Synaptically Regulated Protein Synthesis. To examine this phenomenon further, we developed a synaptoneurosome preparation following the method of Hollingsworth (11); this preparation consisted, as confirmed by electron microscopy, of pinched-off and resealed presynaptic terminals attached to resealed postsynaptic processes, along with other membrane-bound compartments of less identifiable origin. We found that stimulation (by K+ depolarization or glutamate administration) of such synaptoneurosomes from young rat cerebral cortex resulted in a rapid rise in the association of ribosomes with mRNAs, accompanied by a brief acceleration in protein translation, as shown in Fig. 1 (12). This effect was not blocked by antagonists to N-methyl-D-aspartate or aminomethyl phosphonic acid-kainate receptors or by extracellular calcium chelators. It was driven by specific agonists for group I mGluRs and was blocked by intracellular calcium chelators (13). Thus, this neurotransmitter-evoked protein synthesis is not based on enzymatic reactions occurring in the suspension buffer; indeed, disruption of the synaptoneurosomes, either by sonication or by flash freezing, completely abrogated the response.
The mGluR1 postsynaptic signaling response is well understood; it involves G protein-linked activation of phospholipase C, which hydrolyzes membrane phosphatidyl inositol into inositol triphosphate (which in turn liberates Ca2+ ion from stores in the endoplasmic reticulum) and diacylglycerol, which activates protein kinase C. We were able to mimic this kinase cascade by administering phorbol ester as a protein kinase C activator or alternatively by administering a membrane-permeable analog of diacylglycerol, 1-oleoyl-2-acetyl glycerol. The protein kinase C blocker calphostin reduced the strength of the response (13). The synaptoneurosome suspension contains less than 20% glial components (11), in contrast with total brain homogenates; these glia are rich in polyribosomes. Group I mGluR5 have been reported to be present on glial cells (14–16), such that a contribution to observed polyribosomal aggregation or protein synthesis by glial contaminants cannot be ruled out entirely. There are also fragments of dendrites in the preparation, and a contribution from the nonsynaptic mGluRs seen in dendritic membranes and dendritic ribosomes (15) is also possible.
We reasoned that only a subset of mRNAs would likely be involved in this response of increased translation. If we examined the polyribosomes by fractionating them on a continuous sucrose gradient and by using labeled oligonucleotides to probe the RNA along the gradient, we could identify mRNAs that were present in small polyribosomes at a higher level after stimulation than before. For this purpose, we used cDNA clones from a library of mRNA isolated from distal processes of cultured hippocampal neurons by Jim Eberwine’s group (17). Among these clones was one that showed a striking increase in polyribosomal association after mGluR1 stimulation and that showed sequence homology to both FMR-1 (the fragile X mental retardation gene) and the related molecule FXR1. An oligonucleotide probe made to the 3' region of FMR1 (nucleotides 2,023–2,070), a region that has no homology to other known fragile X-related family members, also revealed a shift of mRNA into polyribosomes after mGluR1 stimulation (18). Thus, we concluded that the FMR-1 mRNA is taken up into translational complexes in response to mGluR1 agonist application.
Fragile X syndrome is the most common form of inherited mental retardation, affecting, by one recent estimate, nearly 1 in 2,000 males and roughly half as many females (19). It is caused by the insertion of extra repeats of (CGG)n DNA into the 5' untranslated region, which in turn leads to hypermethylation of CpG residues and transcriptional silencing of the FMR-1 gene. Phenotypic traits include facial abnormalities, macroorchidism, developmental delay, mental retardation, and autistic-like behaviors (20).
To test whether the mRNA shift was accompanied by translation of the FMR protein, we took samples from fresh synaptoneurosome suspensions at short intervals after stimulation by the mGluR1 agonist dihydroxyphenylglycine and compared them with unstimulated samples by Western blot analysis with the antibody 1C3 and by comparing staining intensity standardized to lane loading by restaining the same samples with antibody to glial fibrillary acidic protein. In repeated experiments, we consistently observed an increase in FMRP within 2–5 min after stimulation (18). Six subsequent experiments have all replicated these findings; in the presence of the protein synthesis inhibitor cycloheximide, the effect is not observed. It has been objected (21) that, because mRNA for FMRP has not been observed by in situ hybridization (although it has been detected by reverse transcription-PCR; C.Bagni, personal communication, and by in situ hybridization with multiple probes; ref. 22), the amount of protein must likewise be small; this assertion is a reasonable one. However, it in no way implies that the amount of protein cannot