bims-auttor Biomed News
on Autophagy and mTOR
Issue of 2020‒03‒01
33 papers selected by
Viktor Korolchuk
Newcastle University

  1. Aging Cell. 2020 Feb 25. e13126
      GGGGCC repeat expansion in C9orf72 is the most common genetic cause in both frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), two neurodegenerative disorders in association with aging. Bidirectional repeat expansions in the noncoding region of C9orf72 have been shown to produce dipeptide repeat (DPR) proteins through repeat-associated non-ATG (RAN) translation and to reduce the expression level of the C9orf72 gene product, C9orf72 protein. Mechanisms underlying C9orf72-linked neurodegeneration include expanded RNA repeat gain of function, DPR toxicity, and C9orf72 protein loss of function. In the current study, we focus on the cellular function of C9orf72 protein. We report that C9orf72 can regulate lysosomal biogenesis and autophagy at the transcriptional level. We show that loss of C9orf72 leads to striking accumulation of lysosomes, autophagosomes, and autolysosomes in cells, which is associated with suppressed mTORC1 activity and enhanced nuclear translocation of MiT/TFE family members MITF, TFE3, and TFEB, three master regulators of lysosomal biogenesis and autophagy. We demonstrate that the DENN domain of C9orf72 specifically binds to inactive Rag GTPases, but not active Rag GTPases, thereby affecting the function of Rag/raptor/mTOR complex and mTORC1 activity. Furthermore, active Rag GTPases, but not inactive Rag GTPases or raptor rescued the impaired activity and lysosomal localization of mTORC1 in C9orf72-deficient cells. Taken together, the present study highlights a key role of C9orf72 in lysosomal and autophagosomal regulation, and demonstrates that Rag GTPases and mTORC1 are involved in C9orf72-mediated autophagy.
    Keywords:  Rag GTPases; autophagy; mTOR complex 1; neurodegenerative diseases; transcription factor EB
  2. Autophagy. 2020 Feb 24. 1-18
      The precise degradation of dysfunctional mitochondria by mitophagy is essential for maintaining neuronal homeostasis. HTT (huntingtin) can interact with numerous other proteins and thereby perform multiple biological functions within the cell. In this study, we investigated the role of HTT during mitophagy and analyzed the impact of the expansion of its polyglutamine (polyQ) tract. HTT is involved in different mitophagy steps, promoting the physical proximity of different protein complexes during the initiation of mitophagy and recruiting mitophagy receptors essential for promoting the interaction between damaged mitochondria and the nascent autophagosome. The presence of the polyQ tract in mutant HTT affects the formation of these protein complexes and determines the negative consequences of mutant HTT on mitophagy, leading to the accumulation of damaged mitochondria and an increase in oxidative stress. These outcomes contribute to general mitochondrial dysfunction and neurodegeneration in Huntington disease.Abbreviations: AMPK: AMP-activated protein kinase; ATG13: autophagy related 13; BECN1: beclin 1, autophagy related; BNIP3: BCL2/adenovirus E1B interacting protein 3; BNIP3L/Nix: BCL2/adenovirus E1B interacting protein 3-like; CCCP: carbonyl cyanide 3-chlorophenyl hydrazone; DMEM: Dulbecco's modified eagle medium; EDTA: ethylene-diamine-tetra-acetic acid; EGFP: enhanced green fluorescent protein; EGTA: ethylene glycol bis(2-aminoethyl ether)tetraacetic acid; FUNDC1: FUN14 domain containing 1; HD: Huntington disease; HRP: horseradish peroxidase; HTT: huntingtin; LC3-II: lipidated form of MAP1LC3/LC3; mtDNA: mitochondrial deoxyribonucleic acid; MTDR: MitoTracker Deep Red; MTOR: mechanistic target of rapamycin kinase; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1, autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; OCR: oxygen consumption rate; OPTN: optineurin; OXPHOS: oxidative phosphorylation; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PINK1: PTEN induced putative kinase 1; PLA: proximity ligation assay; PMSF: phenylmethylsulfonyl fluoride; polyQ: polyglutamine; PtdIns3K: phosphatidylinositol 3-kinase; ROS: reactive oxygen species; Rot: rotenone; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SEM: standard error of the mean; SQSTM1/p62: sequestosome 1; TMRM: tetramethylrhodamine methyl ester; UB: ubiquitin; ULK1: unc-51 like kinase 1.
    Keywords:  Autophagy; Huntington disease; huntingtin; mitochondria; mitophagy
  3. J Mol Biol. 2020 Feb 19. pii: S0022-2836(20)30162-5. [Epub ahead of print]
      Neurodegenerative diseases (NDDs) comprise conditions with impaired neuronal function and loss, and may be associated with build-up of aggregated proteins with altered physicochemical properties (misfolded proteins). There are many disorders, and causes include gene mutations, infections, or exposure to toxins. The autophagy pathway is involved in the removal of unwanted proteins and organelles through lysosomes. While lysosomal storage disorders have been described for many years, it is now recognised that perturbations of the autophagy pathway itself, can also lead to neurodegenerative disease. These include monogenic disorders of key proteins involved in the autophagy pathway, and disorders within pathways that critically control autophagy through monitoring of the supply of nutrients (mTORC1 pathway) or of energy supply in cells (AMPK pathway). This review focuses on childhood onset neurodegenerative disorders with perturbed autophagy, due to defects in the autophagy pathway, or in upstream signalling via mTORC1 and AMPK. The review first provides a short description of autophagy, as related to neurons. It then examines the extended role of autophagy in neuronal function, plasticity and memory. There follows a description of each step of the autophagy pathway in greater detail, illustrated with examples of diseases grouped by the stage of their perturbation of the pathway. Each disease is accompanied by a short clinical description, to illustrate the diversity but also the overlap of symptoms caused by perturbation of key proteins necessary for the proper functioning of autophagy. Finally, there is a consideration of current challenges that need addressing for future therapeutic advances.
    Keywords:  Autophagy; child; neurodegeneration
  4. Autophagy. 2020 Feb 25. 1-3
      A key feature of macroautophagy/autophagy is the formation of a transient de novo compartment called the phagophore, which envelops cytoplasmic material, ultimately enclosing it within an autophagosome, allowing it to be targeted for degradation. Schütter et al describe a novel mechanism that spatiotemporally coordinates phospholipid synthesis to drive phagophore expansion and autophagosome formation. These authors show that during starvation, fatty acids (FAs) are channeled into phospholipid synthesis, and the newly synthesized lipids are directed toward autophagosome biogenesis.Abbreviations: ACS: acyl-CoA synthetase; ER: endoplasmic reticulum; FA: fatty acid; FAS: fatty acid synthetase; MCS: membrane contact sites; PAS: phagophore assembly site.
    Keywords:  Autophagosome; fatty acids; lipids; lysosome; membrane; stress; vacuole
  5. J Mol Biol. 2020 Feb 24. pii: S0022-2836(20)30194-7. [Epub ahead of print]
      Lysosomal dysfunction is considered pathogenic in Alzheimer Disease (AD). Loss of Presenilin-1 (PSEN1) function causing AD impedes acidification via defective vATPase V0a1 subunit delivery to lysosomes. We report that isoproterenol and related β2-adrenergic agonists re-acidify lysosomes in PSEN1 KO cells and fibroblasts from PSEN1 familial AD(FAD) patients, which restores lysosomal proteolysis, calcium homeostasis, and normal autophagy flux. We identify a novel rescue mechanism involving PKA-mediated facilitation of ClC-7 delivery to lysosomes which reverses markedly lowered Cl- content in PSEN1 KO lysosomes. Notably, PSEN1 loss-of-function impedes ER-to-lysosome delivery of ClC-7. Transcriptomics of PSEN1-deficient cells reveal strongly down-regulated ER-to-lysosome transport pathways and reversibility by isoproterenol thus accounting for lysosomal Cl- deficits that compound pH elevation due to deficient vATPase and rescue by β2-adrenergic agonists. Our findings uncover a broadened PSEN1 role in lysosomal ion homeostasis and novel pH modulation of lysosomes through β2-adrenergic regulation of ClC-7, which can potentially be modulated therapeutically.
    Keywords:  Acidification; Alzheimer disease; Chloride; ClC-7; Isoproterenol; Lysosome; PKA; PSEN1; RNAseq; β2-adrenergic receptor
  6. Cell Rep. 2020 Feb 25. pii: S2211-1247(20)30122-4. [Epub ahead of print]30(8): 2807-2819.e4
      The class III phosphoinositide 3-kinase vacuolar protein sorting 34 (VPS34) is a core protein of autophagy initiation, yet the regulatory mechanisms responsible for its stringent control remain poorly understood. Here, we report that the E3 ubiquitin ligase NEDD4-1 promotes the autophagy flux by targeting VPS34. NEDD4-1 undergoes lysine 29 (K29)-linked auto-ubiquitination at K1279 and serves as a scaffold for recruiting the ubiquitin-specific protease 13 (USP13) to form an NEDD4-1-USP13 deubiquitination complex, which subsequently stabilizes VPS34 to promote autophagy through removing the K48-linked poly-ubiquitin chains from VPS34 at K419. Knockout of either NEDD4-1 or USP13 increased K48-linked ubiquitination and degradation of VPS34, thus attenuating the formation of the autophagosome. Our results identify an essential role for NEDD4-1 in regulating autophagy, which provides molecular insights into the mechanisms by which ubiquitination regulates autophagy flux.
    Keywords:  NEDD4-1; USP13; VPS34; auto-ubiquitination; autophagy; deubiquitination complex
  7. Autophagy. 2020 Feb 25. 1-25
      Macroautophagy/autophagy is an auto-digestive pro-survival pathway activated in response to stress to target cargo for lysosomal degradation. In recent years, autophagy has become prominent as an innate antiviral defense mechanism through multiple processes, such as targeting virions and viral components for elimination. These exciting findings have encouraged studies on the ability of autophagy to restrict HIV. However, the role of autophagy in HIV infection remains unclear. Whereas some reports indicate that autophagy is detrimental for HIV, others have claimed that HIV deliberately activates this pathway to increase its infectivity. Moreover, these contrasting findings seem to depend on the cell type investigated. Here, we show that autophagy poses a hurdle for HIV replication, significantly reducing virion production. However, HIV-1 uses its accessory protein Nef to counteract this restriction. Previous studies have indicated that Nef affects autophagy maturation by preventing the fusion between autophagosomes and lysosomes. Here, we uncover that Nef additionally blocks autophagy initiation by enhancing the association between BECN1 and its inhibitor BCL2, and this activity depends on the cellular E3 ligase PRKN. Remarkably, the ability of Nef to counteract the autophagy block is more frequently observed in pandemic HIV-1 and its simian precursor SIVcpz infecting chimpanzees than in HIV-2 and its precursor SIVsmm infecting sooty mangabeys. In summary, our findings demonstrate that HIV-1 is susceptible to autophagy restriction and define Nef as the primary autophagy antagonist of this antiviral process.Abbreviations: 3-MA: 3-methyladenine; ACTB: actin, beta; ATG16L1: autophagy related 16 like 1; BCL2: bcl2 apoptosis regulator; BECN1: beclin 1; cDNA: complementary DNA; EGFP: enhanced green fluorescence protein; ER: endoplasmic reticulum; Gag/p55: group-specific antigen; GFP: green fluorescence protein; GST: glutathione S transferase; HA: hemagglutinin; HIV: human immunodeficiency virus; IP: immunoprecipitation; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; Nef: negative factor; PRKN: parkin RBR E3 ubiquitin ligase; PtdIns3K: phosphatidylinositol 3 kinase; PtdIns3P: phosphatidylinositol 3 phosphate; PTM: post-translational modification; RT-qPCR: reverse transcription followed by quantitative PCR; RUBCN: rubicon autophagy regulator; SEM: standard error of the mean; SERINC3: serine incorporator 3; SERINC5: serine incorporator 5; SIV: simian immunodeficiency virus; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; UVRAG: UV radiation resistance associated gene; VSV: vesicular stomatitis virus; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.
    Keywords:  Autophagy; BCL2; BECN1; Gag; HIV; Nef; PRKN
  8. Cells. 2020 Feb 21. pii: E495. [Epub ahead of print]9(2):
      Autophagy induction is an attractive therapeutic approach to ameliorate aggregate accumulation in many neurodegenerative diseases. In Huntington's disease (HD) in vivo models, a number of genetic and pharmacological mechanisms aimed to induce autophagy have been successfully tested [1], demonstrating the role of autophagy in promoting the elimination of mutant huntingtin (mHTT) aggregates and its neuroprotective effect. In their recent report in Cells, Vernizzi and colleagues [2] presented a totally new mechanism to induce autophagy, promote the elimination of mHTT aggregates, and ultimately achieve neuroprotection. This novel therapy is based on the overexpression of glutamine synthetase 1 (GS1), an enzyme that catalyzes the synthesis of L-glutamine from L-glutamate as part of the glutamate glutamine cycle (GGC), a physiological process between glia and neurons that controls glutamate homeostasis [3].[...].
  9. Nat Commun. 2020 Feb 25. 11(1): 1032
      The AMP-activated protein kinase (AMPK) is a master sensor of the cellular energy status that is crucial for the adaptive response to limited energy availability. AMPK is implicated in the regulation of many cellular processes, including autophagy. However, the precise mechanisms by which AMPK controls these processes and the identities of relevant substrates are not fully understood. Using protein microarrays, we identify Cyclin Y as an AMPK substrate that is phosphorylated at Serine 326 (S326) both in vitro and in cells. Phosphorylation of Cyclin Y at S326 promotes its interaction with the Cyclin-dependent kinase 16 (CDK16), thereby stimulating its catalytic activity. When expressed in cells, Cyclin Y/CDK16 is sufficient to promote autophagy. Moreover, Cyclin Y/CDK16 is necessary for efficient AMPK-dependent activation of autophagy. This functional interaction is mediated by AMPK phosphorylating S326 of Cyclin Y. Collectively, we define Cyclin Y/CDK16 as downstream effector of AMPK for inducing autophagy.
  10. J Mol Biol. 2020 Feb 19. pii: S0022-2836(20)30163-7. [Epub ahead of print]
      Cells have evolved molecular chaperones that modulate phase separation and misfolding of amyloidogenic proteins to prevent neurodegenerative diseases. Protein disulfide isomerase (PDI), mainly located at the endoplasmic reticulum and also present in the cytosol, acts as both an enzyme and a molecular chaperone. PDI is observed to be S-nitrosylated in brains of Alzheimer's disease patients, but the mechanism has remained elusive. We herein report that both wild-type PDI and its quadruple cysteine mutant only having chaperone activity significantly inhibit pathological phosphorylation and abnormal aggregation of Tau in cells, and significantly decrease the mitochondrial damage and Tau cytotoxicity resulting from Tau aberrant aggregation, highlighting the chaperone property of PDI. More importantly, we show that wild-type PDI is selectively recruited by liquid droplets of Tau, which significantly inhibits phase separation and stress granule formation of Tau, whereas S-nitrosylation of PDI abrogates the recruitment and inhibition. These findings demonstrate how phase separation of Tau is physiologically regulated by PDI and how S-nitrosylation of PDI, a perturbation in this regulation, leads to disease.
    Keywords:  S-nitrosylation; Tau protein; molecular chaperone; protein aggregation; protein disulfide isomerase; protein phase separation
  11. Mol Nutr Food Res. 2020 Feb 26. e1900768
      Improper adipose tissue lipid storage and secretory functions as found in visceral obesity and lipodystrophy, for instance, are generally associated with the development of several metabolic diseases namely insulin resistance, nonalcoholic fatty liver disease (NAFLD), cardiovascular disease and certain types of cancer, among others. Evidence gathered in the last decades suggests that lipotoxicity and inflammation are the main factors connecting adipose tissue dysfunction to the development of the aforementioned diseases. Mechanistic Target Of Rapamycin (mTOR) is a serine threonine kinase that functions as the catalytic entity of two multiprotein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). These complexes are important components of signaling pathways activated by nutrients, growth factors and inflammatory mediators and therefore directly involved in the regulation of adipocyte and macrophage metabolism and function. In this article, we reviewed studies that evaluated the involvement of both mTORC1 and 2 in the regulation of macrophage and adipocyte function and their implication in the development of metabolic disease-associated adipose tissue dysfunction. This article is protected by copyright. All rights reserved.
    Keywords:  adipose tissue; inflammation; lipotoxicity.; mTOR
  12. Sci Rep. 2020 Feb 28. 10(1): 3735
      Elevation of the levels of reactive oxygen species (ROS) is a major tissue-degenerative phenomenon involved in aging and aging-related diseases. The detailed mechanisms underlying aging-related ROS generation remain unclear. Presently, the expression of microRNA (miR)-142-5p was significantly upregulated in bone marrow mesenchymal stem cells (BMMSCs) of aged mice. Overexpression of miR-142 and subsequent observation revealed that miR-142 involved ROS accumulation through the disruption of selective autophagy for peroxisomes (pexophagy). Mechanistically, attenuation of acetyltransferase Ep300 triggered the upregulation of miR-142 in aged BMMSCs, and miR-142 targeted endothelial PAS domain protein 1 (Epas1) was identified as a regulatory protein of pexophagy. These findings support a novel molecular mechanism relating aging-associated ROS generation and organelle degradation in BMMSCs, and suggest a potential therapeutic target for aging-associated disorders that are accompanied by stem cell degeneration.
  13. BMC Biol. 2020 Feb 24. 18(1): 18
      BACKGROUND: Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown.RESULTS: We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosage-dependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells.
    CONCLUSIONS: Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types.
    Keywords:  Autophagy; Cryptic variation; Natural genetic variation; Polyglutamine; Protein aggregation; Proteostasis; Regulatory variation
  14. Cell Mol Life Sci. 2020 Feb 22.
      Hypoxia-inducible factors (HIFs) mediate metabolic reprogramming in response to hypoxia. However, the role of HIFs in branched-chain amino acid (BCAA) metabolism remains unknown. Here we show that hypoxia upregulates mRNA and protein levels of the BCAA transporter LAT1 and the BCAA metabolic enzyme BCAT1, but not their paralogs LAT2-4 and BCAT2, in human glioblastoma (GBM) cell lines as well as primary GBM cells. Hypoxia-induced LAT1 protein upregulation is mediated by both HIF-1 and HIF-2 in GBM cells. Although both HIF-1α and HIF-2α directly bind to the hypoxia response element at the first intron of the human BCAT1 gene, HIF-1α is exclusively responsible for hypoxia-induced BCAT1 expression in GBM cells. Knockout of HIF-1α and HIF-2α significantly reduces glutamate labeling from BCAAs in GBM cells under hypoxia, which provides functional evidence for HIF-mediated reprogramming of BCAA metabolism. Genetic or pharmacological inhibition of BCAT1 inhibits GBM cell growth under hypoxia. Together, these findings uncover a previously unrecognized HIF-dependent metabolic pathway that increases GBM cell growth under conditions of hypoxic stress.
    Keywords:  Branched-chain amino acid; Gene regulation; Glioblastoma; Hypoxia; Hypoxia-inducible factor; Metabolism
  15. Elife. 2020 Feb 26. pii: e50580. [Epub ahead of print]9
      Cilia both receive and send information, the latter in the form of extracellular vesicles (EVs). EVs are nano-communication devices that influence cell, tissue, and organism behavior. Mechanisms driving ciliary EV biogenesis are almost entirely unknown. Here, we show that the ciliary G-protein Rab28, associated with human autosomal recessive cone-rod dystrophy, negatively regulates EV levels in the sensory organs of Caenorhabditis elegans in a cilia specific manner. Sequential targeting of lipidated Rab28 to periciliary and ciliary membranes is highly dependent on the BBSome and the prenyl-binding protein phosphodiesterase 6 subunit delta (PDE6D), respectively, and BBSome loss causes excessive and ectopic EV production. We also find that EV defective mutants display abnormalities in sensory compartment morphogenesis. Together, these findings reveal that Rab28 and the BBSome are key in vivo regulators of EV production at the periciliary membrane and suggest that EVs may mediate signaling between cilia and glia to shape sensory organ compartments. Our data also suggest that defects in the biogenesis of cilia-related EVs may contribute to human ciliopathies.
    Keywords:  BBSome; C. elegans; PKD2; Rab28; cell biology; cilia; ciliopathy; extracellular vesicles
  16. Toxicol Lett. 2020 Feb 25. pii: S0378-4274(20)30060-6. [Epub ahead of print]
      Simultaneous detection of autophagy and apoptosis is important in drug discovery and signaling studies. Here we report, a real-time reporter cell line for the simultaneous detection of apoptosis and autophagy at single-cell level employing stable integration of two fluorescent protein reporters of apoptosis and autophagy. Cells stably expressing EGFP-LC3 fusion was developed initially as a marker for autophagy and subsequently stably expressed with inter-mitochondrial membrane protein SMAC with RFP fusion to detect mitochondrial permeabilization event of apoptosis. The cell lines faithfully reported the LC3 punctae formation and release of intermembrane proteins in response to diverse apoptotic and autophagic stimuli.
  17. J Mol Biol. 2020 Feb 24. pii: S0022-2836(20)30196-0. [Epub ahead of print]
      Considerable evidences suggest a link between autophagy dysfunction, protein aggregation and neurodegenerative diseases. Given that autophagy is a conserved intracellular housekeeping process, modulation of autophagy flux in various model organisms have highlighted its importance for maintaining proteostasis. In postmitotic cells such as neurons, compromised autophagy is sufficient to cause accumulation of ubiquitinated aggregates, neuronal dysfunction, degeneration and loss of motor coordination-all hallmarks of neurodegenerative diseases. Reciprocally, enhanced autophagy flux augments cellular and organismal health, in addition to extending life span. These genetic studies not-withstanding, a plethora of small molecule modulators of autophagy flux have been reported that alleviate disease symptoms in models of neurodegenerative diseases. This review summarizes the potential of such molecules to be, perhaps, one of the first autophagy drugs for treating these currently incurable diseases.
    Keywords:  Aggrephagy; Aggresome; Autophagy modulators; Misfolded proteins; Neuroprotection
  18. Cell. 2020 Feb 25. pii: S0092-8674(20)30156-2. [Epub ahead of print]
      Mammalian tissues engage in specialized physiology that is regulated through reversible modification of protein cysteine residues by reactive oxygen species (ROS). ROS regulate a myriad of biological processes, but the protein targets of ROS modification that drive tissue-specific physiology in vivo are largely unknown. Here, we develop Oximouse, a comprehensive and quantitative mapping of the mouse cysteine redox proteome in vivo. We use Oximouse to establish several paradigms of physiological redox signaling. We define and validate cysteine redox networks within each tissue that are tissue selective and underlie tissue-specific biology. We describe a common mechanism for encoding cysteine redox sensitivity by electrostatic gating. Moreover, we comprehensively identify redox-modified disease networks that remodel in aged mice, establishing a systemic molecular basis for the long-standing proposed links between redox dysregulation and tissue aging. We provide the Oximouse compendium as a framework for understanding mechanisms of redox regulation in physiology and aging.
    Keywords:  ROS; aging; cysteine; proteomics; reactive oxygen species
  19. Biochem Biophys Res Commun. 2020 Feb 20. pii: S0006-291X(20)30362-4. [Epub ahead of print]
      To examine the cell-protective role of podocyte autophagy against glomerular endothelial dysfunction in diabetes, we analyzed the renal phenotype of tamoxifen (TM)-inducible podocyte-specific Atg5-deficient (iPodo-Atg5-/-) mice with experimental endothelial dysfunction. In both control and iPodo-Atg5-/- mice, high fat diet (HFD) feeding induced glomerular endothelial damage characterized by decreased urinary nitric oxide (NO) excretion, collapsed endothelial fenestrae, and reduced endothelial glycocalyx. HFD-fed control mice showed slight albuminuria and nearly normal podocyte morphology. In contrast, HFD-fed iPodo-Atg5-/- mice developed massive albuminuria accompanied by severe podocyte injury that was observed predominantly in podocytes adjacent to damaged endothelial cells by scanning electron microscopy. Although podocyte-specific autophagy deficiency did not affect endothelial NO synthase deficiency-associated albuminuria, it markedly exacerbated albuminuria and severe podocyte morphological damage when the damage was induced by intravenous neuraminidase injection to remove glycocalyx from the endothelial surface. Furthermore, endoplasmic reticulum stress was accelerated in podocytes of iPodo-Atg5-/- mice stimulated with neuraminidase, and treatment with molecular chaperone tauroursodeoxycholic acid improved neuraminidase-induced severe albuminuria and podocyte injury. In conclusion, podocyte autophagy plays a renoprotective role against diabetes-related structural endothelial damage, providing an additional insight into the pathogenesis of massive proteinuria in diabetic nephropathy.
    Keywords:  Autophagy; Diabetic kidney disease; Massive proteinuria; Mitochondria; Podocytes
  20. Proc Natl Acad Sci U S A. 2020 Feb 24. pii: 201912864. [Epub ahead of print]
      Terminal oligopyrimidine (TOP) motifs are sequences at the 5' ends of mRNAs that link their translation to the mTOR Complex 1 (mTORC1) nutrient-sensing signaling pathway. They are commonly regarded as discrete elements that reside on ∼100 mRNAs that mostly encode translation factors. However, the full spectrum of TOP sequences and their prevalence throughout the transcriptome remain unclear, primarily because of uncertainty over the mechanism that detects them. Here, we globally analyzed translation targets of La-related protein 1 (LARP1), an RNA-binding protein and mTORC1 effector that has been shown to repress TOP mRNA translation in a few specific cases. We establish that LARP1 is the primary translation regulator of mRNAs with classical TOP motifs genome-wide, and also that these motifs are extreme instances of a broader continuum of regulatory sequences. We identify the features of TOP sequences that determine their potency and quantify these as a metric that accurately predicts mTORC1/LARP1 regulation called a TOPscore. Analysis of TOPscores across the transcriptomes of 16 mammalian tissues defines a constitutive "core" set of TOP mRNAs, but also identifies tissue-specific TOP mRNAs produced via alternative transcription initiation sites. These results establish the central role of LARP1 in TOP mRNA regulation on a transcriptome scale and show how it connects mTORC1 to a tunable and dynamic program of gene expression that is tailored to specific biological contexts.
    Keywords:  LARP1; TOP mRNA; mTORC1; translation
  21. J Mater Chem B. 2020 Feb 26.
      Cysteine (Cys) is one of the most important essential biothiols in lysosomes. Highly selective probes for specific detection and imaging of lysosomal Cys over other biological thiols are rare. Herein, we developed a lysosome-targeted near-infrared fluorescent probe SHCy-C based on a novel NIR-emitting thioxanthene-indolium dye. Due to the turn-on fluorescence response elicited by the intramolecular charge transfer (ICT) processes before and after the reaction with Cys, probe SHCy-C exhibits high selectivity and sensitivity (16 nM) for the detection of Cys. More importantly, probe SHCy-C is found to precisely target lysosomes and achieves the "turn-on" detection and imaging of endogenous Cys in lysosomes.
  22. Mol Neurodegener. 2020 Feb 24. 15(1): 13
      BACKGROUND: The C9ORF72 hexanucleotide repeat expansion is the most common known genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two fatal age-related neurodegenerative diseases. The C9ORF72 expansion encodes five dipeptide repeat proteins (DPRs) that are produced through a non-canonical translation mechanism. Among the DPRs, proline-arginine (PR), glycine-arginine (GR), and glycine-alanine (GA) are the most neurotoxic and increase the frequency of DNA double strand breaks (DSBs). While the accumulation of these genotoxic lesions is increasingly recognized as a feature of disease, the mechanism(s) of DPR-mediated DNA damage are ill-defined and the effect of DPRs on the efficiency of each DNA DSB repair pathways has not been previously evaluated.METHODS AND RESULTS: Using DNA DSB repair assays, we evaluated the efficiency of specific repair pathways, and found that PR, GR and GA decrease the efficiency of non-homologous end joining (NHEJ), single strand annealing (SSA), and microhomology-mediated end joining (MMEJ), but not homologous recombination (HR). We found that PR inhibits DNA DSB repair, in part, by binding to the nucleolar protein nucleophosmin (NPM1). Depletion of NPM1 inhibited NHEJ and SSA, suggesting that NPM1 loss-of-function in PR expressing cells leads to impediments of both non-homologous and homology-directed DNA DSB repair pathways. By deleting NPM1 sub-cellular localization signals, we found that PR binds NPM1 regardless of the cellular compartment to which NPM1 was directed. Deletion of the NPM1 acidic loop motif, known to engage other arginine-rich proteins, abrogated PR and NPM1 binding. Using confocal and super-resolution immunofluorescence microscopy, we found that levels of RAD52, a component of the SSA repair machinery, were significantly increased iPSC neurons relative to isogenic controls in which the C9ORF72 expansion had been deleted using CRISPR/Cas9 genome editing. Western analysis of post-mortem brain tissues confirmed that RAD52 immunoreactivity is significantly increased in C9ALS/FTD samples as compared to controls.
    CONCLUSIONS: Collectively, we characterized the inhibitory effects of DPRs on key DNA DSB repair pathways, identified NPM1 as a facilitator of DNA repair that is inhibited by PR, and revealed deficits in homology-directed DNA DSB repair pathways as a novel feature of C9ORF72-related disease.
    Keywords:  Amyotrophic lateral sclerosis; CRISPR; DNA damage; DNA double strand break repair; Homology-directed repair; Induced pluripotent stem cells; RAD52; Single-strand annealing
  23. Nat Cell Biol. 2020 Feb 24.
      During endoplasmic-reticulum-associated protein degradation (ERAD), misfolded proteins are polyubiquitinated, extracted from the ER membrane and degraded by the proteasome1-4. In a process called retrotranslocation, misfolded luminal proteins first need to traverse the ER membrane before ubiquitination can occur in the cytosol. It was suggested that the membrane-embedded ubiquitin ligase Hrd1 forms a retrotranslocation pore regulated by cycles of auto- and deubiquitination5-8. However, the mechanism by which auto-ubiquitination affects Hrd1 and allows polypeptides to cross the membrane and whether Hrd1 forms a membrane-spanning pore remained unknown. Here, using purified Hrd1 incorporated into different model membranes, we show that Hrd1 auto-ubiquitination leads to the opening of a pore. Substrate binding increases the pore size and its activity, whereas deubiquitination closes the pore and renders it unresponsive to substrate. We identify two binding sites for misfolded proteins in Hrd1, a low-affinity luminal site and a high-affinity cytoplasmic site formed following auto-ubiquitination of specific lysine residues in Hrd1's RING domain. We propose that the affinity difference between the luminal and cytoplasmic binding sites provides the initial driving force for substrate movement through Hrd1.
  24. Nat Commun. 2020 Feb 28. 11(1): 1121
      Macropinocytic cancer cells scavenge amino acids from extracellular proteins. Here, we show that consuming necrotic cell debris via macropinocytosis (necrocytosis) offers additional anabolic benefits. A click chemistry-based flux assay reveals that necrocytosis provides not only amino acids, but sugars, fatty acids and nucleotides for biosynthesis, conferring resistance to therapies targeting anabolic pathways. Indeed, necrotic cell debris allow macropinocytic breast and prostate cancer cells to proliferate, despite fatty acid synthase inhibition. Standard therapies such as gemcitabine, 5-fluorouracil (5-FU), doxorubicin and gamma-irradiation directly or indirectly target nucleotide biosynthesis, creating stress that is relieved by scavenged nucleotides. Strikingly, necrotic debris also render macropinocytic, but not non-macropinocytic, pancreas and breast cancer cells resistant to these treatments. Selective, genetic inhibition of macropinocytosis confirms that necrocytosis both supports tumor growth and limits the effectiveness of 5-FU in vivo. Therefore, this study establishes necrocytosis as a mechanism for drug resistance.
  25. Redox Biol. 2020 Feb 07. pii: S2213-2317(19)31498-3. [Epub ahead of print]32 101457
      Nitric oxide (NO) is well established as a regulator of neurogenesis. NO increases the proliferation of neural stem cells (NSC), and is essential for hippocampal injury-induced neurogenesis following an excitotoxic lesion. One of the mechanisms underlying non-classical NO cell signaling is protein S-nitrosylation. This post-translational modification consists in the formation of a nitrosothiol group (R-SNO) in cysteine residues, which can promote formation of other oxidative modifications in those cysteine residues. S-nitrosylation can regulate many physiological processes, including neuronal plasticity and neurogenesis. In this work, we aimed to identify S-nitrosylation targets of NO that could participate in neurogenesis. In NSC, we identified a group of proteins oxidatively modified using complementary techniques of thiol redox proteomics. S-nitrosylation of some of these proteins was confirmed and validated in a seizure mouse model of hippocampal injury and in cultured hippocampal stem cells. The identified S-nitrosylated proteins are involved in the ERK/MAPK pathway and may be important targets of NO to enhance the proliferation of NSC.
    Keywords:  Hippocampus; Neural stem cells; Neurogenesis; Nitric oxide; S-nitrosylation; Seizures
  26. Cell Rep. 2020 Feb 25. pii: S2211-1247(20)30105-4. [Epub ahead of print]30(8): 2729-2742.e4
      Pancreatic ductal adenocarcinoma (PDAC) features a near-universal mutation in KRAS. Additionally, the tumor suppressor PTEN is lost in ∼10% of patients, and in mouse models, this dramatically accelerates tumor progression. While oncogenic KRAS and phosphatidylinositol 3-kinase (PI3K) cause divergent metabolic phenotypes individually, how they synergize to promote tumor metabolic alterations and dependencies remains unknown. We show that in KRAS-driven murine PDAC cells, loss of Pten strongly enhances both mTOR signaling and macropinocytosis. Protein scavenging alleviates sensitivity to mTOR inhibition by rescuing AKT phosphorylation at serine 473 and consequently cell proliferation. Combined inhibition of mTOR and lysosomal processing of internalized protein eliminates the macropinocytosis-mediated resistance. Our results indicate that mTORC2, rather than mTORC1, is an important regulator of protein scavenging and that protein-mediated resistance could explain the lack of effectiveness of mTOR inhibitors in certain genetic backgrounds. Concurrent inhibition of mTOR and protein scavenging might be a valuable therapeutic approach.
    Keywords:  AKT; cancer metabolism; mTORC2; macropinocytosis; metabolic scavenging; pancreatic ductal adenocarcinoma
  27. Int J Mol Sci. 2020 Feb 24. pii: E1548. [Epub ahead of print]21(4):
      Persistent signalling via the PI3K/AKT/mTOR pathway is a major driver of malignancy in NF1-associated malignant peripheral nerve sheath tumours (MPNST). Nevertheless, single targeting of this pathway is not sufficient to inhibit MPNST growth. In this report, we demonstrate that combined treatment with the allosteric pan-AKT inhibitor MK-2206 and the mTORC1/mTORC2 inhibitor AZD8055 has synergistic effects on the viability of MPNST cell lines in comparison to the treatment with each compound alone. However, when treating animals bearing experimental MPNST with the combined AKT/mTOR regime, no influence on tumour growth was observed. Further analysis of the MPNST xenograft tumours resistant to AKT/mTOR treatment revealed a reactivation of both AKT and mTOR in several tumour samples. Additional targeting of the RAS/RAF/MEK/MAPK pathway with the allosteric MEK1/2 inhibitor AZD6244 showed synergistic effects on the viability of MPNST cell lines in vitro in comparison to the dual AKT/mTOR inhibition. In summary, these data indicate that combined treatment with AKT and mTOR inhibitors is effective on MPNST cells in vitro but tumour resistance can occur rapidly in vivo by restoration of AKT/mTOR signalling. Our data further suggest that a triple treatment with inhibitors against AKT, mTORC1/2 and MEK1/2 may be a promising treatment option that should be further analysed in an experimental MPNST mouse model in vivo.
    Keywords:  AKT; MPNST; neurofibromatosis Type 1; signaling; targeted therapy; xenograft model
  28. Aging (Albany NY). 2020 Feb 23. 12
      Numerous genetic manipulations that extend lifespan in mice have been discovered over the past two decades, the most robust of which has arguably been the down regulation of growth hormone (GH) signaling. However, while decreased GH signaling has been associated with improved health and lifespan, many of the underlying physiological changes and molecular mechanisms associated with GH signaling have yet to be elucidated. To this end, we have completed the first transcriptomic and metabolomic study on long-lived growth hormone releasing hormone knockout (GHRH-KO) and wild-type mice in brown adipose tissue (transcriptomics) and blood serum (metabolomics). We find that GHRH-KO mice have increased transcript levels of mitochondrial and amino acid genes with decreased levels of extracellular matrix genes. Concurrently, mitochondrial metabolites are differentially regulated in GHRH-KO. Furthermore, we find a strong signal of genotype-by-sex interactions, suggesting the sexes have differing physiological responses to GH deficiency. Overall, our results point towards a strong influence of mitochondrial metabolism in GHRH-KO mice which potentially is tightly intertwined with their extended lifespan phenotype.
    Keywords:  aging; growth hormone; metabolite; mouse; transcriptomics
  29. Comp Biochem Physiol B Biochem Mol Biol. 2020 Feb 20. pii: S1096-4959(20)30018-X. [Epub ahead of print] 110424
      In all eukaryotic organisms, the control of growth, metabolism, reproduction, and lifespan is realized by interactions of genetic and environmental signals. An important player in the regulatory network is the target of rapamycin (TOR) signaling pathway, which is triggered by nutritional cues. Given the pivotal role of TOR in regulating multiple processes in organisms, we inhibited TOR by inducible expression of specific RNAi in Drosophila intestinal stem and progenitor cells or progenitor cells alone. We found that TOR inhibition in stem and progenitor cells shortened the lifespan on both regular diet and under malnutrition. Moreover, flies became more short-lived under starvation or oxidative stress conditions if TOR was inhibited. TOR-RNAi expression resulted in a decrease in body glycogen and TAG levels. All these physiological and metabolic changes might be partially explained by significant changes in mRNA levels for genes encoding the Drosophila insulin-like peptides (dilp2, dilp3 and dilp5) with subsequent effects on insulin signaling to modulate gene expression in peripheral tissues (e.g. tobi and pepck transcripts). In the gut, a strong increase in transcript levels of cytokines upd2, upd3 and downstream target socs36e of the JAK/STAT signaling pathway in the gut indicate an important role for this signaling pathway when TOR is inhibited.
    Keywords:  Fruit fly; Intestinal stem cell; Lifespan; Metabolism; TOR
  30. Mol Ther. 2020 Feb 12. pii: S1525-0016(20)30091-5. [Epub ahead of print]
      Lysosomal storage diseases (LSDs) are inherited disorders caused by lysosomal deficiencies and characterized by dysfunction of the autophagy-lysosomal pathway (ALP) often associated with neurodegeneration. No cure is currently available to treat neuropathology in LSDs. By studying a mouse model of mucopolysaccharidosis (MPS) type IIIA, one of the most common and severe forms of LSDs, we found that multiple amyloid proteins including α-synuclein, prion protein (PrP), Tau, and amyloid β progressively aggregate in the brain. The amyloid deposits mostly build up in neuronal cell bodies concomitantly with neurodegeneration. Treating MPS-IIIA mice with CLR01, a "molecular tweezer" that acts as a broad-spectrum inhibitor of amyloid protein self-assembly reduced lysosomal enlargement and re-activates autophagy flux. Restoration of the ALP was associated with reduced neuroinflammation and amelioration of memory deficits. Together, these data provide evidence that brain deposition of amyloid proteins plays a gain of neurotoxic function in a severe LSD by affecting the ALP and identify CLR01 as new potent drug candidate for MPS-IIIA and likely for other LSDs.
    Keywords:  amyloid aggregation; autophagy; lysosomal storage disease; molecular tweezers; mucopolysaccharidosis type IIIA
  31. Autophagy. 2020 Feb 26.
      Primary cilium-dependent macroautophagy/autophagy is induced by the urinary flow in epithelial cells of the kidney proximal tubule. A major physiological outcome of this cascade is the control of cell size. Some components of the ATG machinery are recruited at the primary cilium to generate autophagic structures. Shear stress induced by the liquid flow promotes PtdIns3P synthesis at the primary cilium, and this lipid is required both for ciliogenesis and initiation of autophagy. We showed that PtdIns3P is generated by PIK3C2A, but not by PIK3C3/VPS34, during flow-associated primary cilium-dependent autophagy, in a ULK1-independent manner. Along the same line BECN1 (beclin 1), a partner of PIK3C3 in starvation-induced autophagy, is not recruited at the primary cilium under shear stress. Thus, kidney epithelial cells mobilize different PtdIns 3-kinases, i.e., PIK3C2A or PIK3C3, to produce PtdIns3P in order to initiate autophagy depending on the stimuli (shear stress or starvation).
    Keywords:  PIK3C2A; PtdIns3P; autophagy; primary cilium; shear stress
  32. Antioxid Redox Signal. 2020 Feb 27.
      AIMS: Mitochondrial stress and dysfunction within the intestinal epithelium are known to contribute to the pathogenesis of inflammatory bowel disease (IBD). However, the importance of mitophagy during intestinal inflammation remains poorly understood. The primary aim of this study is to investigate how the mitophagy protein BCL2/adenovirus E1B 19 kDa protein-interacting protein 3-like (BNIP3L/NIX) mitigates mitochondrial damage during intestinal inflammation in the hopes that these data will allow us to target mitochondrial health in the intestinal epithelium as an adjunct to immune-based treatment strategies.RESULTS: In the intestinal epithelium of patients with ulcerative colitis (UC), we found that NIX was upregulated and targeted to the mitochondria. We obtained similar findings in wild-type (WT) mice undergoing experimental colitis. An increase in NIX expression was found to depend upon stabilization of hypoxia-inducible factor-1 alpha (HIF1α), which binds to the Nix promoter region. Using the reactive oxygen species (ROS) scavenger MitoTEMPO, we were able to attenuate disease and inhibit both HIF1α stabilization and subsequent NIX expression, suggesting that mitochondrially-derived ROS are crucial to initiating the mitophagic response during intestinal inflammation. We subjected a global Nix-/- mouse to DSS colitis and found that these mice developed worse disease. Additionally, Nix-/- mice were found to exhibit increased mitochondrial mass, likely due to the inability to clear damaged or dysfunctional mitochondria.
    INNOVATION: These results demonstrate the importance of mitophagy within the intestinal epithelium during IBD pathogenesis.
    CONCLUSION: NIX-mediated mitophagy is required to maintain intestinal homeostasis during inflammation, highlighting the impact of mitochondrial damage on IBD progression.
  33. Sci Rep. 2020 Feb 25. 10(1): 3418
      The Insulin/IGF-1 signalling (IIS) pathway plays an essential role in the regulation of glucose and lipid homeostasis. At the same time, a reduction in the IIS pathway activity can extend lifespan and healthspan in various model organisms. Amongst a number of body organs that sense and respond to insulin/IGF-1, the adipose tissue has a central role in both the metabolic and lifespan effects of IIS at the organismal level. Genetic inactivation of IIS components specifically in the adipose tissue has been shown before to improve metabolic profile and extend lifespan in various model organisms. We sought to identify conserved molecular mechanisms that may underlie the beneficial effects of IIS inhibition in the adipose tissue, specifically at the level of phosphoinositide 3-kinase (PI3K), a key IIS effector molecule. To this end, we inactivated PI3K by genetic means in the fly fat body and by pharmacological inhibition in mammalian adipocytes. Gene expression studies revealed changes to metabolism and upregulation of mitochondrial activity in mouse adipocytes and fly fat bodies with downregulated PI3K, which were confirmed by biochemical assays in mammalian adipocytes. These data suggest that PI3K inactivation has a conserved effect of upregulating mitochondrial metabolism in both fly and mammalian adipose tissue, which likely contributes to the health- and life-span extending effect of IIS pathway downregulation.