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The Journal of Neuroscience, May 16, 2007, 27(20):5338-5348; doi:10.1523/JNEUROSCI.0937-07.2007
Previous Article | Next Article
Cellular/Molecular
Dysregulated Metabotropic Glutamate Receptor-Dependent Translation of AMPA Receptor and Postsynaptic Density-95 mRNAs at Synapses in a Mouse Model of Fragile X Syndrome
Ravi S. Muddashetty,1 * Sofija Keli ,3 *
Christina Gross,1 Mei Xu,2 andGary J. Bassell1,2 1Departments of Cell Biology and 2Neurology, Emory University, Atlanta, Georgia 30322, and 3Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461
Abstract
Top
Abstract
Introduction
Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that is hypothesized to regulate local mRNA translation in dendrites downstream of gp1 metabotropic glutamate receptors (mGluRs). However, specific FMRP-associated mRNAs that localize to dendrites in vivo and show altered mGluR-dependent translation at synapses of Fmr1 knock-out mice are unknown so far. Using fluorescence in situ hybridization, we discovered that GluR1/2 and postsynaptic density-95 (PSD-95) mRNAs are localized to dendrites of cortical and hippocampal neurons in vivo. Quantitative analyses of their dendritic mRNA levels in cultured neurons and synaptoneurosomes did not detect differences between wild-type and Fmr1 knock-out (KO) mice. In contrast, PSD-95, GluR1/2, and calcium/calmodulin-dependent kinase II(CaMKII
Key words: fragile X syndrome; fragile X mental retardation protein; local protein synthesis; synaptoneurosomes; dendritic mRNA localization; AMPA receptor; postsynaptic density-95
Introduction
Glutamate receptor activation stimulates the local synthesis of postsynaptic components that are critical for synaptic structure and function (Klann and Dever, 2004; Sutton and Schuman, 2005
A potentially important regulator of mRNA at the synapse is the fragile X mental retardation protein (FMRP), whose inherited absence leads to fragile X syndrome, a common form of mental retardation that is linked to autism and epilepsy (Garber et al., 2006
FMRP is hypothesized to repress translation downstream of gp1 metabotropic GluRs (mGluRs) (Bear et al., 2004
In this study, we discovered that the mRNAs encoding GluR1/2 and postsynaptic density-95 (PSD-95) are localized to dendrites in vivo and, in addition to CaMKII
Materials and Methods
Tissue preparation. Mice (C57BL/6 and Fmr1 knock-out, backcrossed in C57BL/6J) at the age of postnatal day 21 (P21) were perfused transcardially with 0.1x PBS, pH 7.4, followed by 4% paraformaldehyde (PFA) under deep anesthesia with pentobarbital sodium (150 mg/kg, i.p.). Brain tissues were extracted from the skull, postfixed with 4% PFA overnight, and then transferred to 30% sucrose/0.1 M PBS at 4°C. The tissues were frozen and sectioned at 20 µm. A set of sections were used for in situ hybridization and their adjacent sections were processed for Nissl staining.Primary neuronal culture. Cerebral cortices were dissected and cultured from postnatal day 1 wild-type (WT) and Fmr1 knock-out mice. Dissociated neurons were plated on poly-L-lysine coated coverslips (1.0 mg/ml). Neurons were attached to the substrate in minimal essential medium with FBS (10%) for 2 h, inverted onto dishes containing astroglia, and grown in defined Neurobasal Medium (Invitrogen, Eugene, OR) with Glutamax (Invitrogen) and B-27 supplements (Invitrogen) (Goslin and Banker, 1998
Fluorescence in situ hybridization. Antisense and sense (control) oligonucleotide probes were designed to mouse sequences for CaMKII
For fluorescence in situ hybridization (FISH) analysis of tissue samples using DNA oligonucleotide probes (supplemental Figs. 3–5, available at www.jneurosci.org as supplemental material), postfixed brain sections were washed in PBS, acetylated, dehydrated in 70, 95, and 100% ethanol, and delipidized in 100% chloroform. Preincubation in hybridization buffer (25% dextran sulfate, 2x SSC, 0.4% bovine serum albumin, 20 mM ribonucleoside vanadyl complex and 0.1x PBS) was done before the sections were hybridized with DIG-labeled oligonucleotide probes in hybridization buffer containing 40% formamide at 42°C overnight. After hybridization, the sections were washed with 1x SSC. DIG-labeling was detected by using Cy3-conjugated monoclonal antibody against DIG (1:200; Jackson ImmunoResearch).
To generate DIG-labeled antisense and sense riboprobes, cDNA fragments of CaMKII
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Figure 1. FISH detection of dendritic mRNAs in the cortex of wild-type and Fmr1 knock-out mice. a, b, Coronal brain slices from wild-type (a) and Fmr1 knock-out mice (b) were hybridized with digoxigenin-labeled antisense riboprobes specific for CaMKII
The sections were viewed with a Zeiss (Oberkochen, Germany) LSM510 confocal microscope.Quantification of FISH signals. To determine relative FISH intensities for CaMKII
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Figure 2. Quantitative analysis of FISH signals for CaMKII
Synaptoneurosome preparation. Synaptoneurosomes were prepared from 18- to 21-d-old wild-type and Fmr1 knock-out mice in a similar way as described previously (Hollingsworth et al., 1985Electron microscopy. The synaptoneurosome preparations were fixed with 2.5% glutaraldehyde in 100 mM cacodylic acid, pH 7.4, pelleted at 48,000 x g for 30 min in fixative, secondary-fixed in osmium tetroxide, dehydrated, embedded in resin, and processed for electron microscopy. Examination was done using JEOL (Peabody, MA) 1200 EX TEM.
Western blot analyses. Equal amounts of proteins were resolved on 8% polyacrylamide gels, transferred to nitrocellulose, and probed with antibodies against FMRP (mouse anti-FMRP antibody; Millipore), NMDA receptor (NMADR; rabbit anti-NMDAR1 antibody against splice variants NR-1a, NR1–1b, NR-1–2a, and NR1–2b; Millipore), CaMKII
Synaptoneurosome stimulation. Synaptoneurosome preparations in triplicate samples were resuspended to 1 mg protein/ml in homogenization buffer, preincubated at 37°C for 10 min, and stimulated with either KCl (50 mM), NMDA/glutamate (50 µM/10 µM; Sigma), glutamate (10 µM), or (RS)-3,5-dihydroxyphenylglycine (DHPG; 20 µM; Tocris Bioscience, Bristol, UK). Effects of cycloheximide (50 µg/ml; Sigma) or rapamycin (5 nM; Tocris Bioscience) on protein synthesis were studied by pretreating the unstimulated samples with these protein synthesis inhibitors for 15 min (cycloheximide) or 30 min (rapamycin) after prewarming. Additional sets of samples were incubated in the presence of either NMDAR antagonist APV (120 µM; Tocris Bioscience), or mGluR antagonists
TCA precipitation. An equal volume of ice-cold 10% TCA was added to synaptoneurosome preparations for 1 h. Insoluble material was then pelleted by centrifugation and rinsed in ice-cold 5% TCA (three times for 20 min) and ice-cold methanol (three times for 20 min), air-dried at room temperature, and solubilized in either 0.1N NaOH for scintillation counting or in SDS sample buffer (Bio-Rad, Hercules, CA) for PAGE. Lysates were analyzed by 8% SDS-PAGE. Gels were treated with fluorographic reagent Amplify (Amersham) before drying and exposure, and radioactive proteins were detected using BioMax-MR film with appropriate intensifying screens (Kodak).
Radioimmunoprecipitation. Synaptoneurosome preparations were pelleted by centrifugation at 1000 x g at 4°C for 15 min and solubilized in cold lysis buffer (LB)/washing buffer 1 [WB1; 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Nonidet P-40 (NP40), 0.5% sodium deoxycholate, Complete protease inhibitors]. Samples were mixed with 1–4 µg of each of the following antibodies against CaMKII
Quantitative measurement of mRNAs by real-time PCR. mRNAs were reverse transcribed with random hexamer primers by superscript II (Invitrogen) according to manufacturer's instructions. mRNA quantification was done by the relative quantification method (Nakamoto et al., 2005
Immunoprecipitation for qRT-PCR. Immunoprecipitation was performed as described previously (Brown et al., 2001
Polysome assay. Synaptoneurosomes prepared from P21 wild-type and Fmr1 knock-out mice (as described previously) were treated with 100 µg/ml cycloheximide, 1 mM puromycin, or 30 mM EDTA and incubated at 37°C for 30 min. For DHPG stimulation, synaptoneurosomes were prewarmed at 37°C for 5 min. Samples were incubated with 50 µM DHPG (Tocris Bioscience) for 5 or 15 min at 37°C. Synaptoneurosomes were lysed using a buffer (20 mM Tris-HCl 7.5, 150 mM NaCl, 5 mM MgCl2, protease and RNase inhibitors) containing 1% NP40 and membranous structures were removed by spinning at 20,000 x g for 40 min. Resulting supernatant was loaded on a 15–45% linear sucrose gradient (prepared in 20 mM Tris-HCl 7.5, 100 mM KCl, and 5 mM MgCl2) and spun at 38,000 rpm for 90 min in SW41 rotor (Stefani et al., 2004
Statistical analysis. Data are expressed as the average ± SD from at least three independent experiments. For quantification of dendritic mRNA localization (see Fig. 2d), relative signal intensities in wild-type and Fmr1 knock-out neurons were compared separately for each mRNA by two-tailed unpaired t test. To compare the extent of dendritic localization of CaMKII
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Figure 3. Interaction of FMRP with mRNAs in synaptoneurosomes. a, The relative amounts of mRNAs were measured from wild-type (wt) and Fmr1 knock-out (ko) synaptoneurosomes by qRT-PCR. No significant difference was observed (unpaired t test, p > 0.05). b, Quantitative measurement of FMRP immunoprecipitated mRNAs from synaptoneurosomes shows that CaMKII
Results
Dendritic in vivo localization of mRNAs encoding AMPA receptor subunits GluR1 and GluR2, and PSD-95 in wild-type and Fmr1 knock-out mice
Although previous studies suggest or indicate local regulation of the synthesis of AMPAR subunits and the associated postsynaptic scaffolding protein PSD-95 at synapses (Akama and McEwen, 2003Additionally, we were able to independently demonstrate these findings by using a different fluorescent in situ hybridization method using digoxigenin-labeled oligonucleotide probes, which offered somewhat higher resolution yet lower sensitivity (supplemental Figs. 3–5, available at www.jneurosci.org as supplemental material). IF detection of PSD-95 mRNA was observed in numerous cortical neurons within each optical section (supplemental Fig. 3b,c, available at www.jneurosci.org as supplemental material). Hybridization signal was often detected in proximal dendrites at distances up to 40 µm. Dendritic mRNA localization for PSD-95 mRNA was also detected in the molecular layer of the dentate gyrus (supplemental Fig. 3e, available at www.jneurosci.org as supplemental material) and striatum (supplemental Fig. 3g, available at www.jneurosci.org as supplemental material). A similar pattern of dendritic GluR1 mRNA localization was observed for many cortical neurons in optical sections (0.5 µm) (supplemental Fig. 4a, available at www.jneurosci.org as supplemental material). Labeling of GluR1 mRNA in apical dendrites was noted at distances of >40 µm in these optical sections (supplemental Fig. 4b, available at www.jneurosci.org as supplemental material). Analysis of three separate optical sections from a z-series illustrate that numerous neurons are positive for GluR1 mRNA in at least one of the optical sections (supplemental Fig. 4c–c", available at www.jneurosci.org as supplemental material). In contrast to detection of GluR1 and PSD-95 mRNA signal in dendrites, hybridization of DIG-labeled probes to tubulin mRNA did not reveal any frequent localization into dendrites and fluorescence signal was mostly restricted to the cell body (supplemental Fig. 5b, available at www.jneurosci.org as supplemental material), as shown previously (Paradies and Steward, 1997
Next, we sought to assess the role of FMRP in dendritic mRNA localization in vivo. In situ hybridization analyses on brains from Fmr1 knock-out mice revealed no obvious differences in localization of these mRNAs in the absence of FMRP (Fig. 1b, supplemental Figs. 1b, 2b, available at www.jneurosci.org as supplemental material). To substantiate this observation we examined the mRNA distribution of CaMKII
FMRP interacts with a subset of synaptoneurosomal mRNAs
We used synaptoneurosomes as a model system to investigate interactions of specific mRNAs with FMRP in wild-type neurons, and to assess their possible altered translation in Fmr1 KO mice. The details on isolation of synaptoneurosomes and their validation are described in Material and Methods (see Fig. 4). Quantitative real-time PCR was used to analyze relative mRNA levels in synaptoneurosomes of wild-type and Fmr1 KO mice (Fig. 3a). Consistent with the FISH data, we did not detect any significant differences in the relative abundance of several mRNAs (CaMKII
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Figure 4. Total protein synthesis is increased after stimulation of synaptoneurosomes isolated from mouse cortex at P21. a, b, Characterization of synaptoneurosome preparations was done by electron microscopy (a) and Western blot analysis (b). a, At the ultrastructural level, we frequently observed presynaptic particles with vesicles that were apposed to sealed particle containing a postsynaptic density and internal membranes. b, There was a threefold enrichment of PSD-95 in synaptoneurosome preparations (S) compared with the total cortex protein extracts (T) as shown by Western blot. c, RT-PCR was used to detect CaMKII
Rapid glutamate-induced protein synthesis in mouse synaptoneurosomes using metabolic labeling with [35S]methionine
Rat brain synaptoneurosomes have been used to demonstrate glutamate-stimulation of protein synthesis using radiolabeled amino acids (Rao and Steward, 1991When equal amounts of proteins from unstimulated samples and samples stimulated with 10 µM glutamate for 15 min were separated on an SDS-PAGE gel, dried, and autoradiographed, a general increase in total protein synthesis was observed (Fig. 4d) in the stimulated samples. After very short time intervals (5 and 15 min), there was an obvious, robust increase in protein synthesis in WT synaptoneurosomes, either with 50 mM KCl, 50 µM NMDA/10 µM glutamate, or 20 µM DHPG, as estimated by TCA-precipitation (Fig. 4d,e). After 15 min incubation in the presence of 50 µg/ml cycloheximide, total protein synthesis of unstimulated samples was reduced by 80% (Fig. 4f), as observed previously by Scheetz et al. (2000)
The rapid induction of protein synthesis by NMDA/glutamate or DHPG is absent in synaptoneurosomes from Fmr1 KO mice
To examine whether there were any differences in activity-induced total protein synthesis, we have quantified[35S]methionine incorporation after both NMDA/glutamate and DHPG stimulations of WT and Fmr1 KO synaptoneurosomes. NMDA/glutamate stimulated total protein synthesis in synaptoneurosomes from WT, yet this response was absent in Fmr1 KO (Fig. 5a). As NMDA/glutamate-induced synthesis was only partly blocked by the NMDAR antagonist APV (Fig. 5b), this suggests possible involvement of other types of glutamate receptors. Activation of group 1 metabotropic glutamate receptors with DHPG also stimulated total protein synthesis in WT, but not in Fmr1 KO synaptoneurosomes (Fig. 5c). Here, we also show that DHPG-stimulation of total protein synthesis was completely blocked by the broad spectrum mGluR antagonist (MCPG), but only partly blocked by an mGluR1 antagonist (LY-367385) or an mGluR5 antagonist (MPEP), suggesting a role of both types of mGluR receptors in regulated synaptic protein synthesis (Fig. 5d).
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Figure 5. Rapid glutamate-regulated increase in total synaptic protein synthesis was absent in synaptoneurosomes from Fmr1 KO mice. a–d, Synaptoneurosome stimulation by either NMDA/glutamate (a, b) or the group I mGluR agonist DHPG (c, d) induced rapid acceleration of protein synthesis in WT mice, but not in Fmr1 KO mice. b, Pretreatment of the synaptoneurosome preparation with the NMDAR antagonist APV did not completely block stimulation of protein synthesis by the NMDA/glutamate mixture. d, Stimulation of protein synthesis by the group I mGluR agonist DHPG was blocked by MCPG, a general group I antagonist, but was only partially blocked by the specific GluR5 antagonist MPEP or the mGluR1 antagonist LY 367385. These data indicate that more than one type of glutamate receptor regulates activity-induced protein synthesis at synapses. Asterisks denote significant stimulation of protein synthesis (p < 0.01, Student's t test) when stimulated (black bars) conditions are compared with unstimulated (open bars) conditions. Error bars represent SD.
We then examined for alterations in the synthesis of specific proteins in synaptoneurosomes from Fmr1 KO. Radioimmunoprecipitation was used as a sensitive method to quantify rapid protein synthesis (Fig. 6a). The specific synaptic proteins CaMKII
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Figure 6. Deficient DHPG-stimulated synthesis of
Although previous studies have revealed protein differences between WT and Fmr1 KO by Western blot (Zalfa et al., 2003Translation dysregulation of CaMKII
Translational efficiency of the mRNAs was assessed by their distribution in polysomal fractions on a linear sucrose gradient (Zalfa et al., 2003
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Figure 7. Polysomal incorporation of PSD-95 mRNA in the presence of cycloheximide, puromycin, and EDTA. Synaptoneurosomal extracts from wild-type mice were treated with 100 µg/ml cycloheximide (CHX), 1 mM puromycin, or 30 mM EDTA and the lysate was separated on 15–45% sucrose density gradient. a, A254 absorbance profiles from sucrose density gradients. b, qRT-PCR for PSD-95 mRNA in each fraction. c, Results pooled into three groups: mRNP/monosomes, light polysomes (L-poly), and heavy polysomes (H-poly). These results indicate that, after EDTA treatment, most of the mRNA is in mRNP fraction and cycloheximide results in mRNA predominantly in heavy polysomes, whereas puromycin treatment considerably decreases the mRNA in heavy polysome fraction.
In Fmr1 knock-out synaptoneurosomes at the basal level there was an enhanced polysomal incorporation of several mRNAs when compared with wild-type. PSD-95 (28%) and GluR1 (39%) mRNAs were significantly increased in the actively translating polysomal fraction from Fmr1 knock-out synaptoneurosomes, indicating an exaggerated basal translation of these mRNAs (Fig. 8a). DHPG treatment enhanced the polysomal incorporation of several synaptoneurosomal mRNAs from wild-type mice (Fig. 8b, supplemental Fig. 7, available at www.jneurosci.org as supplemental material). DHPG stimulation for 15 min increased the polysomal incorporation of PSD-95 (37%), GluR1 (53%), and GluR2 (24%) mRNAs significantly compared with unstimulated samples, although the shift in CaMKII
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Figure 8. Polyribosome association of mRNAs from Fmr1 KO synaptoneurosomes indicates their dysregulated translation. a, Polysomal incorporation of mRNAs was compared between wild-type (wt) and Fmr1 knock-out (ko) synaptoneurosomal preparations. At the basal level, several mRNAs (PSD95, GluR1, and GluR2) have higher polysomal incorporation in KO samples, indicating a higher basal translation. b, After DHPG stimulation in wt (50 µM for 15 min), several mRNAs (PSD-95, GluR1, and GluR2) had increased polysomal incorporation. c, In Fmr1 knock-out, stimulation with DHPG induced an opposite effect on polysomal incorporation of synaptoneurosomal mRNAs. There was a significant decrease in their polysomal incorporation compared with basal level and also to the corresponding wild-type synaptoneurosomal mRNAs (*p < 0.05, **p < 0.01, ***p < 0.005, unpaired t test; n = 3). Error bars represent SD.
Discussion
Localization of GluR1/2 and PSD-95 mRNA in dendrites in vitro and in vivo
Here, we used FISH methods to enable detection of PSD-95, GluR1, and GluR2 mRNAs within dendrites of cultured neurons (Fig. 2) and brain sections from the cerebral cortex, dentate gyrus, and hippocampus (Fig. 1, supplemental Figs. 1–3, available at www.jneurosci.org as supplemental material). The dendritic localization of PSD-95 mRNA has not been reported previously in vitro or in vivo. The dendritic localization of GluR1/2 mRNAs was first reported in cultured hippocampal neurons (Grooms et al., 2006Does FMRP play a role in dendritic mRNA localization?
FMRP has long been hypothesized to play a role in dendritic mRNA localization, although direct evidence has been lacking (Steward et al., 1998FMRP association with CaMKII
CaMKIIRole of FMRP in glutamate receptor-stimulated protein synthesis
The requirement of FMRP or any mRNA binding protein in the glutamate-regulated synthesis of specific proteins at synapses has not been demonstrated previously. Here, we show that synaptoneurosomes from Fmr1 KO mice were deficient in their ability to induce total protein synthesis after stimulation of glutamate receptors (Fig. 5). These findings are consistent with a previous report showing deficient mGluR-dependent translation (Weiler et al., 2004Dysregulation of PSD-95, GluR1/2, and CaMKII
Previous studies have reported increased mRNA levels in brain polyribosomes for CaMKIIHere, we have investigated the consequence of excess basal translation in Fmr1 KO on the ability to elicit local protein synthesis after activation of glutamate receptors. In WT synaptoneurosomes, PSD-95, GluR1, and GluR2 mRNAs were all recruited into active polyribosomes after 15 min DHPG stimulation (Fig. 8b). In WT, a shift of CaMKII
mRNA dysregulation in active polysomes from Fmr1 KO mice are consistent with the known role of FMRP as a translational repressor. FMRP may repress translation by stalling ribosomes, which may be disinhibited by synaptic stimulation and dephosphorylation of FMRP (Ceman et al., 2003
FMRP may regulate control of postsynaptic protein composition during synaptic plasticity
Protein synthesis-dependent increases in PSD-95 expression have been described in cultured neurons, hippocampal slices, and synaptoneurosomes (Akama and McEwen, 2003Here, we show impaired mGluR-dependent translation of CaMKII
Locally synthesized GluR1 subunits can be inserted into the plasma membrane (Kacharimina et al., 2000
Our findings for dysregulated synaptic translation of CaMKII
Footnotes
Received Aug. 15, 2005; revised April 3, 2007; accepted April 6, 2007.*R.S.M. and S.K. contributed equally to this work.
This work was supported by National Institutes of Health Grant NS39641 and the Dana Foundation (G.J.B.), Department of Neurology, Emory University Grant 2T32NS007480 (M.X.), and the Conquer Fragile X Foundation (S.K.). We thank Stephen Warren for helpful discussions, and providing the 7G1 antibody and FMR1 knock-out mice. We thank Yue Feng and Mika Nakamoto for helpful discussions, Tiesha Murray for mouse breeding, and Michelle Kline and Xiaodi Yao for technical assistance. We thank Claudia Mahlke for sharing FISH-TSA protocols.
Correspondence should be addressed to either of the following: Gary J. Bassell, Department of Cell Biology, Emory University, 615 Michael Street, Atlanta, GA 30322, Email: gbassel{at}emory.edu; or Sofija Kelic at her present address, Hoffman-La Roche, 340 Kingsland Street, Nutley, NJ 07110, Email: sofija.kelic{at}roche.com
Copyright © 2007 Society for Neuroscience 0270-6474/07/275338-11$15.00/0
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