Research ArticleFRAGILE X SYNDROME

Functional changes of AMPA responses in human induced pluripotent stem cell–derived neural progenitors in fragile X syndrome

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Science Signaling  16 Jan 2018:
Vol. 11, Issue 513, eaan8784
DOI: 10.1126/scisignal.aan8784
  • Fig. 1 Increased differentiation of cells expressing AMPARs in human FXS neural progenitors.

    (A) Representative images of iPSCs, neurospheres, and neurospheres immunostained for MAP2 (red) at days 1 and 7 of differentiation. DAPI, 4′,6-diamidino-2-phenylindole. (B) Fluorescence image of a neurosphere and representative cell responses to NMDA, KA, and DHPG after stimulation as indicated. (C and D) Pie charts showing the proportions of cells responsive to DHPG and KA at day 1 and to KA and NMDA at day 7 of differentiation (responsive cells/total cell number). n = 239 cells (CTRL) and 389 cells (FXS) at day 1, and n = 498 cells (CTRL) and 803 cells (FXS) at day 7; 40 to 100 cells per neurosphere. Data are from four independent experiments with three control and four FXS cell lines. (E to G) Representative cell responses (E), proportion of responsive cells (F), and average amplitude of responses (G) to KA. Data are means ± SEM of the experiments performed in (C) and (D). (H) Comparison of KA and AMPA responses. n = 174 cells (CTRL) and 166 cells (FXS); 40 to 100 cells per neurosphere. Data are means ± SEM of three independent experiments with two different control and FXS cell lines. Scale bars, 50 μm (A) and 100 μm (B). *P < 0.05, **P < 0.01, ***P < 0.001 by Fisher’s exact test (F) and Student’s unpaired t test (G and H).

  • Fig. 2 Decreased GluA2 expression increases Ca2+ permeability and reduces neurite outgrowth in human neural progenitors.

    (A) Representative single-cell recordings showing fully inhibited KA-induced [Ca2+]i response by philanthotoxin (PhTx). (B and C) Proportion of KA-responsive cells (B) and the cell population (C) divided to cells with and without NMDA responses, showing fully inhibited responses by PhTx at day 7 of differentiation. n = 185 cells (CTRL) and 172 cells (FXS); 40 to 100 cells per neurosphere of HEL46.11, HEL23.3, HEL100.1, and HEL69.5 cell lines. Data are means ± SEM from two independent experiments. (D) Representative single-cell recording showing complete inhibition of AMPA-induced Ca2+ response by Naspm. (E and F) Proportion of AMPA-responsive cells (E) and the cell population (F) divided to cells with and without NMDA responses, showing fully inhibited AMPA responses by Naspm at day 7 of differentiation. n = 134 cells (CTRL) and 156 cells (FXS); 40 to 100 cells per neurosphere of HEL46.11, HEL11.4, HEL100.2, and HEL70.3 cell lines. Data are means ± SEM from two independent experiments. (G) Neurite length of MAP2-immunopositive neurons with and without Naspm treatment at day 1 of differentiation. n = 20 cells in each group. Data are means ± SEM from two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by Fisher’s exact test (B, C, E, and F) or Mann-Whitney test (G).

  • Fig. 3 Enhanced differentiation of CP-AMPAR–expressing cells in FMRP-deficient mouse neural progenitors.

    (A and B) Pie charts showing the proportions of cells responsive to DHPG and KA at day 1 of differentiation (A) and to KA, NMDA, and both KA and NMDA at day 7 of differentiation (B). n = 262 cells [wild-type (WT)] and 316 cells (Fmr1-KO) at day 1, and n = 370 cells (WT) and 396 cells (Fmr1-KO) at day 7; 55 to 100 cells per neurosphere. Data are from four independent experiments. (C) Representative single-cell response to KA during the time periods indicated. (D) Proportion of cells responding to KA (cells responsive/total cell number). Data are means ± SEM of the experiments performed in (A) and (B). (E) Representative single-cell recording showing complete inhibition of KA-induced [Ca2+]i response by PhTx. (F) Proportion of cells fully inhibited by PhTx and Naspm in WT and Fmr1-KO neurospheres at day 7 of differentiation. n = 245 cells (WT + PhTx), 252 cells (Fmr1-KO + PhTx), 185 cells (WT + Naspm), and 208 cells (Fmr1-KO + Naspm); 55 to 100 cells per neurosphere. Data are means ± SEM from three independent experiments. **P < 0.01, ***P < 0.001 by Fisher’s exact test (D and F).

  • Fig. 4 Increased rectification of AMPARs in Fmr1-KO mouse progenitors.

    (A) Representative current traces evoked by glutamate in the presence of cyclothiazide (C) and AMPAR blockers NBQX (NB) and Naspm (NA) at day 7 of differentiation in WT and Fmr1-KO neurospheres. Horizontal bar indicates the drug application. (B) Inhibition of maximal currents (1 mM glutamate and 100 μM cyclothiazide) by NBQX and Naspm in the indicated cells and conditions. n = 10 cells (WT + NBQX), 9 cells (Fmr1-KO + NBQX), 13 cells (WT + Naspm), and 10 cells (Fmr1-KO + Naspm). Data are means ± SEM from four independent experiments. (C) Representative single-cell recording traces of I-V relationship in the WT and Fmr1-KO neurospheres. Horizontal bar indicates the agonist application. (D) I-V relationship (−60 to 40 mV) of normalized currents in WT (red) and Fmr1-KO (black) progenitors. The dashed line indicates the linear I-V relationship. (E) Inward rectification (IR) index defined as the ratio of current amplitudes at +40 and −60 mV recorded from WT and Fmr1-KO cells. n = 12 cells (WT) and 9 cells (Fmr1-KO). Data are means ± SEM from four independent experiments. *P < 0.05 by Mann-Whitney test (B and E).

  • Fig. 5 Altered expression of GluA1 and GluA2 in human and mouse FXS neural progenitors.

    (A) Immunofluorescence images showing GluA2 (green) and relative GluA2 protein expression in human neural progenitors at day 7 of differentiation (n = 100 cells per group). Data are means ± SEM of two independent experiments with two different control and FXS cell lines. (B) Representative images and analysis of GluA2 staining (red) in mouse neural progenitors at day 7 of differentiation. Data are means ± SEM of two independent experiments (n = 100 cells per group). (C) Relative mRNA expression of GRIA1 in proliferating (Prolif.) and differentiated (at days 7 and 14) human neural progenitors. Data are means ± SEM of three independent experiments performed in triplicate; n = 3 cell lines (CTRL) and 3 cell lines (FXS). (D and E) Relative mRNA expression of Gria1 and Gria2 in (D) mouse neural progenitors at day 7 of differentiation and (E) the prefrontal cortex of Fmr1-KO and WT mice (n = 8 brain samples each). Data are means ± SEM of at least three independent experiments performed in triplicate. Scale bars, 100 μm (A) and 100 μm (10×) and 10 μm (63×) (B). *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney test (A, D, and E) or Fisher’s exact test (B).

  • Fig. 6 Expression profiling and increased miR-181a expression in FXS progenitors.

    (A) Highest ranked pathways of genes up-regulated in human FXS iPSC-derived neural progenitors compared with controls at day 1 of differentiation in the transcriptome analysis. n = 3 cell lines each. (B and C) Relative expression of miR-181a-5p (B) and miR-181a-3p (C) in human neural progenitors at days 1 and 7 of differentiation. Data are means ± SEM of three independent experiments performed in triplicate; n = 3 cell lines each. *P < 0.05, **P < 0.01, ***P < 0.001 by Mann-Whitney test (B and C).

  • Fig. 7 Intracellular calcium responses to KA/AMPA in subpopulations of glutamate-responsive FXS progenitors.

    (A) Average amplitude of [Ca2+]i responses to DHPG in subpopulations of cells responsive to AMPA blocked totally and partially with Naspm in human neural progenitors at day 7 of differentiation. n = 134 cells (CTRL) and 156 cells (FXS) from HEL46.11, HEL11.4, HEL100.2, and HEL70.3 cell lines. Data are means ± SEM of two independent experiments. (B) Average amplitude of responses to KA in subpopulations of cells responsive to KA, DHPG, and NMDA in human neural progenitors at day 7 of differentiation. n = 498 cells (CTRL) and 803 cells (FXS). Data are means ± SEM of four independent experiments with three control and four FXS cell lines. (C) Amplitude of [Ca2+]i response to AMPA in mouse neural progenitors at day 7 of differentiation. n = 185 cells (WT) and 208 cells (Fmr1-KO); 55 to 100 cells per neurosphere. Data are means ± SEM from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way analysis of variance (ANOVA) with post hoc Tukey analysis (A) or Student’s unpaired t test (B).

Supplementary Materials

  • Supplementary Materials for:

    Functional changes of AMPA responses in human induced pluripotent stem cell–derived neural progenitors in fragile X syndrome

    Venkat Swaroop Achuta, Tommi Möykkynen, Ulla-Kaisa Peteri, Giorgio Turconi, Claudio Rivera, Kari Keinänen, Maija L. Castrén*

    *Corresponding author. Email: maija.castren{at}helsinki.fi

    This PDF file includes:

    • Fig. S1. Characterization of human iPSC lines.
    • Table S1. Primer sequences used in real-time quantitative PCR.

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    Citation: V. S. Achuta, T. Möykkynen, U.-K. Peteri, G. Turconi, C. Rivera, K. Keinänen, M. L. Castrén, Functional changes of AMPA responses in human induced pluripotent stem cell–derived neural progenitors in fragile X syndrome. Sci. Signal. 11, eaan8784 (2018).

    © 2018 American Association for the Advancement of Science

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