Article Figures & Data
Figures
- Figure 1.
A–E , Exaggerated PI3K activity and signaling at Fmr1 KO synapses. A , PI3K activity is threefold increased in Fmr1 KO SNS compared with WT SNS, as assessed by the amount of radiolabeled PI3-P on autoradiographies from in vitro PI3K assays (n = 8, *p = 0.035, paired t test) (control experiment shown in supplemental Fig. S1A, available at www.jneurosci.org as supplemental material). B , Quantification of phospho-ELK-1 specific Western blots of in vitro ERK1/2 kinase assays from WT and Fmr1 KO SNS using recombinant ELK-1 as a substrate shows no significant change in ERK1/2 activity in the absence of FMRP (n = 8, p = 0.20, paired t test) (control experiment shown in supplemental Fig. S1B, available at www.jneurosci.org as supplemental material). FMRP-specific Western blot analyses of the lysate used as starting material for the assay are shown below; tubulin served as loading control. C , Quantitative analysis of recombinant RFP–PH(Akt) domain colocalized with synaptophysin in hippocampal neurons (9–10 DIV) demonstrates increased synaptic localization of RFP–PH(Akt) in Fmr1 KO neurons, suggesting elevated PI3-P3 levels at Fmr1 KO synapses, which lead to a translocation of the fluorescently labeled PH domain (scale bar, 5 μm) (n = 5 independent experiments, 6–12 cells each, ***p = 0.00002, paired t test). In contrast, total dendritic RFP–PH(Akt) levels were not different between WT and Fmr1 KO neurons (supplemental Fig. S1C, available at www.jneurosci.org as supplemental material). D , E , Phosphorylation of the PI3K downstream signaling molecule Akt ( D ), but not ERK1/2 ( E ), is significantly increased in Fmr1 KO SNS compared with WT. Phosphorylation levels were assessed by densitometric analysis of Western blots, and phospho-specific signals were normalized to total levels of the respective proteins ( D , n = 6, *p = 0.014; E , n = 7, p = 0.18; paired t tests). Representative Western blots are shown below. F–I , PI3K activity and signaling is increased in FMRP-deficient HEK293T cells. In contrast to cortical SNS, HEK293T cells do not express detectable levels of mGluR1 and mGluR5 receptors (Western blot analyses shown in supplemental Fig. S1D, available at www.jneurosci.org as supplemental material). F , G , siRNA-mediated knockdown of Fmr1 in HEK293T cells increases PI3K activity significantly ( F , n = 7, *p = 0.039, paired t test), whereas in vitro ERK1/2 assays showed no significant change in activity ( G , n = 7, p = 0.32, paired t test). H , I , Quantification of Akt and ERK1/2 phosphorylation after Fmr1 knockdown demonstrate significantly enhanced phospho-Akt levels ( H , n = 5, *p = 0.018, paired t test) but not phospho-ERK1/2 levels ( I , n = 6, p = 0.29, paired t test). Genotype or siRNA-mediated knockdown was confirmed by Western blotting with an FMRP-specific antibody and a tubulin antibody as loading control (shown below for each experiment). All error bars represent SEM. a.u., Arbitrary unit.
- Figure 2.
Excess PI3K activity in Fmr1 KO cortical neurons can be reduced by inhibition of gp1 mGluR-mediated signaling. A , Treatment with the mGluR5 antagonist MPEP significantly reduces PI3K activity in Fmr1 KO SNS but not in WT SNS (n = 4, two-way ANOVA: genotype, p = 0.953; treatment, p = 0.425; interaction of genotype and treatment, *p = 0.006; LSD post hoc analyses: p wtctr–wtMPEP = 0.327, *p wtctr–koctr = 0.033, *p koctr–koMPEP = 0.012). B , Disruption of the mGluR5–Homer complex with a tat-fused mGluR5 C-terminal peptide but not with a mutated peptide (CT and MUT, respectively; 5 μm) decreases PI3K activity in both WT and Fmr1 KO SNS. Peptide treatment led to a significant decrease in PI3K activity in both WT and KO, but PI3K activity was still significantly increased in KO compared with WT after treatment with the mGluR5 C-terminal peptide (n = 4, two-way-ANOVA; effect of peptide, *p < 0.001; effect of genotype, *p = 0.014; interaction of peptide and genotype, p = 0.143). Moreover, there was neither a significant genotype-specific difference in PI3K activity in KO samples compared with WT after disruption of the mGluR complex, nor a significant difference in the ratios of KO versus WT PI3K activity when treated with mutated compared to C-terminal peptides (analyses shown in supplemental Fig. S2A,B, available at www.jneurosci.org as supplemental material). C , PI3K activity in cultured cortical Fmr1 KO neurons is increased compared with WT, and short-hairpin-mediated knockdown of the PI3K enhancer PIKE-L (shPIKE-L) significantly reduces PI3K activity in Fmr1 KO (n = 3; two-way ANOVA: genotype, *p = 0.001; treatment, *p = 0.008; interaction of genotype and treatment, *p = 0.014; Tukey's HSD post hoc analyses: *p wtctr–koctr = 0.002, *p koctr–koPIKE = 0.007). shPIKE-L did not lead to a complete rescue of PI3K activity to WT levels (KO, 139 ± 14% of WT after PIKE knockdown). Western blots below show knockdown of PIKE-L in WT and KO neurons; tubulin was used as loading control. Additional data in supplemental Figure S2, C and D, show association of PIKE-L mRNA with FMRP in coimmunoprecipitations from brain lysates, as well as increased PIKE-L levels in Fmr1 KO SNS (available at www.jneurosci.org as supplemental material). D , E , Activation of gp1 mGluRs with DHPG (10 min, 100 μm) leads to a twofold increase in PI3K activity in WT SNS ( D , n = 4, *p = 0.033, paired t test) but to decreased PI3K activity in Fmr1 KO SNS ( E , n = 4, *p = 0.049, paired t test).
- Figure 3.
p110β protein expression and translation is regulated by FMRP. A , B , Immunocytochemical analysis of p110β expression at synapses in WT ( A ) and Fmr1 KO ( B ) neurons shows that p110β (red) colocalizes with the synaptic marker synaptophysin (green), as indicated by yellow signal; specificity of antibody is shown in supplemental Figure S3A (available at www.jneurosci.org as supplemental material). Colocalized signal is shown in white (bottom). Scale bar, 20 μm. C , Quantification of p110β signal intensities overlapping with synaptophysin reveals a significant increase of overlap in Fmr1 KO neurons compared with WT neurons (WT, 27% overlap; KO, 34.9% overlap; n = 43 dendrites each for WT and KO, 3 independent hippocampal cultures, **p = 0.008, independent t test) (additional analyses shown in supplemental Fig. S3B–D, available at www.jneurosci.org as supplemental material). Representative images of dendrites from WT and Fmr1 KO that were analyzed for colocalization are shown on the left, with colocalized signal superimposed in white. Scale bar, 3 μm. D , Total levels of synaptophysin were unchanged in Fmr1 KO compared with WT dendrites (n = 43, p = 0.73, independent t test). E , Quantification of fluorescent p110β-specific signal within three-dimensional reconstructed synaptophysin punctae reveals increased mean intensity of p110β-specific signal in synaptophysin punctae in Fmr1 KO dendrites, suggesting specific enrichment of p110β at single synapses (n = 43, **p = 0.004, independent t test). In contrast, the relative number of p110β-positive synapses was not changed in Fmr1 KO neurons (supplemental Fig. S3C, available at www.jneurosci.org as supplemental material). Example of three-dimensional reconstruction of synaptophysin punctae is shown on the left, and p110β signal within these punctae is indicated by arrows. Scale bar, 3 μm. Additional analysis is shown in supplemental Figure. S3D (available at www.jneurosci.org as supplemental material). F , Densitometric analysis of p110β-specific Western blots demonstrates increased p110β protein levels in SNS from Fmr1 KO cortices compared with WT. Signal intensities were normalized to tubulin (n = 5, *p = 0.043, paired t test); a representative western blot is shown at the right. G , siRNA-mediated reduction of FMRP expression in HEK293T cells leads to increased p110β protein levels. Signal intensities were normalized to tubulin (n = 5, *p = 0.005, paired t test). H , FMRP-specific quantitative coimmunoprecipitation from WT and Fmr1 KO brain lysates demonstrates a specific enrichment of p110β mRNA in WT immunoprecipitations, whereas NR1 mRNA is not enriched. mRNA levels were quantified by quantitative real-time PCR (n = 6, two-way ANOVA: p mRNA = 0.488, p genotype = 0.372, *p between subjects = 0.034; Tukey's HSD post hoc analyses: *p p110 β = 0.003, p NR1 = 0.884). I , Recombinant p110β 3′ UTR fused to EGFP expressed in HEK293T cells is significantly enriched in anti-flag pulldowns with coexpressed flag-tagged mCherry–FMRP but not with flag-tagged mCherry. No specific enrichment can be detected with β-actin 3′ UTR. mRNA levels in pulldowns were quantified by qRT-PCR with EGFP-specific primers and normalized to input (n = 4, two-way ANOVA: *p mRNA = 0.006, *p rec.protein = 0.001, *p between subjects = 0.01; Tukey's HSD post hoc analyses: *p p110 β = 0.001, p β -actin = 0.798). J , K , qRT-PCR quantification of mRNA levels in sucrose gradients from SNS shows that p110β mRNA in polysomal fractions is puromycin sensitive ( J , n = 3, *p = 0.033, paired t test) and significantly increased in these fractions from Fmr1 KO SNS compared with WT; PSD95 and NR1 mRNAs served as positive and negative controls, respectively ( K , n = 5; PSD95, *p = 0.029; NR1, p = 0.89; p110β, *p = 0.043, paired t tests; results were normalized to WT). For experimental details, see supplemental Fig. S3, E and F (available at www.jneurosci.org as supplemental material). L , Likewise p110β mRNA is shifted into actively translating polysomes after Fmr1 knockdown in HEK293T cells (n = 3, two-way ANOVA: *p genotype = 0.002, *p treatment < 0.001, *p between subjects = 0.002; Tukey's HSD post hoc analyses: *p ctruntr–ctrpuro = 0.002, *p ctruntr–KDuntr = 0.001, *p KDuntr–KDpuro < 0.001). All error bars represent SEM.
- Figure 4.
Dysregulated gp1 mGluR-dependent p110β expression in Fmr1 KO. A , B , DHPG treatment (15 min, 50 μm) increases p110β protein levels in WT SNS ( A , normalized to control; n = 6, *p = 0.008, paired t test) and leads to a shift of p110β mRNA into puromycin-sensitive fractions ( B , percentage of total mRNA in polysomal fractions; n = 6, *p = 0.028, paired t test). C , D , In Fmr1 KO SNS, DHPG treatment does not increase protein expression ( C , normalized to control, n = 5, p = 0.613, paired t test) or enhance association of p110β mRNA with puromycin-sensitive polysomes ( D , percentage of total mRNA in polysomal fractions, n = 3, p = 0.21, paired t test).
- Figure 5.
PI3K antagonists rescue dysregulated basal and stimulus-induced synaptic translation rates in Fmr1 KO SNS. A , Basal translation in cortical Fmr1 KO SNS is increased ∼30% compared with WT. Translation rates were analyzed by metabolic labeling of SNS with [35S]methionine, and radioactivity incorporation during a 5 min time period was quantified (n = 6, *p = 0.03, paired t test). B , C , Pretreatment of SNS with an mGluR5 antagonist ( B : MPEP, 10 μm) or an mGluR1 antagonist ( C : LY367385, 10 μm) significantly decreased translation rates in Fmr1 KO but not in WT SNS (MPEP: n = 8, *p wt–ko untreated = 0.043, *p ko untreated–treated = 0.002; LY367385: n = 5, *p wt–ko untreated = 0.04, *p ko untreated–treated = 0.007; Tukey's HSD post hoc tests; two-way ANOVAs detect significant interaction between genotype and treatment; MPEP: *p < 0.001; LY367385: *p < 0.001). D , Treatment with a specific NMDAR antagonist (APV, 50 μm) affected translation similarly across genotypes (n = 3; two-way ANOVA: no significant interaction between genotype and treatment; p = 0.782) (also see supplemental Fig. S5A,B, available at www.jneurosci.org as supplemental material). E , F , Treatment of SNS with two different PI3K antagonists, LY294002 ( E ; 50 μm) and wortmannin ( F ; 100 nm), significantly reduced amino acid incorporation rates in Fmr1 KO but not in WT (n = 6; E , LY294002: *p wt–ko untreated = 0.001, *p ko treated–untreated = 0.026; F , wortmannin: *p wt–ko untreated < 0.001, *p ko treated–untreated = 0.002; Tukey's HSD post hoc tests; two-way ANOVA: significant interaction between genotype and treatment; LY294002: *p < 0.001; wortmannin: *p < 0.001). LY303511, an inactive analog of LY294002, did not alter translation rates (supplemental Fig. S5C, available at www.jneurosci.org as supplemental material). G , An ERK1/2 antagonist (U0126, 20 μm) did not show a genotype-specific effect on translation rates [n = 5, two-way ANOVA: significant effects of treatment (*p = 0.001) and genotype (*p = 0.001) but no significant interaction between genotype and treatment (p = 0.68)] (also see supplemental Fig. S5D,E, available at www.jneurosci.org as supplemental material). H , DHPG-induced translational activation in Fmr1 KO SNS is occluded and can be rescued by antagonizing PI3K signaling (wortmannin) but not by ERK1/2 inhibition (U0126) (wortmannin: n = 4, one-way ANOVA, *p = 0.012, Tukey's HSD post hoc tests: *p untrctr–wortctr = 0.045, p wortctr–wortDHPG = 0.010; U0126: n = 4, one-way ANOVA, *p = 0.001, Tukey's HSD post hoc tests: *p untrctr–untrDHPG = 0.029, *p untrctr–U0126ctr/DHPG = 0.006/0.001, *p untrDHPG–U0126ctr/DHPG = 0.019/0.004). I , Inhibitors of PI3K signaling (Wort) and ERK1/2 signaling (U0126) abolish DHPG-induced (15 min, 100 μm) translational activation in WT SNS (wortmannin: n = 4, one-way ANOVA, *p = 0.002, Tukey's HSD post hoc tests: *p untrctr–untrDHPG = 0.006, *p wortctr–wortDHPG = 0.004; U0126: n = 3, one-way ANOVA, *p < 0.001, Tukey's HSD post hoc tests: *p untrctr–untrDHPG = 0.029, *p untrctr/DHPG–U0126DHPG = 0.022/0.001). Results were normalized to control. All error bars represent SEM.
- Figure 6.
A PI3K antagonist rescues increased GluR1 endocytosis in Fmr1 KO neurons. A , Representative images illustrate surface and internalized GluR1 staining in WT and Fmr1 KO primary hippocampal neurons under control conditions and after treatment with the PI3K inhibitor LY294002. Scale bar, 50 μm. B , Box-and-whisker plot of the distribution of constitutive endocytosis of AMPARs in distal dendrites shows that enhanced constitutive endocytosis of AMPARs in Fmr1 KO neurons is corrected by application with the PI3K inhibitor LY294002 (50 μm for 1 h; median: WT, 35.7; KO, 44.5; WT + LY294002, 35.3; KO + LY294002, 35.6; n = 30 each; one-way ANOVA with Bonferroni's post hoc tests: *p < 0.0001, **p < 0.00001). C , Scatter plot of correlations between FMRP signals and endocytosis of AMPARs in distal dendrites shows that AMPAR internalization in Fmr1 KO is reduced by LY294002 (see red symbols, compressed on left side). Scatter plot of signals in each distal dendrite of WT neurons show substantial variation of FMRP signals. Note however that the enhanced endocytosis of AMPARs, detected if FMRP signals are relatively low, is also corrected with LY294002 application. D , Box-and-whisker plot of the distribution of constitutive endocytosis of AMPARs in distal dendrites show that enhanced constitutive endocytosis of AMPARs in Fmr1 KO neurons is not affected by application of the ERK1/2 inhibitor U0126 (20 μm for 1 h; median: WT, 40.2; KO, 50.5; WT + U0126, 43.8; KO + U0126, 53.0; n = 30 each; one-way ANOVA with Bonferroni's post hoc test: ***p < 0.0001). E , Scatter plot of correlations between FMRP signals and endocytosis of AMPARs in distal dendrites show substantial variation of FMRP signals in WT. Note that the enhanced endocytosis of AMPARs, detected if FMRP signals are relatively low, is not affected with U0126 application.
- Figure 7.
A PI3K antagonist rescues increased dendritic spine density in Fmr1 KO neurons. A , Three-dimensional reconstruction of representative dendrites from WT and Fmr1 KO neurons (18 DIV) after 3 d of treatment with vehicle (ctr) or LY294002 (LY, 10 μm) illustrates that increased protrusion density in Fmr1 KO is rescued by PI3K inhibition. Scale bar, 5 μm. B , Automated quantification using FilamentTracer software (Imaris, Bitplane) shows significantly increased protrusion density in vehicle-treated Fmr1 KO, which can be restored to WT levels by LY294002 treatment, but does not change spine morphology in WT. Bar diagrams represent average spine number per 100 μm (WT control, 38.2; WT LY294002, 38.7; KO control, 46.0; KO LY294002, 33.6; n = 30, 2 independent cultures, two-way ANOVA: significant interaction between genotype and treatment, *p = 0.001; LSD post hoc tests: *p wtctr–koctr = 0.024, ***p koctr–koLY294002 < 0.0001, p wtctr–wtLY294002 = 0.996; error bars represent SEM). C , Examples of a dendrite that was analyzed with FilamentTracer (Imaris; Bitplane) illustrate accuracy of the applied method to identify protrusions. Top, Three-dimensional reconstruction of fluorescent signals from GFP–Lifeact transfected hippocampal dendrite; middle, traced and rebuilt dendrite (white) with spines (blue); bottom, overlay of rebuilt dendrite with original image. Scale bar, 10 μm.
- Figure 8.
Proposed model for dysregulated mGluR signaling in FXS. A , B Regulation of the gp1 mGluR-dependent signal pathways PLC/ERK and PI3K/mTOR in WT. A , Under basal conditions, FMRP puts the break on PI3K activity. This is partially attributable to FMRP-mediated repression of p110β mRNA translation and synaptic localization of the catalytic subunit p110β (1). Additional mechanisms might include regulation of p110β-modulating subunits such as p85β (2) and PIKE (3) by FMRP. B , gp1 mGluR-mediated activation of PLC and PI3K pathways at the synapse. PLC and PI3K share the same substrate PIP2 in the membrane to produce either IP3 and DAG, or PIP3, respectively. The PLC product DAG can activate PKC, leading to induction of the mitogen-activate protein kinase kinase (MEK)/ERK pathway, whereas the PI3K product PIP3 recruits PH-containing kinases PDK1 and Akt to the membrane, thereby inducing their phosphorylation followed by activation of downstream signaling molecules including mTOR. Both pathways induce protein synthesis. During gp1 mGluR stimulation, PLC is activated by small G-proteins. PI3K was shown to be activated by at least two different mechanisms, the Homer–PIKE complex (4) and small G-proteins (5). Furthermore, gp1 mGluR stimulation leads to transient removal of FMRP-mediated translational inhibition by dephosphorylation of FMRP (Ceman et al., 2003; Narayanan et al., 2007, 2008). We hypothesize that, during this time window, synapses experience a twofold “boost” of PI3K activity composed of newly synthesized catalytic p110β subunits (1) as well as activation of preexisting and newly synthesized PI3K subunit molecules via PIKE and G-proteins (4 and 5). Together, enhanced ERK and PI3K activity lead to increased synaptic protein synthesis. C , The “molecular brake” FMRP is absent in Fmr1 KO, and FMRP-mediated inhibition of p110β translation and PI3K activity is removed constitutively. Increased p110β protein levels at synapses (1), which can be activated by basal levels of mGluR signaling (4 and 5), contribute to exaggerated PI3K signaling. Additionally, PI3K activity could be increased by dysregulation of p110β-modulating subunits (2 and 3), especially PIKE, in the absence of FMRP. DHPG-mediated transient increase in p110β protein expression is abolished and may partially account for loss of gp1 mGluR-dependent activation of PI3K signaling. Loss of this combined “brake” on PI3K signaling in FXS would elevate PI3K-dependent protein synthesis to a saturated level, which cannot be further increased by gp1 mGluR stimulation.
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