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. 2012 Apr 25;32(17):5924-36.
doi: 10.1523/JNEUROSCI.4650-11.2012.

Evidence for a fragile X mental retardation protein-mediated translational switch in metabotropic glutamate receptor-triggered Arc translation and long-term depression

Farr Niere  1 , Julia R Wilkerson, Kimberly M Huber
Affiliations

Evidence for a fragile X mental retardation protein-mediated translational switch in metabotropic glutamate receptor-triggered Arc translation and long-term depression

Farr Niere et al. J Neurosci. .

Abstract

Group 1 metabotropic glutamate receptor (mGluR)-stimulated protein synthesis and long-term synaptic depression (mGluR-LTD) are altered in the mouse model of fragile X syndrome, Fmr1 knock-out (KO) mice. Fmr1 encodes fragile X mental retardation protein (FMRP), a dendritic RNA binding protein that functions, in part, as a translational suppressor. It is unknown whether and how FMRP acutely regulates LTD and/or the rapid synthesis of new proteins required for LTD, such as the activity-regulated cytoskeletal-associated protein (Arc). The protein phosphatase PP2A dephosphorylates FMRP, which contributes to translational activation of some target mRNAs. Here, we report that PP2A and dephosphorylation of FMRP at S500 are required for an mGluR-induced, rapid (5 min) increase in dendritic Arc protein and LTD in rat and mouse hippocampal neurons. In Fmr1 KO neurons, basal, dendritic Arc protein levels and mGluR-LTD are enhanced, but mGluR-triggered Arc synthesis is absent. Lentiviral-mediated expression of wild-type FMRP in Fmr1 KO neurons suppresses basal dendritic Arc levels and mGluR-LTD, and restores rapid mGluR-triggered Arc synthesis. A phosphomimic of FMRP (S500D) suppresses steady-state dendritic Arc levels but does not rescue mGluR-induced Arc synthesis. A dephosphomimic of FMRP (S500A) neither suppresses dendritic Arc nor supports mGluR-induced Arc synthesis. Accordingly, S500D-FMRP expression in Fmr1 KO neurons suppresses mGluR-LTD, whereas S500A-FMRP has no effect. These data support a model in which phosphorylated FMRP functions to suppress steady-state translation of Arc and LTD. Upon mGluR activation of PP2A, FMRP is rapidly dephosphorylated, which contributes to rapid new synthesis of Arc and mGluR-LTD.

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Figures

Figure 1.
Figure 1.
PP2A inhibitors reveal a rapid, early phase of mGluR-induced LTD and synthesis of dendritic Arc. A, Incubation of acute rat hippocampal slices in the PP2A inhibitor, OA (100 nm; 1–3 h), significantly reduces mGluR-LTD by DHPG (100 μm; 5 min) at 30–40 min (ACSF, 76 ± 2%; OA, 86 ± 3%; *p < 0.05) but not at 50–60 min (ACSF, 76 ± 3%; OA, 80 ± 2%). Plotted are average (±SEM) initial slope values of FPs normalized to the pre-DHPG baseline. Inset, FP waveforms from a representative experiment are taken at the time points indicated on the graph (1, 2, and 3). Calibration: 0.2 mV, 10 ms. B, Pretreatment of acute rat hippocampal slices with 100 nm OA (3 h) increased the phosphorylated (S499) FMRP (P-FMRP) and phosphorylated Akt (P-Akt). Top panel, Representative Western blot of P-FMRP and total FMRP, and P-Akt and total Akt from vehicle (H2O)- and OA-treated slices. Bottom panel, Quantification of P-FMRP normalized to total FMRP (Veh, 1.00 ± 0.05; OA, 1.51 ± 0.19; n = 3 rats) and P-Akt normalized to total Akt (Veh, 1.00 ± 0.19; OA, 1.73 ± 0.17; n = 2 rats). Statistical analysis was by Student's t test, p < 0.05. C, Application of OA (10 nm; 1 h) prevents the rapid but not late increase in dendritic Arc in response to the Gp1 mGluR agonist DHPG (100 μm; 5 min) in dissociated rat hippocampal neurons (DIV 18–21). C1, Representative images of Arc immunofluorescence at 5 min after DHPG application. Scale bars: 20 and 5 μm. C2, C3, Group averages (±SEM) of quantified dendritic Arc immunofluorescence intensity at 5 min (C2) and 60 min (C3) after treatment with vehicle (H2O) or DHPG for each condition. Arc levels are normalized to basal, vehicle-treated neurons of the same culture preparation. C2, Vehicle, 1.00 ± 0.06; DHPG (normalized to basal vehicle), 1.46 ± 0.08; OA, 0.92 ± 0.06; OA plus DHPG, 0.99 ± 0.07. C3, Vehicle, 1.00 ± 0.05; DHPG, 1.30 ± 0.05; OA, 1.05 ± 0.05; OA plus DHPG, 1.30 ± 0.05. D, Pretreatment of neuronal cultures with a structurally distinct PP2A inhibitor, fostriecin (100 nm; 1 h), lowers basal Arc and prevents the rapid increase in dendritic Arc in response to DHPG. D1, Representative images of Arc staining in dissociated hippocampal neurons (DIV 18–21) at 5 min after DHPG application. Scale bars, 20 and 5 μm. D2, Quantification of dendritic Arc immunofluorescence at 5 min after treatment with vehicle (H2O) or DHPG for each condition. Arc levels are normalized to basal, vehicle-treated cells. Vehicle, 1.00 ± 0.06; DHPG, 1.47 ± 0.09; Fos, 0.75 ± 0.07; Fos plus DHPG, 0.82 ± 0.07. For group data in C and D, N = number of cells per condition. Ten to 15 cells/culture were obtained for each condition, and the experiment was repeated in three to four independent culture preparations. Statistical analysis was as follows: two-way ANOVA, Bonferroni's post hoc comparison, *p < 0.05, ***p < 0.001. E, Representative Arc immunofluorescence (Synaptic Systems α-Arc antibody) and DIC images from cultures prepared from WT and Arc KO littermates demonstrating the specificity of the antibody. Scale bar, 20 μm.
Figure 2.
Figure 2.
FMRP suppresses dendritic Arc protein levels. A, Total Arc levels in Fmr1 KO are unchanged as assessed by Western blotting of hippocampal homogenates from WT and Fmr1 KO mice. Left panel, Representative Western blot. Right panel, Group average of Arc/actin levels normalized to WT. N = 12 mice per genotype. B, Dendritic Arc immunofluorescence is greater in Fmr1 KO neurons. B1, Representative images of Arc immunofluorescence in dissociated hippocampal neurons (DIV 18–21) from WT and Fmr1 KO. Scale bars, 20 and 5 μm. B2, Group averages of quantified somatic and dendritic Arc immunofluorescence levels. Soma: WT, 1.00 ± 0.02; Fmr1 KO, 0.78 ± 0.02. Dendrite: WT, 1.00 ± 0.13; Fmr1 KO, 2.88 ± 0.18. C, Double immunofluorescence images of GFP (green) and FMRP (red) in hippocampal CA1 tissue sections of GFP/Fmr1 mosaic mice demonstrating the coexpression of GFP and FMRP. Scale bar, 20 μm. D, Basal dendritic Arc levels are elevated in Fmr1 KO neurons of GFP/Fmr1 mosaic mice. D1, Double immunofluorescence images of GFP (green) and Arc (red) in dissociated hippocampal neurons (DIV 18–21). The white arrows indicate Fmr1 KO (GFP−) cell. D2, Left panel, Quantification of somatic and dendritic Arc immunofluorescence levels from neighboring WT and Fmr1 KO neurons using a rabbit polyclonal Arc antibody from Synaptic Systems in GFP/Fmr1 mosaic cultures. Values are normalized to WT (GFP+) basal [vehicle (H2O)-treated] neurons of the same culture. Soma: WT, 1.00 ± 0.03; KO, 0.91 ± 0.04; dendrite: WT, 1.00 ± 0.08; KO, 3.21 ± 0.32. DHPG treatment of GFP/Fmr1 mosaic cultures increases dendritic Arc levels in WT neurons, but not in neighboring Fmr1 KO neurons, where Arc levels are greatly elevated in basal conditions. WT (DHPG), 1.62 ± 0.13; KO (DHPG), 2.84 ± 0.23. Right panel, Quantification of the ratio of dendritic Arc/MAP2 immunofluorescence in neighboring WT and Fmr1 KO neurons from GFP/Fmr1 mosaic cultures. D3, Quantification of somatic and dendritic Arc immunofluorescence levels from neighboring WT and Fmr1 KO neurons cultured from GFP/Fmr1 mosaic mice using different Arc polyclonal antibody provided by Dr. Paul Worley (Lyford et al., 1995). Soma: WT, 1.00 ± 0.03; KO, 0.83 ± 0.02. Dendrite: WT, 1.00 ± 0.11; KO, 2.5 ± 0.87. E, FISH reveals similar dendritic Arc mRNA levels in dissociated cultured WT and Fmr1 KO neurons prepared from hippocampi of GFP/Fmr1 mosaic mice. α-GFP column, GFP staining identifies WT (GFP+) and Fmr1 KO (GFP−) cells. α-dig column, Digoxigenin staining identifies hybridized digoxigenin-labeled antisense and sense (control) oligo-probes. Arc sense probes are unstained. DIC column, DIC images of dendrites. Overlay column, Overlay images of digoxigenin-labeled probes (red) on DIC images of respective dendrites. Scale bar, 5 μm. F, Neuronal activity does not affect the difference in basal dendritic Arc levels between WT and Fmr1 KO. Left panel, Representative images of Arc staining in WT and Fmr1 KO neurons from GFP/Fmr1 mosaic mice. Scale bar, 5 μm. Right panel, Quantification of basal dendritic Arc levels in vehicle (H2O) and TTX (1 μm; 14–18 h). Values are normalized to WT in vehicle. Veh: WT, 1.00 ± 0.06; Fmr1 KO, 1.90 ± 0.18; TTX: WT, 0.61 ± 0.05; Fmr1 KO, 1.30 ± 0.10. For group data in B, D, and F, N = number of cells per condition. Ten to 15 cells/culture were obtained for each condition, and experiment was repeated in three to four independent culture preparations. Statistical analysis was by two-way ANOVA, Bonferroni's post hoc comparison: *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 3.
Figure 3.
FMRP and PP2A are necessary for a rapid, mGluR-induced increase in dendritic Arc expression. A, Application of OA (10 nm; 1 h) blocks the rapid but not late increase in dendritic Arc protein in response to mGluR activation by DHPG (100 μm; 5 min) in dissociated WT mouse hippocampal neurons. A1, Representative images of Arc immunofluorescence at 5 min after DHPG or vehicle (H2O) treatment. Scale bars, 20 and 5 μm. Quantification of dendritic Arc levels at 5 min (A2) and 60 min (A3) after addition of DHPG. Values are normalized to basal, vehicle-treated cells. A2, Vehicle, 1.00 ± 0.08; DHPG, 1.43 ± 0.08; OA, 1.09 ± 0.06; OA plus DHPG, 1.04 ± 0.07. A3, Vehicle, 1.00 ± 0.04; DHPG, 1.31 ± 0.04; OA, 0.99 ± 0.05; OA plus DHPG, 1.19 ± 0.05. A4, The translation inhibitor, anisomycin (20 μm; 20 min before DHPG application), blocks mGluR-induced, late expression of dendritic Arc by DHPG. Values are normalized to basal, vehicle-treated cells. Vehicle, 1.00 ± 0.04; DHPG, 1.34 ± 0.05; Aniso, 1.13 ± 0.06; Aniso plus DHPG, 0.95 ± 0.05. B, Pretreatment of OA (10 nm; 1 h) prevents DHPG-induced, rapid, total Arc synthesis in dissociated WT mouse hippocampal neurons. Top panel, Representative Western blot of Arc and actin. Bottom panel, Quantification of Arc levels normalized to basal, vehicle-treated cells. Vehicle, 1.00 ± 0.03; DHPG, 1.49 ± 0.13; OA, 1.20 ± 0.12; OA plus DHPG, 1.07 ± 0.11; n = 6 cultures. Actin was used as a loading control. C, P-FMRP level is elevated in OA-treated (10 nm; 1 h), dissociated WT mouse hippocampal neurons. Top panel, Western blot of whole-cell lysates from WT neurons in the presence of vehicle or OA. Bottom panel, Quantification of P-FMRP/tubulin normalized to vehicle-treated cells. Veh, 1.00 ± 0.12; OA, 1.62 ± 0.23. Statistical analysis was by Student's t test, p < 0.05. D, mGluR-induced, rapid synthesis of dendritic Arc by DHPG (100 μm, 5 min) is absent in Fmr1 KO. D1, Representative images of Arc immunofluorescence at 5 min after DHPG in vehicle- or OA-pretreated neurons. Scale bars, 20 and 5 μm. Quantification of dendritic Arc levels at 5 min (D2) and 60 min (D3) after addition of DHPG. Values are normalized to basal, vehicle-treated cells. D2, Vehicle, 1.00 ± 0.06; DHPG, 1.05 ± 0.07; OA, 1.11 ± 0.09; OA plus DHPG, 1.10 ± 0.07. D3, Effects of mGluR stimulation on dendritic Arc levels at 1 h after DHPG in vehicle- (basal, 1.00 ± 0.06; DHPG, 1.26 ± 0.05) or OA-treated cultures (OA, 1.09 ± 0.05; OA plus DHPG, 1.24 ± 0.06; p = 0.058). D4, Effects of mGluR stimulation on dendritic Arc levels at 1 h after DHPG in vehicle- (basal, 1.00 ± 0.04; DHPG, 1.17 ± 0.04) or anisomycin-treated cultures (Aniso, 0.92 ± 0.04; Aniso plus DHPG, 1.04 ± 0.05). Values are normalized to basal, vehicle-treated cells. For all immunofluorescence quantification figures in A and D, N = number of cells per condition. Ten to 15 cells/culture were obtained for each condition, and experiment was repeated in three to four independent culture preparations. For all four group comparisons (A, B, D), statistical analysis was by two-way ANOVA, Bonferroni's post hoc comparison: *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 4.
Figure 4.
Acute expression of wild-type FMRP in Fmr1 KO neurons lowers basal dendritic Arc and rescues mGluR-induced, rapid expression of Arc. A–C, Fmr1 KO neuronal cultures were transfected using lentivirus expressing GFP (control) or an N-terminally GFP tagged FMRP (FMRPGFP). For quantification, values are normalized to GFP-infected, vehicle-treated (basal, H2O) cells, except B3. A1, Representative images of Arc and GFP immunofluorescence of Fmr1 KO neurons transfected with either GFP or FMRPGFP and then treated with vehicle or DHPG (100 μm; 5 min). Scale bars, 20 and 5 μm. A2, Quantification of dendritic Arc levels. Basal (GFP), 1.00 ± 0.08; DHPG (GFP), 0.99 ± 0.08; basal (FMRPGFP), 0.67 ± 0.08; DHPG (FMRPGFP), 0.97 ± 0.09. B1, Representative Western blot of Arc and actin from Fmr1 KO cultures transfected with either GFP or FMRPGFP. B2, GFP- and FMRP-transfected cells express similar levels of total Arc. GFP, 1.00 ± 0.02; FMRP, 0.92 ± 0.12. B3, mGluR activation increases total Arc levels in FMRPGFP-transfected Fmr1 KO cells, but not GFP-transfected. Values are normalized to respective basal conditions. Basal (GFP), 1.00 ± 0.02; DHPG (GFP), 1.11 ± 0.06; basal (FMRPGFP), 1.00 ± 0.03; DHPG (FMRPGFP), 1.29 ± 0.13; n = 5 independent cultures. C1, Representative images of double immunofluorescence for β3-tubulin (red) and GFP (green) of infected cells treated with vehicle or DHPG. Scale bars, 20 and 5 μm. C2, Tubulin levels are similar in GFP- and FMRP-infected cells. Basal (GFP), 1.00 ± 0.10; DHPG (GFP), 0.89 ± 0.08; basal (FMRPGFP), 0.90 ± 0.08; DHPG (FMRPGFP), 0.92 ± 0.09. For immunofluorescence quantification (A, C), N = number of cells per condition. Ten to 15 cells/culture were obtained for each condition, and experiment was repeated in three to four independent culture preparations. For all four group comparisons (A, B3, C), statistical analysis by two-way ANOVA, Bonferroni's post hoc comparison: *p < 0.05; **p < 0.01. For B2, Student's t test was used.
Figure 5.
Figure 5.
Phosphomutant constructs of S500 FMRP differentially affect basal dendritic Arc and do not rescue mGluR-induced, rapid dendritic Arc expression. A, B, Fmr1 KO neuron cultures were infected with lentivirus expressing either GFP (control) a phosphomimetic or dephosphomimetic of FMRPGFP (S500D- or S500A-FMRPGFP). For quantification of basal dendritic Arc, values are normalized to GFP-infected, vehicle-treated (basal, H2O) cells. mGluR activation was induced by applying DHPG (100 μm; 5 min). Scale bars, 20 and 5 μm. A, S500D-FMRPGFP, a phosphomimic form of FMRP, lowers basal dendritic Arc, but does not rescue mGluR-induced increase in dendritic Arc. Left panel, Representative images of infected cells stained for Arc (red) and GFP (green) under basal condition. Right panel: Basal (GFP), 1.00 ± 0.05; DHPG (GFP), 0.87 ± 0.05; basal (S500D-FMRPGFP), 0.76 ± 0.04; DHPG (S500D-FMRPGFP), 0.71 ± 0.05. B, S500A, a dephosphomimic form of FMRP, does not affect dendritic Arc levels under basal condition or upon mGluR stimulation with DHPG. Left panel, Representative images of infected cells stained for Arc (red) and GFP (green) under basal conditions. Right panel, Basal (GFP), 1.00 ± 0.05; DHPG (GFP), 0.99 ± 0.06; basal (S500A-FMRPGFP), 1.00 ± 0.05; DHPG (S500A-FMRPGFP), 0.96 ± 0.04. For immunofluorescence quantification, N = number of cells per condition. Ten to 15 cells/culture were obtained for each condition, and experiment was repeated in three to four independent culture preparations. Statistical analysis was by two-way ANOVA, Bonferroni's post hoc comparison: ***p < 0.001.
Figure 6.
Figure 6.
FMRP and its phosphorylation state at S500 affect mGluR-induced depression of mEPSCs in dissociated hippocampal neurons. A, Effects of brief mGluR activation DHPG (100 μm; 5 min) on mEPSC frequency (measured at 5–15 min after DHPG onset) in cultured untransfected WT or Fmr1 KO and Fmr1 KO neurons with lentiviral-mediated transfection of FMRPGFP S500A-FMRPGFP, or S500D-FMRPGFP. mEPSC frequency is normalized to a 5 min pre-DHPG baseline. WT untransfected, 105 ± 8%; Fmr1 KO untransfected, 72 ± 6%; Fmr1 KO transfected with the following: GFP, 76 ± 9%; FMRPGFP, 114 ± 6%; S500A-FMRPGFP, 81 ± 6%; S500D-FMRPGFP, 125 ± 17%. Statistical analysis was as follows: paired t test, *p < 0.05, **p < 0.01. For comparison among groups, one-way ANOVA, Newman–Keuls post hoc comparison was used: *p < 0.05, **p < 0.01. B, Representative mEPSC recordings during baseline and 10 min after DHPG application from WT, Fmr1 KO, and Fmr1 KO neurons transfected with GFP, FMRPGFP (FMRP), S500A-FMRPGFP (S500A FMRP), or S500D-FMRPGFP (S500D FMRP). Calibration: 10 pA, 100 ms.
Figure 7.
Figure 7.
Working model of the acute role of FMRP regulation of Arc translation and mGluR-LTD. A, In wild-type mice, phosphorylated FMRP functions to suppress Arc translation in dendrites under basal or unstimulated conditions. As yet unknown for Arc mRNA, the translational suppression by FMRP may occur at the initiation or elongation step. Gp1 mGluR agonism stimulates endocytosis of AMPA receptors (GluRA1 and 2) as well as PP2A-mediated dephosphorylation of FMRP (Narayanan et al., 2007). FMRP dephosphorylation contributes to activation of new synthesis of Arc (Figs. 1, 3, 5), which is required to maintain the persistent endocytosis of AMPARs that underlie LTD (Fig. 1) (Park et al., 2008; Waung et al., 2008). B, In Fmr1 KO mice, the loss of FMRP-mediated translational suppression of Arc leads to elevated steady-state levels of dendritic Arc (Fig. 2). Gp1 mGluR stimulation causes endocytosis of AMPARs; but because of the elevated basal dendritic Arc levels in Fmr1 KO dendrites (Fig. 2), no mGluR-stimulated synthesis of Arc is required to maintain LTD (Hou et al., 2006; Nosyreva and Huber, 2006).
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References

    1. Abraham WC, Bear MF. Metaplasticity: the plasticity of synaptic plasticity. Trends Neurosci. 1996;19:126–130. - PubMed
    1. Abrahams BS, Geschwind DH. Advances in autism genetics: on the threshold of a new neurobiology. Nat Rev Genet. 2008;9:341–355. - PMC - PubMed
    1. Antar LN, Afroz R, Dictenberg JB, Carroll RC, Bassell GJ. Metabotropic glutamate receptor activation regulates fragile X mental retardation protein and FMR1 mRNA localization differentially in dendrites and at synapses. J Neurosci. 2004;24:2648–2655. - PMC - PubMed
    1. Banko JL, Hou L, Poulin F, Sonenberg N, Klann E. Regulation of eukaryotic initiation factor 4E by converging signaling pathways during metabotropic glutamate receptor-dependent long-term depression. J Neurosci. 2006;26:2167–2173. - PMC - PubMed
    1. Bassell GJ, Warren ST. Fragile X syndrome: loss of local mRNA regulation alters synaptic development and function. Neuron. 2008;60:201–214. - PMC - PubMed

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