transfection, western blot and immunofluorescence methods. On this basis, we constructed β1-AR and β2-AR knockout mice, and used receptor knockout mice, β1-AR selective blocker (atenolol), and the β2-AR/Gi-biased agonist ICI 118551 to show that β1-AA upregulated Epac1 expression through β1-AR and β2-AR to inhibit autophagy, and biased activation of β2-AR/Gi signaling downregulated myocardial Epac1 expression to reverse β1-AA-induced myocardial autophagy inhibition. This study aimed to test the hypothesis that Epac1 acts as another effector downstream of cAMP on β1-AA-induced reduction in cardiomyocyte autophagy, and β1-AA upregulates myocardial Epac1 expression through β1-AR and β2-AR, and biased activation of the β2-AR/Gi signaling pathway can reverse β1-AA-induced myocardial autophagy inhibition. This study provides new ideas and therapeutic targets for the prevention and treatment of cardiovascular diseases related to dysregulated autophagy.
    Keywords:  Autoantibody; Autophagy; Epac1; β(1)-Adrenoreceptor; β(2)-Adrenoreceptor
    DOI:  https://doi.org/10.1016/j.bbamcr.2023.119512
  56. EMBO Rep. 2023 Jun 15. e55895
    C9orf72 protein quality control by UBR5-mediated heterotypic ubiquitin chains.
    Julia Jülg, Dieter Edbauer, Christian Behrends.
      Hexanucleotide repeat expansions within C9orf72 are a frequent cause of amyotrophic lateral sclerosis and frontotemporal dementia. Haploinsufficiency leading to reduced C9orf72 protein contributes to disease pathogenesis. C9orf72 binds SMCR8 to form a robust complex that regulates small GTPases, lysosomal integrity, and autophagy. In contrast to this functional understanding, we know far less about the assembly and turnover of the C9orf72-SMCR8 complex. Loss of either subunit causes the concurrent ablation of the respective partner. However, the molecular mechanism underlying this interdependence remains elusive. Here, we identify C9orf72 as a substrate of branched ubiquitin chain-dependent protein quality control. We find that SMCR8 prevents C9orf72 from rapid degradation by the proteasome. Mass spectrometry and biochemical analyses reveal the E3 ligase UBR5 and the BAG6 chaperone complex as C9orf72-interacting proteins, which are components of the machinery that modifies proteins with K11/K48-linked heterotypic ubiquitin chains. Depletion of UBR5 results in reduced K11/K48 ubiquitination and increased C9orf72 when SMCR8 is absent. Our data provide novel insights into C9orf72 regulation with potential implication for strategies to antagonize C9orf72 loss during disease progression.
    Keywords:  BAG6 complex; C9orf72; K11/K48-linked ubiquitin; UBR5; heterotypic ubiquitin chains
    DOI:  https://doi.org/10.15252/embr.202255895
  57. Cell Tissue Res. 2023 Jun 17.
    mTORC1/rpS6 and mTORC2/PKC regulate spermatogenesis through Arp3-mediated actin microfilament organization in Eriocheir sinensis.
    Zhen-Fang Li, Hong-Yu Qi, Jia-Ming Wang, Zhan Zhao, Fu-Qing Tan, Wan-Xi Yang.
      Mammalian target of rapamycin (mTOR) is a crucial signaling protein regulating a range of cellular events. Numerous studies have reported that the mTOR pathway is related to spermatogenesis in mammals. However, its functions and underlying mechanisms in crustaceans remain largely unknown. mTOR exists as two multimeric functional complexes termed mTOR complex 1 (mTORC1) and mTORC2. Herein, we first cloned ribosomal protein S6 (rpS6, a downstream molecule of mTORC1) and protein kinase C (PKC, a downstream effector of mTORC2) from the testis of Eriocheir sinensis. The dynamic localization of rpS6 and PKC suggested that both proteins may be essential for spermatogenesis. rpS6/PKC knockdown and Torin1 treatment led to defects in spermatogenesis, including germ cell loss, retention of mature sperm and empty lumen formation. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was disrupted in the rpS6/PKC knockdown and Torin1 treatment groups, accompanied by changing in expression and distribution of junction proteins. Further study demonstrated that these findings may result from the disorganization of filamentous actin (F-actin) networks, which were mediated by the expression of actin-related protein 3 (Arp3) rather than epidermal growth factor receptor pathway substrate 8 (Eps8). In summary, our study illustrated that mTORC1/rpS6 and mTORC2/PKC regulated spermatogenesis via Arp3-mediated actin microfilament organization in E. sinensis.
    Keywords:  Cell junctions; Eriocheir sinensis; Microfilaments; Spermatogenesis; mTORC1/C2
    DOI:  https://doi.org/10.1007/s00441-023-03795-1
  58. Methods Mol Biol. 2023 ;2683 201-212
    Dynamic Measurement of Endosome-Lysosome Fusion in Neurons Using High-Content Imaging.
    Qing Ouyang, Michael Schmidt, Eric M Morrow.
      Endocytosis is a dynamic cellular process that actively transports particles into a cell. Late endosome fusion with the lysosome is a crucial step in the delivery of newly synthesized lysosomal proteins and endocytosed cargo for degradation. Disturbing this step in neurons is associated with neurological disorders. Thus, studying endosome-lysosome fusion in neurons will provide new insight into the mechanisms of these diseases and open new possibilities for therapeutic treatment. However, measuring endosome-lysosome fusion is challenging and time consuming, which limits the research in this area. Here we developed a high throughput method using pH-insensitive dye-conjugated dextrans and the Opera Phenix® High Content Screening System. By using this method, we successfully separated endosomes and lysosomes in neurons, and time-lapse images were collected to capture endosome-lysosome fusion events in hundreds of cells. Both assay set-up and analysis can be completed in an expeditious and efficient manner.
    Keywords:  Dye-conjugated dextran; Endosome-lysosome fusion; High throughput assay; Neurological disorders; Opera Phenix®; Time-lapse images
    DOI:  https://doi.org/10.1007/978-1-0716-3287-1_16
  59. Metabolism. 2023 Jun 10. pii: S0026-0495(23)00233-0. [Epub ahead of print]145 155629
    High levels of intracellular endotrophin in adipocytes mediate COPII vesicle supplies to autophagosome to impair autophagic flux and contribute to systemic insulin resistance in obesity.
    Jiyoung Oh, Chanho Park, Sahee Kim, Min Kim, Chu-Sook Kim, Woobeen Jo, Sungho Park, Gwan-Su Yi, Jiyoung Park.
      BACKGROUND AND AIMS: Extracellular matrix (ECM) homeostasis plays a crucial role in metabolic plasticity and endocrine function of adipose tissue. High levels of intracellular endotrophin, a cleavage peptide of type VI collagen alpha 3 chain (Col6a3), have been frequently observed in adipocyte in obesity and diabetes. However, how endotrophin intracellularly traffics and influences metabolic homeostasis in adipocyte remains unknown. Therefore, we aimed to investigate the trafficking of endotrophin and its metabolic effects in adipocytes depending on lean or obese condition.METHODS: We used doxycycline-inducible adipocyte-specific endotrophin overexpressed mice for a gain-of-function study and CRISPR-Cas9 system-based Col6a3-deficient mice for a loss-of-function study. Various molecular and biochemical techniques were employed to examine the effects of endotrophin on metabolic parameters.
    RESULTS: In adipocytes during obesity, the majority of endosomal endotrophin escapes lysosomal degradation and is released into the cytosol to mediate direct interactions between SEC13, a major component of coat protein complex II (COPII) vesicles, and autophagy-related 7 (ATG7), leading to the increased formation of autophagosomes. Autophagosome accumulation disrupts the balance of autophagic flux, resulting in adipocyte death, inflammation, and insulin resistance. These adverse metabolic effects were ameliorated by either suppressing ATG7 with siRNA ex vivo or neutralizing endotrophin with monoclonal antibodies in vivo.
    CONCLUSIONS: High levels of intracellular endotrophin-mediated autophagic flux impairment in adipocyte contribute to metabolic dysfunction such as apoptosis, inflammation, and insulin resistance in obesity.
    Keywords:  Adipocyte; Autophagy; COPII vesicles; Endotrophin; Insulin resistance; Obesity
    DOI:  https://doi.org/10.1016/j.metabol.2023.155629
  60. Oncol Res. 2022 ;30(2): 89-97
    SPINK1 contributes to proliferation and clonal formation of HT29 cells through Beclin1 associated enhanced autophagy.
    N A Hu, Shiqing Zhang, Aquan Jin, Lianying Guo, Zhenyun Qu, Jun Wang.
      We aimed to explore the molecular mechanism that were involved in SPINK1-induced proliferation and clonogenic survival of human colorectal carcinoma (CRC) HT29 cells. Initially, we generated HT29 cells either permanently silencing or overexpressing SPINK1 protein. The results showed that SPINK1 overexpression (OE) significantly stimulated the proliferation and clonal formation of HT29 cells at the varied time points. Secondly, we found SPINK1 OE enhanced the ratio of LC3II/LC3I and the level of autophagy-related gene 5 (ATG5), whereas SPINK1 knockdown (Kd) reversed the above outcome under normal culturing and/or fasting condition in the cells, indicating its role in autophagy enhancement. Moreover, LC3-GFP-transfected SPINK1-OE HT29 cells strengthened the fluorescence intensity compared with the untransfected control. Chloroquine (CQ) significantly decreased the level of autophagy in both control and SPINK1-OE HT29 cells. The autophagy inhibitors, CQ and 3-Methyladenine (3-MA), remarkably inhibited the proliferation and colony formation of SPINK1-OE HT29 cells, while ATG5 upregulation resulted in the growth of the cells, suggesting the important function of autophagy in cell's growth. Thirdly, SPINK1-induced autophagy was independently of mTOR signaling as p-RPS6 and p-4EBP1 were activated in SPINK1-OE HT29 cells. Instead, Beclin1 up and down regulation were clearly observed in SPINK1-OE and SPINK1 Kd HT29 cells, respectively. Moreover, Beclin1 silencing apparently reduced autophagy in SPINK1-OE HT29 cells, indicating that SPINK1-induced autophagy was closely associated with Beclin1. Collectively, SPINK1-promoted proliferation and clonal formation of HT29 cells were closely associated with Beclin1 associated enhanced autophagy. The above findings would open a new window for probing the role of SPINK1-related autophagic signaling in the pathogenesis of CRC.
    Keywords:  Autophagy; Beclin1; Colorectal carcinoma; SPINK1
    DOI:  https://doi.org/10.32604/or.2022.027058
  61. Nat Chem Biol. 2023 Jun 15.
    Monitoring lysosomal acidity.
    Kangqiang Qiu, Zhiqi Tian, Jiajie Diao.
      
    DOI:  https://doi.org/10.1038/s41589-023-01348-9
  62. Int J Mol Sci. 2023 May 24. pii: 9198. [Epub ahead of print]24(11):
    mTOR Signaling Pathway in Bone Diseases Associated with Hyperglycemia.
    Shuangcheng Wang, Jiale Wang, Shuangwen Wang, Ran Tao, Jianru Yi, Miao Chen, Zhihe Zhao.
      The interplay between bone and glucose metabolism has highlighted hyperglycemia as a potential risk factor for bone diseases. With the increasing prevalence of diabetes mellitus worldwide and its subsequent socioeconomic burden, there is a pressing need to develop a better understanding of the molecular mechanisms involved in hyperglycemia-mediated bone metabolism. The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that senses extracellular and intracellular signals to regulate numerous biological processes, including cell growth, proliferation, and differentiation. As mounting evidence suggests the involvement of mTOR in diabetic bone disease, we provide a comprehensive review of its effects on bone diseases associated with hyperglycemia. This review summarizes key findings from basic and clinical studies regarding mTOR's roles in regulating bone formation, bone resorption, inflammatory responses, and bone vascularity in hyperglycemia. It also provides valuable insights into future research directions aimed at developing mTOR-targeted therapies for combating diabetic bone diseases.
    Keywords:  bone complications; diabetes; mTOR signaling
    DOI:  https://doi.org/10.3390/ijms24119198
  63. Sci Total Environ. 2023 Jun 13. pii: S0048-9697(23)03410-1. [Epub ahead of print] 164787
    Activation of NLRP3 signaling contributes to cadmium-induced bone defects, associated with autophagic flux obstruction.
    Renjie Hu, Huigen Luo, Yuna Ji, Zheng Wang, Pengchao Zheng, Huiya Ouyang, Xinchen Wang, Yun Wang, Baicheng Bao, Guiqing Liao, Baoshan Xu.
      Cadmium (Cd) is a widespread environmental and industrial pollutant to cause various bone metabolic diseases. Our former study reported that Cd promoted adipogenesis and inhibited osteogenic differentiation of primary bone marrow-derived mesenchymal stem cells (BMSCs) by NF-κB inflammation signaling and oxidative stress, and Cd induced osteoporosis of long bone and compromised repair of cranial bone defect in vivo. However, the underlying mechanisms of Cd-induced bone damage remain elusive. In this study, we used Sprague Dawley (SD) rat and NLRP3-knockout mouse models to elucidate the exact effects and molecular mechanisms of Cd-induced bone damage and aging. Herein we found that the exposure of Cd preferentially targeted a few specific tissues such as bone and kidney. Cd triggered NLRP3 inflammasome pathways and the accumulation of autophagosomes of primary BMSCs, and also Cd stimulated the differentiation and bone resorption function of primary osteoclasts. Moreover, Cd not only activated ROS/NLRP3/caspase-1/p20/IL-1β pathways, but also influenced Keap1/Nrf2/ARE signaling. The data revealed that autophagy dysfunction and NLRP3 pathways synergistically mediated the impairments of Cd in bone tissues. Loss of NLRP3 function partially alleviated Cd-induced osteoporosis and craniofacial bone defect in the NLRP3-knockout mouse model. Furthermore, we characterized the protective effects and potential therapeutic targets of the combined treatment of anti-aging agents (rapamycin+melatonin+NLRP3 selective inhibitor MCC950) on Cd-induced bone damage and inflammatory aging. These results illuminate that ROS/NLRP3 pathways and autophagic flux obstruction are involved in the Cd-induced toxic actions of bone tissues. Collectively, our study unveils some therapeutic targets and the regulatory mechanism to prevent Cd-caused bone rarefaction. The findings improve the mechanistic understanding of environmental Cd exposure-caused bone metabolism disorders and tissue damage.
    Keywords:  Autophagy; Bone aging; Bone marrow-derived mesenchymal stem cells (BMSCs); Cadmium (cd); NLRP3 inflammasome; Osteoporosis
    DOI:  https://doi.org/10.1016/j.scitotenv.2023.164787
  64. Indian J Dermatol Venereol Leprol. 2023 May 03. pii: 10.25259/IJDVL_555_2022. [Epub ahead of print] 1-4
    Mammalian target of rapamycin (mTOR) inhibitors in dermatology.
    Anand Mannu, Shekhar Neema, Biju Vasudevan, Siddharth Bhatt.
      
    DOI:  https://doi.org/10.25259/IJDVL_555_2022
  65. Acta Pharmacol Sin. 2023 Jun 14.
    Chloroquine enhances the efficacy of chemotherapy drugs against acute myeloid leukemia by inactivating the autophagy pathway.
    Han-Lin Wang, Jia-Nan Li, Wei-Juan Kan, Gao-Ya Xu, Guang-Hao Luo, Ning Song, Wen-Biao Wu, Bo Feng, Jing-Feng Fu, Yu-Tong Tu, Min-Min Liu, Ran Xu, Yu-Bo Zhou, Gang Wei, Jia Li.
      Current therapy for acute myeloid leukemia (AML) is largely hindered by the development of drug resistance of commonly used chemotherapy drugs, including cytarabine, daunorubicin, and idarubicin. In this study, we investigated the molecular mechanisms underlying the chemotherapy drug resistance and potential strategy to improve the efficacy of these drugs against AML. By analyzing data from ex vivo drug-response and multi-omics profiling public data for AML, we identified autophagy activation as a potential target in chemotherapy-resistant patients. In THP-1 and MV-4-11 cell lines, knockdown of autophagy-regulated genes ATG5 or MAP1LC3B significantly enhanced AML cell sensitivity to the chemotherapy drugs cytarabine, daunorubicin, and idarubicin. In silico screening, we found that chloroquine phosphate mimicked autophagy inactivation. We showed that chloroquine phosphate dose-dependently down-regulated the autophagy pathway in MV-4-11 cells. Furthermore, chloroquine phosphate exerted a synergistic antitumor effect with the chemotherapy drugs in vitro and in vivo. These results highlight autophagy activation as a drug resistance mechanism and the combination therapy of chloroquine phosphate and chemotherapy drugs can enhance anti-AML efficacy.
    Keywords:  acute myeloid leukemia; autophagy; chloroquine phosphate; cytarabine; daunorubicin; drug resistant; idarubicin; multi-omics
    DOI:  https://doi.org/10.1038/s41401-023-01112-8
  66. Nat Cell Biol. 2023 Jun 12.
    Autophagy prevents microglial senescence.
    Sadaf Amin, Bangyan Liu, Li Gan.
      
    DOI:  https://doi.org/10.1038/s41556-023-01168-y
  67. PLoS Biol. 2023 Jun 15. 21(6): e3002159
    Autophagy prevents early proinflammatory responses and neutrophil recruitment during Mycobacterium tuberculosis infection without affecting pathogen burden in macrophages.
    Rachel L Kinsella, Jacqueline M Kimmey, Asya Smirnov, Reilly Woodson, Margaret R Gaggioli, Sthefany M Chavez, Darren Kreamalmeyer, Christina L Stallings.
      The immune response to Mycobacterium tuberculosis infection determines tuberculosis disease outcomes, yet we have an incomplete understanding of what immune factors contribute to a protective immune response. Neutrophilic inflammation has been associated with poor disease prognosis in humans and in animal models during M. tuberculosis infection and, therefore, must be tightly regulated. ATG5 is an essential autophagy protein that is required in innate immune cells to control neutrophil-dominated inflammation and promote survival during M. tuberculosis infection; however, the mechanistic basis for how ATG5 regulates neutrophil recruitment is unknown. To interrogate what innate immune cells require ATG5 to control neutrophil recruitment during M. tuberculosis infection, we used different mouse strains that conditionally delete Atg5 in specific cell types. We found that ATG5 is required in CD11c+ cells (lung macrophages and dendritic cells) to control the production of proinflammatory cytokines and chemokines during M. tuberculosis infection, which would otherwise promote neutrophil recruitment. This role for ATG5 is autophagy dependent, but independent of mitophagy, LC3-associated phagocytosis, and inflammasome activation, which are the most well-characterized ways that autophagy proteins regulate inflammation. In addition to the increased proinflammatory cytokine production from macrophages during M. tuberculosis infection, loss of ATG5 in innate immune cells also results in an early induction of TH17 responses. Despite prior published in vitro cell culture experiments supporting a role for autophagy in controlling M. tuberculosis replication in macrophages, the effects of autophagy on inflammatory responses occur without changes in M. tuberculosis burden in macrophages. These findings reveal new roles for autophagy proteins in lung resident macrophages and dendritic cells that are required to suppress inflammatory responses that are associated with poor control of M. tuberculosis infection.
    DOI:  https://doi.org/10.1371/journal.pbio.3002159
  68. J Nutr. 2023 Jun 14. pii: S0022-3166(23)72417-6. [Epub ahead of print]
    Effects of leucine ingestion and contraction on the Sestrin/GATOR2 pathway and mTORC1 activation in rat fast-twitch muscle.
    Keita Kanzaki, Masanobu Wada.
      BACKGROUND: Leucine activates the mechanistic/mammalian target of rapamycin complex 1 (mTORC1) in mammalian skeletal muscle. Recent studies show that Sestrin, a leucine sensor, might play a role in this process. However, it remains unknown whether Sestrin dissociates from GATOR2 in a dose- and time-dependent manner and whether an acute bout of muscle contraction augments this dissociation.OBJECTIVE: This study aimed to examine the effects of leucine ingestion and muscle contraction on the interaction between Sestrin1/2 and GATOR2 and on mTORC1 activation.
    METHODS: Male Wistar rats were randomly assigned to a control (C), a leucine 3 (L3), or a leucine 10 (L10) group. Intact gastrocnemius muscles were subjected to 30 repetitive unilateral contractions. The L3 and L10 groups were then orally administered 3 and 10 mmol/kg body weight of L-leucine 2 h after the end of the contractions, respectively. Blood and muscle samples were collected 30, 60, or 120 min after the administration.
    RESULTS: The blood and muscle leucine concentrations increased in a dose-dependent manner. The ratio of phosphorylated ribosomal protein S6 kinase (S6K) to total S6K (which indicates mTORC1 signaling activation) was markedly increased by muscle contraction and increased in a dose-dependent manner only in rested muscle. Leucine ingestion but not muscle contraction increased Sestrin1 dissociation from GATOR2 and Sestrin2 association with GATOR2. A negative relationship was observed between the blood and muscle leucine concentrations and the Sestrin1 association with GATOR2.
    CONCLUSION: The results suggest that Sestrin1, but not Sestrin2, regulates leucine-related mTORC1 activation via its dissociation from GATOR2 and that acute exercise-induced mTORC1 activation involves pathways other than the leucine-related Sestrin1/GATOR2 pathway.
    Keywords:  amino acid sensing; dose-dependence; leucine sensor; skeletal muscle; time course
    DOI:  https://doi.org/10.1016/j.tjnut.2023.06.011
  69. Int J Mol Sci. 2023 May 24. pii: 9171. [Epub ahead of print]24(11):
    Genetic Ablation of LAT1 Inhibits Growth of Liver Cancer Cells and Downregulates mTORC1 Signaling.
    Sun-Yee Kim, Qunxiang Ong, Yilie Liao, Zhaobing Ding, Alicia Qian Ler Tan, Ler Ting Rachel Lim, Hui Min Tan, Siew Lan Lim, Qian Yi Lee, Weiping Han.
      Through a comprehensive analysis of the gene expression and dependency in HCC patients and cell lines, LAT1 was identified as the top amino acid transporter candidate supporting HCC tumorigenesis. To assess the suitability of LAT1 as a HCC therapeutic target, we used CRISPR/Cas9 to knockout (KO) LAT1 in the epithelial HCC cell line, Huh7. Knockout of LAT1 diminished its branched chain amino acid (BCAA) transport activity and significantly reduced cell proliferation in Huh7. Consistent with in vitro studies, LAT1 ablation led to suppression of tumor growth in a xenograft model. To elucidate the mechanism underlying the observed inhibition of cell proliferation upon LAT1 KO, we performed RNA-sequencing analysis and investigated the changes in the mTORC1 signaling pathway. LAT1 ablation resulted in a notable reduction in phosphorylation of p70S6K, a downstream target of mTORC1, as well as its substrate S6RP. This reduced cell proliferation and mTORC1 activity were rescued when LAT1 was overexpressed. These findings imply an essential role of LAT1 for maintenance of tumor cell growth and additional therapeutic angles against liver cancer.
    Keywords:  LAT1; amino acid transport; branched chain amino acids; liver cancer; mTORC; slc7a5
    DOI:  https://doi.org/10.3390/ijms24119171
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