Research ArticleFragile X Syndrome

The fragile X mutation impairs homeostatic plasticity in human neurons by blocking synaptic retinoic acid signaling

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Science Translational Medicine  01 Aug 2018:
Vol. 10, Issue 452, eaar4338
DOI: 10.1126/scitranslmed.aar4338
  • Fig. 1 RA-dependent homeostatic synaptic plasticity in human neurons (iN cells) differentiated from human H1 ES cell line.

    (A) Representative images showing expression of pan-neuronal markers (MAP2 and Tuj1) and postsynaptic [postsynaptic density protein 95 (PSD95)] and presynaptic [synapsin-1 (SYN1)] markers in iN cells generated from control hES (H1) cells and cocultured with mouse glia for 4 weeks. Scale bars, 50 μm (MAP2 and Tuj1) and 5 μm (PSD95/SYN1). (B) Example traces (left) and quantification (right) for mEPSC amplitudes recorded from iN cells cocultured with mouse glia and treated with dimethyl sulfoxide (DMSO) or RA (30 to 45 min plus 90-min washout) (***P < 0.001, Student’s t test). Scale bars, 20 pA and 1 s (left) and 5 pA and 5 ms (right). (C) Quantification for mEPSC amplitudes recorded from iN cells cocultured with mouse cortical neurons and treated with DMSO or RA (***P < 0.001, Student’s t test). Scale bar, 5 pA and 5 ms. (D) Quantification for mIPSC amplitudes recorded from iN cells cocultured with mouse cortical neurons (***P < 0.001, Student’s t test). Scale bar, 5 pA and 5 ms. (E and F) Quantification for mEPSC amplitudes (E) and mIPSCs (F) recorded from iN cells cocultured with mouse cortical neurons and treated with DMSO (24 hours), TTX + CNQX (24 hours), or TTX + CNQX + DEAB (24 hours) [***P < 0.001, one-way analysis of variance (ANOVA)]. Scale bars, 5 pA and 50 ms. n/N = number of neurons/number of independent experiments. In all graphs, data represent average values ± SEM.

  • Fig. 2 Generation and characterization of FMR1 cKO human neurons.

    (A) Schematics of the gene targeting strategy used to generate FMR1 cKO hES cells. (B) Homologous recombination validation by polymerase chain reaction (PCR) using S1 and S5 external primers. F33 is the original targeted clone, and F33.F1, F33.F2, and F33.F3 are the subclones generated after Flp-mediated loop out of the selection cassette. (C) Immunoblot analysis of FMRP protein in H1, FMR1 KO (F33), FXS iPS cell #1 (SC135), and FMR1 cKO (F33.F3) cells. (D) Immunostaining of FMRP in H1 and FMR1 KO (F33) cells. Scale bar, 50 μm. (E) Quantitative PCR analysis of FMR1 mRNA expression in iN cells differentiated from F33.F3 cKO ES cells transduced with mCre (WT, control) or Cre (KO) and in brain tissues. (F) Immunoblot of FMRP protein in iN cells differentiated from F33.F3 cKO ES cells transduced with mCre or Cre. (G) Quantification of the iN conversion efficiency of F33.F3 cKO ES cells after doxycycline induction. (H) Representative immunofluorescence images of F33.F3 cKO ES cells transduced with mCre-GFP (WT, control; right) or Cre-GFP (KO; left) (red, MAP2). Scale bar, 50 μm. (I) Quantification of general neuronal morphology in WT (mCre) and FMR1 KO (Cre) iN cells. (J) Images of dendrites immunolabeled with SYN1, PSD95, and MAP2 from WT (mCre) and FMR1 KO (Cre) iN cells. Scale bar, 5 μm. (K) Quantification of puncta density, intensity, and size for SYN1 and PSD95 immunofluorescent signals. In all graphs, data represent average values ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 3 Impaired RA synaptic signaling and homeostatic synaptic plasticity in human neurons lacking FMR1 expression.

    (A) Example traces and quantification for mEPSC amplitudes recorded from iN cells differentiated from FXS cKO #1 F33 ES cells, infected with mCre (WT) or Cre (KO) viruses, cocultured with mouse glia, and treated with DMSO or RA (**P < 0.01, Student’s t test). Scale bars, 10 pA and 1 s (top traces) and 5 pA and 5 ms (inset traces). (B) Quantification for mEPSC amplitudes recorded from WT or KO iN cells differentiated from FXS cKO #1 F33 ES cells, cocultured with mouse cortical neurons, and treated with DMSO or RA (**P < 0.01, Student’s t test). Scale bars, 5 pA and 5 ms. (C) Quantification for mIPSC amplitudes recorded from WT or KO iN cells treated with DMSO or RA (**P < 0.01, Student’s t test). Scale bars, 5 pA and 50 ms. (D) Example traces and quantification for mEPSC amplitudes recorded from WT or KO iN cells differentiated from FXS cKO #1 F33 ES cells treated with DMSO (24 hours), TTX + CNQX (24 hours), or TTX + CNQX + DEAB (24 hours) (***P < 0.001, one-way ANOVA). Scale bars, 20 pA and 1 s (long traces) and 5 pA and 5 ms (single-response traces). (E) Example traces and quantification for mIPSC amplitudes recorded from WT or KO iN cells treated with DMSO (24 hours), TTX + CNQX (24 hours), or TTX + CNQX + DEAB (24 hours) (***P < 0.001, one-way ANOVA). Scale bars, 20 pA and 1 s (left) and 5 pA and 50 ms (right). In all graphs, data represent average values ± SEM.

  • Fig. 4 Biochemical and morphological characterization of human neurons differentiated from control and FXS patient iPS cell lines.

    (A) Quantification of OCT4 and FMR1 mRNA expression in H1 ES cell and FXS iPS cells. (B) Immunostaining images of FMRP showing lack of FMRP expression in control and FXS iPS cell lines. Scale bar, 20 μm. (C) Western blot analysis of FMRP expression in iN cells differentiated from H1 ES cell, FXS iPS cells #1 and #2, and control iPS cells #1 and #2. (D) Representative images and quantification of morphological aspects of iN cells differentiated from both control and FXS iPS cell lines (Ctrl #1 EB1, Ctrl #2 Skl, FXS #1 SC135, and FXS #2 SC153) (*P < 0.05, one-way ANOVA). Scale bar, 50 μm. (E) Representative images showing expression of pan-neuronal markers (MAP2 and Tuj1) and presynaptic (SYN1) and postsynaptic (PSD95) markers in iN cells generated from control human iPS cells (Ctrl #1 EB1 and Ctrl #2 Skl) and FXS human iPS cells (FXS #1 SC135 and FXS #2 SC153) and cocultured with mouse glia for 4 weeks. Scale bars, 50 μm (top images) and 5 μm (bottom higher-magnification images). In all graphs, data represent average values ± SEM.

  • Fig. 5 Impaired synaptic RA signaling in human neurons derived from FXS patients cocultured with mouse glia or mouse neurons.

    (A and B) Example traces and quantification for mEPSC amplitudes recorded from iN cells differentiated from control #1 iPS cells (EB1) (A) and FXS iPS cell #1 (SC135) (B), cocultured with mouse glia, and treated with DMSO or RA (***P < 0.001, Student’s t test). Scale bars, 20 pA and 1 s (left) and 5 pA and 5 ms (right). (C) Quantification for mEPSC amplitudes recorded from iN cells differentiated from control iPS cell #1 (left) or FXS iPS cell #1 (right), cocultured with mouse neurons, and treated with DMSO or RA (***P < 0.001, Student’s t test). Scale bars, 5 pA and 5 ms. (D) Quantification for mIPSC amplitudes recorded from control and FXS iN cells treated with DMSO or RA (***P < 0.001, Student’s t test). Scale bars, 5 pA and 50 ms. (E) Representative traces and quantification of evoked EPSCs recorded from iN cells differentiated from control iPS cell #1 and FXS iPS cell #1 lines after DMSO or RA treatment. Scale bars, 200 pA and 10 ms. (F) Representative traces and quantification of evoked IPSCs recorded from control and FXS iN cells after DMSO or RA treatment. Scale bars, 200 pA and 20 ms. In all graphs, data represent average values ± SEM.

  • Fig. 6 Properties of RA signaling in human neurons differentiated from control patient iPS cells.

    (A) RA dose-dependent responses of mEPSC in iN cells differentiated from control iPS cell #2 line (*P < 0.05, ***P < 0.001, one-way ANOVA). (B) Quantification of mEPSCs recorded from iN cells differentiated from FXS iPS cell #1 line and treated with a maximum dose of RA (10 μM). (C) Quantification of mEPSC amplitudes and frequencies in iN cells differentiated from control iPS cell #2 line treated with RA and NASPM (***P < 0.001, Student’s t test). In all graphs, data represent average values ± SEM.

  • Fig. 7 Impaired homeostatic synaptic plasticity induced by synaptic silencing in human neurons differentiated from FXS patient iPS cells.

    (A and B) Example traces and quantification for mEPSC amplitudes recorded from iN cells differentiated from control iPS cell #1 line (A) or FXS iPS cell #1 line (B), cocultured with mouse cortical neurons, and treated with DMSO (24 hours), TTX + CNQXTTX + CNQX (24 hours), or TTX + CNQX + DEAB (24 hours) (***P < 0.001, one-way ANOVA). Scale bars, 20 pA and 1 s (left) and 5 pA and 5 ms (right). (C and D) Example traces and quantification for mIPSC amplitudes recorded from iN cells differentiated from control iPS cell #1 line (C) and FXS iPS cell #1 line (D) treated with DMSO (24 hours), TTX + CNQX (24 hours), or TTX + CNQX + DEAB (24 hours) (**P < 0.01, one-way ANOVA). Scale bars, 50 pA and 1 s (left) and 5 pA and 50 ms (right). (E) Example traces of evoked EPSCs recorded from control and FXS iN cells under various treatment conditions. Scale bars, 200 pA and 10 ms. (F and G) Quantification of evoked EPSC amplitudes from control (F) and FXS (G) iN cells (**P < 0.01, one-way ANOVA). (H) Example traces of evoked IPSCs recorded from control and FXS iN cells under various treatment conditions. Scale bar, 200 pA and 20 ms. (I and J) Quantification of evoked IPSC amplitudes from control (I) and FXS (J) iN cells (**P < 0.01, one-way ANOVA). In all graphs, data represent average values ± SEM.

  • Fig. 8 Rescues of synaptic RA signaling in human neurons induced from FXS iPS cells by CRISPR/Cas9-mediated removal of excessive CGG repeats upstreams of FMR1 gene.

    (A) Sequence alignment diagram showing the region within exon 1 flanking the CGG repeats upstream of ATG of FMR1 gene from a control hES cell line (H1) and a FXS patient iPS cell line before and after the CRISPR/Cas9-mediated removal of excessive CGG repeats (red-highlighted). (B) Immunoblot of FMRP expression in FXS iPS cell line before and after the repair. (C and D) Quantification of mEPSC (C) and mIPSC (D) amplitudes and frequencies in neurons derived from repaired FXS iPS cells treated with DMSO or RA (**P < 0.01, ***P < 0.001, Student’s t test). In all graphs, data represent average values ± SEM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/452/eaar4338/DC1

    Methods

    Fig. S1. RA treatment changes mEPSC and mIPSC amplitudes but not frequencies of neurons differentiated from ES cells.

    Fig. S2. Electrophysiological characterization of FMR1 cKO iN cells.

    Fig. S3. Removal of FMR1 expression results in impaired RA synaptic signaling and homeostatic synaptic plasticity in iN cells differentiated from FMR1 cKO ES line #1 F33 cocultured with mouse glia or mouse neurons.

    Fig. S4. Removal of FMR1 expression results in impaired RA synaptic signaling and homeostatic synaptic plasticity in iN cells differentiated from FMR1 cKO ES line #2 F17 cocultured with mouse glia or mouse neurons.

    Fig. S5. Electrophysiological characterization of iN cells differentiated from normal and FXS patient iPS cell lines.

    Fig. S6. Homeostatic synaptic plasticity induced by RA is impaired in iN cells derived from FXS patients, when cocultured with mouse glia or cortical neurons.

    Fig. S7. Effect of RA on synapse density and morphology.

    Fig. S8. Homeostatic synaptic plasticity induced by synaptic silencing is impaired in iN cells differentiated from FXS patient iPS cells, when cocultured with mouse cortical neurons.

    Fig. S9. Homeostatic synaptic plasticity induced by synaptic silencing occludes RA-induced synaptic changes in iN cells differentiated from normal patient iPS cells.

    Fig. S10. Model of the molecular mechanisms underlying disruption of RA-dependent homeostatic synaptic plasticity in FXS as analyzed in human neurons.

    Table S1. Raw data (provided as separate Excel file).

    References (9095)

  • The PDF file includes:

    • Methods
    • Fig. S1. RA treatment changes mEPSC and mIPSC amplitudes but not frequencies of neurons differentiated from ES cells.
    • Fig. S2. Electrophysiological characterization of FMR1 cKO iN cells.
    • Fig. S3. Removal of FMR1 expression results in impaired RA synaptic signaling and homeostatic synaptic plasticity in iN cells differentiated from FMR1 cKO ES line #1 F33 cocultured with mouse glia or mouse neurons.
    • Fig. S4. Removal of FMR1 expression results in impaired RA synaptic signaling and homeostatic synaptic plasticity in iN cells differentiated from FMR1 cKO ES line #2 F17 cocultured with mouse glia or mouse neurons.
    • Fig. S5. Electrophysiological characterization of iN cells differentiated from normal and FXS patient iPS cell lines.
    • Fig. S6. Homeostatic synaptic plasticity induced by RA is impaired in iN cells derived from FXS patients, when cocultured with mouse glia or cortical neurons.
    • Fig. S7. Effect of RA on synapse density and morphology.
    • Fig. S8. Homeostatic synaptic plasticity induced by synaptic silencing is impaired in iN cells differentiated from FXS patient iPS cells, when cocultured with mouse cortical neurons.
    • Fig. S9. Homeostatic synaptic plasticity induced by synaptic silencing occludes RA-induced synaptic changes in iN cells differentiated from normal patient iPS cells.
    • Fig. S10. Model of the molecular mechanisms underlying disruption of RA-dependent homeostatic synaptic plasticity in FXS as analyzed in human neurons.
    • Legend for table S1
    • References (9095)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1. Raw data (provided as separate Excel file).

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