The mGluR theory of fragile X syndrome (FXS) states that the functional consequences of mGluR-dependent protein synthesis are exaggerated when the fragile X protein (FMRP) is absent. Our previous studies funded by FRAXA are consistent with this theory in that it appears that the baseline levels of proteins that are synthesized (translated from mRNA) in response to mGluR stimulation during mGluR-LTD are elevated in FXS model mice. However, our data also suggest that although there is exaggerated basal translation of these mRNAs into protein, there is no further mGluR-dependent protein synthesis during mGluR-dependent long-term depression (mGluR-LTD) in the FXS model mice.

mTOR is a protein kinase that is critical for the initiation of translation, which it controls by phosphorylating several substrates, including p70 S6 kinase1 (S6K1). As mentioned above, we have found that baseline levels of proteins that are synthesized in response to mGluR stimulation during mGluR-LTD are elevated in FXS model mice, and that there is no further mGluR-dependent synthesis of these proteins during mGluR-LTD in the FXS model mice. These findings suggest that there may be an upregulation of mTOR-dependent translational control pathways in FXS model mice. Consistent with this idea, in preliminary studies conducted in collaboration with Ali Sharma and Suzanne Zukin at Albert Einstein School of Medicine we have found that FXS model mice exhibit an upregulation in mTOR-dependent translational control, including an increase in S6K1 phosphorylation.

The preliminary results described above have provided important information concerning the elevation of basal translational control in FXS model mice. These results have led us to hypothesize that genetic manipulations that reduce the elevated basal levels of S6K1 phosphorylation in the FXS model mice will reverse aberrant neuronal morphology, enhanced mGluR-LTD, and abnormal behaviors observed in these mice. These experiments will test several aspects of the mGluR theory of FXS put forward by Mark Bear and colleagues, and will provide critical information concerning the therapeutic potential of S6K1 antagonists for treatment of patients with FXS.

Summary of Previous Work

In our first series of studies funded by FRAXA, we identified several signaling pathways that couple mGluRs to the protein translation machinery during metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD). These pathways pointed to potential mRNAs that could be rapidly translated after the induction of LTD. We observed that rapid translation of several mRNAs occurs during LTD in normal mice, and that translation of these mRNAs, including MAP1b, was misregulated in mice that model fragile X syndrome (FXS). In complementary studies, we studied mGluR-LTD in mice that overexpress human FMRP (YAC FMR1 transgenic mice) and found that LTD was inhibited in the YAC FMR1 transgenic mice. Finally, we found that mGluR-LTD was not only associated with the rapid translation of FMRP, but also a rapid degradation of FMRP that was required for mGluR-LTD. These findings were published in Neuron in August 2006 (Neuron 51: 441-454).

As mentioned above, we identified several proteins whose levels are rapidly increased during mGluR-LTD in wild-type mice. In each case, the baseline levels of these proteins were increased in the brains of FXS model mice. Furthermore, we found that mGluR-LTD in FXS mice did not result in an increase in the levels of these proteins. One of these proteins was MAP1b. In a second series of studies funded by FRAXA, we focused on mGluR5-dependent regulation of MAP1b during mGluR-LTD in FXS model mice. We used mGluR5 and MAP1b knockout mice to determine whether the enhanced mGluR-LTD in FXS model mice could be prevented when the levels of mGluR5 and MAP1b were decreased by genetic manipulations by 50%. We found that when FXS model mice were bred to either mGluR5 or MAP1b heterozygous knockout mice, the enhanced mGluR-LTD observed in the FXS model mice was prevented. These findings, conducted in collaborations with Dr. Richard Paylor at Baylor College of Medicine, are in preparation for submission for publication.

Studies have shown that FMRP is a mRNA binding protein, and that one of its "targets" is the mRNA for a protein called MAP1b. The fmr1 knockout mouse, which lacks FMRP, has much more MAP1b in neurons than control animals. The KO mouse also shows much more Long Term Depression (LTD) in response to stimulation of specific glutamate receptors (mGluRs). This suggests that mGluR-LTD may be enhanced in Fmr1 knockout mice because of an increase in the translation of specific mRNAs, such as MAP1b. Our studies are designed to investigate this possibility.

Our current experiments build upon our previous studies funded by FRAXA. We have identified several proteins whose levels are rapidly increased during mGluR-LTD in wildtype mice. In each case, the baseline levels of these proteins are increased in the brain of KO mice, as compared to their wildtype littermates. Furthermore, we found that mGluR-LTD in Fmr1 knockout mice did not result in an increase in the levels of these proteins. One of these proteins is MAP1b. Our current studies focus on mGluR5-dependent regulation of MAP1b during mGluR-LTD in Fmr1 knockout mice. We will use mGluR5 and MAP1b knockout mice to determine whether the enhanced mGluR-LTD in Fmr1 knockout mice can be reversed. We believe that these studies will provide important information concerning the defective synaptic plasticity observed in mouse models of fragile X syndrome - information that could be relevant for treatment of patients with fragile X.

Previous studies indicate that FMRP binds to certain mRNAs and may regulate the translation of these mRNAs into proteins. Other studies show that mGluR-LTD is enhanced in mice that lack FMRP. Taken together, these two findings suggest the intriguing possibility that mGluRLTD may be enhanced in Fragile X mice because of an increase in the translation of specific mRNAs. We are investigating this possibility.

We have found that several signaling pathways couple mGluRs to the protein translation machinery during mGluR-LTD. These pathways point to candidate mRNAs that may be rapidly translated after the induction of LTD. We have observed that rapid translation of several mRNAs occurs during LTD in normal mice, and that, in contrast, translation of these mRNAs is altered in Fragile X mice. In complementary studies, we have begun to study mGluR-LTD in mice that overexpress human FMRP (YAC FMR1 transgenic mice) to determine whether there are differences in LTD-induced mRNA translation between wildtype mice and YAC FMR1 transgenic mice.

We believe that identifying mRNAs translated in response to mGluR activation, and finding out whether their translation is altered during LTD in FMR1 knockout mice and/or YAC FMR1 transgenic mice, will be helpful in designing therapeutic agents for the treatment of patients with Fragile X.