Fragile X syndrome (FXS) is the most common form of inherited mental retardation, with genetic links to autism and epilepsy. FXS is caused by the lack of a single protein, the Fragile X Mental Retardation protein (FMRP). A major challenge for research is to understand the normal function of FMRP in the brain. FMRP is an mRNA binding protein that appears to play an important role at synapses, which are the junctions where neurons communicate with each other. Brain synapses have structural and functional defects in Fragile X. The objective of this research project was to use state-of-the-art methods in brain imaging to visualize the FMRP protein in the nerve cells of normal mice and to try to determine its function at synapses. Imaging experiments were also done on a mouse model for Fragile X syndrome that was previously developed using molecular genetic methods to delete the gene encoding for FMRP. During this project, Dr. Bassell’s laboratory developed microscopic imaging tools that permitted the direct visualization of FMRP and they were able to track the movements of FMRP along the dendrites to the site of the synapse.
Experimental findings demonstrated a role FMRP as a dendritic shuttle bus that carries specific mRNAs to the synapse to permit local synthesis of the encoded proteins. Several mRNA and protein molecules were discovered to have altered expression in dendrites and at synapses in the FXS mouse model. This study allows us to propose a dynamic process of “on-site” and “on-demand” protein synthesis at the synapse that is critical to brain development and learning, and is altered in FXS. Our research findings will have important clinical applications in the design of drugs that may modulate specific brain signaling pathways that are imbalanced in Fragile X, and possibly other brain disorders such as autism, epilepsy, and mental illness.
The goal of this project was to further develop fluorescence imaging methods to permit high-resolution analysis of the localization of the Fragile X Mental Retardation protein and associated mRNAs in neurons. It was anticipated that the refinement of this technology would lead to the discovery of novel dendritically localized mRNAs, and their possible dysregulation in a mouse model of fragile x syndrome.
Toward aim-1, we discovered a new function for the fragile X mental retardation protein (FMRP) in the rapid delivery of several novel dendritic mRNAs important for synaptogenesis and synapse plasticity that are implicated in fragile X syndrome (FXS). Specific mRNAs in neurons from FMR1 KO mice were deficient in glutamatergic signaling-induced dendritic localization, and direct observation of mRNA granules in live neurons revealed impaired transport dynamics. Acute suppression of FMRP and target mRNA transport in WT neurons resulted in altered filopodia-spine morphology that mimicked the FXS phenotype. These findings highlight a novel mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to developmental morphologic plasticity.
Toward aim-2, we discovered that GluR1/2 and PSD-95 mRNAs are localized to dendrites in vivo and associated with FMRP. The steady state levels of GluR1/2 and PSD-95 mRNAs in dendrites did not differ between wild type and FMR1 knockout mice. In contrast, GluR1/2 and PSD-95 mRNAs were translated in excess at basal states in FMR1, yet were dysregulated at synapses in response to mGluR activation. In addition, FMR1 KO mice exhibited an overall increase in the rate of basal protein synthesis at synapses; yet showed loss of mGluR-stimulated protein synthesis. These findings reveal a novel mechanism whereby FMRP regulates the local synthesis of AMPAR subunits and PSD-95 downstream of mGluR-activation. Collectively, our findings implicate a diverse role for FMRP in both the regulation of dendritic mRNA transport and synaptic protein synthesis. In addition, we have made advancements in the FISH technology suitable for discovery and analysis of other dendritic mRNAs in cultured neurons and brain sections.