bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒12‒20
forty papers selected by
Viktor Korolchuk
Newcastle University

  1. Dev Cell. 2020 Dec 07. pii: S1534-5807(20)30925-4. [Epub ahead of print]
      Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function, and neurodegeneration. The compositional and functional alterations in NPC lysosomes and nature of aberrant cholesterol-mTORC1 signaling contribution to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion, enhanced membrane damage, and defective mitophagy. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.
    Keywords:  ESCRT; NPC1; autophagy; cholesterol; lysosome; mTORC1; mitochondria; proteolysis; proteomics
  2. Front Oncol. 2020 ;10 597743
      Tumor cells can employ epithelial-mesenchymal transition (EMT) or autophagy in reaction to microenvironmental stress. Importantly, EMT and autophagy negatively regulate each other, are able to interconvert, and both have been shown to contribute to drug-resistance in glioblastoma (GBM). EMT has been considered one of the mechanisms that confer invasive properties to GBM cells. Autophagy, on the other hand, may show dual roles as either a GBM-promoter or GBM-suppressor, depending on microenvironmental cues. The Wingless (WNT) signaling pathway regulates a plethora of developmental and biological processes such as cellular proliferation, adhesion and motility. As such, GBM demonstrates deregulation of WNT signaling in favor of tumor initiation, proliferation and invasion. In EMT, WNT signaling promotes induction and stabilization of different EMT activators. WNT activity also represses autophagy, while nutrient deprivation induces β-catenin degradation via autophagic machinery. Due to the importance of the WNT pathway to GBM, and the role of WNT signaling in EMT and autophagy, in this review we highlight the effects of the WNT signaling in the regulation of both processes in GBM, and discuss how the crosstalk between EMT and autophagy may ultimately affect tumor biology.
    Keywords:  WNT signaling; autophagy; chaperone-mediated autophagy; epithelial-mesenchymal transition; glioblastoma; metabolic reprograming; microautophagy
  3. Elife. 2020 12 15. pii: e63694. [Epub ahead of print]9
      Mitophagy plays an important role in mitochondrial homeostasis. In yeast, the phosphorylation of the mitophagy receptor Atg32 by casein kinase 2 is essential for mitophagy. This phosphorylation is counteracted by the yeast equivalent of the STRIPAK complex consisting of the PP2A-like protein phosphatase Ppg1 and Far3-7-8-9-10-11 (Far complex), but the underlying mechanism remains elusive. Here we show that two subpopulations of the Far complex reside in the mitochondria and endoplasmic reticulum, respectively, and play distinct roles; the former inhibits mitophagy via Atg32 dephosphorylation, and the latter regulates TORC2 signaling. Ppg1 and Far11 form a subcomplex, and Ppg1 activity is required for the assembling integrity of Ppg1-Far11-Far8. The Far complex preferentially interacts with phosphorylated Atg32, and this interaction is weakened by mitophagy induction. Furthermore, the artificial tethering of Far8 to Atg32 prevents mitophagy. Taken together, the Ppg1-mediated Far complex formation and its dissociation from Atg32 are crucial for mitophagy regulation.
    Keywords:  Atg32; Far complex; Ppg1; S. cerevisiae; STRIPAK complex; autophagy; cell biology; mitophagy
  4. Brain Sci. 2020 Dec 09. pii: E958. [Epub ahead of print]10(12):
      O-GlcNAc transferase (OGT) is a ubiquitous enzyme that regulates the addition of β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of target proteins. Autophagy is a cellular process of self-digestion, in which cytoplasmic resources, such as aggregate proteins, toxic compounds, damaged organelles, mitochondria, and lipid molecules, are degraded and recycled. Here, we examined how three different OGT inhibitors, alloxan, BXZ2, and OSMI-1, modulate O-GlcNAcylation in rat cortical neurons, and their autophagic effects were determined by immunoblot and immunofluorescence assays. We found that the treatment of cortical neurons with an OGT inhibitor decreased O-GlcNAcylation levels and increased LC3-II expression. Interestingly, the pre-treatment with rapamycin, an mTOR inhibitor, further increased the expression levels of LC3-II induced by OGT inhibition, implicating the involvement of mTOR signaling in O-GlcNAcylation-dependent autophagy. In contrast, OGT inhibitor-mediated autophagy was significantly attenuated by 3-methyladenine (3-MA), a blocker of autophagosome formation. However, when pre-treated with chloroquine (CQ), a lysosomotropic agent and a late-stage autophagy inhibitor, OGT inhibitors significantly increased LC3-II levels along with LC3 puncta formation, indicating the stimulation of autophagic flux. Lastly, we found that OGT inhibitors significantly decreased the levels of the autophagy substrate p62/SQSTM1 while increasing the expression of lysosome-associated membrane protein 1 (LAMP1). Together, our study reveals that the modulation of O-GlcNAcylation by OGT inhibition regulates mTOR-dependent autophagy in rat cortical neurons.
    Keywords:  LC3 puncta; O-GlcNAc transferase (OGT); O-GlcNAcylation; autophagy; cortical neuron; mTOR
  5. Neuropathol Appl Neurobiol. 2020 Dec 18.
      AIMS: Limb-girdle muscular dystrophy R9 (LGMDR9) is an autosomal recessive disorder caused by mutations in the fukutin-related protein gene (FKRP), encoding a glycosyltransferase involved in α-dystroglycan modification. Muscle atrophy, a significant feature of LGMDR9, occurs by a change in the normal balance between protein synthesis and protein degradation. The ubiquitin-proteasome system (UPS) and autophagy-lysosomal system play a key role in protein degradation in skeletal muscle cells, but their involvement in the pathology of LGMDR9 is still largely unknown. We have aimed at clarifying whether proteolysis through the UPS and the autophagy-lysosomal pathway is dysregulated in LGMDR9 patients.METHODS: Vastus lateralis biopsies from 8 normal controls and 12 LGMDR9 patients harbouring the c.826C>A/c.826C>A FKRP genotype were assessed for protein markers related to UPS, the autophagy-lysosomal system and endoplasmic reticulum (ER) stress/unfolded protein response (UPR), followed by ultrastructural analysis by transmission electron microscopy (TEM).
    RESULTS: Protein levels of E3 ubiquitin ligases Atrogin-1 and MuRF1 showed a pattern similar to normal controls. Elevation of the autophagy markers Atg7, LC3B-II, decreased level of p62 as well as downregulation of the negative autophagy regulator mTORC1, indicated an activation of autophagy in LGMDR9. Mitophagy markers Bnip3 and Parkin were decreased. TEM analysis demonstrated accumulation of autophagosome-like structures in LGMDR9 muscle. There was also an increase in the expression of ER stress/UPR markers PDI, peIF2α and CHOP and a decrease of IRE1α. However, GRP94, Bip and Calnexin remained unchanged.
    CONCLUSION: Our findings indicate that autophagy and ER stress are induced in LGMDR9 muscle.
    Keywords:  ER stress; LGMD2I; LGMDR9; UPS; autophagy; muscle atrophy
  6. Cell Mol Life Sci. 2020 Dec 19.
      The complex morphology of neurons, the specific requirements of synaptic neurotransmission and the accompanying metabolic demands create a unique challenge for proteostasis. The main machineries for neuronal protein synthesis and degradation are localized in the soma, while synaptic junctions are found at vast distances from the cell body. Sophisticated mechanisms must, therefore, ensure efficient delivery of newly synthesized proteins and removal of faulty proteins. These requirements are exacerbated at presynaptic sites, where the demands for protein turnover are especially high due to synaptic vesicle release and recycling that induces protein damage in an intricate molecular machinery, and where replacement of material is hampered by the extreme length of the axon. In this review, we will discuss the contribution of the two major pathways in place, autophagy and the endolysosomal system, to presynaptic protein turnover and presynaptic function. Although clearly different in their biogenesis, both pathways are characterized by cargo collection and transport into distinct membrane-bound organelles that eventually fuse with lysosomes for cargo degradation. We summarize the available evidence with regard to their degradative function, their regulation by presynaptic machinery and the cargo for each pathway. Finally, we will discuss the interplay of both pathways in neurons and very recent findings that suggest non-canonical functions of degradative organelles in synaptic signalling and plasticity.
    Keywords:  Autophagy; Axonal boutons; Endolysosomal system; Proteostasis; Synaptic plasticity
  7. Sci Rep. 2020 Dec 18. 10(1): 22334
      Even though K63-linked polyubiquitin chains do not target proteins for proteasomal degradation, they play nevertheless a complementary protective role in maintaining protein homeostasis by directing malfunctioning proteins and organelles to inclusion bodies or autophagosomes. A paradigm for this process is the sequestration and autophagic degradation of dysfunctional mitochondria. Although studies have shown that K63-ubiquitylation of mitochondrial proteins by the ubiquitin ligase Parkin is important in this process, it is presently not clear if this modification also suffices to initiate this cascade of events. To address this question, we have engineered the ubiquitin ligase ProxE3, which in an inducible manner synthesizes K63-linked ubiquitin chains on the surface of mitochondria. We found that the presence of K63-linked ubiquitin chains on mitochondria resulted in the recruitment of the ubiquitin adaptor p62 and induced a dramatic redistribution of mitochondria, which was reminiscent to the Parkin-facilitated sequestration in response to mitochondrial uncoupler. However, ProxE3 did not induce autophagic degradation of mitochondria. Our data show that K63-linked ubiquitin chains at the mitochondrial membrane are sufficient for the induction of mitochondrial sequestration, but not mitophagy, without the need of extrinsically inflicting mitochondrial dysfunction.
  8. Front Cell Dev Biol. 2020 ;8 607392
      Parkinson's disease (PD) is a complex neurodegenerative disease with pathological hallmarks including progressive neuronal loss from the substantia nigra pars compacta and α-synuclein intraneuronal inclusions, known as Lewy bodies. Although the etiology of PD remains elusive, mitochondrial damage has been established to take center stage in the pathogenesis of PD. Mitochondria are critical to cellular energy production, metabolism, homeostasis, and stress responses; the association with PD emphasizes the importance of maintenance of mitochondrial network integrity. To accomplish the pleiotropic functions, mitochondria are dynamic not only within their own network but also in orchestrated coordination with other organelles in the cellular community. Through physical contact sites, signal transduction, and vesicle transport, mitochondria and intracellular organelles achieve the goals of calcium homeostasis, redox homeostasis, protein homeostasis, autophagy, and apoptosis. Herein, we review the finely tuned interactions between mitochondria and surrounding intracellular organelles, with focus on the nucleus, endoplasmic reticulum, Golgi apparatus, peroxisomes, and lysosomes. Participants that may contribute to the pathogenic mechanisms of PD will be highlighted in this review.
    Keywords:  Parkinson's disease; endoplasmic reticulum; golgi apparatus; interorganelle communication; lysosome; mitochondria; mitophagy; peroxisome
  9. Biochim Biophys Acta Mol Cell Res. 2020 Dec 11. pii: S0167-4889(20)30284-6. [Epub ahead of print] 118926
      Cellular homeostasis and adaptation to various environmental conditions are importantly regulated by the sophisticated mechanism of autophagy and its crosstalk with Wnt signaling and other developmental pathways. Both autophagy and Wnt signaling are involved in embryogenesis and differentiation. Autophagy is responsible for degradation and recycling of cytosolic materials by directing them to lysosomes through the phagophore compartment. A dual feedback mechanism regulates the interface between autophagy and Wnt signaling pathways. During nutrient deprivation, β-catenin and Dishevelled (essential Wnt signaling proteins) are targeted for autophagic degradation by LC3. When Wnt signaling is activated, β-catenin acts as a corepressor of one of the autophagy proteins, p62. In contrast, another key Wnt signaling protein, GSK3β, negatively regulates the Wnt pathway and has been shown to induce autophagy by phosphorylation of the TSC complex. This article reviews the interplay between autophagy and Wnt signaling, describing how β-catenin functions as a key cellular integration point coordinating proliferation with autophagy, and it discusses the clinical importance of the crosstalk between these mechanisms.
    Keywords:  Autophagy; Wnt signaling pathway; differentiation; regulatory feedback mechanism
  10. J Exp Med. 2021 Mar 01. pii: e20201416. [Epub ahead of print]218(3):
      White adipose tissues (WAT) play crucial roles in maintaining whole-body energy homeostasis, and their dysfunction can contribute to hepatic insulin resistance and type 2 diabetes mellitus (T2DM). However, the mechanisms underlying these alterations remain unknown. By analyzing the transcriptome landscape in human adipocytes based on available RNA-seq datasets from lean, obese, and T2DM patients, we reveal elevated mitochondrial reactive oxygen species (ROS) pathway and NF-κB signaling with altered fatty acid metabolism in T2DM adipocytes. Mice with adipose-specific deletion of mitochondrial redox Trx2 develop hyperglycemia, hepatic insulin resistance, and hepatic steatosis. Trx2-deficient WAT exhibited excessive mitophagy, increased inflammation, and lipolysis. Mechanistically, mitophagy was induced through increasing ROS generation and NF-κB-dependent accumulation of autophagy receptor p62/SQSTM1, which recruits damaged mitochondria with polyubiquitin chains. Importantly, administration of ROS scavenger or NF-κB inhibitor ameliorates glucose and lipid metabolic disorders and T2DM progression in mice. Taken together, this study reveals a previously unrecognized mechanism linking mitophagy-mediated adipose inflammation to T2DM with hepatic insulin resistance.
  11. Life Sci Alliance. 2021 Feb;pii: e202000965. [Epub ahead of print]4(2):
      Hunger drives food-seeking behaviour and controls adaptation of organisms to nutrient availability and energy stores. Lipids constitute an essential source of energy in the cell that can be mobilised during fasting by autophagy. Selective degradation of proteins by autophagy is made possible essentially by the presence of LIR and KFERQ-like motifs. Using in silico screening of Drosophila proteins that contain KFERQ-like motifs, we identified and characterized the adaptor protein Arouser, which functions to regulate fat storage and mobilisation and is essential during periods of food deprivation. We show that hypomorphic arouser mutants are not satiated, are more sensitive to food deprivation, and are more aggressive, suggesting an essential role for Arouser in the coordination of metabolism and food-related behaviour. Our analysis shows that Arouser functions in the fat body through nutrient-related signalling pathways and is degraded by endosomal microautophagy. Arouser degradation occurs during feeding conditions, whereas its stabilisation during non-feeding periods is essential for resistance to starvation and survival. In summary, our data describe a novel role for endosomal microautophagy in energy homeostasis, by the degradation of the signalling regulatory protein Arouser.
  12. Biol Cell. 2020 Dec 11.
      The regulation of nutrient uptake into cells is important, as it allows to either increase biomass for cell growth or to preserve homeostasis. A key strategy to adjust cellular nutrient uptake is the reconfiguration of the nutrient transporter repertoire at the plasma membrane by the addition of nutrient transporters through the secretory pathway and by their endocytic removal. In this review we focus on the mechanisms that regulate selective nutrient transporter endocytosis, which is mediated by the α-arrestin protein family. In the budding yeast Saccharomyces cerevisiae, 14 different α-arrestins (also named arrestin-related trafficking adaptors, ARTs) function as adaptors for the ubiquitin ligase Rsp5. They instruct Rsp5 to ubiquitinate subsets of nutrient transporters to orchestrate their endocytosis. The ART proteins are under multilevel control of the major nutrient sensing systems, including amino acid sensing by the general amino acid control (GAAC) and target of rapamycin (TOR) pathways, and energy sensing by 5'-adenosine-monophosphate-dependent kinase (AMPK). The function of the six human α-arrestins is comparably under-characterized. Here, we summarize the current knowledge about the function, regulation and substrates of yeast ARTs and human α-arrestins, and highlight emerging communalities and general principles. This article is protected by copyright. All rights reserved.
    Keywords:  Arrestin; Endocytosis; Metabolism; Nutrient transporter; Ubiquitin; Ubiquitin ligase; α-arrestin
  13. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Dec 14. pii: S1388-1981(20)30260-2. [Epub ahead of print] 158868
      Autophagy is mediated by membrane-bound organelles and it is an intrinsic catabolic and recycling process of the cell, which is very important for the health of organisms. The biogenesis of autophagic membranes is still incompletely understood. In vitro studies suggest that Atg2 protein transports lipids presumably from the ER to the expanding autophagic structures. Autophagy research has focused heavily on proteins and very little is known about the lipid composition of autophagic membranes. Here we describe a method for immunopurification of autophagic structures from Drosophila melanogaster (an excellent model to study autophagy in a complete organism) for subsequent lipidomic analysis. Western blots of several organelle markers indicate the high purity of the isolated autophagic vesicles, visualized by various microscopy techniques. Mass spectrometry results show that phosphatidylethanolamine (PE) is the dominant lipid class in wild type (control) membranes. We demonstrate that in Atg2 mutants (Atg2-), phosphatidylinositol (PI), negatively charged phosphatidylserine (PS), and phosphatidic acid (PA) with longer fatty acyl chains accumulate on stalled, negatively charged phagophores. Tandem mass spectrometry analysis of lipid species composing the lipid classes reveal the enrichment of unsaturated PE and phosphatidylcholine (PC) in controls versus PI, PS and PA species in Atg2-. Significant differences in the lipid profiles of control and Atg2- flies suggest that the lipid composition of autophagic membranes dynamically changes during their maturation. These lipidomic results also point to the in vivo lipid transport function of the Atg2 protein, pointing to its specific role in the transport of short fatty acyl chain PE species.
    Keywords:  Atg2; Drosophila melanogaster; autophagic membranes; autophagy; lipid composition; lipidomics
  14. Plant Signal Behav. 2020 Dec 17. 1861769
      Chloroplasts and mitochondria serve as intracellular energy production sites that are powered by the electron transport chain in their membranes. These organelles constantly accumulate damage, as their energetic reactions generate reactive oxygen species. To prevent the accumulation of damaged organelles and perturbation of cellular homeostasis, eukaryotic cells must remove damaged mitochondria and chloroplasts. Autophagy is the main route by which organelles are degraded. A type of mitochondrion-targeted autophagy known as mitophagy removes damaged mitochondria in mammalian cells; dysfunctional mitochondria that lose their membrane potential are marked by protein ubiquitination, becoming targets of selective mitophagy. Studies of the quality control system for chloroplasts in plants revealed the involvement of both autophagy and ubiquitination in the degradation of damaged chloroplasts. We recently assessed the relationship between chloroplast-associated ubiquitination mediated by PLANT U-BOX4 (PUB4) and chloroplast-targeted autophagy (chlorophagy) in the turnover of oxidatively damaged chloroplasts. Multiple assays using an Arabidopsis thaliana mutant revealed that PUB4-associated ubiquitination is dispensable for the induction of chlorophagy. Here, we describe the parallel functions of PUB4 and chlorophagy in chloroplast turnover and plant growth.
    Keywords:  PLANT U-BOX4; autophagy; chlorophagy; chloroplast; photodamage; ubiquitination
  15. Med Res Rev. 2020 Dec 13.
      Macroautophagy (autophagy) is an evolutionarily conserved and dynamic degradation/recycling pathway in which portions of the cytoplasm, such as dysfunctional proteins and surplus organelles, are engulfed by double-membrane bound vesicles through a lysosome-dependent process. As the only proteolytic enzyme of the core mammalian autophagy proteins, autophagy-related protein 4 (ATG4) primes newly synthesized pro-light chain 3 (LC3) to form LC3-I that attaches to phosphatidylethanolamine and delipidates LC3-PE to LC3-I for recycling. Besides autophagy, ATG4 has been shown to be involved in regulating various biological and pathological processes. The roles of ATG4 in cancer therapy, a methodology for ATG4 activity detection, and the discovery of chemical modulators have been well-reviewed. However, a comprehensive summary on how ATG4 is regulated by multiple factors and, thereby, how ATG4 influences autophagy or other pathways remains lacking. In this paper, we summarize multiple processes and molecules that regulate the activity of ATG4, such as micro-RNAs, posttranslational modifications, and small molecules. Additionally, we focus on the relationship between ATG4 and diverse diseases, including cancer, neurodegeneration, microbial infection, and other diseases. It provides insight regarding potential ATG4-targeted therapeutic opportunities, which could be beneficial for future studies and human health.
    Keywords:  ATG4; autophagy; disease; inhibitor; regulatory factor
  16. Nature. 2020 Dec 16.
      Autophagy, a process of degradation that occurs via the lysosomal pathway, has an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microorganisms, and host protection against infectious diseases1,2. Autophagy is known to be induced by stimuli such as nutrient deprivation and suppression of mTOR, but little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. Here, using genome-wide short interfering RNA screens, we find that the endosomal protein sorting nexin 5 (SNX5)3,4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We show that SNX5 deletion increases cellular susceptibility to viral infection in vitro, and that Snx5 knockout in mice enhances lethality after infection with several human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing class III phosphatidylinositol-3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of phosphatidylinositol-3-phosphate (PtdIns(3)P) and recruitment of the PtdIns(3)P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism-SNX5-dependent PI3KC3-C1 activation at endosomes-for initiation of autophagy during viral infection.
  17. J Cell Mol Med. 2020 Dec 12.
      Yes-associated protein (YAP), a major effector of the Hippo signalling pathway, is widely implicated in vascular pathophysiology processes. Here, we identify a new role of YAP in the regulation of vascular senescence. The inhibition or deficiency and overexpression of YAP were performed in human umbilical vein endothelial cells (HUVECs) and isolated vascular tissues. Cellular and vascular senescence was assessed by analysis of the senescence-associated β-galactosidase (SA-β-gal) and expression of senescence markers P16, P21, P53, TERT and TRF1. We found that YAP was highly expressed in old vascular tissues, inhibition and knockdown of YAP decreased senescence, while overexpression of YAP increased the senescence in both HUVECs and vascular tissues. In addition, autophagic flux blockage and mTOR pathway activation were observed during YAP-induced HUVECs and vascular senescence, which could be relieved by the inhibition and knockdown of YAP. Moreover, YAP-promoted cellular and vascular senescence could be relieved by mTOR inhibition. Collectively, our findings indicate that YAP may serve as a potential therapeutic target for ageing-associated cardiovascular disease.
    Keywords:  YAP; autophagy; mTOR; vascular senescence
  18. Int J Oral Sci. 2020 Dec 14. 12(1): 34
      Epithelial-mesenchymal transition (EMT) is involved in both physiological and pathological processes. EMT plays an essential role in the invasion, migration and metastasis of tumours. Autophagy has been shown to regulate EMT in a variety of cancers but not in head and neck squamous cell carcinoma (HNSCC). Herein, we investigated whether autophagy also regulates EMT in HNSCC. Analyses of clinical data from three public databases revealed that higher expression of fibronectin-1 (FN1) correlated with poorer prognosis and higher tumour pathological grade in HNSCC. Data from SCC-25 cells demonstrated that rapamycin and Earle's balanced salt solution (EBSS) promoted autophagy, leading to increased FN1 degradation, while 3-methyladenine (3-MA), bafilomycin A1 (Baf A1) and chloroquine (CQ) inhibited autophagy, leading to decreased FN1 degradation. On the other hand, autophagic flux was blocked in BECN1 mutant HNSCC Cal-27 cells, and rapamycin did not promote autophagy in Cal-27 cells; also in addition, FN1 degradation was inhibited. Further, we identified FN1 degradation through the lysosome-dependent degradation pathway using the proteasome inhibitor MG132. Data from immunoprecipitation assays also showed that p62/SQSTM1 participated as an autophagy adapter in the autophagy-lysosome pathway of FN1 degradation. Finally, data from immunoprecipitation assays demonstrated that the interaction between p62 and FN1 was abolished in p62 mutant MCF-7 and A2780 cell lines. These results indicate that autophagy significantly promotes the degradation of FN1. Collectively, our findings clearly suggest that FN1, as a marker of EMT, has adverse effects on HNSCC and elucidate the autophagy-lysosome degradation mechanism of FN1.
  19. Cardiovasc Res. 2020 Dec 17. pii: cvaa340. [Epub ahead of print]
      Mitochondria, the primary ATP-producing organelles, are highly abundant in cardiomyocytes. Mitochondrial function readily deteriorates in the presence of stress and, thus, maintenance of mitochondrial quality is essential for sustaining pump function in the heart. Cardiomyocytes under stress attempt to maintain mitochondrial quality primarily through dynamic changes in their morphology, namely fission and fusion, degradation, and biogenesis. Mitophagy, a mitochondria-specific form of autophagy, is a major mechanism of degradation. The level of mitophagy is altered in stress conditions, which, in turn, significantly affects mitochondrial function, cardiomyocyte survival, and death and cardiac function. Thus, mitophagy has been emerging as a promising target for treatment of cardiac conditions. To develop specific interventions, modulating the activity of mitophagy in the heart, understanding how mitochondria are degraded in a given condition is important. Increasing lines of evidence suggest that there are multiple mechanisms by which mitochondria are degraded through mitophagy in the heart. For example, in addition to the well-established mechanism commonly utilized by general autophagy, involving Atg7 and LC3, recent evidence suggests that an alternative mechanism, independent of Atg7 and LC3, also mediates mitophagy in the heart. Here, we describe molecular mechanisms through which mitochondria are degraded in the heart and discuss their functional significance. We also discuss molecular interventions to modulate the activity of mitophagy and their potential applications for cardiac conditions.
    Keywords:  Alternative mitophagy; Drp1; Parkin; Rab9;  Mitophagy
  20. J Cell Mol Med. 2020 Dec 13.
      Leucine zipper-EF-hand-containing transmembrane protein 1 (LETM1) is a mitochondrial inner membrane protein that is highly expressed in various cancers. Although LETM1 is known to be associated with poor prognosis in colorectal cancer (CRC), its roles in autophagic cell death in CRC have not been explored. In this study, we examined the mechanisms through which LETM1 mediates autophagy in CRC. Our results showed that LETM1 was highly expressed in CRC tissues and that down-regulation of LETM1 inhibited cell proliferation and induced S-phase arrest. LETM1 silencing also suppressed cancer stem cell-like properties and induced autophagy in CRC cells. Additionally, the autophagy inhibitor 3-methyladenine reversed the inhibitory effects of LETM1 silencing on proliferation and stemness, whereas the autophagy activator rapamycin had the opposite effects. Mechanistically, suppression of LETM1 increased the levels of reactive oxygen species (ROS) and mitochondrial ROS by regulation of SOD2, which in turn activated AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), initiated autophagy, and inhibited proliferation and stemness. Our findings suggest that silencing LETM1 induced autophagy in CRC cells by triggering ROS-mediated AMPK/mTOR signalling, thus blocking CRC progression, which will enhance our understanding of the molecular mechanism of LETM1 in CRC.
    Keywords:  LETM1; autophagy; colorectal cancer; reactive oxygen species
  21. Glia. 2020 Dec 14.
      Vacuolar ATPase (v-ATPase) is the main proton pump that acidifies vesicles such as lysosomes. Disruption in the lysosomal localization of v-ATPase leads to lysosomal dysfunction, thus contributing to the pathogenesis of lysosomal storage disorders and neurodegenerative diseases such as Alzheimer's disease. Recent studies showed that increases in cyclic AMP (cAMP) levels acidify lysosomes and consequently enhance autophagy flux. Although the upregulation of v-ATPase function may be the key mechanism underlying the cAMP-mediated lysosomal acidification, it is unknown whether a mechanism independent of v-ATPase may be contributing to this phenomenon. In the present study, we modeled v-ATPase dysfunction in brain cells by blocking lysosomal acidification in cortical astrocytes through treatment with bafilomycin A1, a selective v-ATPase inhibitor. We observed that cAMP reversed the pH changes via the activation of protein kinase A; interestingly, cAMP also increased autophagy flux even in the presence of bafilomycin A1, suggesting the presence of an alternative route of proton entry. Notably, pharmacological inhibitors and siRNAs of H+ /K+ -ATPase markedly shifted the lysosomal pH toward more alkaline values in bafilomycin A1/cAMP-treated astrocytes, suggesting that H+ /K+ -ATPase may be the alternative route of proton entry for lysosomal acidification. Furthermore, the cAMP-mediated reversal of lysosomal pH was nullified in the absence of ZnT3 that interacts with H+ /K+ -ATPase. Our results suggest that the H+ /K+ -ATPase/ZnT3 complex is recruited to lysosomes in a cAMP-dependent manner and functions as an alternative proton pump for lysosomes when the v-ATPase function is downregulated, thus providing insight into the potential development of a new class of lysosome-targeted therapeutics in neurodegenerative diseases.
    Keywords:  H+/K+-ATPase; astrocytes; autophagy; bafilomycin A1; cAMP; lysosome; zinc transport 3
  22. Cancer Discov. 2020 Dec 16. pii: CD-20-0652. [Epub ahead of print]
      KRAS-mutant colorectal cancers (CRC) are resistant to therapeutics, presenting a significant problem for ~40% of cases. Rapalogs, which inhibit mTORC1 and thus protein synthesis, are significantly less potent in KRAS-mutant CRC. Using Kras-mutant mouse models and mouse- and patient-derived organoids we demonstrate that KRAS with G12D mutation fundamentally rewires translation to increase both bulk and mRNA-specific translation initiation. This occurs via the MNK/eIF4E pathway culminating in sustained expression of c-MYC. By genetic and small molecule targeting of this pathway, we acutely sensitize KRASG12D models to rapamycin via suppression of c-MYC. We show that 45% of CRCs have high signaling through mTORC1 and the MNKs, with this signature correlating with a 3.5-year shorter cancer-specific survival in a subset of patients. This work provides a c-MYC-dependent co-targeting strategy with remarkable potency in multiple Kras-mutant mouse models and metastatic human organoids and identifies a patient population who may benefit from its clinical application.
  23. Front Mol Biosci. 2020 ;7 214
      Alzheimer's Disease is driven by protein aggregation and is characterized by accumulation of Tau protein into neurofibrillary tangles. In healthy neurons the cellular protein quality control is successfully in charge of protein folding, which raises the question to which extent this control is disturbed in disease. Here, we describe that brain cells in Alzheimer's Disease show very specific derailment of the protein quality control network. We performed a meta-analysis on the Alzheimer's Disease Proteome database, which provides a quantitative assessment of disease-related proteome changes in six brain regions in comparison to age-matched controls. We noted that levels of all paralogs of the conserved Hsp90 chaperone family are reduced, while most other chaperones - or their regulatory co-chaperones - do not change in disease. The notable exception is a select group consisting of the stress inducible HSP70, its nucleotide exchange factor BAG3 - which links the Hsp70 system to autophagy - and neuronal small heat shock proteins, which are upregulated in disease. They are all members of a cascade controlled in the stress response, channeling proteins towards a pathway of chaperone assisted selective autophagy. Together, our analysis reveals that in an Alzheimer's brain, with exception of Hsp90, the players of the protein quality control are still present in full strength, even in brain regions most severely affected in disease. The specific upregulation of small heat shock proteins and HSP70:BAG3, ubiquitous in all brain areas analyzed, may represent a last, unsuccessful attempt to advert cell death.
    Keywords:  Alzheimer’s disease; autophagy; chaperones; proteomics; proteostasis; stress response
  24. Plants (Basel). 2020 Dec 14. pii: E1771. [Epub ahead of print]9(12):
      The endoplasmic reticulum (ER) is the largest continuous membrane-bound cellular organelle and plays a central role in the biosynthesis of lipids and proteins and their distribution to other organelles. Autophagy is a conserved process that is required for recycling unwanted cellular components. Recent studies have implicated the ER as a membrane source for the formation of autophagosomes, vesicles that transport material to the vacuole during autophagy. When unfolded proteins accumulate in the ER and/or the ER lipid bilayer is disrupted, a condition known as ER stress results. During ER stress, ER membranes can also be engulfed through autophagy in a process termed ER-phagy. An interplay between ER stress responses and autophagy thus maintains the functions of the ER to allow cellular survival. In this review, we discuss recent progress in understanding ER-phagy in plants, including identification of regulatory factors and selective autophagy receptors. We also identify key unanswered questions in plant ER-phagy for future study.
    Keywords:  ER stress; ER-phagy; autophagy; endoplasmic reticulum; unfolded protein response
  25. Front Cell Neurosci. 2020 ;14 603710
      Multiple sclerosis (MS) is a complex auto-immune disorder of the central nervous system (CNS) that involves a range of CNS and immune cells. MS is characterized by chronic neuroinflammation, demyelination, and neuronal loss, but the molecular causes of this disease remain poorly understood. One cellular process that could provide insight into MS pathophysiology and also be a possible therapeutic avenue, is autophagy. Autophagy is an intracellular degradative pathway essential to maintain cellular homeostasis, particularly in neurons as defects in autophagy lead to neurodegeneration. One of the functions of autophagy is to maintain cellular homeostasis by eliminating defective or superfluous proteins, complexes, and organelles, preventing the accumulation of potentially cytotoxic damage. Importantly, there is also an intimate and intricate interplay between autophagy and multiple aspects of both innate and adaptive immunity. Thus, autophagy is implicated in two of the main hallmarks of MS, neurodegeneration, and inflammation, making it especially important to understand how this pathway contributes to MS manifestation and progression. This review summarizes the current knowledge about autophagy in MS, in particular how it contributes to our understanding of MS pathology and its potential as a novel therapeutic target.
    Keywords:  autophagy; inflammation; multiple sclerosis; neurodegeneration; resolution
  26. Exp Ther Med. 2021 Feb;21(2): 118
      Myocardial ischemia/reperfusion injury (MIRI) is an inevitable and unsolved clinical problem in the treatment of ischemic heart diseases. Compound DT-010 is a novel danshensu/tetramethylpyrazine derivative and was examined as a candidate for treating MIRI. In the present study, MTT, lactate dehydrogenase assay and Hoechst staining data indicated that DT-010 attenuated tert-butylhydroperoxide (t-BHP)-induced oxidative damage by increasing cell survival, reducing cell damage and decreasing apoptosis in H9c2 cardiomyocytes. Autophagy was assessed by western blotting for microtubule-associated protein 1A/1B-light chain 3 (LC3-II and LC3-I) expression, acridine orange and monodansylcadaverine staining for autophagosome formation and the monomeric red fluorescent protein-green fluorescent protein-LC3 assay for autophagic flow. t-BHP-induced cell damage was aggravated by the autophagy agonist rapamycin and alleviated by the autophagy blocker hydroxy-chloroquine, suggesting that autophagy was involved in t-BHP-induced cardiomyocyte injury. DT-010 pretreatment significantly prevented t-BHP-induced cell damage, which was partially but significantly abolished by rapamycin and significantly improved by hydroxy-chloroquine treatment. DT-010 treatment inhibited t-BHP-induced autophagy in H9c2 cells, reduced phosphorylation of 5'-AMP-activated protein kinase (AMPK) and promoted the phosphorylation of mTOR and unc-51 like autophagy activating kinase 1 (Ulk1). To conclude, DT-010 can serve as a potential candidate for myocardial ischemia-reperfusion injury therapy. The cardioprotective effects of DT-010 could be partially attributed to its inhibition of autophagy via the AMPK-mTOR-Ulk1 signaling pathway.
    Keywords:  5'-AMP-activated protein kinase/mTOR/unc-51 like autophagy activating kinase 1 signaling pathway; autophagy; cardioprotection; danshensu/tetramethylpyrazine derivative; oxidative stress
  27. FASEB J. 2021 Jan;35(1): e21225
      Studies of neuroglial interaction largely depend on cell-specific gene knockout (KO) experiments using Cre recombinase. However, genes known as glial-specific genes have recently been reported to be expressed in neuroglial stem cells, leading to the possibility that a glia-specific Cre driver results in unwanted gene deletion in neurons, which may affect sound interpretation. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) is generally considered to be an oligodendrocyte (OL) marker. Accordingly, Cnp promoter-controlled Cre recombinase has been used to create OL-specific gene targeting mice. However, in this study, using Rosa26-tdTomato-reporter/Cnp-Cre mice, we found that many forebrain neurons and cerebellar Purkinje neurons belong to the lineages of Cnp-expressing neuroglial stem cells. To answer whether gene targeting by Cnp-Cre can induce neuron-autonomous defects, we conditionally deleted an essential autophagy gene, Atg7, in Cnp-Cre mice. The Cnp-Cre-mediated Atg7 KO mice showed extensive p62 inclusion in neurons, including cerebellar Purkinje neurons with extensive neurodegeneration. Furthermore, neuronal areas showing p62 inclusion in Cnp-Cre-mediated Atg7 KO mice overlapped with the neuronal lineage of Cnp-expressing neuroglial stem cells. Moreover, Cnp-Cre-mediated Atg7-KO mice did not develop critical defects in myelination. Our results demonstrate that a large population of central neurons are derived from Cnp-expressing neuroglial stem cells; thus, conditional gene targeting using the Cnp promoter, which is known to be OL-specific, can induce neuron-autonomous phenotypes.
    Keywords:  CNP; myelination; neurodegeneration; oligodendrocyte; p62
  28. Front Immunol. 2020 ;11 573646
      During their lifespan, dendritic cells (DCs) are exposed to different pO2 levels that affect their differentiation and functions. Autophagy is one of the adaptive responses to hypoxia with important implications for cell survival. While the autophagic machinery in DCs was shown to impact signaling of TLRs, its regulation by the MD-2/TLR4 ligand LPS is still unclear. The aim of this study was to evaluate whether LPS can induce autophagy in DCs exposed to either aerobic or hypoxic conditions. Using human monocyte-derived DCs and the combination of immunofluorescence confocal analysis, measure of mitochondrial membrane potential, Western blotting, and RT-qPCR, we showed that the ability of LPS to modulate autophagy was strictly dependent upon pO2 levels. Indeed, LPS inhibited autophagy in aerobic conditions whereas the autophagic process was induced in a hypoxic environment. Under hypoxia, LPS treatment caused a significant increase of functional lysosomes, LC3B and Atg protein upregulation, and reduction of SQSTM1/p62 protein levels. This selective regulation was accompanied by activation of signalling pathways and expression of cytokines typically associated with DC survival. Bafilomycin A1 and chloroquine, which are recognized as autophagic inhibitors, confirmed the induction of autophagy by LPS under hypoxia and its impact on DC survival. In conclusion, our results show that autophagy represents one of the mechanisms by which the activation of the MD-2/TLR4 ligand LPS promotes DC survival under hypoxic conditions.
    Keywords:  (macroautophagy); autophagy; dendritic cell; hypoxia; hypoxia-inducible factor (HIF)-1α; lipopolysaccharide (LPS)
  29. Life Sci. 2020 Dec 10. pii: S0024-3205(20)31637-4. [Epub ahead of print] 118884
      AIMS: Growing evidence indicates insufficient autophagy is crucial to airway remodeling in asthma. However, it is uncertain whether p62, an autophagy major regulator, mediates the airway remodeling process. This study aimed to evaluate the role and underlying mechanism of p62 in airway remodeling in asthma.MATERIALS AND METHODS: Airway remodeling was confirmed via histopathology. Western blotting and RT-PCR were used to detect the expression of autophagic and glycolytic proteins, as well as glycolytic genes. Glycolysis was measured by glucose consumption and lactate production. Cell proliferation was analyzed by CCK8 assays while and the scratch test and transwell method were used for cell migration.
    KEY FINDINGS: We found that insufficient autophagic flux and increased p62 expression existed in chronic asthma mice. Additionally, knockdown of p62 inhibited asthmatic human bronchial smooth muscle cells (BSMCs) proliferation and migration in vitro. To elucidate the underlying mechanism of p62-mediated autophagy flux in directing BSMCs function, we demonstrated that knockdown of p62 decreased the glucose consumption and lactate production in BSMCs, whereas p62 overexpression had the opposite effect. Furthermore, we showed that p62 regulated glycolysis in BSMCs by the mTOR/c-Myc/hexokinase 2 (HK2) pathway.
    SIGNIFICANCE: Our findings suggest that p62 is involved in BSMCs proliferation and migration via the mTOR/c-Myc/HK2-mediated glycolysis, thereby providing a new target for airway remodeling treatment.
    Keywords:  Aerobic glycolysis; Airway remodeling; Autophagy; hexokinase2; p62
  30. Hepatology. 2020 Dec 13.
      Upon mild liver injury, pre-existing hepatocytes replicate. However, if hepatocyte proliferation is compromised, such as in chronic liver diseases, biliary epithelial cells (BECs) contribute to hepatocytes through liver progenitor cells (LPCs), thereby restoring hepatic mass and function. Recently, augmenting innate BEC-driven liver regeneration has garnered attention as an alternative to liver transplantation, the only reliable treatment for patients with end-stage liver diseases. Despite this attention, the molecular basis of BEC-driven liver regeneration remains poorly understood. By performing a chemical screen with the zebrafish hepatocyte ablation model, in which BECs robustly contribute to hepatocytes, we identified farnesoid X receptor (FXR) agonists as inhibitors of BEC-driven liver regeneration. Here we show that FXR activation blocks the process via the FXR-PTEN-PI3K-AKT-mTOR axis. We found that FXR activation blocked LPC-to-hepatocyte differentiation, but not BEC-to-LPC dedifferentiation. FXR activation also suppressed LPC proliferation and increased its death. These defects were rescued by suppressing PTEN activity with its chemical inhibitor and ptena/b mutants, indicating PTEN as a critical downstream mediator of FXR signaling in BEC-driven liver regeneration. Consistent with the role of PTEN in inhibiting the PI3K-AKT-mTOR pathway, FXR activation reduced the expression of pS6, a marker of mTORC1 activation, in LPCs of regenerating livers. Importantly, suppressing PI3K and mTORC1 activities with their chemical inhibitors blocked BEC-driven liver regeneration, as did FXR activation. Conclusion: FXR activation impairs BEC-driven liver regeneration by enhancing PTEN activity; the PI3K-AKT-mTOR pathway controls the regeneration process. Given clinical trials and usage of FXR agonists for multiple liver diseases due to their beneficial effects on steatosis and fibrosis, the detrimental effects of FXR activation on LPCs suggest a rather personalized use of the agonists in the clinic.
    Keywords:  FXR; GW4064; Nr1h4; biliary epithelial cells
  31. J Cell Sci. 2020 Dec 11. pii: jcs243139. [Epub ahead of print]133(23):
      Neurodegenerative diseases are characterised by the irreversible degeneration of neurons in the central or peripheral nervous systems. These include amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD) and prion diseases. Small extracellular vesicles (sEVs), a type of EV involved in cellular communication, have been well documented as propagating neurodegenerative diseases. These sEVs carry cargo, such as proteins and RNA, to recipient cells but are also capable of promoting protein misfolding, thus actively contributing to the progression of these diseases. sEV secretion is also a compensatory process for lysosomal dysfunction in the affected cells, despite inadvertently propagating disease to recipient cells. Despite this, sEV miRNAs have biomarker potential for the early diagnosis of these diseases, while stem cell-derived sEVs and those generated through exogenous assistance demonstrate the greatest therapeutic potential. This Review will highlight novel advancements in the involvement of sEVs as propagators of neuropathology, biomarkers and potential therapeutics in neurodegenerative diseases.
    Keywords:  Alzheimer's disease; Amyotrophic lateral sclerosis; Autophagy; Exosomes; Extracellular vesicles; Lysosomal dysfunction; Neurogenerative disease; Parkinson's disease; Prion disease; miRNA biomarkers
  32. Elife. 2020 Dec 15. pii: e57950. [Epub ahead of print]9
      Vaccines are powerful tools to develop immune memory to infectious diseases and prevent excess mortality. In older adults, however vaccines are generally less efficacious and the molecular mechanisms that underpin this remain largely unknown. Autophagy, a process known to prevent aging, is critical for the maintenance of immune memory in mice. Here, we show that autophagy is specifically induced in vaccine-induced antigen-specific CD8+ T cells in healthy human volunteers. In addition, reduced IFNγ secretion by RSV-induced T cells in older vaccinees correlates with low autophagy levels. We demonstrate that levels of the endogenous autophagy-inducing metabolite spermidine fall in human T cells with age. Spermidine supplementation in T cells from old donors recovers their autophagy level and function, similar to young donors' cells, in which spermidine biosynthesis has been inhibited. Finally, our data show that endogenous spermidine maintains autophagy via the translation factor eIF5A and transcription factor TFEB. In summary, we have provided evidence for the importance of autophagy in vaccine immunogenicity in older humans and uncovered two novel drug targets that may increase vaccination efficiency in the aging context.
    Keywords:  TFEB; autophagy; human; human T cells; immunology; inflammation; spermidine; vaccine
  33. Biochem Biophys Res Commun. 2020 Dec 12. pii: S0006-291X(20)32197-5. [Epub ahead of print]534 128-133
      The tumor suppressor p53 is involved in cadmium (Cd)-induced apoptosis and autophagy. However, the regulatory mechanisms of p53 in Cd-induced kidney injury are not well established. Here, we report the role of autophagy in Cd-induced p53 induction in human proximal tubular cells (HK-2). HK-2 cells treated with Cd induced the expression of p53, DNA damage autophagy modulator (DRAM), and Bcl-2-associated X protein (BAX), as well as caused poly [ADP-ribose] polymerase 1 (PARP-1) cleavage. Cd exposure also induced autophagy with the accumulation of monomeric p62 and multiple high molecular weight form (HMW)-p62. The expression levels of p53, p62, microtubule-associated protein 1A/1B-light chain 3 (LC3)-1, and LC3-II were similar in the sense that they increased up to 12 h and then gradually decreased. DRAM and BAX levels began to increase post autophagy induction and continued to increase, indicating that autophagy preceded apoptosis. While the genetic knockdown of p53 downregulated HWM-p62, DRAM, and BAX, the expression levels of these proteins were upregulated by p53 overexpression. The genetic knockdown of p62 downregulated p53, autophagy, DRAM, and BAX. The inhibition of autophagy through pharmacological and genetic knockdown reduced p53 and inhibited Cd-induced apoptosis. Collectively, Cd induces apoptosis through p53-mediated DRAM-BAX signaling, which can be regulated by autophagy.
    Keywords:  Autophagy; BAX; Cadmium; DRAM; Human proximal tubular cell; p53
  34. Biochem Biophys Res Commun. 2020 Dec 11. pii: S0006-291X(20)32178-1. [Epub ahead of print]534 107-113
      Measurement of autophagic flux in vivo is critical to understand how autophagy can be used to combat disease. Neurodegenerative diseases have a special relationship with autophagy, which makes measurement of autophagy in the brain a significant research priority. Currently, measurement of autophagic flux is possible through use of transgenic constructs, or application of a lysosomal inhibitor such as chloroquine. Unfortunately, chloroquine is not useful for measuring autophagic flux in the brain and the use of transgenic animals necessitates cross-breeding of transgenic strains and maintenance of lines, which is costly. To find a drug that could block lysosomal function in the brain for the measurement of autophagic flux, we selected compounds from the literature that appeared to have similar properties to chloroquine and tested their ability to inhibit autophagic flux in cell culture and in mice. These chemicals included chloroquine, quinacrine, mefloquine, promazine and trifluoperazine. As expected, chloroquine blocked lysosomal degradation of the autophagic protein LC3B-II in cell culture. Quinacrine also inhibited autophagic flux in cell culture. Other compounds tested were not effective. When injected into mice, chloroquine caused accumulation of LC3B-II in heart tissue, and quinacrine was effective at blocking LC3B-II degradation in male, but not female skeletal muscle. None of the compounds tested were useful for measuring autophagic flux in the brain. During this study we also noted that the vehicle DMSO powerfully up-regulated LC3B-II abundance in tissues. This study shows that chloroquine and quinacrine can both be used to measure autophagic flux in cells, and in some peripheral tissues. However, measurement of flux in the brain using lysosomal inhibitors remains an unresolved research challenge.
    Keywords:  Autophagy; Brain; Chloroquine; LC3; Lysosome; Quinacrine
  35. J Biomed Nanotechnol. 2020 Jul 01. 16(7): 1144-1159
      Oxidative stress is one of the principal causes of hypoxia-induced kidney injury. The ceria nanoparticle (CNP) is known to exhibit free radical scavenger and catalytic activities. When zirconia is attached to CNPs (CZNPs), the ceria atom tends to remain in a Ce3+ form and its efficacy as a free radical scavenger thus increases. We determined the effectiveness of CNP and CZNP antioxidant activities against hypoxia-induced acute kidney injury (AKI) and observed that these nanoparticles suppress the apoptosis of hypoxic HK-2 cells by restoring autophagy flux and alleviating mitochondrial damage. In vivo experiments revealed that CZNPs effectively attenuate hypoxia-induced AKI by preserving renal structures and glomerulus function. These nanoparticles can successfully diffuse into HK-2 cells and effectively counteract reactive oxygen species (ROS) to block hypoxia-induced AKI. This suggests that these particles represent a novel approach to controlling this condition.
  36. Membranes (Basel). 2020 Dec 09. pii: E406. [Epub ahead of print]10(12):
      Lysosomes are acidic cell compartments containing a large set of hydrolytic enzymes. These lysosomal hydrolases degrade proteins, lipids, polysaccharides, and nucleic acids into their constituents. Materials to be degraded can reach lysosomes either from inside the cell, by autophagy, or from outside the cell, by different forms of endocytosis. In addition to their degradative functions, lysosomes are also able to extracellularly release their contents by lysosomal exocytosis. These organelles move from the perinuclear region along microtubules towards the proximity of the plasma membrane, then the lysosomal and plasma membrane fuse together via a Ca2+-dependent process. The fusion of the lysosomal membrane with plasma membrane plays an important role in plasma membrane repair, while the secretion of lysosomal content is relevant for the remodelling of extracellular matrix and release of functional substrates. Lysosomal storage disorders (LSDs) and age-related neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases, share as a pathological feature the accumulation of undigested material within organelles of the endolysosomal system. Recent studies suggest that lysosomal exocytosis stimulation may have beneficial effects on the accumulation of these unprocessed aggregates, leading to their extracellular elimination. However, many details of the molecular machinery required for lysosomal exocytosis are only beginning to be unravelled. Here, we are going to review the current literature on molecular mechanisms and biological functions underlying lysosomal exocytosis, to shed light on the potential of lysosomal exocytosis stimulation as a therapeutic approach.
    Keywords:  TFEB; TRPLML1; cellular clearance; lysosomal exocytosis; lysosomes; mTOR
  37. Trends Biochem Sci. 2020 Dec 09. pii: S0968-0004(20)30275-9. [Epub ahead of print]
      To identify the appropriate times for growth and development, organisms must sense and process information about the availability of nutrients, energy status, and environmental cues. For sessile eukaryotes such as plants, integrating such information can be critical in life or death decisions. For nearly 30 years, the conserved phosphatidylinositol 3-kinase-related protein kinases (PIKKs) target of rapamycin (TOR) has been established as a central hub for integrating external and internal metabolic cues. Despite the functional conservation across eukaryotes, the TOR complex has evolved specific functional and mechanistic features in plants. Here, we present recent findings on the plant TOR complex that highlight the conserved and unique nature of this critical growth regulator and its role in multiple aspects of plant life.
    Keywords:  autophagy; cellular nutrition and homeostasis; circadian cycle; energy sensing; photosynthesis; phytohormones; symbiotic relationships
  38. Cell Death Differ. 2020 Dec 17.
      Oleate, the most abundant endogenous and dietary cis-unsaturated fatty acid, has the atypical property to cause the redistribution of microtubule-associated proteins 1A/1B light chain 3B (referred to as LC3) to the trans-Golgi network (TGN), as shown here. A genome-wide screen identified multiple, mostly Golgi transport-related genes specifically involved in the oleate-induced relocation of LC3 to the Golgi apparatus. Follow-up analyses revealed that oleate also caused the retention of secreted proteins in the TGN, as determined in two assays in which the secretion of proteins was synchronized, (i) an assay involving a thermosensitive vesicular stomatitis virus G (VSVG) protein that is retained in the endoplasmic reticulum (ER) until the temperature is lowered, and (ii) an isothermic assay involving the reversible retention of the protein of interest in the ER lumen and that was used both in vitro and in vivo. A pharmacological screen searching for agents that induce LC3 aggregation at the Golgi apparatus led to the identification of "oleate mimetics" that share the capacity to block conventional protein secretion. In conclusion, oleate represents a class of molecules that act on the Golgi apparatus to cause the recruitment of LC3 and to stall protein secretion.
  39. Front Cell Dev Biol. 2020 ;8 608388
      Chondrocytes are the only cell type in normal cartilage. The pathological changes of osteoarthritis (OA) mostly revolve around the apoptosis and dysfunction of chondrocytes. Autophagy, as an intracellular degradation system that maintains the steady state of energy metabolism in cells, has been shown to restore the function of damaged chondrocytes, alleviating the occurrence and progression of OA. In this review, we explored the relationship between autophagy and OA and the key molecules of autophagy pathway that regulate the progression of OA, providing new ideas for OA treatment by targeting autophagy.
    Keywords:  apoptosis; autophagy; cartilage; chondrocyte; osteoarthritis
  40. Front Cell Dev Biol. 2020 ;8 602574
      Liraglutide, a glucagon-like peptide-1 receptor agonist (GLP-1RA), has been demonstrated to alleviate non-alcoholic fatty liver disease (NAFLD). However, the underlying mechanism has not been fully elucidated. Increasing evidence suggests that autophagy is involved in the pathogenesis of hepatic steatosis. In this study, we examined whether liraglutide could alleviate hepatic steatosis through autophagy-dependent lipid degradation and investigated the underlying mechanisms. Herein, the effects of liraglutide on NAFLD were evaluated in a high-fat diet (HFD)-induced mouse model of NAFLD as well as in mouse primary and HepG2 hepatocytes exposed to palmitic acid (PA). The expression of the GLP-1 receptor (GLP-1R) was measured in vivo and in vitro. Oil red O staining was performed to detect lipid accumulation in hepatocytes. Electron microscopy was used to observe the morphology of autophagic vesicles and autolysosomes. Autophagic flux activity was measured by infecting HepG2 cells with mRFP-GFP-LC3 adenovirus. The roles of GLP-1R and transcription factor EB (TFEB) in autophagy-lysosomal activation were explored using small interfering RNA. Liraglutide treatment alleviated hepatic steatosis in vivo and in vitro. In models of hepatic steatosis, microtubule-associated protein 1B light chain-3-II (LC3-II) and SQSTM1/P62 levels were elevated in parallel to blockade of autophagic flux. Liraglutide treatment restored autophagic activity by improving lysosomal function. Furthermore, treatment with autophagy inhibitor chloroquine weakened liraglutide-induced autophagy activation and lipid degradation. TFEB has been identified as a key regulator of lysosome biogenesis and autophagy. The protein levels of nuclear TFEB and its downstream targets CTSB and LAMP1 were decreased in hepatocytes treated with PA, and these decreases were reversed by liraglutide treatment. Knockdown of TFEB expression compromised the effects of liraglutide on lysosome biogenesis and hepatic lipid accumulation. Mechanistically, GLP-1R expression was decreased in HFD mouse livers as well as PA-stimulated hepatocytes, and liraglutide treatment reversed the downregulation of GLP-1R expression in vivo and in vitro. Moreover, GLP-1R inhibition could mimic the effect of the TFEB downregulation-mediated decrease in lysosome biogenesis. Thus, our findings suggest that liraglutide attenuated hepatic steatosis via restoring autophagic flux, specifically the GLP-1R-TFEB-mediated autophagy-lysosomal pathway.
    Keywords:  TFEB; autophagy-lysosome; liraglutide; lysosomal biogenesis; non-alcoholic fatty liver disease