Fragile X syndrome (FXS) is a common inherited form of mental retardation that is caused, in the vast majority of cases, by the transcriptional silencing of a single gene, fmr1. The encoded protein, FMRP, regulates mRNA translation in neuronal dendrites, and it is thought that changes in translation-dependent forms of synaptic plasticity lead to many symptoms of FXS. However, little is known about the potentially extensive changes in synaptic protein content that accompany loss of FMRP. Here, we describe the development of a high-throughput quantitative proteomic method to identify differences in synaptic protein expression between wild-type and fmr1−/− mouse cortical neurons. The method is based on stable isotope labeling by amino acids in cell culture (SILAC), which has been used to characterize differentially expressed proteins in dividing cells, but not in terminally differentiated cells because of reduced labeling efficiency. To address the issue of incomplete labeling, we developed a mathematical method to normalize protein ratios relative to a reference based on the labeling efficiency. Using this approach, in conjunction with multidimensional protein identification technology (MudPIT), we identified >100 proteins that are up- or down-regulated. These proteins fall into a variety of functional categories, including those regulating synaptic structure, neurotransmission, dendritic mRNA transport, and several proteins implicated in epilepsy and autism, two endophenotypes of FXS. These studies provide insights into the potential origins of synaptic abnormalities in FXS and a demonstration of a methodology that can be used to explore neuronal protein changes in neurological disorders.

Fragile X syndrome results from the transcriptional silencing of a gene, Fmr1, that codes for an mRNA-binding protein (fragile X mental retardation protein, FMRP) present in neuronal dendrites. FMRP can act as a translational suppressor, and its own translation in dendrites is regulated by group I metabotropic glutamate receptors (mGluRs). Multiple lines of evidence suggest that mGluR-induced translation is exaggerated in Fragile X syndrome because of a lack of translational inhibition normally provided by FMRP. We characterized the role of FMRP in the regulation of mRNA granules, which sediment as a heavy peak after polysomes on sucrose gradients. In WT mouse brain, FMRP distributed with polysomes and granules. EM and biochemical analyses suggested that the granule fraction itself contained clusters of polysomes. In Fmr1 knockout brain, we observed a significant decrease in the amount of mRNA granules relative to WT mice. This difference appeared to be due to a role of FMRP in regulating the activation of granules during mGluR-induced translation; in vivo administration of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine increased granule content in Fmr1 knockout mouse brain to levels comparable with those seen in WT brain. In accord with a role of mGluR5 in the regulation of ongoing translation in vivo, we observed that the phosphorylation of several initiation factors in response to application of the mGluR1/5 agonist S-3,5-dihydroxyphenylglycine in vitro was blocked by methyl-6-(phenylethynyl)pyridine. Together, these data suggest that although large, polysome-containing granules can form in the absence of FMRP, their use in response to mGluR-induced translation is exaggerated.

In many cell types, translation can be regulated by a redistribution of translation initiation factors to actin-based cytoskeletal compartments that contain bound mRNAs. This process is evoked by extracellular signals and is regulated by determinants of cytoskeletal organization, such as integrins. In the present experiments, we provide evidence that similar mechanisms regulate local translation in dendrites during synaptic plasticity. Treatment of various neuronal preparations with the brain-derived neurotrophic factor (BDNF) resulted in redistribution of the critical eukaryotic initiation factor 4E (eIF4E) to an mRNA granule-rich cytoskeletal fraction isolated from detergent-solubilized samples. eIF4E linkage to cap structures mediates the recruitment of other translation factors in the initiation of translation events. Immunoprecipitation studies confirmed that eIF4E associates with mRNA granules and that BDNF increased this association. BDNF-induced redistribution of eIF4E was blocked by preincubation with either a peptide (GRGDSP) that inhibits integrin-matrix interactions or by a high concentration (20 μM) of the F-actin depolymerizing agent latrunculin A. Immunohistochemical studies in cultured neurons demonstrated that BDNF facilitated translocation of eIF4E into dendritic spines. Together, the findings suggest that BDNF regulates translation in dendrites by altering the localization of eIF4E relative to cytoskeletally bound mRNA granules. Integrins, which are known to be essential for stabilizing certain forms of synaptic plasticity, may be critical regulators of local translation events at synapses.

Changes in the morphology of dendritic spines are correlated with synaptic plasticity and may relate mechanistically to its expression and stabilization. Recent work has shown that spine length can be altered by manipulations that affect intracellular calcium, and spine length is abnormal in genetic conditions affecting protein synthesis in neurons. We have investigated how ligands of group 1 metabotropic glutamate receptors (mGluRs) affect spine shape; stimulation of these receptors leads both to calcium release from intracellular stores and to dendritic protein synthesis. Thirty-minute incubation of cultured hippocampal slices and dissociated neurons with the selective group 1 mGluR agonist (S)-3,5-dihydroxyphenylglycine (DHPG) induced a significant increase in the average length of dendritic spines. This elongation resulted mainly from the growth of existing spines and was also seen even in the presence of antagonists of ionotropic receptors, indicating that activation of these receptors by mGluR-induced glutamate release was not required. Prolonged antagonism of group 1 mGluRs with (S)-α-methyl-4-carboxyphenylglycine (MCPG) did not result in shorter average spine length. Elongation of dendritic spines induced by DHPG was blocked by calcium chelation and by preincubation with the protein synthesis inhibitor puromycin. The results suggest that in vivo activation of group 1 mGluRs by synaptically released glutamate affects spine shape in a protein synthesis-dependent manner.

Protein synthesis in neurons is essential for the consolidation of memory and for the stabilization of activity-dependent forms of synaptic plasticity such as long-term potentiation (LTP). Activity-dependent translation of dendritically localized mRNAs has been proposed to be a critical source of new proteins necessary for synaptic change. mRNA for the activity-regulated cytoskeletal protein, Arc, is transcribed during LTP and learning, and disruption of its translation gives rise to deficits in both. We have found that selective translation of Arc in a synaptoneurosomal preparation is induced by the brain-derived neurotrophic factor, a neurotrophin that is released during high-frequency stimulation patterns used to elicit LTP. This effect involves signaling through the TrkB receptor and is blocked by the N-methyl-d-aspartate-type glutamate receptor antagonist, MK801. The results suggest there is a synergy between neurotrophic and ionotropic mechanisms that may influence the specificity and duration of changes in synaptic efficacy at glutamatergic synapses.