bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒09‒13
forty-six papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

  1. Dev Cell. 2020 Aug 31. pii: S1534-5807(20)30666-3. [Epub ahead of print]
    Lee C, Lamech L, Johns E, Overholtzer M.
      Lysosome function is essential for cellular homeostasis, but quality-control mechanisms that maintain healthy lysosomes remain poorly characterized. Here, we developed a method to measure lysosome turnover and use this to identify a selective mechanism of membrane degradation that involves lipidation of the autophagy protein LC3 onto lysosomal membranes and the formation of intraluminal vesicles through microautophagy. This mechanism is induced in response to metabolic stress resulting from glucose starvation or by treatment with pharmacological agents that induce osmotic stress on lysosomes. Cells lacking ATG5, an essential component of the LC3 lipidation machinery, show reduced ability to regulate lysosome size and degradative capacity in response to activation of this mechanism. These findings identify a selective mechanism of lysosome membrane turnover that is induced by stress and uncover a function for LC3 lipidation in regulating lysosome size and activity through microautophagy.
    Keywords:  ATG5; LAP; LC3; ammonium; autophagy; glucose; glutamine; lysosome; metabolism; microautophagy
  2. Cell Signal. 2020 Sep 08. pii: S0898-6568(20)30251-5. [Epub ahead of print] 109774
    Suzuki K, Honda T, Akatsu A, Yamaguchi N, Yamaguchi N.
      Src-family kinases (SFKs), such as c-Src, Lyn and Fyn, belong to non-receptor-type tyrosine kinases and play key roles in cell proliferation, adhesion, and migration. SFKs are anchored to the plasma membrane, Golgi membranes and lysosomal membranes through lipid modifications. Although the functions of SFKs being localized to the plasma membrane are intensively studied, those of SFKs being localized to organelle membranes are poorly understood. Here, we show that, among SFKs, c-Src in particular is involved in a decrease in the amount of LC3-II. c-Src and non-palmitoylated Lyn [Lyn(C3S) (cysteine-3 → serine-3)], which are localized onto lysosomes, decrease the amount of LC3-II and treatment with SFK inhibitors increases the amount of LC3-II, suggesting the importance of SFKs' lysosomal localization for a change of autophagic flux in a kinase activity-dependent manner. Colocalization of LC3-II with the lysosome-associated membrane protein LAMP1 shows that lysosome-localized SFKs promote the fusion of autophagosomes with lysosomes. Lysosome-localized SFKs play a positive role in the maintenance of cell viability under starvation conditions, which is further supported by knockdown of c-Src. Therefore, our results suggest that autophagosome-lysosome fusion is promoted by lysosome-localized c-Src, leading to cell survival under starvation conditions.
    Keywords:  Autophagosome; Autophagy; C-Src; LC-3; Lysosome; Src-family tyrosine kinase
  3. Commun Biol. 2020 Sep 09. 3(1): 498
    Olson LJ, Misra SK, Ishihara M, Battaile KP, Grant OC, Sood A, Woods RJ, Kim JP, Tiemeyer M, Ren G, Sharp JS, Dahms NM.
      The cation-independent mannose 6-phosphate receptor (CI-MPR, IGF2 receptor or CD222), is a multifunctional glycoprotein required for normal development. Through the receptor's ability to bind unrelated extracellular and intracellular ligands, it participates in numerous functions including protein trafficking, lysosomal biogenesis, and regulation of cell growth. Clinically, endogenous CI-MPR delivers infused recombinant enzymes to lysosomes in the treatment of lysosomal storage diseases. Although four of the 15 domains comprising CI-MPR's extracellular region bind phosphorylated glycans on lysosomal enzymes, knowledge of how CI-MPR interacts with ~60 different lysosomal enzymes is limited. Here, we show by electron microscopy and hydroxyl radical protein footprinting that the N-terminal region of CI-MPR undergoes dynamic conformational changes as a consequence of ligand binding and different pH conditions. These data, coupled with X-ray crystallography, surface plasmon resonance and molecular modeling, allow us to propose a model explaining how high-affinity carbohydrate binding is achieved through allosteric domain cooperativity.
  4. Dev Cell. 2020 Sep 03. pii: S1534-5807(20)30630-4. [Epub ahead of print]
    Yang S, Zhang Y, Ting CY, Bettedi L, Kim K, Ghaniam E, Lilly MA.
      The dysregulation of the metabolic regulator TOR complex I (TORC1) contributes to a wide array of human pathologies. Tuberous sclerosis complex (TSC) is a potent inhibitor of TORC1. Here, we demonstrate that the Rag GTPase acts in both the amino-acid-sensing and growth factor signaling pathways to control TORC1 activity through the regulation of TSC dynamics in HeLa cells and Drosophila. We find that TSC lysosomal-cytosolic exchange increases in response to both amino acid and growth factor restriction. Moreover, the rate of exchange mirrors TSC function, with depletions of the Rag GTPase blocking TSC lysosomal mobility and rescuing TORC1 activity. Finally, we show that the GATOR2 complex controls the phosphorylation of TSC2, which is essential for TSC exchange. Our data support the model that the amino acid and growth factor signaling pathways converge on the Rag GTPase to inhibit TORC1 activity through the regulation of TSC dynamics.
    Keywords:  AKT1; Drosophila; GATOR2; HeLa cell; Rag GTPase; TORC1; TSC; amino acid signaling; growth factor signaling; lysosome
  5. Genes (Basel). 2020 Sep 03. pii: E1043. [Epub ahead of print]11(9):
    Bjedov I, Rallis C.
      Ageing is a complex trait controlled by genes and the environment. The highly conserved mechanistic target of rapamycin signalling pathway (mTOR) is a major regulator of lifespan in all eukaryotes and is thought to be mediating some of the effects of dietary restriction. mTOR is a rheostat of energy sensing diverse inputs such as amino acids, oxygen, hormones, and stress and regulates lifespan by tuning cellular functions such as gene expression, ribosome biogenesis, proteostasis, and mitochondrial metabolism. Deregulation of the mTOR signalling pathway is implicated in multiple age-related diseases such as cancer, neurodegeneration, and auto-immunity. In this review, we briefly summarise some of the workings of mTOR in lifespan and ageing through the processes of transcription, translation, autophagy, and metabolism. A good understanding of the pathway's outputs and connectivity is paramount towards our ability for genetic and pharmacological interventions for healthy ageing and amelioration of age-related disease.
    Keywords:  Drosophila; TORC1; TORC2; ageing; autophagy; metabolism; nutrient-response; transcription; translation; yeast
  6. Neurochem Res. 2020 Sep 11.
    Nascimento AC, Erustes AG, Reckziegel P, Bincoletto C, Ureshino RP, Pereira GJS, Smaili SS.
      Although the etiology of Parkinson's disease (PD) is multifactorial, it has been linked to abnormal accumulation of α-synuclein (α-syn) in dopaminergic neurons, which could lead to dysfunctions on intracellular organelles, with potential neurodegeneration. Patients with familial early-onset PD frequently present mutation in the α-syn gene (SNCA), which encodes mutant α-syn forms, such as A30P and A53T, which potentially regulate Ca2+ unbalance. Here we investigated the effects of overexpression of wild-type α-syn (WT) and the mutant forms A30P and A53T, on modulation of lysosomal Ca2+ stores and further autophagy activation. We found that in α-syn-overexpressing cells, there was a decrease in Ca2+ released from endoplasmic reticulum (ER) which is related to the increase in lysosomal Ca2+ release, coupled to lysosomal pH alkalization. Interestingly, α-syn-overexpressing cells showed lower LAMP1 levels, and a disruption of lysosomal morphology and distribution, affecting autophagy. Interestingly, all these effects were more evident with A53T mutant isoform when compared to A30P and WT α-syn types, indicating that the pathogenic phenotype for PD is potentially related to impairment of α-syn degradation. Taken together, these events directly impact PD-related dysfunctions, being considered possible molecular targets for neuroprotection.
    Keywords:  Calcium; Lysosomes; Parkinson’s disease; α-Synuclein
  7. Genes (Basel). 2020 Sep 04. pii: E1045. [Epub ahead of print]11(9):
    Fu W, Hall MN.
      Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase and a master regulator of cell growth and metabolism, forms two structurally and functionally distinct complexes, mTOR complex 1 (mTORC1) and mTORC2. While mTORC1 signaling is well characterized, mTORC2 is relatively poorly understood. mTORC2 appears to exist in functionally distinct pools, but few mTORC2 effectors/substrates have been identified. Here, we review recent advances in our understanding of mTORC2 signaling, with particular emphasis on factors that control mTORC2 activity.
    Keywords:  Akt; mTOR; mTORC2 signaling; signaling crosstalk
  8. JIMD Rep. 2020 Sep;55(1): 68-74
    Chin SJ, Saville JT, McDermott BK, Zankl A, Fletcher JM, Fuller M.
      Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism characterized by a deficiency of the lysosomal enzyme, N-acetylgalactosamine 6-sulphatase (GALNS). Consequently, partially degraded GAG, chondroitin 6-sulfate (CS) and keratan sulfate (KS), accumulate in the lysosomes of affected cells, primarily in cartilage resulting in skeletal disease. Excessive urinary excretion of these GAG is often used as the initial biochemical parameter to inform a laboratory diagnosis. Here we present the utility of a CS-disaccharide with a non-reducing 6-sulfated N-acetylgalactosamine residue (HNAc-UA (1S))-the enzyme's substrate-for the diagnosis and biochemical monitoring of MPS IVA patients. Following implementation of this method into the diagnostic laboratory, we identified one MPS IVA patient over 3 years of MPS urine screening, with no false positive results from 2050 urines tested. Uniquely, urinary concentrations of HNAc-UA (1S) are independent of age meaning that age-related reference ranges are not required. Urinary HNAc-UA (1S) was also able to identify two MPS IVA siblings who remained misdiagnosed with spondyloepiphyseal dysplasia for 5 years because of normal urinary GAG. HNAc-UA (1S) could also be used as a biomarker for monitoring response to enzyme replacement therapy (ERT) as there was a drop in urinary concentration following the administration of ERT in all 12 patients and concentrations correlated with urinary KS (R 2 = 0.92). In conclusion, HNAc-UA (1S) is a reliable, sensitive and specific biomarker for the diagnosis of MPS IVA and can be used to biochemically monitor the response to ERT.
    Keywords:  Morquio syndrome; biomarker; chondroitin sulfate; glycosaminoglycan; lysosomal storage disorder; mucopolysaccharidosis
  9. J Physiol. 2020 Sep 07.
    Ogasawara R, Knudsen JR, Li J, Ato S, Jensen TE.
      KEY POINTS: Muscle contractions increase protein synthesis in a mechanistic target of rapamycin (mTOR)-dependent manner, yet it is unclear which/how mTOR complexes regulate muscle protein synthesis. We investigated the requirement of mTOR Complex 2 (mTORC2) in contraction-stimulated muscle protein synthesis. mTORC2 inhibition by muscle-specific Rictor knockout (Rictor mKO) did not prevent contraction-induced muscle protein synthesis. Rapamycin prevented contraction-induced muscle protein synthesis in Rictor mKO but not wild-type mice.ABSTRACT: Protein synthesis increases following muscle contractions. Previous studies showed that the mechanistic target of rapamycin complex 1 (mTORC1) inhibition suppressed the early but not late muscle protein synthesis-response, while the inhibition of both mTORC1 and mTORC2 abolished both effects. Therefore, we hypothesized that mTORC2 regulates muscle protein synthesis following muscle contractions. To test this, we investigated the effect of mTORC2 inhibition by mouse muscle-specific Rictor knockout (Rictor mKO) on muscle protein synthesis 3h post-contraction. The right gastrocnemius muscles of Rictor mKO mice and wild-type (WT) mice were isometrically contracted using percutaneous electrical stimulation, while the left gastrocnemius muscles served as controls. Vehicle or the mTORC1 inhibitor rapamycin (1.5 mg/kg) was injected intraperitoneally 1 h before contraction. Treatment of WT mice with rapamycin and Rictor mKO lowered protein synthesis in general, but the response to contractions was intact 3h post contractions in both conditions. Rapamycin treatment in Rictor mKO prevented contraction-stimulated muscle protein synthesis. Notably, signalling traditionally associated with mTORC1 was increased by muscle contractions despite rapamycin treatment. In rapamycin-treated Rictor mKO mice, the same mTORC1 signalling was blocked following contractions. Our results indicate that although neither rapamycin-sensitive mTOR/mTORC1 nor mTORC2 regulates contraction-induced muscle protein synthesis, combined inhibition of rapamycin-sensitive mTOR/mTORC1 and mTORC2 synergistically inhibits contraction-induced muscle protein synthesis. This article is protected by copyright. All rights reserved.
    Keywords:  cell signalling; exercisemTORC1; mTORC2; protein translation
  10. Front Cell Dev Biol. 2020 ;8 671
    Sukumaran A, Choi K, Dasgupta B.
      The Adenosine Monophosphate-activated Protein Kinase (AMPK) and the Mechanistic Target of Rapamycin (mTOR) are two evolutionarily conserved kinases that together regulate nearly every aspect of cellular and systemic metabolism. These two kinases sense cellular energy and nutrient levels that in turn are determined by environmental nutrient availability. Because AMPK and mTOR are kinases, the large majority of studies remained focused on downstream substrate phosphorylation by these two proteins, and how AMPK and mTOR regulate signaling and metabolism in normal and disease physiology through phosphorylation of their substrates. Compared to the wealth of information known about the signaling and metabolic pathways modulated by these two kinases, much less is known about how the transcription of AMPK and mTOR pathway genes themselves are regulated, and the extent to which AMPK and mTOR regulate gene expression to cause durable changes in phenotype. Acute modification of cellular systems can be achieved through phosphorylation, however, induction of chronic changes requires modulation of gene expression. In this review we will assemble evidence from published studies on transcriptional regulation by AMPK and mTOR and discuss about the putative transcription factors that regulate expression of AMPK and mTOR complex genes.
    Keywords:  AMPK; mTOR; metabolism; signaling; transcription
  11. Neuropharmacology. 2020 Sep 02. pii: S0028-3908(20)30365-8. [Epub ahead of print] 108297
    Theilmann W, Gericke B, Schidlitzki A, Muneeb Anjum SM, Borsdorf S, Harries T, Roberds SL, Aguiar DJ, Brunner D, Leiser SC, Song D, Fabbro D, Hillmann P, Wymann MP, Löscher W.
      Mechanistic target of rapamycin (mTOR) regulates cell proliferation, growth and survival, and is activated in cancer and neurological disorders, including epilepsy. The rapamycin derivative ("rapalog") everolimus, which allosterically inhibits the mTOR pathway, is approved for the treatment of partial epilepsy with spontaneous recurrent seizures (SRS) in individuals with tuberous sclerosis complex (TSC). In contrast to the efficacy in TSC, the efficacy of rapalogs on SRS in other types of epilepsy is equivocal. Furthermore, rapalogs only poorly penetrate into the brain and are associated with peripheral adverse effects, which may compromise their therapeutic efficacy. Here we compare the antiseizure efficacy of three novel, brain-permeable ATP-competitive and selective mTORC1/2 inhibitors PQR620 and PQR626, and the selective dual pan-PI3K/mTORC1/2 inhibitor PQR530 in two mouse models of chronic epilepsy with SRS, the intrahippocampal kainate (IHK) mouse model of acquired temporal lobe epilepsy and Tsc1GFAP CKO mice, a well-characterized mouse model of epilepsy in TSC. During prolonged treatment of IHK mice with rapamycin, everolimus, PQR620, PQR626, or PQR530; only PQR620 exerted a transient antiseizure effect on SRS, at well tolerated doses whereas the other compounds were ineffective. In contrast, all of the examined compounds markedly suppressed SRS in Tsc1GFAP CKO mice during chronic treatment at well tolerated doses. Thus, against our expectation, no clear differences in antiseizure efficacy were found across the three classes of mTOR inhibitors examined in mouse models of genetic and acquired epilepsies. The main advantage of the novel 1,3,5-triazine derivatives is their excellent tolerability compared to rapalogs, which would favor their development as new therapies for TORopathies such as TSC.
    Keywords:  Acquired epilepsies; Antiseizure drugs; Genetic epilepsies; TORopathies; Tolerability
  12. Biochem Biophys Res Commun. 2020 Sep 08. pii: S0006-291X(20)31725-3. [Epub ahead of print]
    Tokumura K, Iwahashi S, Park G, Ochiai S, Okayama Y, Fusawa H, Fukasawa K, Iezaki T, Hinoi E.
      The mechanistic/mammalian target of rapamycin (mTOR) regulates various cellular processes, in part through incorporation into distinct protein complexes. The mTOR complex 1 (mTORC1) contains the Raptor subunit, while mTORC2 specifically contains the Rictor subunit. Mouse genetic studies, including ours, have revealed a critical role for mTOR in skeletogenesis through its expression in undifferentiated mesenchymal cells. In addition, we have recently revealed that mTORC1 expression in chondrocytes is crucial for skeletogenesis. Recent work indicates that mTOR regulates cellular functions, depending on the context, through both complex-dependent (canonical pathway) and complex-independent roles (noncanonical pathway). Here, we determined that mTOR regulates skeletal development through the noncanonical pathway, as well as the canonical pathway, in a cell-type and context-specific manner. Inactivation of Mtor in undifferentiated mesenchymal cells or chondrocytes led to either severe hypoplasia in appendicular skeletons or a severe and generalized chondrodysplasia, respectively. Moreover, Rictor deletion in undifferentiated mesenchymal cells or chondrocytes led to mineralization defects in some skeletal components. Finally, we revealed that simultaneous deletion of Raptor and Rictor in undifferentiated mesenchymal cells recapitulated the appendicular skeletal phenotypes of Mtor deficiency, whereas chondrocyte-specific Raptor and Rictor double-mutants exhibited milder hypoplasia of appendicular and axial skeletons than those seen upon Mtor deletion. These findings indicate that mTOR regulates skeletal development mainly through the canonical pathway in undifferentiated mesenchymal cells, but at least in part through the noncanonical pathway in chondrocytes.
    Keywords:  Canonical pathway; Chondrocytes; Mesenchymal cells; Noncanonical pathway; Skeletogenesis; mTOR
  13. Front Neurosci. 2020 ;14 775
    Norwitz NG, Querfurth H.
      The mechanistic target of rapamycin protein complex, mTORC1, has received attention in recent years for its role in aging and neurodegenerative diseases, such as Alzheimer's disease. Numerous excellent reviews have been written on the pathways and drug targeting of this keystone regulator of metabolism. However, none have specifically highlighted several important nuances of mTOR regulation as relates to neurodegeneration. Herein, we focus on six such nuances/open questions: (1) "Antagonistic pleiotropy" - Should we weigh the beneficial anabolic functions of mTORC1 against its harmful inhibition of autophagy? (2) "Early/late-stage specificity" - Does the relative importance of these neuroprotective/neurotoxic actions change as a disease progresses? (3) "Regional specificity" - Does mTOR signaling respond differently to the same interventions in different brain regions? (4) "Disease specificity" - Could the same intervention to inhibit mTORC1 help in one disease and cause harm in another disease? (5) "Personalized therapy" - Might genetically-informed personalized therapies that inhibit particular nodes in the mTORC1 regulatory network be more effective than generalized therapies? (6) "Lifestyle interventions" - Could specific diets, micronutrients, or exercise alter mTORC1 signaling to prevent or improve the progression neurodegenerative diseases? This manuscript is devoted to discussing recent research findings that offer insights into these gaps in the literature, with the aim of inspiring further inquiry.
    Keywords:  Alzheimer’s disease; Parkinson’s disease; antagonistic pleiotropy; autophagy; insulin/Akt; mTOR
  14. Steroids. 2020 Sep 02. pii: S0039-128X(20)30151-3. [Epub ahead of print]164 108725
    Ramirez CM, Taylor AM, Lopez AM, Repa JJ, Turley SD.
      Lipids present in lipoproteins cleared from the circulation are processed sequentially by three major proteins within the late endosomal/lysosomal (E/L) compartment of all cells: lysosomal acid lipase (LAL), Niemann-Pick (NPC) C2 and NPC1. When all three of these proteins are functioning normally, unesterified cholesterol (UC) exits the E/L compartment and is used in plasma membrane maintenance and various pathways in the endoplasmic reticulum including esterification by sterol O-acyltransferase 2 (SOAT2) or SOAT1 depending partly on cell type. Mutations in either NPC2 or NPC1 result in continual entrapment of UC and glycosphingolipids leading to neurodegeneration, pulmonary dysfunction, splenomegaly and liver damage. To date, the most effective agent for promoting release of entrapped UC in nearly all organs of NPC1-deficient mice and cats is 2-hydroxypropyl-β-cyclodextrin (2HPβCD). The cytotoxic nature of the liberated UC triggers various defenses including suppression of sterol synthesis and increased esterification. The present studies, using the Npc1-/-nih mouse model, measured the comparative quantitative importance of these two responses in the liver versus the spleen of Npc1-/-: Soat2+/+ and Npc1-/-: Soat2-/- mice in the 24 h following a single acute treatment with 2HPβCD. In the liver but not the spleen of both types of mice suppression of synthesis alone or in combination with increased esterification provided the major defense against the rise in unsequestered cellular UC content. These findings have implications for systemic 2HPβCD treatment in NPC1 patients in view of the purportedly low levels of SOAT2 activity in human liver.
    Keywords:  Cholesterol synthesis and esterification; Liver; Lysosomal storage disease; Spleen; Unesterified cholesterol sequestration
  15. Drugs. 2020 Sep 07.
    Nunnery SE, Mayer IA.
      Approximately 70% of invasive breast cancers have some degree of dependence on the estrogen hormone for cell proliferation and growth. These tumors have estrogen and/or progesterone receptors (ER/PR+), generally referred to as hormone receptor positive (HR+) tumors, as indicated by the presence of positive staining and varying intensity levels of estrogen and/or progesterone receptors on immunohistochemistry. Therapies that inhibit ER signaling pathways, such as aromatase inhibitors (letrozole, anastrozole, exemestane), selective ER modulators (tamoxifen), and ER down-regulators (fulvestrant), are the mainstays of treatment for hormone-receptor-positive breast cancers. However, de novo or acquired resistance to ER targeted therapies is present in many tumors, leading to disease progression. The PI3K/AKT/mTOR pathway is implicated in sustaining endocrine resistance and has become the target of many new drugs for ER+ breast cancer. This article reviews the function of the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway and the various classes of PI3K pathway inhibitors that have been developed to disrupt this pathway signaling for the treatment of hormone-receptor-positive breast cancer.
  16. J Anat. 2020 Sep 07.
    Jiang Z, Lau YK, Wu M, Casal ML, Smith LJ.
      The mucopolysaccharidoses (MPS) are a family of lysosomal storage disorders characterized by deficient activity of enzymes that degrade glycosaminoglycans (GAGs). Abnormal development of the vertebrae and long bones is a hallmark of skeletal disease in several MPS subtypes; however, the underlying cellular mechanisms remain poorly understood. The objective of this study was to conduct an ultrastructural examination of how lysosomal storage differentially affects major skeletal cell types in MPS I and VII using naturally occurring canine disease models. We showed that both bone and cartilage cells from MPS I and VII dog vertebrae exhibit significantly elevated storage from early in postnatal life, with storage generally greater in MPS VII than MPS I. Storage was most striking for vertebral osteocytes, occupying more than forty percent of cell area. Secondary to storage, dilation of the rough endoplasmic reticulum (ER), a marker of ER stress, was observed most markedly in MPS I epiphyseal chondrocytes. Significantly elevated immunostaining of light chain 3B (LC3B) in MPS VII epiphyseal chondrocytes suggested impaired autophagy, while significantly elevated apoptotic cell death in both MPS I and VII chondrocytes was also evident. The results of this study provide insights into how lysosomal storage differentially effects major skeletal cell types in MPS I and VII, and suggests a potential relationship between storage, ER stress, autophagy, and cell death in the pathogenesis of MPS skeletal defects.
    Keywords:  bone; canine; cartilage; electron microscopy; endoplasmic reticulum stress; lysosomal storage; mucopolysaccharidosis
  17. J Exp Bot. 2020 Sep 09. pii: eraa410. [Epub ahead of print]
    Kazibwe Z, Soto-Burgos J, MacIntosh GC, Bassham DC.
      The Arabidopsis thaliana T2 family endoribonuclease RNS2 localizes to the vacuole and functions in rRNA degradation. Loss of RNS2 activity impairs rRNA turnover and leads to constitutive autophagy, a process for degradation of cellular components. Autophagy is normally activated during environmental stress and is important for stress tolerance and homeostasis. Here we show that restoration of cytosolic purine nucleotide levels rescues the constitutive autophagy phenotype of rns2-2 seedlings, whereas inhibition of purine synthesis induces autophagy in wild-type seedlings. rns2-2 seedlings have reduced activity of the target of rapamycin (TOR) kinase complex, a negative regulator of autophagy, and this phenotype is rescued by addition of inosine to increase purine levels. Activation of TOR in rns2-2 by exogenous auxin blocks the enhanced autophagy, indicating a possible involvement of the TOR signaling pathway in the activation of autophagy in the rns2-2 mutant. Our data suggest a model in which loss of rRNA degradation in rns2-2 leads to a reduction in cytoplasmic nucleotide concentrations, which in turn inhibits TOR activity, leading to activation of autophagy to restore homeostasis.
    Keywords:  Arabidopsis; RNS2; TOR; autophagy; inosine; nucleotides; rRNA; ribonuclease
  18. J Neurosci. 2020 Sep 11. pii: JN-RM-1759-19. [Epub ahead of print]
    Adnan G, Rubikaite A, Khan M, Reber M, Suetterlin P, Hindges R, Drescher U.
      Interstitial axon branching is an essential step during the establishment of neuronal connectivity. However, the exact mechanisms on how the number and position of branches are determined are still not fully understood. Here, we investigated the role of Arl8B, an adaptor molecule between lysosomes and kinesins. In chick retinal ganglion cells, downregulation of Arl8B reduces axon branch density and shifts their location more proximally, while Arl8B overexpression leads to increased density and more distal positions of branches. These alterations correlate with changes in the location and density of lysosomes and autophagosomes along the axon shaft. Diminishing autophagy directly by knockdown of atg7, a key autophagy gene, reduces branch density, while induction of autophagy by rapamycin increases axon branching, indicating that autophagy plays a prominent role in axon branch formation. In vivo, local inactivation of autophagy in the retina using a mouse conditional knockout approach disturbs retino-collicular map formation which is dependent on the formation of interstitial axon branches. These data suggest that Arl8B plays a principal role in the positioning of axon branches by spatially controlling autophagy, thus directly controlling formation of neural connectivity in the brain.SIGNIFICANCE STATEMENTThe formation of interstitial axonal branches plays a prominent role in numerous places of the developing brain during neural circuit establishment. We show here that the GTPase Arl8B controls density and location of interstitial axon branches, and at the same time controls also density and location of the autophagy machinery. Up- or down-regulation of autophagy in vitro promotes or inhibits axon branching. Local disruption of autophagy in vivo disturbs retino-collicular mapping. Our data suggest that Arl8B controls axon branching by controlling locally autophagy. This work is one of the first reports showing a role of autophagy during early neural circuit development and suggests that autophagy in general plays a much more prominent role during brain development than previously anticipated.
  19. Nutr Metab Cardiovasc Dis. 2020 Jul 25. pii: S0939-4753(20)30297-0. [Epub ahead of print]
    Ding Y, Feng Y, Zou Y, Wang F, Liu H, Liu C, Zhang Y.
      BACKGROUND AND AIM: Abnormal aggregation of oxidized low-density lipoprotein (Ox-LDL) in vascular endothelial cells (VECs) is one of the major pathological changes in atherosclerotic lesions. Our research aimed to assess the mechanism of humanin (HN) in promoting autophagic degradation of Ox-LDL in HUVECs.METHODS AND RESULTS: Flow cytometry and lipid quantitation results showed that Ox-LDL caused lipid and cholesterol accumulation in HUVECs. Western blot results showed that Ox-LDL increased the expression of autophagy-related proteins P62 and LC3-II in a concentration-dependent manner. The cathepsin D activity assay showed that Ox-LDL inhibited the function of cathepsin D. HNG pretreatment reduced lipid and cholesterol aggregation in HUVECs induced by Ox-LDL, increased LC3-II protein level, decreased P62 protein content, and reversed Ox-LDL-induced cathepsin D functional impairment. Inhibition of the FPRL1 pathway by FPRL1 siRNA or the FPRL1-specific inhibitor Boc-MLF blocked all HNG's protective effects. These results indicate that HNG could restore cathepsin D activity and protein level in HUVECs to repair lysosomal functional damage induced by Ox-LDL, further repairing Ox-LDL-induced autophagic damage in HUVECs.
    CONCLUSION: HNG restores the activity of Ox-LDL-induced damaged lysosomal enzyme cathepsin D through its membrane protein receptor FPRL1 to promote autophagic degradation of Ox-LDL in HUVECs.
    Keywords:  Atherosclerosis; Cathepsin D; FPRL1; HNG; HUVECs; Ox-LDL
  20. Retin Cases Brief Rep. 2020 Aug 31.
    Hasegawa A, Noda K, Fujiya A, Hirooka K, Anzai T, Ishida S.
      PURPOSE: To report outer retinal abnormalities evaluated using high-resolution imaging modalities in a patient with Danon disease.METHODS: Case report.
    RESULTS: A 26-year-old woman, diagnosed with Danon disease based on genetic testing, was referred to our department for further evaluation of ocular findings. Her best-corrected visual acuity was 20/20, and color vision was normal. Fundus examination revealed pigmentary changes consisting of mottled depigmentation and pigmentation in the peripheral retina of both eyes. Spectral-domain optical coherence tomography (SD-OCT) revealed disruptions of the ellipsoid and interdigitation zones, irregularity of the retinal pigment epithelium (RPE), and hyperreflectivity of the outer nuclear layer. In addition, an adaptive optics retinal camera demonstrated the ambiguous macular cone mosaic pattern.
    CONCLUSION: Danon disease is caused by a primary deficiency in lysosomal associated membrane protein 2 (LAMP-2), an important constituent of the lysosomal membrane that plays a crucial role in the process of autophagy. It is possible that the findings of SD-OCT and adaptive optics retinal camera are early changes associated with the accumulation of autophagosomes and/or phagosomes due to LAMP-2 dysfunction in the photoreceptors, eventually followed by outer retinal degeneration, such as thinning of both the photoreceptor and RPE layers at the fovea.
  21. Cell Metab. 2020 Aug 31. pii: S1550-4131(20)30424-1. [Epub ahead of print]
    Kim CS, Ding X, Allmeroth K, Biggs LC, Kolenc OI, L'Hoest N, Chacón-Martínez CA, Edlich-Muth C, Giavalisco P, Quinn KP, Denzel MS, Eming SA, Wickström SA.
      Stem cells reside in specialized niches that are critical for their function. Upon activation, hair follicle stem cells (HFSCs) exit their niche to generate the outer root sheath (ORS), but a subset of ORS progeny returns to the niche to resume an SC state. Mechanisms of this fate reversibility are unclear. We show that the ability of ORS cells to return to the SC state requires suppression of a metabolic switch from glycolysis to oxidative phosphorylation and glutamine metabolism that occurs during early HFSC lineage progression. HFSC fate reversibility and glutamine metabolism are regulated by the mammalian target of rapamycin complex 2 (mTORC2)-Akt signaling axis within the niche. Deletion of mTORC2 results in a failure to re-establish the HFSC niche, defective hair follicle regeneration, and compromised long-term maintenance of HFSCs. These findings highlight the importance of spatiotemporal control of SC metabolic states in organ homeostasis.
    Keywords:  Akt; Hif1; cell fate; glutamine; hair follicle; hypoxia; mTOR; mTORC2; metabolism; stem cell
  22. Carcinogenesis. 2020 Sep 07. pii: bgaa096. [Epub ahead of print]
    N Haines C, Klingensmith HD, Komara M, Burd CJ.
      Over 70% of breast cancers express the estrogen receptor (ER) and depend on ER activity for survival and proliferation. While hormone therapies that target receptor activity are initially effective, patients invariably develop resistance which is often associated with activation of the PI3K/Akt/mTOR pathway. While the mechanism by which estrogen regulates proliferation is not fully understood, one gene target of ER, growth regulation by estrogen in breast cancer 1 (GREB1), is required for hormone-dependent proliferation. However, the molecular function by which GREB1 regulates proliferation is unknown. Herein, we validate that knockdown of GREB1 results in growth arrest and that exogenous GREB1 expression initiates senescence, suggesting that an optimal level of GREB1 expression is necessary for proliferation of breast cancer cells. Under both of these conditions, GREB1 is able to regulate signaling through the PI3K/Akt/mTOR pathway. GREB1 acts intrinsically through PI3K to regulate PIP3 levels and Akt activity. Critically, growth suppression of estrogen-dependent breast cancer cells by GREB1 knockdown is rescued by expression of constitutively activated Akt. Together, these data identify a novel molecular function by which GREB1 regulates breast cancer proliferation through Akt activation and provides a mechanistic link between estrogen signaling and the PI3K pathway.
  23. Curr Dev Nutr. 2020 Sep;4(9): nzaa131
    Soliman GA, Shukla SK, Etekpo A, Gunda V, Steenson SM, Gautam N, Alnouti Y, Singh PK.
      Background: The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing pathway and a key regulator of amino acid and glucose metabolism. Dysregulation of the mTOR pathways is implicated in the pathogenesis of metabolic syndrome, obesity, type 2 diabetes, and pancreatic cancer.Objectives: We investigated the impact of inhibition of mTORC1/mTORC2 and synergism with metformin on pancreatic tumor growth and metabolomics.
    Methods: Cell lines derived from pancreatic tumors of the KPC (KrasG12D/+; p53R172H/+; Pdx1-Cre) transgenic mice model were implanted into the pancreas of C57BL/6 albino mice (n = 10/group). Two weeks later, the mice were injected intraperitoneally with daily doses of 1) Torin 2 (mTORC1/mTORC2 inhibitor) at a high concentration (TH), 2) Torin 2 at a low concentration (TL), 3) metformin at a low concentration (ML), 4) a combination of Torin 2 and metformin at low concentrations (TLML), or 5) DMSO vehicle (control) for 12 d. Tissues and blood samples were collected for targeted xenometabolomics analysis, drug concentration, and cell signaling.
    Results: Metabolomic analysis of the control and treated plasma samples showed differential metabolite profiles. Phenylalanine was significantly elevated in the TLML group compared with the control (+426%, P = 0.0004), whereas uracil was significantly lower (-38%, P = 0.009). The combination treatment reduced tumor growth in the orthotopic mouse model. TLML significantly decreased pancreatic tumor volume (498 ± 104 mm3; 37%; P < 0.0004) compared with control (1326 ± 134 mm3; 100%), ML (853 ± 67 mm3; 64%), TL (745 ± 167 mm3; 54%), and TH (665 ± 182 mm3; 50%) (ANOVA and post hoc tests). TLML significantly decreased tumor weights (0.66 ± 0.08 g; 52%) compared with the control (1.28 ± 0.19 g; 100%) (P < 0.002).
    Conclusions: The combination of mTOR dual inhibition by Torin 2 and metformin is associated with an altered metabolomic profile and a significant reduction in pancreatic tumor burden compared with single-agent therapy, and it is better tolerated.
    Keywords:  KPC mouse model; antidiabetic drug; glycolysis; metabolomics; metformin; pancreatic cancer; phenylalanine; tricarboxylic acid cycle; xenometabolomics
  24. Cancers (Basel). 2020 Sep 04. pii: E2515. [Epub ahead of print]12(9):
    Shin J, Bae J, Park S, Kang HG, Shin SM, Won G, Kim JS, Cho SG, Choi Y, Oh SM, Shin J, Kim JS, Park HW.
      Oncogenic activation of the mammalian target of rapamycin complex 1 (mTORC1) leads to endometrial cancer cell growth and proliferation. Sestrin2 (SESN2), a highly conserved stress-inducible protein, is involved in homeostatic regulation via inhibition of reactive oxygen species (ROS) and mTORC1. However, the role of SESN2 in human endometrial cancer remains to be investigated. Here, we investigated expression, clinical significance, and underlying mechanisms of SESN2 in endometrial cancer. SESN2 was upregulated more in endometrial cancer tissues than in normal endometrial tissues. Furthermore, upregulation of SESN2 statistically correlated with shorter overall survival and disease-free survival in patients with endometrial cancer. SESN2 expression strongly correlated with mTORC1 activity, suggesting its impact on prognosis in endometrial cancer. Additionally, knockdown of SESN2 promoted cell proliferation, migration, and ROS production in endometrial cancer cell lines HEC-1A and Ishikawa. Treatment of these cells with mTOR inhibitors reversed endometrial cancer cell proliferation, migration, and epithelial-mesenchymal transition (EMT) marker expression. Moreover, in a xenograft nude mice model, endometrial cancer growth increased by SESN2 knockdown. Thus, our study provides evidence for the prognostic significance of SESN2, and a relationship between SESN2, the mTORC1 pathway, and endometrial cancer growth, suggesting SESN2 as a potential therapeutic target in endometrial cancer.
    Keywords:  endometrial cancer; mTORC1; migration; proliferation; reactive oxygen species (ROS); sestrin2
  25. Molecules. 2020 Sep 03. pii: E4025. [Epub ahead of print]25(17):
    Weber P, Thonhofer M, Averill S, Davies GJ, Santana AG, Müller P, Nasseri SA, Offen WA, Pabst BM, Paschke E, Schalli M, Torvisco A, Tschernutter M, Tysoe C, Windischhofer W, Withers SG, Wolfsgruber A, Wrodnigg TM, Stütz AE.
      Glycosidase inhibitors have shown great potential as pharmacological chaperones for lysosomal storage diseases. In light of this, a series of new cyclopentanoid β-galactosidase inhibitors were prepared and their inhibitory and pharmacological chaperoning activities determined and compared with those of lipophilic analogs of the potent β-d-galactosidase inhibitor 4-epi-isofagomine. Structure-activity relationships were investigated by X-ray crystallography as well as by alterations in the cyclopentane moiety such as deoxygenation and replacement by fluorine of a "strategic" hydroxyl group. New compounds have revealed highly promising activities with a range of β-galactosidase-compromised human cell lines and may serve as leads towards new pharmacological chaperones for GM1-gangliosidosis and Morquio B disease.
    Keywords:  4-epi-isofagomine; GM1-gangliosidosis; aminocyclopentane; carbasugar; galactosidase inhibitor; iminoalditol; pharmacological chaperone
  26. Biochem Biophys Res Commun. 2020 Sep 07. pii: S0006-291X(20)31691-0. [Epub ahead of print]
    Leng Y, Chen R, Chen R, He S, Shi X, Zhou X, Zhang Z, Chen AF.
      Endothelial cells injury and pro-inflammation cytokines release are the initial steps of hyperhomocysteinemia (HHcy)-associated vascular inflammation. Pyroptosis is a newly identified pro-inflammation form of programmed cell death, causing cell lysis and IL-1β release, and characterized by the caspases-induced cleavage of its effector molecule gasdermins (GSDMs). However, the effect of homocysteine (Hcy) on endothelial cells pyroptosis and the underlying mechanisms have not been fully defined. We have previously reported that Hcy induces vascular endothelial inflammation accompanied by the increase of high mobility group box-1 protein (HMGB1) and lysosomal cysteine protease cathepsin V in endothelial cells, and other studies have shown that HMGB1 or cathepsins are involved in activation of NLRP3 inflammasome and caspase-1. Here, we investigated the role of HMGB1 and cathepsin V in the process of Hcy-induced pyroptosis. We observed an increase in plasma IL-1β levels in HHcy patients and mice models, cathepsin V inhibitor reduced the plasma IL-1β levels and cleavage of GSDMD full-length into GSDMD N-terminal in the thoracic aorta of hyperhomocysteinemia mice. Using cultured HUVECs, we observed that Hcy promoted GSDMD N-terminal expression, silencing GSDMD or HMGB1 rescued Hcy-induced pyroptosis. HMGB1 also increased GSDMD N-terminal expression, and silencing cathepsin V reversed HMGB1-induced pyroptosis. HMGB1 could increase lysosome permeability, and silencing cathepsin V attenuated HMGB1-induced activation of caspase-1. In conclusion, this study has delineated a novel mechanism that HMGB1 mediated Hcy-induced endothelial cells pyroptosis partly via cathepsin V-dependent pathway.
    Keywords:  Cathepsin V; Endothelial cells; High mobility group box-1 protein; Homocysteine; Pyroptosis
  27. J Neuroinflammation. 2020 Sep 07. 17(1): 265
    Vardi A, Ben-Dor S, Cho SM, Kalinke U, Spanier J, Futerman AH.
      BACKGROUND: The type 1 interferon (IFN) response is part of the innate immune response and best known for its role in viral and bacterial infection. However, this pathway is also induced in sterile inflammation such as that which occurs in a number of neurodegenerative diseases, including neuronopathic Gaucher disease (nGD), a lysosomal storage disorder (LSD) caused by mutations in GBA.METHODS: Mice were injected with conduritol B-epoxide, an irreversible inhibitor of acid beta-glucosidase, the enzyme defective in nGD. MyTrMaSt null mice, where four adaptors of pathogen recognition receptors (PRRs) are deficient, were used to determine the role of the IFN pathway in nGD pathology. Activation of inflammatory and other pathways was analyzed by a variety of methods including RNAseq.
    RESULTS: Elevation in the expression of PRRs associated with the IFN response was observed in CBE-injected mice. Ablation of upstream pathways leading to IFN production had no therapeutic benefit on the lifespan of nGD mice but attenuated neuroinflammation. Primary and secondary pathological pathways (i.e., those associated or not with mouse survival) were distinguished, and a set of ~210 genes including those related to sphingolipid, cholesterol, and lipoprotein metabolism, along with a number of inflammatory pathways related to chemokines, TNF, TGF, complement, IL6, and damage-associated microglia were classified as primary pathological pathways, along with some lysosomal and neuronal genes.
    CONCLUSIONS: Although IFN signaling is the top elevated pathway in nGD, we demonstrate that this pathway is not related to mouse viability and is consequently defined as a secondary pathology pathway. By elimination, we defined a number of critical pathways that are directly related to brain pathology in nGD, which in addition to its usefulness in understanding pathophysiological mechanisms, may also pave the way for the development of novel therapeutic paradigms by targeting such pathways.
    Keywords:  Gaucher disease; Lipid metabolism; Lysosomal storage diseases; Neurodegenerative diseases; Pathogen recognition receptors; Type 1 interferon
  28. Am J Transl Res. 2020 ;12(8): 4807-4818
    Guan X, Shen J, Xu Y, Feng X, Zhou R.
      Lead (Pb), a highly poisonous heavy metal and an important occupational hazard, is currently a widespread environmental pollutant. The kidney is especially susceptible to the toxic effects of Pb because of its major role in Pb excretion. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme that can mitigate cellular injury. However, its role in Pb-elicited nephrotoxicity remains uncertain. This study was designed to examine the role of HO-1 in lead acetate (PbAc)-induced renal tubular cell injury in vitro. PbAc injury was found to suppress HO-1 expression and impair cell viability, with concomitant depletion of the autophagy proteins LC3-II and Beclin 1. Overexpression of HO-1 dramatically restored autophagy and protected cells against PbAc-induced apoptosis. In addition, pretreatment with 3-methyladenine, an inhibitor of autophagy, aggravated apoptosis and abolished renoprotection by HO-1, suggesting that the anti-apoptotic effect of HO-1 in Pb-induced nephrotoxicity is dependent on enhanced autophagy. Furthermore, HO-1 overexpression abrogated the inhibitory effect of PbAc on the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTORC1) signaling pathway. Pretreatment with an AMPK agonist, 5-aminoimidazole-4-carboxamide-1-β-D ribofuranoside, markedly enhanced autophagic activity and diminished apoptosis. Conversely, inhibition of AMPK phosphorylation abolished the pro-autophagic and anti-apoptotic effects of HO-1 in PbAc-injured cells. Our findings suggest that HO-1 alleviates Pb-induced nephrotoxicity via enhanced autophagy, which involves activation of the AMPK/mTORC1 signaling pathway.
    Keywords:  AMPK; Heme oxygenase-1; autophagy; lead acetate; mTORC1; nephrotoxicity
  29. Nat Commun. 2020 Sep 09. 11(1): 4510
    Ham DJ, Börsch A, Lin S, Thürkauf M, Weihrauch M, Reinhard JR, Delezie J, Battilana F, Wang X, Kaiser MS, Guridi M, Sinnreich M, Rich MM, Mittal N, Tintignac LA, Handschin C, Zavolan M, Rüegg MA.
      With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (, as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging.
  30. PLoS One. 2020 ;15(9): e0238624
    Wu C, Iwamoto T, Hossain MA, Akiyama K, Igarashi J, Miyajima T, Eto Y.
      BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive disorder caused by mutations of NPC1 or NPC2, which encode the proteins that are responsible for intracellular cholesterol trafficking. Loss of this function results in the accumulation of cholesterol-related products, such as oxysterols, sphingolipids, and NPC-related bile acids, which were recently used as biochemical biomarkers for the diagnosis of NPC. Bile acid-408 is a new significant compound we found in Japanese NPC patients, and it likely belongs to the category of bile acids. However, the diagnosis of NPC using a single biomarker is not satisfactory for clinical application because of the high instance of false negatives or positives. Therefore, we proposed an application of NPC diagnosis using a combination of 7-ketocholesterol (7-KC), lysosphingomyelin (lysoSM), bile acid-408 and/or glucosylsphingosine (lysoGL-1).METHODS AND FINDINGS: 7-KC, lysoSM and lysoGL-1 in sera and bile acid-408 in dried blood spots (DBS) were quantified within 17 minutes using tandem mass spectrometry and high-resolution mass spectrometry, respectively. We measured these biomarkers in NPC patients (n = 19), X-linked adrenoleukodystrophy (X-ALD) patients (n = 5), patients with other lysosomal diseases (n = 300), newborns (n = 124) and healthy people (n = 74). Our results showed a promising accuracy (97%) for NPC diagnosis using the combination of 7-KC, lysoSM and bile acid-408. However, contrary to our expectations, lysoGL-1 levels did not present at a significantly greater amount in NPC patients than other patients and negative controls.
    CONCLUSIONS: The combination of 7-KC, lysoSM and bile acid-408 improves the accuracy of NPC diagnosis and is feasible for mass screening due to its simple sample preparation and measurement. Future research should investigate the chemical structure of bile acid-408 to further facilitate its advantage in diagnosis.
  31. EMBO Rep. 2020 Sep 07. e50845
    Malecki M, Kamrad S, Ralser M, Bähler J.
      When glucose is available, many organisms repress mitochondrial respiration in favour of aerobic glycolysis, or fermentation in yeast, that suffices for ATP production. Fission yeast cells, however, rely partially on respiration for rapid proliferation under fermentative conditions. Here, we determined the limiting factors that require respiratory function during fermentation. When inhibiting the electron transport chain, supplementation with arginine was necessary and sufficient to restore rapid proliferation. Accordingly, a systematic screen for mutants growing poorly without arginine identified mutants defective in mitochondrial oxidative metabolism. Genetic or pharmacological inhibition of respiration triggered a drop in intracellular levels of arginine and amino acids derived from the Krebs cycle metabolite alpha-ketoglutarate: glutamine, lysine and glutamic acid. Conversion of arginine into these amino acids was required for rapid proliferation when blocking the respiratory chain. The respiratory block triggered an immediate gene expression response diagnostic of TOR inhibition, which was muted by arginine supplementation or without the AMPK-activating kinase Ssp1. The TOR-controlled proteins featured biased composition of amino acids reflecting their shortage after respiratory inhibition. We conclude that respiration supports rapid proliferation in fermenting fission yeast cells by boosting the supply of Krebs cycle-derived amino acids.
    Keywords:   S. pombe ; arginine; cellular metabolism; fermentation; respiration
  32. Nat Commun. 2020 Sep 08. 11(1): 4496
    Shi D, Xia X, Cui A, Xiong Z, Yan Y, Luo J, Chen G, Zeng Y, Cai D, Hou L, McDermott J, Li Y, Zhang H, Han JJ.
      Aging is characterized by the loss of homeostasis and the general decline of physiological functions, accompanied by various degenerative diseases and increased rates of mortality. Aging targeting small molecule screens have been performed many times, however, few have focused on endogenous metabolic intermediates-metabolites. Here, using C. elegans lifespan assays, we conducted a worm metabolite screen and identified an eukaryotes conserved metabolite, myo-inositol (MI), to extend lifespan, increase mobility and reduce fat content. Genetic analysis of enzymes in MI metabolic pathway suggest that MI alleviates aging through its derivative PI(4,5)P2. MI and PI(4,5)P2 are precursors of PI(3,4,5)P3, which is negatively related to longevity. The longevity effect of MI is dependent on the tumor suppressor gene, daf-18 (homologous to mouse Pten), independent of its classical pathway downstream genes, akt or daf-16. Furthermore, we found MI effects on aging and lifespan act through mitophagy regulator PTEN induced kinase-1 (pink-1) and mitophagy. MI's anti-aging effect is also conserved in mouse, indicating a conserved mechanism in mammals.
  33. FEBS J. 2020 Sep 09.
    Kobayashi T, Kageyama R.
      Quiescence is a cellular strategy for maintaining somatic stem cells in a specific niche in a low metabolic state without senescence for a long period of time. During development, neural stem cells actively proliferate and self-renew, and their progeny differentiate into both neurons and glial cells to form mature brain tissues. On the other hand, most neural stem cells in the adult brain are quiescent and arrested in G0/G1 phase of the cell cycle. Quiescence is essential in order to avoid the precocious exhaustion of neural stem cells, ensuring a sustainable source of available stem cells in the brain throughout the lifespan. After receiving activation signals, quiescent neural stem cells reenter the cell cycle and generate new neurons. This switching between quiescence and proliferation is tightly regulated by diverse signaling pathways. Recent studies suggest significant involvement of cellular proteostasis (homeostasis of the proteome) in the quiescent state of neural stem cells. Proteostasis is the result of integrated regulation of protein synthesis, folding, and degradation. In this review, we discuss regulation of quiescence by multiple signaling pathways, especially BMP and Notch signaling, and focus on the functional involvement of the lysosome, an organelle governing cellular degradation, in quiescence of adult neural stem cells.
    Keywords:  adult neural stem cell; lysosome; proteostasis; quiescence; signaling
  34. CEN Case Rep. 2020 Sep 09.
    Hirashio S, Kagawa R, Tajima G, Masaki T.
      Fabry disease is an X-linked recessive disease of glycosphingolipid metabolism caused by deficiency or reduced activity of α-galactosidase A. Fabry disease phenotypes are known to consist of a classic variant and a late-onset variant. In patients with Fabry disease, the phenotype is generally considered to be defined (at least partially) by the genotype. However, patients with the classic variant have been encountered in families with mutations that are expected to produce the late-onset variant. Here, we describe a 4-year-old boy with a classic variant of Fabry disease in a family with the M296I late-onset variant. The patient's grandfather, mother, and aunt experienced late-onset disease, characteristic of the M296I variant. Conversely, the patient experienced typical disease symptoms in childhood. He had symptoms of hypohidrosis and associated heat accumulation. He cried at night due to the occurrence of severe acroparaesthesia. This symptom became more pronounced in warmer climates. Although the patient's family had a late-onset variant mutation of Fabry disease, we determined that the patient's symptoms were similar to those of classic Fabry disease. Therefore, the patient began enzyme replacement therapy, which alleviated his symptoms. Notably, enzyme replacement therapy led to rapid improvement of the patient's subjective symptoms. Thus, we presumed that the patient's symptoms supported a diagnosis of classic Fabry disease. These findings suggest that childhood symptoms may occur in patients with Fabry disease, even in families with late-onset variant mutations. The genotype-phenotype correlation in Fabry disease remains controversial.
    Keywords:  Classic variant; Fabry disease; Genotype; Late-onset variant; M296I mutation; Phenotype
  35. Genes Dev. 2020 Sep 10.
    Van Nostrand JL, Hellberg K, Luo EC, Van Nostrand EL, Dayn A, Yu J, Shokhirev MN, Dayn Y, Yeo GW, Shaw RJ.
      Despite being the frontline therapy for type 2 diabetes, the mechanisms of action of the biguanide drug metformin are still being discovered. In particular, the detailed molecular interplays between the AMPK and the mTORC1 pathway in the hepatic benefits of metformin are still ill defined. Metformin-dependent activation of AMPK classically inhibits mTORC1 via TSC/RHEB, but several lines of evidence suggest additional mechanisms at play in metformin inhibition of mTORC1. Here we investigated the role of direct AMPK-mediated serine phosphorylation of RAPTOR in a new Raptor AA mouse model, in which AMPK phospho-serine sites Ser722 and Ser792 of RAPTOR were mutated to alanine. Metformin treatment of primary hepatocytes and intact murine liver requires AMPK regulation of both RAPTOR and TSC2 to fully inhibit mTORC1, and this regulation is critical for both the translational and transcriptional response to metformin. Transcriptionally, AMPK and mTORC1 were both important for regulation of anabolic metabolism and inflammatory programs triggered by metformin treatment. The hepatic transcriptional response in mice on high-fat diet treated with metformin was largely ablated by AMPK deficiency under the conditions examined, indicating the essential role of this kinase and its targets in metformin action in vivo.
    Keywords:  AMPK; RAPTOR; STAT3; TSC2; mTOR; metformin
  36. Curr Opin Cell Biol. 2020 Sep 02. pii: S0955-0674(20)30101-0. [Epub ahead of print]67 46-55
    Llamas E, Alirzayeva H, Loureiro R, Vilchez D.
      The proteostasis network adjusts protein composition and maintains protein integrity, which are essential processes for cell function and viability. Current efforts, given their intrinsic characteristics, regenerative potential and fundamental biological functions, have been directed to define proteostasis of stem cells. These insights demonstrate that embryonic stem cells and induced pluripotent stem cells exhibit an endogenous proteostasis network that not only modulates their pluripotency and differentiation but also provides a striking ability to suppress aggregation of disease-related proteins. Moreover, recent findings establish a central role of enhanced proteostasis to prevent the aging of somatic stem cells in adult organisms. Notably, proteostasis is also required for the biological purpose of adult germline stem cells, that is to be passed from one generation to the next. Beyond these links between proteostasis and stem cell function, we also discuss the implications of these findings for disease, aging, and reproduction.
    Keywords:  Adult somatic stem cells; Aging; Autophagy; Cell reprogramming; Differentiation; Embryonic stem cells; Germline stem cells; Hematopoietic stem cells; Induced pluripotent stem cells; Muscle stem cells; Neural stem cells; Protein chaperones; Protein synthesis; Proteostasis; RNA-binding proteins; Ubiquitin proteasome system
  37. BMC Med Genet. 2020 Sep 11. 21(1): 180
    He S, Lv N, Bao H, Wang X, Li J.
      BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal-dominant hereditary disease characterized by hamartomas of multiple organ systems, including the brain, skin, heart, kidney and lung. Genetically, TSC is caused by pathogenic variants in the TSC1 or TSC2 gene.CASE PRESENTATION: We reported a sporadic case of a 32-year-old Han Chinese male diagnosed with TSC, whose spouse had a history of two spontaneous miscarriages and an induced abortion of a 30-week fetus identified with cardiac rhabdomyoma by ultrasound. A novel heterozygous missense variant in the TSC2 gene (Exon35:c.4511 T > C:p.L1504P) was identified in the male patient and the aborted fetus by next-generation sequencing, but not in his wife or both his parents. According to the ACMG/AMP criteria, this variant was classified as a "likely pathogenic" variant.
    CONCLUSION: The novel TSC2:c.4511 T > C variant identified was highly likely associated with TSC and could potentially lead to adverse reproductive outcomes. IVF-ET and pre-implantation genetic diagnosis for TSC are recommended for this patient in the future to prevent fetal TSC.
    Keywords:  Next generation sequencing; Novel variant; Preimplantation genetic testing; TSC1; TSC2; Tuberous sclerosis complex
  38. JIMD Rep. 2020 Sep;55(1): 15-21
    Santoro L, Zampini L, Padella L, Monachesi C, Zampieri S, Dardis A, Cordiali R, Galeazzi T, Catassi C.
      Alpha mannosidosis is an ultrarare pathology with variable phenotypic manifestations, characterized by the deficiency of lysosomal alpha mannosidase which causes accumulation of neutral oligosaccharides. Until recently, the hematopoietic stem cell transplantation was the only clinical feasible therapeutic option. Only in 2018, the European Medicines Agency's committee approved the recombinant enzyme velmanase alfa for long-term treatment of non-neurological manifestations in mild and moderate forms of alpha-mannosidosis. In this study, the very early biochemical effects of enzyme replacement therapy in in a 7-month-old patient with alpha-mannosidosis were described. Velmanase alpha was administered as supporting therapy awaiting for hematopoietic stem cell transplantation, the treatment chosen for the patient because of the early onset form. The results showed that the enzyme replacement therapy was able to reduce the content of three different mannosyl-oligosaccharides monitored by tandem mass spectrometry after 2 months of treatment. In particular, the mean relative changes from baseline values were -67% in urine and -53% in serum at the latest observation. The study also showed that the enzymatic activity detected in serum 1 week after the first infusion was four times higher than the normal values and constant in the following points of observation. These findings led us to assume that velmanase alfa might be biologically active in this young patient.
    Keywords:  Hematopoietic cell transplantation; alpha mannosidosis; enzyme replacement therapy; liquid chromatography coupled with tandem mass spectrometry; oligosaccharides; thin layer chromatography
  39. Cells. 2020 Sep 03. pii: E2028. [Epub ahead of print]9(9):
    Scalise M, Console L, Rovella F, Galluccio M, Pochini L, Indiveri C.
      Cancer cells perform a metabolic rewiring to sustain an increased growth rate and compensate for the redox stress caused by augmented energy metabolism. The metabolic changes are not the same in all cancers. Some features, however, are considered hallmarks of this disease. As an example, all cancer cells rewire the amino acid metabolism for fulfilling both the energy demand and the changed signaling routes. In these altered conditions, some amino acids are more frequently used than others. In any case, the prerequisite for amino acid utilization is the presence of specific transporters in the cell membrane that can guarantee the absorption and the traffic of amino acids among tissues. Tumor cells preferentially use some of these transporters for satisfying their needs. The evidence for this phenomenon is the over-expression of selected transporters, associated with specific cancer types. The knowledge of the link between the over-expression and the metabolic rewiring is crucial for understanding the molecular mechanism of reprogramming in cancer cells. The continuous growth of information on structure-function relationships and the regulation of transporters will open novel perspectives in the fight against human cancers.
    Keywords:  amino acids; metabolism; transporters; tumors
  40. Eur J Med Chem. 2020 Jul 19. pii: S0223-5234(20)30466-9. [Epub ahead of print]206 112494
    Yin L, Hu Q.
      Ubiquitin-proteasome system, autophagy-lysosome pathway and N-end rule pathway are crucial protein quality control mechanisms in human body. Hijacking these endogenous protein degrading measures by chimera degraders could be a revolutionary strategy for the discovery of small-molecule drugs. As the most advanced chimera degraders, PROTACs have demonstrated the potential by delivering two drug candidates into clinical trials. The development of chimera degraders exploiting these three pathways are reviewed, a focus is given on the chemical structures and their influences on biological effects from a viewpoint of medicinal chemistry.
    Keywords:  Autophagy-lysosome pathway; Chimera degraders; Drug discovery; N-end rule; PROTACs; Ubiquitin–proteasome system
  41. Mov Disord. 2020 Sep 12.
    Tantawy AAG, Adly AAM, Hashem NH, Ebeid WM, Abdeen MSED, Salah NY.
      BACKGROUND: Patients with Gaucher disease (GD) have an increased risk for parkinsonism. Retinal thinning has been described in parkinsonism as an early nonmotor feature. Scarce reports have addressed retinal thickness changes in GD.OBJECTIVES: The objectives of this study were to compare ganglion cell complex (GCC) thickness in adolescents and young adults (AYAs) with GD with healthy control subjects, and to correlate it with the presence of parkinsonian features (PFs), clinical prodromal markers of parkinsonism, severity score index (SSI), and glucosylsphingosine (Lyso-GL-1).
    METHODS: This study included 48 AYAs with GD (11-29 years), 11 with manifest PFs (Group 1) and 37 with no PFs (Group 2), and 48 matched healthy control subjects (Group 3). Age of GD onset, disease duration, medication history, history of constipation, SSI, and hematological assessment were done. Neurocognitive evaluation included Parts I, II, and III of the Unified Parkinson's Disease Rating Scale (UPDRS), Wechsler Adult and Intelligence Scale and Wechsler Intelligence Scale for Children, Beck Depression Inventory (BDI), rapid eye movement sleep behavior disorder (RBD) scale, Munich Parasomnia Screening scale, and the olfactory dysfunction scale. Molecular analyses of the acid GBA gene and Lyso-GL-1 were done. Participants underwent full ophthalmological examination and optical coherence tomography with GCC thickness measurement.
    RESULTS: GCC was significantly thinner in Group 1 than in Groups 2 and 3 (P < 0.001), whereas no significant difference was found between Groups 2 and 3 (P = 0.977). In addition, a significant interocular GCC thickness difference was found among the studied AYAs with GD (P = 0.007). GCC correlated positively with total intelligence quotient (P < 0.001) and negatively with Lyso-GL-1 (P = 0.019), UPDRS (P = 0.004), and BDI (P = 0.029), but not with SSI (P = 0.874), GD type (P = 0.85), or genotype (P = 0.842). A significant negative relationship was found between GCC thickness and PFs (P = 0.001), parasomnia (P = 0.003), constipation (P = 0.031), RBD (P = 0.044), and hyposmia (P = 0.033).
    CONCLUSIONS: GCC thinning may be a promising biomarker for central nervous system neurodegeneration that has the potential to monitor early PFs among people with GD. © 2020 International Parkinson and Movement Disorder Society.
    Keywords:  Gaucher disease; ganglion cell complex; glucosylsphingosine; lysosomal storage disorders; neurodegeneration; optical coherence tomography; parkinsonian features; retinal thinning
  42. J Bone Miner Res. 2020 Sep 11.
    Li K, Yang P, Zhang Y, Zhang Y, Cao H, Liu P, Huang B, Xu S, Lai P, Lei G, Liu J, Tang Y, Bai X, Zou Z.
      Endoplasmic reticulum (ER) stress has been shown to promote chondrocyte apoptosis and osteoarthritis (OA) progression, but the precise mechanisms via which ER stress is modulated in OA remain unclear. Here we report that DEPTOR (DEP domain-containing mTOR-interacting protein) negatively regulated ER stress and OA development independent of mTOR signaling. DEPTOR is ubiquitinated in articular chondrocytes and its expression is markedly reduced along with OA progression. Deletion of DEPTOR in chondrocytes significantly promoted destabilized medial meniscus (DMM) surgery-induced OA development, whereas intra-articular injection of lentivirus expressing DEPTOR delayed OA progression in mice. Proteomics analysis revealed that DEPTOR interplayed with TRC8, which promoted TRC8 auto-ubiquitination and degraded by the ubiquitin-proteasome system (UPS) in chondrocytes. Loss of DEPTOR leaded to TRC8 accumulation and excessive ER stress, with subsequent chondrocytes apoptosis and OA progression. Importantly, an inhibitor of ER stress eliminated chondrocyte DEPTOR deletion-exacerbated OA in mice. Together, these findings establish a novel mechanism essential for OA pathogenesis, where decreasing DEPTOR in chondrocytes during OA progression relieves the auto-ubiquitination of TRC8, resulting in TRC8 accumulation, excessive ER stress and OA progression. Targeting this pathway has promising therapeutic potential for OA treatment. This article is protected by copyright. All rights reserved.
    Keywords:  DEPTOR; ER stress; TRC8; osteoarthritis; ubiquitination
  43. Oncol Rep. 2020 Sep 04.
    Nishikawa R, Osaki M, Sasaki R, Ishikawa M, Yumioka T, Yamaguchi N, Iwamoto H, Honda M, Kabuta T, Takenaka A, Okada F.
      Sunitinib, a tyrosine kinase inhibitor, is among the first‑line treatments for metastatic or advanced stage renal cell carcinoma (RCC). However, patients with RCC develop resistance to sunitinib. We have previously demonstrated that lysosome‑associated membrane protein 2 (LAMP‑2), which has three splice variants with different functions (LAMP‑2A, LAMP‑2B, and LAMP‑2C), is involved in RCC. In the present study, we examined which splice variants of LAMP‑2 contributed to sunitinib resistance in RCC cells. In vitro analysis using ACHN, human RCC cell line, revealed that the IC50 of sunitinib was significantly increased by overexpression of LAMP‑2A and LAMP‑2B, but not LAMP‑2C (P<0.01). Kaplan‑Meier survival analysis using clinical samples revealed an association between shorter survival and high expression of LAMP‑2A and LAMP‑2B, but not LAMP‑2C, in patients with RCC treated with sunitinib (P=0.01). Furthermore, high expression of LAMP‑2A and LAMP‑2B in RCC revealed a weak to moderate inverse correlation with the tumor shrinkage rate and progression‑free survival, respectively. Thus, high expression of LAMP‑2A and LAMP‑2B contributed to the acquisition of sunitinib resistance, indicating that the expression of these two variants can predict the efficacy of sunitinib treatment in patients with RCC.
  44. J Hum Genet. 2020 Sep 11.
    Meng Y, Yu C, Chen M, Yu X, Sun M, Yan H, Zhao W, Yu S.
      Genetic testing of TSC1 and TSC2 is important for the diagnosis of tuberous sclerosis complex (TSC), an autosomal dominant neurocutaneous disease. This study retrospectively reviewed 347 samples from patients with clinically suspected TSC being tested for mutations in TSC1 and TSC2 genes using next-generation sequencing and multiplex ligation-dependent probe amplification. Two hundred eighty-one patients (80.98%) were classified as definite/possible/uncertain diagnosis of TSC and the mutational spectrum of TSC1/TSC2 was described. Two hundred eighteen unique nonsynonymous SNVs/Indels (64 in TSC1, 154 in TSC2) and 13 copy number variants (CNVs) were identified in 241 samples (85.77%), including 82 novel variants. CNVs involving 12 large deletions and one duplication were detected exclusively in TSC2. Both TSC1 and TSC2 mutations were nearly uniformly distributed in their protein-coding regions. Furthermore, a string of non-TSC1/TSC2 deleterious variants in 12 genes was identified in the patients, especially overwhelmingly present in the patients with no mutation identified (NMI) in TSC1/TSC2. Our study provides a comprehensive TSC1/TSC2 mutation landscape and reveal some potential risk non-TSCs variants present in patients with NMI.
  45. Cancers (Basel). 2020 Sep 03. pii: E2492. [Epub ahead of print]12(9):
    Petővári G, Dankó T, Tőkés AM, Vetlényi E, Krencz I, Raffay R, Hajdu M, Sztankovics D, Németh K, Vellai-Takács K, Jeney A, Kulka J, Sebestyén A.
      In spite of tremendous developments in breast cancer treatment, the relatively high incidence of relapsing cases indicates a great need to find new therapeutic strategies in recurrent, metastatic and advanced cases. The bioenergetic needs of growing tumours at the primary site or in metastases-accumulating genomic alterations and further heterogeneity-are supported by metabolic rewiring, an important hallmark of cancer. Adaptation mechanisms as well as altered anabolic and catabolic processes balance according to available nutrients, energy, oxygen demand and overgrowth or therapeutic resistance. Mammalian target of rapamycin (mTOR) hyperactivity may contribute to this metabolic plasticity and progression in breast carcinomas. We set out to assess the metabolic complexity in breast cancer cell lines and primary breast cancer cases. Cellular metabolism and mTOR-related protein expression were characterised in ten cell lines, along with their sensitivity to specific mTOR and other metabolic inhibitors. Selected immunohistochemical reactions were performed on ~100 surgically removed breast cancer specimens. The obtained protein expression scores were correlated with survival and other clinicopathological data. Metabolic and mTOR inhibitor mono-treatments had moderate antiproliferative effects in the studied cell lines in a subtype-independent manner, revealing their high adaptive capacity and survival/growth potential. Immunohistochemical analysis of p-S6, Rictor, lactate dehydrogenase A, glutaminase, fatty acid synthase and carnitine palmitoyltransferase 1A in human samples identified high mTOR activity and potential metabolic plasticity as negative prognostic factors for breast cancer patients, even in subtypes generally considered as low-risk. According to our results, breast cancer is characterised by considerable metabolic diversity, which can be targeted by combining antimetabolic treatments and recent therapies. Alterations in these pathways may provide novel targets for future drug development in breast cancer. We also propose a set of immunostainings for scoring metabolic heterogeneity in individual cases in order to select patients who may benefit from more accurate follow-up and specific therapies.
    Keywords:  breast cancer; mTOR activity; metabolic characterisation; metabolic plasticity; metabolism
  46. ACS Nano. 2020 Sep 09.
    Ling B, Lee J, Maresca D, Lee-Gosselin A, Malounda D, Swift MB, Shapiro MG.
      Phagocytic clearance and lysosomal processing of pathogens and debris are essential functions of the innate immune system. However, the assessment of these functions in vivo is challenging because most nanoscale contrast agents compatible with non-invasive imaging techniques are made from non-biodegradable synthetic materials that do not undergo regular lysosomal degradation. To overcome this challenge, we describe the use of an all-protein contrast agent to directly visualize and quantify phagocytic and lysosomal activities in vivo by ultrasound imaging. This contrast agent is based on gas vesicles (GVs), a class of air-filled protein nanostructures naturally expressed by buoyant microbes. Using a combination of ultrasound imaging, pharmacology, immunohistology and live-cell optical microscopy, we show that after intravenous injection, GVs are cleared from circulation by liver-resident macrophages. Once internalized, the GVs undergo lysosomal degradation, resulting in the elimination of their ultrasound contrast. By non-invasively monitoring the temporal dynamics of GV-generated ultrasound signal in circulation and in the liver and fitting them with a pharmacokinetic model, we can quantify the rates of phagocytosis and lysosomal degradation in living animals. We demonstrate the utility of this method by showing how these rates are perturbed in two models of liver dysfunction: phagocyte deficiency and non-alcoholic fatty liver disease. The combination of proteolytically-degradable nanoscale contrast agents and quantitative ultrasound imaging thus enables non-invasive functional imaging of cellular degradative processes.