Dysregulated metabotropic glutamate receptor-dependent translation of AMPA receptor and postsynaptic density-95 mRNAs at synapses in a mouse model of fragile X syndrome

Abstract

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 IIalpha (CaMKIIalpha) mRNA levels in actively translating polyribosomes were dysregulated in synaptoneurosomes from Fmr1 knock-out mice in response to mGluR activation. [35S]methionine incorporation into newly synthesized proteins similarly revealed impaired stimulus-induced protein synthesis of CaMKIIalpha and PSD-95 in synaptoneurosomes from Fmr1 KO mice. Quantitative analysis of mRNA levels in FMRP-specific immunoprecipitations from synaptoneurosomes demonstrated the association of FMRP with CaMKIIalpha, PSD-95, and GluR1/2 mRNAs. These findings suggest a novel mechanism whereby FMRP regulates the local synthesis AMPA receptor (AMPAR) subunits, PSD-95, and CaMKIIalpha downstream of mGluR-activation. Dysregulation of local translation of AMPAR and associated factors at synapses may impair control of the molecular composition of the postsynaptic density and consequently alter synaptic transmission, causing impairments of neuronal plasticity observed in Fmr1 knock-out mice and fragile X syndrome.

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 CaMKIIα, GluR1, GluR2, PSD-95, and β-tubulin mRNAs (see Materials and Methods), detected with fluorescein-based tyramide signal amplification and analyzed by confocal microscopy. Shown are photomicrographs of layers 2/3 within the somatosensory cortex. As reported previously, CaMKIIα mRNA can be detected throughout the entire apical dendrites of cortical neurons. Furthermore, GluR1, GluR2, and PSD-95 mRNAs show a significant dendritic localization, which can even be detected in distal parts of the dendrites (highlighted by arrows). In contrast, in situ hybridization signals for β-tubulin mRNA are restricted to cell bodies. No obvious differences in mRNA localization between wild-type and Fmr1 knock-out mice could be detected. c, Control in situ hybridizations with sense riboprobes produced no specific signals. Exposure times and image processing were identical for each sample.

Figure 2.

Quantitative analysis of FISH signals for CaMKIIα, GluR1, and PSD-95 mRNAs in cultured cortical neurons from wild-type and Fmr1 knock-out mice. a–c, e, Postnatal cortical neurons (14 d in vitro) from wild-type (a) and Fmr1 knock-out (b) neurons were hybridized with digoxigenin-labeled oligonucleotide probes to specific mRNAs (see Materials and Methods) and detected with Cy3-conjugated antidigoxigenin antibodies. CaMKIIα mRNAs were distributed throughout dendrites in the form of granules. A similar pattern was observed for GluR1 mRNA and PSD-95 mRNA. c, Low background staining was detected with sense probes. Exposure times and image processing were identical for each sample. Top, Corresponding differential interference contrast (DIC) image; bottom, FISH signals. d, Quantification of FISH signals in dendrites from wild-type and Fmr1 knock-out mice. Columns display mean fluorescent intensities in dendrites normalized to the signal intensity in cell bodies. Error bars represent SEM. No significant differences in normalized dendritic signal intensities between wild-type and Fmr1 knock-out neurons could be detected for CaMKIIα, GluR1, and PSD-95 mRNAs, as shown by two-tailed unpaired t tests (CaMKIIα, n_wt = 18, n_ko = 15, p = 0.97; GluR1, n_wt = 20, n_ko = 17, p = 0.98; PSD-95, n_wt = 19, n_ko = 19, p = 0.94). Normalized dendritic intensities for CaMKIIα mRNAs are significantly higher than for GluR1 and PSD-95 mRNAs in both wild-type (black lines) and Fmr1 knock-out neurons (dashed lines) (*p < 0.05, **p < 0.01, one-way ANOVA and Games–Howell post hoc test). e, Control for the specificity of dendritic FISH signals. In situ hybridizations with probes for α-tubulin mRNA display a clear staining in the cell body, but only faint staining in proximal dendrites, whereas CaMKIIα specific FISH signals are strong in the cell body and throughout the entire dendrites. Left, FISH signals; right, corresponding DIC image.

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 CaMKIIα, GluR1, GluR2, and PSD95 were enriched in the pellet from wild-type synaptoneurosomal preparation compared with that of Fmr1 KO. β-Actin and NR1 mRNAs were not enriched, although present in the preparation. Error bars represent SD. n = 3 for all experiments; *p < 0.05 by Student's t test.

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 CaMKIIα mRNA in both total (T) and synaptoneurosome (S) fractions. In contrast, GFAP mRNA was reduced in synaptoneurosome fractions compared with total. MBP mRNA was not detectable in synaptoneurosome fractions. d, Stimulation of synaptoneurosomes with glutamate at 5 and 15 min increased [³⁵S]methionine incorporation into newly synthesized proteins, as tested by gel electrophoresis and autoradiography. e, TCA-precipitations quantified by scintillation counting show that different types of synaptoneurosome stimulations (KCl, NMDA, glutamate, DHPG) similarly increased total protein synthesis at 5 min (gray columns) or 15 min (black columns) time points. f, Pretreatment of synaptoneurosome preparations with either cyclohexamide or rapamycin markedly reduced total protein synthesis. Error bars represent SD. n = 4.

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.

Figure 6.

Deficient DHPG-stimulated synthesis of α-CaMKII and PSD-95 in Fmr1 KO synaptoneurosomes. a, Schematic of radioimmunoprecipitation method. b, After 5 min of DHPG-stimulation in WT synaptoneurosomes, CaMKIIα and PSD-95 protein synthesis were significantly increased. The levels of NMDAR and β-actin proteins did not change. c, In FMR1 KO mice, CaMKIIα and PSD-95 synthesis was not increased in response to DHPG. All experiments were done in duplicate samples in each of four experiments (n = 4; p < 0.05, unpaired Student's t test). Error bars represent SD.

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.

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.