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Fragile X syndrome is caused by the absence of the fragile X mental-retardation protein (FMRP), an mRNA-binding protein, which may play important roles in the regulation of dendritic mRNA localization and/or synaptic protein synthesis. We have recently applied high-resolution fluorescence imaging methods to document the presence, motility and activity-dependent regulation of FMRP granule trafficking in dendrites and spines of cultured hippocampal neurons. In this study, we show that FMRP granules distribute to F-actin-rich compartments, including filopodia, spines and growth cones during the staged development of hippocampal neurons in culture. Fragile X mental-retardation protein granules were shown to colocalize with ribosomes, ribosomal RNA and MAP1B mRNA, a known FMRP target, which encodes a protein important for microtubule and actin stabilization. The levels of FMRP within dendrites were reduced by disruption of microtubule dynamics, but not by disruption of F-actin. Direct measurements of FMRP transport kinetics using fluorescence recovery after photobleaching in living neurons showed that microtubules were required to induce the mGluR-dependent translocation into dendrites. This study provides further characterization of the composition and regulated trafficking of FMRP granules in dendrites of hippocampal neurons.
The fragile X mental-retardation protein (FMRP) is an mRNA-binding protein with diverse functions in varied regions of the neuron (Antar & Bassell 2003). Absence of the FMRP causes fragile X syndrome (FXS), the most common heritable form of mental retardation. With four RNA-binding domains, a nuclear localization sequence and a nuclear export sequence (Bardoni et al. 1997; Eberhart et al. 1996), FMRP may play a role in the nucleocytoplasmic shuttling of mRNA (Feng et al. 1997b), dendritic mRNA localization (Miyashiro et al. 2003) or synaptic protein synthesis (Weiler et al. 1997; Zalfa et al. 2003). Fragile X mental retardation protein is also tightly associated with polyribosomes in neurons (Khandjian et al. 2004; Stefani et al. 2004). Many studies on FMRP use synaptosomal preparations (Comery et al. 1997). While these studies have provided critical insight into understanding FMRP synthesis, these techniques do not precisely reveal the spatial compartmentalization of FMRP and dynamic aspects of its regulated trafficking to various subcellular compartments. In addition, the use of sectioned material from brain has been important for documenting the dendritic localization of FMRP (Feng et al. 1997a); yet these immunocytochemical approaches are limited in their spatial resolution.
We have previously used cultured hippocampal neurons as a model system to study aspects of FMRP localization, with high spatial resolution, and defined physiological signals involved in regulated trafficking (Antar et al. 2004). Fragile X mental-retardation protein was observed in granules within dendrites and spines. Depolarization of neurons using KCl resulted in the rapid localization of FMRP to dendrites. The increased levels of FMRP in dendrites, in response to depolarization, were not dependent on protein synthesis. The rapid, activity-dependent trafficking of FMRP, fused to enhanced green fluorescent protein (EGFP), was also observed in live neurons. The KCl-induced trafficking was blocked by antagonists of metabotropic glutamate receptors, including MPEP, which is an antagonist of mGluR5. Previous work has shown that a particular type of LTD is enhanced in hippocampal slices from FMR1-knockout mice (Huber et al. 2002). As this type of LTD requires mGluR5 activation and protein synthesis (Huber et al. 2001), these studies taken together suggest that FMRP trafficking may be involved in mediating a type of synaptic plasticity that is altered in FXS. Further work is needed to elucidate the mechanism of regulated FMRP transport and to identify the associated mRNAs that are cotransported, as this would provide important insight into the role of FMRP in protein synthesis-dependent plasticity.
mRNAs are frequently transported in granules, self-contained functional packets, that carry most, but not all, of the molecular machinery necessary for localization and local synthesis (Krichevsky & Kosik 2001). This may include mRNA-binding proteins, bound mRNAs, ribosomes, adapter proteins, molecular motors and other machinery that the granule will utilize at a distal target for local translation (Willemsen et al. 2004). A recent study has shown that RNA granules, which contain FMRP and many other mRNA-binding proteins, can be coprecipitated with conventional kinesin heavy chain in the form of a large complex (Kanai et al. 2004). However, this study did not identify any direct molecular interactions between mRNA-binding proteins contained in the granules and kinesin. Previous work has shown that EGFP–FMRP was localized in granules within neurites of PC12 cells (De Diego Otero et al. 2002). Motile granules displayed both oscillatory and unidirectional transport, the latter of which moved at average rates of 0.2 µm/second which were microtubule dependent.
In this study, we use quantitative and high-resolution fluorescence methods in fixed and live cultured hippocampal neurons to further characterize the composition of the FMRP granule and the role of microtubules in mGluR-dependent trafficking in dendrites. We show that FMRP granules colocalized with MAP1B mRNA and ribosomal RNA (rRNA) in dendrites, suggesting a large complex that may need to be transported for local translation at synapses. Microtubules were shown to be required for the mGluR-dependent transport of EGFP–FMRP in live neurons.