bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒12‒11
48 papers selected by
Viktor Korolchuk, Newcastle University

  1. Cell Commun Signal. 2022 Dec 06. 20(1): 192
      BACKGROUND: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species. Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on catalase during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice.METHODS: We performed experiments on WT and catalase KO younger (9 weeks) and mature adult (53 weeks) male mice and Mouse embryonic fibroblasts isolated from WT and KO mice from E13.5 embryos as in vivo and in ex-vivo respectively. Mouse phenotyping studies were performed with controls, and a minimum of two independent experiments were performed with more than five mice in each group.
    RESULTS: We found that at the age of 53 weeks (mature adult), catalase-KO mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that mature adult catalase-KO mice induced leaky lysosome by progressive accumulation of lysosomal content, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation. However, the antioxidant N-acetyl-L-cysteine and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient mature adult mice.
    CONCLUSIONS: This study unveils the new role of catalase and its role in lysosomal function during aging. Video abstract.
    Keywords:  Aging; Catalase; Lysosome; ROS; mTORC1
  2. Cell Rep. 2022 Dec 06. pii: S2211-1247(22)01524-8. [Epub ahead of print]41(10): 111653
      The endosomal-lysosomal system is a series of organelles in the endocytic pathway that executes trafficking and degradation of proteins and lipids and mediates the internalization of nutrients and growth factors to ensure cell survival, growth, and differentiation. Here, we reveal regulatory, non-proteolytic ubiquitin signals in this complex system that are controlled by the enigmatic deubiquitinase USP32. Knockout (KO) of USP32 in primary hTERT-RPE1 cells results among others in hyperubiquitination of the Ragulator complex subunit LAMTOR1. Accumulation of LAMTOR1 ubiquitination impairs its interaction with the vacuolar H+-ATPase, reduces Ragulator function, and ultimately limits mTORC1 recruitment. Consistently, in USP32 KO cells, less mTOR kinase localizes to lysosomes, mTORC1 activity is decreased, and autophagy is induced. Furthermore, we demonstrate that depletion of USP32 homolog CYK-3 in Caenorhabditis elegans results in mTOR inhibition and autophagy induction. In summary, we identify a control mechanism of the mTORC1 activation cascade at lysosomes via USP32-regulated LAMTOR1 ubiquitination.
    Keywords:  CP: Cell biology; LAMTOR1; Rab7; Ragulator complex; USP32; autophagy; deubiquitinase (DUB); mTORC1; ubiquitin; v-ATPase
  3. Autophagy. 2022 Dec 05.
      Autophagosomes are crucial components of the cellular recycling machinery that form at endoplasmic reticulum (ER)-associated sites. As the autophagosome membrane is largely devoid of transmembrane proteins, autophagosome biogenesis is thought to be largely regulated by lipid transfer and lipid modifications, as well as membrane-associated proteins. While the membrane origin of autophagosomes and their lipid composition are still incompletely understood, previous studies have found the autophagosome membrane to be enriched in unsaturated fatty acids and have little cholesterol, suggesting that cholesterol removal is an integral step during autophagosome biogenesis. In our study, we demonstrate that short term cholesterol depletion leads to a rapid induction of autophagy and identify the ER-localized cholesterol transport protein GRAMD1C as a negative regulator of starvation-induced macroautophagy/autophagy.
    Keywords:  Aster; GRAMD1C; VASt; autophagy; ccRCC; cholesterol
  4. Cell Stress. 2022 Dec;6(12): 93-107
      (Macro)autophagy is a major lysosome-dependent degradation mechanism which engulfs, removes and recycles unwanted cytoplasmic material, including damaged organelles and toxic protein aggregates. Although a few studies implicate autophagy in CNS demyelinating pathologies, its role, particularly in mature oligodendrocytes and CNS myelin, remains poorly studied. Here, using both pharmacological and genetic inhibition of the autophagic machinery, we provide evidence that autophagy is an essential mechanism for oligodendrocyte maturation in vitro. Our study reveals that two core myelin proteins, namely proteolipid protein (PLP) and myelin basic protein (MBP) are incorporated into autophagosomes in oligodendrocytes, resulting in their degradation. Furthermore, we ablated atg5, a core gene of the autophagic machinery, specifically in myelinating glial cells in vivo by tamoxifen administration (plp-Cre ERT2 ; atg5 f/f ) and showed that myelin maintenance is perturbed, leading to PLP accumulation. Significant morphological defects in myelin membrane such as decompaction accompanied with increased axonal degeneration are observed. As a result, the mice exhibit behavioral deficits. In summary, our data highlight that the maintenance of adult myelin homeostasis in the CNS requires the involvement of a fully functional autophagic machinery.
    Keywords:  CNS; MBP; PLP; autophagy; myelin; oligodendrocyte
  5. MicroPubl Biol. 2022 ;2022
      The highly conserved complexes of Target of Rapamycin (TORC1 and TORC2) are central regulators to many vital cellular processes including growth and autophagy in response to nutrient availability. Previous research has extensively elucidated exogenous nutrient control on TORC1/TORC2; however, little is known about the potential alteration of nutrient pools from mutations in biosynthesis pathways and their impact on Tor pathway activity. Here, we analyze the impacts of heterozygous mutations in aromatic amino acid biosynthesis genes on TOR signaling via differential expression of genes downstream of TORC1 and autophagy induction for TORC1 and TORC2 activity.
  6. Biochem Soc Trans. 2022 Dec 09. pii: BST20220726. [Epub ahead of print]
      Quality control of mitochondria is essential for their homeostasis and function. Light chain 3 (LC3) associated autophagosomes-mediated mitophagy represents a canonical mitochondrial quality control pathway. Alternative quality control processes, such as mitochondrial-derived vesicles (MDVs), have been discovered, but the intact mitochondrial quality control remains unknown. We recently discovered a novel mitolysosome exocytosis mechanism for mitochondrial quality control in flunarizine (FNZ)-induced mitochondria clearance, where autophagosomes are not required, but rather mitochondria are engulfed directly by lysosomes, mediating mitochondrial secretion. As FNZ results in parkinsonism, we propose that excessive mitolysosome exocytosis is the cause.
    Keywords:  extracellular vesicles; lysosome; mitochondria-free; mitochondrial quality control; parkinsonism
  7. Small. 2022 Dec 04. e2204310
      Silica nanoparticles (SiNPs) are one of the most common inorganic nanomaterials. Autophagy is the predominant biological response to nanoparticles and transcription factor EB (TFEB) is a master regulator of the autophagy-lysosome pathway. Previous studies show that SiNPs induce autophagosome accumulation, yet the precise underlying mechanisms remain uncertain. The present study investigates the role of TFEB during SiNP-induced autophagy. SiNP-induced TFEB nuclear translocation is verified using immunofluorescence and western blot assay. The regulation of TFEB is proved to be via EIF2AK3 pathway. A TFEB knockout (KO) cell line is constructed to validate the TFEB involvement in SiNP-induced autophagy. The transcriptomes of wild-type and TFEB KO cells are compared using RNA-sequencing to identify genes of the TFEB-mediated autophagy and lysosome pathways affected by SiNPs. Based on these data and the Human Autophagy Database, four candidate autophagic genes are identified, including HSPB8, ATG4D, CTSB and CTSD. Specifically, that the chaperone HSPB8 is upregulated through SiNP-mediated TFEB activation and forms a chaperone-assisted selective autophagy (CASA) complex with BAG3 and HSC70, triggering HSPB8-assisted selective autophagy, is found. Thus, this study characterizes a novel mechanism underlying SiNP-induced autophagy that helps pave the way for further research on the toxicity and risk assessment of SiNPs.
    Keywords:  EIF2AK3; HSPB8; autophagy; silica nanoparticles; transcription factor EB
  8. Aging (Albany NY). 2022 Dec 01. 14
      During the aging process damaged/dysfunctional proteins and organelles accumulate and contribute to organ dysfunction. Luckily, there is a conserved intracellular process to reuse and recycle these dysregulated cellular components termed macroautophagy (autophagy). Unfortunately, strong evidence indicates autophagy is compromised with aging, protein quality control is jeopardized, and resultant proteotoxicity can contribute significantly to age-associated organ dysfunction. Are there interventions that can re-establish autophagic flux that is otherwise impaired with aging? With particular regard to the heart, here we review evidence that caloric-restriction, the polyamine spermidine, and the mTOR inhibitor rapamycin, even when initiated late-in-life, restore cardiomyocyte autophagy to an extent that lessens age-associated cardiac dysfunction. Cho et al. provide a physiological intervention to this list i.e., regular physical exercise initiated late-in-life boosts cardiomyocyte autophagic flux and rejuvenates cardiac function in male mice. While this study provides strong evidence for a mechanism whereby heightened physical activity can lead to improved heart health in the context of aging, (i) only male mice were studied; (ii) the intensity of exercise-training might not be suitable for all; and (iii) mice with aging-associated comorbidities were not investigated. Nonetheless, Cho et al. provide robust evidence that a low-cost and simple behavioral intervention initiated late-in-life improves cardiomyocyte autophagic flux and rejuvenates cardiac function.
    Keywords:  autophagy; cardiac function; exercise training; heart
  9. ACS Chem Biol. 2022 Dec 05.
      Autophagy is a conserved metabolic pathway that is central to many diseases. Recently, there has been a lot of interest in targeting autophagy with small molecule inhibitors as a possible therapeutic strategy. However, many of the compounds used for autophagy are nonselective. Here, we explored the inhibition of autophagy in pancreatic cancer cells using established selective small molecule inhibitors and discovered an unexpected link between the autophagy pathway and progression through the cell cycle. Our findings revealed that treatments with inhibitors that have different autophagy pathway targets block cell replication and activate other metabolic pathways to compensate for the blockade in autophagy. An unbiased screen looking for known drugs that might synergize with autophagy inhibition revealed new combination treatments that might provide a blueprint for therapeutic approaches to pancreatic cancer. The drugs quizartinib and THZ1 showed a strong synergistic effect in pancreatic cells with autophagy inhibition.
  10. Amino Acids. 2022 Dec 06.
      The activation of the mechanistic target of rapamycin complex 1 (mTORC1), a master regulator of protein synthesis, by anabolic stimuli (such as muscle contraction or essential amino acids) involves its translocation to the cell periphery. Leucine is generally considered the most anabolic of amino acids for its ability to independently modulate muscle protein synthesis. However, it is currently unknown if free leucine impacts region-specific mTORC1-mediated phosphorylation events and protein-protein interactions. In this clinical trial (NCT03952884; registered May 16, 2019), we used immunofluorescence methods to investigate the role of dietary leucine on the postprandial regulation of mTORC1 and ribosomal protein S6 (RPS6), an important downstream readout of mTORC1 activity. Eight young, healthy, recreationally active males (n = 8; 23 ± 3 yrs) ingested 2 g of leucine with vastus lateralis biopsies collected at baseline, 30, 60, and 180 min postprandial. Leucine promoted mTOR translocation to the periphery (~ 18-29%; p ≤ 0.012) and enhanced mTOR localization with the lysosome (~ 16%; both p = 0.049) at 30 and 60 min post-feeding. p-RPS6Ser240/244 staining intensity, a readout of mTORC1 activity, was significantly elevated at all postprandial timepoints in both the total fiber (~ 14-30%; p ≤ 0.032) and peripheral regions (~ 16-33%; p ≤ 0.014). Additionally, total and peripheral p-RPS6Ser240/244 staining intensity at 60 min was positively correlated (r = 0.74, p = 0.036; r = 0.80, p = 0.016, respectively) with rates of myofibrillar protein synthesis over 180 min. The ability of leucine to activate mTORC1 in peripheral regions favors an enhanced rate of MPS, as this is the intracellular space thought to be replete with the cellular machinery that facilitates this anabolic process.
    Keywords:  Amino acids; Anabolism; Immunofluorescence; Muscle protein synthesis; Protein trafficking; mRNA translation
  11. Autophagy. 2022 Dec 05. 1-3
      Viruses are masters at using cellular pathways to aid their replication. Cryo-electron tomography of poliovirus-infected cells revealed how it utilizes macroautophagy to its advantage. Assembly of these non-enveloped virions takes place directly on membranes and requires PIK3C3/VPS34 activity to be completed, whereas the canonical autophagy inducer ULK1 restricts virus assembly. The tomograms further revealed that enterovirus-induced autophagy is selective for RNA-loaded virions, which may help ensure maximum infectivity of the virus-laden vesicles released through secretory autophagy.
    Keywords:  Autophagy; cryo-EM; cryo-electron tomography; enteroviruses; membrane trafficking; membranes; poliovirus; virology; virus replication
  12. J Biochem. 2022 Dec 07. pii: mvac094. [Epub ahead of print]
      Mechanistic target of rapamycin complex 1 (mTORC1) is a serine-threonine kinase that is activated by extracellular signals such as nutrients and growth factors. It plays a key role in the control of various biological processes such as protein synthesis and energy metabolism by mediating or regulating the phosphorylation of multiple target molecules, some of which remain to be identified. We have here reanalyzed a large-scale phosphoproteomics data set for mTORC1 target molecules and identified pre-B cell leukemia transcription factor 2 (PBX2) as such a novel target that is dephosphorylated downstream of mTORC1. We confirmed that PBX2, but not other members of the PBX family, is dephosphorylated in an mTORC1 activity-dependent manner. Furthermore, pharmacological and gene knockdown experiments revealed that glycogen synthase kinase 3 (GSK3) and protein phosphatase 1 (PP1) are responsible for the phosphorylation and dephosphorylation of PBX2, respectively. Our results thus suggest that the balance between the antagonistic actions of GSK3 and PP1 determines the phosphorylation status of PBX2 and its regulation by mTORC1.
    Keywords:  glycogen synthase kinase 3 (GSK3); mechanistic target of rapamycin complex 1 (mTORC1); phosphorylation; pre–B cell leukemia transcription factor 2 (PBX2); protein phosphatase 1 (PP1)
  13. Br J Pharmacol. 2022 Dec 07.
      BACKGROUND AND PURPOSE: Acetaminophen (APAP)-induced hepatotoxicity is the leading cause of drug-induced liver injury worldwide. Autophagy is a degradative process by which various cargoes are collected by the autophagic receptors such as p62/SQSTSM-1/Sequestosome-1 for lysosomal degradation. Here, we investigated the protective role of p62-dependent autophagy in APAP-induced liver injury.EXPERIMENTAL APPROACH: APAP-induced hepatotoxicity was induced by a single i.p. injection of APAP (500 mg/kg) in C57/BL6 male mice. YTK-2205 (20 mg/kg), a p62 agonist targeting ZZ domain, was co- or post-administered with APAP. Western blotting and immunocytochemistry were performed to explore the mechanism.
    KEY RESULTS: N-terminal arginylation of the molecular chaperone calreticulin retro-translocated from the endoplasmic reticulum (ER) was induced in the livers undergoing APAP-induced hepatotoxicity, and YTK-2205 exhibited notable therapeutic efficacy in acute hepatotoxicity as assessed by the levels of serum alanine aminotransferase and hepatic necrosis. This efficacy was significantly attributed to accelerated degradation of ubiquitin (Ub) conjugates as well as damaged mitochondria (mitophagy) and ER (ER-phagy). In primary murine hepatocytes treated with APAP, YTK-2205 induced the co-localization of p62+ LC3+ phagophores to the sites of mitophagy and ER-phagy. A similar activity of YTK-2205 was observed with N-acetyl-p-benzoquinone imine, a putative toxic APAP metabolite in Hep3B cells.
    CONCLUSION AND IMPLICATIONS: Our results elucidated that p62-dependent autophagy plays a key role in the removal of cytotoxic materials such as damaged mitochondria in APAP-induced hepatotoxicity. Small molecule ligands to p62 may be developed into drugs to treat this pathological condition.
    Keywords:  Acetaminophen; ZZ ligand; hepatotoxicity; p62; ubiquitination
  14. Autophagy. 2022 Dec 05. 1-22
      The ubiquitin (Ub) kinase-ligase pair PINK1-PRKN mediates the degradation of damaged mitochondria by macroautophagy/autophagy (mitophagy). PINK1 surveils mitochondria and upon stress accumulates on the mitochondrial surface where it phosphorylates serine 65 of Ub to activate PRKN and to drive mitochondrial turnover. While loss of either PINK1 or PRKN is genetically linked to Parkinson disease (PD) and activating the pathway seems to have great therapeutic potential, there is no formal proof that stimulation of mitophagy is always beneficial. Here we used biochemical and cell biological methods to study single nucleotide variants in the activation loop of PINK1 to modulate the enzymatic function of this kinase. Structural modeling and in vitro kinase assays were used to investigate the molecular mechanism of the PINK1 variants. In contrast to the PD-linked PINK1G411S mutation that diminishes Ub kinase activity, we found that the PINK1G411A variant significantly boosted Ub phosphorylation beyond levels of PINK1 wild type. This resulted in augmented PRKN activation, mitophagy rates and increased viability after mitochondrial stress in midbrain-derived, gene-edited neurons. Mechanistically, the G411A variant stabilizes the kinase fold of PINK1 and transforms Ub to adopt the preferred, C-terminally retracted conformation for improved substrate turnover. In summary, we identify a critical role of residue 411 for substrate receptivity that may now be exploited for drug discovery to increase the enzymatic function of PINK1. The genetic substitution of Gly411 to Ala increases mitophagy and may be useful to confirm neuroprotection in vivo and might serve as a critical positive control during therapeutic development.Abbreviations: ATP: adenosine triphosphate; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; Ub-CR: ubiquitin with C-terminally retracted tail; CTD: C-terminal domain (of PINK1); ELISA: enzyme-linked immunosorbent assay; HCI: high-content imaging; IB: immunoblot; IF: immunofluorescence; NPC: neuronal precursor cells; MDS: molecular dynamics simulation; PD: Parkinson disease; p-S65-Ub: ubiquitin phosphorylated at Ser65; RMSF: root mean scare fluctuation; TOMM: translocase of outer mitochondrial membrane; TVLN: ubiquitin with T66V and L67N mutation, mimics Ub-CR; Ub: ubiquitin; WT: wild-type.
    Keywords:  Mitophagy; PINK1; PRKN; parkin; parkinson disease; ubiquitin
  15. Nat Commun. 2022 Dec 06. 13(1): 7526
      The glycolytic enzyme, pyruvate kinase Pyk1 maintains telomere heterochromatin by phosphorylating histone H3T11 (H3pT11), which promotes SIR (silent information regulator) complex binding at telomeres and prevents autophagy-mediated Sir2 degradation. However, the exact mechanism of action for H3pT11 is poorly understood. Here, we report that H3pT11 directly inhibits Dot1-catalyzed H3K79 tri-methylation (H3K79me3) and uncover how this histone crosstalk regulates autophagy and telomere silencing. Mechanistically, Pyk1-catalyzed H3pT11 directly reduces the binding of Dot1 to chromatin and inhibits Dot1-catalyzed H3K79me3, which leads to transcriptional repression of autophagy genes and reduced autophagy. Despite the antagonism between H3pT11 and H3K79me3, they work together to promote the binding of SIR complex at telomeres to maintain telomere silencing. Furthermore, we identify Reb1 as a telomere-associated factor that recruits Pyk1-containing SESAME (Serine-responsive SAM-containing Metabolic Enzyme) complex to telomere regions to phosphorylate H3T11 and prevent the invasion of H3K79me3 from euchromatin into heterochromatin to maintain telomere silencing. Together, these results uncover a histone crosstalk and provide insights into dynamic regulation of silent heterochromatin and autophagy in response to cell metabolism.
  16. Ther Adv Respir Dis. 2022 Jan-Dec;16:16 17534666221140972
      Idiopathic pulmonary fibrosis (IPF) is an interstitial pulmonary disease with an extremely poor prognosis. Autophagy is a fundamental intracellular process involved in maintaining cellular homeostasis and regulating cell survival. Autophagy deficiency has been shown to play an important role in the progression of pulmonary fibrosis. This review focused on the six steps of autophagy, as well as the interplay between autophagy and other seven pulmonary fibrosis related mechanisms, which include extracellular matrix deposition, myofibroblast differentiation, epithelial-mesenchymal transition, pulmonary epithelial cell dysfunction, apoptosis, TGF-β1 pathway, and the renin-angiotensin system. In addition, this review also summarized autophagy-related signaling pathways such as mTOR, MAPK, JAK2/STAT3 signaling, p65, and Keap1/Nrf2 signaling during the development of IPF. Furthermore, this review also illustrated the commonly used autophagy detection methods, the currently approved antifibrotic drugs pirfenidone and nintedanib, and several prospective compounds targeting autophagy for the treatment of IPF.
    Keywords:  autophagy; autophagy detection; idiopathic pulmonary fibrosis; therapies
  17. Autophagy. 2022 Dec 06.
      Macroautophagy/autophagy, a stress-responsive cellular survival mechanism, plays important and context-dependent roles in cancer, and its inhibition has been implicated as a promising cancer therapeutic approach. The detailed mechanisms underlying the function of autophagy in cancer have not been fully understood. In this study, we show that autophagy inhibition promotes both the efficacy of chemotherapy for the treatment of glioblastoma (GBM) and therapy-induced senescence of GBM cells. As a specific cell fate characterized by permanent cell cycle arrest, senescence is also associated with the expression of a panel of specific secreted protein factors known as senescence-associated secretory phenotype (SASP). Intriguingly, we found that autophagy inhibition not only quantitatively enhanced GBM cell senescence but also qualitatively altered the spectrum of SASP. The altered SASP had increased potent activity to induce paracrine senescence of neighboring GBM cells, to skew macrophage polarization toward the anti-tumor M1 state, and to block the recruitment of pro-tumor neutrophils to GBM tumor tissues. Taken together, this study reveals novel functional communication between autophagy and senescence and suggests cancer therapeutic approaches harnessing autophagy blockage in inducing senescence-mediated antitumor immunity.
    Keywords:  SASP; antitumor immunity; autophagy; glioblastoma multiform; senescence
  18. Exp Hematol. 2022 Dec 05. pii: S0301-472X(22)00811-6. [Epub ahead of print]
      Although establishing and maintaining mitochondria are essential to produce massive amounts of heme in erythroblasts, mitochondria must be degraded upon terminal differentiation to red blood cells, thus creating a bi-phasic regulatory process. Previously, we reported that iron deficiency in mice promotes mitochondria retention in red blood cells, suggesting that the proper amount of iron and/or heme is necessary for the degradation of mitochondria during erythroblast maturation. Since the transcription factor GATA1 regulates autophagy in erythroid cells, which involves mitochondrial clearance (mitophagy), we investigated the relationship between iron, heme and mitophagy by analyzing the expression of genes related to GATA1 and autophagy and the impact of iron or heme restriction on the amount of mitochondria. We found that heme promotes the expression of GATA1-regulated mitophagy-related genes and induction of mitophagy. GATA1 might induce the expression of autophagy-related genes Atg4d and Stk11 for mitophagy through a heme-dependent mechanism in murine erythroleukemia (MEL) cells and in a genetic rescue system with G1E-ER-GATA1 erythroblast cells derived from Gata1-null murine embryonic stem cells. These results provide evidence for a bi-phasic mechanism in which mitochondria are essential for heme generation, and the heme generated during differentiation promotes mitophagy and mitochondria disposal. This mechanism provides a molecular framework for understanding this fundamentally important cell biological process.
    Keywords:  GATA transcription factor; autophagy; erythropoiesis; heme; iron; mitochondria
  19. Signal Transduct Target Ther. 2022 Dec 09. 7(1): 388
      Chemoresistance has long been the bottleneck of ovarian cancer (OC) prognosis. It has been shown that mitochondria play a crucial role in cell response to chemotherapy and that dysregulated mitochondrial dynamics is intricately linked with diseases like OC, but the underlying mechanisms remain equivocal. Here, we demonstrate a new mechanism where CRL4CUL4A/DDB1 manipulates OC cell chemoresistance by regulating mitochondrial dynamics and mitophagy. CRL4CUL4A/DDB1 depletion enhanced mitochondrial fission by upregulating AMPKαThr172 and MFFSer172/Ser146 phosphorylation, which in turn recruited DRP1 to mitochondria. CRL4CUL4A/DDB1 loss stimulated mitophagy through the Parkin-PINK1 pathway to degrade the dysfunctional and fragmented mitochondria. Importantly, CRL4CUL4A/DDB1 loss inhibited OC cell proliferation, whereas inhibiting autophagy partially reversed this disruption. Our findings provide novel insight into the multifaceted function of the CRL4 E3 ubiquitin ligase complex in regulating mitochondrial fission, mitophagy, and OC chemoresistance. Disruption of CRL4CUL4A/DDB1 and mitophagy may be a promising therapeutic strategy to overcome chemoresistance in OC.
  20. Front Pharmacol. 2022 ;13 999017
      Bone homeostasis depends on a precise dynamic balance between bone resorption and bone formation, involving a series of complex and highly regulated steps. Any imbalance in this process can cause disturbances in bone metabolism and lead to the development of many associated bone diseases. Autophagy, one of the fundamental pathways for the degradation and recycling of proteins and organelles, is a fundamental process that regulates cellular and organismal homeostasis. Importantly, basic levels of autophagy are present in all types of bone-associated cells. Due to the cyclic nature of autophagy and the ongoing bone metabolism processes, autophagy is considered a new participant in bone maintenance. Novel therapeutic targets have emerged as a result of new mechanisms, and bone metabolism can be controlled by interfering with autophagy by focusing on certain regulatory molecules in autophagy. In parallel, several studies have reported that various natural products exhibit a good potential to mediate autophagy for the treatment of metabolic bone diseases. Therefore, we briefly described the process of autophagy, emphasizing its function in different cell types involved in bone development and metabolism (including bone marrow mesenchymal stem cells, osteoblasts, osteocytes, chondrocytes, and osteoclasts), and also summarized research advances in natural product-mediated autophagy for the treatment of metabolic bone disease caused by dysfunction of these cells (including osteoporosis, rheumatoid joints, osteoarthritis, fracture nonunion/delayed union). The objective of the study was to identify the function that autophagy serves in metabolic bone disease and the effects, potential, and challenges of natural products for the treatment of these diseases by targeting autophagy.
    Keywords:  autophagy; bone metabolism; fracture nonunion/delayed union; natural products; osteoarthritis; osteoporosis; rheumatoid arthritis
  21. Cancer Res. 2022 Dec 08. pii: CAN-22-2370. [Epub ahead of print]
      The drug-tolerant persister (DTP) state enables cancer cells to evade cytotoxic stress from anti-cancer therapy. However, the mechanisms governing DTP generation remain poorly understood. Here, we observed that lung adenocarcinoma (LUAD) cells and organoids entered a quiescent DTP state to survive MAPK inhibitor treatment. DTP cells following MAPK inhibition underwent a metabolic switch from glycolysis to oxidative phosphorylation (OXPHOS). PTEN-induced kinase 1 (PINK1), a serine/threonine kinase that initiates mitophagy, was upregulated to maintain mitochondrial homeostasis during DTP generation. PINK1-mediated mitophagy supported DTP cell survival and contributed to poor prognosis. Mechanistically, MAPK pathway inhibition resulted in MYC-dependent transcriptional upregulation of PINK1, leading to mitophagy activation. Mitophagy inhibition using either clinically applicable chloroquine or depletion of PINK1 eradicated drug tolerance and allowed complete response to MAPK inhibitors. This study uncovers PINK1-mediated mitophagy as a novel tumor protective mechanism for DTP generation, providing a therapeutic opportunity to eradicate DTP and achieve complete responses.
  22. J Orthop Translat. 2023 Jan;38 229-240
      Background: Glucocorticoid (GC) is one of frequently used anti-inflammatory agents, but its administration is unfortunately accompanied with bone loss. Although sporadic studies indicated that osteocytes are subject to a series of pathological changes under GC stress, including overexpression of cathepsin K, the definite role of osteocytes in GC-induced bone loss remains largely unclear.Methods: Gene expression of Ctsk and protein levels of cathepsin K were assessed in MLO-Y4 cell lines exposed to dexamethasone (Dex) of different time (0, 12, 24 hours) and dose (0, 10-8 and 10-6 M) courses by RT-qPCR and western blotting, respectively. Confocal imaging and immunostaining were then performed to evaluate the effects of osteocyte-derived cathepsin K on type I collagen in a primary osteocyte ex vivo culture system. MitoTracker Red was used to stain mitochondria for mitochondria morphology assessment and JC-1 assay was employed to evaluate the mitochondria membrane potential in MLO-Y4 cells following Dex treatment. Activation of PINK1-mediated mitophagy was evaluated by immunostaining of the PINK1 protein and CytoID assay. Mdivi-1 was used to inhibit mitophagy and siRNAs were used for the inhibition of Pink1 and Atg5.
    Results: GC triggered osteocytes to produce excessive cathepsin K which in turn led to the degradation of type I collagen in the extracellular matrix in a primary osteocyte ex vivo culture system. Meanwhile, GC administration increased mitochondrial fission and membrane depolarization in osteocytes. Further, the activation of PINK1-mediated mitophagy was demonstrated to be responsible for the diminishment of dysfunctional mitochondria in osteocytes. Examination of relationship between mitophagy and cathepsin K production revealed that inhibition of mitophagy via knocking down Pink1 gene abolished the GC-triggered cathepsin K production. Interestingly, GC's activation effect towards cathepsin K via mitophagy was found to be independent on the canonical autophagy as this effect was not impeded when inhibiting the canonical autophagy via Atg5 suppression.
    Conclusion: GC-induced PINK1-mediated mitophagy substantially modulates the production of cathepsin K in osteocytes, which could be an underlying mechanism by which osteocytes contribute to the extracellular matrix degradation during bone loss.
    The Translational potential of this article: Findings of the current study indicate a possible role of osteocyte mitophagy in GC-induced bone loss, which provides a potential therapeutic approach to alleviate GC-induced osteoporosis by targeting PINK1-mediated osteocytic mitophagy.
    Keywords:  CCCP, Carbonyl cyanide chlorophenylhydrazone; CTSK, cathepsin K; CTX, carboxyterminal telopeptide of type I collagen; Cathepsin K; Drp1, dynamin-related protein 1; GC, glucocorticoid; Glucocorticoid; MTR, MitoTracker Red; Mfn2, mitofusin-2; Mitophagy; Osteocyte; PINK1; PINK1, PTEN-induced putative kinase 1; TFEB, transcription factor EB; TRAP, tartrate-resistant acid phosphatase; ΔѰm, mitochondria membrane potential
  23. Nat Commun. 2022 Dec 05. 13(1): 7493
      Although light is essential for photosynthesis, it has the potential to elevate intracellular levels of reactive oxygen species (ROS). Since high ROS levels are cytotoxic, plants must alleviate such damage. However, the cellular mechanism underlying ROS-induced leaf damage alleviation in peroxisomes was not fully explored. Here, we show that autophagy plays a pivotal role in the selective removal of ROS-generating peroxisomes, which protects plants from oxidative damage during photosynthesis. We present evidence that autophagy-deficient mutants show light intensity-dependent leaf damage and excess aggregation of ROS-accumulating peroxisomes. The peroxisome aggregates are specifically engulfed by pre-autophagosomal structures and vacuolar membranes in both leaf cells and isolated vacuoles, but they are not degraded in mutants. ATG18a-GFP and GFP-2×FYVE, which bind to phosphatidylinositol 3-phosphate, preferentially target the peroxisomal membranes and pre-autophagosomal structures near peroxisomes in ROS-accumulating cells under high-intensity light. Our findings provide deeper insights into the plant stress response caused by light irradiation.
  24. Front Mol Neurosci. 2022 ;15 1039135
      Recent evidence has shown that G protein-coupled receptors (GPCRs) are direct sensors of the autophagic machinery and opioid receptors regulate neuronal plasticity and neurotransmission with an as yet unclarified mechanism. Using in vitro and in vivo experimental approaches, this study aims to clarify the potential role of autophagy and κ-opioid receptor (κ-OR) signaling in synaptic alterations. We hereby demonstrate that the selective κ-OR agonist U50,488H, induces autophagy in a time-and dose-dependent manner in Neuro-2A cells stably expressing the human κ-OR by upregulating microtubule-associated protein Light Chain 3-II (LC3-II), Beclin 1 and Autophagy Related Gene 5 (ATG5). Pretreatment of neuronal cells with pertussis toxin blocked the above κ-OR-mediated cellular responses. Our molecular analysis also revealed a κ-OR-driven upregulation of becn1 gene through ERK1,2-dependent activation of the transcription factor CREB in Neuro-2A cells. Moreover, our studies demonstrated that sub-chronic U50,488H administration in mice causes profound increases of specific autophagic markers in the hippocampus with a concomitant decrease of several pre-and post-synaptic proteins, such as spinophilin, postsynaptic density protein 95 (PSD-95) and synaptosomal associated protein 25 (SNAP25). Finally, using acute stress, a stimulus known to increase the levels of the endogenous κ-OR ligand dynorphin, we are demonstrating that administration of the κ-ΟR selective antagonist, nor-binaltorphimine (norBNI), blocks the induction of autophagy and the stress-evoked reduction of synaptic proteins in the hippocampus. These findings provide novel insights about the essential role of autophagic machinery into the mechanisms through which κ-OR signaling regulates brain plasticity.
    Keywords:  Beclin 1; CREB; ERK1,2; Gi/o; autophagy; dynorphin; synaptic alterations; κ-opioid receptor
  25. Front Oncol. 2022 ;12 1013500
      ATF-4 is a master regulator of transcription of genes essential for cellular-adaptive function. In response to the quantum and duration of stress, ATF-4 diligently responds to both pro-apoptotic and pro-survival signals converging into either autophagy or apoptosis/senescence. Despite emerging cues implying a relationship between autophagy and senescence, how these two processes are controlled remains unknown. Herein, we demonstrate β-(4-fluorobenzyl) Arteannuin B (here after Arteannuin 09), a novel semisynthetic derivative of Arteannuin B, as a potent ER stress inducer leading to the consistent activation of ATF-4. Persistent ATF-4 expression at early time-points facilitates the autophagy program and consequently by upregulating p21 at later time-points, the signaling is shifted towards G2/M cell cycle arrest. As bZIP transcription factors including ATF-4 are obligate dimers, and because ATF-4 homodimers are not highly stable, we hypothesized that ATF-4 may induce p21 expression by physically interacting with another bZIP family member i.e., C/EBPβ. Our co-immunoprecipitation and co-localization studies demonstrated that ATF-4 is principally responsible for the autophagic potential of Arteannuin 09, while as, induction of both ATF-4 and C/EBPβ is indispensable for the p21 regulated-cell cycle arrest. Interestingly, inhibition of autophagy signaling switches the fate of Arteannuin 09 treated cells from senescence to apoptosis. Lastly, our data accomplished that Arteannuin 09 is a potent inhibitor of tumor growth and inducer of premature senescence in vivo.
    Keywords:  ATF-4; C/EBPβ; arteannuin B; autophagy; breast cancer; senescence
  26. Neurobiol Dis. 2022 Dec 05. pii: S0969-9961(22)00333-3. [Epub ahead of print]176 105941
      The protein DJ-1 is mutated in rare familial forms of recessive Parkinson's disease and in parkinsonism accompanied by amyotrophic lateral sclerosis symptoms and dementia. DJ-1 is considered a multitasking protein able to confer protection under various conditions of stress. However, the precise cellular function still remains elusive. In the present work, we evaluated fruit flies lacking the expression of the DJ-1 homolog dj-1β as compared to control aged-matched individuals. Behavioral evaluations included lifespan, locomotion in an open field arena, sensitivity to oxidative insults, and resistance to starvation. Molecular analyses were carried out by analyzing the mitochondrial morphology and functionality, and the autophagic response. We demonstrated that dj-1β null mutant flies are hypoactive and display higher sensitivity to oxidative insults and food deprivation. Analysis of mitochondrial homeostasis revealed that loss of dj-1β leads to larger and more circular mitochondria, characterized by impaired complex-I-linked respiration while preserving ATP production capacity. Additionally, dj-1β null mutant flies present an impaired autophagic response, which is suppressed by treatment with the antioxidant molecule N-Acetyl-L-Cysteine. Overall, our data point to a mechanism whereby DJ-1 plays a critical role in the maintenance of energy homeostasis, by sustaining mitochondrial homeostasis and affecting the autophagic flux through the maintenance of the cellular redox state. In light of the involvement of DJ-1 in neurodegenerative diseases and considering that neurons are highly energy-demanding cells, particularly sensitive to redox stress, our study sheds light on a key role of DJ-1 in the maintenance of cellular homeostasis.
    Keywords:  Autophagy; DJ-1; Energy balance; Mitochondria; Redox homeostasis
  27. In Vitro Cell Dev Biol Anim. 2022 Dec 09.
      Benzophenones (BPs) are endocrine disruptors frequently used in sunscreens and food packaging as UV blockers. Our goal was to assess the effect of benzophenone 2 (BP2) and 3 (BP3) on gene expression related to autophagy process and ER stress response in pancreatic beta cells. To that end, the mouse pancreatic beta cell line MIN6B1 was treated with 10 µM BP2 or BP3 in the presence or absence of the autophagy-inhibitor chloroquine (CQ, 10 µM) or the autophagy-inducer rapamycin (RAPA, 50 nM) during 24 h. BP3 inhibited the expression of the autophagic gene Ulk1, and additional effects were uncovered when autophagy was modified by CQ and RAPA. BP3 counteracted CQ-induced Lamp2 expression but did not compensate CQ-induced Sqstm1/p62 gene transcription, neither BP2. Nevertheless, the BPs did not alter the autophagic flux. In relation to ER stress, BP3 inhibited unspliced and spliced Xbp1 mRNA levels in the presence or absence of CQ, totally counteracted CQ-induced Chop gene expression, and partially reverted CQ-induced Grp78/Bip mRNA levels, while BP2 also partially inhibited Grp78/Bip mRNA induction by CQ. In conclusion, BPs, principally BP3, affect cellular adaptive responses related to autophagy, lysosomal biogenesis, and ER stress in pancreatic beta cells, indicating that BP exposure could lead to beta cell dysfunction.
    Keywords:  Autophagy; Benzophenone 2; Benzophenone 3; Endocrine disruptor; Endoplasmic reticulum stress; Pancreatic beta cells
  28. Neurochem Res. 2022 Dec 05.
      We previously showed that kaempferol (KAE) could exert neuroprotective effects against PD. It has been demonstrated that abnormal autophagy plays a key role in the development of PD. Mitochondrial dysfunction, involved in the development of PD, can damage dopaminergic neurons. Whether the protective effects of KAE were exerted via regulating autophagy remains largely undefined, however. This study aimed to investigate whether KAE could protect dopaminergic neurons via autophagy and the underlying mechanisms using a MPTP/MPP+-stimulated PD model. Cell viability was detected by cell counting kit-8 (CCK-8) assay, and protein levels of autophagy mediators along with mTOR signaling pathway molecules were investigated by immunohistochemistry and Western blot analyses. The results showed that KAE could ameliorate the behavioral impairments of mice, reduce the loss of tyrosine hydroxylase (TH)-positive neurons in the substantia nigra pars compacta, and reduce α-synuclein (α-syn) levels. Furthermore, KAE upregulated levels of autophagy effector protein of Beclin-1 and autophagy microtubule associated protein of light chain 3 (LC3) in the substantia nigra (SN) while rescuing mitochondrial integrity, and downregulated levels of ubiquitin binding protein p62 and cleaved caspase-3, probably by decreasing the mammalian target of rapamycin (mTOR) signaling pathway. Further in vitro experiments demonstrated similar results. In conclusion, KAE exerts neuroprotective effects against PD potentially by promoting autophagy via inhibiting the mTOR signaling pathway.
    Keywords:  Autophagy; Kaempferol; Parkinson’s disease; mTOR signaling pathway
  29. Food Funct. 2022 Dec 09.
      In recent years, the effect of lipid metabolism on health has attracted more and more attention. Ginseng is a traditional Chinese herbal medicine in China and is widely used as food in Asia. Ginsenoside Rb1 (Gs-Rb1) is the most abundant ingredient in ginsenoside, which has a variety of biological activities. In this study, we found that Gs-Rb1 can reduce lipid accumulation in mice and HepG2 cells induced by a high-fat diet (HFD) and palmitic acid (PA). At the same time, we also found that Gs-Rb1 could stimulate the autophagic flux of HFD-fed mice and PA-treated HepG2 cells, and it is further verified by adding the autophagy activator rapamycin (Rapa) and autophagy inhibitor chloroquine (CQ). Furthermore, we found that Gs-Rb1 promoted the nucleus translocation of the transcription factor EB (TFEB) and the target role of miR-128, thus stimulating autophagic flux. Therefore, our results showed that Gs-Rb1 enhanced the transcription of TFEB and its downstream lysosome-related genes by inhibiting miR-128, improved the degradation ability of lysosomes to autophagosomes, and then promoted autophagic lipid degradation.
  30. Front Cardiovasc Med. 2022 ;9 921829
      Improving the survival rate of cardiomyocytes is the key point to treat most of the heart diseases, and targeting autophagy is a potential advanced therapeutic approach. Monitoring autophagic activity in cardiomyocytes in situ will be useful for studying autophagy-related heart disease and screening autophagy-modulating drugs. Zebrafish, Danio rerio, has been proven as an animal model for studying heart diseases in situ. Taken the advantage of zebrafish, especially the imaging of intact animals, here we generated two stable transgenic zebrafish lines that specifically expressed EGFP-map1lc3b or mRFP-EGFP-map1lc3b in cardiomyocytes under the promoter of myosin light chain 7. We first used a few known autophagy-modulating drugs to confirm their usefulness. By quantifying the density of autophagosomes and autolysosomes, autophagy inducers and inhibitors showed their regulatory functions, which were consistent with previous studies. With the two lines, we then found a significant increase in the density of autophagosomes but not autolysosomes in zebrafish cardiomyocytes at the early developmental stages, indicating the involvement of autophagy in early heart development. To prove their applicability, we also tested five clinical statins by the two lines. And we found that statins did not change the density of autophagosomes but reduced the density of autolysosomes in cardiomyocytes, implying their regulation in autophagic flux. Our study provides novel animal models for monitoring autophagic activity in cardiomyocytes in situ, which could be used to study autophagy-related cardiomyopathy and drug screening.
    Keywords:  animal model; autophagy; myocardial cell; statins; zebrafish
  31. Cell Death Differ. 2022 Dec 08.
      Cisplatin and other platinum-based anticancer agents are among the most widely used chemotherapy drugs in the treatment of different types of cancer. However, it is common to find patients who respond well to treatment at first but later relapse due to the appearance of resistance to cisplatin. Among the mechanisms responsible for this phenomenon is the increase in DNA damage repair. Here, we elucidate the effect of cisplatin on the MRN (MRE11-RAD50-NBS1) DNA damage sensor complex. We found that the tumor suppressor FBXW7 is a key factor in controlling the turnover of the MRN complex by inducing its degradation through lysosomes. Inhibition of lysosomal enzymes allowed the detection of the association of FBXW7-dependent ubiquitylated MRN with LC3 and the autophagy adaptor p62/SQSTM1 and the localization of MRN in lysosomes. Furthermore, cisplatin-induced cell death increased MRN degradation, suggesting that this complex is one of the targets that favor cell death. These findings open the possibility of using the induction of the degradation of the MRN complex after genotoxic damage as a potential therapeutic strategy to eliminate tumor cells.
  32. Drug Dev Res. 2022 Dec 07.
      Urolithin A (UA) is a microbial metabolite of natural polyphenols ellagitannins and ellagic acid with well-established antitumor properties against various malignancies. However, the exact role of UA in gastric cancer (GC) progression remains largely unclear. In the present study, we investigated the effects and potential mechanisms of UA in GC in vitro and in vivo. Our results revealed that UA could suppress GC cell proliferation, inhibit migration and invasion, promote apoptosis, and induce autophagy via the phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin pathway in vitro. The autophagy inhibitors 3-methyladenine and chloroquine augmented the inhibitory effect of UA on proliferation and promoted apoptosis, implying that UA mediated the cytoprotective role of autophagy. Meanwhile, the in vivo experiments showed that UA effectively suppressed tumor growth, enhanced the therapeutic effects, and alleviated chemotherapy toxicity in xenograft models. Overall, these findings offer novel insights into the role of UA in tumor therapy and suggest that UA may possess potential therapeutic applications for GC.
    Keywords:  PI3K/Akt/mTOR pathway; autophagy; gastric cancer; urolithin A
  33. Sci Rep. 2022 Dec 08. 12(1): 21265
      Solid tumors often contain regions with very low oxygen concentrations or hypoxia resulting from altered metabolism, uncontrolled proliferation, and abnormal tumor blood vessels. Hypoxia leads to resistance to both radio- and chemotherapy and a predisposition to tumor metastases. Under hypoxia, sequestosome 1 (SQSTM1/p62), a multifunctional stress-inducible protein involved in various cellular processes, such as autophagy, is down-regulated. The hypoxic depletion of p62 is mediated by autophagic degradation. We herein demonstrated that hypoxia down-regulated p62 in the hepatoma cell line Hep3B at the transcriptional and post-translational levels. At the transcriptional level, hypoxia down-regulated p62 mRNA by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2). The overexpression of Nrf2 and knockdown of Siah2, a negative regulator of Nrf2 under hypoxia, diminished the effects of hypoxia on p62 mRNA. At the post-translational level, the proteasome inhibitor MG132, but not the lysosomal inhibitors ammonium chloride and bafilomycin, prevented the hypoxic depletion of p62, suggesting the involvement of the proteasome pathway. Under hypoxia, the expression of the E3 ubiquitin ligase Parkin was up-regulated in a hypoxia-inducible factor 1α-dependent manner. Parkin ubiquitinated p62 and led to its proteasomal degradation, ensuring low levels of p62 under hypoxia. We demonstrated that the effects of Parkin on p62 required heat shock cognate 71 kDa protein (Hsc70). We also showed that the overexpression of Nrf2 and knockdown of Parkin or Hsc70 induced the accumulation of p62 and reduced the viability of cells under hypoxia. We concluded that a decrease in p62, which involves regulation at the transcriptional and post-translational levels, is critical for cell survival under hypoxia. The present results show the potential of targeting Nrf2/Parkin-Hsc70-p62 as a novel strategy to eradicate hypoxic solid tumors.
  34. Mol Divers. 2022 Dec 08.
      DNA damage response (DDR) and autophagy are concerned with maintaining cellular homeostasis and dysregulation of these two pathways lead to pathologic conditions including tumorigenesis. Autophagy is activated as a protective mechanism during DDR which is indicative of their functional cooperativity but the molecular mechanism leading to the convergence of these two pathways during genotoxic stress remains elusive. In this study, through in silico analysis, we have shown an interaction between the Mediator of DNA damage checkpoint 1 (MDC1), an important DDR-associated protein, and Beclin-1, an autophagy inducer. MDC1 is an adaptor or scaffold protein known to regulate DDR, apoptosis, and cell cycle progression. While, Beclin-1 is involved in autophagosome nucleation and exhibits affinity for binding to Fork-head-associated domain (FHA) containing proteins. The FHA domain is commonly conserved in DDR-related proteins including MDC1. Through molecular docking, we have predicted the modeled complex between the MDC1 FHA domain and the Beclin-1 Coiled coil domain (CCD). The docking complex was modeled using ClusPro2.0, based on the crystal structure for the dimerized MDC1 FHA domain and Beclin-1 CCD. The complex stability and binding affinities were assessed using a Ramachandran plot, MD simulation, MM/GBSA, and PRODIGY webserver. Finally, the hot-spot residues at the interface were determined using computational alanine scanning by the DrugScorePPI webserver. Our analysis unveils significant interaction between MDC1 and Beclin-1, involving hydrogen bonds, non-bonded contacts, and salt bridges and indicates MDC1 possibly recruits Beclin-1 to the DSBs, as a consequence of which Beclin-1 is able to modulate DDR.
    Keywords:  And tumor; Autophagy; Beclin-1; DNA damage response; Mediator of DNA damage checkpoint 1
  35. J Reprod Immunol. 2022 Nov 24. pii: S0165-0378(22)00295-9. [Epub ahead of print]155 103766
      Inhibition of autophagy contributes to the pathophysiology of preeclampsia. Although chloroquine (CHQ) is an autophagy inhibitor, it can reduce the occurrence of preeclampsia in women with systemic lupus erythematosus. To clarify this important clinical question, this study aimed to address the safety of CHQ in trophoblast cells from the viewpoint of homeostasis, in which the anti-oxidative stress (OS) response and autophagy are involved. We used Western blotting to evaluate the protein levels in the trophoblast cells. The expression levels of heme oxygenase-1 (HO-1), an anti-OS enzyme, mediate resistance to OS induced by hydrogen peroxide (H2O2) in trophoblast cell lines. Among the autophagy modulators, bafilomycin A1 (BAF), an autophagy inhibitor, but not autophagy activators, suppressed HO-1 expression in BeWo cells; CHQ did not suppress HO-1 expression in BeWo cells. To clarify the role of autophagy in HO-1 induction, we observed no difference in HO-1 induction by H2O2 between autophagy-normal and autophagy-deficient cells. As for the mechanism of HO-1 induction by OS, BAF suppressed HO-1 induction by downregulating the expression of neighbor of BRCA1 gene 1 (NBR1) in the selective p62-NBR1-nuclear factor erythroid 2-related factor 2 (Nrf2) autophagy pathway. CHQ did not inhibit HO-1 expression by sustaining NBR1 expression in human villous tissues compared to BAF treatment. In conclusion, CHQ is a safer medicine than BAF for sustaining NBR1, which resist against OS in trophoblasts by connecting selective autophagy and the anti-OS response.
    Keywords:  Autophagy; Chloroquine; HO-1; NBR1; Oxidative stress
  36. Biofouling. 2022 Dec 07. 1-14
      Autophagy is a highly conserved catabolic pathway that is vital for cells; however, the effects of autophagy on the biofilm formation and antifungal resistance of Candida albicans are still unknown. In this study, the potential molecular mechanisms of autophagy in biofilm formation and antifungal resistance were investigated. It was found that 3536 genes were differentially expressed between biofilm and planktonic C. albicans. ATG gene expression and autophagy activity were higher in biofilm than in planktonic C. albicans. Autophagic activities were higher in matured biofilms than that in pre-matured biofilms. Autophagy was involved in C. albicans biofilm formation and its activity increased during biofilm maturation. Further, ALP activity, AO staining cells, and autophagosomes inside cells were obviously reduced in biofilms of atg13Δ/Δ and atg27Δ/Δ strains; moreover, biofilm formation and antifungal resistance were also significantly decreased. Lastly, autophagy regulates biofilm formation and drug resistance of C. albicans and could be served as a new molecular target to the C. albicans biofilm infections.
    Keywords:  ATG13; ATG27; C. albicans biofilm; antifungal resistance; autophagy; planktonic C. albicans; transcriptome analysis
  37. Cell Death Dis. 2022 Dec 09. 13(12): 1031
      The cell-cell interaction between hepatocytes and Kupffer cells (KCs) is crucial for maintaining liver homeostasis, and the loss of KCs and hepatocytes is known to represent a common pathogenic phenomenon in autoimmune hepatitis. Until now, the mechanisms of cell-cell interaction between hepatocytes and KCs involved in immune-mediated hepatitis remains unclear. Here we dissected the impact of activated mTORC1 on the cell-cell interaction of KCs and hepatocyte in immune-mediated hepatitis. In the liver from patients with AIH and mice administrated with Con-A, mTORC1 was activated in both KCs and hepatocytes. The activated mTORC1 signal in hepatocytes with Con-A challenge caused a markedly production of miR-329-3p. Upregulated miR-329-3p inhibited SGMS1 expression in KCs through paracrine, resulting in the death of KCs. Most of maintained KCs were p-S6 positive and distributed in hepatocyte mTORC1 negative area. The activation of mTORC1 enabled KCs expressed complement factor B (CFB) to enhance the complement alternative system, which produced more complement factors to aggravate liver injury. Our findings remonstrate a heterogeneous role of mTORC1 in specific cell type for maintaining tolerogenic liver environment, and will form the basis for the development of new interventions against immune-mediated hepatitis.
  38. Clin Exp Med. 2022 Dec 06.
      Cancer is a dysregulated cellular level pathological condition that results in tumor formation followed by metastasis. In the heterogeneous tumor architecture, cancer stem cells (CSCs) are essential to push forward the progression of tumors due to their strong pro-tumor properties such as stemness, self-renewal, plasticity, metastasis, and being poorly responsive to radiotherapy and chemotherapeutic agents. Cancer stem cells have the ability to withstand various stress pressures by modulating transcriptional and translational mechanisms, and adaptable metabolic changes. Owing to CSCs heterogeneity and plasticity, these cells display varied metabolic and redox profiles across different types of cancers. It has been established that there is a disparity in the levels of Reactive Oxygen Species (ROS) generated in CSCs vs Non-CSC and these differential levels are detected across different tumors. CSCs have unique metabolic demands and are known to change plasticity during metastasis by passing through the interchangeable epithelial and mesenchymal-like phenotypes. During the metastatic process, tumor cells undergo epithelial to mesenchymal transition (EMT) thus attaining invasive properties while leaving the primary tumor site, similarly during the course of circulation and extravasation at a distant organ, these cells regain their epithelial characteristics through Mesenchymal to Epithelial Transition (MET) to initiate micrometastasis. It has been evidenced that levels of Reactive Oxygen Species (ROS) and associated metabolic activities vary between the epithelial and mesenchymal states of CSCs. Similarly, the levels of oxidative and metabolic states were observed to get altered in CSCs post-drug treatments. As oxidative and metabolic changes guide the onset of autophagy in cells, its role in self-renewal, quiescence, proliferation and response to drug treatment is well established. This review will highlight the molecular mechanisms useful for expanding therapeutic strategies based on modulating redox regulation and autophagy activation to targets. Specifically, we will account for the mounting data that focus on the role of ROS generated by different metabolic pathways and autophagy regulation in eradicating stem-like cells hereafter referred to as cancer stem cells (CSCs).
    Keywords:  Autophagy; Cancer therapy; Mitophagy; Oxidative stress; Reactive oxygen species; Redox regulation; Redox-active compounds; Warburg effect
  39. Transl Psychiatry. 2022 Dec 10. 12(1): 508
      Impaired proteostasis is associated with normal aging and is accelerated in neurodegeneration. This impairment may lead to the accumulation of protein, which can be toxic to cells and tissue. In a subset of frontotemporal lobar degeneration with tau pathology (FTLD-tau) cases, pathogenic mutations in the microtubule-associated protein tau (MAPT) gene are sufficient to cause tau accumulation and neurodegeneration. However, the pathogenic events triggered by the expression of the mutant tau protein remain poorly understood. Here, we show that molecular networks associated with lysosomal biogenesis and autophagic function are disrupted in brains from FTLD-tau patients carrying a MAPT p.R406W mutation. We then used human induced pluripotent stem cell (iPSC)-derived neurons and 3D cerebral organoids from patients carrying the MAPT p.R406W mutation and CRISPR/Cas9, corrected controls to evaluate proteostasis. MAPT p.R406W was sufficient to induce morphological and functional deficits in the lysosomal pathway in iPSC-neurons. These phenotypes were reversed upon correction of the mutant allele with CRISPR/Cas9. Treatment with mTOR inhibitors led to tau degradation specifically in MAPT p.R406W neurons. Together, our findings suggest that MAPT p.R406W is sufficient to cause impaired lysosomal function, which may contribute to disease pathogenesis and serve as a cellular phenotype for drug screening.
  40. DNA Cell Biol. 2022 Dec 06.
      Despite activated autophagy ameliorating hepatocyte steatosis and metabolic associated fatty liver disease (MAFLD), mechanisms underlying the beneficial roles of autophagy in hepatic deregulation of lipid metabolism remain undefined. We explored whether autophagy can ameliorate oleic acid (OA)-induced hepatic steatosis by suppressing pyroptosis. Pyroptosis is involved in hepatocyte steatosis induced by OA. In addition, autophagy flux was blocked in OA-treated hepatocytes. Treatment with OA induced lipid accumulation in liver cell line L-02, which was attenuated by rapamycin (Rap), an autophagy agonist, while aggravated by autophagy inhibitor bafilomycin A1 (Baf A1). Inversely, treatment with pyroptotic agonist Nigericin aggravated OA-induced hepatic steatosis, while pyroptosis antagonist disulfiram ameliorated this effect. Mechanistically, treatment with Rap downregulated the expression of pyroptosis-related proteins, including NLRP3, Caspase-1, IL-18, GSDMD expression evoked by OA, thus improving pyroptosis in hepatic steatosis. Significantly, overexpression of ATG5 obviously downregulated cleaved caspase-1 expressions without altering the total caspase1 expressions in hepatic cell steatosis. Taken together, our studies strongly demonstrated that the activation of ATG5 inhibits pyroptosis to improve hepatic steatosis and suggest autophagy activation as a potential therapeutic strategy for pyroptosis-mediated MAFLD.
    Keywords:  ATG5; autophagy; hepatocyte steatosis; lipotoxicity; pyroptosis
  41. Biol Trace Elem Res. 2022 Dec 05.
      Fluoride can cause developmental neurotoxicity; however, the precise mechanism has yet to be determined. We aimed to explore the possible role and mechanism of fluoride-induced developmental neurotoxicity, specifically the significance of the lysosomal stress response. As an in vivo model, Sprague Dawley rats were exposed to sodium fluoride (NaF) from embryo to 2 months of age. We found that NaF caused autophagic flux blockage and apoptosis in the rat hippocampus. These results were validated in human neuroblastoma (SH-SY5Y) cells in vitro. In addition, in SH-SY5Y cells, NaF hindered autophagosome-lysosome fusion, decreased lysosomal degradation, and elevated lysosomal pH, which is the most prominent hallmark of a lysosomal stress response. Interestingly, rapamycin promoted autophagosome-lysosome fusion, effectively restoring autophagic flux and reducing apoptosis. Notably, bafilomycin A1, a lysosomal lumen alkalizer, unsurprisingly exacerbated the NaF-induced increase in lysosomal pH and decreased lysosomal degradability, as well as enhanced apoptosis of SH-SY5Y cells. In conclusion, our results suggest that NaF exposure initiates excessive lysosomal stress response, resulting in elevated lysosomal pH, decreased lysosomal degradation, and blocked autophagic flux, which leads to neuronal apoptosis. Thus, the lysosomal stress response may be a promising target for the prevention and treatment of fluoride-induced developmental neurotoxicity.
    Keywords:  Autophagy; Developmental neurotoxicity; Fluoride; Lysosomal stress response
  42. J Exp Clin Cancer Res. 2022 Dec 09. 41(1): 340
      BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematological cancer resulting from uncontrolled proliferation of differentiation-blocked myeloid cells. Seventy percent of AML patients are currently not cured with available treatments, highlighting the need of novel therapeutic strategies. A promising target in AML is the mammalian target of rapamycin complex 1 (mTORC1). Clinical inhibition of mTORC1 is limited by its reactivation through compensatory and regulatory feedback loops. Here, we explored a strategy to curtail these drawbacks through inhibition of an important effector of the mTORC1signaling pathway, the eukaryotic initiation factor 4A (eIF4A).METHODS: We tested the anti-leukemic effect of a potent and specific eIF4A inhibitor (eIF4Ai), CR-1-31-B, in combination with cytosine arabinoside (araC) or the BCL2 inhibitor venetoclax. We utilized the MOLM-14 human AML cell line to model chemoresistant disease both in vitro and in vivo. In eIF4Ai-treated cells, we assessed for changes in survival, apoptotic priming, de novo protein synthesis, targeted intracellular metabolite content, bioenergetic profile, mitochondrial reactive oxygen species (mtROS) and mitochondrial membrane potential (MMP).
    RESULTS: eIF4Ai exhibits anti-leukemia activity in vivo while sparing non-malignant myeloid cells. In vitro, eIF4Ai synergizes with two therapeutic agents in AML, araC and venetoclax. EIF4Ai reduces mitochondrial membrane potential (MMP) and the rate of ATP synthesis from mitochondrial respiration and glycolysis. Furthermore, eIF4i enhanced apoptotic priming while reducing the expression levels of the antiapoptotic factors BCL2, BCL-XL and MCL1. Concomitantly, eIF4Ai decreases intracellular levels of specific metabolic intermediates of the tricarboxylic acid cycle (TCA cycle) and glucose metabolism, while enhancing mtROS. In vitro redox stress contributes to eIF4Ai cytotoxicity, as treatment with a ROS scavenger partially rescued the viability of eIF4A inhibition.
    CONCLUSIONS: We discovered that chemoresistant MOLM-14 cells rely on eIF4A-dependent cap translation for survival in vitro and in vivo. EIF4A drives an intrinsic metabolic program sustaining bioenergetic and redox homeostasis and regulates the expression of anti-apoptotic proteins. Overall, our work suggests that eIF4A-dependent cap translation contributes to adaptive processes involved in resistance to relevant therapeutic agents in AML.
    Keywords:  AML; BCL-XL; BCL2; Bioenergetics; MCL1; Metabolism; ROS; Venetoclax; araC; eIF4A; mTORC1
  43. Ageing Res Rev. 2022 Dec 05. pii: S1568-1637(22)00258-6. [Epub ahead of print] 101816
      The protein dyshomeostasis is identified as the hallmark of many age-related neurodegenerative disorders including Parkinson's disease (PD). The diseased brain shows the deposition of Lewy bodies composed of α-synuclein protein aggregates. Functional proteostasis is characterized by the well-coordinated signaling network constituting unfolded protein response (UPR), the ubiquitin-proteasome system (UPS), and the autophagy-lysosome pathway (ALP). These networks ensure proper synthesis, folding, confirmation, and degradation of protein i.e., α-synuclein protein in PD. The proper functioning the of intricately woven proteostasis network is quite resilient to sustain under the influence of stressors. The synuclein protein turnover is hugely influenced by the autosomal dominant, recessive, and X-linked mutational changes of a gene involved in UPR, UPS, and ALP. The methylation, acetylation-related epigenetic modifications of DNA and histone proteins along with microRNA-mediated transcriptional changes also lead to extensive proteostasis dysregulation. The result of defective proteostasis is the deposition of many proteins which start appearing in the biofluids and can be identified as potential biomarkers for early diagnosis of PD. The therapeutic intervention targeted at different strata of proteostasis machinery holds great possibilities for delaying the age-related accumulation of pathological hallmarks.
    Keywords:  Chaperones; Dopamine; Endoplasmic reticulum stress; substantia nigra pars compacta
  44. Cell Death Discov. 2022 Dec 05. 8(1): 481
      Previous studies suggest that mesenchymal stem cells may represent a promising cellular therapy for acute lung injury (ALI); however, the underlying relevant molecular mechanisms remain unclear. Adipose-derived mesenchymal stem cells (ADSCs) were isolated and characterized by alizarin red staining, oil red staining, and flow cytometry. Lung injury and inflammatory cell infiltration were determined using the Evans blue method, wet/dry weight ratio, and H&E staining. An ELISA was used to detect the concentrations of IFN-γ, IL-2, and TNF-α. Autophagy was detected with an mRFP-GFP-LC3 dual-fluorescence autophagy indicator system, Western blotting, and electron microscopy. We first demonstrated that ADSCs did alleviate the inflammatory responses and tissue damage in lipopolysaccharide (LPS)-induced ALI. Next, we further demonstrated in vivo that autophagy plays a key role in the maintenance of ADSC therapeutic efficacy. In vitro experiments demonstrated that ADSCs co-cultured with alveolar epithelial cells depend on autophagy for significant anti-inflammatory functions. Moreover, the mammalian target of rapamycin (mTOR) is a key regulator of autophagy. Taken together, our findings demonstrate that the effect of ADSC on ALI, especially on alveolar epithelial cells, is dependent on mTOR-mediated autophagy maintenance. The significance of our study for ALI therapy is discussed with respect to a more complete understanding of the therapeutic strategy paradigm.
  45. J Reprod Immunol. 2022 Nov 29. pii: S0165-0378(22)00310-2. [Epub ahead of print]155 103781
      Autophagy is a lysosomal degradation pathway that supports metabolic adaptation and energy cycling. It is essential for cell homeostasis, differentiation, development, and survival. Recent studies have shown that autophagy could influence immune responses by regulating immune cell functions. Reciprocally, immune cells strongly influence autophagy. Immune cells at the maternal-fetal interface are thought to play essential roles in pregnancy. Here, we review the induction of autophagy at the maternal-fetal interface and its role in decidualization and placental development. Additionally, we emphasize the role of autophagy in the immune microenvironment at the maternal-fetal interface, including innate immunity, adaptive immunity, and immune tolerance molecules. It also suggests new research directions and prospects.
    Keywords:  Autophagy; adaptive immunity; immune tolerance molecules; innate immunity; maternal-fetal interface
  46. WIREs Mech Dis. 2022 Dec 09. e1591
      Alzheimer's disease (AD) is one of the progressive neurodegenerative disorders and the most common cause of dementia in the elderly worldwide causing difficulties in the daily life of the patient. AD is characterized by the aberrant accumulation of β-amyloid plaques and tau protein-containing neurofibrillary tangles (NFTs) in the brain giving rise to neuroinflammation, oxidative stress, synaptic failure, and eventual neuronal cell death. The total cost of care in AD treatment and related health care activities is enormous and pharmaceutical drugs approved by Food and Drug Administration have not manifested sufficient efficacy in protection and therapy. In recent years, there are growing studies that contribute a fundamental understanding to AD pathogenesis, AD-associated risk factors, and pharmacological intervention. However, greater molecular process-oriented research in company with suitable experimental models is still of the essence to enhance the prospects for AD therapy and cell lines as a disease model are still the major part of this milestone. In this review, we provide an insight into molecular mechanisms, particularly the recent concept in gut-brain axis, vascular dysfunction and autophagy, and current models used in the study of AD. Here, we emphasized the importance of therapeutic strategy targeting multiple mechanisms together with utilizing appropriate models for the discovery of novel effective AD therapy. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.
    Keywords:  C. elegans; amyloidosis; autophagy; gut microbiota; stem cells
  47. Stem Cell Reports. 2022 Nov 22. pii: S2213-6711(22)00537-9. [Epub ahead of print]
      Following acute genotoxic stress, both normal and tumorous stem cells can undergo cell-cycle arrest to avoid apoptosis and later re-enter the cell cycle to regenerate daughter cells. However, the mechanism of protective, reversible proliferative arrest, "quiescence," remains unresolved. Here, we show that mitophagy is a prerequisite for reversible quiescence in both irradiated Drosophila germline stem cells (GSCs) and human induced pluripotent stem cells (hiPSCs). In GSCs, mitofission (Drp1) or mitophagy (Pink1/Parkin) genes are essential to enter quiescence, whereas mitochondrial biogenesis (PGC1α) or fusion (Mfn2) genes are crucial for exiting quiescence. Furthermore, mitophagy-dependent quiescence lies downstream of mTOR- and PRC2-mediated repression and relies on the mitochondrial pool of cyclin E. Mitophagy-dependent reduction of cyclin E in GSCs and in hiPSCs during mTOR inhibition prevents the usual G1/S transition, pushing the cells toward reversible quiescence (G0). This alternative method of G1/S control may present new opportunities for therapeutic purposes.
    Keywords:  PRC2; cyclin E; epigenetic; mTOR; mitochondria; mitophagy; pluripotent stem cells
  48. EMBO J. 2022 Dec 07. e113046
      In their recent article, Polyansky et al identify phosphatidylcholine (PC) as the most abundant lipid in the autophagosome membrane and demonstrate that eliminating de novo PC synthesis sharply impairs autophagic processing. In the absence of PC synthesis, open cup-like structures accumulate, implicating PC as a key component in the closure of autophagosomes.