A kinesin adapter directly mediates dendritic mRNA localization during neural development in mice.

Motor protein-based active transport is essential for mRNA localization and local translation in animal cells, yet how mRNA granules interact with motor proteins remains poorly understood. Using an unbiased yeast two-hybrid screen for interactions between murine RNA-binding proteins (RBPs) and motor proteins, here we identified protein interaction with APP tail-1 (PAT1) as a potential direct adapter between zipcode-binding protein 1 (ZBP1, a ß-actin RBP) and the kinesin-I motor complex. The amino acid sequence of mouse PAT1 is similar to that of the kinesin light chain (KLC), and we found that PAT1 binds to KLC directly. Studying PAT1 in mouse primary hippocampal neuronal cultures from both sexes and using structured illumination microscopic imaging of these neurons, we observed that brain-derived neurotrophic factor (BDNF) enhances co-localization of dendritic ZBP1 and PAT1 within granules that also contain kinesin-I. PAT1 is essential for BDNF-stimulated neuronal growth cone development and dendritic protrusion formation, and we noted that ZBP1 and PAT1 co-locate along with ß-actin mRNA in actively transported granules in living neurons. Acute disruption of the PAT1-ZBP1 interaction in neurons with PAT1 siRNA or a dominant-negative ZBP1 construct diminished localization of ß-actin mRNA but not of Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) mRNA in dendrites. The aberrant ß-actin mRNA localization resulted in abnormal dendritic protrusions and growth cone dynamics. These results suggest a critical role for PAT1 in BDNF-induced ß-actin mRNA transport during postnatal development and reveal a new molecular mechanism for mRNA localization in vertebrates.

Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1.

Localization of beta-actin messenger RNA to sites of active actin polymerization modulates cell migration during embryogenesis, differentiation and possibly carcinogenesis. This localization requires the oncofetal protein ZBP1 (Zipcode binding protein 1), which binds to a conserved 54-nucleotide element in the 3'-untranslated region of the beta-actin mRNA known as the 'zipcode'. ZBP1 promotes translocation of the beta-actin transcript to actin-rich protrusions in primary fibroblasts and neurons. It is not known how the ZBP1-RNA complex achieves asymmetric protein sorting by localizing beta-actin mRNA. Here we show that chicken ZBP1 modulates the translation of beta-actin mRNA. ZBP1 associates with the beta-actin transcript in the nucleus and prevents premature translation in the cytoplasm by blocking translation initiation. Translation only occurs when the ZBP1-RNA complex reaches its destination at the periphery of the cell. At the endpoint of mRNA transport, the protein kinase Src promotes translation by phosphorylating a key tyrosine residue in ZBP1 that is required for binding to RNA. These sequential events provide both temporal and spatial control over beta-actin mRNA translation, which is important for cell migration and neurite outgrowth.

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.

Slitrk5 Mediates BDNF-Dependent TrkB Receptor Trafficking and Signaling.

Recent studies in humans and in genetic mouse models have identified Slit- and NTRK-like family (Slitrks) as candidate genes for neuropsychiatric disorders. All Slitrk isotypes are highly expressed in the CNS, where they mediate neurite outgrowth, synaptogenesis, and neuronal survival. However, the molecular mechanisms underlying these functions are not known. Here, we report that Slitrk5 modulates brain-derived neurotrophic factor (BDNF)-dependent biological responses through direct interaction with TrkB receptors. Under basal conditions, Slitrk5 interacts primarily with a transsynaptic binding partner, protein tyrosine phosphatase δ (PTPδ); however, upon BDNF stimulation, Slitrk5 shifts to cis-interactions with TrkB. In the absence of Slitrk5, TrkB has a reduced rate of ligand-dependent recycling and altered responsiveness to BDNF treatment. Structured illumination microscopy revealed that Slitrk5 mediates optimal targeting of TrkB receptors to Rab11-positive recycling endosomes through recruitment of a Rab11 effector protein, Rab11-FIP3. Thus, Slitrk5 acts as a TrkB co-receptor that mediates its BDNF-dependent trafficking and signaling.

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? A report on the 23rd Annual International Symposium of the Center for the Study of Gene Structure and Function.

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.

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.

Dynamic association of the fragile X mental retardation protein as a messenger ribonucleoprotein between microtubules and polyribosomes.

The fragile X mental retardation protein (FMRP) is a selective RNA-binding protein that regulates translation and plays essential roles in synaptic function. FMRP is bound to specific mRNA ligands, actively transported into neuronal processes in a microtubule-dependent manner, and associated with polyribosomes engaged in translation elongation. However, the biochemical relationship between FMRP-microtubule association and FMRP-polyribosome association remains elusive. Here, we report that although the majority of FMRP is incorporated into elongating polyribosomes in the soluble cytoplasm, microtubule-associated FMRP is predominantly retained in translationally dormant, polyribosome-free messenger ribonucleoprotein (mRNP) complexes. Interestingly, FMRP-microtubule association is increased when mRNPs are dynamically released from polyribosomes as a result of inhibiting translation initiation. Furthermore, the I304N mutant FMRP that fails to be incorporated into polyribosomes is associated with microtubules in mRNP particles and transported into neuronal dendrites in a microtubule-dependent, 3,5-dihydroxyphenylglycine-stimulated manner with similar kinetics to that of wild-type FMRP. Hence, polyribosome-free FMRP-mRNP complexes travel on microtubules and wait for activity-dependent translational derepression at the site of function. The dual participation of FMRP in dormant mRNPs and polyribosomes suggests distinct roles of FMRP in dendritic transport and translational regulation, two distinct phases that control local protein production to accommodate synaptic plasticity.

Imaging mRNA movement from transcription sites to translation sites.

RNA localization is one mechanism to temporally and spatially restrict protein synthesis to specific subcellular compartments in response to extracellular stimuli. To understand the mechanisms of mRNA localization, a number of methods have been developed to follow the path of these molecules in living cells including direct labeling of target mRNAs, the MS2-GFP system, and molecular beacons. We review advances in these methods with the goal of identifying the particular strengths and weaknesses of the various approaches in their ability to follow the movements of mRNAs from transcription sites to translation sites.

Hyperlipidemic effects of trans fatty acids are accentuated by dietary cholesterol in gerbils.

Trans isomers of dietary fatty acids, generated during the commercial hydrogenation of unsaturated fats, may contribute to coronary heart disease (CHD) in humans by interfering with lipid metabolism. To examine this possibility in a fat-sensitive model, the Mongolian gerbil (Meriones unguiculatus) was used to compare the cholesterolemic and triglyceridemic potential of modest increments of trans fatty acids from partially hydrogenated soybean oil with other saturated fatty acids in the presence and absence of dietary cholesterol. Age-, dose-, and time-dependent effects were examined in weanling, 6-month-old, and 1-year-old gerbils. Although lipoprotein metabolism in weanling gerbils was initially refractory to trans fat, even as perturbations by saturated fatty acids were demonstrable, these gerbils eventually (after 16 weeks) developed a trans-induced hypercholesterolemia that was intermediate between the response to 16:0 and 12:0 + 14:0. The hepatic and plasma 18:1/18:2 cholesteryl ester (CE) ratio was depressed by trans in a manner similar to saturated fatty acids. The 6-month-old gerbils readily developed hypertriglyceridemia but not hypercholesterolemia, again revealing a decrease in the plasma 18:1/18:2 CE ratio. The 1-year-old gerbils revealed a dose-related (0, 5, 10%en as trans) elevation in total cholesterol (TC), and especially triglycerides (TG), that was accentuated by 0.04% dietary cholesterol. Increases in plasma lipids were again accompanied by a significant decrease in the mass of hepatic esterified cholesterol, particularly 18:1-cholesteryl esters. Thus, dietary trans-fatty acids induce age-, time-, and dose-dependent modulations in gerbil plasma lipids associated with decreased 18:1 cholesteryl esters. Further investigation with gerbils may reveal mechanisms by which trans fat consumption disturbs lipoprotein metabolism.

The fragile X syndrome: from molecular genetics to neurobiology.

Since the identification of the FMR1 gene basic research has been focused on the molecular characterization of the FMR1 gene product, the fragile X mental retardation protein (FMRP). Recent developments in fragile X research have provided new insights and knowledge about the physiological function of FMRP in the cell and the nerve cell in particular. Currently, compelling evidence suggests a role for FMRP in the transport/translation of dendritically localized mRNAs. In addition, the identification of some of the target mRNAs of FMRP have led to an increased interest in the neurobiology of the syndrome. This review highlights the role of FMRP in dendritic mRNA transport/translation in relation to synaptic plasticity, a molecular mechanism implicated in learning and memory.