bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒08‒16
twenty-nine papers selected by
Viktor Korolchuk
Newcastle University

  1. Sci Rep. 2020 Aug 14. 10(1): 13810
      Cell signaling important for homeostatic regulation of colonic epithelial cells (CECs) remains poorly understood. Mammalian target of rapamycin complex 1 (mTORC1), a protein complex that contains the serine-threonine kinase mTOR, mediates signaling that underlies the control of cellular functions such as proliferation and autophagy by various external stimuli. We here show that ablation of tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, specifically in intestinal epithelial cells of mice resulted in increased activity of mTORC1 of, as well as increased proliferative activity of, CECs. Such Tsc2 ablation also reduced the population of Lgr5-positive colonic stem cells and the expression of Wnt target genes in CECs. The stimulatory phosphorylation of the kinase Akt and inhibitory phosphorylation of glycogen synthase kinase 3β were both markedly decreased in the colon of the Tsc2 conditional knockout (CKO) mice. Development of colonic organoids with cryptlike structures was enhanced for Tsc2 CKO mice compared with control mice. Finally, Tsc2 CKO mice manifested increased susceptibility to dextran sulfate sodium-induced colitis. Our results thus suggest that mTORC1 activity promotes the proliferation of, as well as the expression of Wnt target genes in, CECs and thereby contributes to colonic organogenesis and homeostasis.
  2. Int J Biol Sci. 2020 ;16(14): 2675-2691
      Bone metabolic disorders include osteolysis, osteoporosis, osteoarthritis and rheumatoid arthritis. Osteoblasts and osteoclasts are two major types of cells in bone constituting homeostasis. The imbalance between bone formation by osteoblasts and bone resorption by osteoclasts has been shown to have a direct contribution to the onset of these diseases. Recent evidence indicates that autophagy and mitophagy, the selective autophagy of mitochondria, may play a vital role in regulating the proliferation, differentiation and function of osteoblasts and osteoclasts. Several signaling pathways, including PINK1/Parkin, SIRT1, MAPK8/FOXO3, Beclin-1/BECN1, p62/SQSTM1, and mTOR pathways, have been implied in the regulation of autophagy and mitophagy in these cells. Here we review the current progress about the regulation of autophagy and mitophagy in osteoblasts and osteoclasts in these bone metabolic disorders, as well as the molecular signaling activated or deactivated during this process. Together, we hope to draw attention to the role of autophagy and mitophagy in bone metabolic disorders, and their potential as a new target for the treatment of bone metabolic diseases and the requirements of further mechanism studies.
    Keywords:  autophagy; bone metabolic disorder; mitophagy; osteoblast; osteoclast
  3. Stem Cell Reports. 2020 Jul 30. pii: S2213-6711(20)30290-3. [Epub ahead of print]
      Mutations and loss of activity in PARKIN, an E3 ubiquitin ligase, play a role in the pathogenesis of Parkinson's disease (PD). PARKIN regulates many aspects of mitochondrial quality control including mitochondrial autophagy (mitophagy) and mitochondrial biogenesis. Defects in mitophagy have been hypothesized to play a predominant role in the loss of dopamine (DA) neurons in PD. Here, we show that although there are defects in mitophagy in human DA neurons lacking PARKIN, the mitochondrial deficits are primarily due to defects in mitochondrial biogenesis that are driven by the upregulation of PARIS and the subsequent downregulation of PGC-1α. CRISPR/Cas9 knockdown of PARIS completely restores the mitochondrial biogenesis defects and mitochondrial function without affecting the deficits in mitophagy. These results highlight the importance mitochondrial biogenesis versus mitophagy in the pathogenesis of PD due to inactivation or loss of PARKIN in human DA neurons.
    Keywords:  PARIS; PARKIN; PGC-1α; Parkinson’s disease; ZNF746; dopamine; human IPSC; isogenic; mitochondrial biogenesis; mitophagy
  4. Cells. 2020 Aug 08. pii: E1858. [Epub ahead of print]9(8):
      The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.
    Keywords:  C. elegans; autophagy; crosstalk; tissue specificity; ubiquitin–proteasome system
  5. Elife. 2020 Aug 10. pii: e59099. [Epub ahead of print]9
      The selective autophagy pathways of xenophagy and mitophagy are initiated when the adaptor NDP52 recruits the ULK1 complex to autophagic cargo. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to map the membrane and NDP52 binding sites of the ULK1 complex to unique regions of the coiled coil of the FIP200 subunit. Electron microscopy of the full-length ULK1 complex shows that the FIP200 coiled coil projects away from the crescent-shaped FIP200 N-terminal domain dimer. NDP52 allosterically stimulates membrane-binding by FIP200 and the ULK1 complex by promoting a more dynamic conformation of the membrane-binding portion of the FIP200 coiled coil. Giant unilamellar vesicle (GUV) reconstitution confirmed that membrane recruitment by the ULK1 complex is triggered by NDP52 engagement. These data reveal how the allosteric linkage between NDP52 and the ULK1 complex could drive the first membrane recruitment event of phagophore biogenesis in xenophagy and mitophagy.
    Keywords:  biochemistry; cell biology; chemical biology; human
  6. Nat Commun. 2020 Aug 12. 11(1): 4029
      In autosomal dominant optic atrophy (ADOA), caused by mutations in the mitochondrial cristae biogenesis and fusion protein optic atrophy 1 (Opa1), retinal ganglion cell (RGC) dysfunction and visual loss occur by unknown mechanisms. Here, we show a role for autophagy in ADOA pathogenesis. In RGCs expressing mutated Opa1, active 5' AMP-activated protein kinase (AMPK) and its autophagy effector ULK1 accumulate at axonal hillocks. This AMPK activation triggers localized hillock autophagosome accumulation and mitophagy, ultimately resulting in reduced axonal mitochondrial content that is restored by genetic inhibition of AMPK and autophagy. In C. elegans, deletion of AMPK or of key autophagy and mitophagy genes normalizes the axonal mitochondrial content that is reduced upon mitochondrial dysfunction. In conditional, RGC specific Opa1-deficient mice, depletion of the essential autophagy gene Atg7 normalizes the excess autophagy and corrects the visual defects caused by Opa1 ablation. Thus, our data identify AMPK and autophagy as targetable components of ADOA pathogenesis.
  7. Aging Cell. 2020 Aug 11. e13211
      Accumulation of PINK1 on the outer mitochondrial membrane (OMM) is necessary for PINK-mediated mitophagy. The proton ionophores, like carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), inhibit PINK1 import into mitochondrial matrix and induce PINK1 OMM accumulation. Here, we show that the CHCHD4/GFER disulfide relay system in the mitochondrial intermembrane space (IMS) is required for PINK1 stabilization when mitochondrial membrane potential is lost. Activation of CHCHD4/GFER system by mitochondrial oxidative stress or inhibition of CHCHD4/GFER system with antioxidants can promote or suppress PINK1 accumulation, respectively. Thus data suggest a pivotal role of CHCHD4/GFER system in PINK1 accumulation. The amyotrophic lateral sclerosis-related superoxide dismutase 1 mutants dysregulated redox state and CHCHD4/GFER system in the IMS, leading to inhibitions of PINK1 accumulation and mitophagy. Thus, the redox system in the IMS is involved in PINK1 accumulation and damaged mitochondrial clearance, which may play roles in mitochondrial dysfunction-related neurodegenerative diseases.
    Keywords:  PINK1; autophagy; mitochondrion; mitophagy; neurodegenerative diseases
  8. Expert Rev Proteomics. 2020 Aug 10.
      INTRODUCTION: Autophagy is an evolutionarily conserved cellular clearance process, by which cytosolic components are delivered to autolysosomes for breakdown and recycling to maintain cellular homeostasis. During the past decades, autophagy has been found to be tightly implicated in various physiological and pathological progresses. Unravelling the regulatory mechanisms of the autophagy process will contribute to the development of emerging autophagy-targeting strategies for the treatment of various diseases. Recently, the rapid development of proteomics approaches has enabled the use of large-scale unbiased strategies to unravel autophagy machinery.AREAS COVERED: In this review, we will highlight the recent contributions of proteomics strategies in clarifying the autophagy machinery, with an emphasis on the three different types of autophagy (namely macroautophagy, microautophagy and chaperone-mediated autophagy). We will also discuss the emerging role of proteomics approaches in investigating the mechanism of autophagy-based unconventional secretory pathway (secretory autophagy).
    EXPERT OPINION: Proteomics has provided an effective strategy for the comprehensive analysis of the autophagy process, which will broaden our understanding of autophagy machinery and holds great promise for developing clinical therapies targeting autophagy.
    Keywords:  Autophagy; CMA; LDELS; Microautophagy; Proteomics; Secretory autophagy
  9. Am J Transl Res. 2020 ;12(7): 3964-3973
      BACKGROUND: Cyclophilin A (CyPA) plays an important role in the progression of atherosclerosis. Additionally, it has been reported that lysosomal function is markedly impaired in atherosclerosis induced by oxidized low-density lipoprotein (ox-LDL). As the CyPA degradation pathway remains to be elucidated, we aimed to uncover the role of lysosomes and ox-LDL in the degradation of CyPA.METHODS: We exploited RNA interference (RNAi) in combination with either the lysosomal inhibitor chloroquine (CQ) or the proteasomal inhibitor MG-132 to examine CyPA turnover. We also investigated the role of ox-LDL in lysosomal function and the CyPA degradation pathway and determined whether CyPA interacts with the selective autophagy adaptor p62.
    RESULTS: CQ markedly reversed the CyPA downregulation induced by RNAi and increased intracellular levels of LC3 and p62. MG-132 significantly suppressed polyubiquitinated protein degradation but did not inhibit RNAi-induced CyPA downregulation. Additionally, neither CQ nor MG-132 influenced the gene-silencing efficiency of CyPA siRNA. Moreover, ox-LDL induced cytosolic accumulation of p62 was inconsistent with increased expression of LC3-II. Meanwhile, ox-LDL inhibited RNAi-induced downregulation of CyPA. Immunofluorescence indicated colocalization of endogenous CyPA with ubiquitin and with p62 in response to CQ treatment, and co-immunoprecipitation analysis confirmed interaction between CyPA and p62.
    CONCLUSION: CyPA is degraded by a lysosome-dependent pathway that may involve p62-mediated selective autophagy. Furthermore, ox-LDL modulates the degradation of CyPA via its inhibitory role in lysosomes, contributing to increased expression of CyPA in atherosclerotic plaques.
    Keywords:  Oxidized low-density lipoprotein; RNA interference; atherosclerotic plaques; cycloheximide; cyclophilin A; lysosome
  10. Biochem Biophys Res Commun. 2020 Aug 10. pii: S0006-291X(20)31526-6. [Epub ahead of print]
      Melanosomes are specialized membrane-bound organelles that are involved in melanin synthesis. Unlike melanosome biogenesis, the melanosome degradation pathway is poorly understood. Among the cellular processes, autophagy controls degradation of intracellular components by cooperating with lysosomes. In this study, we showed that ursolic acid inhibits skin pigmentation by promoting melanosomal autophagy, or melanophagy, in melanocytes. We found that B16F1 cells treated with ursolic acid suppressed alpha-melanocyte stimulating hormone (α-MSH) stimulated increase in melanin content and activated autophagy. In addition, we found that treatment with ursolic acid promotes melanosomal degradation, and bafilomycin A1 inhibition of autophagosome-lysosome fusion blocked the removal of melanosomes in α-MSH-stimulated B16F1 cells. Furthermore, depletion of the autophagy-related gene 5 (ATG5) resulted in significant suppression of ursolic acid-mediated anti-pigmentation activity and autophagy in α-MSH-treated B16F1 cells. Taken together, our results suggest that ursolic acid inhibits skin pigmentation by increasing melanosomal degradation in melanocytes.
    Keywords:  Autophagy; B16F1 cells; Melanophagy; Melanosome; TPC2; Ursolic acid
  11. J Biol Chem. 2020 Aug 11. pii: jbc.RA120.014687. [Epub ahead of print]
      Autophagy is a conserved process that recycles cellular contents to promote survival. Although nitrogen limitation is the canonical inducer of autophagy, recent studies have revealed several other nutrients important to this process. In this study, we used a quantitative, high-throughput assay to identify potassium starvation as a new and potent inducer of autophagy in the yeast Saccharomyces cerevisiae We found that potassium-dependent autophagy requires the core pathway kinases Atg1, Atg5, Vps34, as well as other components of the phosphatidylinositol 3-kinase complex. Transmission electron microscopy revealed abundant autophagosome formation in response to both stimuli. RNA sequencing indicated distinct transcriptional responses - nitrogen affects transport of ions such as copper while potassium targets the organization of other cellular components. Thus, nitrogen and potassium share the ability to influence molecular supply and demand but do so in different ways. Both inputs promote catabolism through bulk autophagy, but result in distinct mechanisms of cellular remodeling and synthesis.
    Keywords:  RNA sequencing; autophagy; electron microscopy (EM); phosphatidylinositide 3-kinase (PI 3-kinase); potassium; starvation; transcriptomics; yeast
  12. Differentiation. 2020 Jul - Aug;114:pii: S0301-4681(20)30035-9. [Epub ahead of print]114 58-66
      Osteoclasts are terminally multinucleated cells that are regulated by nuclear factor-activated T cells c1 (NFATc1), and are responsible for bone resorption while the tartrate resistant acid phosphatase (TRAP) enzymes releases into bone resorption lacunae. Furthermore, tumor suppressor p53 is a negative regulator during osteoclastogenesis. Osteoprotegerin (OPG) inhibits osteoclastogenesis and bone resorption by activating autophagy, however, whether p53 is involved in OPG-mediated inhibition of osteoclastogenesis remains unclear. In the current study, OPG could enhance the expression of p53 and tuberin sclerosis complex 2 (TSC2). Moreover, the expression of p53 is regulated by autophagy during OPG-mediated inhibition of osteoclastogenesis. Inhibition of p53 by treated with pifithrin-α (PFTα) causing augments of osteoclastogenesis and bone resorption, also reversed OPG-mediated inhibition of osteoclastogenesis by reducing the expression of TSC2. In addition, knockdown of TSC2 using siRNA could rescue OPG-mediated inhibition of osteoclastogenesis by reducing autophagy, which is manifested by the decrease of the expression of Beclin1 and the phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase beta 1 (S6K1, also known as p70S6K). Collectively, p53 plays a critical role during OPG-mediated inhibition of osteoclastogenesis via regulating the TSC2-induced autophagy in vitro.
    Keywords:  OPG; Osteoclastogenesis; PFTα; TSC2; p53
  13. Oxid Med Cell Longev. 2020 ;2020 4908162
      The skeletal muscle plays an important role in maintaining whole-body mechanics, metabolic homeostasis, and interorgan crosstalk. However, during aging, functional and structural changes such as fiber integrity loss and atrophy can occur across different species. A commonly observed hallmark of aged skeletal muscle is the accumulation of oxidatively modified proteins and protein aggregates which point to an imbalance in proteostasis systems such as degradation machineries. Recently, we showed that the ubiquitin-proteasomal system was impaired. Specifically, the proteasomal activity, which was declining in aged M. soleus (SOL) and M. extensor digitorum longus (EDL). Therefore, in order to understand whether another proteolytic system would compensate the decline in proteasomal activity, we aimed to investigate age-related changes in the autophagy-lysosomal system (ALS) in SOL, mostly consisting of slow-twitch fibers, and EDL, mainly composed of fast-twitch fibers, from young (4 months) and old (25 months) C57BL/6JRj mice. Here, we focused on changes in the content of modified proteins and the ALS. Our results show that aged SOL and EDL display high levels of protein modifications, particularly in old SOL. While autophagy machinery appears to be functional, lysosomal activity declines gradually in aged SOL. In contrast, in old EDL, the ALS seems to be affected, demonstrated by an increased level of key autophagy-related proteins, which are known to accumulate when their delivery or degradation is impaired. In fact, lysosomal activity was significantly decreased in old EDL. Results presented herein suggest that the ALS can compensate the high levels of modified proteins in the more oxidative muscle, SOL, while EDL seems to be more prone to ALS age-related alterations.
  14. Biochimie. 2020 Aug 10. pii: S0300-9084(20)30172-3. [Epub ahead of print]
      Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PA). The η isozyme of DGK is abundantly expressed in C2C12 myoblasts. However, the role of DGKη in skeletal muscle cells remains unknown. In the present study, we showed that DGKη was downregulated at an early stage of myogenic differentiation. The knockdown of DGKη by siRNAs significantly inhibited C2C12 myoblast proliferation but did not inhibit differentiation. Moreover, the suppression of DGKη expression decreased the expression levels of mammalian target of rapamycin (mTOR), which is a key regulator of cell proliferation, and fatty acid synthase (FASN), which catalyzes the de novo synthesis of fatty acids for cell proliferation and is transcriptionally regulated via mTOR signaling. Furthermore, the knockdown of mTOR or raptor, which is a component of mTOR complex 1 (mTORC1), decreased the amount of FASN. These results indicate that DGKη regulates myoblast proliferation through the mTOR (mTORC1)-FASN pathway. Interestingly, the knockdown of mTOR reduced the expression levels of DGKη, implying mutual regulation between DGKη and mTOR. In DGKη-knockdown myoblasts, C30-C36-PA species, mTOR activators, were decreased, suggesting that the modulation of mTOR activity through these PA species also plays an important role in myoblast proliferation.
    Keywords:  Diacylglycerol kinase; Fatty acid synthase; Myoblast proliferation; Skeletal muscle; mTOR
  15. Front Cell Dev Biol. 2020 ;8 460
      Autophagy starts with the initiation and nucleation of isolation membranes, which further expand and seal to form autophagosomes. The regulation of isolation membrane closure remains poorly understood. CK1δ is a member of the casein kinase I family of serine/threonine specific kinases. Although CK1δ is reported to be involved in various cellular processes, its role in autophagy is unknown. Here, we show that CK1δ regulates the progression of autophagy from the formation of isolation membranes to autophagosome closure, and is essential for macroautophagy. CK1δ depletion results in impaired autophagy flux and the accumulation of unsealed isolation membranes. The association of LC3 with ATG9A, ATG14L, and ATG16L1 was found to be increased in CK1δ-depleted cells. The role of CK1δ in autophagosome completion appears to be conserved between yeasts and humans. Our data reveal a key role for CK1δ/Hrr25 in autophagosome completion.
    Keywords:  CK1δ; Hrr25; autophagosome closure; autophagy; isolation membrane
  16. Elife. 2020 Aug 10. pii: e56811. [Epub ahead of print]9
      The use of cannabis is rapidly expanding worldwide. Thus, innovative studies aimed to identify, understand and potentially reduce cannabis-evoked harms are warranted. Here, we found that Δ9-tetrahydrocannabinol, the psychoactive ingredient of cannabis, disrupts autophagy selectively in the striatum, a brain area that controls motor behavior, both in vitro and in vivo. Boosting autophagy, either pharmacologically (with temsirolimus) or by dietary intervention (with trehalose), rescued the Δ9-tetrahydrocannabinol-induced impairment of motor coordination in mice. The combination of conditional knockout mouse models and viral vector-mediated autophagy-modulating strategies in vivo showed that cannabinoid CB1 receptors located on neurons belonging to the direct (striatonigral) pathway are required for the motor-impairing activity of Δ9-tetrahydrocannabinol by inhibiting local autophagy. Taken together, these findings identify inhibition of autophagy as an unprecedented mechanistic link between cannabinoids and motor performance, and suggest that activators of autophagy might be considered as potential therapeutic tools to treat specific cannabinoid-evoked behavioral alterations.
    Keywords:  autophagy; cannabinoid; drug abuse; mTOR; motor behaviour; mouse; neuroscience; striatum
  17. Elife. 2020 Aug 14. pii: e58504. [Epub ahead of print]9
      Intracellular transport undergoes remodeling upon cell differentiation, which involves cell type-specific regulators. Bone morphogenetic protein 2-inducible kinase (BMP2K) has been potentially implicated in endocytosis and cell differentiation but its molecular functions remained unknown. We discovered that its longer (L) and shorter (S) splicing variants regulate erythroid differentiation in a manner unexplainable by their involvement in AP-2 adaptor phosphorylation and endocytosis. However, both variants interact with SEC16A and could localize to the juxtanuclear secretory compartment. Variant-specific depletion approach showed that BMP2K isoforms constitute a BMP2K-L/S regulatory system that controls the distribution of SEC16A and SEC24B as well as SEC31A abundance at COPII assemblies. Finally, we found L to promote and S to restrict autophagic degradation and erythroid differentiation. Hence, we propose that BMP2K-L and BMP2K-S differentially regulate abundance and distribution of COPII assemblies as well as autophagy, possibly thereby fine-tuning erythroid differentiation.
    Keywords:  cell biology; human; mouse
  18. Redox Biol. 2020 Jul 27. pii: S2213-2317(20)30866-1. [Epub ahead of print]36 101661
      Both iron metabolism and mitophagy, a selective mitochondrial degradation process via autolysosomal pathway, are fundamental for the cellular well-being. Mitochondria are the major site for iron metabolism, especially the biogenesis of iron-sulfur clusters (ISCs) via the mitochondria-localized ISCs assembly machinery. Here we report that mitochondrial ISCs biogenesis is coupled with receptor-mediated mitophagy in mammalian cells. Perturbation of mitochondrial ISCs biogenesis, either by depleting iron with the iron chelator or by knocking down the core components of the mitochondrial ISCs assembly machinery, triggers FUNDC1-dependent mitophagy. IRP1, one of the cellular iron sensors to maintain iron homeostasis, is crucial for iron stresses induced mitophagy. Knockdown of IRP1 disturbed iron stresses induced mitophagy. Furthermore, IRP1 could bind to a newly characterized IRE in the 5' untranslated region of the Bcl-xL mRNA and suppress its translation. Bcl-xL is an intrinsic inhibitory protein of the mitochondrial phosphatase PGAM5, which catalyzes the dephosphorylation of FUNDC1 for mitophagy activation. Alterations of the IRP1/Bcl-xL axis navigate iron stresses induced mitophagy. We conclude that ISCs serve as physiological signals for mitophagy activation, thus coupling mitophagy with iron metabolism.
    Keywords:  Bcl-xL; FUNDC1; ISCs; Mitophagy
  19. Cell Death Dis. 2020 Aug 14. 11(8): 624
      Although peripheral artery disease (PAD) is a major health problem, there have been limited advances in medical therapies. In PAD patients, angiogenesis is regarded as a promising therapeutic strategy to promote new arterial vessels and improve perfusion of ischemic tissue. Autophagy plays a critical role in catabolic processes for cell survival under normal and stressful conditions and plays fundamental biological roles in various cellular functions. In the present study, we showed that autophagy in endothelial cells is important for the repair and regeneration of damaged tissues. In a hindlimb ischemia mouse model, autophagy was stimulated in endothelial cells of the quadriceps muscle, and adjacent cells proliferated and regenerated. The autophagy pathway was induced under prolonged hypoxia in endothelial cells, and autophagy increased angiogenic activities. Moreover, conditioned media from endothelial cells blocked autophagy and inhibited the proliferation of muscle cells, suggesting that autophagic stimulation in endothelial cells affects the survival of adjacent cells, such as muscle. Collectively, hypoxia/ischemia-induced autophagy angiogenesis, and the damaged tissue surrounded by neo-vessels was regenerated in an ischemia model. Therefore, we strongly suggest that stimulation of autophagy in endothelial cells may be a potent therapeutic strategy in severe vascular diseases, including PAD.
  20. Sci Adv. 2020 Jul;6(31): eabb8725
      Autophagy is involved in the occurrence and development of tumors. Here, a pH-responsive polymersome codelivering hydroxychloroquine (HCQ) and tunicamycin (Tuni) drugs is developed to simultaneously induce endoplasmic reticulum (ER) stress and autophagic flux blockade for achieving an antitumor effect and inhibiting tumor metastasis. The pH response of poly(β-amino ester) and HCQ synergistically deacidifies the lysosomes, thereby blocking the fusion of autophagosomes and lysosomes and lastly blocking autophagic flux. The function mechanism of regulating autophagy was systematically investigated on orthotopic luciferase gene-transfected, 4T1 tumor-bearing BALB/c mice through Western blot and immunohistochemistry analyses. The Tuni triggers ER stress to regulate the PERK/Akt signaling pathway to increase the autophagic level. The "autophagic stress" generated by triggering ER stress-induced autophagy and blocking autophagic flux is effective against tumors. The reduced expression of matrix metalloproteinase-2 due to ER stress and reduced focal adhesions turnover due to the blockade of autophagic flux synergistically inhibit tumor metastasis.
  21. Autophagy. 2020 Aug 11. 1-16
      BURKHOLDERIA PSEUDOMALLEI: which causes melioidosis with high mortality in humans, has become a global public health concern. Recently, infection-driven lipid droplet accumulation has been related to the progression of host-pathogen interactions, and its contribution to the pathogenesis of infectious disease has been investigated. Here, we demonstrated that B. pseudomallei infection actively induced a time-dependent increase in the number and size of lipid droplets in human lung epithelial cells and macrophages. We also found that lipid droplet accumulation following B. pseudomallei infection was associated with downregulation of PNPLA2/ATGL (patatin like phospholipase domain containing 2) and lipophagy inhibition. Functionally, lipid droplet accumulation, facilitated via PNPLA2 downregulation, inhibited macroautophagic/autophagic flux and, thus, hindered autophagy-dependent inhibition of B. pseudomallei infection in lung epithelial cells. Mechanistically, we further revealed that nuclear receptor NR1D2 might be involved in the suppression of PNPLA2 after cell exposure to B. pseudomallei. Taken together, our findings unraveled an evolutionary strategy, by which B. pseudomallei interferes with the host lipid metabolism, to block autophagy-dependent suppression of infection. This study proposes potential targets for clinical therapy of melioidosis.ABBREVIATIONS: 3-MA: 3-methyladenine; ACTB: actin beta; ATG7: autophagy related 7; B. pseudomallei: Burkholderia pseudomallei; CFU: colony-forming unit; DG: diglyceride; FASN: fatty acid synthase; GFP: green fluorescent protein; LAMP1: lysosomal associated membrane protein 1; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LD: lipid droplet; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MG: monoglyceride; MOI: multiplicity of infection; mRFP: monomeric red fluorescent protein; NR1D2: nuclear receptor subfamily 1 group D member 2; p.i., post-infection; PLIN2/ADRP: perilipin 2; PNPLA2/ATGL: patatin like phospholipase domain containing 2; Rapa: rapamycin; SQSTM1/p62: sequestosome 1; shRNA: short hairpin RNA; TEM: transmission electron microscopy; TG: triglyceride.
    Keywords:   Burkholderia pseudomallei ; Autophagy; NR1D2; PNPLA2/ATGL; lipid droplet; lipid metabolism; lipophagy
  22. Alcohol. 2020 Aug 07. pii: S0741-8329(20)30263-9. [Epub ahead of print]
      Prenatal alcohol exposure causes fetal neurodevelopmental damage and growth restriction. Among regions of the brain, the cerebellum is the most vulnerable to developmental alcohol exposure. Despite vast research in the field, there is still a need to identify specific mechanisms by which alcohol causes this damage in order to design effective therapeutic interventions. Mechanistic target of rapamycin (mTOR) is known to be associated with axonal regeneration, dendritic arborization, synaptic plasticity, cellular growth, autophagy and many other cellular processes. Glutamine and glutamine related amino acids play a key role in fetal development and are known to alter the mTOR pathway; recent research has shown that disturbances in their bioavailability and signaling pathways may mediate adverse effects of prenatal alcohol exposure. This study investigated the role of the mTOR signaling pathway in the fetal cerebellum and skeletal muscle after third trimester-equivalent prenatal alcohol exposure and examined the effect on the mTOR pathway of maternal L-glutamine (GLN) supplementation concurrent with alcohol exposure. Pregnant sheep were assigned to: saline control, GLN control, alcohol, and alcohol + GLN groups. Treatment was administered on 3 consecutive days followed by a 4-day treatment-free interval, to mimic a weekend binge drinking pattern. Fetal cerebella and skeletal muscles were sampled for western blot analysis of mTOR and its downstream targets S6 kinase and eukaryotic initiation factor 4E-bindin protein (4E-BP1). Maternal alcohol exposure decreased the overall expression of fetal cerebellar total mTOR in the alcohol and alcohol+GLN group and the ratio of phosphorylated mTOR to total mTOR was elevated in the alcohol+GLN group compared to saline and GLN groups. Alcohol exposure increased the ratio of phosphorylated S6K to total S6K in fetal cerebellum and no significant effect of GLN supplementation was observed. Maternal GLN supplementation reduced the activation of mTOR and S6K in fetal skeletal muscle, possibly to make GLN and other amino acids available for use by other organs. These findings suggest prenatal alcohol exposure and maternal GLN supplementation during the third trimester-equivalent alters the mTOR signaling cascade which plays a possible key role in alcohol-induced developmental damage.
    Keywords:  Cerebellum; alcohol; glutamine; mTOR; teratogenicity
  23. Autophagy. 2020 Aug 10. 1-3
      Chaperone-mediated autophagy (CMA), as one of the main pathways of lysosomal catabolism, plays essential roles for the maintenance of cellular homeostasis. To date, the absence of any identifiable LAMP2A - the necessary and limiting protein required for CMA - in non-tetrapod lineages, led to the paradigm that this cellular process was restricted to mammals and birds. The recent findings of Lescat et al., demonstrating the existence of a CMA activity in fish, now reshuffle the cards regarding how the entire evolution of CMA function should be considered and appreciated across metazoans. Hence, beyond challenging the current tetrapod-centered accepted view, the work of Lescat et al. tackles the possibility - or the compelling need - of using complementary and powerful genetic models, such as zebrafish or medaka, for studying this fundamental function from an evolutionary perspective.
    Keywords:   Chaperone-mediated autophagy; CMA; Lamp2a; evolution; fish; medaka; zebrafish
  24. Sci Adv. 2020 Jul;6(31): eaay9131
      Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.
  25. Nat Commun. 2020 Aug 13. 11(1): 4056
      Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid β-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.
  26. J Clin Med. 2020 Aug 11. pii: E2596. [Epub ahead of print]9(8):
      Mitochondrial dysfunction is emerging as an important contributory factor to the pathophysiology of lysosomal storage disorders (LSDs). The cause of mitochondrial dysfunction in LSDs appears to be multifactorial, although impaired mitophagy and oxidative stress appear to be common inhibitory mechanisms shared amongst these heterogeneous disorders. Once impaired, dysfunctional mitochondria may impact upon the function of the lysosome by the generation of reactive oxygen species as well as depriving the lysosome of ATP which is required by the V-ATPase proton pump to maintain the acidity of the lumen. Given the reported evidence of mitochondrial dysfunction in LSDs together with the important symbiotic relationship between these two organelles, therapeutic strategies targeting both lysosome and mitochondrial dysfunction may be an important consideration in the treatment of LSDs. In this review we examine the putative mechanisms that may be responsible for mitochondrial dysfunction in reported LSDs which will be supplemented with morphological and clinical information.
    Keywords:  autophagy; inflammation; lysosomal storage diseases; mitochondrial dysfunction; mitophagy and cytokine; oxidative stress; reactive oxygen species
  27. J Cell Sci. 2020 Aug 11. pii: jcs.247817. [Epub ahead of print]
      In Schizosaccharomyces pombe, a general strategy for survival in response to environmental changes is sexual differentiation, which is triggered by TORC1 inactivation. However, mechanisms of TORC1 regulation in fission yeast remain poorly understood. In this study, we found that Pef1, which is an ortholog of mammalian CDK5, regulates the initiation of sexual differentiation through positive regulation of TORC1 activity. Conversely, deletion of pef1 leads to activation of autophagy and subsequent excessive TORC1 reactivation during the early phases of the nitrogen starvation response. This excessive TORC1 reactivation results in the silencing of the Ste11-Mei2 pathway and mating defects. Additionally, we found that pef1 genetically interacts with tsc1/2 in TORC1 regulation, and physically interacts with three types of cyclins, Clg1, Pas1, and Psl1. The double deletion of clg1 and pas1 promotes activation of autophagy and TORC1 during nitrogen starvation, similar to pef1Δ cells. Overall, our work suggests that Pef1-Clg1 and Pef1-Pas1 complexes regulate initiation of sexual differentiation through control of the TSC-TORC1 pathway and autophagy.
    Keywords:  Autophagy; CDK5; Cyclin; Pef1; Sexual differentiation; TORC1
  28. Sci Rep. 2020 Aug 11. 10(1): 13523
      Autophagy, an integral part of the waste recycling process, plays an important role in cellular physiology and pathophysiology. Impaired autophagic flux causes ectopic lipid deposition, which is defined as the accumulation of lipids in non-adipose tissue. Ectopic lipid accumulation is observed in patients with cardiometabolic syndrome, including obesity, diabetes, insulin resistance, and cardiovascular complications. Metformin is the first line of treatment for type 2 diabetes, and one of the underlying mechanisms for the anti-diabetic effect of metformin is mediated by the stimulation of AMP-activated protein kinase (AMPK). Because the activation of AMPK is crucial for the initiation of autophagy, we hypothesize that metformin reduces the accumulation of lipid droplets by increasing autophagic flux in vascular endothelial cells. Incubation of vascular endothelial cells with saturated fatty acid (SFA) increased the accumulation of lipid droplets and impaired autophagic flux. We observed that the accumulation of lipid droplets was reduced, and the autophagic flux was enhanced by treatment with metformin. The knock-down of AMPKα by using siRNA blunted the effect of metformin. Furthermore, treatment with SFA or inhibition of autophagy increased leukocyte adhesion, whereas treatment with metformin decreased the SFA-induced leukocyte adhesion. The results suggest a novel mechanism by which metformin protects vascular endothelium from SFA-induced ectopic lipid accumulation and pro-inflammatory responses. In conclusion, improving autophagic flux may be a therapeutic strategy to protect endothelial function from dyslipidemia and diabetic complications.