Jason B. Dictenberg

Metabotropic glutamate receptor activation regulates Fragile X mental retardation protein and Fmr1 mRNA localization differentially in dendrites and at synapses

Fragile X syndrome is caused by the absence of the mRNA-binding protein Fragile X mental retardation protein (FMRP), which may play a role in activity-regulated localization and translation of mRNA in dendrites and at synapses. We investigated whether neuronal activity and glutamatergic signals regulate trafficking of FMRP and its encoding Fmr1 mRNA into dendrites or at synapses. Using high-resolution fluorescence and digital imaging microscopy in cultured hippocampal neurons, FMRP and Fmr1 mRNA were localized in granules throughout dendrites and within spines. KCl depolarization rapidly increased FMRP and Fmr1 mRNA levels in dendrites. Metabotropic glutamate receptor (mGluR) activation, in particular mGluR5 activation, was necessary for localization of FMRP into dendrites. Blockade of either PKC or internal calcium prevented mGluR-dependent localization of both FMRP and Fmr1 mRNA in dendrites. The activity-dependent localization of FMRP was not dependent on protein synthesis. Fluorescence recovery after photobleaching analysis of live neurons transfected with enhanced green fluorescent protein–FMRP revealed increased granule trafficking in response to KCl depolarization. In contrast to its dendritic localization, mGluR activation diminished FMRP, but not Fmr1 mRNA, localization at synapses. These results demonstrate regulation of FMRP and Fmr1 mRNA trafficking in dendrites and synapses in response to specific glutamatergic signals.

A Direct Role for FMRP in Activity-Dependent Dendritic mRNA Transport Links Filopodial-Spine Morphogenesis to Fragile X Syndrome

The function of local protein synthesis in synaptic plasticity and its dysregulation in fragile X syndrome (FXS) is well studied, however the contribution of regulated mRNA transport to this function remains unclear. We report a function for the fragile X mental retardation protein (FMRP) in the rapid, activity-regulated transport of mRNAs important for synaptogenesis and plasticity. mRNAs were deficient in glutamatergic signaling-induced dendritic localization in neurons from Fmr1 KO mice, and single mRNA particle dynamics in live neurons revealed diminished kinesis. Motor-dependent translocation of FMRP and cognate mRNAs involved the C terminus of FMRP and kinesin light chain, and KO brain showed reduced kinesin-associated mRNAs. 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 mechanism for stimulus-induced dendritic mRNA transport and link its impairment in a mouse model of FXS to altered developmental morphologic plasticity.

Localization of FMRP-associated mRNA granules and requirement of microtubules for activity-dependent trafficking in hippocampal neurons

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.

A Role for Dendritic mGluR5-Mediated Local Translation of Arc/Arg3.1 in MEF2-Dependent Synapse Elimination

Experience refines synaptic connectivity through neural activity-dependent regulation of transcription factors. Although activity-dependent regulation of transcription factors has been well described, it is unknown whether synaptic activity and local, dendritic regulation of the induced transcripts are necessary for mammalian synaptic plasticity in response to transcription factor activation. Neuronal depolarization activates the myocyte enhancer factor 2 (MEF2) family of transcription factors that suppresses excitatory synapse number. We report that activation of metabotropic glutamate receptor 5 (mGluR5) on the dendrites, but not cell soma, of hippocampal CA1 neurons is required for MEF2-induced functional and structural synapse elimination. We present evidence that mGluR5 is necessary for synapse elimination to stimulate dendritic translation of the MEF2 target gene Arc/Arg3.1. Activity-regulated cytoskeletal-associated protein (Arc) is required for MEF2-induced synapse elimination, where it plays an acute, cell-autonomous, and postsynaptic role. This work reveals a role for dendritic activity in local translation of specific transcripts in synapse refinement.

Genetic encoding of fluorescent RNA ensures a bright future for visualizing nucleic acid dynamics

Recently RNA localization has been appreciated as an essential post-transcriptional mechanism to program local proteome composition and function. Although RNA has been visualized using diverse techniques, the use of the bacteriophage MS2 method to encode genetically fluorescent RNA has revolutionized the study of RNA dynamics in living cells. Here, I highlight the strength of MS2 compared to other techniques, and how further evolution of this system will enable the visualization of RNA in the context of complex live-cell dynamics. Although the generation of MS2-fluorescence resonance energy transfer (FRET) and MS2-bifluorescence complementation (BiFC) will require further development, it has the potential to increase significantly the signal-to-noise ratio, which is the major obstacle to rapid live-cell imaging of RNA.

Genes, brain, and behavior: development gone awry in autism?

Autism and its highly variable symptomology were the themes of the 23rd Annual International Symposium of the Center for the Study of Gene Structure and Function at Hunter College in New York City, held 15 January 2010. The meeting explored the extensive research on autism from several perspectives—integrating research on genetics, neuroscience, and behavior—from researchers presenting new and innovative approaches to understanding the autism spectrum. Early diagnosis, intervention, and genetics were major themes because they are seen as essential areas in which progress is needed before the rise in numbers of cases of autism throughout the world, which some describe as approaching an epidemic, can be stemmed. Several genetic, neurobiological, and behavioral markers of autism have been identified that may ultimately provide the basis for early identification, and that presently define the key areas requiring intensive intervention.

Dendritic RNA Transport: Dynamic Spatio-Temporal Control of Neuronal Gene Expression

The localization and translation of messenger RNA (mRNA) within dendrites is a specialized form of gene expression which enables neurons to locally manage information flow and provides a mechanism for the spatiotemporal regulation of structural synapse plasticity that contributes to learning and memory. The delivery of mRNA from the nucleus into dendrites involves complex formation between cis-acting elements and numerous trans-acting proteins, which display dynamic movements and play essential roles in the maintenance of several forms of synaptic plasticity in the mammalian brain. The diverse composition of these posttranscriptional operons is presented in the context of activity-regulated dendritic mRNA transport with regard to their role in synaptic plasticity and defects in their regulation that result in neuronal disease. An example of the analysis of rapid dendritic transport in living hippocampal neurons is presented that shows calcium/calmodulin-dependent protein kinase II (CaMKII)-alpha mRNA movements at rates tenfold greater than previously estimated using improved imaging techniques.