Fragile X mental retardation protein is necessary for neurotransmitter-activated protein translation at synapses

Abstract

Fragile X mental retardation is caused by absence of the RNA-binding protein fragile X mental retardation protein (FMRP), encoded by the FMR1 gene. There is increasing evidence that FMRP regulates transport and modulates translation of some mRNAs. We studied neurotransmitter-activated synaptic protein synthesis in fmr1-knockout mice. Synaptoneurosomes from knockout mice did not manifest accelerated polyribosome assembly or protein synthesis as it occurs in wild-type mice upon stimulation of group I metabotropic glutamate receptors. Direct activation of protein kinase C did not compensate in the knockout mouse, indicating that the FMRP-dependent step is further along the signaling pathway. Visual cortices of young knockout mice exhibited a lower proportion of dendritic spine synapses containing polyribosomes than did the cortices of wild-type mice, corroborating this finding in vivo. This deficit in rapid neurotransmitter-controlled local translation of specific proteins may contribute to morphological and functional abnormalities observed in patients with fragile X syndrome.

Fig. 1.

K⁺ or DHPG stimulation initiates translation in WT but not in fmr1-KO synaptoneurosomes. Graphs depict mRNA incorporation into polyribosomes (P-mRNA) after stimulation. Each point represents polyribosomal RNA as a fraction of total RNA divided by the baseline proportion (at t = 0). (A) WT levels of P-mRNA after K⁺ stimulation (•, n = 7 experiments) were significantly increased overall and at t = 2 min compared with WT unstimulated samples (○, n = 7) (all samples normalized to t = 0). (B) Response of KO samples stimulated with K⁺ (•, n = 6) did not differ from KO unstimulated samples (○, n = 6) (all samples normalized to t = 0). Because limited sample was available, the 10-min time point was omitted here. (C) When K⁺-stimulated samples are normalized to unstimulated samples, WT synaptoneurosomes (□, n = 7) exhibit increased P-mRNA at t = 2 min compared with KO samples (▪, n = 6). (D) WT P-mRNA levels after DHPG stimulation (•, n = 13 experiments) were significantly increased overall and at t = 5 min compared with WT unstimulated samples (○, n = 9) (all samples normalized to t = 0). (E) Response of KO samples stimulated with DHPG (•, n = 10) did not differ from KO unstimulated samples (○, n = 6) (all samples normalized to t = 0). (F) When DHPG-stimulated samples are normalized to corresponding unstimulated samples, WT synaptoneurosomes (□, n = 8) exhibit increased P-mRNA overall and at t = 5 min compared with KO samples (▪, n = 6). Error bars indicate SEMs; *, P < 0.05.

Fig. 2.

Stimulation of PKC with OAG (diacylglycerol analog) initiates translation in WT but not in fmr1-KO synaptoneurosomes. (A) WT levels of P-mRNA after OAG stimulation (•, n = 12 experiments) were significantly increased overall and at t = 5 min compared with WT unstimulated samples (○, n = 9) (all samples normalized to t = 0). (B) Response of KO samples stimulated with OAG (•, n = 10) did not differ from KO unstimulated samples (○, n = 6) (all samples normalized to t = 0). (C) Western blot analysis reveals that levels of PKC (normalized to actin) do not differ between WT and KO synaptoneurosomes (P > 0.05; n = 4 per group; two per group shown). Error bars indicate SEMs; *, P < 0.05.

Fig. 3.

In vivo translational deficit in fmr1-KO mice. (A) Electron microscopic image of a dendrite in the neuropil of layer IV of visual cortex (FVB.129 sighted WT mouse). A spine on this dendrite forms a synaptic contact and contains a PRA (arrow). Numerous PRAs (arrowheads) are in the dendrite itself. (B) Unbiased stereological estimate of the proportion of axospinous synapses associated with PRAs from the visual cortex neuropil of sighted WT and fmr1-KO mice on postnatal days 15 and 25. KO mice exhibit a significantly lower proportion of synapses with PRAs than do WT mice (ANOVA: main effect of genotype, P < 0.05), indicating a deficit in synaptic protein synthesis. There was no significant effect of age. Error bars indicate SEMs. Preliminary data from this project were published in ref. .

Fig. 4.

A putative role for FMRP in synaptic protein synthesis. (A) FMRP binds to its target mRNAs in the nucleus and helps export them to somatic cytoplasm (7, 42). (B) FMRP and its target mRNA are packaged into transport assemblies and travel, likely by way of microtubules, down dendrites toward synapses (15). (C) FMRP–mRNA transport assemblies may take the form of nontranslating granules near synapses where they await some synaptic signal (43, 44). (D) In response to activation of group 1 mGluRs (stimulated here with DHPG), phosphatidylinositol is cleaved into diacyl glycerol (DAG) and inositol triphosphate (IP₃), initiating the release of intracellular calcium (Ca²⁺ⁱ) and activation of PKC (26). (E) PKC activation triggers an enzyme cascade that, by means of many intermediates (broken arrow), signals nontranslating granules to release FMRP-bound target mRNA for translation.