bims-lysosi Biomed News
on Lysosomes and signaling
Issue of 2020‒08‒16
fifty-five papers selected by
Stephanie Fernandes
Max Planck Institute for Biology of Ageing

  1. J Clin Med. 2020 Aug 11. pii: E2596. [Epub ahead of print]9(8):
    Stepien KM, Roncaroli F, Turton N, Hendriksz CJ, Roberts M, Heaton RA, Hargreaves I.
      Mitochondrial dysfunction is emerging as an important contributory factor to the pathophysiology of lysosomal storage disorders (LSDs). The cause of mitochondrial dysfunction in LSDs appears to be multifactorial, although impaired mitophagy and oxidative stress appear to be common inhibitory mechanisms shared amongst these heterogeneous disorders. Once impaired, dysfunctional mitochondria may impact upon the function of the lysosome by the generation of reactive oxygen species as well as depriving the lysosome of ATP which is required by the V-ATPase proton pump to maintain the acidity of the lumen. Given the reported evidence of mitochondrial dysfunction in LSDs together with the important symbiotic relationship between these two organelles, therapeutic strategies targeting both lysosome and mitochondrial dysfunction may be an important consideration in the treatment of LSDs. In this review we examine the putative mechanisms that may be responsible for mitochondrial dysfunction in reported LSDs which will be supplemented with morphological and clinical information.
    Keywords:  autophagy; inflammation; lysosomal storage diseases; mitochondrial dysfunction; mitophagy and cytokine; oxidative stress; reactive oxygen species
  2. Exp Neurol. 2020 Aug 08. pii: S0014-4886(20)30263-6. [Epub ahead of print] 113432
    Iffland PH, Barnes AE, Baybis M, Crino PB.
      TSC1 or TSC2 mutations cause Tuberous Sclerosis Complex (TSC), and lead to mechanistic target of rapamycin (mTOR) hyperactivation evidenced by hyperphosphorylation of ribosomal S6 protein and 4-elongation factor binding protein (4E-BP1). Amino acid (AA) levels modulate mTOR-dependent S6 and 4E-BP1 phosphorylation in non-neural cells, but this has not been investigated in neurons. The effects of AA levels on mTOR signaling and S6 and 4E-BP1 phosphorylation were analyzed in Tsc2 and Depdc5 (a distinct mTOR regulatory gene associated with epilepsy) CRISPR-edited Neuro2a (N2a) cells and differentiated neurons. Tsc2 or Depdc5 knockout (KO) led to S6 and 4E-BP1 hyperphosphorylation and cell soma enlargement, but while Tsc2 KO N2a cells exhibited reduced S6 phosphorylation (Ser240/244) and cell soma size after incubation in AA free (AAF) media, Depdc5 KO cells did not. Using a CFP/YFP FRET-biosensor coupled to 4E-BP1, we assayed 4E-BP1 phosphorylation in living N2a cells and differentiated neurons following Tsc2 or Depdc5 KO. AAF conditions reduced 4E-BP1 phosphorylation in Tsc2 KO N2a cells but had no effect in Depdc5 KO cells. Rapamycin blocked S6 protein phosphorylation but had no effect on 4E-BP1 phosphorylation, following either Tsc2 or Depdc5 KO. Confocal imaging demonstrated that AAF media promoted movement of mTOR off the lysosome, functionally inactivating mTOR, in Tsc2 KO but not Depdc5 KO cells, demonstrating that AA levels modulate lysosomal mTOR localization and account, in part, for differential effects of AAF conditions following Tsc2 versus Depdc5 KO. AA levels and rapamycin differentially modulate S6 and 4E-BP1 phosphorylation and mTOR lysosomal localization in neurons following Tsc2 KO versus Depdc5 KO. Neuronal mTOR signaling in mTOR associated epilepsies may have distinct responses to mTOR inhibitors and to levels of cellular amino acids.
    Keywords:  Cortical malformations; Epilepsy; GATOR1; Tuberous sclerosis complex; mTORopathies
  3. Am J Transl Res. 2020 ;12(7): 3964-3973
    Su Z, Lin M, Su Y, Li J, Lin R, Wu M, Wang X, Huang L, Chen Y, Shu X, Liang S, Zhang H, Huang T, Nie R, Wang J, Xie S.
      BACKGROUND: Cyclophilin A (CyPA) plays an important role in the progression of atherosclerosis. Additionally, it has been reported that lysosomal function is markedly impaired in atherosclerosis induced by oxidized low-density lipoprotein (ox-LDL). As the CyPA degradation pathway remains to be elucidated, we aimed to uncover the role of lysosomes and ox-LDL in the degradation of CyPA.METHODS: We exploited RNA interference (RNAi) in combination with either the lysosomal inhibitor chloroquine (CQ) or the proteasomal inhibitor MG-132 to examine CyPA turnover. We also investigated the role of ox-LDL in lysosomal function and the CyPA degradation pathway and determined whether CyPA interacts with the selective autophagy adaptor p62.
    RESULTS: CQ markedly reversed the CyPA downregulation induced by RNAi and increased intracellular levels of LC3 and p62. MG-132 significantly suppressed polyubiquitinated protein degradation but did not inhibit RNAi-induced CyPA downregulation. Additionally, neither CQ nor MG-132 influenced the gene-silencing efficiency of CyPA siRNA. Moreover, ox-LDL induced cytosolic accumulation of p62 was inconsistent with increased expression of LC3-II. Meanwhile, ox-LDL inhibited RNAi-induced downregulation of CyPA. Immunofluorescence indicated colocalization of endogenous CyPA with ubiquitin and with p62 in response to CQ treatment, and co-immunoprecipitation analysis confirmed interaction between CyPA and p62.
    CONCLUSION: CyPA is degraded by a lysosome-dependent pathway that may involve p62-mediated selective autophagy. Furthermore, ox-LDL modulates the degradation of CyPA via its inhibitory role in lysosomes, contributing to increased expression of CyPA in atherosclerotic plaques.
    Keywords:  Oxidized low-density lipoprotein; RNA interference; atherosclerotic plaques; cycloheximide; cyclophilin A; lysosome
  4. Sci Rep. 2020 Aug 14. 10(1): 13810
    Kotani T, Setiawan J, Konno T, Ihara N, Okamoto S, Saito Y, Murata Y, Noda T, Matozaki T.
      Cell signaling important for homeostatic regulation of colonic epithelial cells (CECs) remains poorly understood. Mammalian target of rapamycin complex 1 (mTORC1), a protein complex that contains the serine-threonine kinase mTOR, mediates signaling that underlies the control of cellular functions such as proliferation and autophagy by various external stimuli. We here show that ablation of tuberous sclerosis complex 2 (Tsc2), a negative regulator of mTORC1, specifically in intestinal epithelial cells of mice resulted in increased activity of mTORC1 of, as well as increased proliferative activity of, CECs. Such Tsc2 ablation also reduced the population of Lgr5-positive colonic stem cells and the expression of Wnt target genes in CECs. The stimulatory phosphorylation of the kinase Akt and inhibitory phosphorylation of glycogen synthase kinase 3β were both markedly decreased in the colon of the Tsc2 conditional knockout (CKO) mice. Development of colonic organoids with cryptlike structures was enhanced for Tsc2 CKO mice compared with control mice. Finally, Tsc2 CKO mice manifested increased susceptibility to dextran sulfate sodium-induced colitis. Our results thus suggest that mTORC1 activity promotes the proliferation of, as well as the expression of Wnt target genes in, CECs and thereby contributes to colonic organogenesis and homeostasis.
  5. Int J Mol Sci. 2020 Aug 10. pii: E5732. [Epub ahead of print]21(16):
    Coutinho MF, Santos JI, S Mendonça L, Matos L, Prata MJ, S Jurado A, Pedroso de Lima MC, Alves S.
      More than two thirds of Lysosomal Storage Diseases (LSDs) present central nervous system involvement. Nevertheless, only one of the currently approved therapies has an impact on neuropathology. Therefore, alternative approaches are under development, either addressing the underlying enzymatic defect or its downstream consequences. Also under study is the possibility to block substrate accumulation upstream, by promoting a decrease of its synthesis. This concept is known as substrate reduction therapy and may be triggered by several molecules, such as small interfering RNAs (siRNAs). siRNAs promote RNA interference, a naturally occurring sequence-specific post-transcriptional gene-silencing mechanism, and may target virtually any gene of interest, inhibiting its expression. Still, naked siRNAs have limited cellular uptake, low biological stability, and unfavorable pharmacokinetics. Thus, their translation into clinics requires proper delivery methods. One promising platform is a special class of liposomes called stable nucleic acid lipid particles (SNALPs), which are characterized by high cargo encapsulation efficiency and may be engineered to promote targeted delivery to specific receptors. Here, we review the concept of SNALPs, presenting a series of examples on their efficacy as siRNA nanodelivery systems. By doing so, we hope to unveil the therapeutic potential of these nanosystems for targeted brain delivery of siRNAs in LSDs.
    Keywords:  RNA interference (RNAi); lysosomal storage diseases (LSDs); neuropathy; siRNA nanodelivery systems; stable nucleic acid lipid particles (SNALPs); substrate reduction therapy (SRT)
  6. Eur J Pharmacol. 2020 Aug 08. pii: S0014-2999(20)30512-4. [Epub ahead of print] 173420
    Riaz S, Abdulrahman N, Uddin S, Jabeen A, Gadeau AP, Fliegel L, Mraiche F.
      Previous studies have established the role of Na+/H+ exchanger isoform-1 (NHE1) and cathepsin B (Cat B) in the development of cardiomyocyte hypertrophy (CH). Both NHE1 and Cat B are activated under acidic conditions suggesting that their activities might be interrelated. The inhibition of NHE1 has been demonstrated to reduce cardiac hypertrophy but the mechanism that contributes to the anti-hypertrophic effect of NHE1 inhibition still remains unclear. H9c2 cardiomyoblasts were stimulated with Angiotensin (Ang) II in the presence and absence of N-[2-methyl-4,5-bis(methylsulphonyl)-benzoyl]-guanidine, hydrochloride (EMD, EMD 87580), an NHE1 inhibitor or CA-074Me, a Cat B inhibitor, and various cardiac hypertrophic parameters, namely cell surface area, protein content and atrial natriuretic peptide (ANP) mRNA were analyzed. EMD significantly suppressed markers of cardiomyocyte hypertrophy and inhibited Ang II stimulated Cat B protein and gene expression. Cat B is located within the acidic environment of lysosomes. Cat B proteases are released into the cytoplasm upon disintegration of the lysosomes. EMD or CA-074Me prevented the dispersal of the lysosomes induced by Ang II and reduced the ratio of LC3-II to LC3-I, a marker of autophagy. Moreover, Cat B protein expression and MMP-9 activity in the extracellular space were significantly attenuated in the presence of EMD or CA-074Me. Our study demonstrates a novel mechanism for attenuation of the hypertrophic phenotype by NHE1 inhibition that is mediated by a regression in Cat B. The inhibition of Cat B via EMD or CA-074Me attenuates the autosomal-lysosomal pathway and MMP-9 activation.
    Keywords:  Angiotensin; Autophagy; Cardiomyocytes; Cathepsins; Hypertrophy; Matrix metalloproteinases
  7. ACS Appl Mater Interfaces. 2020 Aug 13.
    Liu CG, Tang HX, Zheng X, Yang DY, Zhang Y, Zhang JT, Kankala RK, Wang SB, Liu G, Chen AZ.
      The over-developed lysosomes in cancer cells are gaining increasing attention towards more precise and effective organelle-targeted cancer therapy. It is suggested that rods/plates-like nanomaterials with an appropriate size exhibited a greater quantity and longer-term lysosomal enrichment, as the shape plays a notable role in nanomaterials transmembrane process and subcellular behaviors. Herein, a biodegradable platform based on layered double hydroxide-copper sulfide nanocomposites (LDH-CuS NCs) is successfully prepared via in situ growth of CuS nanodots on LDH nanoplates. The as-prepared LDH-CuS NCs exhibited not only high photothermal conversion, near-infrared (NIR)-induced chemodynamic, and photodynamic therapeutic efficacies, but also could achieve real-time in vivo photoacoustic imaging (PAI) of the entire tumor. LDH-CuS NCs accumulated in lysosomes would then generate extensive subcellular reactive oxygen species (ROS) in situ, leading to lysosomal membrane permeabilization (LMP) pathway-associated cell death both in vitro and in vivo.
  8. J Orthop Res. 2020 Aug 11.
    Fort BP, Dubyak GR, Greenfield EM.
      Wear particles from orthopaedic implants cause aseptic loosening, the leading cause of implant revisions. The particles are phagocytosed by macrophages leading to activation of the NLRP3 inflammasome and release of IL-1β which then contributes to osteoclast differentiation and implant loosening. The mechanism of inflammasome activation by orthopaedic particles is undetermined but other particles cause the cytosolic accumulation of the lysosomal cathepsin-family proteases which can activate the NLRP3 inflammasome. Here, we demonstrate that lysosome membrane disruption causes cathepsin release into the cytoplasm that drives both inflammasome activation and cell death but that these processes occur independently. Using wild-type and genetically-manipulated immortalized murine bone marrow derived macrophages and pharmacologic inhibitors, we found that NLRP3 and gasdermin D are required for particle-induced IL-1β release but not for particle-induced cell death. In contrast, phagocytosis and lysosomal cathepsin release are critical for both IL-1β release and cell death. Collectively, our findings identify the pan-cathepsin inhibitor Ca-074Me and the NLRP3 inflammasome inhibitor MCC950 as therapeutic interventions worth exploring in aseptic loosening of orthopaedic implants. We also found that particle-induced activation of the NLRP3 inflammasome in pre-primed macrophages and cell death are not dependent on pathogen-associated molecular patterns adherent to the wear particles despite such pathogen-associated molecular patterns being critical for all other previously studied wear particle responses, including priming of the NLRP3 inflammasome. This article is protected by copyright. All rights reserved.
    Keywords:  aseptic loosening; cathepsins; gasdermin D; inflammasome; lysosomal disruption
  9. Neural Regen Res. 2021 Jan;16(1): 26-35
    Tran AP, Silver J.
      Proteases comprise a variety of enzymes defined by their ability to catalytically hydrolyze the peptide bonds of other proteins, resulting in protein lysis. Cathepsins, specifically, encompass a class of at least twenty proteases with potent endopeptidase activity. They are located subcellularly in lysosomes, organelles responsible for the cell's degradative and autophagic processes, and are vital for normal lysosomal function. Although cathepsins are involved in a multitude of cell signaling activities, this chapter will focus on the role of cathepsins (with a special emphasis on Cathepsin B) in neuronal plasticity. We will broadly define what is known about regulation of cathepsins in the central nervous system and compare this with their dysregulation after injury or disease. Importantly, we will delineate what is currently known about the role of cathepsins in axon regeneration and plasticity after spinal cord injury. It is well established that normal cathepsin activity is integral to the function of lysosomes. Without normal lysosomal function, autophagy and other homeostatic cellular processes become dysregulated resulting in axon dystrophy. Furthermore, controlled activation of cathepsins at specialized neuronal structures such as axonal growth cones and dendritic spines have been positively implicated in their plasticity. This chapter will end with a perspective on the consequences of cathepsin dysregulation versus controlled, localized regulation to clarify how cathepsins can contribute to both neuronal plasticity and neurodegeneration.
    Keywords:  CSPGs; axon regeneration; cathepsin; extracellular matrix; growth cone; lysosomes; neuronal plasticity; protease; remodeling; spinal cord injury; synaptogenesis
  10. Case Rep Ophthalmol. 2020 May-Aug;11(2):11(2): 306-314
    Fukuyo H, Inoue Y, Takahashi H, Hatano Y, Shibuya T, Sakai N, Kawashima H.
      Galactosialidosis is a rare metabolic disorder resulting from mutations in the CTSA gene. Few studies have reported on the ocular findings of galactosialidosis type IIb in detail. We report on a case of galactosialidosis, the diagnosis of which was suggested by bilateral macular cherry-red spots, which is an indication of lysosomal storage disease. In this case, retinal and systemic dysfunctions were mild. Genetic studies revealed an abnormality of relevant protective proteins, and thus a definitive diagnosis was made. The patient was a 35-year-old man who had blurred vision from young age, but he did not seek any therapy due to good visual acuity. He visited a local clinic after the blurred vision in the left eye worsened and was referred to us for bilateral macular cherry-red spots. He had no family history of note. We observed fine grayish-white deposits in the corneal stroma and fine opacity of the lens. Optical coherence tomography showed a hyperreflective region and a thick bilateral retinal ganglion cell layer. Goldmann perimetry showed focal loss of sensitivity. There was almost no functional decline noted on multifocal electroretinography. Lysosomal storage disease was suspected due to corneal clouding and macular cherry-red spots, and so further evaluation was performed. Though neurological abnormality was mild, we made a diagnosis of galactosialidosis because of decreased activity of β-galactosidase and sialidase. Genetic studies revealed an abnormality of relevant protective proteins. Since the onset was later in life and clinical symptoms were mild, we expect that the ophthalmological findings will remain stable. Long-term observation is necessary for this case.
    Keywords:  CTSA; Cherry-red spots; Galactosialidosis; Lysosomal storage disease
  11. Mol Ther Methods Clin Dev. 2020 Sep 11. 18 558-570
    Piras G, Montiel-Equihua C, Chan YA, Wantuch S, Stuckey D, Burke D, Prunty H, Phadke R, Chambers D, Partida-Gaytan A, Leon-Rico D, Panchal N, Whitmore K, Calero M, Benedetti S, Santilli G, Thrasher AJ, Gaspar HB.
      Pompe disease is a lysosomal storage disorder caused by malfunctions of the acid alpha-glucosidase (GAA) enzyme with a consequent toxic accumulation of glycogen in cells. Muscle wasting and hypertrophic cardiomyopathy are the most common clinical signs that can lead to cardiac and respiratory failure within the first year of age in the more severe infantile forms. Currently available treatments have significant limitations and are not curative, highlighting a need for the development of alternative therapies. In this study, we investigated the use of a clinically relevant lentiviral vector to deliver systemically GAA through genetic modification of hematopoietic stem and progenitor cells (HSPCs). The overexpression of GAA in human HSPCs did not exert any toxic effect on this cell population, which conserved its stem cell capacity in xenograft experiments. In a murine model of Pompe disease treated at young age, we observed phenotypic correction of heart and muscle function with a significant reduction of glycogen accumulation in tissues after 6 months of treatment. These findings suggest that lentiviral-mediated HSPC gene therapy can be a safe alternative therapy for Pompe disease.
    Keywords:  GAA; Pompe disease; acid alpha-glucosidase; erythroid-specific enhancer; hematopoietic stem cell gene therapy; lentiviral vector; lysosomal storage disorders; personalized medicine
  12. Mol Ther Methods Clin Dev. 2020 Sep 11. 18 607-619
    Yasuda M, Huston MW, Pagant S, Gan L, St Martin S, Sproul S, Richards D, Ballaron S, Hettini K, Ledeboer A, Falese L, Cao L, Lu Y, Holmes MC, Meyer K, Desnick RJ, Wechsler T.
      Fabry disease is an X-linked lysosomal storage disorder caused by mutations in the alpha-galactosidase A (GLA) gene, which encodes the exogalactosyl hydrolase, alpha-galactosidase A (α-Gal A). Deficient α-Gal A activity results in the progressive, systemic accumulation of its substrates, globotriaosylceramide (Gb3) and globotriaosylsphingosine (Lyso-Gb3), leading to renal, cardiac, and/or cerebrovascular disease and early demise. The current standard treatment for Fabry disease is enzyme replacement therapy, which necessitates lifelong biweekly infusions of recombinant enzyme. A more long-lasting treatment would benefit Fabry patients. Here, a gene therapy approach using an episomal adeno-associated viral 2/6 (AAV2/6) vector that encodes the human GLA cDNA driven by a liver-specific expression cassette was evaluated in a Fabry mouse model that lacks α-Gal A activity and progressively accumulates Gb3 and Lyso-Gb3 in plasma and tissues. A detailed 3-month pharmacology and toxicology study showed that administration of a clinical-scale-manufactured AAV2/6 vector resulted in markedly increased plasma and tissue α-Gal A activities, and essentially normalized Gb3 and Lyso-Gb3 at key sites of pathology. Further optimization of vector design identified the clinical lead vector, ST-920, which produced several-fold higher plasma and tissue α-Gal A activity levels with a good safety profile. Together, these studies provide the basis for the clinical development of ST-920.
    Keywords:  AAV gene therapy; Fabry; GLA; GLAKO mouse; alpha galactosidase A; globotriaosylceramide; preclinical studies
  13. Am J Med Genet C Semin Med Genet. 2020 Aug 07.
    Schiff ER, Varela MD, Robson AG, Pierpoint K, Ba-Abbad R, Nutan S, Zein WM, Ullah E, Huryn LA, Tuupanen S, Mahroo OA, Michaelides M, Burke D, Harvey K, Arno G, Hufnagel RB, Webster AR.
      Pathogenic variants in the gene HGSNAT (heparan-α-glucosaminide N-acetyltransferase) have been reported to underlie two distinct recessive conditions, depending on the specific genotype, mucopolysaccharidosis type IIIC (MPSIIIC)-a severe childhood-onset lysosomal storage disorder, and adult-onset nonsyndromic retinitis pigmentosa (RP). Here we describe the largest cohort to-date of HGSNAT-associated nonsyndromic RP patients, and describe their retinal phenotype, leukocyte enzymatic activity, and likely pathogenic genotypes. We identified biallelic HGSNAT variants in 17 individuals (15 families) as the likely cause of their RP. None showed any other symptoms of MPSIIIC. All had a mild but significant reduction of HGSNAT enzyme activity in leukocytes. The retinal condition was generally of late-onset, showing progressive degeneration of a concentric area of paramacular retina, with preservation but reduced electroretinogram responses. Symptoms, electrophysiology, and imaging suggest the rod photoreceptor to be the cell initially compromised. HGSNAT enzymatic testing was useful in resolving diagnostic dilemmas in compatible patients. We identified seven novel sequence variants [p.(Arg239Cys); p.(Ser296Leu); p.(Phe428Cys); p.(Gly248Ala); p.(Gly418Arg), c.1543-2A>C; c.1708delA], three of which were considered to be retina-disease-specific alleles. The most prevalent retina-disease-specific allele p.(Ala615Thr) was observed heterozygously or homozygously in 8 and 5 individuals respectively (7 and 4 families). Two siblings in one family, while identical for the HGSNAT locus, but discordant for retinal disease, suggest the influence of trans-acting genetic or environmental modifying factors.
    Keywords:  HGSNAT; inherited retinal disease; retinopathy
  14. Pharmaceutics. 2020 Aug 08. pii: E747. [Epub ahead of print]12(8):
    Kubo Y, Yamada M, Konakawa S, Akanuma SI, Hosoya KI.
      Lysosomal trapping at the blood-retinal barrier (BRB) was investigated through quinacrine and fluorescence-labeled verapamil (EFV) uptake. Quinacrine uptake by conditionally immortalized rat retinal capillary endothelial (TR-iBRB2) cells suggested saturable and non-saturable transport processes in the inner BRB. The reduction of quinacrine uptake by bafilomycin A1 suggested quinacrine distribution to the acidic intracellular compartments of the inner BRB, and this notion was also supported in confocal microscopy. In the study using the lysosome-enriched fraction of TR-iBRB2 cells, quinacrine uptake was inhibited by bafilomycin A1, suggesting the lysosomal trapping of quinacrine in the inner BRB. Pyrilamine, clonidine, and nicotine had no effect on quinacrine uptake, suggesting the minor role of lysosomal trapping in their transport across the inner BRB. Bafilomycin A1 had no effect on EFV uptake, and lysosomal trapping driven by the acidic interior pH was suggested as a minor mechanism for EFV transport in the inner BRB. The minor contribution of lysosomal trapping was supported by the difference in inhibitory profiles between EFV and quinacrine uptakes. Similar findings were observed in the outer BRB study with the fraction of conditionally immortalized rat retinal pigment epithelial (RPE-J) cells. These results suggest the usefulness of lysosome-enriched fractions in studying lysosomal trapping at the BRB.
    Keywords:  blood-retinal barrier; cationic drug; lysosomal trapping; transport
  15. Mediators Inflamm. 2020 ;2020 5437175
    Du J, Li G, Hua M, Han J, Hao Y, Zeng H, Li A, Kong Y.
      The cecal ligation and perforation (CLP) model is the gold standard for the polymicrobial sepsis. In the CLP mice, the myeloid cells play an important role in septic shock. The phenotypes and the activation state of the macrophage and neutrophil correlate with their metabolism. In the present study, we generated the specific myeloid deletion of PDK1 and mTOR mice, which was the important regulator of metabolic signaling. We found that the deletion of PDK1 in the myeloid cells could aggravate the early septic shock in the CLP mice, as well as the deletion of mTORC1 and mTORC2. Moreover, PDK1 deletion attenuated the inflammation induced by LPS in the late stage on CLP mice, which was exacerbated in mTORC1 and mTORC2 knockout mice. Both PDK1 and mTORC1/2 could not only regulate the cellular metabolism but also play important roles on the myeloid cells in the secondary stimulation of sepsis. The present study will provide a theoretical prospect for the therapy of the septic shock in different stages.
  16. J Vet Intern Med. 2020 Aug 12.
    Faller KME, Ridyard AE, Gutierrez-Quintana R, Rupp A, Kun-Rodrigues C, Orme T, Tylee KL, Church HJ, Guerreiro R, Bras J.
      BACKGROUND: Mucopolysaccharidosis type I (MPS-I) is a lysosomal storage disorder caused by a deficiency of the enzyme α-l-iduronidase, leading to accumulation of undegraded dermatan and heparan sulfates in the cells and secondary multiorgan dysfunction. In humans, depending upon the nature of the underlying mutation(s) in the IDUA gene, the condition presents with a spectrum of clinical severity.OBJECTIVES: To characterize the clinical and biochemical phenotypes, and the genotype of a family of Golden Retriever dogs.
    ANIMALS: Two affected siblings and 11 related dogs.
    METHODS: Family study. Urine metabolic screening and leucocyte lysosomal enzyme activity assays were performed for biochemical characterization. Whole genome sequencing was used to identify the causal mutation.
    RESULTS: The clinical signs shown by the proband resemble the human attenuated form of the disease, with a dysmorphic appearance, musculoskeletal, ocular and cardiac defects, and survival to adulthood. Urinary metabolic studies identified high levels of dermatan sulfate, heparan sulfate, and heparin. Lysosomal enzyme activities demonstrated deficiency in α-l-iduronidase activity in leucocytes. Genome sequencing revealed a novel homozygous deletion of 287 bp resulting in full deletion of exon 10 of the IDUA gene (NC_006585.3(NM_001313883.1):c.1400-76_1521+89del). Treatment with pentosan polyphosphate improved the clinical signs until euthanasia at 4.5 years.
    CONCLUSION AND CLINICAL IMPORTANCE: Analysis of the genotype/phenotype correlation in this dog family suggests that dogs with MPS-I could have a less severe phenotype than humans, even in the presence of severe mutations. Treatment with pentosan polyphosphate should be considered in dogs with MPS-I.
    Keywords:  Hurler; Scheie; iduronidase; lysosomal storage disease
  17. Oxid Med Cell Longev. 2020 ;2020 4908162
    Fernando R, Castro JP, Flore T, Deubel S, Grune T, Ott C.
      The skeletal muscle plays an important role in maintaining whole-body mechanics, metabolic homeostasis, and interorgan crosstalk. However, during aging, functional and structural changes such as fiber integrity loss and atrophy can occur across different species. A commonly observed hallmark of aged skeletal muscle is the accumulation of oxidatively modified proteins and protein aggregates which point to an imbalance in proteostasis systems such as degradation machineries. Recently, we showed that the ubiquitin-proteasomal system was impaired. Specifically, the proteasomal activity, which was declining in aged M. soleus (SOL) and M. extensor digitorum longus (EDL). Therefore, in order to understand whether another proteolytic system would compensate the decline in proteasomal activity, we aimed to investigate age-related changes in the autophagy-lysosomal system (ALS) in SOL, mostly consisting of slow-twitch fibers, and EDL, mainly composed of fast-twitch fibers, from young (4 months) and old (25 months) C57BL/6JRj mice. Here, we focused on changes in the content of modified proteins and the ALS. Our results show that aged SOL and EDL display high levels of protein modifications, particularly in old SOL. While autophagy machinery appears to be functional, lysosomal activity declines gradually in aged SOL. In contrast, in old EDL, the ALS seems to be affected, demonstrated by an increased level of key autophagy-related proteins, which are known to accumulate when their delivery or degradation is impaired. In fact, lysosomal activity was significantly decreased in old EDL. Results presented herein suggest that the ALS can compensate the high levels of modified proteins in the more oxidative muscle, SOL, while EDL seems to be more prone to ALS age-related alterations.
  18. Genes (Basel). 2020 Aug 06. pii: E899. [Epub ahead of print]11(8):
    Grissom JH, Segarra VA, Chi RJ.
      Saccharomyces cerevisiae is one of the best model organisms for the study of endocytic membrane trafficking. While studies in mammalian cells have characterized the temporal and morphological features of the endocytic pathway, studies in budding yeast have led the way in the analysis of the endosomal trafficking machinery components and their functions. Eukaryotic endomembrane systems were thought to be highly conserved from yeast to mammals, with the fusion of plasma membrane-derived vesicles to the early or recycling endosome being a common feature. Upon endosome maturation, cargos are then sorted for reuse or degraded via the endo-lysosomal (endo-vacuolar in yeast) pathway. However, recent studies have shown that budding yeast has a minimal endomembrane system that is fundamentally different from that of mammalian cells, with plasma membrane-derived vesicles fusing directly to a trans-Golgi compartment which acts as an early endosome. Thus, the Golgi, rather than the endosome, acts as the primary acceptor of endocytic vesicles, sorting cargo to pre-vacuolar endosomes for degradation. The field must now integrate these new findings into a broader understanding of the endomembrane system across eukaryotes. This article synthesizes what we know about the machinery mediating endocytic membrane fusion with this new model for yeast endomembrane function.
    Keywords:  Golgi; SNARE; clathrin; endocytosis; membrane fusion; membrane trafficking; yeast
  19. Mol Genet Genomic Med. 2020 Aug 11. e1371
    Fischetto R, Palladino V, Mancardi MM, Giacomini T, Palladino S, Gaeta A, Di Rocco M, Zampini L, Lassandro G, Favia V, Tripaldi ME, Strisciuglio P, Romano A, Severino M, Morrone A, Giordano P.
      BACKGROUND: In GM1 gangliosidosis the lack of function of β-galactosidase results in an accumulation of GM1 ganglioside and related glycoconjugates in visceral organs, and particularly in the central nervous system, leading to severe disability and premature death. In the type 2 form of the disease, early intervention would be important to avoid precocious complications. To date, there are no effective therapeutic options in preventing progressive neurological deterioration. Substrate reduction therapy with Miglustat, a N-alkylated sugar that inhibits the enzyme glucosylceramide synthase, has been proposed for the treatment of several lysosomal storage disorders such as Gaucher type 1 and Niemann Pick Type C diseases. However, data on Miglustat therapy in patients with GM1 gangliosidosis are still scarce.METHODS: We report here the results of Miglustat administration in four Italian children (average age: 55 months, range 20-125) affected by GM1 gangliosidosis type 2 treated in three different Italian pediatric hospitals specialized in metabolic diseases.
    CONCLUSION: This treatment was safe and relatively well tolerated by all patients, with stabilization and/or slowing down of the neurological progression in three subjects.
    Keywords:  GM1 gangliosidosis; Miglustat; pediatric
  20. Biochimie. 2020 Aug 10. pii: S0300-9084(20)30172-3. [Epub ahead of print]
    Sakai H, Murakami C, Usuki T, Lu Q, Matsumoto KI, Urano T, Sakane F.
      Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to produce phosphatidic acid (PA). The η isozyme of DGK is abundantly expressed in C2C12 myoblasts. However, the role of DGKη in skeletal muscle cells remains unknown. In the present study, we showed that DGKη was downregulated at an early stage of myogenic differentiation. The knockdown of DGKη by siRNAs significantly inhibited C2C12 myoblast proliferation but did not inhibit differentiation. Moreover, the suppression of DGKη expression decreased the expression levels of mammalian target of rapamycin (mTOR), which is a key regulator of cell proliferation, and fatty acid synthase (FASN), which catalyzes the de novo synthesis of fatty acids for cell proliferation and is transcriptionally regulated via mTOR signaling. Furthermore, the knockdown of mTOR or raptor, which is a component of mTOR complex 1 (mTORC1), decreased the amount of FASN. These results indicate that DGKη regulates myoblast proliferation through the mTOR (mTORC1)-FASN pathway. Interestingly, the knockdown of mTOR reduced the expression levels of DGKη, implying mutual regulation between DGKη and mTOR. In DGKη-knockdown myoblasts, C30-C36-PA species, mTOR activators, were decreased, suggesting that the modulation of mTOR activity through these PA species also plays an important role in myoblast proliferation.
    Keywords:  Diacylglycerol kinase; Fatty acid synthase; Myoblast proliferation; Skeletal muscle; mTOR
  21. PLoS Genet. 2020 Aug 10. 16(8): e1008966
    Cools M, Lissoir S, Bodo E, Ulloa-Calzonzin J, DeLuna A, Georis I, André B.
      The vacuole of the yeast Saccharomyces cerevisiae plays an important role in nutrient storage. Arginine, in particular, accumulates in the vacuole of nitrogen-replete cells and is mobilized to the cytosol under nitrogen starvation. The arginine import and export systems involved remain poorly characterized, however. Furthermore, how their activity is coordinated by nitrogen remains unknown. Here we characterize Vsb1 as a novel vacuolar membrane protein of the APC (amino acid-polyamine-organocation) transporter superfamily which, in nitrogen-replete cells, is essential to active uptake and storage of arginine into the vacuole. A shift to nitrogen starvation causes apparent inhibition of Vsb1-dependent activity and mobilization of stored vacuolar arginine to the cytosol. We further show that this arginine export involves Ypq2, a vacuolar protein homologous to the human lysosomal cationic amino acid exporter PQLC2 and whose activity is detected only in nitrogen-starved cells. Our study unravels the main arginine import and export systems of the yeast vacuole and suggests that they are inversely regulated by nitrogen.
  22. Anal Chem. 2020 Jul 28.
    Khaledi H, Gelb MH.
      The mucopolysaccharidoses (MPSs) are a class of inborn errors of metabolism caused by deficiency of each of the enzymes involved in the lysosomal degradation of mucopolysaccharides. Newborn screening panels worldwide have been recently expanded to include one or more MPS disorders as treatments are available and are most efficacious if initiated early in life. Here we report the first multiplex assay of 10 enzymatic activities in dried blood spots and fibroblast lysates that allow newborn screening and diagnosis of all MPS disorders except the ultra-rare MPS-IX. The assay consists of incubation of enzyme-specific substrates with dried blood spot punches or fibroblast lysate followed by quantification of enzymatic products using liquid chromatography-tandem mass spectrometry (LC-MS/MS) together with internal standards. Assay of all MPS enzymes using fluorimetric or other methods has not been possible. The steps of the LC-MS/MS assay are sufficiently simple and rapid to be used in newborn screening and diagnostic laboratories. Assays showed acceptable precision, and enzymatic activities measured in confirmed MPS samples are well below the reference range.
  23. Oxid Med Cell Longev. 2020 ;2020 5296341
    Abokyi S, Shan SW, To CH, Chan HH, Tse DY.
      Trehalose is a natural dietary molecule that has shown antiaging and neuroprotective effects in several animal models of neurodegenerative diseases. The role of trehalose in the management of age-related macular degeneration (AMD) is yet to be investigated and whether trehalose could be a remedy for the treatment of diseases linked to oxidative stress and NRF2 dysregulation. Here, we showed that incubation of human retinal pigment epithelial (RPE) cells with trehalose enhanced the mRNA and protein expressions of TFEB, autophagy genes ATG5 and ATG7, as well as protein expressions of macroautophagy markers, LC3B and p62/SQTM1, and the chaperone-mediated autophagy (CMA) receptor LAMP2. Cathepsin D, a hydrolytic lysosomal enzyme, was also increased by trehalose, indicating higher proteolytic activity. Moreover, trehalose upregulated autophagy flux evident by an increase in the endogenous LC3B level, and accumulation of GFP-LC3B puncta and free GFP fragments in GFP-LC3 - expressing cells in the presence of chloroquine. In addition, the mRNA levels of key molecular targets implicated in RPE damage and AMD, such as vascular endothelial growth factor- (VEGF-) A and heat shock protein 27 (HSP27), were downregulated, whereas NRF2 was upregulated by trehalose. Subsequently, we mimicked in vitro AMD conditions using hydroquinone (HQ) as the oxidative insult on RPE cells and evaluated the cytoprotective effect of trehalose compared to vehicle treatment. HQ depleted NRF2, increased oxidative stress, and reduced the viability of cells, while trehalose pretreatment protected against HQ-induced toxicity. The cytoprotection by trehalose was dependent on autophagy but not NRF2 activation, since autophagy inhibition by shRNA knockdown of ATG5 led to a loss of the protective effect. The results support the transcriptional upregulation of TFEB and autophagy by trehalose and its protection against HQ-induced oxidative damage in RPE cells. Further investigation is, therefore, warranted into the therapeutic value of trehalose in alleviating AMD and retinal diseases associated with impaired NRF2 antioxidant defense.
  24. Sci Adv. 2020 Jul;6(31): eabb8725
    Xu F, Li X, Huang X, Pan J, Wang Y, Zhou S.
      Autophagy is involved in the occurrence and development of tumors. Here, a pH-responsive polymersome codelivering hydroxychloroquine (HCQ) and tunicamycin (Tuni) drugs is developed to simultaneously induce endoplasmic reticulum (ER) stress and autophagic flux blockade for achieving an antitumor effect and inhibiting tumor metastasis. The pH response of poly(β-amino ester) and HCQ synergistically deacidifies the lysosomes, thereby blocking the fusion of autophagosomes and lysosomes and lastly blocking autophagic flux. The function mechanism of regulating autophagy was systematically investigated on orthotopic luciferase gene-transfected, 4T1 tumor-bearing BALB/c mice through Western blot and immunohistochemistry analyses. The Tuni triggers ER stress to regulate the PERK/Akt signaling pathway to increase the autophagic level. The "autophagic stress" generated by triggering ER stress-induced autophagy and blocking autophagic flux is effective against tumors. The reduced expression of matrix metalloproteinase-2 due to ER stress and reduced focal adhesions turnover due to the blockade of autophagic flux synergistically inhibit tumor metastasis.
  25. Autophagy. 2020 Aug 10. 1-3
    Herpin A, Lescat L, Bobe J, Jenny A, Seiliez I.
      Chaperone-mediated autophagy (CMA), as one of the main pathways of lysosomal catabolism, plays essential roles for the maintenance of cellular homeostasis. To date, the absence of any identifiable LAMP2A - the necessary and limiting protein required for CMA - in non-tetrapod lineages, led to the paradigm that this cellular process was restricted to mammals and birds. The recent findings of Lescat et al., demonstrating the existence of a CMA activity in fish, now reshuffle the cards regarding how the entire evolution of CMA function should be considered and appreciated across metazoans. Hence, beyond challenging the current tetrapod-centered accepted view, the work of Lescat et al. tackles the possibility - or the compelling need - of using complementary and powerful genetic models, such as zebrafish or medaka, for studying this fundamental function from an evolutionary perspective.
    Keywords:   Chaperone-mediated autophagy; CMA; Lamp2a; evolution; fish; medaka; zebrafish
  26. Sci Rep. 2020 Aug 13. 10(1): 13688
    Telpoukhovskaia MA, Liu K, Sayed FA, Etchegaray JI, Xie M, Zhan L, Li Y, Zhou Y, Le D, Bahr BA, Bogyo M, Ding S, Gan L.
      Patients with frontotemporal dementia (FTD) resulting from granulin (GRN) haploinsufficiency have reduced levels of progranulin and exhibit dysregulation in inflammatory and lysosomal networks. Microglia produce high levels of progranulin, and reduction of progranulin in microglia alone is sufficient to recapitulate inflammation, lysosomal dysfunction, and hyperproliferation in a cell-autonomous manner. Therefore, targeting microglial dysfunction caused by progranulin insufficiency represents a potential therapeutic strategy to manage neurodegeneration in FTD. Limitations of current progranulin-enhancing strategies necessitate the discovery of new targets. To identify compounds that can reverse microglial defects in Grn-deficient mouse microglia, we performed a compound screen coupled with high throughput sequencing to assess key transcriptional changes in inflammatory and lysosomal pathways. Positive hits from this initial screen were then further narrowed down based on their ability to rescue cathepsin activity, a critical biochemical readout of lysosomal capacity. The screen identified nor-binaltorphimine dihydrochloride (nor-BNI) and dibutyryl-cAMP, sodium salt (DB-cAMP) as two phenotypic modulators of progranulin deficiency. In addition, nor-BNI and DB-cAMP also rescued cell cycle abnormalities in progranulin-deficient cells. These data highlight the potential of a transcription-based platform for drug screening, and advance two novel lead compounds for FTD.
  27. Front Cell Dev Biol. 2020 ;8 460
    Li Y, Chen X, Xiong Q, Chen Y, Zhao H, Tahir M, Song J, Zhou B, Wang J.
      Autophagy starts with the initiation and nucleation of isolation membranes, which further expand and seal to form autophagosomes. The regulation of isolation membrane closure remains poorly understood. CK1δ is a member of the casein kinase I family of serine/threonine specific kinases. Although CK1δ is reported to be involved in various cellular processes, its role in autophagy is unknown. Here, we show that CK1δ regulates the progression of autophagy from the formation of isolation membranes to autophagosome closure, and is essential for macroautophagy. CK1δ depletion results in impaired autophagy flux and the accumulation of unsealed isolation membranes. The association of LC3 with ATG9A, ATG14L, and ATG16L1 was found to be increased in CK1δ-depleted cells. The role of CK1δ in autophagosome completion appears to be conserved between yeasts and humans. Our data reveal a key role for CK1δ/Hrr25 in autophagosome completion.
    Keywords:  CK1δ; Hrr25; autophagosome closure; autophagy; isolation membrane
  28. Cells. 2020 Aug 08. pii: E1858. [Epub ahead of print]9(8):
    Jha S, Holmberg CI.
      The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Several studies have reported on the interplay between the UPS and ALP, however it remains largely unknown how compromised autophagy affects UPS function in vivo. Here, we have studied the crosstalk between the UPS and ALP by investigating the tissue-specific effect of autophagy genes on the UPS at an organismal level. Using transgenic Caenorhabditis elegans expressing fluorescent UPS reporters, we show that the downregulation of the autophagy genes lgg-1 and lgg-2 (ATG8/LC3/GABARAP), bec-1 (BECLIN1), atg-7 (ATG7) and epg-5 (mEPG5) by RNAi decreases proteasomal degradation, concomitant with the accumulation of polyubiquitinated proteasomal substrates in a tissue-specific manner. For some of these genes, the changes in proteasomal degradation occur without a detectable alteration in proteasome tissue expression levels. In addition, the lgg-1 RNAi-induced reduction in proteasome activity in intestinal cells is not dependent on sqst-1/p62 accumulation. Our results illustrate that compromised autophagy can affect UPS in a tissue-specific manner, and demonstrate that UPS does not function as a direct compensatory mechanism in an animal. Further, a more profound understanding of the multilayered crosstalk between UPS and ALP can facilitate the development of therapeutic options for various disorders linked to dysfunction in proteostasis.
    Keywords:  C. elegans; autophagy; crosstalk; tissue specificity; ubiquitin–proteasome system
  29. Int J Clin Pract. 2020 Aug 10. e13672
    Reynolds TM, Tylee K, Booth K, Wierzbicki AS, .
      AIMS: Lysosomal α-galactosidase A deficiency (Fabry disease (FD)) was considered an X-linked recessive disorder but is now viewed as a variable penetrance dominant trait. The prevalence of FD is 1 in 40000-117,000 but ascertainment of late-onset cases and degree of female penetrance makes this unclear. Its prevalence in the general population, especially in patients with abnormal renal function is unclear. This study attempted to identify the prevalence of FD in patients with abnormal renal function results from laboratory databases.METHODS: Electronic laboratory databases were interrogated to identify from clinical biochemistry records patients with a phenotype of reduced estimated glomerular filtration rate categorised by age on one occasion or more over a 3-year time interval. Patients were recalled and a dried blood spot sample was collected for determination of α-galactosidase A activity by fluorimetric enzyme assay in men and mass spectrometry assays of α-galactosidase A and lyso-globotriaosylceramide (lyso-GL-3) concentrations in women.
    RESULTS: Samples were obtained from 1084 patients identified with reduced renal function. No cases of FD were identified in 505 men. From 579 women one subject with reduced α-galactosidase activity (1.5µmol/l/hr) and increased Lyso-GL-3 (5.5ng/ml) was identified and shown to be heterozygous for a likely FD pathogenic variant (GLA c.898C>T; p.L300F; Leu300Phe). It was later confirmed she was a relative of a known affected patient.
    CONCLUSIONS: Pathology databases hold routine information that can be used to identify patients with inherited errors of metabolism. Biochemical screening using reduced eGFR alone has a low yield for unidentified cases of Fabry Disease.
    Keywords:  Fabry Disease; Inherited Metabolic Disease; Screening
  30. Sci Rep. 2020 Aug 07. 10(1): 13334
    Jiang D, Lee H, Pardridge WM.
      Niemann-Pick C1 (NPC1) is a lysosomal cholesterol storage disorder, that severely affects the brain, and is caused by mutations in the NPC1 gene, which encodes an intracellular membrane transporter of non-esterified cholesterol. Therapeutic options for NPC1 are few, and classical enzyme replacement therapy with the recombinant protein is not possible as the NPC1 gene product is an insoluble membrane protein, which increases the need for development of gene therapy for NPC1. While viral based gene therapy is under development, it is important to investigate alternative approaches to brain gene therapy without viral vectors. The present work develops a plasmid DNA approach to gene therapy of NPC1 using Trojan horse liposomes (THLs), wherein the plasmid DNA is encapsulated in 100 nm pegylated liposomes, which are targeted to organs with a monoclonal antibody against the mouse transferrin receptor. THLs were encapsulated with a 8.0 kb plasmid DNA encoding the 3.9 kb human NPC1 open reading frame, under the influence of a 1.5 kb platelet derived growth factor B (PDGFB) promoter. THLs were administered weekly beginning at 6-7 weeks in the NPC1-/- null mouse, and delivery of the plasmid DNA, and NPC1 mRNA expression in brain, spleen, and liver were confirmed by quantitative PCR. THL treatment reduced tissue inclusion bodies in brain, and peripheral organs, but did not prolong lifespan in these mice. The work suggests that early treatment after birth may be required to reverse this disease model with NPC1 gene replacement therapy.
  31. Dev Cell. 2020 Aug 06. pii: S1534-5807(20)30579-7. [Epub ahead of print]
    Miao ZF, Lewis MA, Cho CJ, Adkins-Threats M, Park D, Brown JW, Sun JX, Burclaff JR, Kennedy S, Lu J, Mahar M, Vietor I, Huber LA, Davidson NO, Cavalli V, Rubin DC, Wang ZN, Mills JC.
      Differentiated cells can re-enter the cell cycle to repair tissue damage via a series of discrete morphological and molecular stages coordinated by the cellular energetics regulator mTORC1. We previously proposed the term "paligenosis" to describe this conserved cellular regeneration program. Here, we detail a molecular network regulating mTORC1 during paligenosis in both mouse pancreatic acinar and gastric chief cells. DDIT4 initially suppresses mTORC1 to induce autodegradation of differentiated cell components and damaged organelles. Later in paligenosis, IFRD1 suppresses p53 accumulation. Ifrd1-/- cells do not complete paligenosis because persistent p53 prevents mTORC1 reactivation and cell proliferation. Ddit4-/- cells never suppress mTORC1 and bypass the IFRD1 checkpoint on proliferation. Previous reports and our current data implicate DDIT4/IFRD1 in governing paligenosis in multiple organs and species. Thus, we propose that an evolutionarily conserved, dedicated molecular network has evolved to allow differentiated cells to re-enter the cell cycle (i.e., undergo paligenosis) after tissue injury. VIDEO ABSTRACT.
    Keywords:  ADM; Drosophila; SPEM; Schizosaccharomyces pombe; acinar-ductal metaplasia; regeneration; spasmolytic polypeptide-expressing metaplasia
  32. Blood. 2020 Aug 12. pii: blood.2019004713. [Epub ahead of print]
    Fontan Gabas L, Goldstein RL, Casalena G, Durant M, Teater MR, Wilson J, Phillip JM, Xia M, Shah S, Us I, Shinglot H, Singh A, Inghirami GG, Melnick A.
      MALT1 inhibitors are promising therapeutic agents for B-cell lymphomas dependent on constitutive or aberrant signaling pathways. However, a potential limitation for signal transduction targeted therapies is the occurrence of feedback mechanisms that enable escape from the full impact of such drugs. Here, we used a functional genomics screen in ABC-DLBCL cells treated with a small molecule irreversible inhibitor of MALT1 to identify genes that might confer resistance or enhance the activity of MALT1 inhibition. We find that loss of BCR and PI3K activating proteins enhanced sensitivity, while loss of negative regulators of these pathways (e.g. TRAF2, TNFAIP3) promoted resistance. These findings were validated by knockdown of individual genes and a combinatorial drug screen focused on BCR and PI3K pathway targeting drugs. Among these, the most potent combinatorial effect was observed with PI3Kd inhibitors against ABC-DLBCLs in vitro and in vivo, but which led to an adaptive increase in p-S6 and eventual disease progression. Along these lines, MALT1 inhibition promoted increased MTORC1 activity and phosphorylation of S6K1-T389 and S6-S235/6, an effect that was only partially blocked by PI3Kd inhibition in vitro and in vivo. In contrast, simultaneous inhibition of MALT1 and MTORC1 prevented S6 phosphorylation, yielded potent activity against DLBCL cell lines and primary patient specimens, and resulted in more profound tumor regression and significantly improved survival of ABC-DLBCLs in vivo as compared to PI3K inhibitors. These findings provide a basis for maximal therapeutic impact of MALT1 inhibitors in the clinic, by disrupting feedback mechanisms that might otherwise limit their efficacy.
  33. J Clin Lipidol. 2020 Jul 11. pii: S1933-2874(20)30213-0. [Epub ahead of print]
    Nidorf SM, Fiolet A, Abela GS.
      Reducing the residual risk of cardiovascular (CV) events in patients with atherosclerosis continues to be a challenge. Thus, understanding how cholesterol spontaneously self assembles into metastable structures that evolve into flat plate cholesterol crystals (CCs) in atherosclerotic plaque, and why they fundamentally change the nature of the disease provides a paradigm for the development of additional therapies. Specifically, flat plate CCs that form within lysosomes of macrophages may become large enough to disrupt lysosomal membranes leading to the release of cathepsin B and CCs fragments directly into the cytosol. In the cytosol, the surface of flat plate CCs can be recognized by complosome that together with cathepsin B may trigger pyrin domain-containing inflammasome. In addition, flat plate CCs in the cytosol may trigger caspase 8 initiating apoptosis. In the interstitial space, the surface of flat plate CCs can be recognized by complement and receptors on proinflammatory cells, and larger fragments can induce "frustrated phagocytosis" that together perpetuate inflammatory injury. In addition, rapid transition of metastable CCs into large flat plate CCs within lipid rich plaques can lead to traumatic injury by expansion of the plaque's necrotic core causing plaque disruption or rupture that may precipitate further inflammation. Other crystalloids in plaque including monosodium urate and calcium phosphate crystals can augment these processes. Thus, therapies that further limit the deposition of cholesterol in the vascular bed, slow the formation of flat plate CCs and inhibit crystal-induced inflammation may lead to further reduce CV risk in patients with established CV disease.
    Keywords:  Acute coronary syndromes; Atherosclerosis; Lipids and cholesterol
  34. ACS Chem Biol. 2020 Aug 13.
    Sahni A, Qian Z, Pei D.
      Cell-penetrating peptides (CPPs) are capable of delivering membrane-impermeable cargoes (including small molecules, peptides, proteins, nucleic acids, and nanoparticles) into the cytosol of mammalian cells and have the potential to revolu-tionize biomedical research and drug discovery. However, the mechanism of action of CPPs has remained poorly under-stood, especially how they escape from the endosome into the cytosol following endocytic uptake. We show herein that CPPs exit the endosome by inducing budding and collapse of CPP-enriched vesicles from the endosomal membrane. This mechanism provides a theoretical basis for designing CPPs and other delivery vehicles of improved efficiencies.
  35. Clin Nutr. 2020 Jul 31. pii: S0261-5614(20)30380-0. [Epub ahead of print]
    Moinard C, Fontaine E.
      Muscle protein synthesis (MPS) is a complex and finely-regulated mechanism that plays a key role in muscle homeostasis. Amino acid bioavailability is widely considered a major driver of MPS regulation via mTOR pathway activation. However, recent results suggest that amino acid bioavailability affects cellular energy status. Whatever the tool used to modulate energy status (amino acid depletion or mild mitochondrial uncoupling), a decrease in cellular energy status decreases MPS, without necessarily involving the mTOR pathway. Here we propose that energy status directly regulates one or several energy-consuming step(s) during MPS. This new paradigm modifies our vision of protein metabolism and raises prospects for new advances in therapeutics.
    Keywords:  ATP; Amino acids; Energy status; Malnutrition; Muscle protein synthesis
  36. Mol Genet Genomic Med. 2020 Aug 14. e1454
    Luo Y, Wu D, Shen M.
      OBJECTIVE: Fabry disease (FD) is a rare X-linked lysosomal storage disorder due to the absent or deficient activity of lysosomal hydrolase a-galactosidase A (α-Gal A), which leads to the accumulation of its substrates in various organs and tissues. Classic clinical manifestations include angiokeratomas, proteinuria, renal failure, neuropathic pain, and left ventricular hypertrophy. Fever is one of the rare symptoms that may occur during FD.METHODS: Three Chinese Han patients with FD referred to Peking Union Medical College Hospital were reported. The complete medical records were established, and detailed data were collected. Whole-exome sequencing by next-generation sequencing and α-Gal A enzyme activity assay were performed to confirm the diagnosis.
    RESULTS: These three patients all presented with recurrent fever of unknown origin initially, accompanied with arthralgia/arthritis and other symptoms. We identified two known variants in the GLA gene, c.1176_1179delGAAG and c.782G>A (p.G261D), and a novel variant c.440G>A (p.G147E) which is likely pathogenic in our patient.
    CONCLUSIONS: FD should be considered as a rare cause of recurrent fever of unknown origin. The coexistence of gene variants related to systemic autoinflammatory diseases may make the clinical phenotypes of FD more complex and prone to recurrent fever.
    Keywords:  Fabry disease; fever; fever of unknown origin
  37. Ther Adv Med Oncol. 2020 ;12 1758835920940939
    du Rusquec P, Blonz C, Frenel JS, Campone M.
      Recently many therapeutic classes have emerged in advanced hormone receptor-positive breast cancer, which is the leading cause of cancer death in women. In absence of visceral crisis, treatment relies on endocrine therapy combined with cyclin dependent kinase 4 and 6 inhibitor. Many mechanisms lead to resistance to endocrine therapy, including the activation of intracellular signaling pathways critical for cell survival. Approximately 70% of breast tumors harbor an alteration in the phosphoinositide 3 kinase (PI3K)/Akt pathway, leading to its hyper activation. This pathway is involved in the regulation of growth, proliferation and cell survival as well as in angiogenesis and is consequently a major target in the oncogenesis. An aberrant PIK3CA mutation is a common phenomenon in breast cancer and found in approximately 40% of patients with advanced hormone receptor-positive breast cancer. For the moment, the only positive trials showing a progression free survival benefit in this population are BOLERO-2 (2012), SOLAR-1 (2019), which tested everolimus, a mammalian target of rapamycin inhibitor, and alpelisib, a PI3K inhibitor, and led to their marketing authorization. However, many other inhibitors of this pathway are promising; nevertheless their development is actually limited by toxicity, mainly cutaneous (rash), digestive (diarrhea) and endocrine (diabetes).
    Keywords:  HR positive advanced breast cancer; PI3K/Akt/mTOR inhibitor; PI3K/Akt/mTOR pathway; PIK3 mutations; endocrine resistance
  38. Sci Adv. 2020 Jul;6(31): eaay9131
    Shin SH, Lee JS, Zhang JM, Choi S, Boskovic ZV, Zhao R, Song M, Wang R, Tian J, Lee MH, Kim JH, Jeong M, Lee JH, Petukhov M, Lee SW, Kim SG, Zou L, Byun S.
      Despite considerable efforts, mTOR inhibitors have produced limited success in the clinic. To define the vulnerabilities of mTORC1-addicted cancer cells and to find previously unknown therapeutic targets, we investigated the mechanism of piperlongumine, a small molecule identified in a chemical library screen to specifically target cancer cells with a hyperactive mTORC1 phenotype. Sensitivity to piperlongumine was dependent on its ability to suppress RUVBL1/2-TTT, a complex involved in chromatin remodeling and DNA repair. Cancer cells with high mTORC1 activity are subjected to higher levels of DNA damage stress via c-Myc and displayed an increased dependency on RUVBL1/2 for survival and counteracting genotoxic stress. Examination of clinical cancer tissues also demonstrated that high mTORC1 activity was accompanied by high RUVBL2 expression. Our findings reveal a previously unknown role for RUVBL1/2 in cell survival, where it acts as a functional chaperone to mitigate stress levels induced in the mTORC1-Myc-DNA damage axis.
  39. Front Psychiatry. 2020 ;11 718
    Niemeyer C, Matosin N, Kaul D, Philipsen A, Gassen NC.
      Cathepsins are proteases with functions in cellular homeostasis, lysosomal degradation and autophagy. Their role in the development of neurodegenerative diseases has been extensively studied. It is well established that impairment of proper cathepsin function plays a crucial role in the pathophysiology of neurodegenerative diseases, and in recent years a role for cathepsins in mental disorders has emerged given the involvement of cathepsins in memory function, hyperactivity, and in depression- and anxiety-like behavior. Here we review putative cathepsin functions with a special focus on their role in the pathophysiology of psychiatric diseases. Specifically, cathepsins are crucial for maintaining cellular homeostasis, particularly as part of the autophagy machinery of neural strategies underlying acute stress response. Disruption of cathepsin functions can lead to psychiatric diseases such as major depressive disease (MDD), bipolar disorder, and schizophrenia. Specifically, cathepsins can be excreted via a process called secretory autophagy. Thereby, they are able to regulate extracellular factors such as brain-derived neurotrophic factor and perlecan c-terminal fragment LG3 providing maintenance of neuronal homeostasis and mediating neuronal plasticity in response to acute stress or trauma. In addition, impairment of proper cathepsin function can result in impaired synaptic transmission by compromised recycling and biogenesis of synaptic vesicles. Taken together, further investigations on cathepsin functions and stress response, neuroplasticity, and synaptic transmission will be of great interest in understanding the pathophysiology of psychiatric disorders.
    Keywords:  autophagy; cathepsins; memory function; neuroinflammation; neuronal plasticity
  40. Pediatr Endocrinol Rev. 2020 Aug;17(4): 317-326
    Hampe CS, Polgreen LE, Lund TC, McIvor RS.
      Mucopolysaccharidosis type I (MPS I) is a rare autosomal recessive disorder, caused by deficiency of α-L-iduronidase, and consequent accumulation of dermatan and heparan sulfates. Severity of the disease ranges from mild (Scheie) to moderate (Hurler-Scheie) to severe (Hurler or MPS-IH). A prominent clinical manifestation of MPS-IH is dysostosis multiplex, a constellation of skeletal abnormalities. We performed a retrospective review comparing manifestations of dysostosis multiplex in patients presenting with MPSIH and relevant animal models. Dog, cat and mouse models of MPS-IH are extensively studied to better understand the pathology of the disease. While all animal models display certain characteristics of human MPSIH, species-specific manifestations must be considered when evaluating skeletal abnormalities. Moreover, some skeletal abnormalities emerge at species-specific developmental stages, e.g. thoracolumbar kyphosis is an early manifestation in humans, while it appears late in the mouse model. The choice of the appropriate diagnostic test is of importance to avoid misleading conclusions.
    Keywords:  Animal models; Bone remodeling biomarkers; Dysostosis multiplex; Mucopolysaccharidosis type I; α-L-iduronidase
  41. Clin Genet. 2020 Aug 12.
    Naseri N, Sharma M, Velinov M.
      The Neuronal Ceroid Lipofuscinoses (NCLs) are at least 13 distinct progressive neurodegenerative disorders unified by the accumulation of lysosomal auto-fluorescent material called lipofuscin. The only form that occurs via autosomal-dominant inheritance exhibits adult onset and is sometimes referred to as Parry type NCL. The manifestations may include behavioral symptoms followed by seizures, ataxia, dementia, and early death. Mutations in the gene DNAJC5 that codes for the presynaptic co-chaperone cysteine string protein-α (CSPα) were recently reported in sporadic adult-onset cases and in families with dominant inheritance. The mutant CSPα protein may lead to disease progression by both loss and gain of function mechanisms. Iron chelation therapy may be considered as a possible pharmaceutical intervention based on our recent mechanism-based proposal of CSPα oligomerization via ectopic Fe-S cluster-binding, summarized in this review.
    Keywords:  Autosomal Dominant Neuronal Ceroid Lipofuscinosis; CSPα; DNAJC5; Deferiprone; Kufs disease; Parry type NCL
  42. ACS Omega. 2020 Aug 04. 5(30): 19131-19139
    Natarajan N, Shaik A, Thiruvenkatam V.
      Tuberous sclerosis complex (TSC) is a neurological syndrome manifested by non-cancerous tumors in several organs. Mutations in either TSC1 or TSC2 tumor suppressor gene cause the disease. In the cell, TSC1 is known to form a heterodimer with TSC2 because of which an active complex is formed that negatively regulates the mTORC1 activity during cellular stress. Hence, mutation in TSC1 or TSC2 is manifested by excess proliferation of the cells leading to the development of numerous benign tumors. The TSC1 and TSC2 complex is known to interact with several protein-binding partners. One such significant interaction of this complex is with the molecular chaperone HSP70. The role of TSC1 in that interaction is still elusive. Here, we have expressed and purified TSC1 (302-420 residues) in a bacterial expression system and have shown that this region directly interacts with HSP70. We have shown that TSC1 increases the ATPase activity of Escherichia coli DnaK, a HSP70 homologue. On the contrary, TSC1 was found to show inhibitory activity toward human HSP70. Our result suggests that TSC1 (302-420 aa) shows differential interaction between the HSP70 homologues. This points toward the evolutionary significance of chaperoning system and the importance of eukaryotic tetratricopeptide repeat domain interaction motif -EEVD. Our study shows the evidence that TSC1 interacts with HSP70 and has a role to play in the chaperoning activity to maintain cellular homeostasis.
  43. Biochim Biophys Acta Mol Cell Biol Lipids. 2020 Aug 07. pii: S1388-1981(20)30179-7. [Epub ahead of print] 158787
    Abe A, Hiraoka M, Matsuzawa F, Aikawa SI, Niimura Y.
      Side-chain oxysterols produced from cholesterol either enzymatically or non-enzymatically show various bioactivities. Lecithin-cholesterol acyltransferase (LCAT) esterifies the C3-hydroxyl group of these sterols as well as cholesterol. Lysosomal phospholipase A2 (LPLA2) is related to LCAT but does not catalyze esterification of cholesterol. First, esterification of side-chain oxysterols by LPLA2 was investigated using recombinant mouse LPLA2 and dioleoyl-PC/sulfatide/oxysterol liposomes under acidic conditions. TLC and LC-MS/MS showed that the C3 and C27-hydroxyl groups of 27-hydroxycholesterol could be individually esterified by LPLA2 to form a monoester with the C27-hydroxyl preference. Cholesterol did not inhibit this reaction. Also, LPLA2 esterified other side-chain oxysterols. Their esterifications by mouse serum containing LCAT supported the idea that their esterifications by LPLA2 occur at the C3-hydroxyl group. N-acetylsphingosine (NAS) acting as an acyl acceptor in LPLA2 transacylation inhibited the side-chain oxysterol esterification by LPLA2. This suggests a competition between hydroxycholesterol and NAS on the acyl-LPLA2 intermediate formed during the reaction. Raising cationic amphiphilic drug concentration or ionic strength in the reaction mixture evoked a reduction of the side-chain oxysterol esterification by LPLA2. This indicates that the esterification could progress via an interfacial interaction of LPLA2 with the lipid membrane surface through an electrostatic interaction. The docking model of acyl-LPLA2 intermediate and side-chain oxysterol provided new insight to elucidate the transacylation mechanism of sterols by LPLA2. Finally, exogenous 25-hydroxycholesterol esterification within alveolar macrophages prepared from wild-type mice was significantly higher than that from LPLA2 deficient mice. This suggests that there is an esterification pathway of side-chain oxysterols via LPLA2.
    Keywords:  Lecithin-cholesterol acyltransferase; acyl-CoA:choleserol acyltransferase; esterification; lysosomal phospholipase A2; side-chain oxysterols; transacylation
  44. Elife. 2020 Aug 10. pii: e59099. [Epub ahead of print]9
    Shi X, Chang C, Yokom AL, Jensen LE, Hurley JH.
      The selective autophagy pathways of xenophagy and mitophagy are initiated when the adaptor NDP52 recruits the ULK1 complex to autophagic cargo. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to map the membrane and NDP52 binding sites of the ULK1 complex to unique regions of the coiled coil of the FIP200 subunit. Electron microscopy of the full-length ULK1 complex shows that the FIP200 coiled coil projects away from the crescent-shaped FIP200 N-terminal domain dimer. NDP52 allosterically stimulates membrane-binding by FIP200 and the ULK1 complex by promoting a more dynamic conformation of the membrane-binding portion of the FIP200 coiled coil. Giant unilamellar vesicle (GUV) reconstitution confirmed that membrane recruitment by the ULK1 complex is triggered by NDP52 engagement. These data reveal how the allosteric linkage between NDP52 and the ULK1 complex could drive the first membrane recruitment event of phagophore biogenesis in xenophagy and mitophagy.
    Keywords:  biochemistry; cell biology; chemical biology; human
  45. ACS Omega. 2020 Aug 04. 5(30): 19014-19019
    Zhang E, Yu C, Tang H, Li Y, Zuo D, Yang R, Ma C, Miao J, Liu P, Su X.
      Current tumor imaging agents are often limited by their liability to dissipate from tumor tissues. As cell sugar sorting enables exogenous sugars to be delivered into predetermined subcellular locations, we synthesized sialic acid (Sia) derivatives with rhodamine-X conjugated at C-9 (ROXSia), which hitchhikes cell sialic acid sorting to target tumor cell lysosomes, exhibiting pH-independent long-term probe retention in lysosomes. ROXSia gives selective, bright, and endured fluorescence signals in subcutaneous tumors and orthotopic tumors in mice models. These results indicate the potential of ROXSia as a lysosome-targeted optical agent for fluorescence-guided tumor resection.
  46. Int J Mol Sci. 2020 Aug 09. pii: E5708. [Epub ahead of print]21(16):
    Murase D, Kusaka-Kikushima A, Hachiya A, Fullenkamp R, Stepp A, Imai A, Ueno M, Kawabata K, Takahashi Y, Hase T, Ohuchi A, Nakamura S, Yoshimori T.
      Autophagy is a membrane traffic system that provides sustainable degradation of cellular components for homeostasis, and is thus considered to promote health and longevity, though its activity declines with aging. The present findings show deterioration of autophagy in association with premature skin aging. Autophagy flux was successfully determined in skin tissues, which demonstrated significantly decreased autophagy in hyperpigmented skin such as that seen in senile lentigo. Furthermore, an exacerbated decline in autophagy was confirmed in xerotic hyperpigmentation areas, accompanied by severe dehydration and a barrier defect, which showed correlations with skin physiological conditions. The enhancement of autophagy in skin ex vivo ameliorated skin integrity, including pigmentation and epidermal differentiation. The present results indicate that the restoration of autophagy can contribute to improving premature skin aging by various intrinsic and extrinsic factors via the normalization of protein homeostasis.
    Keywords:  aging; autophagy; hyperpigmentation; keratinocyte; melanosome; skin
  47. Differentiation. 2020 Jul - Aug;114:pii: S0301-4681(20)30035-9. [Epub ahead of print]114 58-66
    Tong X, Gu J, Chen M, Wang T, Zou H, Song R, Zhao H, Bian J, Liu Z.
      Osteoclasts are terminally multinucleated cells that are regulated by nuclear factor-activated T cells c1 (NFATc1), and are responsible for bone resorption while the tartrate resistant acid phosphatase (TRAP) enzymes releases into bone resorption lacunae. Furthermore, tumor suppressor p53 is a negative regulator during osteoclastogenesis. Osteoprotegerin (OPG) inhibits osteoclastogenesis and bone resorption by activating autophagy, however, whether p53 is involved in OPG-mediated inhibition of osteoclastogenesis remains unclear. In the current study, OPG could enhance the expression of p53 and tuberin sclerosis complex 2 (TSC2). Moreover, the expression of p53 is regulated by autophagy during OPG-mediated inhibition of osteoclastogenesis. Inhibition of p53 by treated with pifithrin-α (PFTα) causing augments of osteoclastogenesis and bone resorption, also reversed OPG-mediated inhibition of osteoclastogenesis by reducing the expression of TSC2. In addition, knockdown of TSC2 using siRNA could rescue OPG-mediated inhibition of osteoclastogenesis by reducing autophagy, which is manifested by the decrease of the expression of Beclin1 and the phosphorylation of mammalian target of rapamycin (mTOR) and ribosomal protein S6 kinase beta 1 (S6K1, also known as p70S6K). Collectively, p53 plays a critical role during OPG-mediated inhibition of osteoclastogenesis via regulating the TSC2-induced autophagy in vitro.
    Keywords:  OPG; Osteoclastogenesis; PFTα; TSC2; p53
  48. Nat Commun. 2020 Aug 13. 11(1): 4056
    Bosc C, Broin N, Fanjul M, Saland E, Farge T, Courdy C, Batut A, Masoud R, Larrue C, Skuli S, Espagnolle N, Pagès JC, Carrier A, Bost F, Bertrand-Michel J, Tamburini J, Récher C, Bertoli S, Mansat-De Mas V, Manenti S, Sarry JE, Joffre C.
      Autophagy has been associated with oncogenesis with one of its emerging key functions being its contribution to the metabolism of tumors. Therefore, deciphering the mechanisms of how autophagy supports tumor cell metabolism is essential. Here, we demonstrate that the inhibition of autophagy induces an accumulation of lipid droplets (LD) due to a decrease in fatty acid β-oxidation, that leads to a reduction of oxidative phosphorylation (OxPHOS) in acute myeloid leukemia (AML), but not in normal cells. Thus, the autophagic process participates in lipid catabolism that supports OxPHOS in AML cells. Interestingly, the inhibition of OxPHOS leads to LD accumulation with the concomitant inhibition of autophagy. Mechanistically, we show that the disruption of mitochondria-endoplasmic reticulum (ER) contact sites (MERCs) phenocopies OxPHOS inhibition. Altogether, our data establish that mitochondria, through the regulation of MERCs, controls autophagy that, in turn finely tunes lipid degradation to fuel OxPHOS supporting proliferation and growth in leukemia.
  49. Ann Transl Med. 2020 Jul;8(14): 865
    Dong ZY, Wang Q, Lin SP, Chen P, Liu JN, Liu SW, Cai GY, Chen XM, Hong Q.
      Background: Fabry disease (FD) is an X-linked recessive inheritance lysosomal storage disorder due to mutations in the GLA gene leading to deficiency of lysosomal α-galactosidase A (α-Gal A) and has a wide range of clinical presentations. Over 900 GLA gene mutations are currently known and of those most are thought not to be clinically significant, some with doubtful clinical significance, posing diagnostic and prognostic difficulties for the clinician.Methods: Whole-exome sequencing (WES) was performed to detect the mutation in family members with Fabry disease. The function of g.1170C>T mutation was confirmed by dual luciferase system.
    Results: A total of 1,375 variants were found in a Chinese family with FD. A missense variants c.1025C>T (p.Arg342Gln) which have been previously reported in association with FD and g.1170C>T single-nucleotide polymorphism (SNP) in the GLA gene were found in five patients. The g.1170C>T SNP affects transcription of GLA gene, presumably the transcription start site. Female patients only have hypohidrosis and neuropathic pain, while male patients have severe symptoms with simultaneous renal impairment.
    Conclusions: Two simultaneous variants in cis of the GLA gene, c.1025C>T (p.Arg342Gln) and g.1170C>T, were verified in Chinese individuals, and the corresponding clinical symptoms were described. The disease severity in male patients is worse than in female patients. These results may be helpful for genetic counseling, diagnosis and prognosis of patients with FD.
    Keywords:  5'UTR; Fabry disease; GLA gene; mutation; p.Arg342Gln
  50. Am J Transl Res. 2020 ;12(7): 3302-3310
    Zheng S, Liu J, Zhao Z, Song R.
      STAT3/mTOR pathway plays an important role in inflammation, cell growth, and proliferation. However, the role of STAT3/mTOR pathway in chronic kidney injury remains unclear. Folic acid was used to induce kidney injury C57BL/6 mouse model followed by analysis of serum creatinine, renal weight ratio changes, renal pathological changes and STAT3/mTOR pathway changes. Glomerular mesangial cells were divided into control group, model group, STAT3 inhibitor (S3I-201) group followed by analysis of cell proliferation by MTT assay, cell apoptosis by flow cytometry, formation of autophagosomes by electron microscopy, expression of STAT3/mTOR signaling proteins and autophagy proteins LC3II and p62 by Western blot, expression of E-cadherin and Vimentin by immunofluorescence. The serum creatinine and renal weight ratio was increased with obvious lesions and upregulated STAT3 and p-mTOR level. Compared with control group, the difference was statistically significant (P < 0.05). Folic acid-induced injury of mesangial cells showed inhibited cell proliferation, promoted apoptosis, increased LC3II expression, decreased p62 expression, increased autophagic vacuoles and expression of STAT3 and p-mTOR as well as decreased E-cadherin expression and increased Vimentin expression. The difference was statistically significant compared with control group (P < 0.05). All above changes were significantly reversed after treatment with STAT3 inhibitor S3I-201 (P < 0.05). Activated STAT3/mTOR pathway, enhanced autophagy, promoted apoptosis of mesangial cells and inhibited cell proliferation were found in mice with renal injury. Inhibition of STAT3/mTOR activation inhibits autophagy and cell apoptosis and promotes cell proliferation.
    Keywords:  Kidney injury; STAT3/mTOR; apoptosis; autophagy; proliferation
  51. Breast Cancer Res. 2020 Aug 14. 22(1): 89
    O'Brien NA, McDermott MSJ, Conklin D, Luo T, Ayala R, Salgar S, Chau K, DiTomaso E, Babbar N, Su F, Gaither A, Hurvitz SA, Linnartz R, Rose K, Hirawat S, Slamon DJ.
      BACKGROUND: Combined targeting of CDK4/6 and ER is now the standard of care for patients with advanced ER+/HER2- breast cancer. However, acquired resistance to these therapies frequently leads to disease progression. As such, it is critical to identify the mechanisms by which resistance to CDK4/6-based therapies is acquired and also identify therapeutic strategies to overcome resistance.METHODS: In this study, we developed and characterized multiple in vitro and in vivo models of acquired resistance to CDK4/6-based therapies. Resistant models were screened by reverse phase protein array (RPPA) for cell signaling changes that are activated in resistance.
    RESULTS: We show that either a direct loss of Rb or loss of dependence on Rb signaling confers cross-resistance to inhibitors of CDK4/6, while PI3K/mTOR signaling remains activated. Treatment with the p110α-selective PI3K inhibitor, alpelisib (BYL719), completely blocked the progression of acquired CDK4/6 inhibitor-resistant xenografts in the absence of continued CDK4/6 inhibitor treatment in models of both PIK3CA mutant and wild-type ER+/HER2- breast cancer. Triple combination therapy against PI3K:CDK4/6:ER prevented and/or delayed the onset of resistance in treatment-naive ER+/HER2- breast cancer models.
    CONCLUSIONS: These data support the clinical investigation of p110α-selective inhibitors of PI3K, such as alpelisib, in patients with ER+/HER2- breast cancer who have progressed on CDK4/6:ER-based therapies. Our data also support the investigation of PI3K:CDK4/6:ER triple combination therapy to prevent the onset of resistance to the combination of endocrine therapy plus CDK4/6 inhibition.
    Keywords:  Alpelisib; Palbociclib; Translational
  52. J Cell Sci. 2020 Aug 11. pii: jcs.247817. [Epub ahead of print]
    Matsuda S, Kikkawa U, Uda H, Nakashima A.
      In Schizosaccharomyces pombe, a general strategy for survival in response to environmental changes is sexual differentiation, which is triggered by TORC1 inactivation. However, mechanisms of TORC1 regulation in fission yeast remain poorly understood. In this study, we found that Pef1, which is an ortholog of mammalian CDK5, regulates the initiation of sexual differentiation through positive regulation of TORC1 activity. Conversely, deletion of pef1 leads to activation of autophagy and subsequent excessive TORC1 reactivation during the early phases of the nitrogen starvation response. This excessive TORC1 reactivation results in the silencing of the Ste11-Mei2 pathway and mating defects. Additionally, we found that pef1 genetically interacts with tsc1/2 in TORC1 regulation, and physically interacts with three types of cyclins, Clg1, Pas1, and Psl1. The double deletion of clg1 and pas1 promotes activation of autophagy and TORC1 during nitrogen starvation, similar to pef1Δ cells. Overall, our work suggests that Pef1-Clg1 and Pef1-Pas1 complexes regulate initiation of sexual differentiation through control of the TSC-TORC1 pathway and autophagy.
    Keywords:  Autophagy; CDK5; Cyclin; Pef1; Sexual differentiation; TORC1
  53. Int J Mol Sci. 2020 Aug 10. pii: E5715. [Epub ahead of print]21(16):
    Pena-Leon V, Perez-Lois R, Seoane LM.
      Mammalian, or mechanic, target of rapamycin (mTOR) signaling is a crucial factor in the regulation of the energy balance that functions as an energy sensor in the body. The present review explores how the mTOR/S6k intracellular pathway is involved in modulating the production of different signals such as ghrelin and nesfatin-1 in the gastrointestinal tract to regulate food intake and body weight. The role of gastric mTOR signaling in different physiological processes was studied in depth through different genetic models that allow the modulation of mTOR signaling in the stomach and specifically in gastric X/A type cells. It has been described that mTOR signaling in X/A-like gastric cells has a relevant role in the regulation of glucose and lipid homeostasis due to its interaction with different organs such as liver and adipose tissue. These findings highlight possible therapeutic strategies, with the gut-brain axis being one of the most promising targets in the treatment of obesity.
    Keywords:  gastrointestinal tract; ghrelin; homeostasis; mTOR; nesfatin-1; obesity
  54. J Allergy Clin Immunol. 2020 Aug 06. pii: S0091-6749(20)31102-7. [Epub ahead of print]
    Kracht M, Müller-Ladner U, Schmitz ML.
      The NF-κB signaling system, a key regulator of immunological processes, also affects a plethora of metabolic changes associated with inflammation and the immune response. NF-κB-regulating signaling cascades, in concert with NF-κB-mediated transcriptional events control the metabolism at several levels. NF-κB modulates apical components of metabolic processes including metabolic hormones such as insulin and glucagon, the cellular master switches AMPK and mTOR and also numerous metabolic enzymes and their respective regulators. Vice versa, metabolic enzymes and their products also exert multi-level control of NF-κB activity, thereby creating a highly connected regulatory network. These insights have resulted in the identification of the noncanonical IKK kinases IKKɛ and TBK1, which are upregulated by overnutrition, and may therefore be suitable potential therapeutic targets for metabolic syndromes. An inhibitor interfering with the activity of both kinases reduces obesity-related metabolic dysfunctions in mouse models and the encouraging results from a recent clinical trial indicate that targeting these NF-κB pathway components improves glucose homeostasis in a subset of patients with type 2 diabetes.
    Keywords:  NF-κB; cancer; inflammation; metabolism