The cellular basis of FMRP function

Fragile X syndrome is the most common form of mental retardation that can be inherited from ones? parents. It is caused by the absence of a single protein called the fragile X mental retardation protein or FMRP. As the same implies, the gene that makes FMRP is located on the X chromosome. Men have one copy of the X chromosome whereas women have two copies, each containing a copy of the FMRP gene. Interestingly, the two X-chromosomes in females do not express their genes equally; during early foetal development each cell makes a decision to inactivate one X-chromosome (in a process known as X-inactivation) apparently at random. In the case of females with FXS, if a cell inactivates the chromosome containing the normal gene, the cell will not make FMRP. Similarly, if the cell inactivates the X-chromosome containing the mutant FMR1 gene, the cell will make normal levels of FMRP. Because X-inactivation is random, the severity of neurological defects in females varies depending on the relative number of mutant and normal cells. In mice, we can exploit the fact that females have normal and abnormal cells to learn about how FMRP regulates brain development and whether or not the disease is treatable to certain types of drug treatments. For example the presence of cells with normal levels of FMRP may secrete some factors that rescue the cells that do not express FMRP. Women would only show symptoms if the proportion of normal cells is too low to effectively rescue the mutant cells. In this case identification of the factors by which rescue occurs would provide excellent prospective drug therapies for male and female FXS sufferers. Alternatively, the presence of normal cells may not be able to rescue the mutant cells because the mutant cells do not have the ability to respond to factors made by the normal cells. In this case, alternative forms of treatments that either directly alter the genetics of the mutant cells or that can bypass the normal role of FMRP would have to be pursued. All of the experiments will focus on neurons of the cerebral cortex and hippocampus, brain regions that mediate cognition and memory in mammals. These experiments will therefore guide future research into treatments of FXS. Furthermore, they will be the first experiments to directly examine the development of FXS in females.

Fragile X syndrome (FXS) is the most common genetically inherited form of mental retardation. FXS results from the silencing of the fragile X mental retardation 1 gene (FMR1) and loss of the protein it encodes, FMRP. Humans and mice lacking FMRP show abnormalities in dendritic branching, spine density and morphology, and synaptic plasticity. Preliminary evidence from our laboratory has also revealed a defect in the differentiation of the primary somatosensory cortex of mice lacking FMRP. These developmental morphological and physiological changes are likely to underlie the cognitive deficits associated with FXS.

As its name implies, FXS is an X chromosome-linked disorder. Although females have two X-chromosomes, the process of X-chromosome inactivation results in one of these being silenced. The result is that females heterozygous for the mutant FMR1 allele are mosaics, with some cells expressing normal levels of FMRP (FMR1+), while others lack FMRP (FMR1-). By examining Fmr1- and Fmr1+ cells in Fmr1+/- mosaic female mice we will determine whether the mutant phenotypes that result from deletion of the Fmr1 gene result from cell autonomous or cell non-autonomous processes. This distinction will give important insight into the cellular processes regulated by FMRP and will have implications for treatments for the disease as has been shown for other X-linked dieases (Guy et al., 2007). Furthermore, this is will be the first study to fully characterize the developmental aetiology of mutant phenotypes in female mice lacking FMRP.

We will take advantage of the stereotypical progression of sensory map formation in primary somatosensory cortex to study early developmental processes. Since much work on the roles of FMRP have used hippocampus, we will also focus on the well-characterised CA3 to CA1 connection to examine juvenile neuronal development and plasticity. In both cases all analysis will be conducted at the level of individual neurons.
Specific Aim 1: To determine whether the Fmr1- and Fmr1+ cells contribute equally to the differentiation of the primary somatosensory cortex in Fmr1+/- female mice.
Specific Aim 2: To determine whether Fmr1- cells have defects in dendrite and spine development in cortical and hippocampal neurons of Fmr1+/- female mice.
Specific Aim 3: To determine whether synaptic plasticity in the hippocampus is altered in Fmr1- cells in Fmr1+/- female animals.

These studies will give valuable insight into the normal function of FMRP in developing neurons and will provide valuable information that will guide future research aimed at finding theapeutic agents to treat FXS.