bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2023–04–02
fifty-six papers selected by
Viktor Korolchuk, Newcastle University
  1. TFEB-dependent lysosome biogenesis is required for senescence.
  2. The ER membrane protein complex governs lysosomal turnover of a mitochondrial tail-anchored protein, BNIP3, to restrict mitophagy.
  3. An affinity tool for the isolation of endogenous active mTORC1 from various cellular sources.
  4. Epigenetic regulation of autophagy by histone-modifying enzymes under nutrient stress.
  5. Autophagy at the synapse, an early site of dysfunction in neurodegeneration.
  6. Role of autophagy and mitophagy in neurodegenerative disorders.
  7. Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation.
  8. Reduction of Spermine Synthase Suppresses Tau Accumulation Through Autophagy Modulation in Tauopathy.
  9. TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.
  10. Developing a comprehensive solution aimed to disrupt LARS1/RagD protein-protein interaction.
  11. Autophagy Function and Benefits of Autophagy Induction in Models of Spinocerebellar Ataxia Type 3.
  12. Endothelial Autophagy Dysregulation in Diabetes.
  13. The Role of Autophagy in the Female Reproduction System: For Beginners to Experts in This Field.
  14. The skeletal muscle fiber periphery: A nexus of mTOR-related anabolism.
  15. Enhanced Levels of Peroxisome-Derived H2O2 Do Not Induce Pexophagy but Impair Autophagic Flux in HEK-293 and HeLa Cells.
  16. Autophagy pathways in autoimmune diseases.
  17. The Drp1-Mediated Mitochondrial Fission Protein Interactome as an Emerging Core Player in Mitochondrial Dynamics and Cardiovascular Disease Therapy.
  18. The N-degron pathway: From basic science to therapeutic applications.
  19. A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway.
  20. NAADP-Evoked Ca2+ Signaling Leads to Mutant Huntingtin Aggregation and Autophagy Impairment in Murine Astrocytes.
  21. Mammalian Target of Rapamycin (mTOR) Signalling Pathway a Potential Target for Cancer Intervention: a Short Overview.
  22. KNO1-mediated autophagic degradation of the Bloom syndrome complex component RMI1 promotes homologous recombination.
  23. CDK5-PRMT1-WDR24 signaling cascade promotes mTORC1 signaling and tumor growth.
  24. RIP1 Mediates Manzamine-A-Induced Secretory Autophagy in Breast Cancer.
  25. Differential Role of Active Compounds in Mitophagy and Related Neurodegenerative Diseases.
  26. PSMD2 contributes to the progression of esophageal squamous cell carcinoma by repressing autophagy.
  27. A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells.
  28. Tactics with Prebiotics for the Treatment of Metabolic Dysfunction-Associated Fatty Liver Disease via the Improvement of Mitophagy.
  29. Crosstalk between Autophagy and RLR Signaling.
  30. Autophagy is the key to making chronic wounds acute in skin wound healing.
  31. The adhesion G-protein-coupled receptor VLGR1/ADGRV1 controls autophagy.
  32. Progranulin Deficiency Induces Mitochondrial Dysfunction in Frontotemporal Lobar Degeneration with TDP-43 Inclusions.
  33. The small GTPase Rit2 modulates LRRK2 kinase activity, is required for lysosomal function and protects against alpha-synuclein neuropathology.
  34. Spermidine Rescues Bioenergetic and Mitophagy Deficits Induced by Disease-Associated Tau Protein.
  35. The role of mitochondrial quality surveillance in skin aging: Focus on mitochondrial dynamics, biogenesis and mitophagy.
  36. Inhibition of Sphingosine Kinase 2 Results in PARK2-Mediated Mitophagy and Induces Apoptosis in Multiple Myeloma.
  37. Mechanotransduction Impairment in Primary Fibroblast Model of Krabbe Disease.
  38. Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.
  39. DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages.
  40. Transcriptomic changes predict metabolic alterations in LC3 associated phagocytosis in aged mice.
  41. ROS-AMPK/mTOR-dependent enterocyte autophagy is involved in the regulation of Giardia infection-related tight junction protein and nitric oxide levels.
  42. Total Chemical Synthesis of LC3A and LC3B Activity-Based Probes.
  43. Determination of the autophagic flux in murine and human peripheral blood mononuclear cells.
  44. Bcat1 is controlled by Tsc2/mTORC1 pathway at expression levels and its deficiency together with Bcat2 inactivation suppresses the growth of a Tsc2-/- tumor cell line.
  45. Mitochondrial Oxidative Stress and Mitophagy Activation Contribute to TNF-Dependent Impairment of Myogenesis.
  46. Alpha 2-macroglobulin acts as a clearance factor in the lysosomal degradation of extracellular misfolded proteins.
  47. Lithium engages autophagy for neuroprotection and neuroplasticity: translational evidence for therapy.
  48. Curcumin Modulates p62-Keap1-Nrf2-Mediated Autophagy in Rotenone-Induced Parkinson's Disease Mouse Models.
  49. Destabilization of microrchidia family CW-type zinc finger 2 via the cyclin-dependent kinase 1-chaperone-mediated autophagy pathway promotes mitotic arrest and enhances cancer cellular sensitivity to microtubule-targeting agents.
  50. Metastasis in Neuroblastoma and Its Link to Autophagy.
  51. Transcriptional profiling of the response to starvation and fattening reveals differential regulation of autophagy genes in mammals.
  52. The mtDNA-STING pathway plays an important role in both navitoclax- and S63845-induced autophagy and enhances cell death.
  53. The essential effect of mTORC1-dependent lipophagy in non-alcoholic fatty liver disease.
  54. A multi-organ analysis of the role of mTOR in fetal alcohol spectrum disorders.
  55. The plant unique ESCRT component FREE1 regulates autophagosome closure.
  56. Mechanistic Insights into the Role of OPN in Mediating Brain Damage via Triggering Lysosomal Damage in Microglia/Macrophage.
  1. EMBO J. 2023 Mar 27. e111241
    TFEB-dependent lysosome biogenesis is required for senescence.
    Rachel Curnock, Katy Yalci, Johan Palmfeldt, Marja Jäättelä, Bin Liu, Bernadette Carroll.
      The accumulation of senescent cells is recognised as a driver of tissue and organismal ageing. One of the gold-standard hallmarks of a senescent cell is an increase in lysosomal content, as measured by senescence-associated β-galactosidase (Senβ-Gal) activity. The lysosome plays a central role in integrating mitogenic and stress cues to control cell metabolism, which is known to be dysregulated in senescence. Despite this, little is known about the cause and consequence of lysosomal biogenesis in senescence. We find here that lysosomes in senescent cells are dysfunctional; they have higher pH, increased evidence of membrane damage and reduced proteolytic capacity. The significant increase in lysosomal content is however sufficient to maintain degradative capacity of the cell to a level comparable to proliferating control cells. We demonstrate that increased nuclear TFEB/TFE3 supports lysosome biogenesis, is a hallmark of multiple forms of senescence and is required for senescent cell survival. TFEB/TFE3 are hypo-phosphorylated and show constitutive nuclear localisation in senescence. Evidence suggests that several pathways may contribute to TFEB/TFE3 dysregulation in senescence.
    Keywords:  TFEB; autophagy; lysosome; senescence
    DOI:  https://doi.org/10.15252/embj.2022111241
  2. bioRxiv. 2023 Mar 24. pii: 2023.03.22.533681. [Epub ahead of print]
    The ER membrane protein complex governs lysosomal turnover of a mitochondrial tail-anchored protein, BNIP3, to restrict mitophagy.
    Jose M Delgado, Logan Wallace Shepard, Sarah W Lamson, Samantha L Liu, Christopher J Shoemaker.
      Lysosomal degradation of autophagy receptors is a common proxy for selective autophagy. However, we find that two established mitophagy receptors, BNIP3 and BNIP3L/NIX, violate this assumption. Rather, BNIP3 and NIX are constitutively delivered to lysosomes in an autophagy-independent manner. This alternative lysosomal delivery of BNIP3 accounts for nearly all of its lysosome-mediated degradation, even upon mitophagy induction. To identify how BNIP3, a tail-anchored protein in the outer mitochondrial membrane, is delivered to lysosomes, we performed a genome-wide CRISPR screen for factors influencing BNIP3 flux. By this approach, we revealed both known modifiers of BNIP3 stability as well as a pronounced reliance on endolysosomal components, including the ER membrane protein complex (EMC). Importantly, the endolysosomal system regulates BNIP3 alongside, but independent of, the ubiquitin-proteosome system (UPS). Perturbation of either mechanism is sufficient to modulate BNIP3-associated mitophagy and affect underlying cellular physiology. In short, while BNIP3 can be cleared by parallel and partially compensatory quality control pathways, non-autophagic lysosomal degradation of BNIP3 is a strong post-translational modifier of BNIP3 function. More broadly, these data reveal an unanticipated connection between mitophagy and TA protein quality control, wherein the endolysosomal system provides a critical axis for regulating cellular metabolism. Moreover, these findings extend recent models for tail-anchored protein quality control and install endosomal trafficking and lysosomal degradation in the canon of pathways that ensure tight regulation of endogenous TA protein localization.
    DOI:  https://doi.org/10.1101/2023.03.22.533681
  3. J Biol Chem. 2023 Mar 23. pii: S0021-9258(23)00286-7. [Epub ahead of print] 104644
    An affinity tool for the isolation of endogenous active mTORC1 from various cellular sources.
    Yasir H Ibrahim, Spyridon Pantelios, Anders P Mutvei.
      The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of mammalian cell growth that is dysregulated in a number of human diseases, including metabolic syndromes, aging and cancer. Structural, biochemical and pharmacological studies that have increased our understanding of how mTORC1 executes growth control often relied upon purified mTORC1 protein. However, current immunoaffinity-based purification methods are expensive, inefficient, and do not necessarily isolate endogenous mTORC1, hampering their overall utility in research. Here we present a simple tool to isolate endogenous mTORC1 from various cellular sources. By recombinantly expressing and isolating mTORC1-binding Rag GTPases from E. Coli and using them as affinity probes, we demonstrate that mTORC1 can be isolated from mouse, bovine and human sources. Our results indicate that mTORC1 isolated by this relatively inexpensive method is catalytically active and amenable to scaling. Collectively, this tool may be utilized to isolate mTORC1 from various cellular sources, organs, and disease contexts, aiding mTORC1-related research.
    DOI:  https://doi.org/10.1016/j.jbc.2023.104644
  4. Cell Death Differ. 2023 Mar 30.
    Epigenetic regulation of autophagy by histone-modifying enzymes under nutrient stress.
    Young Suk Yu, Hyunkyung Kim, Keun Il Kim, Sung Hee Baek.
      Autophagy is an evolutionarily conserved catabolic process that is induced in response to various stress factors in order to protect cells and maintain cellular homeostasis by degrading redundant components and dysfunctional organelles. Dysregulation of autophagy has been implicated in several conditions such as cancer, neurodegenerative diseases, and metabolic disorders. Although autophagy has been commonly considered as a cytoplasmic process, accumulating evidence has revealed that epigenetic regulation within the nucleus is also important for regulation of autophagy. In particular, when energy homeostasis is disrupted, for instance due to nutrient deprivation, cells increase autophagic activity at the transcriptional level, thereby also increasing the extent of overall autophagic flux. The transcription of genes associated with autophagy is strictly regulated by epigenetic factors through a network of histone-modifying enzymes along with histone modifications. A better understanding of the complex regulatory mechanisms of autophagy could reveal potential new therapeutic targets for autophagy-related diseases. In this review, we discuss the epigenetic regulation of autophagy in response to nutrient stress, focusing on histone-modifying enzymes and histone modifications.
    DOI:  https://doi.org/10.1038/s41418-023-01154-9
  5. Curr Opin Physiol. 2023 Apr;pii: 100631. [Epub ahead of print]32
    Autophagy at the synapse, an early site of dysfunction in neurodegeneration.
    Hilary Grosso Jasutkar, Ai Yamamoto.
      Macroautophagy, herein referred to as autophagy, has long been implicated in the pathophysiology of neurodegenerative diseases. However, an incomplete understanding of how autophagy contributes to disease pathogenesis has limited progress in acting on this potential target for the development of disease modifying therapeutics. Research in the past few decades has revealed that autophagy plays a specialized role in the synapse, a site of early dysfunction in multiple neurodegenerative diseases. In this review we discuss the evidence suggesting that inadequate autophagy at the synapse may contribute to neurodegeneration, and why the functions of autophagy may be particularly relevant for synaptic function.
    Keywords:  autophagy; neurodegeneration; selective autophagy; synapse
    DOI:  https://doi.org/10.1016/j.cophys.2023.100631
  6. CNS Neurol Disord Drug Targets. 2023 Mar 27.
    Role of autophagy and mitophagy in neurodegenerative disorders.
    Lakshay Kapil, Vishal Kumar, Simranjit Kaur, Deepali Sharma, Charan Singh, Arti Singh.
      Autophagy is a self-destructive cellular process that removes essential metabolites and waste from inside the cell to maintain cellular health. Mitophagy is the process by which autophagy causes disruption inside mitochondria and the total removal of damaged or stressed mitochondria, hence enhancing cellular health. The mitochondria are the powerhouses of the cell, performing essential functions such as ATP (adenosine triphosphate) generation, metabolism, Ca2+ buffering, and signal transduction. Many different mechanisms, including endosomal and autophagosomal transport, bring these substrates to lysosomes for processing. Autophagy and endocytic processes each have distinct compartments, and they interact dynamically with one another to complete digestion. Since mitophagy is essential for maintaining cellular health and using genetics, cell biology, and proteomics techniques, it is necessary to understand its beginning, particularly in ubiquitin and receptor-dependent signalling in injured mitochondria. Despite their similar symptoms and emerging genetic foundations, Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS) have all been linked to abnormalities in autophagy and endolysosomal pathways associated with neuronal dysfunction. Mitophagy is responsible for normal mitochondrial turnover and, under certain physiological or pathological situations, may drive the elimination of faulty mitochondria. Due to their high energy requirements and post-mitotic origin, neurons are especially susceptible to autophagic and mitochondrial malfunction. This article focused on the importance of autophagy and mitophagy in neurodegenerative illnesses and how they might be used to create novel therapeutic approaches for treating a wide range of neurological disorders.
    Keywords:  Alzheimer’s disease; Amyotrophic lateral sclerosis; Autophagy; Huntington’s disease; Parkinson’s disease; mitophagy; neurodegenerative disorders
    DOI:  https://doi.org/10.2174/1871527322666230327092855
  7. Int J Mol Sci. 2023 Mar 22. pii: 5984. [Epub ahead of print]24(6):
    Impaired Autophagy in Krabbe Disease: The Role of BCL2 and Beclin-1 Phosphorylation.
    Nadia Papini, Roberta Todisco, Paola Giussani, Michele Dei Cas, Rita Paroni, Chiara Giallanza, Cristina Tringali.
      Autophagic impairment was identified in many lysosomal storage diseases and adult neurodegenerative diseases. It seems that this defect could be directly related to the appearance of a neurodegenerative phenotype and could contribute to worsen metabolite accumulation and lysosomal distress. Thus, autophagy is becoming a promising target for supportive therapies. Autophagy alterations were recently identified also in Krabbe disease. Krabbe disease is characterized by extensive demyelination and dysmyelination and it is due to the genetic loss of function of the lysosomal enzyme galactocerebrosidase (GALC). This enzyme leads to the accumulation of galactosylceramide, psychosine, and secondary substrates such as lactosylceramide. In this paper, we induced autophagy through starvation and examined the cellular response occurring in fibroblasts isolated from patients. We demonstrated that the inhibitory AKT-mediated phosphorylation of beclin-1 and the BCL2-beclin-1 complex concur to reduce autophagosomes formation in response to starvation. These events were not dependent on the accumulation of psychosine, which was previously identified as a possible player in autophagic impairment in Krabbe disease. We believe that these data could better elucidate the capability of response to autophagic stimuli in Krabbe disease, in order to identify possible molecules able to stimulate the process.
    Keywords:  BCL2; Krabbe disease; autophagy; beclin-1
    DOI:  https://doi.org/10.3390/ijms24065984
  8. bioRxiv. 2023 Mar 18. pii: 2023.03.17.533015. [Epub ahead of print]
    Reduction of Spermine Synthase Suppresses Tau Accumulation Through Autophagy Modulation in Tauopathy.
    Xianzun Tao, Jiaqi Liu, Zoraida Diaz-Perez, Jackson R Foley, Tracy Murray Stewart, Robert A Casero, R Grace Zhai.
      Tauopathy, including Alzheimer Disease (AD), is characterized by Tau protein accumulation and autophagy dysregulation. Emerging evidence connects polyamine metabolism with the autophagy pathway, however the role of polyamines in Tauopathy remains unclear. In the present study we investigated the role of spermine synthase (SMS) in autophagy regulation and tau protein processing in Drosophila and human cellular models of Tauopathy. Our previous study showed that Drosophila spermine synthase ( dSms ) deficiency impairs lysosomal function and blocks autophagy flux. Interestingly, partial loss-of-function of SMS in heterozygous dSms flies extends lifespan and improves the climbing performance of flies with human Tau (hTau) overexpression. Mechanistic analysis showed that heterozygous loss-of-function mutation of dSms reduces hTau protein accumulation through enhancing autophagic flux. Measurement of polyamine levels detected a mild elevation of spermidine in flies with heterozygous loss of dSms . SMS knock-down in human neuronal or glial cells also upregulates autophagic flux and reduces Tau protein accumulation. Proteomics analysis of postmortem brain tissue from AD patients showed a significant albeit modest elevation of SMS protein level in AD-relevant brain regions compared to that of control brains consistently across several datasets. Taken together, our study uncovers a correlation between SMS protein level and AD pathogenesis and reveals that SMS reduction upregulates autophagy, promotes Tau clearance, and reduces Tau protein accumulation. These findings provide a new potential therapeutic target of Tauopathy.
    DOI:  https://doi.org/10.1101/2023.03.17.533015
  9. EMBO Mol Med. 2023 Mar 29. e16877
    TFEB and TFE3 drive kidney cystogenesis and tumorigenesis.
    Chiara Di Malta, Angela Zampelli, Letizia Granieri, Claudia Vilardo, Rossella De Cegli, Laura Cinque, Edoardo Nusco, Salvatore Pece, Daniela Tosoni, Francesca Sanguedolce, Nicolina Cristina Sorrentino, Maria J Merino, Deborah Nielsen, Ramaprasad Srinivasan, Mark W Ball, Christopher J Ricketts, Cathy D Vocke, Martin Lang, Baktiar Karim, Luisa Lanfrancone, Laura S Schmidt, W Marston Linehan, Andrea Ballabio.
      Birt-Hogg-Dubé (BHD) syndrome is an inherited familial cancer syndrome characterized by the development of cutaneous lesions, pulmonary cysts, renal tumors and cysts and caused by loss-of-function pathogenic variants in the gene encoding the tumor-suppressor protein folliculin (FLCN). FLCN acts as a negative regulator of TFEB and TFE3 transcription factors, master controllers of lysosomal biogenesis and autophagy, by enabling their phosphorylation by the mechanistic Target Of Rapamycin Complex 1 (mTORC1). We have previously shown that deletion of Tfeb rescued the renal cystic phenotype of kidney-specific Flcn KO mice. Using Flcn/Tfeb/Tfe3 double and triple KO mice, we now show that both Tfeb and Tfe3 contribute, in a differential and cooperative manner, to kidney cystogenesis. Remarkably, the analysis of BHD patient-derived tumor samples revealed increased activation of TFEB/TFE3-mediated transcriptional program and silencing either of the two genes rescued tumorigenesis in human BHD renal tumor cell line-derived xenografts (CDXs). Our findings demonstrate in disease-relevant models that both TFEB and TFE3 are key drivers of renal tumorigenesis and suggest novel therapeutic strategies based on the inhibition of these transcription factors.
    Keywords:  BHD; TFE3; TFEB; cysts; kidney cancer
    DOI:  https://doi.org/10.15252/emmm.202216877
  10. J Biomol Struct Dyn. 2023 Mar 30. 1-12
    Developing a comprehensive solution aimed to disrupt LARS1/RagD protein-protein interaction.
    Alexey Raevsky, Oksana Kovalenko, Elijah Bulgakov, Mohsen Sharifi, Dmityi Volochnyuk, Michael Tukalo.
      Aminoacyl-tRNA synthetases are crucial enzymes involved in protein synthesis and various cellular physiological reactions. Aside from their standard role in linking amino acids to the corresponding tRNAs, they also impact protein homeostasis by controlling the level of soluble amino acids within the cell. For instance, leucyl-tRNA synthetase (LARS1) acts as a leucine sensor for the mammalian target of rapamycin complex 1 (mTORC1), and may also function as a probable GTPase-activating protein (GAP) for the RagD subunit of the heteromeric activator of mTORC1. In turn, mTORC1 regulates cellular processes, such as protein synthesis, autophagy, and cell growth, and is implicated in various human diseases including cancer, obesity, diabetes, and neurodegeneration. Hence, inhibitors of mTORC1 or a deregulated mTORC1 pathway may offer potential cancer therapies. In this study, we investigated the structural requirements for preventing the sensing and signal transmission from LARS to mTORC1. Building upon recent studies on mTORC1 regulation activation by leucine, we lay the foundation for the development of chemotherapeutic agents against mTORC1 that can overcome resistance to rapamycin. Using a combination of in-silico approaches to develop and validate an alternative interaction model, discussing its benefits and advancements. Finally, we identified a set of compounds ready for testing to prevent LARS1/RagD protein-protein interactions. We establish a basis for creating chemotherapeutic drugs targeting mTORC1, which can conquer resistance to rapamycin. We utilize in-silico methods to generate and confirm an alternative interaction model, outlining its advantages and improvements, and pinpoint a group of novel substances that can prevent LARS1/RagD interactions.Communicated by Ramaswamy H. Sarma.
    Keywords:  AlphaFold; LARS1; leucyl-tRNA synthetase; mTORC1 signaling; rapamycin; resistance
    DOI:  https://doi.org/10.1080/07391102.2023.2194996
  11. Cells. 2023 Mar 14. pii: 893. [Epub ahead of print]12(6):
    Autophagy Function and Benefits of Autophagy Induction in Models of Spinocerebellar Ataxia Type 3.
    Maxinne Watchon, Luan Luu, Stuart K Plenderleith, Kristy C Yuan, Angela S Laird.
       BACKGROUND: Spinocerebellar ataxia 3 (SCA3, also known as Machado Joseph disease) is a fatal neurodegenerative disease caused by the expansion of the trinucleotide repeat region within the ATXN3/MJD gene. The presence of this genetic expansion results in an ataxin-3 protein containing a polyglutamine repeat region, which renders the ataxin-3 protein aggregation prone. Formation of ataxin-3 protein aggregates is linked with neuronal loss and, therefore, the development of motor deficits.
    METHODS: Here, we investigated whether the autophagy protein quality control pathway, which is important in the process of protein aggregate removal, is impaired in a cell culture and zebrafish model of SCA3.
    RESULTS: We found that SH-SY5Y cells expressing human ataxin-3 containing polyglutamine expansion exhibited aberrant levels of autophagy substrates, including increased p62 and decreased LC3II (following bafilomycin treatment), compared to the controls. Similarly, transgenic SCA3 zebrafish showed signs of autophagy impairment at early disease stages (larval), as well as p62 accumulation at advanced age stages (18 months old). We then examined whether treating with compounds known to induce autophagy activity, would aid removal of human ataxin-3 84Q and improve the swimming of the SCA3 zebrafish larvae. We found that treatment with loperamide, trehalose, rapamycin, and MG132 each improved the swimming of the SCA3 zebrafish compared to the vehicle-treated controls.
    CONCLUSION: We propose that signs of autophagy impairment occur in the SH-SY5Y model of SCA3 and SCA3 zebrafish at larval and advanced age stages. Treatment of the larval SCA3 zebrafish with various compounds with autophagy induction capacity was able to produce the improved swimming of the zebrafish, suggesting the potential benefit of autophagy-inducing compounds for the treatment of SCA3.
    Keywords:  autophagy; polyglutamine; zebrafish
    DOI:  https://doi.org/10.3390/cells12060893
  12. Cells. 2023 Mar 21. pii: 947. [Epub ahead of print]12(6):
    Endothelial Autophagy Dysregulation in Diabetes.
    Yann Salemkour, Olivia Lenoir.
      Diabetes mellitus is a major public health issue that affected 537 million people worldwide in 2021, a number that is only expected to increase in the upcoming decade. Diabetes is a systemic metabolic disease with devastating macro- and microvascular complications. Endothelial dysfunction is a key determinant in the pathogenesis of diabetes. Dysfunctional endothelium leads to vasoconstriction by decreased nitric oxide bioavailability and increased expression of vasoconstrictor factors, vascular inflammation through the production of pro-inflammatory cytokines, a loss of microvascular density leading to low organ perfusion, procoagulopathy, and/or arterial stiffening. Autophagy, a lysosomal recycling process, appears to play an important role in endothelial cells, ensuring endothelial homeostasis and functions. Previous reports have provided evidence of autophagic flux impairment in patients with type I or type II diabetes. In this review, we report evidence of endothelial autophagy dysfunction during diabetes. We discuss the mechanisms driving endothelial autophagic flux impairment and summarize therapeutic strategies targeting autophagy in diabetes.
    Keywords:  autophagy; diabetes; endothelial cells
    DOI:  https://doi.org/10.3390/cells12060947
  13. Biology (Basel). 2023 Feb 26. pii: 373. [Epub ahead of print]12(3):
    The Role of Autophagy in the Female Reproduction System: For Beginners to Experts in This Field.
    Akitoshi Nakashima, Atsushi Furuta, Kiyotaka Yamada, Mihoko Yoshida-Kawaguchi, Akemi Yamaki-Ushijima, Ippei Yasuda, Masami Ito, Satoshi Yamashita, Sayaka Tsuda, Satoshi Yoneda, Shibin Cheng, Surendra Sharma, Tomoko Shima.
      Autophagy is a fundamental process involved in regulating cellular homeostasis. Autophagy has been classically discovered as a cellular process that degrades cytoplasmic components non-selectively to produce energy. Over the past few decades, this process has been shown to work in energy production, as well as in the reduction of excessive proteins, damaged organelles, and membrane trafficking. It contributes to many human diseases, such as neurodegenerative diseases, carcinogenesis, diabetes mellitus, development, longevity, and reproduction. In this review, we provide important information for interpreting results related to autophagic experiments and present the role of autophagy in this field.
    Keywords:  ER stress; LC3; NBR1; TFEB; autophagy; autophagy flux; decidualization; oxidative stress; p62; syncytiotrophoblasts
    DOI:  https://doi.org/10.3390/biology12030373
  14. Sports Med Health Sci. 2023 Mar;5(1): 10-19
    The skeletal muscle fiber periphery: A nexus of mTOR-related anabolism.
    Cassidy T Tinline-Goodfellow, Matthew J Lees, Nathan Hodson.
      Skeletal muscle anabolism is driven by numerous stimuli such as growth factors, nutrients (i.e., amino acids, glucose), and mechanical stress. These stimuli are integrated by the mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) signal transduction cascade. In recent years, work from our laboratory and elsewhere has sought to unravel the molecular mechanisms underpinning the mTOR-related activation of muscle protein synthesis (MPS), as well as the spatial regulation of these mechanisms within the skeletal muscle cell. These studies have suggested that the skeletal muscle fiber periphery is a region of central importance in anabolism (i.e., growth/MPS). Indeed, the fiber periphery is replete with the substrates, molecular machinery, and translational apparatus necessary to facilitate MPS. This review provides a summary of the mechanisms underpinning the mTOR-associated activation of MPS from cell, rodent, and human studies. It also presents an overview of the spatial regulation of mTORC1 in response to anabolic stimuli and outlines the factors that distinguish the periphery of the cell as a highly notable region of skeletal muscle for the induction of MPS. Future research should seek to further explore the nutrient-induced activation of mTORC1 at the periphery of skeletal muscle fibers.
    Keywords:  Hypertrophy; Muscle protein synthesis; Periphery; Skeletal muscle; Translation; mTOR
    DOI:  https://doi.org/10.1016/j.smhs.2022.11.004
  15. Antioxidants (Basel). 2023 Mar 02. pii: 613. [Epub ahead of print]12(3):
    Enhanced Levels of Peroxisome-Derived H2O2 Do Not Induce Pexophagy but Impair Autophagic Flux in HEK-293 and HeLa Cells.
    Hongli Li, Celien Lismont, Cláudio F Costa, Mohamed A F Hussein, Myriam Baes, Marc Fransen.
      Peroxisomes are functionally specialized organelles that harbor multiple hydrogen peroxide (H2O2)-producing and -degrading enzymes. Given that this oxidant functions as a major redox signaling agent, peroxisomes have the intrinsic ability to mediate and modulate H2O2-driven processes, including autophagy. However, it remains unclear whether changes in peroxisomal H2O2 (po-H2O2) emission impact the autophagic process and to which extent peroxisomes with a disturbed H2O2 metabolism are selectively eliminated through a process called "pexophagy". To address these issues, we generated and validated HEK-293 and HeLa pexophagy reporter cell lines in which the production of po-H2O2 can be modulated. We demonstrate that (i) po-H2O2 can oxidatively modify multiple selective autophagy receptors and core autophagy proteins, (ii) neither modest nor robust levels of po-H2O2 emission act as a prime determinant of pexophagy, and (iii) high levels of po-H2O2 impair autophagic flux by oxidative inhibition of enzymes involved in LC3II formation. Unexpectedly, our analyses also revealed that the autophagy receptor optineurin can be recruited to peroxisomes, thereby triggering pexophagy. In summary, these findings lend support to the idea that, during cellular and organismal aging, peroxisomes with enhanced H2O2 release can escape pexophagy and downregulate autophagic activity, thereby perpetuating the accumulation of damaged and toxic cellular debris.
    Keywords:  autophagy; hydrogen peroxide; mKeima; optineurin; oxidative stress; peroxisomes; pexophagy
    DOI:  https://doi.org/10.3390/antiox12030613
  16. J Autoimmun. 2023 Mar 29. pii: S0896-8411(23)00039-2. [Epub ahead of print]136 103030
    Autophagy pathways in autoimmune diseases.
    Christian W Keller, Iannis E Adamopoulos, Jan D Lünemann.
      Autophagy comprises a growing range of cellular pathways, which occupy central roles in response to energy deprivation, organelle turnover and proteostasis. Over the years, autophagy has been increasingly linked to governing several aspects of immunity, including host defence against various pathogens, unconventional secretion of cytokines and antigen presentation. While canonical autophagy-mediated antigen processing in thymic epithelial cells supports the generation of a self-tolerant CD4+ T cell repertoire, mounting evidence suggests that deregulated autophagy pathways contribute to or sustain autoimmune responses. In animal models of multiple sclerosis (MS), non-canonical autophagy pathways such as microtubule-associated protein 1 A/1 B-light chain 3 (LC3)-associated phagocytosis can contribute to major histocompatibility complex (MHC) class II presentation of autoantigen, thereby amplifying autoreactive CD4+ T cell responses. In systemic lupus erythematosus (SLE), increased type 1 interferon production is linked to excessive autophagy in plasmacytoid dendritic cells (DCs). In rheumatoid arthritis (RA), autophagy proteins contribute to pathological citrullination of autoantigen. Immunotherapies effective in autoimmune diseases modulate autophagy functions, and strategies harnessing autophagy pathways to restrain autoimmune responses have been developed. This review illustrates recent insights in how autophagy, distinct autophagy pathways and autophagy protein functions intersect with the evolution and progression of autoimmune diseases, focusing on MS, SLE and RA.
    Keywords:  Antigen presentation; Autoimmunity; Autophagy; Multiple sclerosis; Rheumatoid arthritis; Systemic lupus erythematosus
    DOI:  https://doi.org/10.1016/j.jaut.2023.103030
  17. Int J Mol Sci. 2023 Mar 17. pii: 5785. [Epub ahead of print]24(6):
    The Drp1-Mediated Mitochondrial Fission Protein Interactome as an Emerging Core Player in Mitochondrial Dynamics and Cardiovascular Disease Therapy.
    Mulate Zerihun, Surya Sukumaran, Nir Qvit.
      Mitochondria, the membrane-bound cell organelles that supply most of the energy needed for cell function, are highly regulated, dynamic organelles bearing the ability to alter both form and functionality rapidly to maintain normal physiological events and challenge stress to the cell. This amazingly vibrant movement and distribution of mitochondria within cells is controlled by the highly coordinated interplay between mitochondrial dynamic processes and fission and fusion events, as well as mitochondrial quality-control processes, mainly mitochondrial autophagy (also known as mitophagy). Fusion connects and unites neighboring depolarized mitochondria to derive a healthy and distinct mitochondrion. In contrast, fission segregates damaged mitochondria from intact and healthy counterparts and is followed by selective clearance of the damaged mitochondria via mitochondrial specific autophagy, i.e., mitophagy. Hence, the mitochondrial processes encompass all coordinated events of fusion, fission, mitophagy, and biogenesis for sustaining mitochondrial homeostasis. Accumulated evidence strongly suggests that mitochondrial impairment has already emerged as a core player in the pathogenesis, progression, and development of various human diseases, including cardiovascular ailments, the leading causes of death globally, which take an estimated 17.9 million lives each year. The crucial factor governing the fission process is the recruitment of dynamin-related protein 1 (Drp1), a GTPase that regulates mitochondrial fission, from the cytosol to the outer mitochondrial membrane in a guanosine triphosphate (GTP)-dependent manner, where it is oligomerized and self-assembles into spiral structures. In this review, we first aim to describe the structural elements, functionality, and regulatory mechanisms of the key mitochondrial fission protein, Drp1, and other mitochondrial fission adaptor proteins, including mitochondrial fission 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics 49 (Mid49), and mitochondrial dynamics 51 (Mid51). The core area of the review focuses on the recent advances in understanding the role of the Drp1-mediated mitochondrial fission adaptor protein interactome to unravel the missing links of mitochondrial fission events. Lastly, we discuss the promising mitochondria-targeted therapeutic approaches that involve fission, as well as current evidence on Drp1-mediated fission protein interactions and their critical roles in the pathogeneses of cardiovascular diseases (CVDs).
    Keywords:  cardiovascular diseases (CVDs); dynamin-related protein 1 (Drp1); fission; fusion; mitochondrial dynamics; mitochondrial dynamics 49 (Mid49); mitochondrial dynamics 51 (Mid51); mitochondrial fission 1 (Fis1); mitochondrial fission factor (Mff); mitochondrial fission proteins; mitophagy; protein structure; protein–protein interactions (PPIs)
    DOI:  https://doi.org/10.3390/ijms24065785
  18. Biochim Biophys Acta Gene Regul Mech. 2023 Mar 27. pii: S1874-9399(23)00029-9. [Epub ahead of print] 194934
    The N-degron pathway: From basic science to therapeutic applications.
    Ah Jung Heo, Su Bin Kim, Yong Tae Kwon, Chang Hoon Ji.
      The N-degron pathway is a degradative system in which single N-terminal (Nt) amino acids regulate the half-lives of proteins and other biological materials. These determinants, called N-degrons, are recognized by N-recognins that link them to the ubiquitin (Ub)-proteasome system (UPS) or autophagy-lysosome system (ALS). In the UPS, the Arg/N-degron pathway targets the Nt-arginine (Nt-Arg) and other N-degrons to assemble Lys48 (K48)-linked Ub chains by UBR box N-recognins for proteasomal proteolysis. In the ALS, Arg/N-degrons are recognized by the N-recognin p62/SQSTSM-1/Sequestosome-1 to induce cis-degradation of substrates and trans-degradation of various cargoes such as protein aggregates and subcellular organelles. This crosstalk between the UPS and ALP involves reprogramming of the Ub code. Eukaryotic cells developed diverse ways to target all 20 principal amino acids for degradation. Here we discuss the components, regulation, and functions of the N-degron pathways, with an emphasis on the basic mechanisms and therapeutic applications of Arg/N-degrons and N-recognins.
    Keywords:  Autophagy-lysosome system; E3 ligase; N-recognin; Nt-arginylation; The N-degron pathway; Ubiquitin-proteasome system
    DOI:  https://doi.org/10.1016/j.bbagrm.2023.194934
  19. bioRxiv. 2023 Mar 24. pii: 2023.03.22.533675. [Epub ahead of print]
    A Comprehensive Enumeration of the Human Proteostasis Network. 2. Components of the Autophagy-Lysosome Pathway.
    Proteostasis Consortium
      The condition of having a healthy, functional proteome is known as protein homeostasis, or proteostasis. Establishing and maintaining proteostasis is the province of the proteostasis network, approximately 2,700 components that regulate protein synthesis, folding, localization, and degradation. The proteostasis network is a fundamental entity in biology that is essential for cellular health and has direct relevance to many diseases of protein conformation. However, it is not well defined or annotated, which hinders its functional characterization in health and disease. In this series of manuscripts, we aim to operationally define the human proteostasis network by providing a comprehensive, annotated list of its components. We provided in a previous manuscript a list of chaperones and folding enzymes as well as the components that make up the machineries for protein synthesis, protein trafficking into and out of organelles, and organelle-specific degradation pathways. Here, we provide a curated list of 838 unique high-confidence components of the autophagy-lysosome pathway, one of the two major protein degradation systems in human cells.
    DOI:  https://doi.org/10.1101/2023.03.22.533675
  20. Int J Mol Sci. 2023 Mar 15. pii: 5593. [Epub ahead of print]24(6):
    NAADP-Evoked Ca2+ Signaling Leads to Mutant Huntingtin Aggregation and Autophagy Impairment in Murine Astrocytes.
    Cássia Arruda de Souza Pereira, Natalia de Castro Medaglia, Rodrigo Portes Ureshino, Claudia Bincoletto, Manuela Antonioli, Gian Maria Fimia, Mauro Piacentini, Gustavo José da Silva Pereira, Adolfo Garcia Erustes, Soraya Soubhi Smaili.
      Huntington's disease (HD) is a progressive neurodegenerative disease characterized by mutations in the huntingtin gene (mHtt), causing an unstable repeat of the CAG trinucleotide, leading to abnormal long repeats of polyglutamine (poly-Q) in the N-terminal region of the huntingtin, which form abnormal conformations and aggregates. Alterations in Ca2+ signaling are involved in HD models and the accumulation of mutated huntingtin interferes with Ca2+ homeostasis. Lysosomes are intracellular Ca2+ storages that participate in endocytic and lysosomal degradation processes, including autophagy. Nicotinic acid adenine dinucleotide phosphate (NAADP) is an intracellular second messenger that promotes Ca2+ release from the endo-lysosomal system via Two-Pore Channels (TPCs) activation. Herein, we show the impact of lysosomal Ca2+ signals on mHtt aggregation and autophagy blockade in murine astrocytes overexpressing mHtt-Q74. We observed that mHtt-Q74 overexpression causes an increase in NAADP-evoked Ca2+ signals and mHtt aggregation, which was inhibited in the presence of Ned-19, a TPC antagonist, or BAPTA-AM, a Ca2+ chelator. Additionally, TPC2 silencing revert the mHtt aggregation. Furthermore, mHtt has been shown co-localized with TPC2 which may contribute to its effects on lysosomal homeostasis. Moreover, NAADP-mediated autophagy was also blocked since its function is dependent on lysosomal functionality. Taken together, our data show that increased levels of cytosolic Ca2+ mediated by NAADP causes mHtt aggregation. Additionally, mHtt co-localizes with the lysosomes, where it possibly affects organelle functions and impairs autophagy.
    Keywords:  Huntington’s disease; NAADP; lysosome; two-pore channels
    DOI:  https://doi.org/10.3390/ijms24065593
  21. Curr Mol Pharmacol. 2023 Mar 31.
    Mammalian Target of Rapamycin (mTOR) Signalling Pathway a Potential Target for Cancer Intervention: a Short Overview.
    Rajesh Basnet, Buddha Bahadur Basnet, Radheshyam Gupta, TilBahadur Basnet, Sandhya Khadka, Md Shan Alam.
       BACKGROUND: The mammalian role of the rapamycin (mTOR) pathway is the practical nutrient-sensitive regulation of animal growth and plays a central role in physiology, metabolism, and common diseases. The mTOR is activated in response to nutrients, growth factors, and cellular energy. The mTOR pathway activates in various cellular processes and human cancer diseases. Dysfunction of mTOR signal transduction is associated with metabolic disorders, cancer for instance.
    OBJECTIVE: In recent years, significant achievements envisaged in developing targeted drugs for cancer. The global impact of cancer continues to grow. However, the focus of disease-modifying therapies remains elusive. The mTOR is a significant target in cancer to be considered for mTOR inhibitors, even though the costs are high. Despite many mTOR inhibitors, potent, selective inhibitors for mTOR are still limited. Therefore, in this review, the mTOR structure and protein-ligand interactions of utmost importance to provide the basis for molecular modelling and structure-based drug design are discussed.
    CONCLUSION: This review introduces the mTOR, its crystal structure, and the latest research on mTOR.Besides, the role of mTOR in cancer, its function, and its regulation are reviewed. In addition, the mechanistic role of mTOR signalling networks in cancer and interaction with drugs that inhibit the development of mTOR and crystal structures of mTOR and its complexes are explored. Finally, the current status and prospects of mTOR-targeted therapy are addressed.
    Keywords:  Cancer; Cell growth; Crystal structure; PIK3; mTOR; mTOR 2; mTOR1
    DOI:  https://doi.org/10.2174/1874467217666230331081959
  22. EMBO J. 2023 Mar 27. e111980
    KNO1-mediated autophagic degradation of the Bloom syndrome complex component RMI1 promotes homologous recombination.
    Poyu Chen, Nancy De Winne, Geert De Jaeger, Masaki Ito, Maren Heese, Arp Schnittger.
      Homologous recombination (HR) is a key DNA damage repair pathway that is tightly adjusted to the state of a cell. A central regulator of homologous recombination is the conserved helicase-containing Bloom syndrome complex, renowned for its crucial role in maintaining genome integrity. Here, we show that in Arabidopsis thaliana, Bloom complex activity is controlled by selective autophagy. We find that the recently identified DNA damage regulator KNO1 facilitates K63-linked ubiquitination of RMI1, a structural component of the complex, thereby triggering RMI1 autophagic degradation and resulting in increased homologous recombination. Conversely, reduced autophagic activity makes plants hypersensitive to DNA damage. KNO1 itself is also controlled at the level of proteolysis, in this case mediated by the ubiquitin-proteasome system, becoming stabilized upon DNA damage via two redundantly acting deubiquitinases, UBP12 and UBP13. These findings uncover a regulatory cascade of selective and interconnected protein degradation steps resulting in a fine-tuned HR response upon DNA damage.
    Keywords:  DNA damage response; autophagy; homologous recombination; proteasome; ubiquitination
    DOI:  https://doi.org/10.15252/embj.2022111980
  23. Cell Rep. 2023 Mar 29. pii: S2211-1247(23)00327-3. [Epub ahead of print]42(4): 112316
    CDK5-PRMT1-WDR24 signaling cascade promotes mTORC1 signaling and tumor growth.
    Shasha Yin, Liu Liu, Lauren E Ball, Yalong Wang, Mark T Bedford, Stephen A Duncan, Haizhen Wang, Wenjian Gan.
      The mammalian target of rapamycin complex1 (mTORC1) is a central regulator of metabolism and cell growth by sensing diverse environmental signals, including amino acids. The GATOR2 complex is a key component linking amino acid signals to mTORC1. Here, we identify protein arginine methyltransferase 1 (PRMT1) as a critical regulator of GATOR2. In response to amino acids, cyclin-dependent kinase 5 (CDK5) phosphorylates PRMT1 at S307 to promote PRMT1 translocation from nucleus to cytoplasm and lysosome, which in turn methylates WDR24, an essential component of GATOR2, to activate the mTORC1 pathway. Disruption of the CDK5-PRMT1-WDR24 axis suppresses hepatocellular carcinoma (HCC) cell proliferation and xenograft tumor growth. High PRMT1 protein expression is associated with elevated mTORC1 signaling in patients with HCC. Thus, our study dissects a phosphorylation- and arginine methylation-dependent regulatory mechanism of mTORC1 activation and tumor growth and provides a molecular basis to target this pathway for cancer therapy.
    Keywords:  CDK5; CP: Cancer; CP: Molecular biology; GATOR2; HCC; PRMT1; WDR24; amino acids; arginine methylation; mTORC1
    DOI:  https://doi.org/10.1016/j.celrep.2023.112316
  24. Mar Drugs. 2023 Feb 25. pii: 151. [Epub ahead of print]21(3):
    RIP1 Mediates Manzamine-A-Induced Secretory Autophagy in Breast Cancer.
    Xuan Wang, Yuanpeng Liu, Huan Qin, Guocui Qi, Xuehong Chen, Yi Lyu, Yantao Han.
      Cancer-derived small extracellular vesicles (sEVs) serve as critical mediators of cell-to-cell communication. Manzamine A (MA), a unique marine-derived alkaloid with various bioactivities, exerts anticancer effects against several kinds of tumors, but it remains unclear whether it has the same activity against breast cancer. Here, we proved that MA inhibits MDA-MB-231 and MCF-7 cell proliferation, migration, and invasion in a time- and dose-dependent manner. In addition, MA promotes autophagosome formation but suppresses autophagosome degradation in breast cancer cells. Importantly, we also found that MA stimulates sEVs secretion and increases autophagy-related protein accumulation in secreted sEVs, further potentiated by autophagy inhibitor chloroquine (CQ). Mechanistically, MA decreases the expression level of RIP1, the key upstream regulator of the autophagic pathway, and reduces the acidity of lysosome. Overexpression of RIP1 activated AKT/mTOR signaling, thus attenuating MA-induced autophagy and the corresponding secretion of autophagy-associated sEVs. Collectively, these data suggested that MA is a potential inhibitor of autophagy by preventing autophagosome turnover, and RIP1 mediates MA-induced secretory autophagy, which may be efficacious for breast cancer treatment.
    Keywords:  RIP1; breast cancer; manzamine A; sEVs; secretory autophagy
    DOI:  https://doi.org/10.3390/md21030151
  25. Toxins (Basel). 2023 Mar 06. pii: 202. [Epub ahead of print]15(3):
    Differential Role of Active Compounds in Mitophagy and Related Neurodegenerative Diseases.
    Mark Makarov, Eduard Korkotian.
      Neurodegenerative diseases, such as Alzheimer's disease or Parkinson's disease, significantly reduce the quality of life of patients and eventually result in complete maladjustment. Disruption of the synapses leads to a deterioration in the communication of nerve cells and decreased plasticity, which is associated with a loss of cognitive functions and neurodegeneration. Maintaining proper synaptic activity depends on the qualitative composition of mitochondria, because synaptic processes require sufficient energy supply and fine calcium regulation. The maintenance of the qualitative composition of mitochondria occurs due to mitophagy. The regulation of mitophagy is usually based on several internal mechanisms, as well as on signals and substances coming from outside the cell. These substances may directly or indirectly enhance or weaken mitophagy. In this review, we have considered the role of some compounds in process of mitophagy and neurodegeneration. Some of them have a beneficial effect on the functions of mitochondria and enhance mitophagy, showing promise as novel drugs for the treatment of neurodegenerative pathologies, while others contribute to a decrease in mitophagy.
    Keywords:  T-2 toxin; autophagy; haloperidol; mitophagy; neurodegeneration; niclosamide; quercetin; resveratrol; sevoflurane; spermidine; urolithin
    DOI:  https://doi.org/10.3390/toxins15030202
  26. Cell Biosci. 2023 Mar 30. 13(1): 67
    PSMD2 contributes to the progression of esophageal squamous cell carcinoma by repressing autophagy.
    Yachen Liu, Meng Wu, Shuxiang Xu, Xiangjie Niu, Weiling Liu, Chuanwang Miao, Ai Lin, Yang Xu, Lili Yu.
       BACKGROUND: The ubiquitin-proteasome and autophagy-lysosomal systems collaborate in regulating the levels of intracellular proteins. Dysregulation of protein homeostasis is a central feature of malignancy. The gene encoding 26S proteasome non-ATPase regulatory subunit 2 (PSMD2) of the ubiquitin-proteasome system is an oncogene in various types of cancer. However, the detailed role of PSMD2 in autophagy and its relationship to tumorigenesis in esophageal squamous cell carcinoma (ESCC) remain unknown. In the present study, we have investigated the tumor-promoting roles of PSMD2 in the context of autophagy in ESCC.
    METHODS: Molecular approaches including DAPgreen staining, 5-Ethynyl-2'-deoxyuridine (EdU), cell counting kit 8 (CCK8), colony formation, transwell assays, and cell transfection, xenograft model, immunoblotting and Immunohistochemical analysis were used to investigate the roles of PSMD2 in ESCC cells. Data-independent acquisition (DIA) quantification proteomics analysis and rescue experiments were used to study the roles of PSMD2 in ESCC cells.
    RESULTS: We demonstrate that the overexpression of PSMD2 promotes ESCC cell growth by inhibiting autophagy and is correlated with tumor progression and poor prognosis of ESCC patients. DIA quantification proteomics analysis shows a significant positive correlation between argininosuccinate synthase 1 (ASS1) and PSMD2 levels in ESCC tumors. Further studies indicate that PSMD2 activates the mTOR pathway by upregulating ASS1 to inhibit autophagy.
    CONCLUSIONS: PSMD2 plays an important role in repressing autophagy in ESCC, and represents a promising biomarker to predict prognosis and a therapeutic target of ESCC patients.
    Keywords:  ASS1; Autophagy; Esophageal squamous cell carcinoma; PSMD2; Proliferation; Proteomics
    DOI:  https://doi.org/10.1186/s13578-023-01016-4
  27. Curr Issues Mol Biol. 2023 Mar 13. 45(3): 2381-2392
    A Feedback Loop between TGF-β1 and ATG5 Mediated by miR-122-5p Regulates Fibrosis and EMT in Human Trabecular Meshwork Cells.
    Munmun Chakraborthy, Aparna Rao.
      Autophagy is a cell's evolutionary conserved process for degrading and recycling cellular proteins and removing damaged organelles. There has been an increasing interest in identifying the basic cellular mechanism of autophagy and its implications in health and illness during the last decade. Many proteinopathies such as Alzheimer's and Huntington's disease are reported to be associated with impaired autophagy. The functional significance of autophagy in exfoliation syndrome/exfoliation glaucoma (XFS/XFG), remains unknown though it is presumed to be impaired autophagy to be responsible for the aggregopathy characteristic of this disease. In the current study we have shown that autophagy or ATG5 is enhanced in response to TGF-β1 in human trabecular meshwork (HTM) cells and TGF-β1 induced autophagy is necessary for increased expression of profibrotic proteins and epithelial to mesenchymal (EMT) through Smad3 that lead to aggregopathy. Inhibition of ATG5 by siRNA mediated knockdown reduced profibrotic and EMT markers and increased protein aggregates in the presence of TGF-β1 stimulation. The miR-122-5p, which was increased upon TGF exposure, was also reduced upon ATG5 inhibition. We thus conclude that TGF-β1 induces autophagy in primary HTM cells and a positive feedback loop exists between TGF-β1 and ATG5 that regulated TGF downstream effects mainly mediated by Smad3 signaling with miR-122-5p also playing a role.
    Keywords:  ATG5; TGF-β1; feedback loop; miR-122-5p
    DOI:  https://doi.org/10.3390/cimb45030154
  28. Int J Mol Sci. 2023 Mar 13. pii: 5465. [Epub ahead of print]24(6):
    Tactics with Prebiotics for the Treatment of Metabolic Dysfunction-Associated Fatty Liver Disease via the Improvement of Mitophagy.
    Ai Tsuji, Sayuri Yoshikawa, Yuka Ikeda, Kurumi Taniguchi, Haruka Sawamura, Sae Morikawa, Moeka Nakashima, Tomoko Asai, Satoru Matsuda.
      Mitophagy/autophagy plays a protective role in various forms of liver damage, by renovating cellular metabolism linking to sustain liver homeostasis. A characterized pathway for mitophagy is the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1)/Parkin-dependent signaling pathway. In particular, PINK1-mediated mitophagy could play an indispensable role in improving the metabolic dysfunction-associated fatty liver disease (MAFLD) which could precede to steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma. In addition, the PI3K/AKT/mTOR pathway might regulate the various characteristics of cellular homeostasis including energy metabolism, cell proliferation, and/or cell protection. Therefore, targeting mitophagy with the alteration of PI3K/AKT/mTOR or PINK1/Parkin-dependent signaling to eliminate impaired mitochondria might be an attractive strategy for the treatment of MAFLD. In particular, the efficacy of prebiotics for the treatment of MAFLD has been suggested to be useful via the modulation of the PI3K/AKT/mTOR/AMPK pathway. Additionally, several edible phytochemicals could activate mitophagy for the improvement of mitochondrial damages, which could also be a promising option to treat MAFLD with providing liver protection. Here, the potential therapeutics with several phytochemicals has been discussed for the treatment of MAFLD. Tactics with a viewpoint of prospective probiotics might contribute to the development of therapeutic interventions.
    Keywords:  AMPK; MAFLD; NAFLD; PI3K/AKT signaling pathway; ROS; mTOR; mitophagy; prebiotics
    DOI:  https://doi.org/10.3390/ijms24065465
  29. Cells. 2023 Mar 21. pii: 956. [Epub ahead of print]12(6):
    Crosstalk between Autophagy and RLR Signaling.
    Po-Yuan Ke.
      Autophagy plays a homeostatic role in regulating cellular metabolism by degrading unwanted intracellular materials and acts as a host defense mechanism by eliminating infecting pathogens, such as viruses. Upon viral infection, host cells often activate retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling to induce the transcription of type I interferons, thus establishing the first line of the innate antiviral response. In recent years, numerous studies have shown that virus-mediated autophagy activation may benefit viral replication through different actions on host cellular processes, including the modulation of RLR-mediated innate immunity. Here, an overview of the functional molecules and regulatory mechanism of the RLR antiviral immune response as well as autophagy is presented. Moreover, a summary of the current knowledge on the biological role of autophagy in regulating RLR antiviral signaling is provided. The molecular mechanisms underlying the crosstalk between autophagy and RLR innate immunity are also discussed.
    Keywords:  RLR; antiviral response; autophagy; innate immunity; selective autophagy
    DOI:  https://doi.org/10.3390/cells12060956
  30. Autophagy. 2023 Mar 30.
    Autophagy is the key to making chronic wounds acute in skin wound healing.
    Dalibor Mijaljica, Fabrizio Spada, Daniel J Klionsky, Ian P Harrison.
      As a highly regulated and dynamically balanced intracellular degradation mechanism, macroautophagy/autophagy plays an essential housekeeping role in different successive stages of skin wound healing; from the homeostasis and inflammatory stages to the proliferative and remodeling stages. Under both progressive and defective skin wound healing conditions, autophagy operates at different levels with a precise extent of activity, at the interface of inflammation, stress signaling and cell metabolism through a complex spatiotemporal cascade of molecular and cellular events. Depending on the wound healing conditions autophagic activity is fine-tuned and differentially modulated at each stage of skin wound healing in order to cope with stage-specific requirements. Here, we postulate that under favorable conditions autophagy may act as the key modulator of skin wound healing by making chronic wounds acute. Enhancing autophagy through the topical application of pro-autophagy biologics in an appropriate hydrating vehicle/moisturizing base such as hydrogels, onto a chronic skin wound may provide moisture and immune modulation, thus contributing to rapid and efficient skin wound healing. A moist environment is more conducive to skin wound healing as it helps to not only accelerate cell proliferation and migration, and extracellular matrix reorganization, but also promotes autophagy and reduces the incidence of inflammation.
    Keywords:  Acute wound; autophagy modulation; chronic wound; hydration; hydrogel; immunomodulation
    DOI:  https://doi.org/10.1080/15548627.2023.2194155
  31. Basic Clin Pharmacol Toxicol. 2023 Apr 01.
    The adhesion G-protein-coupled receptor VLGR1/ADGRV1 controls autophagy.
    Joshua Linnert, Baran E Güler, Jacek Krzysko, Uwe Wolfrum.
      VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest known adhesion G protein-coupled receptor. Mutations in VLGR1/ADGRV1 cause Usher syndrome (USH), the most common form of hereditary deaf-blindness, and have been additionally linked to epilepsy. Although VLGR1/ADGRV1 is almost ubiquitously expressed, little is known about the subcellular function and signalling of the VLGR1 protein and thus about mechanisms underlying the development of diseases. Using affinity proteomics, we identified key components of autophagosomes as putative interacting proteins of VLGR1. In addition, whole transcriptome sequencing of the retinae of the Vlgr1/del7TM mouse model revealed altered expression profiles of gene-related autophagy. Monitoring autophagy by immunoblotting and immunocytochemistry of the LC3 and p62 as autophagy marker proteins revealed evoked autophagy in VLGR1-deficient hTERT-RPE1 cells and USH2C patient-derived fibroblasts. Our data demonstrate the molecular and functional interaction of VLGR1 with key components of the autophagy process and point to an essential role of VLGR1 in the regulation of autophagy at internal membranes. The close association of VLGR1 with autophagy helps to explain the pathomechanisms underlying human USH and epilepsy-related to VLGR1 defects.
    Keywords:  Adhesion GPCR; Autophagy; Usher syndrome; affinity proteomics; proteostasis
    DOI:  https://doi.org/10.1111/bcpt.13869
  32. Antioxidants (Basel). 2023 Feb 25. pii: 581. [Epub ahead of print]12(3):
    Progranulin Deficiency Induces Mitochondrial Dysfunction in Frontotemporal Lobar Degeneration with TDP-43 Inclusions.
    Guiomar Rodríguez-Periñán, Ana de la Encarnación, Fermín Moreno, Adolfo López de Munain, Ana Martínez, Ángeles Martín-Requero, Carolina Alquézar, Fernando Bartolomé.
      Loss-of-function (LOF) mutations in GRN gene, which encodes progranulin (PGRN), cause frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). FTLD-TDP is one of the most common forms of early onset dementia, but its pathogenesis is not fully understood. Mitochondrial dysfunction has been associated with several neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Here, we have investigated whether mitochondrial alterations could also contribute to the pathogenesis of PGRN deficiency-associated FTLD-TDP. Our results showed that PGRN deficiency induced mitochondrial depolarization, increased ROS production and lowered ATP levels in GRN KD SH-SY5Y neuroblastoma cells. Interestingly, lymphoblasts from FTLD-TDP patients carrying a LOF mutation in the GRN gene (c.709-1G > A) also demonstrated mitochondrial depolarization and lower ATP levels. Such mitochondrial damage increased mitochondrial fission to remove dysfunctional mitochondria by mitophagy. Interestingly, PGRN-deficient cells showed elevated mitochondrial mass together with autophagy dysfunction, implying that PGRN deficiency induced the accumulation of damaged mitochondria by blocking its degradation in the lysosomes. Importantly, the treatment with two brain-penetrant CK-1δ inhibitors (IGS-2.7 and IGS-3.27), known for preventing the phosphorylation and cytosolic accumulation of TDP-43, rescued mitochondrial function in PGRN-deficient cells. Taken together, these results suggest that mitochondrial function is impaired in FTLD-TDP associated with LOF GRN mutations and that the TDP-43 pathology linked to PGRN deficiency might be a key mechanism contributing to such mitochondrial dysfunction. Furthermore, our results point to the use of drugs targeting TDP-43 pathology as a promising therapeutic strategy for restoring mitochondrial function in FTLD-TDP and other TDP-43-related diseases.
    Keywords:  FTLD-TDP; autophagy; bioenergetics; mitochondria; mitophagy; progranulin
    DOI:  https://doi.org/10.3390/antiox12030581
  33. NPJ Parkinsons Dis. 2023 Mar 27. 9(1): 44
    The small GTPase Rit2 modulates LRRK2 kinase activity, is required for lysosomal function and protects against alpha-synuclein neuropathology.
    Julia Obergasteiger, Anne-Marie Castonguay, Sara Pizzi, Stefano Magnabosco, Giulia Frapporti, Evy Lobbestael, Veerle Baekelandt, Andrew A Hicks, Peter P Pramstaller, Claude Gravel, Corrado Corti, Martin Lévesque, Mattia Volta.
      In Parkinson's disease (PD) misfolded alpha-synuclein (aSyn) accumulates in the substantia nigra, where dopaminergic neurons are progressively lost. The mechanisms underlying aSyn pathology are still unclear, but they are hypothesized to involve the autophagy-lysosome pathway (ALP). LRRK2 mutations are a major cause of familial and sporadic PD, and LRRK2 kinase activity has been shown to be involved in pS129-aSyn inclusion modulation. We observed selective downregulation of the novel PD risk factor RIT2 in vitro and in vivo. Rit2 overexpression in G2019S-LRRK2 cells rescued ALP abnormalities and diminished aSyn inclusions. In vivo, viral mediated overexpression of Rit2 operated neuroprotection against AAV-A53T-aSyn. Furthermore, Rit2 overexpression prevented the A53T-aSyn-dependent increase of LRRK2 kinase activity in vivo. On the other hand, reduction of Rit2 levels leads to defects in the ALP, similar to those induced by the G2019S-LRRK2 mutation. Our data indicate that Rit2 is required for correct lysosome function, inhibits overactive LRRK2 to ameliorate ALP impairment, and counteracts aSyn aggregation and related deficits. Targeting Rit2 could represent an effective strategy to combat neuropathology in familial and idiopathic PD.
    DOI:  https://doi.org/10.1038/s41531-023-00484-2
  34. Int J Mol Sci. 2023 Mar 10. pii: 5297. [Epub ahead of print]24(6):
    Spermidine Rescues Bioenergetic and Mitophagy Deficits Induced by Disease-Associated Tau Protein.
    Lauren H Fairley, Imane Lejri, Amandine Grimm, Anne Eckert.
      Abnormal tau build-up is a hallmark of Alzheimer's disease (AD) and more than 20 other serious neurodegenerative diseases. Mitochondria are paramount organelles playing a predominant role in cellular bioenergetics, namely by providing the main source of cellular energy via adenosine triphosphate generation. Abnormal tau impairs almost every aspect of mitochondrial function, from mitochondrial respiration to mitophagy. The aim of our study was to investigate the effects of spermidine, a polyamine which exerts neuroprotective effects, on mitochondrial function in a cellular model of tauopathy. Recent evidence identified autophagy as the main mechanism of action of spermidine on life-span prolongation and neuroprotection, but the effects of spermidine on abnormal tau-induced mitochondrial dysfunction have not yet been investigated. We used SH-SY5Y cells stably expressing a mutant form of human tau protein (P301L tau mutation) or cells expressing the empty vector (control cells). We showed that spermidine improved mitochondrial respiration, mitochondrial membrane potential as well as adenosine triphosphate (ATP) production in both control and P301L tau-expressing cells. We also showed that spermidine decreased the level of free radicals, increased autophagy and restored P301L tau-induced impairments in mitophagy. Overall, our findings suggest that spermidine supplementation might represent an attractive therapeutic approach to prevent/counteract tau-related mitochondrial impairments.
    Keywords:  autophagy; bioenergetics; mitochondria; mitophagy; spermidine; tau
    DOI:  https://doi.org/10.3390/ijms24065297
  35. Ageing Res Rev. 2023 Mar 25. pii: S1568-1637(23)00076-4. [Epub ahead of print]87 101917
    The role of mitochondrial quality surveillance in skin aging: Focus on mitochondrial dynamics, biogenesis and mitophagy.
    Chang Zhang, Xingyu Gao, Minghe Li, Xiao Yu, Fanke Huang, Yiming Wang, Yueqi Yan, Haiying Zhang, Yingai Shi, Xu He.
      The skin is the largest organ of the human body and the first line of defense against environmental hazards. Many factors, including internal factors such as natural aging and external factors such as ultraviolet radiation and air pollution, can lead to skin aging. Mitochondria provide sufficient energy to maintain the high-speed turnover capacity of the skin, so the quality control of mitochondria plays an indispensable role in this process. Mitochondrial dynamics, mitochondrial biogenesis and mitophagy are the key steps in mitochondrial quality surveillance. They are coordinated to maintain mitochondrial homeostasis and restore damaged mitochondrial function. All of the mitochondrial quality control processes are related to skin aging caused by various factors. Therefore, fine-tuning regulation of the above process is of great significance to the skin aging problem that needs to be solved urgently. This article mainly reviews the physiological and environmental factors causing skin aging, the effects of mitochondrial dynamics, mitochondrial biogenesis and mitophagy on skin aging, as well as their specific regulatory mechanisms. Finally, mitochondrial biomarkers for diagnosis of skin aging, and therapeutic approaches of skin aging via mitochondrial quality control were illustrated.
    Keywords:  Mitochondrial biogenesis; Mitochondrial dynamics; Mitophagy; Skin aging; Therapeutic
    DOI:  https://doi.org/10.1016/j.arr.2023.101917
  36. Curr Oncol. 2023 Mar 04. 30(3): 3047-3063
    Inhibition of Sphingosine Kinase 2 Results in PARK2-Mediated Mitophagy and Induces Apoptosis in Multiple Myeloma.
    Jian Wu, Shengjun Fan, Daniel Feinberg, Xiaobei Wang, Shaima Jabbar, Yubin Kang.
      Mitophagy plays an important role in maintaining mitochondrial homeostasis by clearing damaged mitochondria. Sphingosine kinase 2 (SK2), a type of sphingosine kinase, is an important metabolic enzyme involved in generating sphingosine-1-phosphate. Its expression level is elevated in many cancers and is associated with poor clinical outcomes. However, the relationship between SK2 and mitochondrial dysfunction remains unclear. We found that the genetic downregulation of SK2 or treatment with ABC294640, a specific inhibitor of SK2, induced mitophagy and apoptosis in multiple myeloma cell lines. We showed that mitophagy correlates with apoptosis induction and likely occurs through the SET/PP2AC/PARK2 pathway, where inhibiting PP2AC activity may rescue this process. Furthermore, we found that PP2AC and PARK2 form a complex, suggesting that they might regulate mitophagy through protein-protein interactions. Our study demonstrates the important role of SK2 in regulating mitophagy and provides new insights into the mechanism of mitophagy in multiple myeloma.
    Keywords:  PARK2; apoptosis; mitophagy; multiple myeloma; sphingosine kinase 2
    DOI:  https://doi.org/10.3390/curroncol30030231
  37. Biomedicines. 2023 Mar 16. pii: 927. [Epub ahead of print]11(3):
    Mechanotransduction Impairment in Primary Fibroblast Model of Krabbe Disease.
    Roberta Mezzena, Ambra Del Grosso, Roberto Maria Pellegrino, Husam B R Alabed, Carla Emiliani, Ilaria Tonazzini, Marco Cecchini.
      Krabbe disease (KD) is a genetic disorder caused by the absence of the galactosylceramidase (GALC) functional enzyme. No cure is currently available. Here, we investigate the mechanotransduction process in primary fibroblasts collected from the twitcher mouse, a natural KD murine model. Thanks to mechanotransduction, cells can sense their environment and convert external mechanical stimuli into biochemical signals that result in intracellular changes. In GALC-deficient fibroblasts, we show that focal adhesions (FAs), the protein clusters necessary to adhere and migrate, are increased, and that single-cell migration and wound healing are impaired. We also investigate the involvement of the autophagic process in this framework. We show a dysregulation in the FA turnover: here, the treatment with the autophagy activator rapamycin boosts cell migration and improves the clearance of FAs in GALC-deficient fibroblasts. We propose mechanosensing impairment as a novel potential pathological mechanism in twitcher fibroblasts, and more in general in Krabbe disease.
    Keywords:  Krabbe disease; autophagy; focal adhesions; mechanotransduction; migration
    DOI:  https://doi.org/10.3390/biomedicines11030927
  38. Int J Mol Sci. 2023 Mar 14. pii: 5525. [Epub ahead of print]24(6):
    Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.
    Taylor C Harned, Radu V Stan, Ze Cao, Rajarshi Chakrabarti, Henry N Higgs, Catherine C Y Chang, Ta Yuan Chang.
      Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD. Additionally, blocking ACAT1/SOAT1 activity stimulates autophagy and lysosomal biogenesis; however, the exact molecular connection between the ACAT1/SOAT1 blockade and these observed benefits remain unknown. Here, using biochemical fractionation techniques, we observe cholesterol accumulation at the MAM which leads to ACAT1/SOAT1 enrichment in this domain. MAM proteomics data suggests that ACAT1/SOAT1 inhibition strengthens the ER-mitochondria connection. Confocal and electron microscopy confirms that ACAT1/SOAT1 inhibition increases the number of ER-mitochondria contact sites and strengthens this connection by shortening the distance between these two organelles. This work demonstrates how directly manipulating local cholesterol levels at the MAM can alter inter-organellar contact sites and suggests that cholesterol buildup at the MAM is the impetus behind the therapeutic benefits of ACAT1/SOAT1 inhibition.
    Keywords:  ACAT inhibitors; ACAT1; Alzheimer’s disease; F12511; K-604; SOAT1; cholesterol; endoplasmic reticulum; lipid metabolism; mitochondria-associated membrane (MAM)
    DOI:  https://doi.org/10.3390/ijms24065525
  39. Cells. 2023 Mar 07. pii: 828. [Epub ahead of print]12(6):
    DRAM1 Promotes Lysosomal Delivery of Mycobacterium marinum in Macrophages.
    Adrianna Banducci-Karp, Jiajun Xie, Sem A G Engels, Christos Sarantaris, Patrick van Hage, Monica Varela, Annemarie H Meijer, Michiel van der Vaart.
      Damage-Regulated Autophagy Modulator 1 (DRAM1) is an infection-inducible membrane protein, whose function in the immune response is incompletely understood. Based on previous results in a zebrafish infection model, we have proposed that DRAM1 is a host resistance factor against intracellular mycobacterial infection. To gain insight into the cellular processes underlying DRAM1-mediated host defence, here we studied the interaction of DRAM1 with Mycobacterium marinum in murine RAW264.7 macrophages. We found that, shortly after phagocytosis, DRAM1 localised in a punctate pattern to mycobacteria, which gradually progressed to full DRAM1 envelopment of the bacteria. Within the same time frame, DRAM1-positive mycobacteria colocalised with the LC3 marker for autophagosomes and LysoTracker and LAMP1 markers for (endo)lysosomes. Knockdown analysis revealed that DRAM1 is required for the recruitment of LC3 and for the acidification of mycobacteria-containing vesicles. A reduction in the presence of LAMP1 further suggested reduced fusion of lysosomes with mycobacteria-containing vesicles. Finally, we show that DRAM1 knockdown impairs the ability of macrophages to defend against mycobacterial infection. Together, these results support that DRAM1 promotes the trafficking of mycobacteria through the degradative (auto)phagolysosomal pathway. Considering its prominent effect on host resistance to intracellular infection, DRAM1 is a promising target for therapeutic modulation of the microbicidal capacity of macrophages.
    Keywords:  DRAM1; innate immunity; lysosomes; macrophages; mycobacterium; phagosomes; vesicle trafficking; xenophagy
    DOI:  https://doi.org/10.3390/cells12060828
  40. bioRxiv. 2023 Mar 14. pii: 2023.03.14.532586. [Epub ahead of print]
    Transcriptomic changes predict metabolic alterations in LC3 associated phagocytosis in aged mice.
    Anuradha Dhingra, John W Tobias, Kathleen Boesze-Battaglia.
      LC3b ( Map1lc3b ) plays an essential role in canonical autophagy and is one of several components of the autophagy machinery that mediates non-canonical autophagic functions. Phagosomes are often associated with lipidated LC3b, to promote phagosome maturation in a process called LC3-associated phagocytosis (LAP). Specialized phagocytes such as mammary epithelial cells, retinal pigment epithelial (RPE) cells, and sertoli cells utilize LAP for optimal degradation of phagocytosed material, including debris. In the visual system, LAP is critical to maintain retinal function, lipid homeostasis and neuroprotection. In a mouse model of retinal lipid steatosis - mice lacking LC3b ( LC3b -/- ), we observed increased lipid deposition, metabolic dysregulation and enhanced inflammation. Herein we present a non-biased approach to determine if loss of LAP mediated processes modulate the expression of various genes related to metabolic homeostasis, lipid handling, and inflammation. A comparison of the RPE transcriptome of WT and LC3b -/- mice revealed 1533 DEGs, with ∼73% up-regulated and 27% down-regulated. Enriched gene ontology (GO) terms included inflammatory response (up-regulated DEGs), fatty acid metabolism and vascular transport (down-regulated DEGs). Gene set enrichment analysis (GSEA) identified 34 pathways; 28 were upregulated (dominated by inflammation/related pathways) and 6 were downregulated (dominated by metabolic pathways). Analysis of additional gene families identified significant differences for genes in the solute carrier family, RPE signature genes, and genes with potential role in age-related macular degeneration. These data indicate that loss of LC3b induces robust changes in the RPE transcriptome contributing to lipid dysregulation and metabolic imbalance, RPE atrophy, inflammation, and disease pathophysiology.
    DOI:  https://doi.org/10.1101/2023.03.14.532586
  41. Front Immunol. 2023 ;14 1120996
    ROS-AMPK/mTOR-dependent enterocyte autophagy is involved in the regulation of Giardia infection-related tight junction protein and nitric oxide levels.
    Jingxue Wu, Yongwu Yang, Lin Liu, Weining Zhu, Min Liu, Xiran Yu, Wei Li.
      Giardia duodenalis, a cosmopolitan noninvasive protozoan parasite of zoonotic concern and public health importance, infects the upper portions of the small intestine and causes one of the most common gastrointestinal diseases globally termed giardiasis, especially in situations lacking safe drinking water and adequate sanitation services. The pathogenesis of giardiasis is complex and involves multiple factors from the interaction of Giardia and intestinal epithelial cells (IECs). Autophagy is an evolutionarily conserved catabolic pathway that involves multiple pathological conditions including infection. Thus far, it remains uncertain if autophagy occurs in Giardia-infected IECs and if autophagic process is associated with the pathogenic factors of giardiasis, such as tight junction (TJ) barrier defects and nitric oxide (NO) release of IECs. Here Giardia-in vitro exposed IECs showed upregulation of a series of autophagy-related molecules, such as LC3, Beclin1, Atg7, Atg16L1, and ULK1, and downregulation of p62 protein. IEC autophagy induced by Giardia was further assessed by using autophagy flux inhibitor, chloroquine (CQ), with the ratio of LC3-II/LC3-I significantly increased and downregulated p62 significantly reversed. Inhibition of autophagy by 3-methyladenine (3-MA) rather than CQ could markedly reverse Giardia-induced downregulation of TJ proteins (claudin-1, claudin-4, occludin, and ZO-1; also known as epithelial cell markers) and NO release, implying the involvement of early-stage autophagy in TJ/NO regulation. We subsequently confirmed the role of ROS-mediated AMPK/mTOR signaling in modulating Giardia-induced autophagy, TJ protein expression, and NO release. In turn, impairment of early-stage autophagy by 3-MA and late-stage autophagy by CQ both exhibited an exacerbated effect on ROS accumulation in IECs. Collectively, we present the first attempt to link the occurrence of IEC autophagy with Giardia infection in vitro, and provides novel insights into the contribution of ROS-AMPK/mTOR-dependent autophagy to Giardia infection-related downregulation of TJ protein and NO levels.
    Keywords:  Giardia; autophagy; nitric oxide; pathogenesis; reactive oxygen species; tight junction
    DOI:  https://doi.org/10.3389/fimmu.2023.1120996
  42. Biomedicines. 2023 Mar 13. pii: 884. [Epub ahead of print]11(3):
    Total Chemical Synthesis of LC3A and LC3B Activity-Based Probes.
    Yara Huppelschoten, Jens Buchardt, Thomas E Nielsen, Aysegul Sapmaz, Gerbrand J van der Heden van Noort.
      Autophagy is a conserved cellular process involved in the degradation of intercellular materials. During this process, double-membrane vesicles called autophagosomes engulf cytoplasmic components ready for degradation. A key component in the formation of autophagosomes are the autophagy-related (Atg) proteins, including microtubule-associated protein light chain 3A (LC3A) and 3B (LC3B). After the C-terminus of LC3 is conjugated to a phospholipid, it promotes the elongation of the phagosome and provides a docking station for the delivery of proteins ready for degradation. Since dysregulation of the autophagy pathway has been associated with a variety of human diseases, components of this process have been considered as potential therapeutic targets. However, the mechanistic details of LC3-specific ligases and deconjugation enzymes are far from unraveled and chemical tools for activity profiling could aid in affording more insights into this process. Herein, we describe a native chemical ligation approach for the synthesis of two LC3 activity-based probes (ABPs). Initial studies show that the probes covalently interact with the cysteine protease ATG4B, showcasing the potential of these probes to unravel mechanistic and structural details.
    Keywords:  activity-based probe; chemical synthesis; protein chemistry
    DOI:  https://doi.org/10.3390/biomedicines11030884
  43. Front Cell Dev Biol. 2023 ;11 1122998
    Determination of the autophagic flux in murine and human peripheral blood mononuclear cells.
    Sophia Walter, Tobias Jung, Catrin Herpich, Kristina Norman, Olga Pivovarova-Ramich, Christiane Ott.
      The autophagy lysosomal system (ALS) is crucial for cellular homeostasis, contributing to maintain whole body health and alterations are associated with diseases like cancer or cardiovascular diseases. For determining the autophagic flux, inhibition of lysosomal degradation is mandatory, highly complicating autophagy measurement in vivo. To overcome this, herein blood cells were used as they are easy and routinely to isolate. Within this study we provide detailed protocols for determination of the autophagic flux in peripheral blood mononuclear cells (PBMCs) isolated from human and, to our knowledge the first time, also from murine whole blood, extensively discussing advantages and disadvantages of both methods. Isolation of PBMCs was performed using density gradient centrifugation. To minimize changes on the autophagic flux through experimental conditions, cells were directly treated with concanamycin A (ConA) for 2 h at 37°C in their serum or for murine cells in serum filled up with NaCl. ConA treatment decreased lysosomal cathepsins activity and increased Sequestosome 1 (SQSTM1) protein and LC3A/B-II:LC3A/B-I ratio in murine PBMCs, while transcription factor EB was not altered yet. Aging further enhanced ConA-associated increase in SQSTM1 protein in murine PBMCs but not in cardiomyocytes, indicating tissue-specific differences in autophagic flux. In human PBMCs, ConA treatment also decreased lysosomal activity and increased LC3A/B-II protein levels, demonstrating successful autophagic flux detection in human subjects. In summary, both protocols are suitable to determine the autophagic flux in murine and human samples and may facilitate a better mechanistic understanding of altered autophagy in aging and disease models and to further develop novel treatment strategies.
    Keywords:  LC3; NZO; TFEB; aging; cardiomyocytes; concanamycin A; lysosomes; sequestome-1
    DOI:  https://doi.org/10.3389/fcell.2023.1122998
  44. Genes Cells. 2023 Mar 25.
    Bcat1 is controlled by Tsc2/mTORC1 pathway at expression levels and its deficiency together with Bcat2 inactivation suppresses the growth of a Tsc2-/- tumor cell line.
    Keiko Nishikawa, Yoshihiro Mezawa, Toshiyuki Kobayashi.
      The tuberous sclerosis complex (TSC) gene products (TSC1/TSC2) negatively regulate mTORC1. Although mTORC1 inhibitors are used for the treatment of TSC, incomplete tumor elimination and the adverse effects from long-term administration are problems that need to be solved. Branched-chain amino acid (BCAA) metabolism is involved in the growth of many tumor cells via the mTORC1 pathway. However, it remains unclear how BCAA metabolism affects the growth of mTORC1-dysregulated tumors. We show here that the expression of branched-chain amino transferase1 (Bcat1) was suppressed in Tsc2-deficient murine renal tumor cells either by treatment with rapamycin or restoration of Tsc2 expression suggesting that Bcat1 is located downstream of Tsc2-mTORC1 pathway. We also found that gabapentin, a Bcat1 inhibitor suppressed the growth of Tsc2-deficient tumor cells and increased efficacy when combined with rapamycin. We investigate the functional importance of Bcat1 and the mitochondrial isoform Bcat2 by inhibiting each enzyme separately or both together by genome editing and shRNA in Tsc2-deficient cells. We found that deficiency of both enzymes, but not either alone, inhibited cell growth, indicating that BCAA-metabolic reactions support Tsc2-deficient cell proliferation. Our results indicate that inhibition of Bcat1 and Bcat2 by specific drugs should be a useful method for TSC treatment.
    Keywords:  BCAA; Bcat1; Bcat2; Tuberous sclerosis complex; mTORC1
    DOI:  https://doi.org/10.1111/gtc.13027
  45. Antioxidants (Basel). 2023 Mar 01. pii: 602. [Epub ahead of print]12(3):
    Mitochondrial Oxidative Stress and Mitophagy Activation Contribute to TNF-Dependent Impairment of Myogenesis.
    Daniil A Chernyavskij, Olga Yu Pletjushkina, Anastasia V Kashtanova, Ivan I Galkin, Anna Karpukhina, Boris V Chernyak, Yegor S Vassetzky, Ekaterina N Popova.
      Many muscular pathologies are associated with oxidative stress and elevated levels of the tumor necrosis factor (TNF) that cause muscle protein catabolism and impair myogenesis. Myogenesis defects caused by TNF are mediated in part by reactive oxygen species (ROS), including those produced by mitochondria (mitoROS), but the mechanism of their pathological action is not fully understood. We hypothesized that mitoROS act by triggering and enhancing mitophagy, an important tool for remodelling the mitochondrial reticulum during myogenesis. We used three recently developed probes-MitoTracker Orange CM-H2TMRos, mito-QC, and MitoCLox-to study myogenesis in human myoblasts. Induction of myogenesis resulted in a significant increase in mitoROS generation and phospholipid peroxidation in the inner mitochondrial membrane, as well as mitophagy enhancement. Treatment of myoblasts with TNF 24 h before induction of myogenesis resulted in a significant decrease in the myoblast fusion index and myosin heavy chain (MYH2) synthesis. TNF increased the levels of mitoROS, phospholipid peroxidation in the inner mitochondrial membrane and mitophagy at an early stage of differentiation. Trolox and SkQ1 antioxidants partially restored TNF-impaired myogenesis. The general autophagy inducers rapamycin and AICAR, which also stimulate mitophagy, completely blocked myogenesis. The autophagy suppression by the ULK1 inhibitor SBI-0206965 partially restored myogenesis impaired by TNF. Thus, suppression of myogenesis by TNF is associated with a mitoROS-dependent increase in general autophagy and mitophagy.
    Keywords:  SkQ1; TNF; antioxidant; mitochondria; mitophagy; myogenesis; reactive oxygen species (ROS)
    DOI:  https://doi.org/10.3390/antiox12030602
  46. Sci Rep. 2023 Mar 28. 13(1): 4680
    Alpha 2-macroglobulin acts as a clearance factor in the lysosomal degradation of extracellular misfolded proteins.
    Ayaka Tomihari, Mako Kiyota, Akira Matsuura, Eisuke Itakura.
      Proteostasis regulates protein folding and degradation; its maintenance is essential for resistance to stress and aging. The loss of proteostasis is associated with many age-related diseases. Within the cell, molecular chaperones facilitate the refolding of misfolded proteins into their bioactive forms, thus preventing undesirable interactions and aggregation. Although the mechanisms of intracellular protein degradation pathways for intracellular misfolded proteins have been extensively studied, the protein degradation pathway for extracellular proteins remain poorly understood. In this study, we identified several misfolded proteins that are substrates for alpha 2-macroglobulin (α2M), an extracellular chaperone. We also established a lysosomal internalization assay for α2M, which revealed that α2M mediates the lysosomal degradation of extracellular misfolded proteins. Comparative analyses of α2M and clusterin, another extracellular chaperone, indicated that α2M preferentially targets aggregation-prone proteins. Thus, we present the degradation pathway of α2M, which interacts with aggregation-prone proteins for lysosomal degradation via selective internalization.
    DOI:  https://doi.org/10.1038/s41598-023-31104-x
  47. Neurosci Biobehav Rev. 2023 Mar 28. pii: S0149-7634(23)00117-3. [Epub ahead of print] 105148
    Lithium engages autophagy for neuroprotection and neuroplasticity: translational evidence for therapy.
    Stefano Puglisi-Allegra, Gloria Lazzeri, Carla L Busceti, Filippo S Giorgi, Francesca Biagioni, Francesco Fornai.
      Here an overview is provided on therapeutic/neuroprotective effects of Lithium (Li+) in neurodegenerative and psychiatric disorders focusing on the conspicuous action of Li+ through autophagy. The effects on the autophagy machinery remain the key molecular mechanisms to explain the protective effects of Li+ for neurodegenerative diseases, offering potential therapeutic strategies for the treatment of neuropsychiatric disorders and emphasizes a crossroad linking autophagy, neurodegenerative disorders, and mood stabilization. Sensitization by psychostimulants points to several mechanisms involved in psychopathology, most also crucial in neurodegenerative disorders. Evidence shows the involvement of autophagy and metabotropic Glutamate receptors-5 (mGluR5) in neurodegeneration due to methamphetamine neurotoxicity as well as in neuroprotection, both in vitro and in vivo models. More recently, Li+ was shown to modulate autophagy through its action on mGluR5, thus pointing to an additional way of autophagy engagement by Li+ and to a substantial role of mGluR5 in neuroprotection related to neural e neuropsychiatry diseases. We propose Li+ engagement of autophagy through the canonical mechanisms of autophagy machinery and through the intermediary of mGluR5.
    Keywords:  Autophagy; Bipolar Disorder; Dopamine; Lithium; Locus coeruleus; Methamphetamine; Neurodegeneration; Norepinephrine; Sensitization; Ventral Tegmental Area; mGluR5
    DOI:  https://doi.org/10.1016/j.neubiorev.2023.105148
  48. ACS Chem Neurosci. 2023 Mar 29.
    Curcumin Modulates p62-Keap1-Nrf2-Mediated Autophagy in Rotenone-Induced Parkinson's Disease Mouse Models.
    Aaina Singh Rathore, Saumitra Sen Singh, Hareram Birla, Walia Zahra, Priyanka Kumari Keshri, Hagera Dilnashin, Richa Singh, Shekhar Singh, Surya Pratap Singh.
      Autophagy mediates self-digestion of abnormally aggregated proteins and organelles present in the cytoplasm. This mechanism may prove to be neuroprotective against Parkinson's disease (PD) by clearing misfolded α-synuclein (α-syn) aggregates from dopaminergic neurons. p62, an adaptor protein acts as a selective substrate for autophagy and regulates the formation as well as the degradation of protein aggregates. p62 sequesters keap1 freeing Nrf2 and consequently activating the transcription of its target genes. In the present study, we aimed to investigate the anti-parkinsonian activity of curcumin targeting primarily activation of autophagy via the Nrf2-Keap1 pathway. The mice were subcutaneously injected with rotenone (2.5 mg/kg bodyweight) and co-treated with oral administration of curcumin (80 mg/kg bodyweight) for 35 days. Following completion of dosing, motor activities, anti-oxidative potential, mitochondrial dysfunction, and various protein expressions, including Nrf2, Keap1, p62, LC3, Bcl2, Bax, and caspase 3, were assessed. The results revealed that curcumin restored the motor coordination and anti-oxidative activity while improving the mitochondrial functioning in PD mice. Autophagy was evaluated by the change in the expression of autophagic markers, p62 and LC3-II. Reduced p62 and LC3-II expressions in the rotenone mouse model of PD confirmed the compromised autophagy pathway, consequently increasing the aggregation of misfolded protein α-syn. Whereas, curcumin treatment-enhanced autophagy-mediated clearance of misfolded α-syn proteins by increasing the LC3-II expression and blocked apoptotic cascade. Curcumin administration upregulated the Nrf2 expression and normalized the Nrf2-Keap1 pathway, which justifies the improved anti-oxidative activity. Therefore, the findings reveal that curcumin is a Nrf2-inducer and is endowed with neuroprotective potential, which may prove to be a potential candidate for the anti-Parkinson's disease treatment therapy.
    Keywords:  Nrf2−Keap1 pathway; Parkinson’s disease; apoptosis; autophagy; oxidative stress; protein aggregation
    DOI:  https://doi.org/10.1021/acschemneuro.2c00706
  49. Clin Transl Med. 2023 Mar;13(3): e1210
    Destabilization of microrchidia family CW-type zinc finger 2 via the cyclin-dependent kinase 1-chaperone-mediated autophagy pathway promotes mitotic arrest and enhances cancer cellular sensitivity to microtubule-targeting agents.
    Shu-Yuan Hu, Jin-Xian Qian, Shao-Ying Yang, Lisa Andriani, Li Liao, Ling Deng, Min-Ying Huang, Yin-Ling Zhang, Fang-Lin Zhang, Zhi-Min Shao, Da-Qiang Li.
       BACKGROUND: Microtubule-targeing agents (MTAs), such as paclitaxel (PTX) and vincristine (VCR), kill cancer cells through activtion of the spindle assembly checkpoint (SAC) and induction of mitotic arrest, but the development of resistance poses significant clinical challenges.
    METHODS: Immunoblotting and RT-qPCR were used to investigate potential function and related mechanism of MORC2. Flow cytometry analyses were carried out to determine cell cycle distribution and apoptosis. The effect of MORC2 on cellular sensitivity to PTX and VCR was determined by immunoblotting, flow cytometry, and colony formation assays. Immunoprecipitation assays and immunofluorescent staining were utilized to investigate protein-protein interaction and protein co-localization.
    RESULTS: Here, we identified microrchidia family CW-type zinc finger 2 (MORC2), a poorly characterized oncoprotein, as a novel regulator of SAC activation, mitotic progression, and resistance of cancer cells to PTX and VCR. Mechanically, PTX and VCR activate cyclin-dependent kinase 1, which in turn induces MORC2 phosphorylation at threonine 717 (T717) and T733. Phosphorylated MORC2 enhances its interation with HSPA8 and LAMP2A, two essential components of the chaperone-mediated autophagy (CMA) mechinery, resulting in its autophagic degradation. Degradation of MORC2 during mitosis leads to SAC activation through stabilizing anaphase promoting complex/cyclosome activator protein Cdc20 and facilitating mitotic checkpoint complex assembly, thus contributing to mitotic arrest induced by PTX and VCR. Notably, knockdown of MORC2 promotes mitotic arrest induced by PTX and VCR and enhances the sensitivity of cancer cells to PTX and VCR.
    CONCLUSIONS: Collectively, these findings unveil a previously unrecognized function and regulatory mechanism of MORC2 in mitotic progression and resistance of cancer cells to MTAs. These results also provide a new clue for developing combined treatmentstrategy by targeting MORC2 in combination with MTAs against human cancer.
    Keywords:  MORC2; chaperone-mediated autophagy; cyclin-dependent kinase 1; microtubule-targeting agents; mitotic arrest; spindle assembly checkpoint
    DOI:  https://doi.org/10.1002/ctm2.1210
  50. Life (Basel). 2023 Mar 17. pii: 818. [Epub ahead of print]13(3):
    Metastasis in Neuroblastoma and Its Link to Autophagy.
    Leila Jahangiri.
      Neuroblastoma is a paediatric malignancy originating from the neural crest that commonly occurs in the abdomen and adrenal gland, leading to cancer-related deaths in children. Distant metastasis can be encountered at diagnosis in greater than half of these neuroblastoma patients. Autophagy, a self-degradative process, plays a key role in stress-related responses and the survival of cells and has been studied in neuroblastoma. Accordingly, in the early stages of metastasis, autophagy may suppress cancer cell invasion and migration, while its role may be reversed in later stages, and it may facilitate metastasis by enhancing cancer cell survival. To that end, a body of literature has revealed the mechanistic link between autophagy and metastasis in neuroblastoma in multiple steps of the metastatic cascade, including cancer cell invasion and migration, anoikis resistance, cancer cell dormancy, micrometastasis, and metastatic outbreak. This review aims to take a step forward and discuss the significance of multiple molecular players and compounds that may link autophagy to metastasis and map their function to various metastatic steps in neuroblastoma.
    Keywords:  autophagy; metastasis; neuroblastoma; paediatric cancers
    DOI:  https://doi.org/10.3390/life13030818
  51. Proc Biol Sci. 2023 03 29. 290(1995): 20230407
    Transcriptional profiling of the response to starvation and fattening reveals differential regulation of autophagy genes in mammals.
    Margarita Galves, Michal Sperber, Fatima Amer-Sarsour, Ran Elkon, Avraham Ashkenazi.
      Nutrient deprivation (starvation) induced by fasting and hypercaloric regimens are stress factors that can influence cell and tissue homeostasis in mammals. One of the key cellular responses to changes in nutrient availability is the cell survival pathway autophagy. While there has been much research into the protein networks regulating autophagy, less is known about the gene expression networks involved in this fundamental process. Here, we applied a network algorithm designed to analyse omics datasets, to identify sub-networks that are enriched for induced genes in response to starvation. This enabled us to identify two prominent active modules, one composed of key stress-induced transcription factors, including members of the Jun, Fos and ATF families, and the other comprising autophagosome sub-network genes, including ULK1. The results were validated in the brain, liver and muscle of fasting mice. Moreover, differential expression analysis of autophagy genes in the brain, liver and muscle of high-fat diet-exposed mice showed significant suppression of GABARAPL1 in the liver. Finally, our data provide a resource that may facilitate the future identification of regulators of autophagy.
    Keywords:  autophagy; high-fat diet; nutrient deprivation; transcriptional networks
    DOI:  https://doi.org/10.1098/rspb.2023.0407
  52. Cell Biol Toxicol. 2023 Mar 31.
    The mtDNA-STING pathway plays an important role in both navitoclax- and S63845-induced autophagy and enhances cell death.
    Jia Jia, Mengqing Li, Yunjian Li, Jun Xiao, Haiming Dai.
      Targeting BCL2 family proteins to induce cancer cell death has been successful in the treatment of cancer. BH3 mimetics such as ABT-737 not only induce cell death, but also activate autophagy. The molecular mechanism by which the BH3 mimetics induce autophagy is still controversial. In this study, we show that the BCL2/BCLXL/BCLw inhibitor navitoclax and the MCL1 inhibitor S63845 induce both apoptosis and autophagy in mouse embryonic fibroblasts (MEFs) and leukemia cell lines, while autophagy induced by navticlax and S63845 in leukemia cell lines requires the inhibition of caspase activities. Further experiments demonstrate that the autophagy induced by navitoclax or S63845 does not depend on Beclin 1, but downstream of Bax/Bak. Moreover, both navitoclax and S63845 treatment induce mtDNA release in MEFs, which activates STING and thereby induces autophagy, while STING KO inhibits both navitoclax- and S63845-induced autophagy. Furthermore, STING KO diminishes navitoclax- or S63845-induced apoptosis, suggesting that STING activation enhances rather than inhibits apoptosis. Thus, our findings provide new insights into the regulations of navitoclax- or S63845-induced autophagy and cell death.
    Keywords:  Apoptosis; Autophagy; BH3 mimetics; Beclin 1; STING; mtDNA
    DOI:  https://doi.org/10.1007/s10565-023-09804-x
  53. Front Pharmacol. 2023 ;14 1124003
    The essential effect of mTORC1-dependent lipophagy in non-alcoholic fatty liver disease.
    Xiangyun Tan, Xinyu Huang, Zhuhang Lu, Liang Chen, Junjie Hu, Xianxiang Tian, Zhenpeng Qiu.
      Non-alcoholic fatty liver disease (NAFLD) is a chronic progressive liver disease with increasing prevalence. Lipophagy is a type of programmed cell death that plays an essential role in maintaining the body's balance of fatty acid metabolism. However, the livers of NAFLD patients are abnormally dysregulated in lipophagy. mTORC1 is a critical negative regulator of lipophagy, which has been confirmed to participate in the process of lipophagy through various complex mechanisms. Therefore, targeting mTORC1 to restore failed autophagy may be an effective therapeutic strategy for NAFLD. This article reviews the main pathways through which mTORC1 participates in the formation of lipophagy and the intervention effect of mTORC1-regulated lipophagy in NAFLD, providing new therapeutic strategies for the prevention and treatment of NAFLD in the future.
    Keywords:  NAFLD; hepatic fibrosis; hepatic steatosis; insulin resistance; lipophagy; mTORC1
    DOI:  https://doi.org/10.3389/fphar.2023.1124003
  54. FASEB J. 2023 May;37(5): e22897
    A multi-organ analysis of the role of mTOR in fetal alcohol spectrum disorders.
    Alexander L Carabulea, Joseph D Janeski, Vishal D Naik, Kang Chen, Gil Mor, Jayanth Ramadoss.
      Alcohol exposure during gestation can lead to fetal alcohol spectrum disorders (FASD), an array of cognitive and physical developmental impairments. Over the past two and a half decades, Mammalian Target of Rapamycin (mTOR) has emerged at the nexus of many fields of study, and has recently been implicated in FASD etiology. mTOR plays an integral role in modulating anabolic and catabolic activities, including protein synthesis and autophagy. These processes are vital for proper development and can have long lasting effects following alcohol exposure, such as impaired hippocampal and synapse formation, reduced brain size, as well as cognitive, behavioral, and memory impairments. We highlight recent advances in the field of FASD, primarily with regard to animal model discoveries and discuss the interaction between alcohol and mTOR in the context of various tissues, including brain, placenta, bone, and muscle, with respect to developmental alcohol exposure paradigms. The current review focuses on novel FASD research within the context of the mTOR signaling and sheds light on mechanistic etiologies at various biological levels including molecular, cellular, and functional, across multiple stages of development and illuminates the dichotomy between the different mTOR complexes and their unique signaling roles.
    Keywords:  alcohol; autophagy; development; fetal; mTOR; pregnancy
    DOI:  https://doi.org/10.1096/fj.202201865R
  55. Nat Commun. 2023 Mar 30. 14(1): 1768
    The plant unique ESCRT component FREE1 regulates autophagosome closure.
    Yonglun Zeng, Baiying Li, Shuxian Huang, Hongbo Li, Wenhan Cao, Yixuan Chen, Guoyong Liu, Zhenping Li, Chao Yang, Lei Feng, Jiayang Gao, Sze Wan Lo, Jierui Zhao, Jinbo Shen, Yan Guo, Caiji Gao, Yasin Dagdas, Liwen Jiang.
      The energy sensor AMP-activated protein kinase (AMPK) can activate autophagy when cellular energy production becomes compromised. However, the degree to which nutrient sensing impinges on the autophagosome closure remains unknown. Here, we provide the mechanism underlying a plant unique protein FREE1, upon autophagy-induced SnRK1α1-mediated phosphorylation, functions as a linkage between ATG conjugation system and ESCRT machinery to regulate the autophagosome closure upon nutrient deprivation. Using high-resolution microscopy, 3D-electron tomography, and protease protection assay, we showed that unclosed autophagosomes accumulated in free1 mutants. Proteomic, cellular and biochemical analysis revealed the mechanistic connection between FREE1 and the ATG conjugation system/ESCRT-III complex in regulating autophagosome closure. Mass spectrometry analysis showed that the evolutionary conserved plant energy sensor SnRK1α1 phosphorylates FREE1 and recruits it to the autophagosomes to promote closure. Mutagenesis of the phosphorylation site on FREE1 caused the autophagosome closure failure. Our findings unveil how cellular energy sensing pathways regulate autophagosome closure to maintain cellular homeostasis.
    DOI:  https://doi.org/10.1038/s41467-023-37185-6
  56. Cells. 2023 Mar 09. pii: 854. [Epub ahead of print]12(6):
    Mechanistic Insights into the Role of OPN in Mediating Brain Damage via Triggering Lysosomal Damage in Microglia/Macrophage.
    Chengcheng Gai, Yijing Zhao, Danqing Xin, Tingting Li, Yahong Cheng, Zige Jiang, Yan Song, Dexiang Liu, Zhen Wang.
      We previously found that osteopontin (OPN) played a role in hypoxia-ischemia (HI) brain damage. However, its underlying mechanism is still unknown. Bioinformatics analysis revealed that the OPN protein was linked to the lysosomal cathepsin B (CTSB) and galectin-3 (GAL-3) proteins after HI exposure. In the present study, we tested the hypothesis that OPN was able to play a critical role in the lysosomal damage of microglia/macrophages following HI insult in neonatal mice. The results showed that OPN expression was enhanced, especially in microglia/macrophages, and colocalized with lysosomal-associated membrane protein 1 (LAMP1) and GAL-3; this was accompanied by increased LAMP1 and GAL-3 expression, CTSB leakage, as well as impairment of autophagic flux in the early stage of the HI process. In addition, the knockdown of OPN expression markedly restored lysosomal function with significant improvements in the autophagic flux after HI insult. Interestingly, cleavage of OPN was observed in the ipsilateral cortex following HI. The wild-type OPN and C-terminal OPN (Leu152-Asn294), rather than N-terminal OPN (Met1-Gly151), interacted with GAL-3 to induce lysosomal damage. Furthermore, the secreted OPN stimulated lysosomal damage by binding to CD44 in microglia in vitro. Collectively, this study demonstrated that upregulated OPN in microglia/macrophages and its cleavage product was able to interact with GAL-3, and secreted OPN combined with CD44, leading to lysosomal damage and exacerbating autophagosome accumulation after HI exposure.
    Keywords:  cathepsin B; galectin-3; hypoxia–ischemia; lysosomal; microglia/macrophages; osteopontin
    DOI:  https://doi.org/10.3390/cells12060854
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