DETERMINING DEVELOPMENTAL TIMING REQUIREMENTS FOR FMR1 USING INDUCIBLE ALLELES

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Absence of FMRP causes fragile X syndrome, the most common inherited form of mental retardation. Significant advances have been made in understanding the function of FMRP, and numerous mRNA targets of this RNA binding protein have been identified. Stemming from the observation that hippocampal long-term depression (LTD) is enhanced in Fmr1 knockout mice, Huber, Bear and Warren proposed a theory to explain much of the phenotype of fragile X syndrome1. This "mGluR theory" proposes that abnormally elevated responses to glutamate signaling through the metabotropic glutamate receptor(s) (mGluRs) results from absence of FMRP in the dendritic compartment of glutamatergic synapses. This theory suggests new directions in treatment of fragile X patients using inhibitors of mGluR signaling. Very encouraging data have been developed in preclinical testing with both mouse and fly models using MPEP, a specific antagonist of the mGluRS receptor2"4, and additional effort to define this pathway and potential drugs for intervention is proceeding in numerous laboratories. The principal goal of this project is to elucidate the requirements for Fmrp during development. Fragile X syndrome is widely considered to be a developmental disorder; yet direct evidence to support this assertion is limited. We propose to use mouse models that allow expression of Fmrp to be manipulated in a timedependent manner to assess the requirement for Fmrp during development. Temporal deletion of Fmr1 in mice during embryonic and postnatal development will be achieved by in vivo expression of a Tamoxifen-inducible Ore recombinase to ablate the Fmr1 promoter and first exon flanked by /ox P sites. A similar approach will allow restoration of full Fmr1 expression from a gene that has an interfering neomycin cassette in intron 1 that can be removed with Ore recombination. Using these inducible Fmr1 alleles, we will determine whether the phenotypes observed in fragile X mice result from a lack of postnatal FMRP expression or are due to lasting consequences of the absence of FMRP during development. While mGluR antagonists have great promise as a potential treatment for some symptoms seen in fragile X adults, it remains unclear whether the disease results primarily from a lack of postnatal expression of Fmrp or is due to the consequences of early brain development in the absence of Fmrp. The outcome of our proposed studies will allow better definition of expectations with therapeutic treatments, as they should set a "best outcome" target for phenotypic changes. In combination with the projects proposed by Paylor and Warren, which seek to identify and study additional drugs and to understand effects of background mutations, this effort will significantly advance our understanding of the consequences of absence of Fmrp and the potential for treatment in fragile X syndrome. We propose to utilize inducible ablation and restoration models of Fmr1 allowing temporal alterations to the gene by induction of Ore recombinase using tamoxifen to study the potential to rescue phenotypes previously demonstrated in knockout animals.