Dendritic mRNA Translation Research Articles

The Fragile X Syndrome Protein Represses Activity-Dependent Translation through CYFIP1, a New 4E-BP

Strong evidence indicates that regulated mRNA translation in neuronal dendrites underlies synaptic plasticity and brain development. The fragile X mental retardation protein (FMRP) is involved in this process; here, we show that it acts by inhibiting translation initiation. A binding partner of FMRP, CYFIP1/Sra1, directly binds the translation initiation factor eIF4E through a domain that is structurally related to those present in 4E-BP translational inhibitors. Brain cytoplasmic RNA 1 (BC1), another FMRP binding partner, increases the affinity of FMRP for the CYFIP1-eIF4E complex in the brain. Levels of proteins encoded by known FMRP target mRNAs are increased upon reduction of CYFIP1 in neurons. Translational repression is regulated in an activity-dependent manner because BDNF or DHPG stimulation of neurons causes CYFIP1 to dissociate from eIF4E at synapses, thereby resulting in protein synthesis. Thus, the translational repression activity of FMRP in the brain is mediated, at least in part, by CYFIP1.

Dendritic mRNA: transport, translation and function

Many cellular functions require the synthesis of a specific protein or functional cohort of proteins at a specific time and place in the cell. Local protein synthesis in neuronal dendrites is essential for understanding how neural activity patterns are transduced into persistent changes in synaptic connectivity during cortical development, memory storage and other long-term adaptive brain responses. Regional and temporal changes in protein levels are commonly coordinated by an asymmetric distribution of mRNAs. This Review attempts to integrate current knowledge of dendritic mRNA transport, storage and translation, placing particular emphasis on the coordination of regulation and function during activity-dependent synaptic plasticity in the adult mammalian brain.

Dysregulated Metabotropic Glutamate Receptor-Dependent Translation of AMPA Receptor and Postsynaptic Density-95 mRNAs at Synapses in a Mouse Model of Fragile X Syndrome

Fragile X syndrome, a common form of inherited mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP), an mRNA binding protein that is hypothesized to regulate local mRNA translation in dendrites downstream of gp1 metabotropic glutamate receptors (mGluRs). However, specific FMRP-associated mRNAs that localize to dendrites in vivo and show altered mGluR-dependent translation at synapses of Fmr1 knock-out mice are unknown so far. Using fluorescence in situ hybridization, we discovered that GluR1/2 and postsynaptic density-95 (PSD-95) mRNAs are localized to dendrites of cortical and hippocampal neurons in vivo. Quantitative analyses of their dendritic mRNA levels in cultured neurons and synaptoneurosomes did not detect differences between wild-type and Fmr1 knock-out (KO) mice. In contrast, PSD-95, GluR1/2, and calcium/calmodulin-dependent kinase IIalpha (CaMKIIalpha) mRNA levels in actively translating polyribosomes were dysregulated in synaptoneurosomes from Fmr1 knock-out mice in response to mGluR activation. [35S]methionine incorporation into newly synthesized proteins similarly revealed impaired stimulus-induced protein synthesis of CaMKIIalpha and PSD-95 in synaptoneurosomes from Fmr1 KO mice. Quantitative analysis of mRNA levels in FMRP-specific immunoprecipitations from synaptoneurosomes demonstrated the association of FMRP with CaMKIIalpha, PSD-95, and GluR1/2 mRNAs. These findings suggest a novel mechanism whereby FMRP regulates the local synthesis AMPA receptor (AMPAR) subunits, PSD-95, and CaMKIIalpha downstream of mGluR-activation. Dysregulation of local translation of AMPAR and associated factors at synapses may impair control of the molecular composition of the postsynaptic density and consequently alter synaptic transmission, causing impairments of neuronal plasticity observed in Fmr1 knock-out mice and fragile X syndrome.

Impaired motor functions in mice lacking the RNA-binding protein Hzf

Local protein synthesis in dendrites plays an important role in some aspects of neuronal development and synaptic plasticity. Neuronal RNA-binding proteins regulate the transport and/or translation of the localized mRNAs. Previously, we reported that hematopoietic zinc finger (Hzf) is one of the neuronal RNA-binding proteins that regulate these processes. The Hzf protein is highly expressed in neuronal cells including hippocampal pyramidal neurons and cerebellar Purkinje cells, and plays essential roles in the dendritic mRNA localization and translation. In the present study we demonstrated that mice lacking Hzf ( Hzf −/− mice) exhibited severe impairments of motor coordination and cerebellum-dependent motor learning. These findings raise the possibility that the post-transcriptional regulation by Hzf may contribute to some aspects of synaptic plasticity and motor learning in the cerebellum.

Myosin-Va Facilitates the Accumulation of mRNA/Protein Complex in Dendritic Spines

mRNA localization has an essential role in localizing cytoplasmic determinants, controlling the direction of protein secretion, and allowing the local control of protein synthesis in neurons. In neuronal dendrites, the localization and translocation of mRNA is considered as one of the molecular bases of synaptic plasticity. Recent imaging and functional studies revealed that several RNA-binding proteins form a large messenger ribonucleoprotein (mRNP) complex that is involved in transport and translation of mRNA in dendrites. However, the mechanism of mRNA translocation into dendritic spines is unknown. Here, we show that an actin-based motor, myosin-Va, plays a significant role in mRNP transport in neuronal dendrites and spines. Myosin-Va was Ca2+-dependently associated with TLS, an RNA-binding protein, and its target RNA Nd1-L, an actin stabilizer. A dominant-negative mutant or RNAi of myosin-Va in neurons suppressed TLS accumulation in spines and further impaired TLS dynamics upon activation of mGluRs. The TLS translocation into spines was impeded also in neurons prepared from myosin-Va-null dilute-lethal (dl) mice, which exhibit neurological defects. Our results demonstrate that myosin-Va facilitates the transport of TLS-containing mRNP complexes in spines and may function in synaptic plasticity through Ca2+ signaling.

Activity- and mTOR-Dependent Suppression of Kv1.1 Channel mRNA Translation in Dendrites

Mammalian target of rapamycin (mTOR) is implicated in synaptic plasticity and local translation in dendrites. We found that the mTOR inhibitor, rapamycin, increased the Kv1.1 voltage-gated potassium channel protein in hippocampal neurons and promoted Kv1.1 surface expression on dendrites without altering its axonal expression. Moreover, endogenous Kv1.1 mRNA was detected in dendrites. Using Kv1.1 fused to the photoconvertible fluorescence protein Kaede as a reporter for local synthesis, we observed Kv1.1 synthesis in dendrites upon inhibition of mTOR or the N-methyl-d-aspartate (NMDA) glutamate receptor. Thus, synaptic excitation may cause local suppression of dendritic Kv1 channels by reducing their local synthesis.

Channeling Local Translation

Local, activity-dependent changes in excitability are thought to play a role in various aspects of neuromodulation. Translation of dendritically localized messenger RNAs is one way for a neuron to modify its excitability and signal processing machinery at or near active synapses. Raab-Graham et al . used photoconvertible fluorophores to show that potassium channels are locally translated in dendrites. This local translation is regulated by N -methyl- D -aspartate receptor activation via the mammalian target of rapamycin/phosphatidylinositol 3-kinase signaling pathway. K. F. Raab-Graham, P. C. G. Haddick, Y. N. Jan, L. Y. Jan, Activity- and mTOR-dependent suppression of Kv1.1 channel mRNA translation in dendrites. Science 314 , 144-148 (2006). [Abstract] [Full Text]

Synaptic Regulation of Translation of Dendritic mRNAs

The selective localization of protein synthetic machinery at postsynaptic sites makes it possible for the synthesis of particular proteins to be regulated by synaptic signals. Here we consider how the structure of the machinery constrains synthetic capacity and the evidence that mRNA translation is locally controlled by synaptic signals. Since the discovery of protein synthetic machinery at synaptic sites on dendrites (Steward and Levy, 1982), substantial progress has been made in identifying dendritic mRNAs and in showing that dendritic protein synthesis is critical for persistent synaptic modifications like long-term potentiation (LTP) and long-term depression (LTD). Although many pieces of the puzzle have been identified, major questions remain. Here we focus on one of the unknowns: how translational activity at synapses is regulated and whether regulation involves upregulation or downregulation of overall translation or differential regulation of the translation of particular transcripts. It is useful to begin by considering constraints imposed by the nature of the protein synthetic machinery at synapses.

Region-directed phototransfection reveals the functional significance of a dendritically synthesized transcription factor

Multiple nuclear transcription factors including E-26-like protein 1 (Elk-1) have been found in neuronal dendrites, yet the functional significance of such localization has not yet been explained. Here we use a focal transfection procedure, 'phototransfection', to introduce Elk1 mRNA into specific regions of live, intact primary rat neurons. Introduction and translation of Elk1 mRNA in dendrites produced cell death, whereas introduction and translation of Elk1 mRNA in cell bodies did not produce cell death. Elk-1 translated in dendrites was transported to the nucleus, and cell death depended upon transcription, supporting the dendritic imprinting hypothesis and highlighting the importance of the dendritic environment on protein function. Our demonstration of the utility of phototransfection for spatially controlled introduction of mRNAs opens the broader opportunity to use this method to introduce selected quantities of small molecules into discrete regions of live cells to assess their biological functions.

Postsynaptic recruitment of Dendrin depends on both dendritic mRNA transport and synaptic anchoring

Synaptic plasticity and memory formation involve remodeling of the postsynaptic cytoskeleton, a process that is in part based on both local translation of dendritic mRNAs and synaptic recruitment of newly synthesized proteins. The postsynaptic component Dendrin that is encoded by a dendritically localized mRNA is thought to modulate the structure of the synaptic cytoskeleton. However, molecular mechanisms that control extrasomatic Dendrin mRNA transport and postsynaptic protein recruitment are unknown. The data presented here reveal that Dendrin interacts with the cytoskeletal components alpha-actinin and Maguk with inverted orientation (MAGI) or synaptic scaffolding molecule (S-SCAM). The latter retains Dendrin in the cytoplasm of mammalian cells and prevents its nuclear import. Furthermore in neurons, postsynaptic clustering of Dendrin requires dendritic targeting of its messenger RNA (mRNA), a process that is mediated by a sequence motif within the 3' untranslated region. In summary our finding suggest that postsynaptic recruitment of Dendrin appears to critically depend on both local protein synthesis and association with the synaptic scaffolding protein MAGI/S-SCAM. Its nuclear localization capacity further points to a function in retrograde signaling from the synapse to the nucleus.

RNA Reigns in Neurons

A workshop entitled "RNA Control of Neuronal Function" was recently held in Kfar Blum, Israel. The main topics discussed at the meeting included neuronal RNA targeting mechanisms and the contributing codes and components, translational control mechanisms in dendrites and axons, and the relevance of these mechanisms for neuronal development, plasticity, and dysfunction.

Internal initiation of translation of the TrkB mRNA is mediated by multiple regions within the 5' leader

Translational regulation of the dendritically localized mRNA encoding for the neurotrophin receptor TrkB has important ramifications for synaptic function. We examined whether the TrkB mRNA is translated through an internal initiation entry site (IRES). The human TrkB 5′ leaders are derived from the use of alternative promoters and alternative splicing, but all 5′ leaders share a common exon. Insertion of a full-length 5′ leader, as well as the common exon into the intercistronic region of a dicistronic luciferase construct, yielded luciferase activity generated from the second cistron that was either equivalent or higher than that observed from the encephalomyocarditis virus IRES. Moreover, inhibiting cap-dependent translation ex vivo and in in vitro lysates had only a minimal effect on the translation of mRNA containing the TrkB 5′ leader. Dissecting the 5′ leader showed that the IRES is located in the exon common to all TrkB 5′ leaders. Moreover, six regions ranging from 2 to 25 nt were identified that either promoted or inhibited IRES activity. Taken together, these results suggest that the 5′ leader of the human TrkB mRNA contains multiple cis-elements that regulate internal initiation of translation and that this mechanism may contribute significantly to the translation of the TrkB mRNA in neuronal dendrites.

Proteomic Characterization of Messenger Ribonucleoprotein Complexes Bound to Nontranslated or Translated Poly(A) mRNAs in the Rat Cerebral Cortex

Receptor-triggered control of local postsynaptic protein synthesis plays a crucial role for enabling long lasting changes in synaptic functions, but signaling pathways that link receptor stimulation with translational control remain poorly known. Among the putative regulatory factors are mRNA-binding proteins (messenger ribonucleoprotein, mRNP), which control the fate of cytosolic localized mRNAs. Based on the assumption that a subset of mRNA is maintained in an inactive state, mRNP-mRNA complexes were separated into polysome-bound (translated) and polysome-free (nontranslated) fractions by sucrose density centrifugation. Poly(A) mRNA-mRNP complexes were purified from a postmitochondrial extract of rat cerebral cortex by oligo(dT)-cellulose affinity chromatography. The mRNA processing proteins were characterized, from solution, by a nanoflow reverse phase-high pressure liquid chromatography-mu-electrospray ionization mass spectrometry. The majority of detected mRNA-binding proteins was found in both fractions. However, a small number of proteins appeared to be fraction-specific. This subset of proteins is by far the most interesting because the proteins are potentially involved in controlling an activity-dependent onset of translation. They include transducer proteins, kinases, and anchor proteins. This study of the mRNP proteome is the first step in allowing future experimentation to characterize individual proteins responsible for mRNA processing and translation in dendrites.

Metabotropic Glutamate Receptor Activation Regulates Fragile X Mental Retardation Protein andFmr1mRNA 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.

Molecular Insights into Mental Retardation: Multiple Functions for the Fragile X Mental Retardation Protein?

Mental retardation is a frequent cause of intellectual and physical impairment. Several genes associated with mental retardation have been mapped to the X chromosome, among them, there is FMR1. The absence of or mutation in the Fragile Mental Retardation Protein, FMRP, is responsible for the Fragile X syndrome. FMRP is an RNA binding protein that shuttles between the nucleus and the cytoplasm. FMRP binds to several mRNAs including its own mRNA at a sequence region containing a G quartet structure. Some of the candidate downstream genes recently identified encode for synaptic proteins. Neuronal studies indicate that FMRP is located at synapses and loss of FMRP affects synaptic plasticity. At the synapses, FMRP acts as a translational repressor and in particular regulates translation of specific dendritic mRNAs, some of which encode cytoskeletal proteins and signal transduction molecules. This action occurs via a ribonucleoprotein complex that includes a small dendritic non-coding neuronal RNA that determines the specificity of FMRP function via a novel mechanism of translational repression. Since local protein synthesis is required for synaptic development and function, this role of FMRP likely underlies some of the behavioural and developmental symptoms of FRAXA patients. Finally we review recent work on the Drosophila system that connects cytoskeleton remodelling and FMRP function.

N-methyl-D-aspartate receptor signaling results in Aurora kinase-catalyzed CPEB phosphorylation and alpha CaMKII mRNA polyadenylation at synapses.

Activity-dependent local translation of dendritic mRNAs is one process that underlies synaptic plasticity. Here, we demonstrate that several of the factors known to control polyadenylation-induced translation in early vertebrate development [cytoplasmic polyadenylation element-binding protein (CPEB), maskin, poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF) and Aurora] also reside at synaptic sites of rat hippocampal neurons. The induction of polyadenylation at synapses is mediated by the N-methyl-D-aspartate (NMDA) receptor, which transduces a signal that results in the activation of Aurora kinase. This kinase in turn phosphorylates CPEB, an essential RNA-binding protein, on a critical residue that is necessary for polyadenylation-induced translation. These data demonstrate a remarkable conservation of the regulatory machinery that controls signal-induced mRNA translation, and elucidates an axis connecting the NMDA receptor to localized protein synthesis at synapses.

Candidate RNA-binding proteins regulating extrasomatic mRNA targeting and translation in mammalian neurons.

In mammalian neurons, long-lasting changes in the efficacy of individual synapses depend on the synthesis of new proteins. To maintain specificity, neuronal cells have to ensure that these newly synthesized proteins accumulate at the appropriate subpopulation of synapses. One way that neurons have solved this challenge appears to be the local translation of extrasomatic mRNAs in dendrites and at postsynaptic sites. Mechanisms, which regulate the targeting, translation, and stability of dendritic mRNAs, involve an organized interaction between cis-acting elements of localized transcripts and trans-acting RNA-binding proteins. The molecular identity and cellular functions of trans-acting factors that are likely to play an important role in post-transcriptional processing of extrasomatic transcripts in mammalian neurons are now being elucidated.

CPEB-Mediated Cytoplasmic Polyadenylation and the Regulation of Experience-Dependent Translation of α-CaMKII mRNA at Synapses

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. α-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB and mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces α-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses.

The 3'-untranslated region of CaMKII alpha is a cis-acting signal for the localization and translation of mRNA in dendrites.

Neuronal signaling requires that synaptic proteins be appropriately localized within the cell and regulated there. In mammalian neurons, polyribosomes are found not just in the cell body, but also in dendrites where they are concentrated within or beneath the dendritic spine. The alpha subunit of Ca(2+)-calmodulin-dependent protein kinase II (CaMKII alpha) is one of only five mRNAs known to be present within the dendrites, as well as in the soma of neurons. This targeted subcellular localization of the mRNA for CaMKII alpha provides a possible cell biological mechanism both for controlling the distribution of the cognate protein and for regulating independently the level of protein expression in individual dendritic spines. To characterize the cis-acting elements involved in the localization of dendritic mRNA we have produced two lines of transgenic mice in which the CaMKII alpha promoter is used to drive the expression of a lacZ transcript, which either contains or lacks the 3'-untranslated region of the CaMKII alpha gene. Although both lines of mice show expression in forebrain neurons that parallels the expression of the endogenous CaMKII alpha gene, only the lacZ transcripts bearing the 3'-untranslated region are localized to dendrites. The beta-galactosidase protein shows a variable level of expression along the dendritic shaft and within dendritic spines, which suggests that neurons can control the local biochemistry of the dendrite either through differential localization of the mRNA or variations in the translational efficiency at different sites along the dendrite.

Characterization and sleep deprivation-induced expression modulation of dendrin, a novel dendritic protein in rat brain neurons.

We report on the characterization of the novel rat brain protein dendrin which is encoded by the brain-specific transcript 464. On immunoblots, two protein variants (81 kD, 89 kD) were identified in cytosolic and membraneous protein fractions. The variants are most abundant in the hippocampus, notably in apical dendrites of CA1 pyramidal cells. Dendritic and perikaryal immunolabelling is apparent in neurons of the cerebral cortex, dentate gyrus, subiculum, amygdala, and preoptic areas. In cortical and hippocampal dendrites, electron-dense immunoreaction is associated with the endoplasmic reticulum, the plasma membrane, and spine heads. An association of dendrin with polyribosomes and the presence of its mRNA in dendrites both provide evidence for dendritic mRNA translation. In the rat forebrain, dendrin expression is altered after an extended period of wakefulness. Twenty-four-hour sleep deprivation decreases the mRNA and protein concentrations of both variants in subcortical forebrain plus midbrain areas by 24 +/- 11% (P < 0.05) and 40 +/- 14% (P < 0.1), respectively, as measured relative to beta-actin mRNA and neural actin. In the cerebral cortex and hippocampus, the relative mRNA level remains unchanged whereas the cortical protein concentration is reduced by 42 +/- 10% (P < 0.05). Thus, dendrin belongs to a new class of dendritic proteins whose expression is differentially modulated by prolonged behavioral activity.