Research ArticleFRAGILE X SYNDROME

Reducing eIF4E-eIF4G interactions restores the balance between protein synthesis and actin dynamics in fragile X syndrome model mice

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Science Signaling  07 Nov 2017:
Vol. 10, Issue 504, eaan0665
DOI: 10.1126/scisignal.aan0665

Figures

  • Fig. 1 Inhibition of eIF4E-eIF4G interactions restores deficits in context discrimination and increases spine density in FXS model mice.

    (A and B) Mean discrimination ratio [percentage of time spent in freezing behavior in the unpaired (S−) divided by the paired (S+) context] of FXS model mice and wild-type (WT) littermates in the context discrimination test, performed on day 1 during the acquisition phase of the test (A) and on day 2 performed 1 hour after intracerebroventricular infusions of 4EGI-1 (B; 100 μM). n = 10 to 12 mice per treatment; comparisons of genotype (F1,39 = 15.35), treatment (F1,39 = 12.94), and time × treatment interaction (F1,39 = 17.39) were significantly different by two-way analysis of variance (ANOVA), each ***P < 0.005; ***P < 0.005 by Tukey’s multiple comparisons test. (C) Representative images of the dendritic spines of CA1 hippocampal neurons in WT and FXS model mice treated with vehicle or 4EGI-1 (100 μM infused in the lateral ventricles 24 hours before Golgi staining). n = 15 to 20 neurons per mouse; 3 to 4 mice per genotype per treatment. Scale bar, 3 μm. (D) Density of total spines in mice described in (C). Comparisons of genotype (F1,11 = 21.48), treatment (F1,11 = 31.92) (each ***P < 0.005), and time × treatment interaction (F1,11 = 11.38) (**P < 0.01) were significantly different by two-way ANOVA; ***P < 0.005 by Tukey’s multiple comparisons test. (E) Analysis of spine density in mice described in (C), distinguishing immature (filopodia and thin) and mature (stubby, mushroom, and branched) spines. For the immature spine densities, comparisons of genotype (F1,26 = 7.37), treatment (F1,26 = 7.5), and time × treatment interaction (F1,26 = 4.3) were significantly different by two-way ANOVA, each *P < 0.05; **P < 0.01 by Tukey’s multiple comparisons test. Difference among mature spine densities was not significant by two-way ANOVA.

  • Fig. 2 Inhibition of eIF4E-eIF4G interactions normalizes mGluR-LTD independently of protein synthesis in FXS model mice.

    (A and B) mGluR-LTD experiments in hippocampal slices from WT (A) and FXS model (B) mice. mGluR-LTD was elicited by application of DHPG (50 μM; 10 min) 20 min after stable baseline recordings, performed in the presence or absence of 4EGI-1 (100 μM; applied at least 40 min before DHPG) alone or with anisomycin (20 μM; applied at least 10 min before DHPG). Both drugs were present in the perfusion bath until the end of the recordings. mGluR-LTD in slices from WT mice. n = 7 to 12 (WT) or 9 to 13 (FXS) slices per treatment. Representative fEPSPs of baseline (black; assessed before DHPG, denoted by 1 in the graph) and mGluR-LTD (red; assessed after DHPG, denoted by 2 in the graph) are shown on the right of each graph. (C and D) Average mGluR-LTD in slices described in (A) and (B), 10 min before (C) or 50 min after (D) DHPG application. n =7 to 13 slices per genotype per treatment. (C) Not significantly different by two-way ANOVA. (D) Comparisons of treatment (F2,51 = 7.83), genotype (F2,51 = 11.81) (each **P < 0.01), and treatment × genotype interaction (F2,51 = 4.32) (*P < 0.05) were significantly different by two-way ANOVA; *P < 0.05 by Tukey’s multiple comparisons test. Data are means ± SEM normalized to WT controls.

  • Fig. 3 Rac1-PAK1/2-cofilin signaling and the F-actin/G-actin ratio are increased in FXS model mice.

    (A to H) Representative Western blots and quantification assessing the abundance of (A) total Rac1 relative to tubulin (G), (B) Rac1-GTP abundance by PAK-PBD pull-down (t = 3.44), (C) phospho-Ser199/204 PAK1 and Ser192/197 PAK2 relative to total PAK1/2 (E; t = 3.31), (D) phospho-Ser3 cofilin relative to total cofilin (F; t = 2.24), and (H) the F-actin/G-actin ratio (t = 2.51) in the hippocampal CA1 area of FXS model mice and WT littermates. Data are means ± SEM normalized to WT controls. n = 4 to 10 samples per genotype; not significant unless noted; *P < 0.05, **P < 0.01 by Student’s t test.

  • Fig. 4 Rac1-PAK1/2-cofilin signaling pathway is dysregulated in FXS model mice but is normalized by blocking the interaction of eIF4E and eIF4G.

    (A to E) Representative Western blots and quantification of (A) phospho-Ser199/204/Ser192/197 PAK1/2 and (B) phospho-Ser3 cofilin, relative to (D) total PAK1/2 or (E) total cofilin, respectively, and of tubulin (C, loading control), in area CA1 of hippocampal slices obtained from WT or FXS mice treated with DHPG (50 μM for 10 min), 4EGI-1 (100 μM; alone or applied 40 min before DHPG), or the combination DHPG and 4EGI-1. n = 9 to 22 (WT) or 11 to 17 (FXS) samples per treatment. Data are means ± SEM normalized to WT vehicle-treated controls. Effects of DHPG treatment [F1,47 = 4.76 (A) and F1,48 = 7.12 (B)], 4EGI-1 treatment [F1,47 = 5.3 (A) and F1,48 = 5.06 (B)], and DHPG + 4EGI-1 treatment [F1,47 = 5.4 (A) and F1,48 = 6.5 (B)] in samples from WT mice were significant (analyzed by two-way ANOVA, each P < 0.05). ***P < 0.005 effect of the 4EGI-1 treatment in FXS model mice [F1,48 = 12.55 (A) and 18.99 (B)] by two-way ANOVA. (A and B) *P < 0.05 and **P < 0.01 by Tukey’s multiple comparisons test. Data in (C) to (E) are not significantly different.

  • Fig. 5 F-actin/G-actin ratio and preferential binding of CYFIP1 to Rac1 in FXS model mice are normalized by administration of 4EGI-1.

    (A and B) Representative Western blot (A) and quantification (B) of F-actin relative to G-actin abundance in WT or FXS hippocampal slices treated with 4EGI-1 or vehicle. n = 9 to 10 (WT) or 7 (FXS) samples per genotype. t = 2.54 (FXS). *P < 0.05 by Student’s t test. (C and D) Representative Western blots and quantification of pull-down assays with PAK-PBD beads (C) or m7GTP beads (D) performed on lysates of hippocampal slices from WT (WT) or FXS mice. n = 4 (C) or 3 (D) samples per genotype. t = 2.56 (C) and 3.86 (D). *P < 0.05 by Student’s t test. (E and F) Representative Western blots and quantification of pull-down assays performed as described for (C) and (D) in hippocampal slices treated with 4EGI-1 (100 μM or equivalent volume of vehicle). Data are means ± SEM normalized to their respective vehicle controls. n = 4 to 5 (WT) or 6 to 7 (FXS) samples per genotype. (E) t = 2.60 (WT) and 2.40 (FXS); (F) t = 2.46 (WT) and 2.23 (FXS). *P < 0.05, **P < 0.01, Student’s t test. (G and H) Representative Western blots and quantification of pull-down assays performed in hippocampi dissected 1 hour after intracerebroventricular infusions with 4EGI-1 (100 μM) or equivalent volume of vehicle. Data are means ± SEM normalized to WT vehicle controls. n = 4 mice per genotype per treatment. *P < 0.05, ***P < 0.005, †P < 0.05, †††P < 0.005, effects of treatment [F1,11 = 5.03 (G) and F1,12 = 23.12 (H)] and genotype [F1,11 = 8.16 (G) and F1,12 = 23.95 (H)], analyzed by two-way ANOVA.

  • Fig. 6 Proposed model for the coordinated interaction of protein synthesis and dendritic spine dynamics induced by activation of mGluR1/5.

    (A) In area CA1 of the hippocampus in WT mice, CYFIP1 is present in two macromolecular complexes: CYFIP1-FMRP-eIF4E, which represses translation, and CYFIP1-WRC-Rac1-GTP, which regulates actin remodeling. Activation of mGluR1/5 changes the balance between these two complexes by increasing the activation of Rac1 and downstream signaling molecules and inducing protein synthesis via the relocation of CYFIP1 to the WRC-Rac1-GTP complex. The concomitant change in these signaling pathways ensures normal physiological synaptic plasticity and higher brain function. (B) In FXS model mice, the signaling molecules regulating protein synthesis and actin dynamics are no longer regulated by activation of mGluR1/5. Moreover, the absence of FMRP leads to exaggerated protein synthesis, perhaps via enhanced association of eIF4E to eIF4G. The lack of FMRP also results in an alteration in actin dynamics via enhanced Rac1-GTP abundance and increased association of CYFIP1 to WRC-Rac1-GTP. The disruption of these two signaling modules results in aberrant synaptic plasticity, spine morphology, and brain function that are characteristic of FXS. (C) 4EGI-1 restores the balance between protein synthesis and actin dynamics by creating free eIF4E that competes with Rac1-GTP to bind CYFIP1. This normalizes the aberrant synaptic plasticity, spine morphology, and cognitive function exhibited in FXS model mice. Dashed arrows indicate an indirect phosphorylation mechanism. Thick and thin arrows indicate enhanced and reduced interactions, respectively. Red arrows indicate the effect of 4EGI-1.

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