bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2022‒01‒09
fifty-six papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2022 Jan 05. 1-16
      Barth syndrome (BTHS) is an X-linked genetic disorder caused by mutations in the TAFAZZIN/Taz gene which encodes a transacylase required for cardiolipin remodeling. Cardiolipin is a mitochondrial signature phospholipid that plays a pivotal role in maintaining mitochondrial membrane structure, respiration, mtDNA biogenesis, and mitophagy. Mutations in the TAFAZZIN gene deplete mature cardiolipin, leading to mitochondrial dysfunction, dilated cardiomyopathy, and premature death in BTHS patients. Currently, there is no effective treatment for this debilitating condition. In this study, we showed that TAFAZZIN deficiency caused hyperactivation of MTORC1 signaling and defective mitophagy, leading to accumulation of autophagic vacuoles and dysfunctional mitochondria in the heart of Tafazzin knockdown mice, a rodent model of BTHS. Consequently, treatment of TAFAZZIN knockdown mice with rapamycin, a potent inhibitor of MTORC1, not only restored mitophagy, but also mitigated mitochondrial dysfunction and dilated cardiomyopathy. Taken together, these findings identify MTORC1 as a novel therapeutic target for BTHS, suggesting that pharmacological restoration of mitophagy may provide a novel treatment for BTHS.Abbreviations: BTHS: Barth syndrome; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CL: cardiolipin; EIF4EBP1/4E-BP1: eukaryotic translation initiation factor 4E binding protein 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; KD: knockdown; KO: knockout; LAMP1: lysosomal-associated membrane protein 1; LV: left ventricle; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; OCR: oxygen consumption rate; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PINK1: PTEN induced putative kinase 1; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; qRT-PCR: quantitative real-time polymerase chain reaction; RPS6KB/S6K: ribosomal protein S6 kinase beta; SQSTM1/p62: sequestosome 1; TLCL: tetralinoleoyl cardiolipin; WT: wild-type.
    Keywords:  BTHS; MTORC1; TAFAZZIN; cardiolipin; mitophagy; rapamycin
  2. J Cell Sci. 2022 Jan 04. pii: jcs.258687. [Epub ahead of print]
      The human Apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found in cells stably expressing each APOE isoform that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-golgi compartment is degraded by autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that may contribute to AD pathogenesis.
    Keywords:  APOE; APOE4; Alzheimer's disease; Chaperone-mediated autophagy; LC3-associated endocytosis
  3. Plant Cell Rep. 2022 Jan 03.
      KEY MESSAGE: This study reveals that plant roots show a rapid termination of autophagy induction, offering a plant model for studying how excessive autophagy is deterred. In eukaryotes, autophagy is an intracellular mechanism that is important for recycling nutrients by degrading various macromolecules and organelles in vacuoles and lysosomes. Autophagy is induced when the nutrient supply to plant cells is limited. The protein kinase target of rapamycin (TOR) complex negatively regulates autophagy when nutrients are present in adequate amounts. The TOR inhibitor AZD8055 is an autophagy inducer that is useful for studying starvation-induced autophagy in plant cells. The mechanism by which AZD8055 increases the autophagic flux in plant cells has not been studied in detail. Here, we show that AZD8055-induced autophagy requires phosphatidylinositol 3-kinase activity and canonical AUTOPHAGY-RELATED (ATG) genes in Arabidopsis thaliana. Autophagic flux rapidly increased in seedlings treated with AZD8055. Unexpectedly, autophagy induction was transient in root cells and terminated earlier than in cotyledon cells. Transient induction is partly caused by a temporary effect of AZD8055 on phagophore initiation. These findings indicate a TOR-independent mechanism for terminating autophagy induction, thereby paving the way for elucidating how excess autophagy is prevented in plant roots.
    Keywords:  ATG5; ATG8; Phosphoinositide; atg2
  4. Mol Cell Proteomics. 2021 Dec 30. pii: S1535-9476(21)00163-8. [Epub ahead of print] 100191
      Mitophagy, the selective degradation of mitochondria by autophagy, affects defective mitochondria following damage or stress. At the onset of mitophagy, parkin ubiquitylates proteins on the mitochondrial outer membrane (MOM). While the role of parkin at the onset of mitophagy is well understood, less is known about its activity during later stages of the process. Here we used HeLa cells expressing catalytically active or inactive parkin to perform temporal analysis of the proteome, ubiquitylome and phosphoproteome during 18 hours after induction of mitophagy by mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazine (CCCP). Abundance profiles of proteins downregulated in parkin-dependent manner revealed a stepwise, "outside-in" directed degradation of mitochondrial subcompartments. While ubiquitylation of MOM proteins was enriched among early parkin-dependent targets, numerous mitochondrial inner membrane, matrix and cytosolic proteins were also found ubiquitylated at later stages of mitophagy. Phosphoproteome analysis revealed a possible cross-talk between phosphorylation and ubiquitylation during mitophagy on key parkin targets, such as VDAC1/2.
    Keywords:  Mitochondria; Mitophagy; Parkin; Quantitative proteomics; Ubiquitin
  5. Sci Rep. 2022 Jan 07. 12(1): 79
      Autophagic flux can be quantified based on the accumulation of lipidated LC3B in the presence of late-stage autophagy inhibitors. This method has been widely applied to identify novel compounds that activate autophagy. Here we scrutinize this approach and show that bafilomycin A1 (BafA) but not chloroquine is suitable for flux quantification due to the stimulating effect of chloroquine on non-canonical LC3B-lipidation. Significant autophagic flux increase by rapamycin could only be observed when combining it with BafA concentrations not affecting basal flux, a condition which created a bottleneck, rather than fully blocking autophagosome-lysosome fusion, concomitant with autophagy stimulation. When rapamycin was combined with saturating concentrations of BafA, no significant further increase of LC3B lipidation could be detected over the levels induced by the late-stage inhibitor. The large assay window obtained by this approach enables an effective discrimination of autophagy activators based on their cellular potency. To demonstrate the validity of this approach, we show that a novel inhibitor of the acetyltransferase EP300 activates autophagy in a mTORC1-dependent manner. We propose that the creation of a sensitized background rather than a full block of autophagosome progression is required to quantitatively capture changes in autophagic flux.
  6. Adv Biol Regul. 2021 Dec 06. pii: S2212-4926(21)00072-5. [Epub ahead of print] 100854
      Aberrant signaling of mechanistic target of rapamycin (mTOR' aka mammalian target of rapamycin) is shown to be linked to tumorigenesis of numerous malignancies including glioblastoma (GB). Glioblastoma mTOR is a serine threonine kinase that functions by forming two multiprotein complexes. There complexes are named mTORC1 and mTORC2 and downstream activated substrate execute cellular and metabolic functions. This signaling cascade of PI3K/AKT/mTOR is often upregulated due to frequent loss of the tumor suppressor PTEN, a phosphatase that functions antagonistically to PI3K. mTOR regulates cell growth, motility, and metabolism by forming two multiprotein complexes, mTORC1 and mTORC2, which are composed of special binding partners. These complexes are sensitive to distinct stimuli. mTORC1 is sensitive to nutrients and mTORC2 is regulated via PI3K and growth factor signaling. Since rapamycin and it's analogue are less effective in treatment of GB, we used novel ATP-competitive dual inhibitors of mTORC1 and mTORC2, namely, Torin1, Torin2, and XL388. Torin2 caused a concentration dependent pharmacodynamic effects on inhibition of phosphorylation of the mTORC1 substrates S6KSer235/236 and 4E-BP1Thr37/46 as well as the mTORC2 substrate AKTSer473 resulting in suppression of tumor cell proliferation and migration. Torin1 showed similar effects only at higher doses. Another small molecule compound, XL388 suppressed cell proliferation at a higher dose but failed to inhibit cell migration. Torin1 suppressed phosphorylation of PRAS40Thr246, however Torin2 completely abolished it. XL388 treatment inhibited the phosphorylation of PRAS40Thr246 at higher doses only. These findings underscore the use of novel compounds in treatment of cancer. In addition, formulation of third generation mTOR inhibitor "Rapalink-1" may provide new aspects to target mTOR pathways. Numerous inhibitors are currently being used in clinical trials that are aimed to target activated mTOR pathways.
    Keywords:  Glioblastoma; mTOR; mTORC1 mTORC2
  7. EMBO Rep. 2022 Jan 07. e48754
      Mitochondria are unavoidably subject to organellar stress resulting from exposure to a range of reactive molecular species. Consequently, cells operate a poorly understood quality control programme of mitophagy to facilitate elimination of dysfunctional mitochondria. Here, we used a model stressor, deferiprone (DFP), to investigate the molecular basis for stress-induced mitophagy. We show that mitochondrial fission 1 protein (Fis1) is required for DFP-induced mitophagy and that Fis1 is SUMOylated at K149, an amino acid residue critical for Fis1 mitochondrial localization. We find that DFP treatment leads to the stabilization of the SUMO protease SENP3, which is mediated by downregulation of the E3 ubiquitin (Ub) ligase CHIP. SENP3 is responsible for Fis1 deSUMOylation and depletion of SENP3 abolishes DFP-induced mitophagy. Furthermore, preventing Fis1 SUMOylation by conservative K149R mutation enhances Fis1 mitochondrial localization. Critically, expressing a Fis1 K149R mutant restores DFP-induced mitophagy in SENP3-depleted cells. Thus, we propose a model in which SENP3-mediated deSUMOylation facilitates Fis1 mitochondrial localization to underpin stress-induced mitophagy.
    Keywords:  Fis1; SENP3; SUMO; mitophagy; organellar stress
  8. Cell Death Differ. 2022 Jan 06.
      p62/SQSTM1 is a selective autophagy receptor that drives ubiquitinated cargos towards autophagic degradation. This receptor is also a stress-induced scaffold protein that helps cells to cope with oxidative stress through activation of the Nrf2 pathway. Functional disorders of p62 are closely associated with multiple neurodegenerative diseases and cancers. The gene encoding the E3 ubiquitin ligase substrate-binding adapter SPOP is frequently mutated in prostate cancer (PCa), but the molecular mechanisms underlying how SPOP mutations contribute to PCa tumorigenesis remain poorly understood. Here, we report that cytoplasmic SPOP binds and induces the non-degradative ubiquitination of p62 at residue K420 within the UBA domain. This protein modification decreases p62 puncta formation, liquid phase condensation, dimerization, and ubiquitin-binding capacity, thereby suppressing p62-dependent autophagy. Moreover, we show that SPOP relieves p62-mediated Keap1 sequestration, which ultimately decreases Nrf2-mediated transcriptional activation of antioxidant genes. We further show that PCa-associated SPOP mutants lose the capacity to ubiquitinate p62 and instead promote autophagy and the redox response in a dominant-negative manner. Thus, our findings indicate oncogenic roles of autophagy and Nrf2 activation in the tumorigenesis of SPOP-mutated PCa.
  9. Autophagy. 2022 Jan 06. 1-20
      The mitochondrial-anchored deubiquitinating enzyme USP30 (ubiquitin specific peptidase 30) antagonizes PRKN/parkin-mediated mitophagy, making it a potential target for treating Parkinson disease. However, few inhibitors targeting USP30 have been reported. Here, we report a novel peptide (Q14) derived from the transmembrane (TM) domain of USP30 that can target mitochondrial-anchored USP30 directly and increase mitophagy through two intriguing and distinct mechanisms: a novel autoinhibition mechanism in USP30 and accelerated autophagosome formation via the LC3-interacting region (LIR) of the Q14 peptide. We identified the potential binding sites between the Q14 peptide and USP30 and postulated that an allosteric autoinhibition mechanism regulates USP30 activity. Furthermore, the LIR motif in the Q14 peptide offers additional binding with LC3 and accelerated autophagosome formation. The two mechanisms synergistically enhance mitophagy. Our work provides novel insight and direction to the design of inhibitors for USP30 or other deubiquitinating enzymes (DUBs).Abbreviations: 3-MA: 3-methyladenine; ATTEC: autophagosome-tethering compound; BafA1: bafilomycin A1; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; FP: fluorescence polarization; FUNDC1: FUN14 domain containing 1; HCQ: hydroxychloroquine; LIR: LC3-interacting region; MST: microscale thermophoresis; mtDNA: mitochondrial DNA; mtPA-GFP: mitochondria-targeted photoactive fluorescence protein; OMM: outer mitochondrial membrane; PINK1: PTEN induced kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase; Rap: rapamycin; SA: streptavidin; TM: transmembrane; Ub: ubiquitin; Ub-AMC: Ub-7-amido-4-methylcoumarin; UPS: ubiquitin-protease system; USP: ubiquitin specific peptidase; USP30: ubiquitin specific peptidase 30.
    Keywords:  Autoinhibition; USP30; mitophagy; peptide inhibitor; transmembrane
  10. Front Cell Dev Biol. 2021 ;9 790721
      Autophagy is an evolutionary conserved catabolic pathway essential for the maintenance of cellular homeostasis. Defective proteins and organelles are engulfed by autophagosomal membranes which fuse with lysosomes for cargo degradation. In neurons, the orchestrated progression of autophagosome formation and maturation occurs in distinct subcellular compartments. For synapses, the distance from the soma and the oxidative stress generated during intense neuronal activity pose a challenge to maintain protein homeostasis. Autophagy constitutes a crucial mechanism for proper functioning of this unique and vulnerable cellular compartment. We are now beginning to understand how autophagy is regulated at pre-synaptic terminals and how this pathway, when imbalanced, impacts on synaptic function and -ultimately- neuronal survival. We review here the current state of the art of "synaptic autophagy", with an emphasis on the biogenesis of autophagosomes at the pre-synaptic compartment. We provide an overview of the existing knowledge on the signals inducing autophagy at synapses, highlight the interplay between autophagy and neurotransmission, and provide perspectives for future research.
    Keywords:  macroautophagy; neurotransmission; synapse; synaptic autophagy; vesicle cycling
  11. Front Cell Dev Biol. 2021 ;9 814047
      Peroxisomes harbor numerous enzymes that can produce or degrade hydrogen peroxide (H2O2). Depending on its local concentration and environment, this oxidant can function as a redox signaling molecule or cause stochastic oxidative damage. Currently, it is well-accepted that dysfunctional peroxisomes are selectively removed by the autophagy-lysosome pathway. This process, known as "pexophagy," may serve a protective role in curbing peroxisome-derived oxidative stress. Peroxisomes also have the intrinsic ability to mediate and modulate H2O2-driven processes, including (selective) autophagy. However, the molecular mechanisms underlying these phenomena are multifaceted and have only recently begun to receive the attention they deserve. This review provides a comprehensive overview of what is known about the bidirectional relationship between peroxisomal H2O2 metabolism and (selective) autophagy. After introducing the general concepts of (selective) autophagy, we critically examine the emerging roles of H2O2 as one of the key modulators of the lysosome-dependent catabolic program. In addition, we explore possible relationships among peroxisome functioning, cellular H2O2 levels, and autophagic signaling in health and disease. Finally, we highlight the most important challenges that need to be tackled to understand how alterations in peroxisomal H2O2 metabolism contribute to autophagy-related disorders.
    Keywords:  autophagy; disease; hydrogen peroxide; oxidative damage; peroxisomes; pexophagy; thiol-based redox signaling
  12. Elife. 2022 Jan 04. pii: e72466. [Epub ahead of print]11
      Autophagosomes are double-membrane intracellular vesicles that degrade protein aggregates, intracellular organelles, and other cellular components. During the development of the nematode Caenorhabditis elegans, many somatic and germ cells undergo apoptosis. These cells are engulfed and degraded by their neighboring cells. We discovered a novel role of autophagosomes in facilitating the degradation of apoptotic cells using a real-time imaging technique. Specifically, the double-membrane autophagosomes in engulfing cells are recruited to the surfaces of phagosomes containing apoptotic cells and subsequently fuse to phagosomes, allowing the inner vesicle to enter the phagosomal lumen. Mutants defective in the production of autophagosomes display significant defects in the degradation of apoptotic cells, demonstrating the importance of autophagosomes to this process. The signaling pathway led by the phagocytic receptor CED-1, the adaptor protein CED-6, and the large GTPase dynamin (DYN-1) promotes the recruitment of autophagosomes to phagosomes. Moreover, the subsequent fusion of autophagosomes with phagosomes requires the functions of the small GTPase RAB-7 and the HOPS complex components. Further observations suggest that autophagosomes provide apoptotic cell-degradation activities in addition to and in parallel of lysosomes. Our findings reveal that, unlike the single-membrane, LC3-associated phagocytosis (LAP) vesicles reported for mammalian phagocytes, the canonical double-membrane autophagosomes facilitate the clearance of C. elegans apoptotic cells. These findings add autophagosomes to the collection of intracellular organelles that contribute to phagosome maturation, identify novel crosstalk between the autophagy and phagosome maturation pathways, and discover the upstream signaling molecules that initiate this crosstalk.
    Keywords:  C. elegans; cell biology; developmental biology
  13. Sci Total Environ. 2022 Jan 02. pii: S0048-9697(21)07838-4. [Epub ahead of print] 152759
      Exposure to benzo (a)pyrene (BaP) has been confirmed to interfere with embryo implantation. As the primary organ of progesterone synthesis during early pregnancy, the ovarian corpus luteum (CL) is essential for embryo implantation and pregnancy maintenance. We previously demonstrated that BaP impaired luteal function, but the molecular mechanism remains unclear. In CL cells, mitochondria are the main sites of progesterone synthesis. Mitophagy, a particular type of autophagy, regulates mitochondrial quality by degrading damaged mitochondria and ensuring the homeostasis of cell physiology. Therefore, the present study investigated the effects and the potential molecular mechanisms of BaP on ovarian mitophagy during early pregnancy. We found that BaP and its metabolite, BPDE, inhibited autophagy and PINK1/Parkin-mediated mitophagy in the pregnant ovaries and luteinized granulosa cell, KGN. Notably, adenine nucleotide translocator 1 (ANT1), a crucial mediator of PINK1-dependent mitophagy, was suppressed by BaP and BPDE both in vivo and in vitro. The inhibition of ANT1 leads to the decrease in the PINK1 bound to the outer membrane of mitochondria and consequently reduces recruitment of Parkin to the mitochondria, which is required for the subsequent clearance of mitochondria. Meanwhile, exposure to BPDE also damaged mitochondrial function, causing the reduction in mitochondrial potential and ATP production. Overexpression of ANT1 in KGN cells partially relieved the inhibition of mitophagy caused by BPDE, restored mitochondrial function and expression of hormone synthesis-associated genes. Collectively, our study firstly clarified that BaP and BPDE suppress mitophagy of CL cells via the ANT1-PINK1-Parkin pathway, which provides a new insight to explore the detailed mechanism of the BaP-induced ovarian toxicity.
    Keywords:  ANT1; BPDE; Benzo(a)pyrene; Mitophagy; Ovarian corpus luteum
  14. Front Cell Dev Biol. 2021 ;9 772965
      Autophagy is a conservative lysosomal catabolic pathway commonly seen in eukaryotic cells. It breaks down proteins and organelles by forming a two-layer membrane structure of autophagosomes and circulating substances and maintaining homeostasis. Autophagy can play a dual role in viral infection and serve either as a pro-viral factor or an antiviral defense element dependent on the virus replication cycle. Recent studies have suggested the complicated and multidirectional role of autophagy in the process of virus infection. On the one hand, autophagy can orchestrate immunity to curtail infection. On the other hand, some viruses have evolved strategies to evade autophagy degradation, facilitating their replication. In this review, we summarize recent progress of the interaction between autophagy and viral infection. Furthermore, we highlight the link between autophagy and SARS-CoV-2, which is expected to guide the development of effective antiviral treatments against infectious diseases.
    Keywords:  SARS-CoV-2; autophagy; coronavirus; infection; virus
  15. Neurochem Res. 2022 Jan 07.
      Cerebral ischemia is one of the most devastating brain injuries and a primary cause of acquired and persistent disability worldwide. Despite ongoing therapeutic interventions at both the experimental and clinical levels, options for stroke-related brain injury are still limited. Several evidence suggests that autophagy is triggered in response to cerebral ischemia, therefore targeting autophagy-related signaling pathways can provide a new direction for the therapeutic implications in the ischemic injury. Autophagy is a highly conserved lysosomal-dependent pathway that degrades and recycles damaged or non-essential cellular components to maintain neuronal homeostasis. But, whether autophagy activation promotes cell survival against ischemic injury or, on the contrary, causes neuronal death is still under debate. We performed an extensive literature search from PubMed, Bentham and Elsevier for various aspects related to molecular mechanisms and pathobiology involved in autophagy and several pre-clinical studies justifiable further in the clinical trials. Autophagy modulates various downstream molecular cascades, i.e., mTOR, NF-κB, HIF-1, PPAR-γ, MAPK, UPR, and ROS pathways in cerebral ischemic injury. In this review, the various approaches and their implementation in the translational research in ischemic injury into practices has been covered. It will assist researchers in finding a way to cross the unbridgeable chasm between the pre-clinical and clinical studies.
    Keywords:  Autophagy; Bcl2; Cerebral ischemia; Chaperone-mediated autophagy; Endoplasmic reticulum stress; Neuroprotection
  16. Front Physiol. 2021 ;12 807654
      Mitochondria are highly dynamic organelles and play essential role in ATP synthase, ROS production, innate immunity, and apoptosis. Mitochondria quality control is critical for maintaining the cellular function in response to cellular stress, growth, and differentiation Signals. Damaged or unwanted mitochondria are selectively removed by mitophagy, which is a crucial determinant of cell viability. Mitochondria-associated Endoplasmic Reticulum Membranes (MAMs) are the cellular structures that connect the ER and mitochondria and are involved in calcium signaling, lipid transfer, mitochondrial dynamic, and mitophagy. Abnormal mitochondrial quality induced by mitophagy impairment and MAMs dysfunction is associated with many diseases, including cardiovascular diseases (CVDs), metabolic syndrome, and neurodegenerative diseases. As a mitophagy receptor, FUNDC1 plays pivotal role in mitochondrial quality control through regulation of mitophagy and MAMs and is closely related to the occurrence of several types of CVDs. This review covers the regulation mechanism of FUNDC1-mediated mitophagy and MAMs formation, with a particular focus on its role in CVDs.
    Keywords:  FUNDC1; cardiovascular diseases; mitochondria; mitochondria quality/dynamics; mitophagy
  17. FEBS J. 2022 Jan 07.
      The pan-caspase inhibitor Z-VAD-fmk acts as an inhibitor of peptide:N-glycanase (NGLY1); an endoglycosidase which cleaves N-linked glycans from glycoproteins exported from the endoplasmic reticulum (ER) during ER-associated degradation (ERAD). Both pharmacological N-glycanase inhibition by Z-VAD-fmk and siRNA-mediated knockdown (KD) of NGLY1 induce GFP-LC3 positive puncta in HEK 293 cells. Activation of ER stress markers or induction of reactive oxygen species (ROS) are not observed under either condition. Moreover, Ca2+ handling is unaffected when observing release from intracellular stores. Under conditions of pharmacological NGLY1 inhibition or NGLY1 KD, upregulation of autophagosome formation without impairment of autophagic flux is observed. Enrichment of autophagosomes by immunoprecipitation (IP) and mass spectrometry-based proteomics analysis reveal comparable autophagosomal protein content. Gene ontology analysis of proteins enriched in autophagosome IPs shows overrepresentation of factors involved in protein translation, localization and targeting, RNA degradation and protein complex disassembly. Upregulation of autophagy represents a cellular adaptation to NGLY1 inhibition or KD, and ATG13-deficient mouse embryonic fibroblasts (MEFs) show reduced viability under these conditions. In contrast, treatment with pan-caspase inhibitor, Q-VD-OPh does not induce cellular autophagy. Therefore, experiments with Z-VAD-fmk are complicated by the effects of NGLY1 inhibition, including induction of autophagy, and Q-VD-OPh represents an alternative caspase inhibitor free from this limitation.
    Keywords:  NGLY1; Peptide:N-glycanase 1; Z-VAD-fmk; autophagosome proteomics; autophagy
  18. Kidney Int. 2022 Jan;pii: S0085-2538(21)01048-6. [Epub ahead of print]101(1): 13-15
      Acute kidney injury (AKI) increases the risk of chronic kidney disease (CKD), but the mechanisms of CKD development after AKI remain unclear. Recent studies have elucidated that autophagy protects against AKI, but the role of autophagy during the AKI-to-CKD transition is controversial. Beclin1 is a key molecule for autophagy as well as endocytosis and phagocytosis. Shi et al. demonstrate that Beclin1 activates autophagy and is a promising therapeutic target for AKI-to-CKD transition.
  19. Int J Biochem Cell Biol. 2021 Dec 30. pii: S1357-2725(21)00234-X. [Epub ahead of print] 106153
      As part of the glomerular filtration membrane, podocyte is terminally differentiated, structurally unique, and highly specialized in maintaining kidney function. Proteinuria caused by podocyte injury (foot process effacement) is the clinical symptom of various kidney diseases (CKD), including nephrotic syndrome. Podocyte autophagy has become a powerful therapeutic strategy target in ameliorating podocyte injury. Autophagy is known to be associated significantly with sirtuin-1, proteinuria, and podocyte injury. Various key findings in podocyte autophagy were reported in the past ten years, such as the role of endoplasmic reticulum (ER) stress in podocyte autophagy impairment, podocyte autophagy-related gene, essential roles of the signaling pathways: Mammalian Target of Rapamycin (mTOR)/ Phosphoinositide 3-kinase (PI3k)/ serine/threonine kinase 1 (Akt) in podocyte autophagy. These significant factors caused podocyte injury associated with autophagy impairment. Sirtuin-1 was reported to have a vital key role in mTOR signaling, 5'AMP-activated protein kinase (AMPK) regulation, autophagy activation, and various critical pathways associated with podocyte's function and health; it has potential value to podocyte injury pathogenesis investigation. From these findings, podocyte autophagy has become an attractive therapeutic strategy to ameliorate podocyte injury, and this review will provide an in-depth review on therapeutic targets he podocyte autophagy.
    Keywords:  Sirtuin-1; diabetic nephropathy; glomerulopathy; mTOR signaling pathway; podocyte autophagy; podocyte injury
  20. Eur J Pharmacol. 2021 Dec 30. pii: S0014-2999(21)00879-7. [Epub ahead of print] 174723
      Over the past two decades, researchers have revealed the crucial functions of glutamine in supporting the hyperproliferation state of cancer cells. Glutamine acts on maintaining high energy production, supporting redox status and amino acid homeostasis. Therefore, cancer cells exhibit excessive uptake of the extracellular glutamine, synthesize it in some cases, and recycle intracellular and extracellular proteins to provide an additional source of glutamine to satisfy the increasing glutamine demand. On the other hand, autophagy's role is still debated regarding tumor initiation and progression. However, most cancer cells urgently need autophagy to overcome the existential threats during glutamine restriction stress. Downstream to various stress pathways induced during such a condition, autophagy is considered an indispensable cytoprotective tool to maintain cell integrity and survival. However, the overactivation of the autophagy process is related to lethal consequences. This review summarized glutamine pathways to control autophagy and highlighted autophagy's primary activation pathways, and discussed the roles during glutamine deprivation.
    Keywords:  Autophagy; Cancer cell metabolism; Glutamine deprivation
  21. FASEB J. 2022 Feb;36(2): e22145
      Osteoarthritis (OA), the most prevalent joint disease, is characterized by the progressive loss of articular cartilage. Autophagy, a lysosomal degradation pathway, maintains cellular homeostasis, and autophagic dysfunction in chondrocytes is a hallmark of OA pathogenesis. However, the cause of autophagic dysfunction in OA chondrocytes remains incompletely understood. Recent studies have reported that decidual protein induced by progesterone (C10orf10/DEPP) positively regulates autophagic functions. In this study, we found that DEPP was involved in mitochondrial autophagic functions of chondrocytes, as well as in OA pathogenesis. DEPP expression decreased in human OA chondrocytes in the absence or presence of pro-inflammatory cytokines, and was induced by starvation, hydrogen peroxide (H2 O2 ), and hypoxia (cobalt chloride). For functional studies, DEPP knockdown decreased autophagic flux induced by H2 O2 , whereas DEPP overexpression increased autophagic flux and maintained cell viability following H2 O2 treatment. DEPP was downregulated by knockdown of forkhead box class O (FOXO) transcription factors and modulated the autophagic function regulated by FOXO3. In an OA mouse model by destabilization of the medial meniscus, DEPP-knockout mice exacerbated the progression of cartilage degradation with TUNEL-positive cells, and chondrocytes isolated from knockout mice were decreased autophagic flux and increased cell death following H2 O2 treatment. Subcellular fractionation analysis revealed that mitochondria-located DEPP activated mitochondrial autophagy via BCL2 interacting protein 3. Taken together, our data demonstrate that DEPP is a major stress-inducible gene involved in the activation of mitochondrial autophagy in chondrocytes, and maintains chondrocyte viability during OA pathogenesis. DEPP represents a potential therapeutic target for enhancing autophagy in patients with OA.
    Keywords:  C10orf10/DEPP; autophagy; chondrocyte; mitochondria; osteoarthritis
  22. J Cardiovasc Pharmacol. 2021 Dec 27.
      ABSTRACT: The abnormal proliferation of vascular smooth muscle cells (VSMCs) is a key pathological characteristic of vascular proliferative diseases. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays an important role in regulating cell growth, motility, proliferation, and survival, as well as gene expression in response to hypoxia, growth factors, and nutrients. Increasing evidence shows that mTOR also regulates VSMC proliferation in vascular proliferative diseases and that mTOR inhibitors, such as rapamycin, effectively restrain VSMC proliferation. However, the molecular mechanisms linking mTOR to vascular proliferative diseases remain elusive. In our review, we summarize the key roles of the mTOR and the recent discoveries in vascular proliferative diseases, focusing on the therapeutic potential of mTOR inhibitors to target mTOR signaling pathway for the treatment of vascular proliferative diseases. Herein, we discuss mTOR inhibitors as promising candidates to prevent VSMC-associated vascular proliferative diseases.
  23. Front Cell Dev Biol. 2021 ;9 761273
      Autophagy is an evolutionarily conserved lysosomal degradation pathway that maintains metabolism and homeostasis by eliminating protein aggregates and damaged organelles. Many studies have reported that autophagy plays an important role in spinal cord injury (SCI). However, the spatiotemporal patterns of autophagy activation after traumatic SCI are contradictory. Most studies show that the activation of autophagy and inhibition of apoptosis have neuroprotective effects on traumatic SCI. However, reports demonstrate that autophagy is strongly associated with distal neuronal death and the impaired functional recovery following traumatic SCI. This article introduces SCI pathophysiology, the physiology and mechanism of autophagy, and our current review on its role in traumatic SCI. We also discuss the interaction between autophagy and apoptosis and the therapeutic effect of activating or inhibiting autophagy in promoting functional recovery. Thus, we aim to provide a theoretical basis for the biological therapy of SCI.
    Keywords:  autophagy; autophagy-regulated; biological functions; spinal cord injury; therapeutic target
  24. Cell Mol Biol Lett. 2022 Jan 03. 27(1): 2
      Sestrins (Sesns), highly conserved stress-inducible metabolic proteins, are known to protect organisms against various noxious stimuli including DNA damage, oxidative stress, starvation, endoplasmic reticulum (ER) stress, and hypoxia. Sesns regulate metabolism mainly through activation of the key energy sensor AMP-dependent protein kinase (AMPK) and inhibition of mammalian target of rapamycin complex 1 (mTORC1). Sesns also play pivotal roles in autophagy activation and apoptosis inhibition in normal cells, while conversely promoting apoptosis in cancer cells. The functions of Sesns in diseases such as metabolic disorders, neurodegenerative diseases, cardiovascular diseases, and cancer have been broadly investigated in the past decades. However, there is a limited number of reviews that have summarized the functions of Sesns in the pathophysiological processes of human diseases, especially musculoskeletal system diseases. One aim of this review is to discuss the biological functions of Sesns in the pathophysiological process and phenotype of diseases. More significantly, we include some new evidence about the musculoskeletal system. Another purpose is to explore whether Sesns could be potential biomarkers or targets in the future diagnostic and therapeutic process.
    Keywords:  Biological functions; Biomarker; Human diseases; Musculoskeletal system disease; Sestrins; Therapeutic target
  25. Acta Histochem. 2022 Jan 04. pii: S0065-1281(21)00163-X. [Epub ahead of print]124(1): 151841
      After several years of extensive research, the main cause of aging is yet elusive. There are some theories about aging, such as stem cell aging, senescent cells accumulation, and neuro-endocrine theories. None of them is able to explain all changes that happen in cells and body through aging. By finding out the main cause of aging, it will be much easier to control, prevent and even reverse the aging process. Our cells, regardless of their replicative capacity, get old through aging and they have almost the same epigenetic age. Different cell signaling pathways contribute to aging. The most important one is mTORC1 that becomes hyperactive in cells that undergo aging. Other significant changes with age are lysosome accumulation, impaired autophagy, and mitophagy. Immune system undergoes gradual changes through aging including a shift from lymphoid to myeloid lineage production as well as increased IL-6 and TNF-α which lead to age-related weight loss and meta-inflammation. Additionally, our endocrine system also experiences some changes that should be taken into consideration when looking for the main cause of aging in the human body. In this review, we planned to summarize some of the changes that happen in cells and the body through aging.
    Keywords:  Aging; Cell senescence; Hormonal changes; Immuno-senescence; MTORC1; Senolytics
  26. J Immunol. 2022 Jan 07. pii: ji2100684. [Epub ahead of print]
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has seriously threatened global public health. Severe COVID-19 has been reported to be associated with an impaired IFN response. However, the mechanisms of how SARS-CoV-2 antagonizes the host IFN response are poorly understood. In this study, we report that SARS-CoV-2 helicase NSP13 inhibits type I IFN production by directly targeting TANK-binding kinase 1 (TBK1) for degradation. Interestingly, inhibition of autophagy by genetic knockout of Beclin1 or pharmacological inhibition can rescue NSP13-mediated TBK1 degradation in HEK-293T cells. Subsequent studies revealed that NSP13 recruits TBK1 to p62, and the absence of p62 can also inhibit TBK1 degradation in HEK-293T and HeLa cells. Finally, TBK1 and p62 degradation and p62 aggregation were observed during SARS-CoV-2 infection in HeLa-ACE2 and Calu3 cells. Overall, our study shows that NSP13 inhibits type I IFN production by recruiting TBK1 to p62 for autophagic degradation, enabling it to evade the host innate immune response, which provides new insights into the transmission and pathogenesis of SARS-CoV-2 infection.
  27. Acta Physiol (Oxf). 2022 Jan 03. e13757
      Coordinated action among various organelles maintain cellular functions. For instance, mitochondria and lysosomes are the main organelles contributing to cellular metabolism and provide energy for cardiomyocyte contraction. They also provide essential signaling platforms in the cell that regulate many key processes such as autophagy, apoptosis, oxidative stress, inflammation, and cell death. Often, abnormalities in mitochondrial or lysosomal structures and functions bring about cardiovascular disease (CVD). Although the communication between mitochondria and lysosomes throughout the cardiovascular system is intensely studied, the regulatory mechanisms have not been completely understood. We summarize the most recent studies related to mitochondria and lysosomes' role in CVDs and their potential connections and communications under cardiac pathophysiological conditions. Further we discuss limitations and future perspectives regarding diagnosis, therapeutic strategies, and drug discovery in CVDs.
    Keywords:  autophagy; cardiovascular disease; interaction; lysosome; mechanism; metabolism; mitochondrion
  28. Bioact Mater. 2022 May;11 300-316
      Compromised autophagy and defective lysosomal clearance significantly contribute to impaired neuronal proteostasis, which represents a hallmark of Alzheimer's disease (AD) and other age-related neurodegenerative disorders. Growing evidence has implicated that modulating autophagic flux, instead of inducing autophagosome formation alone, would be more reliable to rescue neuronal proteostasis. Concurrently, selectively enhancing drug concentrations in the leision areas, instead of the whole brain, will maximize therapeutic efficacy while reduing non-selective autophagy induction. Herein, we design a ROS-responsive targeted micelle system (TT-NM/Rapa) to enhance the delivery efficiency of rapamycin to neurons in AD lesions guided by the fusion peptide TPL, and facilitate its intracellular release via ROS-mediated disassembly of micelles, thereby maximizing autophagic flux modulating efficacy of rapamycin in neurons. Consequently, it promotes the efficient clearance of intracellular neurotoxic proteins, β-amyloid and hyperphosphorylated tau proteins, and ameliorates memory defects and neuronal damage in 3 × Tg-AD transgenic mice. Our studies demonstrate a promising strategy to restore autophagic flux and improve neuronal proteostasis by rationally-engineered nano-systems for delaying the progression of AD.
    Keywords:  Alzheimer's disease; Autophagic flux; Brain-neuron targeting; ROS-Responsive micelle system; Rapamycin
  29. Open Biol. 2022 Jan;12(1): 210304
      The extracellular matrix (ECM) exists as a dynamic network of biophysical and biochemical factors that maintain tissue homeostasis. Given its sensitivity to changes in the intra- and extracellular space, the plasticity of the ECM can be pathological in driving disease through aberrant matrix remodelling. In particular, cancer uses the matrix for its proliferation, angiogenesis, cellular reprogramming and metastatic spread. An emerging field of matrix biology focuses on proteoglycans that regulate autophagy, an intracellular process that plays both critical and contextual roles in cancer. Here, we review the most prominent autophagic modulators from the matrix and the current understanding of the cellular pathways and signalling cascades that mechanistically drive their autophagic function. We then critically assess how their autophagic functions influence tumorigenesis, emphasizing the complexities and stage-dependent nature of this relationship in cancer. We highlight novel emerging data on immunoglobulin-containing and proline-rich receptor-1, heparanase and thrombospondin 1 in autophagy and cancer. Finally, we further discuss the pro- and anti-autophagic modulators originating from the ECM, as well as how these proteoglycans and other matrix constituents specifically influence cancer progression.
    Keywords:  angiogenesis; collagen VI; decorin; perlecan; proteoglycan
  30. J Appl Toxicol. 2022 Jan 05.
      Quantum dots (QDs), with unique and tunable optical properties, have been are widely used in many fields closely related to our daily lives, such as biomedical application and electronic products. Therefore, the potential toxicity of QDs on the human health should be understood. Autophagy plays an important role in cell survival and death. Endoplasmic reticulum autophagy (ER-phagy), a selective autophagy that degrades ER, responds to the accumulation of misfolded proteins and ER stress. Although many reports have revealed that autophagy can be disturbed by CdTe-QDs and other nanomaterials, there are still lack more detail researches to illustrate the function of autophagy in CdTe-QDs treated cells. And the function of ER-phagy in CdTe-QDs-treated cells remains to be illustrated. On the basis of transcriptome analysis, we explored the effect of CdTe-QDs on Saccharomyces cerevisiae, and firstly illustrated that both of autophagy and ER-phagy were protective mechanisms in CdTe-QDs-treated cells. It was found that CdTe-QDs inhibited the proliferation of yeast cells, disrupted homeostasis of cells, membrane integrity and metabolism process. All of these can be reasons of the reduction of cell viability. The abolish of autophagy and ER-phagy reduce the cell survival, indicating both of them are cell protective mechanisms against CdTe-QDs toxicity in yeast cells. Therefore, our data are significant for the application of CdTe-QDs and provide precious information for understanding of nanomaterials-related ER-phagy.
    Keywords:  CdTe-QDs; ER-phagy; autophagy; toxicity; transcriptome analysis
  31. Exp Cell Res. 2021 Dec 30. pii: S0014-4827(21)00557-7. [Epub ahead of print]411(2): 113001
      Autophagy is involved in the activation of hepatic stellate cells (HSCs) and liver fibrosis. Previous studies have shown that interleukin 10 (IL-10) has a marked therapeutic effect against liver fibrosis. However, few studies have evaluated the effect of IL-10 on autophagy in HSCs and fibrotic livers. The aim of this study was to assess the effect of IL-10 on the autophagy of HSCs in vitro and in vivo and then to explore the underlying pathway. In vitro, The results revealed that IL-10 had inhibitory effects on hydrogen peroxide (H2O2)-induced autophagy, as evidenced by the decreased LC3II/I ratio and Beclin1 expression, increased p62 expression, reduced numbers of autophagosomes, and blocked autophagy initiation in HSCs. Mechanistically, IL-10 significantly promoted the phosphorylation of the signal transducer and activator of transcription 3(STAT3) and mammalian target of rapamycin (mTOR), leading to the activation of STAT3 and mTOR, which in turn inhibited autophagy. In vivo, the increased expression of IL-10 in fibrotic livers inhibited significantly liver fibrosis and decreased the autophagic activity in fibrotic livers and HSCs. Overall, our results indicate that IL-10 suppressed H2O2-induced autophagy in HSCs by activating the STAT3-mTOR signaling pathway. Present study provides a new theoretical basis for the anti-fibrotic effects of IL-10.
    Keywords:  Autophagosome; Hepatic stellate cells; Interleukin 10; Signaling pathway
  32. FASEB J. 2022 Feb;36(2): e22148
      Thymic stromal lymphopoietin (TSLP), a type I cytokine belonging to the IL-2 cytokine family, promotes Th2-mediated inflammatory responses. The aim of this study is to investigate whether TSLP increases inflammatory responses via induction of autophagy using a murine T cell lymphoma cell line, EL4 cells, and lipopolysaccharide (LPS)-injected mice. TSLP increased expression levels of autophagy-related factors, such as Beclin-1, LC3-II, p62, Atg5, and lysosome associated membrane protein 1/2, whereas these factors increased by TSLP disappeared by neutralization of TSLP in EL4 cells. TSLP activated JAK1/JAK2/STAT5/JNK/PI3K, while the blockade of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways reduced the expression levels of Beclin-1, LC3-II, and p62 in TSLP-stimulated EL4 cells. In addition, TSLP simultaneously increased levels of inflammatory cytokines via induction of autophagy by activation of JAK1/JAK2/STAT5/JNK/PI3K signaling pathways. In an LPS-induced acute liver injury (ALI) mouse model, exogenous TSLP increased expression levels of Beclin-1 and LC3-II, whereas functional deficiency of TSLP by TSLP siRNA resulted in lower expression of Beclin-1, LC3-II, and inflammatory cytokines, impairing their ability to form autophagosomes in ALI mice. Thus, our findings show a new role of TSLP between autophagy and inflammatory responses. In conclusion, regulating TSLP-induced autophagy may be a potential therapeutic strategy for inflammatory responses.
    Keywords:  EL4 cells; autophagy; inflammatory responses; thymic stromal lymphopoietin
  33. Anal Chem. 2022 Jan 06.
      Elucidating the intrinsic relationship between mitochondrial pH (pHm) fluctuation and lipid droplets (LDs) formation is vital in cell physiology. The development of small-molecular fluorescent probes for discrimination and simultaneous visualization of pHm fluctuation toward LDs has not yet been reported. In this work, utilizing pH-driven polarity-reversible hemicyanine and rhodamine derivatives, a multifunctional fluorescent probe is developed for selectively identifying mitochondria and LDs under specific pH values via dual-emission channels. This rapid-response probe, Hcy-Rh, has two distinct chemical structures under acidic and alkaline circumstances. In acidic conditions, Hcy-Rh exhibits good hydrophilicity that can target mitochondria and display an intense red fluorescence. Conversely, the probe becomes lipophilic under weakly alkaline conditions and targets LDs, showing a strong blue emission. In this manner, Hcy-Rh can selectively label mitochondria and LDs, exhibiting red and blue fluorescence, respectively. Moreover, this ratiometric probe is applied to map pHm changes in living cells under the stimulus with FCCP, NAC, and H2O2. The interplay of LD-mitochondria under oleic acid treatment and starvation-induced autophagy has been studied using this probe at different pH values. In a word, Hcy-Rh is a potential candidate for further exploring mitochondria-LD interaction mechanisms under pHm fluctuation. Moreover, the polarity-dependent strategy is valuable for designing other functional biological probes in imaging multiple organelles.
  34. Biochemistry. 2022 Jan 05.
      Autophagy-related proteins (Atgs) drive the lysosome-mediated degradation pathway, autophagy, to enable the clearance of dysfunctional cellular components and maintain homeostasis. In humans, this process is driven by the mammalian Atg8 (mAtg8) family of proteins comprising the LC3 and GABARAP subfamilies. The mAtg8 proteins play essential roles in the formation and maturation of autophagosomes and the capture of specific cargo through binding to the conserved LC3-interacting region (LIR) sequence within target proteins. Modulation of interactions of mAtg8 with its target proteins via small-molecule ligands would enable further interrogation of their function. Here we describe unbiased fragment and DNA-encoded library (DEL) screening approaches for discovering LC3 small-molecule ligands. Both strategies resulted in compounds that bind to LC3, with the fragment hits favoring a conserved hydrophobic pocket in mATG8 proteins, as detailed by LC3A-fragment complex crystal structures. Our findings demonstrate that the malleable LIR-binding surface can be readily targeted by fragments; however, rational design of additional interactions to drive increased affinity proved challenging. DEL libraries, which combine small, fragment-like building blocks into larger scaffolds, yielded higher-affinity binders and revealed an unexpected potential for reversible, covalent ligands. Moreover, DEL hits identified possible vectors for synthesizing fluorescent probes or bivalent molecules for engineering autophagic degradation of specific targets.
  35. Comput Struct Biotechnol J. 2022 ;20 107-116
      Ribosome biogenesis is an energetically costly process, and tight regulation is required for stoichiometric balance between components. This requires coordination of RNA polymerases I, II, and III. Lack of nutrients or the presence of stress leads to downregulation of ribosome biogenesis, a process for which mechanistic target of rapamycin complex I (mTORC1) is key. mTORC1 activity is communicated by means of specific transcription factors, and in yeast, which is a primary model system in which transcriptional coordination has been delineated, transcription factors involved in regulation of ribosomal protein genes include Fhl1p and its cofactors, Ifh1p and Crf1p. Ifh1p is an activator, whereas Crf1p has been implicated in maintaining the repressed state upon mTORC1 inhibition. Computational analyses of evolutionary relationships have indicated that Ifh1p and Crf1p descend from a common ancestor. Here, we discuss recent evidence, which suggests that Crf1p also functions as an activator. We propose a model that consolidates available experimental evidence, which posits that Crf1p functions as an alternate activator to prevent the stronger activator Ifh1p from re-binding gene promoters upon mTORC1 inhibition. The correlation between retention of Crf1p in related yeast strains and duplication of ribosomal protein genes suggests that this backup activation may be important to ensure gene expression when Ifh1p is limiting. With ribosome biogenesis as a hallmark of cell growth, failure to control assembly of ribosomal components leads to several human pathologies. A comprehensive understanding of mechanisms underlying this process is therefore of the essence.
    Keywords:  CK2, casein kinase 2; Crf1, corepressor with forkhead like; Crf1p; FHA, forkhead-associated; FHB, forkhead-binding; FKBP, FK506 binding protein; Fhl1, forkhead like; Fpr1, FK506-sensitive proline rotamase; Gene regulation; Hmo1, high mobility group; Ifh1, interacts with forkhead like; Ifh1p; RASTR, ribosome assembly stress response; RP, ribosomal protein; Rap1, repressor/activator protein; RiBi, ribosome biogenesis; Ribosomal protein; Ribosome biogenesis; Sfp1, split finger protein; WGD, whole genome duplication; mTORC1; mTORC1, mechanistic target of rapamycin complex 1
  36. Biol Open. 2022 Jan 07. pii: bio.058728. [Epub ahead of print]
      Fibroblasts are quiescent and tumor suppressive in nature but become activated in wound healing and cancer. The response of fibroblasts to cellular stress has not been extensively investigated however the p53 tumor suppressor has been shown to be activated in fibroblasts during nutrient deprivation. Since the p19 Alternative reading frame (p19Arf) tumor suppressor is a key regulator of p53 activation during oncogenic stress, we investigated the role of p19Arf in fibroblasts during nutrient deprivation. Here we show that prolonged leucine deprivation resulted in increased expression and nuclear localization of p19Arf, triggering apoptosis in primary murine adult lung fibroblasts (ALFs). In contrast, the absence of p19Arf during long-term leucine deprivation resulted in increased ALF proliferation, migration and survival through upregulation of the Integrated Stress Response pathway and increased autophagic flux. Our data implicates a new role for p19Arf in response to nutrient deprivation.
    Keywords:   p19Arf ; Autophagy; Fibroblast; Integrated Stress Response; Leucine Deprivation
  37. Free Radic Biol Med. 2021 Dec 30. pii: S0891-5849(21)01172-2. [Epub ahead of print]
      Oxidative stress is associated with various disease pathologies including Inborn Errors of Metabolism (IEMs), among the most important causes of childhood morbidity and mortality. At least as much as oxidative stress in cells, reductive stress poses a danger to the disruption of cell homeostasis. p62/SQSTM1,protects cells from stress by activation of Nrf2/Keap1 and autophagy pathways. In this study, we tested the role of cellular stress, mitochondrial dysfunction and autophagy via Nrf2/Keap1/p62 pathway in the pathophysiology of three main groups of IEMs. Our results showed that antioxidant and oxidant capacity alone would not be sufficient to reflect the true clinical picture of these diseases. ATP, ROS and mitochondrial membrane potantial (MMP) measurements demonstrated increased cellular stress and bioenergetic imbalance in methylmalonic acidemia (MMA), indicating mild mitochondrial dysfunction. In isovaleric acidemia (IVA), no major change was detected in ATP, ROS and MMP values. Propionic acidemia (PA), mitochondrial diseases (MIT) and mucopolysaccharidosis IV (MPS IV)might point out mitohormesis to cope with chronic reductive stress. Induction of Nrf2/Keap1/p62 pathway and increased expression of HMOX1 were detected in all IEMs.LC3B-II and p62 expression results indicated an impaired autophagic flux in MIT and MPS IV and an induction of autophagic flux in MMA, PA and IVA, but also partial expression of Beclin1, enables autophagy activation, was detected in all IEMs. We conclude that individual diagnosis and treatments are of great importance in IEMs. In addition, we assume that the application of therapeutic antioxidant or preventive treatments without determining the cellular stress status in IEMs may disrupt the sensitive oxidant-antioxidant balance in the cell, leading to the potential to further disrupt the clinical picture, especially in patients with reductive stress. To the best of our knowledge, this is the first study to simultaneously relate IEMs with cellular stress, mitochondrial dysfunction, and autophagy.
    Keywords:  Autophagy; Inborn errors of metabolism; Mitochondrial dysfunction; Nrf2/Keap1/p62 pathway; Oxidative stress; Reductive stress
  38. Proc Natl Acad Sci U S A. 2022 Jan 11. pii: e2115261119. [Epub ahead of print]119(2):
      The target of rapamycin (TOR) kinase is a master regulator that integrates nutrient signals to promote cell growth in all eukaryotes. It is well established that amino acids and glucose are major regulators of TOR signaling in yeast and metazoan, but whether and how TOR responds to carbon availability in photosynthetic organisms is less understood. In this study, we showed that photosynthetic assimilation of CO2 by the Calvin-Benson-Bassham (CBB) cycle regulates TOR activity in the model single-celled microalga Chlamydomonas reinhardtii Stimulation of CO2 fixation boosted TOR activity, whereas inhibition of the CBB cycle and photosynthesis down-regulated TOR. We uncovered a tight link between TOR activity and the endogenous level of a set of amino acids including Ala, Glu, Gln, Leu, and Val through the modulation of CO2 fixation and the use of amino acid synthesis inhibitors. Moreover, the finding that the Chlamydomonas starch-deficient mutant sta6 displayed disproportionate TOR activity and high levels of most amino acids, particularly Gln, further connected carbon assimilation and amino acids to TOR signaling. Thus, our results showed that CO2 fixation regulates TOR signaling, likely through the synthesis of key amino acids.
    Keywords:  CO2; Chlamydomonas; TOR kinase; amino acids
  39. J Clin Invest. 2022 Jan 06. pii: e153157. [Epub ahead of print]
      The bone marrow (BM) microenvironment regulates acute myeloid leukemia (AML) initiation, proliferation and chemotherapy resistance. Following cancer cell death, a growing body of evidence suggests an important role for remaining apoptotic debris in regulating the immunologic response to, and growth of, solid tumors. Here we investigated the role of macrophage LC3-associated phagocytosis (LAP) within the BM microenvironment of AML. Depletion of BM macrophages increased AML growth in-vivo. We showed that LAP is the predominate method of BM macrophage phagocytosis of dead and dying cells in the AML microenvironment. Targeted inhibition of LAP led to accumulation of apoptotic cells (AC) and apoptotic bodies (AB) resulting in accelerated leukemia growth. Mechanistically, LAP of AMLderived-AB by BM macrophages, resulted in STING pathway activation. We identified that AML derived mitochondrial damage associated molecular patterns were processed by BM macrophages via LAP. Moreover, depletion of mitochondrial DNA (mtDNA) in AML derived-AB showed that it is this mtDNA which was responsible for the induction of STING signalling in BM macrophages. Phenotypically we found that STING activation suppressed AML growth through a mechanism related to increased phagocytosis. In summary, we report that macrophage LAP of apoptotic debris in the AML BM microenvironment suppressed tumor growth.
    Keywords:  Autophagy; Hematology; Leukemias; Mitochondria; Oncology
  40. Nat Biomed Eng. 2022 Jan 06.
      A reduced removal of dysfunctional mitochondria is common to aging and age-related neurodegenerative pathologies such as Alzheimer's disease (AD). Strategies for treating such impaired mitophagy would benefit from the identification of mitophagy modulators. Here we report the combined use of unsupervised machine learning (involving vector representations of molecular structures, pharmacophore fingerprinting and conformer fingerprinting) and a cross-species approach for the screening and experimental validation of new mitophagy-inducing compounds. From a library of naturally occurring compounds, the workflow allowed us to identify 18 small molecules, and among them two potent mitophagy inducers (Kaempferol and Rhapontigenin). In nematode and rodent models of AD, we show that both mitophagy inducers increased the survival and functionality of glutamatergic and cholinergic neurons, abrogated amyloid-β and tau pathologies, and improved the animals' memory. Our findings suggest the existence of a conserved mechanism of memory loss across the AD models, this mechanism being mediated by defective mitophagy. The computational-experimental screening and validation workflow might help uncover potent mitophagy modulators that stimulate neuronal health and brain homeostasis.
  41. Cell Rep. 2022 Jan 04. pii: S2211-1247(21)01701-0. [Epub ahead of print]38(1): 110197
      AMP-activated protein kinase (AMPK) regulates the balance between cellular anabolism and catabolism dependent on energy resources to maintain proliferation and survival. Small-compound AMPK activators show anti-cancer activity in preclinical models. Using the direct AMPK activator GSK621, we show that the unfolded protein response (UPR) is activated by AMPK in acute myeloid leukemia (AML) cells. Mechanistically, the UPR effector protein kinase RNA-like ER kinase (PERK) represses oxidative phosphorylation, tricarboxylic acid (TCA) cycle, and pyrimidine biosynthesis and primes the mitochondrial membrane to apoptotic signals in an AMPK-dependent manner. Accordingly, in vitro and in vivo studies reveal synergy between the direct AMPK activator GSK621 and the Bcl-2 inhibitor venetoclax. Thus, selective AMPK-activating compounds kill AML cells by rewiring mitochondrial metabolism that primes mitochondria to apoptosis by BH3 mimetics, holding therapeutic promise in AML.
    Keywords:  AML; AMPK; GSK621; PERK; mitochondrial apoptosis; unfolded protein response; venetoclax
  42. Front Cell Dev Biol. 2021 ;9 821719
    Keywords:  FUS; IL6-receptor; PLA2; PX domains; TOM1; endosomes; lysosomes; v-ATPase
  43. Cell Biosci. 2022 Jan 03. 12(1): 1
      Oxidative stress is mainly caused by intracellular reactive oxygen species (ROS) production, which is highly associated with normal physiological homeostasis and the pathogenesis of diseases, particularly ocular diseases. Autophagy is a self-clearance pathway that removes oxidized cellular components and regulates cellular ROS levels. ROS can modulate autophagy activity through transcriptional and posttranslational mechanisms. Autophagy further triggers transcription factor activation and degrades impaired organelles and proteins to eliminate excessive ROS in cells. Thus, autophagy may play an antioxidant role in protecting ocular cells from oxidative stress. Nevertheless, excessive autophagy may cause autophagic cell death. In this review, we summarize the mechanisms of interaction between ROS and autophagy and their roles in the pathogenesis of several ocular diseases, including glaucoma, age-related macular degeneration (AMD), diabetic retinopathy (DR), and optic nerve atrophy, which are major causes of blindness. The autophagy modulators used to treat ocular diseases are further discussed. The findings of the studies reviewed here might shed light on the development and use of autophagy modulators for the future treatment of ocular diseases.
    Keywords:  Age-related macular degeneration; Autophagy; Diabetic retinopathy; Glaucoma; Optic nerve atrophy; Reactive oxygen species
  44. Inflammation. 2022 Jan 07.
      Melatonin, as an endogenous circadian indoleamine secreted by the pineal gland, executes extensive biological functions, including antioxidant, anti-inflammatory, anti-tumor, and neuroprotective effects. Although melatonin has been reported to serve as a potential therapeutic against many nerve injury diseases, its effect on ropivacaine-induced neurotoxicity remains obscure. Our research aimed to explore the impact and mechanism of melatonin on ropivacaine-induced neurotoxicity. Our results showed that melatonin pretreatment protected the cell viability, morphology, and apoptosis of PC12 and HT22 cells, and it also improved ropivacaine-induced mitochondrial dysfunction and the activation of mitophagy. In addition, we found that autophagy activation with rapamycin significantly weakened the protective effect of melatonin against ropivacaine-induced apoptosis, whereas autophagy inhibition with 3-MA enhanced the effect of melatonin. We also detected the activation of Parkin and PINK1, a canonical mechanism for mitophagy regulation, and results shown that melatonin downregulated the expression of Parkin and PINK1, and upregulated Tomm20 and COXIV proteins, so that those results indicated that melatonin protected ropivacaine-induced apoptosis through suppressing excessive mitophagy by inhibiting the Parkin/PINK1 pathway. Melatonin may be a useful potential therapeutic agent against ropivacaine-induced neurotoxicity.
    Keywords:  apoptosis.; autophagy; melatonin; mitophagy; neurotoxicity; ropivacaine
  45. Front Pharmacol. 2021 ;12 801234
      Background: The mechanistic target of rapamycin complex 1 (mTORC1) signaling has served as a promising target for therapeutic intervention of major depressive disorder (MDD), but the mTORC1 signaling underlying MDD has not been well elucidated. In the present study, we investigated whether mTORC1 signaling pathway mediates synapse loss induced by chronic stress in the hippocampus. Methods: Chronic restraint stress-induced depression-like behaviors were tested by behavior tests (sucrose preference test, forced swim test and tail suspension test). Synaptic proteins and alternations of phosphorylation levels of mTORC1 signaling-associated molecules were measured using Western blotting. In addition, mRNA changes of immediate early genes (IEGs) and glutamate receptors were measured by RT-PCR. Rapamycin was used to explore the role of mTORC1 signaling in the antidepressant effects of fluoxetine. Results: After successfully establishing the chronic restraint stress paradigm, we observed that the mRNA levels of some IEGs were significantly changed, indicating the activation of neurons and protein synthesis alterations. Then, there was a significant downregulation of glutamate receptors and postsynaptic density protein 95 at protein and mRNA levels. Additionally, synaptic fractionation assay revealed that chronic stress induced synapse loss in the dorsal and ventral hippocampus. Furthermore, these effects were associated with the mTORC1 signaling pathway-mediated protein synthesis, and subsequently the phosphorylation of associated downstream signaling targets was reduced after chronic stress. Finally, we found that intracerebroventricular infusion of rapamycin simulated depression-like behavior and also blocked the antidepressant effects of fluoxetine. Conclusion: Overall, our study suggests that mTORC1 signaling pathway plays a critical role in mediating synapse loss induced by chronic stress, and has part in the behavioral effects of antidepressant treatment.
    Keywords:  chronic restraint stress; depression; fluoxetine; mammalian target of rapamycin; postsynaptic density protein 95
  46. Front Pharmacol. 2021 ;12 795058
      Osteoarthritis (OA) is a common degenerative joint disease and is a leading cause of disability and reduced quality of life worldwide. There are currently no clinical treatments that can stop or slow down OA. Drugs have pain-relieving effects, but they do not slow down the course of OA and their long-term use can lead to serious side effects. Therefore, safe and clinically appropriate long-term treatments for OA are urgently needed. Autophagy is an intracellular protective mechanism, and targeting autophagy-related pathways has been found to prevent and treat various diseases. Attenuation of the autophagic pathway has now been found to disrupt cartilage homeostasis and plays an important role in the development of OA. Therefore, modulation of autophagic signaling pathways mediating cartilage homeostasis has been considered as a potential therapeutic option for OA. Phytochemicals are active ingredients from plants that have recently been found to reduce inflammatory factor levels in cartilage as well as attenuate chondrocyte apoptosis by modulating autophagy-related signaling pathways, which are not only widely available but also have the potential to alleviate the symptoms of OA. We reviewed preclinical studies and clinical studies of phytochemicals mediating autophagy to regulate cartilage homeostasis for the treatment of OA. The results suggest that phytochemicals derived from plant extracts can target relevant autophagic pathways as complementary and alternative agents for the treatment of OA if subjected to rigorous clinical trials and pharmacological tests.
    Keywords:  Osteoarthritis; autophagy; chondrocytes; inflammation; phytochemicals
  47. Front Cell Dev Biol. 2021 ;9 798262
      For a long time, lysosomes were considered as mere waste bags for cellular constituents. Thankfully, studies carried out in the past 15 years were brimming with elegant and crucial breakthroughs in lysosome research, uncovering their complex roles as nutrient sensors and characterizing them as crucial multifaceted signaling organelles. This review presents the scientific knowledge on lysosome physiology and functions, starting with their discovery and reviewing up to date ground-breaking discoveries highlighting their heterogeneous functions as well as pending questions that remain to be answered. We also review the roles of lysosomes in anti-cancer drug resistance and how they undergo a series of molecular and functional changes during malignant transformation which lead to tumor aggression, angiogenesis, and metastases. Finally, we discuss the strategy of targeting lysosomes in cancer which could lead to the development of new and effective targeted therapies.
    Keywords:  BORC; TFEB; autophagy; cancer; endocytosis; lysosome; mTOR; reformation
  48. Aging (Albany NY). 2022 Jan 04. 13(undefined):
      Autophagy is an important regulator of cellular homeostasis and its dysregulation often results in cancer. Aberrant glycosylation induced by oncogenic transformation contributes to tumor invasion and metastasis. In a previous study, we have demonstrated that EpCAM, a glycosylation protein, is associated with cell growth and metastasis in breast cancer. But the effect of EpCAM glycosylation on autophagy is not clear. the precise mechanism of regulation remains largely unknown. In this study, breast cancer cells were transfected with N-glycosylation mutation EpCAM plasmid to express deglycosylated EpCAM. The result showed that deglycosylated EpCAM promoted autophagy in breast cancer cells. We further confirmed this conclusion with the activator (Rapamycin, RAP) and inhibitor (Wortmannin) of autophagy. We also found that deglycosylated EpCAM promoted apoptosis and inhibited proliferation through activating autophagy by suppressing Akt/mTOR signaling pathway in breast cancer cells. These findings represent a novel mechanism by which deglycosylated EpCAM inhibits proliferation by enhancing autophagy of breast cancer cells via PI3K/Akt/mTOR pathway. In conclusion, the combination of autophagy modulation and EpCAM targeted therapy is a promising therapeutic strategy in the treatment of breast cancer.
    Keywords:  EpCAM; PI3K/Akt/mTOR; autophagy; breast cancer; glycosylation
  49. Mol Biol Rep. 2022 Jan 08.
      BACKGROUND: Melatonin can regulate apoptosis and autophagy of mouse Leydig cells, but its specific mechanism is still unclear.METHODS: In this study, we used the TM3 cell line as the research object, and used H2O2 to induce autophagy. After adding 10 ng/ml melatonin, we used qRT-PCR and western-blot to detect autophagy-related gene and protein expression, and flow cytometry to detect cellular ROS level.
    RESULTS: The results showed that melatonin can significantly inhibit the occurrence of autophagy, accompanied by a significant decrease in the expression of Becn1, LC3, and FOXO1 (P < 0.05), a significant increase in the expression of p62 and pAKT (P < 0.05), and a significant decrease in ROS level (P < 0.05). After added the inhibitor of AKT perifosine, the effect of melatonin on inhibiting autophagy was reversed. On this basis, we used small RNA interference technology to knock down the expression of FOXO1, and found that there was no significant change of the expression of genes and proteins related to autophagy and ROS level.
    CONCLUSIONS: In summary, melatonin can inhibit H2O2-induced autophagy in TM3 cells through the AKT/FOXO1 pathway.
    Keywords:  AKT; Autophagy; FOXO1; Melatonin; Mouse; TM3 cells
  50. Oncogene. 2022 Jan 07.
      Despite the establishment of novel therapeutic interventions, multiple myeloma (MM) remains invariably incurable due to development of drug resistance and subsequent relapse, which are attributed to activation of oncogenic pathways such as autophagy. Deubiquitinating enzymes (DUBs) are promising targets to overcome resistance to proteasome inhibitor-based treatment. Ubiquitin-specific protease-12 (USP12) is a DUB with a known prognostic value in several cancers. We found that USP12 protein levels were significantly higher in myeloma patient samples than in non-cancerous human samples. Depletion of USP12 suppressed cell growth and clonogenicity and inhibited autophagy. Mechanistic studies showed that USP12 interacted with, deubiquitylated and stabilized the critical autophagy mediator HMGB1 (high mobility group box-1) protein. Knockdown of USP12 decreased the level of HMGB1 and suppressed HMGB1-mediated autophagy in MM. Furthermore, basal autophagy activity associated with USP12/HMGB1 was elevated in bortezomib (BTZ)-resistant MM cell lines. USP12 depletion, concomitant with a reduced expression of HMGB1, suppressed autophagy and increased the sensitivity of resistant cells to BTZ. Collectively, our findings have identified an important role of the deubiquitylase USP12 in pro-survival autophagy and resultant BTZ resistance in MM by stabilizing HMGB1, suggesting that the USP12/HMGB1 axis might be pursued as a potential diagnostic and therapeutic target in human MM.
  51. Mol Ther Nucleic Acids. 2022 Mar 08. 27 147-164
      FilaminC (Flnc) is a member of the actin binding protein family, which is preferentially expressed in the cardiac and skeletal muscle tissues. Although it is known to interact with proteins associated with myofibrillar myopathy, its unique role in skeletal muscle remains largely unknown. In this study, we identify the biological functions of Flnc in vitro and in vivo using chicken primary myoblast cells and animal models, respectively. From the results, we observe that the growth rate and mass of the skeletal muscle of fast-growing chickens (broilers) were significantly higher than those in slow-growing chickens (layers). Furthermore, we find that the expression of Flnc in the skeletal muscle of broilers was higher than that in the layers. Our results indicated that Flnc was highly expressed in the skeletal muscle, especially in the skeletal muscle of broilers than in layers. This suggests that Flnc plays a positive regulatory role in myoblast development. Flnc knockdown resulted in muscle atrophy, whereas the overexpression of Flnc promotes muscle hypertrophy in vivo in an animal model. We also found that Flnc interacted with dishevelled-2 (Dvl2), activated the wnt/β-catenin signaling pathway, and controlled skeletal muscle development. Flnc also antagonized the LC3-mediated autophagy system by decreasing Dvl2 ubiquitination. Moreover, Flnc knockdown activated and significantly increased mitophagy. In summary, these results indicate that the absence of Flnc induces autophagy or mitophagy and regulates muscle atrophy.
    Keywords:  atrophy; autophagy; dishevelled-2; filamin C; mitophagy; skeletal muscle
  52. Redox Biol. 2021 Dec 30. pii: S2213-2317(21)00387-6. [Epub ahead of print]49 102227
      Copper (Cu) is a necessary micronutrient at lower concentration, while excessive Cu exposure or Cu homeostasis disorders can lead to toxicity. The mechanism of male reproductive toxicity induced by Cu is still unknown. This study aims to investigate whether autophagy plays an important role in copper-induced spermatogenesis disorder in vivo and vitro. The present study showed that copper sulfate (CuSO4) might significantly promote autophagy level in the testis and mouse-derived spermatogonia cell line GC-1 spg cells. Concurrently, CuSO4 could induce autophagy via AMPK-mTOR pathway that downregulated p-mTOR/mTOR and subsequently upregulated p-AMPKα/AMPKα as well as p-ULK1/ULK1. In the meanwhile, CuSO4 treatment could also increase expression levels of the autophagy-related proteins. Then, the role of oxidative stress in CuSO4-induced autophagy was investigated. The findings demonstrated that oxidative stress inhibitor (NAC) attenuated CuSO4-induced autophagy in vivo and vitro, reversing the activation for AMPK-mTOR pathway. Additionally, the study also investigated how autophagy worked under the spermatogenesis disorder induced by CuSO4. Inhibition of autophagy could decrease cell viability, and enhance the ROS accumulation and apoptosis in the GC-1 cells, meanwhile, the spermatogenesis disorder, oxidative stress and histopathological changes were increased in the testis. Furthermore, co-treatment with the apoptosis inhibitor (Z-VAD-FMK) could decrease the spermatogenesis disorder but not influence autophagy. Besides, the crosslink between autophagy and ferroptosis were also measured, the data showed that inhibition of autophagy could suppress CuSO4-induced ferroptosis in in vivo and vitro. Altogether, abovementioned results indicated that CuSO4 induced autophagy via oxidative stress-dependent AMPK-mTOR pathway in the GC-1 cells and testis, and autophagy activation possibly led to the generation of protection mechanism through oxidative damage and apoptosis inhibition, however, autophagy also aggravate CuSO4 toxicology through promoting ferroptosis. Overall, autophagy plays a positive role for attenuating CuSO4-induced testicular damage and spermatogenesis disorder. Our study provides a possible targeted therapy for Cu overload-induced reproduction toxicology.
    Keywords:  Apoptosis; Autophagy; CuSO(4); Oxidative stress; Spermatogenesis disorder
  53. PLoS One. 2022 ;17(1): e0262134
      Autophagy drives drug resistance and drug-induced cancer cell cytotoxicity. Targeting the autophagy process could greatly improve chemotherapy outcomes. The discovery of specific inhibitors or activators has been hindered by challenges with reliably measuring autophagy levels in a clinical setting. We investigated drug-induced autophagy in breast cancer cell lines with differing ER/PR/Her2 receptor status by exposing them to known but divergent autophagy inducers each with a unique molecular target, tamoxifen, trastuzumab, bortezomib or rapamycin. Differential gene expression analysis from total RNA extracted during the earliest sign of autophagy flux showed both cell- and drug-specific changes. We analyzed the list of differentially expressed genes to find a common, cell- and drug-agnostic autophagy signature. Twelve mRNAs were significantly modulated by all the drugs and 11 were orthogonally verified with Q-RT-PCR (Klhl24, Hbp1, Crebrf, Ypel2, Fbxo32, Gdf15, Cdc25a, Ddit4, Psat1, Cd22, Ypel3). The drug agnostic mRNA signature was similarly induced by a mitochondrially targeted agent, MitoQ. In-silico analysis on the KM-plotter cancer database showed that the levels of these mRNAs are detectable in human samples and associated with breast cancer prognosis outcomes of Relapse-Free Survival in all patients (RSF), Overall Survival in all patients (OS), and Relapse-Free Survival in ER+ Patients (RSF ER+). High levels of Klhl24, Hbp1, Crebrf, Ypel2, CD22 and Ypel3 were correlated with better outcomes, whereas lower levels of Gdf15, Cdc25a, Ddit4 and Psat1 were associated with better prognosis in breast cancer patients. This gene signature uncovers candidate autophagy biomarkers that could be tested during preclinical and clinical studies to monitor the autophagy process.
  54. iScience. 2022 Jan 21. 25(1): 103574
      Heart disease is the leading cause of death with no method to repair damaged myocardium due to the limited proliferative capacity of adult cardiomyocytes. Curiously, mouse neonates and zebrafish can regenerate their hearts via cardiomyocyte de-differentiation and proliferation. However, a molecular mechanism of why these cardiomyocytes can re-enter cell cycle is poorly understood. Here, we identify a unique metabolic state that primes adult zebrafish and neonatal mouse ventricular cardiomyocytes to proliferate. Zebrafish and neonatal mouse hearts display elevated glutamine levels, predisposing them to amino-acid-driven activation of TOR, and that TOR activation is required for zebrafish cardiomyocyte regeneration in vivo. Through a multi-omics approach with cellular validation we identify metabolic and mitochondrial changes during the first week of regeneration. These data suggest that regeneration of zebrafish myocardium is driven by metabolic remodeling and reveals a unique metabolic regulator, TOR-primed state, in which zebrafish and mammalian cardiomyocytes are regeneration competent.
    Keywords:  Biological sciences; Cell biology; Tissue Engineering