bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2021‒06‒20
thirty-six papers selected by
Viktor Korolchuk
Newcastle University

  1. Mol Aspects Med. 2021 Jun 12. pii: S0098-2997(21)00032-7. [Epub ahead of print] 100972
      Autophagy is the process by which cells can selectively or non-selectively remove damaged proteins and organelles. As the cell's main means of sequestering damaged mitochondria for removal, mitophagy is central to cellular function and survival. Research on autophagy and mitochondrial quality control has increased exponentially in relation to the pathogenesis of numerous disease conditions, from cancer and immune diseases to chronic neurodegenerative diseases like Parkinson's disease (PD). Understanding how components of the autophagic/mitophagic machinery are affected during disease, as well as the contextual relationship of autophagy with determining neuronal health and function, is essential to the goal of designing therapies for human disease. In this review, we will summarize key signaling molecules that consign damaged mitochondria for autophagic degradation, describe the relationship of genes linked to PD to autophagy/mitophagy dysfunction, and discuss additional roles of both mitochondrial and cytosolic pools of PTEN-induced kinase 1 (PINK1) in mitochondrial homeostasis, dendritic morphogenesis and inflammation.
    Keywords:  Autophagy; Mitochondria; Mitophagy; Neurodegeneration; PINK1; Parkinson's disease
  2. EMBO J. 2021 06 14. e105985
      Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.
    Keywords:  PIK3R4; PRKAG2; ULK1; VPS15; p62
  3. Bioessays. 2021 Jun 14. e2100093
      Ferroptosis, a form of regulated cell death triggered by lipid hydroperoxide accumulation, has an important role in a variety of diseases and pathological conditions, such as cancer. Targeting ferroptosis is emerging as a promising means of therapeutic intervention in cancer treatment. Polyunsaturated fatty acids, reactive oxygen species, and labile iron constitute the major underlying triggers for ferroptosis. Other regulators of ferroptosis have also been discovered recently, among them the mechanistic target of rapamycin complex 1 (mTORC1), a central controller of cell growth and metabolism. Inhibitors of mTORC1 have been used in treating diverse diseases, including cancer. In this review, we discuss recent findings linking mTORC1 to ferroptosis, dissect mechanisms underlying the establishment of mTORC1 as a key ferroptosis modulator, and highlight the potential of co-targeting mTORC1 and ferroptosis in cancer treatment. This review will provide valuable insights for future investigations of ferroptosis and mTORC1 in fundamental biology and cancer therapy.
    Keywords:  GPX4; SLC7A11; autophagy; cancer therapy; ferroptosis; lipid peroxidation; mTOR; mTORC1; oncogene
  4. Front Cell Dev Biol. 2021 ;9 621906
      Myocardial ischemia-reperfusion injury (MIRI), characterized by post-ischemic cardiomyocytes death and reperfusion myocardial damage, is a lethal yet unresolved complication in the treatment of acute myocardial infarction (AMI). Previous studies have demonstrated that poly(ADP-ribose) polymerase-1 (PARP1) participates in the progression of various cardiovascular diseases, and various reports have proved that PARP1 can be a therapeutic target in these diseases, but whether it plays a role in MIRI is still unknown. Therefore, in this study, we aimed to explore the role and mechanism of PARP1 in the development of MIRI. Firstly, we demonstrated that PARP1 was activated during MIRI-induced myocardial autophagy in vitro. Moreover, PARP1 inhibition protected cardiomyocytes from MIRI through the inhibition of autophagy. Next, we discovered that specificity protein1 (Sp1), as a transcription factor of PARP1, regulates its target gene PARP1 through binding to its target gene promoter during transcription. Furthermore, silencing Sp1 protected cardiomyocytes from MIRI via the inhibition of PARP1. Finally, the functions and mechanisms of PARP1 in the development of MIRI were also verified in vivo with SD rats model. Based on these findings, we concluded that PARP1 inhibition protects cardiomyocytes from MIRI through the inhibition of autophagy, which is targeted by Sp1 suppression. Therefore, the utilization of PARP1 exhibits great therapeutic potential for MIRI treatment in future.
    Keywords:  PARP1; Sp1; autophagy; myocardial ischemia-reperfusion injury; oxygen-glucose deprivation/reperfusion
  5. Nat Commun. 2021 06 16. 12(1): 3660
      The mechanistic target of rapamycin complex 1 (mTORC1) integrates cellular nutrient signaling and hormonal cues to control metabolism. We have previously shown that constitutive nutrient signaling to mTORC1 by means of genetic activation of RagA (expression of GTP-locked RagA, or RagAGTP) in mice resulted in a fatal energetic crisis at birth. Herein, we rescue neonatal lethality in RagAGTP mice and find morphometric and metabolic alterations that span glucose, lipid, ketone, bile acid and amino acid homeostasis in adults, and a median lifespan of nine months. Proteomic and metabolomic analyses of livers from RagAGTP mice reveal a failed metabolic adaptation to fasting due to a global impairment in PPARα transcriptional program. These metabolic defects are partially recapitulated by restricting activation of RagA to hepatocytes, and revert by pharmacological inhibition of mTORC1. Constitutive hepatic nutrient signaling does not cause hepatocellular damage and carcinomas, unlike genetic activation of growth factor signaling upstream of mTORC1. In summary, RagA signaling dictates dynamic responses to feeding-fasting cycles to tune metabolism so as to match the nutritional state.
  6. Mol Aspects Med. 2021 Jun 10. pii: S0098-2997(21)00033-9. [Epub ahead of print] 100973
      The liver is a highly dynamic metabolic organ that plays critical roles in plasma protein synthesis, gluconeogenesis and glycogen storage, cholesterol metabolism and bile acid synthesis as well as drug/xenobiotic metabolism and detoxification. Research from the past decades indicate that autophagy, the cellular catabolic process mediated by lysosomes, plays an important role in maintaining cellular and metabolic homeostasis in the liver. Hepatic autophagy fluctuates with hormonal cues and the availability of nutrients that respond to fed and fasting states as well as circadian activities. Dysfunction of autophagy in liver parenchymal and non-parenchymal cells can lead to various liver diseases including non-alcoholic fatty liver diseases, alcohol associated liver disease, drug-induced liver injury, cholestasis, viral hepatitis and hepatocellular carcinoma. Therefore, targeting autophagy may be a potential strategy for treating these various liver diseases. In this review, we will discuss the current progress on the understanding of autophagy in liver physiology. We will also discuss several forms of selective autophagy in the liver and the molecular signaling pathways in regulating autophagy of different cell types and their implications in various liver diseases.
    Keywords:  AALD; DILI; HCC; Lipophagy; Mitophagy; NAFLD
  7. Bio Protoc. 2021 Apr 20. 11(8): e3989
      Mammalian target of rapamycin (mTOR) controls many crucial cellular functions, including protein synthesis, cell size, energy metabolism, lysosome and mitochondria biogenesis, and autophagy. Consequently, deregulation of mTOR signaling plays a role in numerous pathological conditions such as cancer, metabolic disorders and neurological diseases. Developing new tools to monitor mTOR spatiotemporal activation is crucial to better understand its roles in physiological and pathological conditions. However, the most widely used method to report mTOR activity relies on the quantification of specific mTOR-phosphorylated substrates by western blot. This approach requires cellular lysate preparation, which restricts the quantification to a single time point. Here, we present a simple protocol to study mTOR activity in living cells in real time using AIMTOR, an intramolecular BRET-based (bioluminescence resonance energy transfer) biosensor that we recently designed ( Bouquier et al., 2020 ). We describe transfection of AIMTOR in the C2C12 cell line and procedures to monitor BRET in a cell population using a plate reader and in single cells by microscopy. Importantly, this protocol is transposable to any cell line and primary cells. In addition, several subcellular compartment-specific versions of AIMTOR have been developed, enabling compartmentalized assessment of mTOR activity. This protocol describes how to use the sensitive AIMTOR biosensor to investigate mTOR signaling dynamics in living cells. Graphic abstract: AIMTOR protocol overview from seeding cells to live BRET recording.
    Keywords:  BRET; Kinase activity; Living cells; Real time imaging; mTOR signaling; mTORC1 biosensor
  8. Autophagy. 2021 Jun 16.
      The autophagosome has two lipid bilayer membranes. The outer membrane fuses with the lysosome, while the inner membrane is degraded to release autophagic contents for degradation. It remains unclear how the inner vesicle of the autophagosome (called the autophagic vesicle) is disintegrated after autophagosome-lysosome fusion. Here, we identified C. elegans LPLA-2/M05B5.4 as a key enzyme that degrades membranous material in lysosomes. LPLA-2 is homologous to human PLA2G15, a lysosomal phospholipase A2 family protein that catalyzes cleavage of membrane phospholipids. We found that loss of LPLA-2 causes accumulation of large membrane whorls in enlarged lysosomes and both phenotypes are suppressed by blocking macroautophagy/autophagy. Moreover, autophagic vesicles persisted in enlarged lysosomes in PLA2G15 knockdown cells and lpla-2(lf) mutants, which suggests that the breakdown of the inner autophagosomal membrane in lysosomes is impaired. lpla-2(lf) mutants exhibit severe defects in both embryonic and larval development. Our data suggest that disintegration of the inner autophagosomal membrane by LPLA-2 promotes the release and subsequent degradation of autophagic contents in lysosomes, which is essential for C. elegans development.
    Keywords:  Autophagic vesicle, C. elegans; development; inner autophagosomal membrane; lysosomal phospholipase A2; lysosome
  9. Int J Biol Macromol. 2021 Jun 07. pii: S0141-8130(21)01222-8. [Epub ahead of print]183 2364-2375
      TANK-binding kinase 1 (TBK1) regulates various biological processes including, NF-κB signaling, immune response, autophagy, cell division, Ras-mediated oncogenesis, and AKT pro-survival signaling. Enhanced TBK1 activity is associated with autoimmune diseases and cancer, suggesting its role in therapeutic targeting of interferonopathies. In addition, dysregulation of TBK1 activity promotes several inflammatory disorders and oncogenesis. Structural and biochemical study reports provide the molecular process of TBK1 activation and recap the substrate selection about TBK1. This review summarizes recent findings on the molecular mechanisms by which TBK1 is involved in cancer signaling. The IKK-ε and TBK1 are together associated with inflammatory diseases by inducing type I IFNs. Furthermore, TBK1 signaling regulates radiation-induced epithelial-mesenchymal transition by controlling phosphorylation of GSK-3β and expression of Zinc finger E-box-binding homeobox 1, suggesting, TBK1 could be targeted for radiotherapy-induced metastasis therapy. Despite a considerable increase in the list of TBK1 inhibitors, only a few has potential to control cancer. Among them, a compound BX795 is considered a potent and selective inhibitor of TBK1. We discussed the therapeutic potential of small-molecule inhibitors of TBK1, particularly those with high selectivity, which will enable further exploration in the therapeutic management of cancer and inflammatory diseases.
    Keywords:  Autophagy; Cell division; Immune infiltration; Inflammation; Kinase inhibitors; Signaling; TANK-binding kinase 1; Targeted therapy
  10. Nat Commun. 2021 06 16. 12(1): 3653
      The Mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway controls several aspects of neuronal development. Mutations in regulators of mTORC1, such as Tsc1 and Tsc2, lead to neurodevelopmental disorders associated with autism, intellectual disabilities and epilepsy. The correct development of inhibitory interneurons is crucial for functional circuits. In particular, the axonal arborisation and synapse density of parvalbumin (PV)-positive GABAergic interneurons change in the postnatal brain. How and whether mTORC1 signaling affects PV cell development is unknown. Here, we show that Tsc1 haploinsufficiency causes a premature increase in terminal axonal branching and bouton density formed by mutant PV cells, followed by a loss of perisomatic innervation in adult mice. PV cell-restricted Tsc1 haploinsufficient and knockout mice show deficits in social behavior. Finally, we identify a sensitive period during the third postnatal week during which treatment with the mTOR inhibitor Rapamycin rescues deficits in both PV cell innervation and social behavior in adult conditional haploinsufficient mice. Our findings reveal a role of mTORC1 signaling in the regulation of the developmental time course and maintenance of cortical PV cell connectivity and support a mechanistic basis for the targeted rescue of autism-related behaviors in disorders associated with deregulated mTORC1 signaling.
  11. Mol Biol Rep. 2021 Jun 16.
      Glioblastoma multiforme (GBM) is one of the aggressive brain cancers with patients having less survival period upto 12-15 months. Mammalian target of rapamycin (mTOR) is a serine/threonine kinase, belongs to the phosphatidylinositol 3-kinases (PI3K) pathway and is involved in various cellular processes of cancer cells. Cancer metabolism is regulated by mTOR and its components. mTOR forms two complexes as mTORC1 and mTORC2. Studies have identified the key component of the mTORC2 complex, Rapamycin-insensitive companion of mammalian target of rapamycin (Rictor) plays a prominent role in the regulation of cancer cell proliferation and metabolism. Apart, growth factor receptor signaling such as epidermal growth factor signaling mediated by epidermal growth factor receptor (EGFR) regulates cancer-related processes. In EGFR signaling various other signaling cascades such as phosphatidyl-inositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR pathway) and Ras/Raf/mitogen-activated protein kinase/ERK kinase (MEK)/extracellular-signal-regulated kinase (ERK) -dependent signaling cross-talk each other. From various studies about GBM, it is very well established that Rictor and EGFR mediated signaling pathways majorly playing a pivotal role in chemoresistance and tumor aggressiveness. Recent studies have shown that non-coding RNAs such as microRNAs (miRs) and long non-coding RNAs (lncRNAs) regulate the EGFR and Rictor and sensitize the cells towards chemotherapeutic agents. Thus, understanding of microRNA mediated regulation of EGFR and Rictor will help in cancer prevention and management as well as a future therapy.
    Keywords:  EGFR; Glioblastoma multiforme (GBM); HDAC; Rictor; Temozolomide (TMZ); mTOR
  12. JCI Insight. 2021 Jun 17. pii: 138835. [Epub ahead of print]
      Cancer cells re-program cellular metabolism to maintain adequate nutrient pools to sustain proliferation. Moreover, autophagy is a regulated mechanism to breakdown dysfunctional cellular components and recycle cellular nutrients. However, the requirement for autophagy and the integration in cancer cell metabolism is not clear in colon cancer. Here we show a cell-autonomous dependency of autophagy for cell growth in colorectal cancer. Loss of epithelial autophagy inhibits tumor growth in both sporadic and colitis associated cancer models. Genetic and pharmacological inhibition of autophagy inhibits cell growth in colon cancer-derived cell lines and patient-derived enteroid models. Importantly, normal colon epithelium and patient-derived normal enteroid growth was not decreased following autophagy inhibition. To couple the role of autophagy to cellular metabolism, a cell culture screen in conjunction with metabolomic analysis was performed. We identified a critical role of autophagy to maintain mitochondrial metabolites for growth. Loss of mitochondrial recycling through inhibition of mitophagy hinders colon cancer cell growth. These findings have revealed a cell-autonomous role of autophagy that plays a critical role in regulating nutrient pools in vivo and in cell models and provides therapeutic targets for colon cancer.
    Keywords:  Colorectal cancer; Gastroenterology; Oncology
  13. J Clin Invest. 2021 Jun 15. pii: 146870. [Epub ahead of print]
      Disordered lysosomal/autophagy pathways initiate and drive pancreatitis, but the underlying mechanisms and links to disease pathology are poorly understood. Here, we show that mannose-6-phosphate (M6P) pathway of hydrolase delivery to lysosomes critically regulates pancreatic acinar cell cholesterol metabolism. Ablation of the Gnptab gene coding for a key enzyme in M6P pathway disrupted acinar cell cholesterol turnover, causing accumulation of non-esterified cholesterol in lysosomes/autolysosomes, its' depletion in the plasma membrane, and upregulation of cholesterol synthesis and uptake. We found similar dysregulation of acinar cell cholesterol, and a decrease in GNPTAB levels, in both WT experimental pancreatitis and human disease. The mechanisms mediating pancreatic cholesterol dyshomeostasis in Gnptab-/- and experimental models involve disordered endolysosomal system, resulting in impaired cholesterol transport through lysosomes and blockage of autophagic flux. By contrast, in Gnptab-/- liver the endolysosomal system and cholesterol homeostasis were largely unaffected. Gnptab-/- mice developed spontaneous pancreatitis. Normalization of cholesterol metabolism by pharmacologic means alleviated responses of experimental pancreatitis, particularly trypsinogen activation, the disease hallmark. The results reveal the essential role of M6P pathway in maintaining exocrine pancreas homeostasis and function, and implicate cholesterol disordering in the pathogenesis of pancreatitis.
    Keywords:  Autophagy; Cell Biology; Gastroenterology; Lysosomes; Mitochondria
  14. Biochim Biophys Acta Mol Cell Biol Lipids. 2021 Jun 08. pii: S1388-1981(21)00116-5. [Epub ahead of print]1866(9): 158988
      Neutral lipases-mediated lipolysis and acid lipases-moderated lipophagy are two main processes for degradation of lipid droplets (LDs). However, the individual and interactive roles of these metabolic pathways are not well known across vertebrates. This study explored the roles of lipolysis and lipophagy from the aspect of neutral and acid lipases in zebrafish. We established zebrafish strains deficient in either adipose triglyceride lipase (atgl-/-; AKO fish) or lysosomal acid lipase (lal-/-; LKO fish) respectively, and then inhibited lipolysis in the LKO fish and lipophagy in the AKO fish by feeding diets supplemented with the corresponding inhibitors Atglistatin and 3-Methyladenine, respectively. Both the AKO and LKO fish showed reduced growth, swimming activity, and oxygen consumption. The AKO fish did not show phenotypes in adipose tissue, but mainly accumulated triacylglycerol (TAG) in liver, also, they had large LDs in the hepatocytes, and did not stimulate lipophagy as a compensation response but maintained basal lipophagy. The LKO fish reduced total lipid accumulation in the body but had high cholesterol content in liver; also, they accumulated small LDs in the hepatocytes, and showed increased lipolysis, especially Atgl expression, as a compensatory mechanism. Simultaneous inhibition of lipolysis and lipophagy in zebrafish resulted in severe liver damage, with the potential to trigger mitophagy. Overall, our study illustrates that lipolysis and lipophagy perform individual and interactive roles in maintaining homeostasis of TAG and cholesterol metabolism. Furthermore, the interactive roles of lipolysis and lipophagy may be essential in regulating the functions and form of mitochondria.
    Keywords:  Interaction; Lipid catabolism; Lipolysis; Lipophagy; Mitochondria; Zebrafish
  15. Nat Rev Mol Cell Biol. 2021 Jun 17.
      The human genome contains more than one million short tandem repeats, and expansion of a subset of these repeat tracts underlies more than 50 human disorders. In this Review, we discuss the four major mechanisms by which expansion of short tandem repeats causes disease: loss of function through transcription repression, RNA-mediated gain of function through gelation and sequestration of RNA-binding proteins, gain of function of canonically translated repeat-harbouring proteins, and repeat-associated non-AUG translation of toxic repeat peptides. Somatic repeat instability amplifies these mechanisms and influences both disease age of onset and tissue specificity of pathogenic features. We focus on the crosstalk between these disease mechanisms, and argue that they often synergize to drive pathogenesis. We also discuss the emerging native functions of repeat elements and how their dynamics might contribute to disease at a larger scale than currently appreciated. Lastly, we propose that lynchpins tying these disease mechanisms and native functions together offer promising therapeutic targets with potential shared applications across this class of human disorders.
  16. Autophagy. 2021 Jun 16.
      Primary dysfunction of autophagy due to Mendelian defects affecting core components of the autophagy machinery or closely related proteins have recently emerged as an important cause of genetic disease. This novel group of human disorders may present throughout life and comprises severe early-onset neurodevelopmental and more common adult-onset neurodegenerative disorders. Early-onset (or congenital) disorders of autophagy often share a recognizable "clinical signature," including variable combinations of neurological, neuromuscular and multisystem manifestations. Structural CNS abnormalities, cerebellar involvement, spasticity and peripheral nerve pathology are prominent neurological features, indicating a specific vulnerability of certain neuronal populations to autophagic disturbance. A typically biphasic disease course of late-onset neurodegeneration occurring on the background of a neurodevelopmental disorder further supports a role of autophagy in both neuronal development and maintenance. In addition, an associated myopathy has been characterized in several conditions. The differential diagnosis comprises a wide range of other multisystem disorders, including mitochondrial, glycogen and lysosomal storage disorders, as well as ciliopathies, glycosylation and vesicular trafficking defects. The clinical overlap between the congenital disorders of autophagy and these conditions reflects the multiple roles of the proteins and/or emerging molecular connections between the pathways implicated and suggests an exciting area for future research. Therapy development for congenital disorders of autophagy is still in its infancy but may result in the identification of molecules that target autophagy more specifically than currently available compounds. The close connection with adult-onset neurodegenerative disorders highlights the relevance of research into rare early-onset neurodevelopmental conditions for much more common, age-related human diseases.
  17. J Biol Chem. 2021 Jun 11. pii: S0021-9258(21)00673-6. [Epub ahead of print] 100873
      Macroautophagy dysregulation is implicated in multiple neurological disorders, such as Parkinson's disease. While autophagy pathways are heavily researched in heterologous cells and neurons, regulation of autophagy in the astrocyte, the most abundant cell type in the mammalian brain, is less well understood. Missense mutations in the Synj1 gene encoding Synaptojanin1 (Synj1), a neuron-enriched lipid phosphatase, have been linked to Parkinsonism with seizures. Our previous study showed that the Synj1 haploinsufficient (Synj1+/-) mouse exhibits age-dependent autophagy impairment in multiple brain regions. Here, we used cultured astrocytes from Synj1-deficient mice to investigate its role in astrocyte autophagy. We report Synj1 is expressed in low levels in astrocytes and represses basal autophagosome formation. We demonstrate using cellular imaging that Synj1-deficient astrocytes exhibit hyperactive autophagosome formation, represented by an increase in the size and the number of GFP-LC3 structures. Interestingly, Synj1 deficiency is also associated with an impairment in stress-induced autophagy clearance. We show, for the first time, that the Parkinsonism-associated R839C mutation impacts autophagy in astrocytes. The impact of this mutation on Synj1's phosphatase function resulted in elevated basal autophagosome formation that mimics Synj1 deletion. We found that the membrane expression of the astrocyte-specific glucose transporter GluT-1 was reduced in Synj1-deficient astrocytes. Consistently, AMPK activity was elevated, suggesting altered glucose sensing in Synj1-deficient astrocytes. Expressing exogenous GluT-1 in Synj1-deficient astrocytes reversed the autophagy impairment, supporting a role for Synj1 in regulating astrocyte autophagy via disrupting glucose-sensing pathways. Thus, our work suggests a novel mechanism for Synj1-related Parkinsonism involving astrocyte dysfunction.
    Keywords:  Autophagy; GluT-1; Parkinson disease; astrocyte; cell culture
  18. Biomol Ther (Seoul). 2021 Jun 15.
      The gastrointestinal (GI) tract is a series of hollow organs that is responsible for the digestion and absorption of ingested foods and the excretion of waste. Any changes in the GI tract can lead to GI disorders. GI disorders are highly prevalent in the population and account for substantial morbidity, mortality, and healthcare utilization. GI disorders can be functional, or organic with structural changes. Functional GI disorders include functional dyspepsia and irritable bowel syndrome. Organic GI disorders include inflammation of the GI tract due to chronic infection, drugs, trauma, and other causes. Recent studies have highlighted a new explanatory mechanism for GI disorders. It has been suggested that autophagy, an intracellular homeostatic mechanism, also plays an important role in the pathogenesis of GI disorders. Autophagy has three primary forms: macroautophagy, microautophagy, and chaperone-mediated autophagy. It may affect intestinal homeostasis, host defense against intestinal pathogens, regulation of the gut microbiota, and innate and adaptive immunity. Drugs targeting autophagy could, therefore, have therapeutic potential for treating GI disorders. In this review, we provide an overview of current understanding regarding the evidence for autophagy in GI diseases and updates on potential treatments, including drugs and complementary and alternative medicines.
    Keywords:  Autophagy; Functional dyspepsia; Inflammation; Irritable bowel syndrome
  19. Crit Rev Biochem Mol Biol. 2021 Jun 17. 1-25
      Mitochondria are organelles present in most eukaryotic cells, where they play major and multifaceted roles. The classical notion of the main mitochondrial function as the powerhouse of the cell per se has been complemented by recent discoveries pointing to mitochondria as organelles affecting a number of other auxiliary processes. They go beyond the classical energy provision via acting as a relay point of many catabolic and anabolic processes, to signaling pathways critically affecting cell growth by their implication in de novo pyrimidine synthesis. These additional roles further underscore the importance of mitochondrial homeostasis in various tissues, where its deregulation promotes a number of pathologies. While it has long been known that mitochondria can move within a cell to sites where they are needed, recent research has uncovered that mitochondria can also move between cells. While this intriguing field of research is only emerging, it is clear that mobilization of mitochondria requires a complex apparatus that critically involves mitochondrial proteins of the Miro family, whose role goes beyond the mitochondrial transfer, as will be covered in this review.
    Keywords:  Miro proteins; Mitochondria; endoplasmic reticulum; intercellular transfer; mitophagy; motor proteins; respiration
  20. Chem Sci. 2020 Feb 11. 11(12): 3152-3163
      Tissue regeneration is a crucial self-renewal capability involving many complex biological processes. Although transgenic techniques and fluorescence immunohistochemical staining have promoted our understanding of tissue regeneration, simultaneous quantification and visualization of tissue regeneration processes is not easy to achieve. Herein, we developed a simple and quantitative method for the real-time and non-invasive observation of the process of tissue regeneration. The synthesized ratiometric aggregation-induced-emission (AIE) probe exhibits high selectivity and reversibility for pH responses, good ability to map lysosomal pH both in vitro and in vivo, good biocompatibility and excellent photostability. The caudal fin regeneration of a fish model (medaka larvae) was monitored by tracking the lysosomal pH change. It was found that the mean lysosomal pH is reduced during 24-48 hpa to promote the autophagic activity for cell debris degradation. Our research can quantify the changes in mean lysosomal pH and also exhibit its distribution during the caudal fin regeneration. We believe that the AIE-active lysosomal pH probe can also be potentially used for long-term tracking of various lysosome-involved biological processes, such as tracking the stress responses of tissue, tracking the inflammatory responses, and so on.
  21. Autophagy. 2021 Jun 16.
      The requirement of macroautophagic/autophagic machinery for filamentous fungal development and pathogenicity has been recognized, but the underlying effects and mechanisms remain elusive. The insect pathogenic fungus Metarhizium robertsii infects hosts by cuticular penetration through the formation of the infection structure appressoria. Here, we show that autophagic fluxes were highly activated during the appressorial formation of M. robertsii. Genome-wide deletion of the autophagy-related genes and insect bioassays identified 10 of 23 encoded MrATG genes with requirements for topical fungal infection of insect hosts. Besides the defect in forming appressoria on insects (two null mutants), these virulence-reduced mutants were largely impaired in penetrating cellophane membrane and insect cuticles, suggesting their failures in generating proper appressorium turgor. We found that the conidial storage of lipid droplets (LDs) had no obvious difference between strains, but autophagic LD degradation was impaired in different mutants. After induction of cell autophagy by nitrogen starvation, we found that LD entry into vacuoles was unaffected in the selected mutant cells with potential failures in forming autophagosomes. The finding therefore reveals a microlipophagy machinery employed in this fungus and that the direct engulfment of LDs occurs without inhibition by the downstream defective lipolysis. Our data first unveil the activation and contribution of microlipophagy to fungal infection biology. The obtained technique may benefit future detection of microlipophagy in different organisms by examining vacuolar or lysosomal engulfment of LDs in core autophagic gene deletion mutants.
    Keywords:  Appressorium; LD engulfment; autophagic body cuticle penetration; fungal virulence; lipid droplets; microlipophagy
  22. Transl Neurodegener. 2021 Jun 15. 10(1): 19
      BACKGROUND: Mitochondrial dysfunction plays a prominent role in the pathogenesis of Parkinson's disease (PD), and several genes linked to familial PD, including PINK1 (encoding PTEN-induced putative kinase 1 [PINK1]) and PARK2 (encoding the E3 ubiquitin ligase Parkin), are directly involved in processes such as mitophagy that maintain mitochondrial health. The dominant p.D620N variant of vacuolar protein sorting 35 ortholog (VPS35) gene is also associated with familial PD but has not been functionally connected to PINK1 and PARK2.METHODS: To better mimic and study the patient situation, we used CRISPR-Cas9 to generate heterozygous human SH-SY5Y cells carrying the PD-associated D620N variant of VPS35. These cells were treated with a protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to induce the PINK1/Parkin-mediated mitophagy, which was assessed using biochemical and microscopy approaches.
    RESULTS: Mitochondria in the VPS35-D620N cells exhibited reduced mitochondrial membrane potential and appeared to already be damaged at steady state. As a result, the mitochondria of these cells were desensitized to the CCCP-induced collapse in mitochondrial potential, as they displayed altered fragmentation and were unable to accumulate PINK1 at their surface upon this insult. Consequently, Parkin recruitment to the cell surface was inhibited and initiation of the PINK1/Parkin-dependent mitophagy was impaired.
    CONCLUSION: Our findings extend the pool of evidence that the p.D620N mutation of VPS35 causes mitochondrial dysfunction and suggest a converging pathogenic mechanism among VPS35, PINK1 and Parkin in PD.
    Keywords:  Mitochondrial membrane potential; Mitophagy; PINK1; Parkin; Parkinson’s disease; VPS35
  23. Commun Biol. 2021 Jun 14. 4(1): 736
      Aggregates of hyperphosphorylated tau protein are a pathological hallmark of more than 20 distinct neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, and frontotemporal dementia. While the exact mechanism of tau aggregation is unknown, the accumulation of aggregates correlates with disease progression. Here we report a genome-wide CRISPR screen to identify modulators of endogenous tau protein for the first time. Primary screens performed in SH-SY5Y cells, identified positive and negative regulators of tau protein levels. Hit validation of the top 43 candidate genes was performed using Ngn2-induced human cortical excitatory neurons. Using this approach, genes and pathways involved in modulation of endogenous tau levels were identified, including chromatin modifying enzymes, neddylation and ubiquitin pathway members, and components of the mTOR pathway. TSC1, a critical component of the mTOR pathway, was further validated in vivo, demonstrating the relevance of this screening strategy. These findings may have implications for treating neurodegenerative diseases in the future.
  24. Front Genet. 2021 ;12 667283
      Discovered in 2009, the DEP-domain containing mTOR-interacting protein, DEPTOR, is a known regulator of the mechanistic target of rapamycin (mTOR), an evolutionarily conserved kinase that regulates diverse cellular processes in response to environmental stimuli. DEPTOR was originally identified as a negative regulator of mTOR complexes 1 (mTORC1) and 2 (mTORC2). However, recent discoveries have started to unravel the roles of DEPTOR in mTOR-independent responses. In the past few years, mTOR emerged as an important regulator of skeletal development, growth, and homeostasis; the dysregulation of its activity contributes to the development of several skeletal diseases, both chronic and genetic. Even more recently, several groups have reported on the relevance of DEPTOR in skeletal biology through its action on mTOR-dependent and mTOR-independent pathways. In this review, we summarize the current understanding of DEPTOR in skeletal development and disease.
    Keywords:  DEPTOR; bone; cartilage; mTOR; skeleton
  25. Cell Rep. 2021 Jun 15. pii: S2211-1247(21)00623-9. [Epub ahead of print]35(11): 109259
      Dysfunction of the endolysosomal-autophagy network is emerging as an important pathogenic process in Alzheimer's disease. Mutations in the sorting receptor-encoding gene SORL1 cause autosomal-dominant Alzheimer's disease, and SORL1 variants increase risk for late-onset AD. To understand the contribution of SORL1 mutations to AD pathogenesis, we analyze the effects of a SORL1 truncating mutation on SORL1 protein levels and endolysosome function in human neurons. We find that truncating mutation results in SORL1 haploinsufficiency and enlarged endosomes in human neurons. Analysis of isogenic SORL1 wild-type, heterozygous, and homozygous null neurons demonstrates that, whereas SORL1 haploinsufficiency results in endosome dysfunction, complete loss of SORL1 leads to additional defects in lysosome function and autophagy. Neuronal endolysosomal dysfunction caused by loss of SORL1 is relieved by extracellular antisense oligonucleotide-mediated reduction of APP protein, demonstrating that PSEN1, APP, and SORL1 act in a common pathway regulating the endolysosome system, which becomes dysfunctional in AD.
    Keywords:  Alzheimer's disease; SORL1; amyloid precursor protein; autophagy; endosome; iPSC; lysosome
  26. Int J Biol Sci. 2021 ;17(8): 1895-1908
      Ovarian cancer is a common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic role remains unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on the regulation of autophagic and apoptotic pathways in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using MTT and colony formation assays in SK-OV-3 cells. PKM2 expression was positively correlated with the severity of the tumor, and expression of pro-apoptotic proteins increased in SK-OV-3 cells following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by mTOR inhibition. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation of SK-OV-3 cells. Compound 3K treatment suppressed tumor progression in an in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected the Warburg effect and induced autophagic cell death. Therefore, use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.
    Keywords:  apoptosis; autophagy; compound 3K; ovarian cancer; pyruvate kinase M2
  27. Front Cell Dev Biol. 2021 ;9 673395
      Parkinson's disease (PD) is a pathological condition characterized by the aggregation and the resultant presence of intraneuronal inclusions termed Lewy bodies (LBs) and Lewy neurites which are mainly composed of fibrillar α-synuclein (α-syn) protein. Pathogenic aggregation of α-syn is identified as the major cause of LBs deposition. Several mutations in α-syn showing varied aggregation kinetics in comparison to the wild type (WT) α-syn are reported in PD (A30P, E46K, H 50Q, G51D, A53E, and A53T). Also, the cell-to-cell spread of pathological α-syn plays a significant role in PD development. Interestingly, it has also been suggested that the pathology of PD may begin in the gastrointestinal tract and spread via the vagus nerve (VN) to brain proposing the gut-brain axis of α-syn pathology in PD. Despite multiple efforts, the behavior and functions of this protein in normal and pathological states (specifically in PD) is far from understood. Furthermore, the etiological factors responsible for triggering aggregation of this protein remain elusive. This review is an attempt to collate and present latest information on α-syn in relation to its structure, biochemistry and biophysics of aggregation in PD. Current advances in therapeutic efforts toward clearing the pathogenic α-syn via autophagy/lysosomal flux are also reviewed and reported.
    Keywords:  Parkinson’s disease; autophagy; gut–brain axis; neurotoxicity; α-synuclein
  28. Nat Commun. 2021 06 15. 12(1): 3651
      Extracellular cytokines are enriched in the tumor microenvironment and regulate various important properties of cancers, including autophagy. However, the precise molecular mechanisms underlying the link between autophagy and extracellular cytokines remain to be elucidated. In the present study, we demonstrate that IL-6 activates autophagy through the IL-6/JAK2/BECN1 pathway and promotes chemotherapy resistance in colorectal cancer (CRC). Mechanistically, IL-6 triggers the interaction between JAK2 and BECN1, where JAK2 phosphorylates BECN1 at Y333. We demonstrate that BECN1 Y333 phosphorylation is crucial for BECN1 activation and IL-6-induced autophagy by regulating PI3KC3 complex formation. Furthermore, we investigate BECN1 Y333 phosphorylation as a predictive marker for poor CRC prognosis and chemotherapy resistance. Combination treatment with autophagy inhibitors or pharmacological agents targeting the IL-6/JAK2/BECN1 signaling pathway may represent a potential strategy for CRC cancer therapy.
  29. Trends Cancer. 2021 Jun 07. pii: S2405-8033(21)00104-7. [Epub ahead of print]
      Autophagy is a catabolic intracellular nutrient-scavenging pathway triggered by nutrient deprivation and stress that captures and degrades intracellular proteins and organelles in lysosomes. The breakdown products are then recycled into metabolic pathways to sustain survival. Organelle turnover by autophagy contributes to quality control and suppresses inflammation. Autophagy is upregulated in many cancers and supports their growth, survival, and malignancy in a tumor cell-autonomous fashion. Host autophagy also promotes tumor growth by maintaining a supply of essential nutrients and suppressing innate and adaptive antitumor immune responses. Autophagy is also upregulated in response to cancer therapy and confers treatment resistance. Thus, autophagy is a cancer vulnerability and its inhibition is under investigation as a novel therapeutic approach.
    Keywords:  T cells; autophagy; cancer; immune response; interferon; metabolism
  30. Front Cell Dev Biol. 2021 ;9 666631
      Autophagy, a lysosomal degradation pathway, serves as a protective cellular mechanism in maintaining cell and tissue homeostasis under mechanical stimulation. As the mechanosensitive cells, periodontal ligament stem cells (PDLSCs) play an important role in the force-induced inflammatory bone remodeling and tooth movement process. However, whether and how autophagy in PDLSCs influences the inflammatory bone remodeling process under mechanical force stimuli is still unknown. In this study, we found that mechanical force stimuli increased the expression of the autophagy protein LC3, the number of M1 macrophages and osteoclasts, as well as the ratio of M1/M2 macrophages in the compression side of the periodontal ligament in vivo. These biological changes induced by mechanical force were repressed by the application of an autophagy inhibitor 3-methyladenine. Moreover, autophagy was activated in the force-loaded PDLSCs, and force-stimulated PDLSC autophagy further induced M1 macrophage polarization in vitro. The macrophage polarization could be partially blocked by the administration of autophagy inhibitor 3-methyladenine or enhanced by the administration of autophagy activator rapamycin in PDLSCs. Mechanistically, force-induced PDLSC autophagy promoted M1 macrophage polarization via the inhibition of the AKT signaling pathway. These data suggest a novel mechanism that force-stimulated PDLSC autophagy steers macrophages into the M1 phenotype via the AKT signaling pathway, which contributes to the inflammatory bone remodeling and tooth movement process.
    Keywords:  AKT signaling; autophagy; bone remodeling; inflammation; macrophage polarization; mechanical force; periodontal ligament stem cells; tooth movement
  31. Front Cell Dev Biol. 2021 ;9 656604
      Skeletal muscle protein synthesis is a highly complex process, influenced by nutritional status, mechanical stimuli, repair programs, hormones, and growth factors. The molecular aspects of protein synthesis are centered around the mTORC1 complex. However, the intricacies of mTORC1 regulation, both up and downstream, have expanded overtime. Moreover, the plastic nature of skeletal muscle makes it a unique tissue, having to coordinate between temporal changes in myofiber metabolism and hypertrophy/atrophy stimuli within a tissue with considerable protein content. Skeletal muscle manages the push and pull between anabolic and catabolic pathways through key regulatory proteins to promote energy production in times of nutrient deprivation or activate anabolic pathways in times of nutrient availability and anabolic stimuli. Branched-chain amino acids (BCAAs) can be used for both energy production and signaling to induce protein synthesis. The metabolism of BCAAs occur in tandem with energetic and anabolic processes, converging at several points along their respective pathways. The fate of intramuscular BCAAs adds another layer of regulation, which has consequences to promote or inhibit muscle fiber protein anabolism. This review will outline the general mechanisms of muscle protein synthesis and describe how metabolic pathways can regulate this process. Lastly, we will discuss how BCAA availability and demand coordinate with synthesis mechanisms and identify key factors involved in intramuscular BCAA trafficking.
    Keywords:  AMPK (5′-AMP activated kinase); BCKD; branch chain amino acids; branched-chain α-ketoacid dehydrogenase; mammalian target of rapamycin; protein synthesis; skeletal muscle
  32. Nat Methods. 2021 Jun 14.
      Lysosomes are critical for cellular metabolism and are heterogeneously involved in various cellular processes. The ability to measure lysosomal metabolic heterogeneity is essential for understanding their physiological roles. We therefore built a single-lysosome mass spectrometry (SLMS) platform integrating lysosomal patch-clamp recording and induced nano-electrospray ionization (nanoESI)/mass spectrometry (MS) that enables concurrent metabolic and electrophysiological profiling of individual enlarged lysosomes. The accuracy and reliability of this technique were validated by supporting previous findings, such as the transportability of lysosomal cationic amino acids transporters such as PQLC2 and the lysosomal trapping of lysosomotropic, hydrophobic weak base drugs such as lidocaine. We derived metabolites from single lysosomes in various cell types and classified lysosomes into five major subpopulations based on their chemical and biological divergence. Senescence and carcinoma altered metabolic profiles of lysosomes in a type-specific manner. Thus, SLMS can open more avenues for investigating heterogeneous lysosomal metabolic changes during physiological and pathological processes.
  33. Biochem J. 2021 Jun 25. 478(12): 2297-2308
      Autophagy is an important component of the innate immune response that restricts infection by different types of pathogens. Viruses have developed multiple strategies to avoid autophagy to complete their replication cycle and promote spreading to new hosts. Here, we report that the ubiquitin deconjugases encoded in the N-terminal domain of the large tegument proteins of Epstein-Barr virus (EBV), Kaposi Sarcoma herpesvirus (KSHV) and human cytomegalovirus (HCMV), but not herpes simplex virus-1 (HSV-1), regulate selective autophagy by inhibiting the activity of the autophagy receptor SQSTM1/p62. We found that all the homologs bind to and deubiquitinate SQSTM1/p62 but with variable efficiency, which correlates with their capacity to prevent the colocalization of light chain 3 (LC3) with SQSTM1/p62 aggregates and promote the accumulation of a model autophagy substrate. The findings highlight important differences in the strategies by which herpesviruses interfere with selective autophagy.
    Keywords:  autophagy; herpesvirus; ubiquitin protease; ubiquitin-proteasome system
  34. Expert Opin Ther Targets. 2021 Jun 17. 1-14
      INTRODUCTION: Autophagy is a cellular catabolic mechanism that helps clear damaged cellular components and is essential for normal cellular and tissue function. The sigma-1 receptor (σ-1R) is a chaperone protein involved in signal transduction, neurite outgrowth, and plasticity, improving memory, and neuroprotection. Recent evidence shows that σ-1R can promote autophagy. Autophagy activation by the σ-1Rs along with other neuroprotective effects makes it an interesting target for the treatment of Alzheimer's disease. AF710B, T-817 MA, and ANAVEX2-73 are some of the σ-1R agonists which have shown promising results and have entered clinical trials. These molecules have also been found to induce autophagy and show cytoprotective effects in cellular models.AREAS COVERED: This review provides insight into the current understanding of σ-1R functions related to autophagy and their role in alleviating AD.
    EXPERT OPINION: We propose a mechanism through which the activation of σ-1R and autophagy could alter amyloid precursor protein processing to inhibit amyloid-β production by reconstituting cholesterol and gangliosides in the lipid raft to offer neuroprotection against AD. Future AD treatment could involve the combined targeting of the σ-1R and autophagy activation. We suggest that future studies investigate the link between autophagy the σ-1R and AD.
    Keywords:  Alzheimer’s disease; anavex2-73; autophagy; avp-786; lipid raft; mitochondrial stress; neurodegenerative disease; non-opioid-sigma-1 receptor; sigma-1 agonists; sigma-1 receptor
  35. J Immunol. 2021 Jun 16. pii: ji2001364. [Epub ahead of print]
      Insufficient autophagic degradation has been implicated in accelerated cellular senescence during chronic obstructive pulmonary disease (COPD) pathogenesis. Aging-linked and cigarette smoke (CS)-induced functional deterioration of lysosomes may be associated with impaired autophagy. Lysosomal membrane permeabilization (LMP) is indicative of damaged lysosomes. Galectin-3 and tripartite motif protein (TRIM) 16 play a cooperative role in recognizing LMP and inducing lysophagy, a lysosome-selective autophagy, to maintain lysosome function. In this study, we sought to examine the role of TRIM16-mediated lysophagy in regulating CS-induced LMP and cellular senescence during COPD pathogenesis by using human bronchial epithelial cells and lung tissues. CS extract (CSE) induced lysosomal damage via LMP, as detected by galectin-3 accumulation. Autophagy was responsible for modulating LMP and lysosome function during CSE exposure. TRIM16 was involved in CSE-induced lysophagy, with impaired lysophagy associated with lysosomal dysfunction and accelerated cellular senescence. Airway epithelial cells in COPD lungs showed an increase in lipofuscin, aggresome and galectin-3 puncta, reflecting accumulation of lysosomal damage with concomitantly reduced TRIM16 expression levels. Human bronchial epithelial cells isolated from COPD patients showed reduced TRIM16 but increased galectin-3, and a negative correlation between TRIM16 and galectin-3 protein levels was demonstrated. Damaged lysosomes with LMP are accumulated in epithelial cells in COPD lungs, which can be at least partly attributed to impaired TRIM16-mediated lysophagy. Increased LMP in lung epithelial cells may be responsible for COPD pathogenesis through the enhancement of cellular senescence.
  36. Sci Immunol. 2021 Jun 18. pii: eabg0791. [Epub ahead of print]6(60):
      The nutrient-sensing mammalian target of rapamycin (mTOR) is integral to cell fate decisions after T cell activation. Sustained mTORC1 activity favors the generation of terminally differentiated effector T cells instead of follicular helper and memory T cells. This is particularly pertinent for T cell responses of older adults who have sustained mTORC1 activation despite dysfunctional lysosomes. Here, we show that lysosome-deficient T cells rely on late endosomes rather than lysosomes as an mTORC1 activation platform, where mTORC1 is activated by sensing cytosolic amino acids. T cells from older adults have an increased expression of the plasma membrane leucine transporter SLC7A5 to provide a cytosolic amino acid source. Hence, SLC7A5 and VPS39 deficiency (a member of the HOPS complex promoting early to late endosome conversion) substantially reduced mTORC1 activities in T cells from older but not young individuals. Late endosomal mTORC1 is independent of the negative-feedback loop involving mTORC1-induced inactivation of the transcription factor TFEB that controls expression of lysosomal genes. The resulting sustained mTORC1 activation impaired lysosome function and prevented lysosomal degradation of PD-1 in CD4+ T cells from older adults, thereby inhibiting their proliferative responses. VPS39 silencing of human T cells improved their expansion to pertussis and to SARS-CoV-2 peptides in vitro. Furthermore, adoptive transfer of CD4+ Vps39-deficient LCMV-specific SMARTA cells improved germinal center responses, CD8+ memory T cell generation, and recall responses to infection. Thus, curtailing late endosomal mTORC1 activity is a promising strategy to enhance T cell immunity.