bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒02‒16
twenty papers selected by
Viktor Korolchuk
Newcastle University

  1. Autophagy. 2020 Feb 10. 1-3
      The degradation of specific cargos such as ubiquitinated protein aggregates and dysfunctional mitochondria via macroautophagy/autophagy is facilitated by SQSTM1/p62, the first described selective autophagy receptor in metazoans. While the general process of autophagy plays crucial roles during aging, it remains unclear whether and how selective autophagy mediates effects on longevity and health. Two recent studies in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster observed gene expression changes of the respective SQSTM1 orthologs in response to environmental stressors or age and showed that overexpression of SQSTM1 is sufficient to extend lifespan and improve proteostasis and mitochondrial function in an autophagy-dependent manner in these model organisms. These findings show that increased expression of the selective autophagy receptor SQSTM1 is sufficient to induce aggrephagy in C. elegans, and mitophagy in Drosophila, and demonstrate an evolutionarily conserved role for SQSTM1 in lifespan determination.
    Keywords:  Aging; C. elegans; Drosophila; SQST-1; SQSTM1; aggrephagy; heat shock; mitophagy; p62; proteostasis; ref(2)P; selective autophagy
  2. J Mol Biol. 2020 Feb 07. pii: S0022-2836(20)30088-7. [Epub ahead of print]
      Autophagy is an intracellular degradation process that is essential for cellular survival, tissue homeostasis and human health. The housekeeping functions of autophagy in mediating the clearance of aggregation-prone proteins and damaged organelles are vital for post-mitotic neurons. Improper functioning of this process contributes to the pathology of myriad human diseases including neurodegeneration. Impairment in autophagy has been reported in several neurodegenerative diseases where pharmacological induction of autophagy has therapeutic benefits in cellular and transgenic animal models. However, emerging studies suggest that the efficacy of autophagy inducers as well as the nature of the autophagy defects may be context-dependent, and therefore, studies in disease-relevant experimental systems may provide more insights for clinical translation to patients. With the advancements in human stem cell technology, it is now possible to establish disease-affected cellular platforms from patients for investigating disease mechanisms and identifying candidate drugs in the appropriate cell-types such as neurons that are otherwise not accessible. Towards this, patient-derived human induced pluripotent stem cells (hiPSCs) have demonstrated considerable promise in constituting a platform for effective disease modelling and drug discovery. Multiple studies have utilized hiPSC models of neurodegenerative diseases to study autophagy and evaluate the therapeutic efficacy of autophagy inducers in neuronal cells. This review provides an overview of the regulation of autophagy, generation of hiPSCs via cellular reprogramming, and neuronal differentiation, and outlines the findings in various neurodegenerative disorders where autophagy has been studied using hiPSC models.
  3. Cell Discov. 2020 ;6 6
      Autophagy is a major intracellular degradation system that derives its degradative abilities from the lysosome. The most well-studied form of autophagy is macroautophagy, which delivers cytoplasmic material to lysosomes via the double-membraned autophagosome. Other forms of autophagy, namely chaperone-mediated autophagy and microautophagy, occur directly on the lysosome. Besides providing the means for degradation, lysosomes are also involved in autophagy regulation and can become substrates of autophagy when damaged. During autophagy, they exhibit notable changes, including increased acidification, enhanced enzymatic activity, and perinuclear localization. Despite their importance to autophagy, details on autophagy-specific regulation of lysosomes remain relatively scarce. This review aims to provide a summary of current understanding on the behaviour of lysosomes during autophagy and outline unexplored areas of autophagy-specific lysosome research.
    Keywords:  Autophagy; Lysosomes
  4. J Cell Biol. 2020 Mar 02. pii: e201902127. [Epub ahead of print]219(3):
      Cellular adaptation in response to nutrient limitation requires the induction of autophagy and lysosome biogenesis for the efficient recycling of macromolecules. Here, we discovered that starvation and TORC1 inactivation not only lead to the up-regulation of autophagy and vacuole proteins involved in recycling but also result in the down-regulation of many vacuole membrane proteins to supply amino acids as part of a vacuole remodeling process. Down-regulation of vacuole membrane proteins is initiated by ubiquitination, which is accomplished by the coordination of multiple E3 ubiquitin ligases, including Rsp5, the Dsc complex, and a newly characterized E3 ligase, Pib1. The Dsc complex is negatively regulated by TORC1 through the Rim15-Ume6 signaling cascade. After ubiquitination, vacuole membrane proteins are sorted into the lumen for degradation by ESCRT-dependent microautophagy. Thus, our study uncovered a complex relationship between TORC1 inactivation and vacuole biogenesis.
  5. Int J Mol Sci. 2020 Feb 11. pii: E1202. [Epub ahead of print]21(4):
      Parkinson's disease (PD) is a common neurodegenerative disorder. Recent identification of genes linked to familial forms of PD has revealed that post-translational modifications, such as phosphorylation and ubiquitination of proteins, are key factors in disease pathogenesis. In PD, E3 ubiquitin ligase Parkin and the serine/threonine-protein kinase PTEN-induced kinase 1 (PINK1) mediate the mitophagy pathway for mitochondrial quality control via phosphorylation and ubiquitination of their substrates. In this review, we first focus on well-characterized PINK1 phosphorylation motifs. Second, we describe our findings concerning relationships between Parkin and HtrA2/Omi, a protein involved in familial PD. Third, we describe our findings regarding inhibitory PAS (Per/Arnt/Sim) domain protein (IPAS), a member of PINK1 and Parkin substrates, involved in neurodegeneration during PD. IPAS is a dual-function protein involved in transcriptional repression of hypoxic responses and the pro-apoptotic activities.
    Keywords:  1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); HtrA2/Omi; IPAS; PINK1; Parkin; Parkinson’s disease
  6. Biochem Biophys Res Commun. 2020 Feb 05. pii: S0006-291X(20)30245-X. [Epub ahead of print]
      Endoplasmic reticulum (ER) stress and autophagy are regulated by shared signaling pathways, and their dysfunction is directly related to pathological conditions. This study investigated the function of the unc-51 like autophagy activating kinase 1 (ULK1)-autophagy related 13 (ATG13) complex in ER stress conditions through a knockout (KO) approach. Unlike other autophagy genes, KO of ULK1 or ATG13 attenuated ER stress and promoted mammalian target of rapamycin complex 1 (mTORC1) activation. Compared with wild type (WT) cells, ULK1 and ATG13 KO cells displayed increased viability, while beclin 1, ATG14, and ULK1/2 KO cells did not. Tunicamycin treatment upregulated the expression of ER stress markers (DNA damage inducible transcript 3, heat shock protein family A (Hsp70) member 5, and phosphorylated eukaryotic translation initiation factor 2 alpha kinase 3, eukaryotic translation initiation factor 2 subunit alpha, and endoplasmic reticulum to nucleus signaling 1); however, these were decreased in ULK1 and ATG13 KO cells. Insulin treatment upregulates the phosphorylation of ribosomal protein S6 kinase B1 (RPS6KB1) and AKT serine/threonine kinase 1 (AKT1), which was suppressed by tunicamycin. Notably, ATG13 and ULK1 deficiency ameliorated tunicamycin-induced insulin resistance, with enhanced RPS6KB1 and AKT1 phosphorylation in KO cells compared to WT cells. Although ULK1 and ATG13 are necessary for autophagy induction after tunicamycin-induced ER stress, autophagy does not seem to directly affect tunicamycin-induced cell death, ER stress, or insulin resistance. Our results indicate that loss of the ULK1-ATG13 complex attenuates ER stress and cell death and increases mTORC1 signaling.
    Keywords:  Apoptosis; Autophagy; Autophagy related 13; Insulin resistance; Tunicamycin; Unc-51 like autophagy activating kinase 1
  7. Autophagy. 2020 Feb 11. 1-14
      Nucleophagy, the mechanism for autophagic degradation of nuclear material, occurs in both a macro- and micronucleophagic manner. Upon nitrogen deprivation, we observed, in an in-depth fluorescence microscopy study, the formation of micronuclei: small parts of superfluous nuclear components surrounded by perinuclear ER. We identified two types of micronuclei associated with a corresponding autophagic mode. Our results showed that macronucleophagy degraded these smaller micronuclei. Engulfed in Atg8-positive phagophores and containing cargo receptor Atg39, macronucleophagic structures revealed finger-like extensions when observed in 3-dimensional reconstitutions of fluorescence microscopy images, suggesting directional growth. Interestingly, in the late stages of phagophore elongation, the adjacent vacuolar membrane showed a reduction of integral membrane protein Pho8. This change in membrane composition could indicate the formation of a specialized vacuolar domain, required for autophagosomal fusion. Significantly larger micronuclei formed at nucleus vacuole junctions and were identified as a substrate of piecemeal microautophagy of the nucleus (PMN), by the presence of the integral membrane protein Nvj1. Micronuclei sequestered by vacuolar invaginations also contained Atg39. A detailed investigation revealed that both Atg39 and Atg8 accumulated between the vacuolar tips. These findings suggest a role for Atg39 in micronucleophagy. Indeed, following the degradation of Nvj1, an exclusive substrate of PMN, in immunoblots, we could confirm the essential role of Atg39 for PMN. Our study thus details the involvement of Atg8 in both macronucleophagy and PMN and identifies Atg39 as the general cargo receptor for nucleophagic processes.Abbreviations: DIC: Differential interference contrast, FWHM: Full width at half maximum, IQR: Interquartile range, MIPA: Micropexophagy-specific membrane apparatus, NLS: Nuclear localization signal, NVJ: Nucleus vacuole junction, PMN: Piecemeal microautophagy of the nucleus, pnER: Perinuclear ER.
    Keywords:  Atg39; Atg8; ER-phagy; autophagosome formation; microautophagy; nucleophagy; nucleus vacuole junction; organellar contact sites
  8. Biochem Biophys Res Commun. 2020 Feb 10. pii: S0006-291X(20)30282-5. [Epub ahead of print]
      Amyloid β (Aβ) oligomers may be a real culprit in the pathogenesis of Alzheimer's disease (AD); therefore, the elimination of these toxic oligomers may be of great significance for AD therapy. Autophagy is the catabolic process by which lysosomes degrade cytosolic components, and heat shock cognate 70 kDa protein (Hsc70) binds to proteins with their KFERQ-like motifs [also known as chaperone-mediated autophagy (CMA) motifs] and carries them to lysosomes through CMA or late endosomes through endosomal microautophagy (eMI) for degradation. In this study, our strategy is to make the pathological Aβ become one selective and suitable substrate for CMA and eMI (termed as Hsc70-based autophagy) by tagging its oligomers with multiple CMA motifs. First, we design and synthesize Aβ oligomer binding peptides with three CMA motifs. Second, we determine that the peptide can help Aβ oligomers enter endosomes and lysosomes, which can be further enhanced by ketone. More importantly, we find that the peptide can dramatically reduce Aβ oligomers in induced pluripotent stem cell (iPSC) cortical neurons derived from AD patient fibroblasts and protect primary cultured cortical neurons against the Aβ oligomer-induced neurotoxicity. In conclusion, we demonstrate that the peptide targeting Hsc70-based autophagy can effectively eliminate Aβ oligomers and have superior neuroprotective activity.
    Keywords:  Alzheimer’s disease; Aβ oligomers; Chaperone-mediated autophagy; Hsc70
  9. J Biol Chem. 2020 Feb 11. pii: jbc.RA119.010734. [Epub ahead of print]
      Oxidative stress-induced DNA damage, the senescence-associated secretory phenotype (SASP), and impaired autophagy all are general features of senescent cells. However, the crosstalk among these events and processes is not fully understood. Here, using NIH3T3 cells exposed to hydrogen peroxide stress, we show that stress-induced DNA damage provokes the SASP largely via cytosolic chromatin fragment (CCF) formation, which activates a cascade comprising cGMP-AMP synthase (cGAS), stimulator of interferon genes protein (STING), NF-κB, and SASP, and that autolysosomal function inhibits this cascade. We found that CCFs accumulate in senescent cells with activated cGAS-STING-NF-κB signaling, promoting SASP and cellular senescence. We also present evidence that the persistent accumulation of CCFs in prematurely senescent cells is partially associated with a defect in DNA- degrading activity in autolysosomes and reduced abundance of activated DNase 2a. Intriguingly, we found that metformin- or rapamycin-induced activation of autophagy significantly lessened the size and levels of CCFs and repressed the activation of the cGAS-STING-NF-κB-SASP cascade and cellular senescence. These effects of autophagy activators indicated that autolysosomal function contributes to CCFs clearance and SASP suppression, further supported by the fact that the lysosome inhibitor bafilomycin A1 blocked the role of autophagy-mediated CCFs clearance and senescence repression. Taken together, these findings confirm the significant role of CCFs formation in the SASP and oxidative stress-induced senescence and reveal that CCF-mediated SASP inversely correlated with autolysosomal function. We conclude that the restoration of autolysosomal function may prevent DNA damage-provoked SASP production and cellular senescence.
    Keywords:  DNA damage; NF-kappa B (NF-KB); autolysosome; autophagy; cGAS-STING pathway; cytosolic chromatin fragment (CCF); oxidative stress; senescence; senescence-associated secretory phenotype (SASP)
  10. Cell Rep. 2020 Feb 11. pii: S2211-1247(20)30066-8. [Epub ahead of print]30(6): 1823-1834.e5
      The tumor suppressor folliculin (FLCN) suppresses nuclear translocation of TFE3, a master transcription factor for lysosomal biogenesis, via regulation of amino-acid-sensing Rag GTPases. However, the importance of this lysosomal regulation in mammalian physiology remains unclear. Following hematopoietic-lineage-specific Flcn deletion in mice, we found expansion of vacuolated phagocytes that accumulate glycogen in their cytoplasm, phenotypes reminiscent of lysosomal storage disorder (LSD). We report that TFE3 acts in a feedback loop to transcriptionally activate FLCN expression, and FLCN loss disrupts this loop, augmenting TFE3 activity. Tfe3 deletion in Flcn knockout mice reduces the number of phagocytes and ameliorates LSD-like phenotypes. We further reveal that TFE3 stimulates glycogenesis by promoting the expression of glycogenesis genes, including Gys1 and Gyg, upon loss of Flcn. Taken together, we propose that the FLCN-TFE3 feedback loop acts as a rheostat to control lysosome activity and prevents excessive glycogenesis and LSD-like phagocyte activation.
    Keywords:  Birt-Hogg-Dubé Syndrome; Folliculin; Lysosomal storage disease; Lysosome; gluconeogenesis; glycogen; glycogenesis; hemophagocytosis
  11. Front Cell Dev Biol. 2019 ;7 373
      The eukaryotic cell has developed intricate machineries that monitor and maintain proteome homeostasis in order to ensure cellular functionality. This involves the carefully coordinated balance between protein synthesis and degradation pathways, which are dynamically regulated in order to meet the constantly changing demands of the cell. Ribosomes, together with the endoplasmic reticulum (ER), are the key drivers of protein synthesis, folding, maturation and sorting, while the proteasome plays a pivotal role in terminating the existence of thousands of proteins that are misfolded, damaged or otherwise obsolete. The synthesis, structure and function of these dedicated machines has been studied for decades, however, much less is understood about the mechanisms that control and execute their own turnover. Autophagy, an evolutionarily conserved catabolic pathway, mediates degradation of a large variety of cytosolic substrates, ranging from single proteins to entire organelles or multi-subunit macromolecular complexes. In this review, we focus on selective autophagy of three key components of the protein homeostasis machinery: ribosomes, ER and proteasomes, through the selective autophagy pathways of ribophagy, ER-phagy, and proteaphagy. We discuss newly discovered mechanisms for the selective clearance of these substrates, which are often stress-dependent and involve specialized signals for cargo recognition by a growing number of receptors. We further discuss the interplay between these pathways and their biological impact on key aspects of proteome homeostasis and cellular function in health and disease.
    Keywords:  ER-phagy; proteaphagy; protein homeostasis; ribophagy; selective autophagy; ubiquitin
  12. Cell Death Differ. 2020 Feb 12.
      Rab5 is a master regulator for endosome biogenesis and transport while its in vivo physiological function remains elusive. Here, we find that Rab5a is upregulated in several in vivo and in vitro myogenesis models. By generating myogenic Rab5a-deficient mice, we uncover the essential roles of Rab5a in regulating skeletal muscle regeneration. We further reveal that Rab5a promotes myoblast differentiation and directly interacts with insulin receptor substrate 1 (IRS1), an essential scaffold protein for propagating IGF signaling. Rab5a interacts with IRS1 in a GTP-dependent manner and this interaction is enhanced upon IGF-1 activation and myogenic differentiation. We subsequently identify that the arginine 207 and 222 of IRS1 and tyrosine 82, 89, and 90 of Rab5a are the critical amino acid residues for mediating the association. Mechanistically, Rab5a modulates IRS1 activation by coordinating the association between IRS1 and the IGF receptor (IGFR) and regulating the intracellular membrane targeting of IRS1. Both myogenesis-induced and IGF-evoked AKT-mTOR signaling are dependent on Rab5a. Myogenic deletion of Rab5a also reduces the activation of AKT-mTOR signaling during skeletal muscle regeneration. Taken together, our study uncovers the physiological function of Rab5a in regulating muscle regeneration and delineates the novel role of Rab5a as a critical switch controlling AKT-mTOR signaling by activating IRS1.
  13. Cells. 2020 Feb 07. pii: E381. [Epub ahead of print]9(2):
      Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disorder that progressively affects motor neurons in the brain and spinal cord. Due to the biological complexity of the disease, its etiology remains unknown. Several cellular mechanisms involved in the neurodegenerative process in ALS have been found, including the loss of RNA and protein homeostasis, as well as mitochondrial dysfunction. Insoluble protein aggregates, damaged mitochondria, and stress granules, which contain RNA and protein components, are recognized and degraded by the autophagy machinery in a process known as selective autophagy. Autophagy is a highly dynamic process whose dysregulation has now been associated with neurodegenerative diseases, including ALS, by numerous studies. In ALS, the autophagy process has been found deregulated in both familial and sporadic cases of the disease. Likewise, mutations in genes coding for proteins involved in the autophagy machinery have been reported in ALS patients, including selective autophagy receptors. In this review, we focus on the role of selective autophagy in ALS pathology.
    Keywords:  NBR1; TBK1; amyotrophic lateral sclerosis; autophagy; optineurin; p62; ubiquilin2
  14. Proc Natl Acad Sci U S A. 2020 Feb 11. pii: 201909814. [Epub ahead of print]
      VDAC1 is a critical substrate of Parkin responsible for the regulation of mitophagy and apoptosis. Here, we demonstrate that VDAC1 can be either mono- or polyubiquitinated by Parkin in a PINK1-dependent manner. VDAC1 deficient with polyubiquitination (VDAC1 Poly-KR) hampers mitophagy, but VDAC1 deficient with monoubiquitination (VDAC1 K274R) promotes apoptosis by augmenting the mitochondrial calcium uptake through the mitochondrial calcium uniporter (MCU) channel. The transgenic flies expressing Drosophila Porin K273R, corresponding to human VDAC1 K274R, show Parkinson disease (PD)-related phenotypes including locomotive dysfunction and degenerated dopaminergic neurons, which are relieved by suppressing MCU and mitochondrial calcium uptake. To further confirm the relevance of our findings in PD, we identify a missense mutation of Parkin discovered in PD patients, T415N, which lacks the ability to induce VDAC1 monoubiquitination but still maintains polyubiquitination. Interestingly, Drosophila Parkin T433N, corresponding to human Parkin T415N, fails to rescue the PD-related phenotypes of Parkin-null flies. Taken together, our results suggest that VDAC1 monoubiquitination plays important roles in the pathologies of PD by controlling apoptosis.
    Keywords:  PINK1; Parkin; Parkinson disease; VDAC1; apoptosis
  15. Cells. 2020 Feb 07. pii: E380. [Epub ahead of print]9(2):
      Poly(ADP-Ribose) polymerases (PARPs) are enzymes that metabolize NAD+. PARP1 and PARP10 were previously implicated in the regulation of autophagy. Here we showed that cytosolic electron-dense particles appear in the cytoplasm of C2C12 myoblasts in which PARP2 is silenced by shRNA. The cytosolic electron-dense bodies resemble autophagic vesicles and, in line with that, we observed an increased number of LC3-positive and Lysotracker-stained vesicles. Silencing of PARP2 did not influence the maximal number of LC3-positive vesicles seen upon chloroquine treatment or serum starvation, suggesting that the absence of PARP2 inhibits autophagic breakdown. Silencing of PARP2 inhibited the activity of AMP-activated kinase (AMPK) and the mammalian target of rapamycin complex 2 (mTORC2). Treatment of PARP2-silenced C2C12 cells with AICAR, an AMPK activator, nicotinamide-riboside (an NAD+ precursor), or EX-527 (a SIRT1 inhibitor) decreased the number of LC3-positive vesicles cells to similar levels as in control (scPARP2) cells, suggesting that these pathways inhibit autophagic flux upon PARP2 silencing. We observed a similar increase in the number of LC3 vesicles in primary PARP2 knockout murine embryonic fibroblasts. We provided evidence that the enzymatic activity of PARP2 is important in regulating autophagy. Finally, we showed that the silencing of PARP2 induces myoblast differentiation. Taken together, PARP2 is a positive regulator of autophagic breakdown in mammalian transformed cells and its absence blocks the progression of autophagy.
    Keywords:  AMPK; ARTD2; LC3; PARP; PARP2; SIRT1; autophagy; mTOR; nicotinamide-riboside
  16. Sci Rep. 2020 Feb 14. 10(1): 2687
      Radiotherapy combined with chemotherapy is the major treatment modality for human glioblastoma multiforme (GBM). GBMs eventually relapse after treatment and the average survival of GBM patients is less than two years. There is some evidence that cannabidiol (CBD) can induce cell death and increases the radiosensitivity of GBM by enhancing apoptosis. Beside initiation of death, CBD has been demonstrated as an inducer of autophagy. In the present study, we address the question whether CBD simultaneously induces a protective effect in GBM by upregulating autophagy. Addition of chloroquine that suppressed autophagic flux to 2D GBM cultures increased CBD-induced cell death, presenting proof for the protective autophagy. Blockage of autophagy upregulated radiation-induced cytotoxicity but only modestly affected the levels of cell death in CBD- or CBD/γ-irradiated 3D GBM cultures. Furthermore, CBD enhanced the pro-apoptotic activities of JNK1/2 and MAPK p38 signaling cascades while partially downregulated the pro-survival PI3K-AKT cascade, thereby changing a balance between cell death and survival. Suppression of JNK activation partially reduced CBD-induced cell death in 3D GBM cultures. In contrast, co-treatment of CBD-targeted cells with inhibitors of PI3K-AKT-NF-κB, IKK-NF-κB or JAK2-STAT3 pathways killed surviving GBM cells in both 2D and 3D cultures, potentially improving the therapeutic ratio of GBM.
  17. Bone. 2020 Feb 06. pii: S8756-3282(20)30045-4. [Epub ahead of print] 115265
      Paget's disease of bone (PDB) is a bone disorder characterized by an increase in bone turnover in a disorganized way with a large increase in bone resorption followed by bone formation. The most important known genetic factor predisposing to PDB is mutation in Sequestosome1 (SQSTM1) gene. We have studied the prevalence of SQSTM1 mutations and examined genotype-phenotype correlations in a Spanish cohort of PDB patients. Also, we have characterized three PDB patients that carry the c.961C>T SQSTM1 gene mutation that it is localized in exon 6 of SQSTM1 gene and it causes the p. R321C mutation. This mutation has been reported in patients with amyotrophic lateral sclerosis and frontotemporal dementia but in our knowledge this is the first time that p62 p. R321C mutation is associated to PDB. We show that p62 p.R321C mutation could induce blockage of autophagy and cell proliferation through NF-kB pathway. These results reinforce the hypothesis of autophagy involvement in Paget's disease of bone.
    Keywords:  Autophagy; Mutation; NF-kB pathway; Paget's disease of bone; SQSTM1; Sequestosome1; p62
  18. Redox Biol. 2020 Jan 28. pii: S2213-2317(19)31180-2. [Epub ahead of print] 101445
      TFEB (transcription factor EB) and TFE3 (transcription factor E3) are "master regulators" of autophagy and lysosomal biogenesis. The stress response p38 mitogen-activated protein (MAP) kinases affect multiple intracellular responses including inflammation, cell growth, differentiation, cell death, senescence, tumorigenesis, and autophagy. Small molecule p38 MAP kinase inhibitors such as SB202190 are widely used in dissection of related signal transduction mechanisms including redox biology and autophagy. Here, we initially aimed to investigate the links between p38 MAP kinase and TFEB/TFE3-mediated autophagy and lysosomal biogenesis. Unexpectedly, we found that only SB202190, rather than several other p38 inhibitors, promotes TFEB and TFE3 to translocate from the cytosol into the nucleus and subsequently enhances autophagy and lysosomal biogenesis. In addition, siRNA-mediated Tfeb and Tfe3 knockdown effectively attenuated SB202190-induced gene expression and lysosomal biogenesis. Mechanistical studies showed that TFEB and TFE3 activation in response to SB202190 is dependent on PPP3/calcineurin rather than on the inhibition of p38 or MTOR signaling, the main pathway for regulating TFEB and TFE3 activation. Importantly, SB202190 increased intracellular calcium levels, and calcium chelator BAPTAP-AM blocked SB202190-induced TFEB and TFE3 activation as well as autophagy and lysosomal biogenesis. Moreover, endoplasmic reticulum (ER) calcium is required for TFEB and TFE3 activation in response to SB202190. In summary, we identified a previously uncharacterized role of SB202190 in activating TFEB- and TFE3-dependent autophagy and lysosomal biogenesis via ER calcium release and subsequent calcium-dependent PPP3/calcineurin activation, leading to dephosphorylation of TFEB and TFE3. Given the importance of p38 MAP kinase invarious conditions including oxidative stress, the findings collectively indicate that SB202190 should not be used as a specific inhibitor for elucidating the p38 MAP kinase biological functions due to its potential effect on activating autophagy-lysosomal axis.
    Keywords:  Autophagy and lysosomal biogenesis; SB202190; SB203580; TFE3; TFEB; p38 inhibitor
  19. Development. 2020 Feb 10. pii: dev.181727. [Epub ahead of print]
      In many eukaryotes, the small GTPase Rheb functions as a switch to toggle activity of TOR complex 1 (TORC1) between anabolism and catabolism, thus controlling lifespan, development, and autophagy. Our CRISPR-generated, fluorescently tagged endogenous C. elegans RHEB-1 and DAF-15/Raptor are expressed ubiquitously and localize to lysosomes. Disruption of LET-363/TOR and DAF-15/Raptor are required for development past the third larval stage (L3). We observed that deletion of RHEB-1 similarly conferred L3 arrest. Unexpectedly, robust RNAi-mediated depletion of TORC1 components caused arrest at stages prior to L3. Accordingly, conditional depletion of endogenous DAF-15/Raptor in the soma revealed that TORC1 is required at each stage of the life cycle to progress to the next stage. Reversal of DAF-15 depletion permits arrested animals to recover to continue development. Our results are consistent with TORC1 functioning as a developmental checkpoint that governs at each stage the decision of the animal to progress through development.
    Keywords:  Ral; RalGAP; TSC; Tuberous sclerosis complex; mTOR; mTORC1
  20. J Clin Invest. 2020 Feb 10. pii: 131048. [Epub ahead of print]
      Smooth muscle cell (SMC) proliferation has been thought to limit the progression of thoracic aortic aneurysm and dissection (TAAD) because loss of medial cells associates with advanced disease. We investigated effects of SMC proliferation in the aortic media by conditional disruption of Tsc1, which hyperactivates mTOR complex 1. Consequent SMC hyperplasia led to progressive medial degeneration and TAAD. In addition to diminished contractile and synthetic functions, fate-mapped SMCs displayed increased proteolysis, endocytosis, phagocytosis, and lysosomal clearance of extracellular matrix and apoptotic cells. SMCs acquired a limited repertoire of macrophage markers and functions via biogenesis of degradative organelles through an mTOR/β-catenin/MITF-dependent pathway, but were distinguishable from conventional macrophages by an absence of hematopoietic lineage markers and certain immune effectors even in the context of hyperlipidemia. Similar mTOR activation and induction of a degradative SMC phenotype in a model of mild TAAD due to Fbn1 mutation greatly worsened disease with near-uniform lethality. The finding of increased lysosomal markers in medial SMCs from clinical TAAD specimens with hyperplasia and matrix degradation further supports the concept that proliferation of degradative SMCs within the media causes aortic disease, thus identifying mTOR-dependent phenotypic modulation as a therapeutic target for combating TAAD.
    Keywords:  Cardiovascular disease; Vascular Biology