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Fragile X Syndrome (FXS) is an inherited neurologic disease caused by loss of Fragile X Mental Retardation Protein (FMRP), which is hypothesized to mediate negative regulation of mRNA translation at synapses. A prominent feature of FXS animal models is exaggerated signaling through group 1 metabotropic glutamate receptors (gp1 mGluRs), and therapeutic strategies to treat FXS are targeted mainly at gp1 mGluRs. Recent studies, however, indicate that a variety of receptor-mediated signal transduction pathways are dysregulated in FXS, suggesting that FMRP acts on a common downstream signaling molecule. Here, we show that deficiency of FMRP results in excess synaptic activity of phosphoinositide 3-kinase (PI3K), a downstream signaling molecule of many cell surface receptors. In Fmr1 knockout neurons, excess PI3K activity can be reduced by perturbation of gp1 mGluR-mediated signaling. Remarkably, increased PI3K activity was also observed in non-neuronal cells in the absence of gp1 mGluRs. Here, we show that FMRP regulates the synthesis and synaptic localization of p110β, the catalytic subunit of PI3K. In wild type, gp1 mGluR activation induces p110β translation, p110β protein expression and PI3K activity. In contrast, both p110β protein synthesis and PI3K activity are elevated and insensitive to gp1 mGluR stimulation in Fmr1 knockout. This suggests that dysregulated PI3K signaling may underlie the synaptic impairments in FXS. In support of this hypothesis, we show that PI3K antagonists rescue three FXS-associated phenotypes: dysregulated synaptic protein synthesis, excess AMPA receptor internalization and increased spine density. Targeting excessive PI3K activity might thus be a potent therapeutic strategy for FXS.
FXS is the most frequent inherited form of intellectual disability. It is caused by loss of function of FMRP, an mRNA binding protein involved in the regulation of translation, transport and stability of target mRNAs (Bassell and Warren, 2008; De Rubeis and Bagni, 2009). FMRP has been shown to act as a translational inhibitor of a few select target mRNAs in vitro (Laggerbauer et al., 2001; Li et al., 2001) and in vivo (Zalfa et al., 2003; Muddashetty et al., 2007). However, these results are insufficient to explain the global dysregulation of basal and neurotransmitter-induced protein synthesis (Weiler et al., 2004; Dolen et al., 2007; Muddashetty et al., 2007), or the loss of a protein synthesis requirement for gp1 mGluR-induced long-term depression (LTD) observed in the absence of FMRP (Nosyreva and Huber, 2006). The molecular mechanism whereby FMRP couples the activation of cell surface receptors to protein synthesis is unclear.
Several studies have shown exaggerated gp1 mGluR signaling in FXS animal models (Pfeiffer and Huber, 2009). The correction of FXS phenotypes by genetic or pharmacologic reduction of mGluR5 signaling (Dolen et al., 2007; D'Hulst and Kooy, 2009) supports the widely accepted ‘mGluR theory of FXS’, which postulates that excessive signaling through gp1 mGluRs underlies synaptic defects observed in the absence of FMRP (Bear et al., 2004). Targeting excess gp1 mGluR signaling is therefore a promising therapeutic strategy (Bear et al., 2008). However, none of these studies reveal how FMRP limits gp1 mGluR-induced signal transduction. While loss of FMRP-mediated regulation of protein synthesis is believed to be the cause, there is little experimental evidence in support of an underlying molecular mechanism.
Other signal transduction pathways besides gp1 mGluR signaling have recently been reported to be dysregulated in the absence of FMRP (Volk et al., 2007; Wang et al., 2008; Weinshenker and Warren, 2008). Thus, FMRP might not exclusively target gp1 mGluRs, but rather regulate a common signaling molecule downstream of multiple membrane receptors. A few studies describe aberrant gp1 mGluR-regulated downstream signaling in FXS (Hou et al., 2006; Ronesi and Huber, 2008a; Sharma et al., 2010); the underlying molecular mechanisms, though, are unknown. Furthermore, the therapeutic value of targeting the intracellular signaling pathway instead of a surface receptor has not been addressed in detail.
These previous studies suggest that FMRP may directly regulate the translation and synaptic localization of a downstream signaling molecule regulating protein synthesis. Here, we show that loss of FMRP leads to excess mRNA translation and synaptic protein expression of p110β, a catalytic subunit of PI3K, which is a key signaling molecule downstream of gp1 mGluRs and other membrane receptors. Remarkably, the basal enzymatic activity of PI3K increased three-fold at FXS synapses, and gp1 mGluR-mediated induction of p110β translation, protein levels and activity at synapses are occluded in Fmr1 KO. Antagonizing PI3K can correct three FXS phenotypes: aberrant synaptic translation, enhanced AMPA receptor endocytosis and excess spine density. Our results broaden the mGluR theory of FXS and suggest aberrant PI3K activity as a promising therapeutic target for FXS.
Puromycin was obtained from Sigma Aldrich; all other drugs were purchased from Tocris Bioscience. Radio-labeled amino acids and trinucleotides were purchased from GE Healthcare Life Sciences and PerkinElmer. L-α-Phosphatidylinositol (liver, bovine; sodium salt) was obtained from Avanti Polar Lipids. All other chemicals were purchased from Sigma Aldrich. tat-mGluR5-CT or tat-mGluR5-MUT peptides were synthesized by Invitrogen (sequence was described previously (Mao et al., 2005)). Stealth™ siRNA (Invitrogen) was designed using BLOCK-iT™ RNAi Designer (Invitrogen, sequence: sifmr1: NM_008031_stealth_298, sense: UGG CGC UUU CUA CAA GGC AUU UGU A, sictr: NM_008031_stealth_control_298, sense: UGG UUU CCA UCG GAA UUA CUG CGU A). siRNA targeting p110β was purchased from Santa Cruz Biotechnology (sc-29447).
Sequences of primers for qRT-PCR were as follows: p110β forward: 5’-ttcttttcagtgttgtgaccaag-3’, reverse: 5’-gccccgaatgtggtaagtt-3’; NR1 forward: 5’-ggcagtaaaccaggccaata-3’, reverse: 5’-agcagagccgtcacattctt-3’; PSD95 forward: 5’-ctatgagacggtgacgcaga-3’, reverse: 5’-cgggaggagacaaagtggta-3’; EGFP forward: 5’-aaggacgacggcaactacaag-3’, reverse: 5’-atgccgttcttctgcttgtcg-3’; PIKE-L forward: 5’- tgtccacaccatctaagactgaa-3’, reverse: 5’- aggattttagtttccacattttgc-3’; βActin forward: 5’-gcactcttccagccttcc-3’, reverse: 5’-ccgctcaggagcaat-3’.
Mouse monoclonal anti-Tubulin, anti-PSD95, anti-GFAP, anti-MAP2 and anti-synaptophysin antibodies, as well as rabbit polyclonal anti-mGluR5 antibodies were purchased from Millipore; mouse monoclonal anti-mGluR1 antibody was purchased from BD Biosciences. Rabbit polyclonal antibody against p110β was purchased from Santa Cruz Biotechnology. Rabbit monoclonal antibodies specific for phosphoAkt(Thr308), Akt(pan), and ERK1/2, as well as mouse monoclonal anti-phosphoERK1/2(Thr202/Tyr204) antibody were purchased from Cell Signaling Technology. Anti-PIKE-L antibody was described previously (Tang et al., 2008). Mouse monoclonal anti-FMRP antibody 7G1-1 (Brown et al., 2001) was obtained from Developmental Studies Hybridoma Bank, University of Iowa. Mouse monoclonal anti-FMRP antibody 2F5-1 (Gabel et al., 2004) (used for western blot analyses) was a kind gift from Jennifer C. Darnell. A rabbit polyclonal antibody raised against the C-terminus of FMRP (as described in (Ferrari et al., 2007)) was used for western blot analysis of polysomal gradients in supplemental Fig. S3F.
Murine flag-mCherry-FMRP (open reading frame) was subcloned from EGFP-FMRP (Antar et al., 2005), murine EGFP-p110β-3’UTR (NM_029094.2; nt 3401-4649) and murine EGFP-βActin-3’UTR (NM_007393.3; nt 1208-1860) were generated by PCR from mouse cDNA and subcloned into pEGFP-C1 (Clontech). The cDNA encoding the PH-domain of mouse Akt1 was generated by PCR from mouse cDNA (GenBank accession no. NM_009652.2, nt 285-725) and cloned into a plasmid containing cDNA encoding monomeric red fluorescent protein (mRFP) (Campbell et al., 2002) . GFPLifeact was a kind gift from Roland Wedler-Soeldner (Riedl et al., 2008).
In vitro PI 3 kinase assays were performed as described previously (Ye et al., 2000) using p110β antibody. In vitro ERK activity assays were performed using the p44/42 MAP Kinase Assay Kit from Cell Signaling Technology according to the manual. HEK293T cells were cultured in DMEM supplemented with 10% FBS. Fmr1 specific siRNA was transfected using LF2000 (Invitrogen) according to the manual. 48 hrs after transfection, cells were scraped from the dish with PBS, divided into two equal parts for PI3K and ERK activity assays, respectively. For treatment with PI3K antagonist cells were incubated with 40 μM LY294002 or LY303552 (inactive analog) for 15 min prior to further processing for PI3K assay. Cortical SNS were prepared as described (Muddashetty et al., 2007). SNS were split in two equal parts for PI3K and ERK activity assays. Cells and SNS were lysed in kinase assay buffers (for PI 3 kinase assay: 50 mM Tris-HCl, pH7.4, 40 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na3VO4, 50 mM NaF, 10 mM sodium pyrophosphate, 10 mM sodium β-glycerol phosphate, supplemented with proteinase inhibitors; for ERK activity assay: lysis buffer provided by Cell Signaling Technology). Lysates were cleared by centrifugation and protein concentrations were determined by the Bradford method. For both kinase assays, the same amount of protein was used for WT and Fmr1 KO SNS, or sifmr1 knockdown and sictr HEK293T cells, respectively. For measuring PI3K activity following various treatments, SNS were incubated for 10 min at 37°C with either 100 μM DHPG or 10 μM MPEP, or for 1 hr at 37°C with tat-mGluR5-CT or tat-mGluR5-MUT peptides (5 μM) prior to lysis (Mao et al., 2005; Ronesi and Huber, 2008a). For PI3K assays with cortical neurons, cells at 11 DIV were transfected with 10 μl control adenovirus or adenovirus carrying shPIKE-L (as described previously (Tang et al., 2008)) for 48 hrs. PI3K assays were performed with 300 μg protein as described above. To confirm PIKE-L knockdown, 80 μg of protein was used for western blot analyses using anti-PIKE-L antibody (Tang et al., 2008).
SDS-PAGE and western blots were performed as described (Muddashetty et al., 2007), and quantified using ImageJ (NIH). Signal intensities of p110β- and FMRP-specific bands were normalized to tubulin signal on the same blot. Signal intensities of phospho-proteins were normalized to total levels of the respective protein on the same blot. For this purpose, western blots were stripped after detection with the phospho-specific antibodies using 100 mM β-Mercaptoethanol, 2% SDS in 62.5 mM Tris-HCl, pH6.8 at 50°C for 30 min, and after extensive washing detected with the respective antibody specific for the total protein.
Hippocampal primary neurons were dissected from WT and Fmr1 KO mice at embryonic day 17 and cultured as described previously (Muddashetty et al., 2007). Neurons were transfected with RFPPH(Akt) at 9 days in vitro (DIV) using Lipofectamine 2000 (LF2000, Invitrogen) according to the manual. Six hours post-transfection, neurons were fixed with 4% PFA, and further processed for immunocytochemistry.
For p110β-specific staining, neurons were fixed at 17 DIV. Cells were permeabilized with 0.3% Triton X100 and blocked with 2% normal donkey serum and 4% BSA. Primary antibodies were incubated at room temperature for 1 hr (anti-p110β 1:50; anti-synaptophysin 1:300), and detected with Cy3-coupled donkey anti-rabbit, and Cy2-coupled donkey anti-mouse antibodies, respectively.
Images of immunocytochemistry on cultured neurons were acquired using a widefield fluorescent Nikon Eclipse inverted microscope with cooled CCD camera and built-in Z-drives. Stacks were deconvolved with AutoQuant X (Media Cybernetics). For quantification of signal intensities, as well as co-localization analyses, an ROI along a dendrite at least 50 μm from the cell body was chosen. Mean fluorescent intensities were determined using IPlab software (BD Biosciences) or Imaris software (Bitplane) and normalized to background staining of the same area on the coverslip. Co-localization analyses were performed using Imaris Coloc software (Bitplane). Images of FISH experiments on tissue were acquired using a Zeiss LSM510 confocal microscope, deconvolved with AutoQuant X (Media Cybernetics), and are displayed as flattened z-stacks (supplemental Fig. S4A), or as single slices (supplemental Fig. 4B,C).
Lysates for RNA Co-IPs were prepared by homogenizing cortices from mice (C57BL/6 and Fmr1 KO, backcrossed in C57BL/6J, postnatal day 17-21) in lysis buffer (20 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1 % NP40, supplemented with proteinase and RNase inhibitors) on ice. RNA Co-IPs and subsequent qRT-PCR were performed as described previously (Muddashetty et al., 2007) using the mouse monoclonal 7G1-1 antibody.
HEK293T cells were transfected with flag-mCherry-FMRP and EGFP-p110β-3'UTR or EGFP-βActin-3'UTR using Lipofectamine 2000 (Invitrogen) according to the manual. For controls, EGFP-constructs were co-transfected with flag-mCherry. 24 hrs after transfection, cells were lysed with co-IP lysis buffer (20 mM Tris-HCl pH7.4, 150 mM NaCl, 5 mM MgCl2, 1% NP40, supplemented with proteinase and RNase inhibitors) on ice. Anti-flag pulldowns were performed with anti-flag M2 affinity gel (Sigma), and analyzed with EGFP-specific qRT-PCRs as described previously (Muddashetty et al., 2007).
Gradient preparation, centrifugation, fractionation, RNA-purification and qRTPCRs were performed as described previously (Muddashetty et al., 2007).
Cortical SNS were prepared as described (Muddashetty et al., 2007), treated with the indicated antagonists or solvent for 5 min at 37°C, for some experiments followed by DHPG (100 μM) for 15 min at 37°C as indicated, and with puromycin (2 mM) for 30 min at 37°C. Metabolic labeling was carried out with 100 μCi Pro-Mix, L-(35S) in vitro cell labeling mix (GE Healthcare) for 5 min. Triplicate samples from time point 0 and 5 min were washed once in homogenization buffer, followed by lysis (100 mM Tris-HCl pH7.4, 150 mM NaCl, 0.5% deoxycholate, 1% Triton X-100, supplemented with proteinase inhibitors) and TCA precipitation. 35S-incorporation levels were estimated by scintillation counting.
The assay was performed as described previously for rat neurons (Nakamoto et al., 2007), with the following modifications: mouse WT or Fmr1 KO cells were pre-incubated with LY294002 (50 μM) or U0126 (20 μM) for 1 hr where indicated. Surface AMPARs in live neurons were labeled with a rabbit polyclonal antibody against the N-terminus of the GluR1 subunit (Calbiochem, 1:5 in conditioned media) for 15 min at 37°C, 0.5% CO2. After incubation with antagonists (1 μM TTX, 10 μM NBQX, and 50 μM APV; Tocris; as described previously (Bhattacharyya et al., 2009)) in conditioned media for 15 min at 37°C, 0.5% CO2, cells were fixed in 4% PFA and processed and analyzed as described previously (Nakamoto et al., 2007).
High density hippocampal neurons from WT and Fmr1 KO were treated for three days with 10 μM LY294002 or an equal amount of vehicle (DMSO) starting at 15 DIV. Every 24 hrs culture media was exchanged with fresh drug in conditioned media. After 48 hrs (17 DIV), cells were transfected (NeuroMag, OZBiociences, according to the manual) with GFP-Lifeact, a peptide that binds to F-Actin (Riedl et al., 2008) and can be used to visualize dendrites and spines. 16-24 hrs after transfection cells were fixed and imaged with a Nikon Eclipse inverted microscope using a 60x Plan-Neofluar objective and a cooled CCD camera (Quantix, Photometrics). Images were taken as Z-stacks (31 stacks, 0.2 μm per stack) and deconvolved using AutoQuant 2X (Media Cybernetics). Dendrites and spines (>40 μm distance from cell body, the analyzed region including a main dendrite of at least 50 μm length) were traced and analyzed using FilamentTracer (Imaris software, Bitplane). Total protrusion density (including spines and filopodia, classified as described previously (Harris et al., 1992)) was displayed as average number per 100 μm.
All statistics were performed using SSPS 17.0. Statistical tests used and n of experiments are indicated in the figure legends and in the text. All t-tests were two-tailed. All data were tested for normal distribution and homogeneity of variances with Levene's tests. Nonparametric tests were used for Fig. S2A,B and S3H, for which the data was not normally distributed. In all other instances, when data sets did not meet the criterion of normal distribution and/or homogenous variances, tests were performed on square-root transformed data (Fig. 2A, S2C, ,3I,3I, ,7B)7B) or lg-transformed data (Fig. 2B, S5F). Effects were considered significant when the p-value was below 0.05. Within each individual experiment, means were normalized to the mean of WT, WT control or control, where applicable, depending on the experiment. Diagrams represent means of n normalized experiments. Error bars represent standard error of the mean (s.e.m.) of n independent experiments.
The molecular mechanisms underlying exaggerated receptor-mediated signaling and protein synthesis in the absence of FMRP are unknown. Some reports have revealed defects in PI3K/mTOR signaling (Ronesi and Huber, 2008a; Sharma et al., 2010), whereas others implicate impaired ERK signaling in FMRP deficient cells (Hou et al., 2006; Kim et al., 2008). Both pathways are crucial to regulate mGluR-induced protein synthesis in normal brain (Klann and Dever, 2004), yet it remains controversial whether one pathway is predominantly dysregulated in FXS. However, no study has directly examined or compared the activity of PI3K and ERK in FXS. Here, we quantified the activity of these two major downstream signaling molecules at synapses from Fmr1 KO mice. PI3K assays were performed with p110β, the predominant neuronal isoform of PI3K catalytic subunits, immunoprecipitated from Fmr1 KO and WT synaptoneurosomes (SNS). The efficiency of the precipitated p110β protein to convert phosphoinositol (PI) into phosphoinositol-3-phosphate (PI-3-P) was assessed by using radio-labeled ATP to quantify PI-3-P production on thin-layer chromatographs (Rong et al., 2003). We show that PI3K activity in Fmr1 KO SNS is markedly increased (threefold) compared to WT (Fig. 1A; n=8, *p=0.035; paired t-test; for control experiment see supplemental Fig. S1A). In contrast, phospho-ERK1/2 immunoprecipitated from Fmr1 KO SNS displayed no difference in the efficiency to phosphorylate recombinant ELK-1 compared to WT SNS (Fig. 1B; n=8, p=0.20; paired t-test; for control experiment see supplemental Fig. S1B). To confirm these initial findings, we further examined PI3K activity at synapses in cultured WT and Fmr1 KO hippocampal neurons. Accumulation of the PI3K products PI-(3,4)-bisphosphate (PIP2) and PI-(3,4,5)-triphosphate (PIP3) leads to recruitment of Pleckstrin homology (PH)-domain-containing proteins to sites of PI3K enzymatic activity (Hawkins et al., 2006), e.g. to synaptic membranes. As an in vivo measure to assess PI3K activity at synapses, we analyzed the subcellular distribution of a recombinant PH-domain of murine Akt1 fused to red fluorescent protein (RFP-PH(Akt)) in hippocampal WT and Fmr1 KO neurons. In Fmr1 KO neurons, co-localization of RFP-PH (Akt) with the synaptic marker synaptophysin was significantly increased, suggesting elevated synaptic PIP3 levels as a consequence of exaggerated PI3K activity at synapses (Fig. 1C; n=5 independent experiments, 6-12 cells each, ***p=0.00002; paired t-test). Total RFP-PH(Akt) levels were not changed in Fmr1 KO neurons compared to WT (supplemental Fig. S1C). Excess synaptic PI3K activity was further corroborated by significantly increased phosphorylation of the downstream signaling molecule Akt in SNS from Fmr1 KO mice (Fig. 1D; n=6, *p=0.014; paired t-test), whereas ERK phosphorylation was not altered (Fig. 1E; n=7, p=0.18; paired t-test). These results indicate that exaggerated gp1 mGluR signaling in FXS does not lead to excess signaling of downstream pathways in general. In contrast, the key downstream signaling molecule PI3K, but not ERK1/2, is specifically overactive at FMRP-deficient synapses.
To investigate whether excess PI3K activity depends on the presence of gp1 mGluRs, we performed a similar set of experiments in the embryonic kidney cell line HEK293T (Fig. 1F-I). HEK293T cells express p110β and FMRP, but neither of the gp1 mGluRs (supplemental Fig. S1D). FMRP expression in these cells was transiently reduced by siRNA-mediated Fmr1 knockdown. Quantification of FMRP levels by western blot analysis showed a significant decrease of FMRP after 48 hrs (57.33±4.57%, n=5, *p=0.0007, paired t-test; FMRP signals were normalized to tubulin signals on the same blot; western blots are shown for each experiment). As shown above for SNS, we also observed significantly increased PI3K enzymatic activity (Fig. 1F; 2.6 fold; n=7, *p=0.039; paired t-test), but not ERK1/2 enzymatic activity (Fig. 1G; n=7; p=0.32, paired t-test) in in vitro assays on lysates from HEK293T cells with reduced FMRP expression. Furthermore, Fmr1 knockdown led to elevated phosphorylation of endogenous Akt (Fig. 1H; n=5, *p=0.018; paired t-test), but not ERK1/2 (Fig. 1I; n=6, p=0.29; paired t-test), similar as shown for SNS (Fig. 1D, E). These results indicate that excess PI3K activity and downstream signaling as a consequence of reduced or absent FMRP can occur independently of gp1 mGluR signaling.
Antagonists of gp1 mGluR signaling can rescue FXS-associated neuronal phenotypes in animal models (McBride et al., 2005; Yan et al., 2005; Nakamoto et al., 2007; de Vrij et al., 2008b). To assess whether gp1 mGluR signaling can regulate excessive PI3K activity at FMRP-deficient synapses, we investigated the effects of different perturbations of mGluR signaling; each showed reduction of PI3K activity in Fmr1 KO. Antagonizing mGluR5-mediated signaling with 2-methyl-6-(phenylethenyl)-pyridine (MPEP) had no significant effect on WT, but significantly reduced PI3K function in Fmr1 KO (Fig. 2A; n=4, 2-way ANOVA with LSD posthoc tests: p(wtctr-wtMPEP)=0.327, *p(wtctr-koctr)=0.033, *p(koctr-koMPEP)=0.012), indicating that inhibition of gp1 mGluRs can correct excessive PI3K activity in the absence of FMRP, which is consistent with the mGluR theory. Synaptic mGluR-dependent induction of PI3K can be regulated by different mechanisms, such as signaling through heterotrimeric G proteins (Hawkins et al., 2006) or a neuronal complex including mGluR5, the scaffolding protein Homer and the PI3K enhancer PIKE-L (Rong et al., 2003; Ronesi and Huber, 2008a). To investigate whether excess PI3K activity in Fmr1 KO SNS is regulated by the mGluR-Homer-PIKE signaling complex, we used two different approaches to interfere with this complex: perturbation of mGluR5-Homer interactions with a peptide comprising the C-terminal sequence of mGluR5 (Mao et al., 2005; Ronesi and Huber, 2008a) (Fig. 2B) and short-hairpin (sh) RNA-mediated knockdown of PIKE-L in cultured cortical neurons (Fig. 2C). Perturbation of mGluR5-Homer associations reduced PI3K activity under basal conditions independently of genotype, although PI3K activity was still increased in Fmr1 KO compared to WT SNS. Quantification of PI3K assays demonstrated a significant effect of the C-terminal peptide, which reduced PI3K activity in both WT and Fmr1 KO, and, in line with our previous results (Fig. 1A, F), a significant effect of genotype, with increased PI3K activity in Fmr1 KO SNS compared to WT regardless of the presence of the C-terminal peptide (Fig. 2B; n=4, 2-way-ANOVA, significant effects of treatment: *p<0.001 and genotype *p=0.014, but no significant interaction between genotype and treatment: p=0.143, additional analyses shown in supplemental Fig. S2A,B). Of interest, quantification of shRNA-mediated PIKE-reduction showed a significant interaction between genotype and treatment to reduce the excess PI3K activity observed in Fmr1 KO. In PIKE-depleted Fmr1 KO neurons, there was a significant decrease in PI3K activity (54.5±9%), but only a slight decrease in PIKE-depleted WT neurons (93±3%) (Fig. 2C; n=3, 2-way ANOVA with Tukey HSD posthoc tests: *p(wtctr-koctr)=0.002, *p(koctr-koPIKE)=0.007). However, PI3K activity is still increased in Fmr1 KO compared to WT after knockdown of PIKE (139±14% of WT), suggesting an additional downstream defect leading to excess PI3K activity in Fmr1 KO.
We hypothesize that excess PI3K activity in Fmr1 KO is due to elevated translation of FMRP associated mRNAs that encode PI3K subunits or modulating proteins. Of interest, PIKE was identified as an FMRP target mRNA in a high-throughput screen (J.Darnell, personal communication), and here, we show by quantitative FMRP-specific co-immuoprecipitation from brain lysates that PIKE-L mRNA specifically associates with FMRP (supplemental Fig. S2C). A previous study has recently reported elevated levels of PIKE-S in hippocampal lysates from Fmr1 KO neurons, and also suggested increased levels of both PIKE-S and -L in postsynaptic densities from Fmr1 KO (Sharma et al., 2010). In line with this previous observation, here we could show that PIKE-L protein levels are significantly enriched in SNS from Fmr1 KO (supplemental Fig. S2D). Further work is needed to assess whether possible dysregulation of PIKE-L mRNA translation in the absence of FMRP may contribute to excess PI3K activity in Fmr1 KO neurons. However, since PI3K activity is exaggerated in non-neuronal cells in the absence of gp1 mGluRs (Fig. S1D) and PIKE-L (our own data not shown, and in (Rong et al., 2003)), our results imply that excess PI3K activity in Fmr1 KO might be partly attributed to a downstream defect within the pathway.
Gp1 mGluR-mediated activation of synaptic translation was shown to be occluded in Fmr1 KO (Muddashetty et al., 2007). Since PI3K is necessary for mGluR-induction of protein synthesis, we investigated whether mGluR-induced PI3K signaling was also dysregulated in the absence of FMRP and might thus underlie dysregulated translation. We analyzed PI3K activity following stimulation of gp1 mGluRs with DHPG in WT and Fmr1 KO SNS. DHPG treatment led to a two-fold increase of PI3K enzymatic activity in WT SNS (Fig. 2D; n=4, *p=0.033; paired t-test), but failed to activate PI3K in KO SNS (Fig. 2E). Based on our findings that PI3K activity is also increased in the absence of gp1 mGluRs this suggests that excess synaptic PI3K activity in Fmr1 KO might not be further inducible by gp1 mGluR stimulation, and thus might underlie the loss of gp1 mGluR-induced protein synthesis. This finding is consistent with an earlier report showing that DHPG does not induce the phosphorylation of PDK1, which is also induced by PI3K, in hippocampal slices from Fmr1 KO mice (Ronesi and Huber, 2008a). Of interest, we observe a significant reduction of PI3K activity in Fmr1 KO SNS upon DHPG stimulation (Fig. 2E; n=4, *p=0.049; paired t-test). The significant reduction in PI3K activity observed in Fmr1 KO SNS could be due to agonist-mediated mGluR5 desensitization, which was shown to decrease PI3K activity (Peavy et al., 2002). Collectively, our findings indicate that mGluR-mediated control of PI3K activity is dysregulated in the absence of FMRP.
p110β is the catalytic subunit of PI3K, and a putative FMRP-target mRNA (Miyashiro et al., 2003). We investigated whether p110β expression was dysregulated in Fmr1 KO, which could contribute to the excess PI3K activity. Synaptic p110β protein levels in cultured hippocampal neurons were analyzed by quantifying p110β co-localization with the synaptic marker synaptophysin (Fig. 3A-E, specificity of the antibody was verified by siRNA-mediated knockdown of FMRP in HEK293T cells, supplemental Fig. S3A). In both WT and Fmr1 KO neurons, p110β showed a punctate distribution within dendrites and co-localized with synaptophysin, which is consistent with its known recruitment to active signaling complexes at synapses (Fig. 3A, B). Quantitative analyses of distal dendrites (>50 μm from cell body) demonstrated significantly increased synaptic localization of p110β protein in Fmr1 KO neurons (Fig. 3C; 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), whereas total synaptophysin levels were unchanged (Fig. 3D; n=43; p=0.73, independent t-test). Furthermore, the Mander's coefficient for p110β, but not synaptophysin is significantly higher in Fmr1 KO dendrites (supplemental Fig. S3B), indicating that the percentage of p110β signal overlapping with synaptophysin is increased in the absence of FMRP. Using 3D-reconstruction, we analyzed p110β protein levels within synaptophysin punctae, here referred to as “synapses”. While the mean intensity of p110β within synapses was increased (Fig. 3E; n=43, **p=0.004, independent t-test), the relative number of p110β-positive synapses was unchanged (supplemental Fig. S3C), implying that there is no imbalance between synapses recruiting PI3K versus synapses not recruiting PI3K in Fmr1 KO neurons. Further quantitative analyses of immunostainings in hippocampal neurons showed that total dendritic p110β levels are also increased in the absence of FMRP (supplemental Fig. S3D).
Western blot analyses of cortical SNS corroborated the above immunocytochemical data by showing that p110β levels were significantly increased in Fmr1 KO (Fig. 3F; n=5, *p=0.042; paired t-test). This is in line with a previous report that showed increased p110β levels in total hippocampal lysates from Fmr1 KO mice, but did not analyze synaptic levels (Sharma et al., 2010). Apart from increased synaptic levels in Fmr1 KO mice, we could also detect increased p110β protein levels in HEK293T cells following siRNA-mediated knockdown of FMRP (Fig. 3G; n=5, *p=0.005; paired t-test), further suggesting that gp1 mGluRs are not necessary for excess p110β expression in the absence of FMRP.
As mentioned above, a previous report suggested that p110β mRNA might be a target mRNA of FMRP, although this could not be validated using specific in vitro assays (Miyashiro et al., 2003). Here, we demonstrate that FMRP associates with p110β mRNA in vivo using FMRP-specific coimmunoprecipitation from brain lysate followed by qRT-PCR analyses (Fig. 3H, n=6, 2-way ANOVA with Tukey HSD posthoc analyses: *p(p110β)=0.003, p(NR1)=0.884). Furthermore, RNA-pulldowns in HEK293T cells using recombinant p110β mRNA and FMRP demonstrated that the 3'UTR of p110β is sufficient to mediate the association of p110β mRNA with FMRP in vitro (Fig. 3I; n=4, 2-way ANOVA with Tukey HSD posthoc analyses: *p(p110β)=0.001, p(βActin)=0.798).
The role of FMRP in p110β mRNA translation has not been studied previously. To investigate whether p110β translation was regulated by FMRP, we quantified translation levels in SNS by analyzing p110β mRNA distribution in polysomal fractions of sucrose gradients (as described previously (Muddashetty et al., 2007), additional analyses of polysomal gradients in cortical SNS shown in supplemental Fig. S3E-G). The association of p110β mRNA with synaptoneurosomal polysomes is sensitive to puromycin treatment, which disrupts translocating ribosomes, indicating that it is actively translated (Fig. 3J; n=3, *p=0.033; paired t-test). In Fmr1 KO SNS, the association of p110β mRNA with heavy polysomes is significantly increased compared to WT (Fig. 3K; n=5, *p(PSD95)=0.029, p(NR1)=0.89,*p(110β)=0.043; paired t-tests), suggesting enhanced basal translation of p110β mRNA in the absence of FMRP. In contrast, total p110β mRNA levels were not altered in Fmr1 KO cortical lysates (supplemental Fig. S3H). Apart from these results in mouse cortical synaptoneurosomes, qRT-PCR analyses of polysomal fractions from HEK293T cells following siRNA-mediated reduction of FMRP also demonstrate that association of p110β mRNA with puromycin-sensitive polysomes is significantly elevated (Fig. 3L; 2-way ANOVA with Tukey HSD posthoc analyses: *p(ctruntr-ctrpuro)=0.002, *p(ctruntr-KDuntr)=0.001, *p(KDuntr-KDpuro)<0.001). Our findings in HEK293T cells support our hypothesis that gp1 mGluRs or other neuron-specific receptors might not be driving the dysregulated p110β mRNA translation and protein expression in FMRP-deficient cells. Of note, the effects on p110β protein levels and translation are larger after transient FMRP-reduction in HEK293T cells than in the Fmr1 KO SNS (protein levels: 2.5fold increase in cells vs. 20% increase in SNS; polysomal RNA: 39% increase in cells vs 28% in SNS). This might be due to additional regulation of p110β expression at cortical synapses and/or compensatory effects in the total Fmr1 KO compared to an acute knockdown of FMRP in HEK293T cells. Nonetheless, our results suggest that excessive translation of the PI3K catalytic subunit p110β in the absence of FMRP leads to increased p110β protein levels at synapses and could therefore contribute to aberrant and dysregulated synaptic PI3K activity. Fluorescent in situ hybridization analyses in cortical and hippocampal mouse brain sections also showed that p110β mRNA can be localized to dendrites in WT and Fmr1 KO (supplemental Fig. S4), consistent with our observations of FMRP-mediated translational regulation of p110β mRNA in synaptic fractions.
Stimulation of gp1 mGluRs leads to activation of PI3K signaling at synapses by recruiting catalytic p110β molecules into receptor protein complexes at the membrane (Rong et al., 2003; Hawkins et al., 2006). FMRP has been shown to play a crucial role for the gp1 mGluR-induced protein synthesis (Muddashetty et al., 2007). We therefore analyzed whether, besides recruitment of existing p110β protein into receptor protein complexes, gp1 mGluR signaling could additionally lead to increased p110β mRNA translation, protein expression and subsequently elevated p110β protein levels at synapses. Treatment of WT SNS with the gp1 mGluR agonist DHPG for 15 min led to increased p110β protein levels (Fig. 4A; n=6, *p=0.008; paired t-test) and induced a significant shift of p110β mRNA into puromycin-sensitive fractions (Fig. 4B; n=6, *p=0.028; paired t-test). In contrast, in Fmr1 KO SNS, gp1 mGluR-mediated increase in p110β protein expression (Fig. 4C) or translation (Fig. 4D) were occluded (C: n=5, p=0.613, D: n=3, p=0.21; paired t-tests). The loss of sensitivity of p110β mRNA translation and protein expression to gp1 mGluR stimulation in the absence of FMRP parallels the loss of PI3K activation upon gp1 mGluR stimulation observed in Fmr1 KO SNS (Fig. 2E). We thus suggest that excess p110β mRNA translation, protein synthesis and consequently activity under basal conditions might occlude gp1 mGluR-induced PI3K activation that is necessary to stimulate protein synthesis.
A prominent phenotype of Fmr1 KO mice is excess and dysregulated protein synthesis (Dolen et al., 2007; Muddashetty et al., 2007), which is hypothesized to underlie many neuronal defects observed in FXS. PI3K activates mTOR and cap-dependent translation (Costa-Mattioli et al., 2009), and is therefore an important upstream regulator of protein synthesis. To examine whether excessive PI3K signaling might cause dysregulated translation in Fmr1 KO, we used metabolic radio-labeling in cortical SNS to quantify the effects of different signal transduction agonists on translation rates. We observed a significant increase of overall synaptic protein synthesis rates in Fmr1 KO SNS compared to WT (Fig. 5A; n=6, *p=0.03; paired t-test), which was similar to the effects observed previously in brain slices from Fmr1 KO mice (Dolen et al., 2007). Pre-incubation of Fmr1 KO SNS with inhibitors of either mGluR5 (MPEP, Fig. 5B) or mGluR1 (LY367385, Fig. 5C) reduced synaptic Fmr1 KO translation to WT levels, indicating that increased translation rates were modulated by gp1 mGluR-dependent signaling pathways (Fig. 5B,C; 2-way ANOVAs, B, MPEP: n=8, *p(wtuntr-kountr)=0.043, *p(kountr-koMPEP)=0.002; C, LY367385: n=5,*p(wtuntr-kountr)=0.04, *p(kountr-koLY367)=0.007; Tukey HSD posthoc tests). This is in line with our results showing that antagonizing gp1 mGluR signaling significantly reduces activity of PI3K, a crucial regulator of protein synthesis, in Fmr1 KO SNS (Fig. 2A-C). In contrast, an NMDA receptor-specific antagonist (APV), failed to reduce augmented translation in KO (Fig. 5D; n=3, 2-way-ANOVA: no significant interaction between genotype and treatment; p=0.782; also see supplemental Fig. S5A, B), further corroborating that excessive synaptic translation in Fmr1 KO is due to exaggerated signaling downstream of gp1 mGluRs. Similar to the effect of Gp1 mGluR antagonists (Fig. 5B,C), antagonizing PI3K signaling with two different drugs, LY294002 (Fig. 5E) and wortmannin (Fig. 5F), could specifically rescue the excessive synaptic translation in Fmr1 KO (Fig. 5E, LY294002: n=6, *p(wtuntr-kountr)=0.001, *p(kountr-koLY294)=0.026; Fig. 5F, wortmannin: n=6, *p(wtuntr-kountr)<0.001, *p(kountrkowort)=0.002; 2-way ANOVAs with Tukey HSD posthoc tests), whereas an inactive analogue of LY294002 (LY303511) did not show any effect (supplemental Fig. S5C). In contrast, a specific antagonist of ERK1/2 signaling (U0126) reduced protein synthesis levels independently of genotype to the same extent (Fig. 5G; n=5, 2-way ANOVA: significant effects of treatment: *p=0.001, and of genotype: *p=0.001, but no significant interaction between genotype and treatment: p=0.68; also see supplemental Fig. S5D, WT: 0.89±0.05, KO: 0.88±0.03), and ratios of KO to WT translation rates were the same before and after U0126 treatment (supplemental Fig. S5E; n=5, ctr: 1.21±0.04, U0126: 1.22±0.12). Together, our results suggest that excess PI3K activity causes excess basal translation in FXS, and might therefore underlie the dysregulated basal and stimulus-induced protein synthesis in the absence of FMRP. Furthermore, treatment with rapamycin, which inhibits the PI3K downstream signaling molecule mTOR, significantly reduced translation in KO, but not in WT, supporting the hypothesis that PI3K signaling is enhanced in the absence of FMRP (supplemental Fig. S5F). This result is also in line with a previous study reporting increased phosphorylation and activity of mTOR in the absence of FMRP (Sharma et al., 2010). Interestingly, this study showed that gp1 mGluR-induced mTOR phosphorylation, which is absent in Fmr1 KO, could be restored with a PI3K inhibitor. Here, we show that the PI3K antagonist wortmannin rescues the translational response of Fmr1 KO SNS to gp1 mGluR stimulation, whereas treatment with an ERK1/2 antagonist did not rescue DHPG-stimulated translation in the absence of FMRP (Fig. 5H; Wort: n=4, one-way ANOVA, *p=0.012, Tukey's posthoc tests: *p(untrctrwortctr)=0.045, *p(wortctr-wortDHPG)=0.010; U0126: n=4, one-way ANOVA, *p=0.001, Tukey's posthoc tests: *p(untrctr- U0126ctr/DHPG)=0.006/0.001, *p(untrDHPG-U0126ctr/DHPG)=0.019/0.004). In WT, inhibitors of either ERK1/2 or PI3K abolished DHPG-induced translational increase (Fig. 5I; Wort: n=4, one-way ANOVA, *p=0.002, Tukey's posthoc tests: p(untrctr-untrDHPG)=0.006, *p(wortctrwortDHPG)=0.004; U0126: n=3, one-way ANOVA, *p<0.001, Tukey's posthoc tests: *p(untrctruntrDHPG)=0.029, *p(untrctr/DHPG-U0126DHPG)=0.022/0.001). These data further corroborate that both ERK- and PI3K/mTOR-pathways are crucial to activate translation in normal brain, yet impaired signaling through PI3K/mTOR underlies the dysregulated translation in FXS.
The observation that PI3K inhibitors, but not ERK1/2 inhibitors, could rescue dysregulated protein synthesis, a fundamental phenotype in FXS believed to underlie neuronal deficits observed in animal models as well as in patients, led us to further investigate the therapeutic potential of PI3K antagonists for FXS-associated defects. We analyzed the effect of the PI3K antagonist LY294002 on two important synaptic phenotypes in Fmr1 KO neurons: (1) aberrant AMPA receptor internalization (Nakamoto et al., 2007), and (2) increased protrusion density in Fmr1 KO dendrites (Antar et al., 2006; de Vrij et al., 2008b).
We have previously shown that siRNA-mediated FMRP-deficiency leads to aberrant internalization of AMPA receptors in rat hippocampal neurons, and that the mGluR5 antagonist MPEP rescued increased levels of internalized AMPARs (Nakamoto et al., 2007). Here, we investigated whether reduction of excess PI3K activity could also correct this phenotype. First we showed that in line with our observations in the rat hippocampal neuron knockdown assay, Fmr1 KO mouse hippocampal neurons similarly display excessive constitutive AMPAR internalization compared to WT neurons (Fig. 6A, B). Treatment with the PI3K inhibitor LY294002 (50 μM, 1 hr) reduced AMPAR endocytosis in Fmr1 KO neurons to WT levels [Fig. 6A-C; n=30, Bonferroni's t- tests: *p< 0.0001 (WTctr-KOctr), **p<0.00001 (KOctr-KO_LY294002)]. Notably, in WT neurons, FMRP expression levels in dendrites correlated with AMPAR internalization, and antagonizing PI3K had an effect on AMPAR surface expression only in neurons with low dendritic expression of FMRP, i.e. high levels of internalized AMPAR (Fig. 6C). As observed for the rescue of excess and dysregulated translation (see above), the effect was specific for PI3K antagonists, whereas the ERK1/2 inhibitor U0126 (20 μM, 1 hr) did not rescue aberrant AMPAR internalization in Fmr1 KO neurons (Fig. 6D, E; n=30, Bonferroni's t-test: *p<0.0001).
A prominent feature of FMRP-deficient neurons in humans (Irwin et al., 2001) and mice (Comery et al., 1997) is altered dendritic spine morphology, including higher density, and immature appearance of spines, which can be corrected by genetic or pharmacologic reduction of mGluR5 signaling (Dolen et al., 2007; de Vrij et al., 2008b). Using automated software (FilamentTracer in Imaris, Bitplane), we analyzed the overall protrusion density in WT and Fmr1 KO hippocampal neurons in vitro following a three day-treatment with the PI3K inhibitor LY294002 (10 μM, 3d) (Fig. 7A-C). To assess a potential value for a therapeutic strategy in FXS patients, a prolonged incubation with low doses of the inhibitor, as opposed to higher doses of shorter duration, was used to minimize effects on WT morphology, as well as to reduce possible side-effects on neuronal function in WT and KO neurons. To visualize spines, cells were transfected with GFP-Lifeact, a small peptide that binds F-Actin without altering actin morphology or dynamics (Riedl et al., 2008; Era et al., 2009; Vidali et al., 2009). We detected a significant increase in dendritic protrusions (including spines and filopodia) in Fmr1 KO neurons, and antagonizing PI3K signaling with LY294002 could rescue the elevated protrusion density observed in Fmr1 KO neurons. In contrast, LY294002 had no effect on WT neurons (Fig. 7B; n=30, LSD post-hoc tests:, *p(WTctr-KOctr)=0.024;***p(koctr-koLY294)<0.0001;, p(wtctr-wtLY294)=0.996). These results indicate that exaggerated PI3K activity at synapses may underlie gp1 mGluR-dependent deficits in synapse morphology in the absence of FMRP.
Here, we report a novel function for FMRP to control the mRNA translation, synaptic localization and enzymatic activity of the catalytic PI3K subunit p110β. These findings have three significant implications. Firstly, this provides an initial indication towards a molecular mechanism underlying exaggerated signaling and dysregulated protein synthesis in FXS. In the absence of FMRP, PI3K is overactive at synapses, leading to excessive protein synthesis and occlusion of agonist-induced protein synthesis. Secondly, this data broadens the mGluR theory of FXS, which posits that FMRP acts as a brake on mGluR signaling regulating protein synthesis. We expand this hypothesis by showing that FMRP restrains the common downstream signaling molecule PI3K, which may explain features of dysregulated translation mediated by other neurotransmitters. Finally, antagonists of PI3K, a number of which are currently being evaluated in clinical oncology trials, may thus be considered as a potential therapeutic modality for FXS.
The mGluR theory of FXS proposes that many FXS-related phenotypes in mice and, by extension, human patients originate in exaggerated signaling through gp1 mGluRs (Huber et al., 2002; Bear et al., 2004). However, the underlying molecular mechanisms have not been revealed so far. Here, we show that FMRP-deficiency results in excessive basal PI3K activity at synapses (Fig. 1A,C), which we propose represents the major cause of the exaggerated mGluR-mediated protein synthesis in FXS. We hypothesize that elevated synaptic p110β protein levels in the absence of FMRP increase the amount of p110β subunits recruited into gp1 mGluR signaling complexes at synapses under basal conditions, and thus contribute to excess PI3K activity resulting in exaggerated mGluR signaling and increased basal protein synthesis in FXS (Fig. 8). This hypothesis is supported by our data showing that excessive PI3K activity in the absence of FMRP is modulated, but not driven or caused by overactive gp1 mGluRs. We show that excess PI3K activity in the absence of FMRP can occur independently of gp1 mGluRs (Fig. 1F). Furthermore, our results demonstrate that perturbation of mGluR-Homer-PIKE signaling decreases PI3K activity in both WT and KO, but cannot reduce the observed exaggerated PI3K activity in Fmr1 KO to WT levels (Fig. 2B). We speculate that upon blockage of the mGluR5-Homer-PIKE pathway in Fmr1 KO, excess p110β protein can still be activated via small G proteins, a known alternative pathway mediating gp1 mGluR-induced activation of PI3K ((Hawkins et al., 2006), see Fig. 8). We speculate that this alternative pathway may be sufficient to induce LTD in Fmr1 KO upon blockage of Homer signaling (Ronesi and Huber, 2008a), due to excess PI3K. However, mGluR-Homer signaling is necessary for LTD in WT because PI3K activity is limited by FMRP.
Finally, our analysis of synaptic p110β localization in cultured Fmr1 KO neurons demonstrates that p110β intensity within synaptophysin punctae is increased in Fmr1 KO, whereas the relative number of p110β positive synapses was unchanged (Fig. 3E, S3C). This data indicates that in the absence of FMRP, the population of synapses recruiting p110β into activated receptor complexes is unchanged, but that, due to increased p110β protein levels in dendrites (Fig. S3D), more p110β subunits are translocated into these p110β-recruiting receptor complexes. This suggests that the number of synapses containing actively signaling gp1 mGluRs is not increased in FMRP-deficient neurons, and is in line with the hypothesis that excess PI3K activity at FXS synapses is not caused by generally elevated mGluR activity, but rather by increased synaptic p110β expression.
This study provides evidence for a molecular mechanism that could also account for other dysregulated neurotransmitter-mediated signaling pathways in FXS. We speculate that aberrant regulation of p110β provides a molecular explanation for several recent studies in Fmr1 KO mice, which reported dysregulated neuronal plasticity and signal transduction downstream of the mGluR1/5-Homer-PIKE complex (Ronesi and Huber, 2008a; Sharma et al., 2010), of D1 dopamine receptors (Wang et al., 2008), of Gq-proteins (Volk et al., 2007), and of Ras (Hu et al., 2008). PI3K regulates all of these pathways. We propose that aberrant signal transduction in FXS is caused by exaggerated p110β protein levels, rather than dysregulated upstream receptors leading to PI3K activation. While up-regulated PIKE-L levels (supplemental Fig. S2C,D, and (Sharma et al., 2010)) could also contribute to excess PI3K activity downstream of gp1 mGluRs (Fig. 2C), they do not explain dysregulation of these other pathways.
Here, we show that gp1 mGluR stimulation increases mRNA translation and protein levels of the catalytic subunit p110β in WT SNS, but not in Fmr1 KO SNS (Fig. 4). This reveals a previously unknown mechanism for gp1 mGluR-mediated stimulation of PI3K activity, suggesting that in WT, gp1 mGluR signaling increases PI3K activity by at least two different mechanisms: (1) recruitment of pre-existing p110β into synaptic receptor protein complexes as shown previously (Rong et al., 2003), and (2) increased synthesis of p110β protein as shown here (Fig. 4A,B). Furthermore, it suggests that increased p110β mRNA translation contributes to the excess PI3K activity in FXS. Our work provides the first experimental indication that the loss of FMRP leads to altered functional activity of a protein encoded by its target mRNA. Apart from the FMRP target mRNA p110β (suggested by (Miyashiro et al., 2003), and shown here), an inhibitory subunit of PI3K, p85β, was identified in a screen for putative FMRP targets (Brown et al., 2001). Furthermore, we show that FMRP associates with PIKE-L mRNA and that PIKE-L protein levels are increased at Fmr1 KO synapses (as shown previously: J. Darnell, personal communication, and (Sharma et al., 2010)). FMRP might thus regulate PI3K activity at multiple steps (Fig. 8C).
In neurons, the PI3K/mTOR pathway and the PKC/ERK pathway represent two main targets downstream of gp1 mGluRs driving the activation of protein synthesis (Banko et al., 2006). Our study provides much experimental evidence that basal ERK1/2 activity is not altered in SNS from Fmr1 KO mice, whereas PI3K activity is significantly increased. Of note, two earlier studies suggested that loss of FMRP leads to dysregulated ERK1/2 phosphorylation (Hou et al., 2006; Kim et al., 2008); however, neither study directly examined and compared PI3K and ERK1/2 activity. Gp1 mGluR activation of ERK1/2 is mediated by PKC which in turn is activated by phospholipase C (PLC) (Fig. 8B). PI3K and PLC compete for the same substrate in the membrane, PI(4,5)P2, and excessive PI3K protein at Fmr1 KO synapses could therefore tip the balance between these two pathways towards PI3K signaling. While our results support the important role played by ERK1/2 for mGluR-stimulated protein synthesis in WT (Fig. 5G), they indicate that it is not the ERK pathway that is primarily dysregulated in FXS. Nevertheless, this does not rule out possible indirect effects on ERK signaling that vary between different cell types or developmental status, and therefore make ERK phosphorylation in lymphocytes, for example, a valid biomarker in FXS patients (Weng et al., 2008). However, we show that interfering with PI3K signaling, but not ERK1/2, rescues dysregulated synaptic translation and corrects exaggerated constitutive internalization of AMPA receptors in Fmr1 KO neurons, supporting the hypothesis that FMRP has a direct effect on PI3K activity, but not on ERK1/2.
A key phenotype in FMRP-deficient neurons is dysregulated synaptic protein synthesis, which results in increased basal protein synthesis in brain slices and at synapses, and loss of neurotransmitter-induced protein synthesis (Weiler et al., 2004; Dolen et al., 2007; Muddashetty et al., 2007) and Fig. 5). We hypothesize that FMRP controls protein synthesis-dependent regulation of synaptic morphology and function through regulation of PI3K signaling. We substantiate this hypothesis by showing that PI3K antagonists can reduce exaggerated basal translation at Fmr1 KO synapses, and restore translational responsiveness to mGluR stimulation. Dysregulated synaptic protein synthesis was proposed to cause many, if not all, neuronal defects in FXS (Ronesi and Huber, 2008b). We therefore suggest that reducing PI3K signaling pathways might be a promising therapeutic strategy to treat FXS in humans. In support of this hypothesis, we show that PI3K antagonists can rescue two protein synthesis-dependent and physiologically relevant phenotypes in hippocampal Fmr1 KO neurons: increased number of internalized AMPA receptors (Nakamoto et al., 2007), and excessive dendritic spine density (Grossman et al., 2006). Altered spine morphology is a prominent FXS-associated phenotype, which is not only observed in all animal models for FXS, but importantly also in human patients (Irwin et al., 2001). Importantly, increased spine density in Fmr1 KO can be rescued using low doses of LY294002 (10 μM, five times lower than the usual dose) in a three day-treatment without affecting WT neurons. The absence of any effect on WT neurons after chronic treatment with a PI3K inhibitor over several days corroborates the potential therapeutic value of antagonizing PI3K. Another study detected rescue of spine morphology 4 hrs after treatment with a very high dose of the mGluR5 inhibitor MPEP (200μM, twenty times more than the usually employed effective dose of 10 μM) (de Vrij et al., 2008a), but the therapeutic value of these findings might be questioned, as for treatment in human patients, drugs would have to be taken chronically, and lower doses are generally beneficial to minimize potential side effects and costs.
PI3K inhibitors have been intensively studied as anti-cancer and anti-inflammation drugs, including the development of isoform-specific p110β inhibitors (Crabbe et al., 2007). Thus targeting p110β directly may be a more precise therapeutic strategy in FXS than the current focus on the upstream gp1 mGluR target.
The authors thank Dr. Ravi Muddashetty for technical assistance with polysome assays and qRT-PCRs, and helpful discussions, Andrew Swanson for technical assistance with microscopes and image processing, Adam Goldberg and Mary Roth for technical assistance, Sharon Swanger for help with co-localization analyses, statistics and critically reading the manuscript, and all members of the Bassell lab for helpful discussions. The authors are grateful to Jennifer Darnell for providing the 2F5-1 antibody. This work was supported by a postdoctoral fellowship from NFXF to C.G., NIH MH085617 to G.J.B., NIH HD020521 to S.T.W. and the Fragile X Center, 3P30HD024064 to S.T.W. and G.J.B.