mTOR-dependent proliferation defect in human ES-derived ... · 1 mTOR-dependent proliferation defect in human ES-derived neural stem cells affected by Myotonic Dystrophy Type1 Jérôme
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mTOR-dependent proliferation defect in human ES-derived neural stem cells affected by Myotonic Dystrophy Type1
Jérôme Alexandre Denisa,d, Morgane Gauthiera*, Latif Rachdib*, Sophie Aubertc*,
Karine Giraud-Triboultc, Pauline Poydenotc, Alexandra Benchouac, Benoite Champonc,
Yves Mauryc, Christine Baldeschia, Raphael Scharfmannb, Geneviève Piétua, Marc
Peschanskia, and Cécile Martinata+
a INSERM/UEVE U-861, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of
Monogenic Diseases, 5 rue Henri Desbruères, 91030 Evry cedex, France b INSERM U-845 Research Center “Growth and Signaling”, Faculty of Medicine Paris
Necker, 156 rue de Vaugirard, 75730 Paris Cedex 15, France c CECS, I-STEM, AFM, Institute for Stem Cell Therapy and Exploration of Monogenic
Diseases, 5 rue Henri Desbruères, 91030 Evry cedex, France d Present address: APHP, Saint Louis-Lariboisière-Fernand Widal University Hospitals,
Lariboisière Hospital, Dept. of Medical Biochemistry and Molecular Biology, Biological
Resource Center, UMRS/INSERM U-942, 2 rue Ambroise Paré 75475 Paris cedex 10,
France.
* Participated equally to the work
+ To whom correspondence should be addressed. Email: [email protected]
Running title: Defect of mTOR signaling pathway in DM1
CES, I-Stem, Evry, France) for technical assistance.
AUTHOR INFORMATION
The authors declare having no competing financial interests.
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Figure Legends Figure 1: Characterization of DM1-NSCs a. Morphology and expression of specific neural markers (SOX2 and Nestin) in NSCs derived from WT-hESC and two DM1-hESC lines. Nuclei are counterstained with DAPI (blue) (Scale bar=20µm). b. Quantification of Nestin expression by FACS c. Quantitative real-time RT-PCR analysis of hESC-derived NSC. RT-PCR levels are presented as the fold change relative to undifferentiated hESC lines after normalization with 18S ribosomal RNA levels. d. Co-detection of mutant RNA with a (CAG)10-Cy3 probe staining and MBNL1 (Muscleblind) protein by immunofluorescence. Nuclei are counterstained using DAPI (blue). The number of foci per nuclei was quantified using ArrayScan and is presented for each cell line as a pie chart. e. Expression of MBNL1 and CUGBP1 proteins measured by western blot analysis after nucleus (N) and cytoplasm (C) subcellular fractioning. Lamin and β-actin were used as loading controls. Figure 2: DM-NSCs exhibit lower proliferative capacity a. Proliferation rate for WT-NSCs and DM1-NSCs detected by quantification of intracytoplasmic ATP during 6 days in culture. b. Analysis of the doubling time for WT-NSCs and DM1-NSCs. c. Decreased number of Ki67 positive DM1-NSCs (VUB03_DM) after 48 hours of culture in comparison with WT-NSCs. The number of Sox2 positive cells was used as a control for NSCs phenotype. Detection and quantification of Ki67 positive cells was done by using the automated ArrayScan Imager. d. Decreased expression of the proliferative marker PCNA in DM1-NSCs in comparison with WT-NSCs, as revealed by real-time RT-PCR analysis. RT-PCR levels are presented as a fold change over control WT-NSCs after normalization with 18S ribosomal RNA levels. e. Decreased expression level of Phosphorylated Rb (S807-811) in DM1-NSCs (VUB03_DM) when compared with WT-NSCs as determined by western blotting. f. Increased expression level of P27 in DM1-NSCs as compared with control NSCs as revealed with western blotting. g. Altered expression of p15ink4B, p21waf1 cell and Cyclin D1 proteins in DM1-NSCs determined by western blot analysis. All western blot analyses were performed using protein extracted from NSCs cells after 48 hours of culture. Band quantification was performed using NIH Image J software. Data are shown as the mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ns: not significant, *: p-value < 0.05, ** p-value < 0.01. *** p-value < 0.001. Figure 3: Induction of autophagy in DM-NSCs a. Detection by phase contrast microscopy of cytoplasmic vacuoles in DM1-NSCs as compared with control after 24 hours of culture. b. Increased expression level of LC3B-II in DM1-NSCs (VUB03_DM) as determined by western blotting after 48 hours of culture c. Nucleofection of DM1-and WT NSCs with a plasmid expressing a LC3-GFP fusion for 48 hours (VUB03_DM and VUB01_WT, respectively). d. Increased number of cells containing LC3-GFP dots in the cytoplasm of DM1-NSCs. EBSS and chloroquine at 50µM were used as a positive control. Data are presented as a percentage of positive cells quantified by the automated ArrayScan Imager. e. Detection by immunostaining of two autophagic markers p62
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and ATG12 in WT and DM1-NSCs (VUB03_DM and VUB01_WT, respectively). Statistical data are expressed as mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ns: not significant, *: p-value < 0.05, ** p-value < 0.01. *** p-value < 0.001. Figure 4: mTOR signaling pathway is defective in DM1-NSCs a. Expression level of key modulators of Akt/mTOR signaling in DM1-NSCs and WT-NSCs including: Phospho(Ser473)-Akt, Phospho(Thr172)-AMPKα, Phospho(Ser21/9)-GSK3α/β, and Phospho(Ser235-236)-Ribosomal protein S6 (rpS6) and the corresponding total forms. Cell lysates were obtained after 48 hours of culture. b. Decreased phosphorylation of S6 Ribosomal protein at Serine 235-236 and Serine 240-245 in DM1-NSCs when compared to control NSCs after 48 hours of culture. c. Effect of the different growth factors required for NSC culture (i.e. EGF (epidermal growth factor), FGF2 (fibroblast growth factor), BDNF (Brain derived neurotrophic factors)) on the phosphorylation of P-rpS6 (Ser235-236). DM1-NSCs and WT-NSCs were growth factor starved for 48 hours and subsequently treated for one hour with or without each of the growth factors. d. FGF2 dose-response analysis after growth factor starvation and treatment with FGF2 from 1 to 100 nM for 1 hour of DM1- and WT-NSCs. The expression level of rpS6 and ERK1/2 was analyzed. e. Kinetic of FGF2 treatment on DM1-and WT-NSCs for the expression of rpS6 and ERK1. Cells were starved for 48 hours and then treated for various durations with FGF2 (10 nM). Western blot were quantified using NIH Image J software and quantitative data are presented as phosphorylated form normalized to their total form. VUB03_DM cells were used as source for DM1-NSCs. Statistical data are expressed as mean ± SEM using three independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ns: not significant, *: p-value < 0.05, ** p-value < 0.01 *** p-value < 0.001. Figure 5: mTOR defect is correlated to decreased proliferation and induction of autophagy in DM-NSCs a. Effect of rapamycin treatment on the expression of key players of the Akt/mTOR signaling pathway in DM1-and WT-NSCs, determined by western blot analysis. b. Detection, by immunofluorescence, of P-rpS6 (Ser235-236) in DM1-and WT-NSCs after treatment with rapamycin (10nM) for 48 hours c. Effect of rapamycin treatment (10 nM for 4 days) on proliferation rate of WT-NSCs as determined by measuring intracytoplasmic ATP. d. Quantification of the proliferation index of the WT-NSCs with or without rapamycin treatment (10 nM) for four days. Data are presented as the mean of signal [ATP] at day x+1/x compared to the control condition (arbitrary 100%) e. Detection of autophagy using anti-LC3B antibody in WT-NSCs treated with rapamycin (10 nM) or treated with chloroquine (50 µM) for 6 hours. f. Quantification of autophagosomes after transfection of WT-NSCs with a plasmid expressing LC3-GFP fusion protein for 48 hours and treatment with rapamycin and/or chloroquine for 6 hours. Data are presented as a percentage of positive cells quantified by the automated ArrayScan Imager. Statistical data are expressed as mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, ** p-value < 0.01. *** p-value < 0.001.
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Figure 6: mTOR signaling defect in DM1-NSCs is related to MBNL1. a. Decreased expression level of P-rpS6 after transfection of WT-NSCs with a plasmid expressing 960 CTG for 24 hours. Western blots were analyzed by NIH Image J software and quantitative data are presented as phosphorylated form normalized to their total form. b. Expression level of the key modulators of the Akt/mTOR signaling pathway in WT-NSCs nucleofected with a SiRNA targeting MBNL1 or with a siScramble RNA for 48 hours. Data are presented as phosphorylated form normalized to their total form. c. Effect of the overexpression of MBNL1 in DM1-NSCs on the expression level of the key modulators of the Akt/mTOR signaling pathway. Cells were nucleofected with a plasmid encoding the 43 kDa isoform of the MBNL1 protein (pMBNL1) or mock nucleofected for 48 hours. Quantitative data are presented as phosphorylated form normalized to their total form.. Western blots were quantified using NIH Image J software d. Effect of the down-expression of MBNL1 in WT-NSCs and the over-expression of MBNL1 in DM1-NSCs on the proliferation rate. Proliferation was measured by immunostaining for Ki67 48 hours after nucleofection. e. Effect of the down-expression of MBNL1 in WT-NSCs and the over-expression of MBNL1 in DM1-NSCs on the induction of autophagy. Cells were co-nucleofected with a plasmid expressing the LC3-GFP protein fusion and autophagy was quantified 48 hours after nucleofection by measuring the number of cells containing LC3-GFP dots. Statistical data are expressed as mean ± SEM using at least 3 independent samples. One-way ANOVA with Dunnett's post hoc test was performed, *: p-value < 0.05, ** p-value < 0.01.