The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology

Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure.     

Dendritic LSm1/CBP80-mRNPs mark the early steps of transport commitment and translational control

Messenger RNA (mRNA) transport to neuronal dendrites is crucial for synaptic plasticity, but little is known of assembly or translational regulation of dendritic messenger ribonucleoproteins (mRNPs). Here we characterize a novel mRNP complex that is found in neuronal dendrites throughout the central nervous system and in some axonal processes of the spinal cord. The complex is characterized by the LSm1 protein, which so far has been implicated in mRNA degradation in nonneuronal cells. In brain, it associates with intact mRNAs. Interestingly, the LSm1-mRNPs contain the cap-binding protein CBP80 that associates with (pre)mRNAs in the nucleus, suggesting that the dendritic LSm1 complex has been assembled in the nucleus. In support of this notion, neuronal LSm1 is partially nuclear and inhibition of mRNA synthesis increases its nuclear localization. Importantly, CBP80 is also present in the dendrites and both LSm1 and CBP80 shift significantly into the spines upon stimulation of glutamergic receptors, suggesting that these mRNPs are translationally activated and contribute to the regulated local protein synthesis.     

Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons

Extension of the endoplasmic reticulum (ER) into dendritic spines of Purkinje neurons is required for cerebellar synaptic plasticity and is disrupted in animals with null mutations in Myo5a, the gene encoding myosin-Va. We show here that myosin-Va acts as a point-to-point organelle transporter to pull ER as cargo into Purkinje neuron spines. Specifically, myosin-Va accumulates at the ER tip as the organelle moves into spines, and hydrolysis of ATP by myosin-Va is required for spine ER targeting. Moreover, myosin-Va is responsible for almost all of the spine ER insertion events. Finally, attenuation of the ability of myosin-Va to move along actin filaments reduces the maximum velocity of ER movement into spines, providing direct evidence that myosin-Va drives ER motility. Thus, we have established that an actin-based motor moves ER within animal cells, and have uncovered the mechanism for ER localization to Purkinje neuron spines, a prerequisite for synaptic plasticity.     

Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells

Staufen1 (Stau1) is an RNA-binding protein involved in transport, localization, decay, and translational control of mRNA. In neurons, it is present in cell bodies and also in RNA granules which are transported along dendrites. Dendritic mRNA localization might be involved in long-term synaptic plasticity and memory. To determine the role of Stau1 in synaptic function, we examined the effects of Stau1 down-regulation in hippocampal slice cultures using small interfering RNA (siRNA). Biolistic transfection of Stau1 siRNA resulted in selective down-regulation of Stau1 in slice cultures. Consistent with a role of Stau1 in transporting mRNAs required for synaptic plasticity, Stau1 down-regulation impaired the late form of chemically induced long-term potentiation (L-LTP) without affecting early-LTP, mGluR1/5-mediated long-term depression, or basal evoked synaptic transmission. Stau1 down-regulation decreased the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting a role in maintaining efficacy at hippocampal synapses. At the cellular level, Stau1 down-regulation shifted spine shape from regular to elongated spines, without changes in spine density. The change in spine shape could be rescued by an RNA interference-resistant Stau1 isoform. Therefore, Stau1 is important for processing and/or transporting in dendrites mRNAs that are critical in regulation of synaptic strength and maintenance of functional connectivity changes underlying hippocampus-dependent learning and memory.     

Functions of Class V Myosins in Neurons

This minireview focuses on recent studies implicating class V myosins in organelle and macromolecule transport within neurons. These studies reveal that class V myosins play important roles in a wide range of fundamental processes occurring within neurons, including the transport into dendritic spines of organelles that support synaptic plasticity, the establishment of neuronal shape, the specification of polarized cargo transport, and the subcellular localization of mRNA.     

Caldendrin-Jacob: a protein liaison that couples NMDA receptor signalling to the nucleus

NMDA (N-methyl-D-aspartate) receptors and calcium can exert multiple and very divergent effects within neuronal cells, thereby impacting opposing occurrences such as synaptic plasticity and neuronal degeneration. The neuronal Ca2+ sensor Caldendrin is a postsynaptic density component with high similarity to calmodulin. Jacob, a recently identified Caldendrin binding partner, is a novel protein abundantly expressed in limbic brain and cerebral cortex. Strictly depending upon activation of NMDA-type glutamate receptors, Jacob is recruited to neuronal nuclei, resulting in a rapid stripping of synaptic contacts and in a drastically altered morphology of the dendritic tree. Jacob's nuclear trafficking from distal dendrites crucially requires the classical Importin pathway. Caldendrin binds to Jacob's nuclear localization signal in a Ca2+-dependent manner, thereby controlling Jacob's extranuclear localization by competing with the binding of Importin-alpha to Jacob's nuclear localization signal. This competition requires sustained synapto-dendritic Ca2+ levels, which presumably cannot be achieved by activation of extrasynaptic NMDA receptors, but are confined to Ca2+ microdomains such as postsynaptic spines. Extrasynaptic NMDA receptors, as opposed to their synaptic counterparts, trigger the cAMP response element-binding protein (CREB) shut-off pathway, and cell death. We found that nuclear knockdown of Jacob prevents CREB shut-off after extrasynaptic NMDA receptor activation, whereas its nuclear overexpression induces CREB shut-off without NMDA receptor stimulation. Importantly, nuclear knockdown of Jacob attenuates NMDA-induced loss of synaptic contacts, and neuronal degeneration. This defines a novel mechanism of synapse-to-nucleus communication via a synaptic Ca2+-sensor protein, which links the activity of NMDA receptors to nuclear signalling events involved in modelling synapto-dendritic input and NMDA receptor-induced cellular degeneration.     

Fragile X related protein 1 clusters with ribosomes and messenger RNAs at a subset of dendritic spines in the mouse hippocampus

The formation and storage of memories in neuronal networks relies on new protein synthesis, which can occur locally at synapses using translational machinery present in dendrites and at spines. These new proteins support long-lasting changes in synapse strength and size in response to high levels of synaptic activity. To ensure that proteins are made at the appropriate time and location to enable these synaptic changes, messenger RNA (mRNA) translation is tightly controlled by dendritic RNA-binding proteins. Fragile X Related Protein 1 (FXR1P) is an RNA-binding protein with high homology to Fragile X Mental Retardation Protein (FMRP) and is known to repress and activate mRNA translation in non-neuronal cells. However, unlike FMRP, very little is known about the role of FXR1P in the central nervous system. To understand if FXR1P is positioned to regulate local mRNA translation in dendrites and at synapses, we investigated the expression and targeting of FXR1P in developing hippocampal neurons in vivo and in vitro. We found that FXR1P was highly expressed during hippocampal development and co-localized with ribosomes and mRNAs in the dendrite and at a subset of spines in mouse hippocampal neurons. Our data indicate that FXR1P is properly positioned to control local protein synthesis in the dendrite and at synapses in the central nervous system.     

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 diffusion-activation model of CaMKII translocation waves in dendrites

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is a key regulator of glutamatergic synapses and plays an essential role in many forms of synaptic plasticity. It has recently been observed that stimulating dendrites locally with a single glutamate/glycine puff induces a local translocation of CaMKII into spines that subsequently spreads in a wave-like manner towards the distal dendritic arbor. Here we present a mathematical model of the diffusion, activation and translocation of dendritic CaMKII. We show how the nonlinear dynamics of CaMKII diffusion-activation generates a propagating translocation wave, provided that the rate of activation is sufficiently fast. We also derive an explicit formula for the wave speed as a function of physiological parameters such as the diffusivity of CaMKII and the density of spines. Our model provides a quantitative framework for understanding the spread of CaMKII translocation and its possible role in heterosynaptic plasticity.     

Ca2+ signalling in postsynaptic dendrites and spines of mammalian neurons in brain slice.

Postsynaptic Ca2+ changes are involved in control of cellular excitability and induction of synaptic long-term changes. We monitored Ca2+ changes in dendrites and spines during synaptic and direct stimulation using high resolution microfluorometry of fura-2 injected into CA3 pyramidal neurons in guinea pig hippocampal slice. When driven by current injection from an intracellular electrode or with synaptic stimulation, postsynaptic Ca2+ accumulations were highest in the proximal dendrites with a pronounced fall-off towards the soma and some fall-off towards more distal dendrites. Muscarinic activation by low concentrations of carbachol strongly increased intradendritic Ca2+ accumulation during directly-evoked repetitive firing. This enhancement occurred in large part because muscarinic activation suppressed the normal Ca(2+)-dependent activation of K-channels that mediates adaptation of firing. Repetitive firing of cholinergic fibers in the slice reproduced the effects of carbachol. Inhibition of acetylcholine-esterase activity by eserine enhanced the effects of repetitive stimulation of chlolinergic fibers. All effects were reversible and were blocked by the muscarinic antagonist atropine. Ca2+ accumulations in postsynaptic spines might be the basis of specificity of synaptic plasticity. With selective stimulation of few associative/comissural fibers, Ca2+ accumulated in single postsynaptic spines but not in the parent dendrite. With strong stimulation, dendrite levels also increased but spine levels were considerably higher. The NMDA-receptor antagonist AP-5 blocked Ca(2+)-peaks in spines, but left Ca2+ changes in dendrite shafts largely unaffected. Sustained steep Ca2+ gradients between single spines and the parent dendrite, often lasting several minutes, developed with repeated stimulation. Our results demonstrate a spine entity that can act independent from the dendrite with respect to Ca(2+)-dependent processes. Muscarinic augmentation of dendritic Ca2+ levels might reduce diffusional loss of Ca2+ from hot spines into the parent dendrite, thus supporting cooperativity and associativity of synaptic 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.     

The transport of Staufen2-containing ribonucleoprotein complexes involves kinesin motor protein and is modulated by mitogen-activated protein kinase pathway

There is increasing evidence showing that mRNA is transported to the neuronal dendrites in ribonucleoprotein (RNP) complexes or RNA granules, which are aggregates of mRNA, rRNA, ribosomal proteins, and RNA-binding proteins. In these RNP complexes, Staufen, a double-stranded RNA-binding protein, is believed to be a core component that plays a key role in the dendritic mRNA transport. This study investigated the molecular mechanisms of the dendritic mRNA transport using green fluorescent protein-tagged Staufen2 produced employing a Sindbis viral expression system. The kinesin heavy chain was found to be associated with Staufen2. The inhibition of kinesin resulted in a significant decrease in the level of dendritic transport of the Staufen2-containing RNP complexes in neurons under non-stimulating or stimulating conditions. This suggests that the dendritic transport of the Staufen2-containing RNP complexes use kinesin as a motor protein. A mitogen-activated protein kinase inhibitor, PD98059, inhibited the activity-induced increase in the amount of both the Staufen2-containing RNP complexes and Ca(2+)/calmodulin-dependent protein kinase II alpha-subunit mRNA in the distal dendrites of cultured hippocampal neurons. Overall, these results suggest that dendritic mRNA transport is mediated via the Staufen2 and kinesin motor proteins and might be modulated by the neuronal activity and mitogen-activated protein kinase pathway.     

Scaffold Protein X11α Interacts with Kalirin-7 in Dendrites and Recruits It to Golgi Outposts

Pyramidal neurons in the mammalian forebrain receive their synaptic inputs through their dendritic trees, and dendritic spines are the sites of most excitatory synapses. Dendritic spine structure is important for brain development and plasticity. Kalirin-7 is a guanine nucleotide-exchange factor for the small GTPase Rac1 and is a critical regulator of dendritic spine remodeling. The subcellular localization of kalirin-7 is thought to be important for regulating its function in neurons. A yeast two-hybrid screen has identified the adaptor protein X11α as an interacting partner of kalirin-7. Here, we show that kalirin-7 and X11α form a complex in the brain, and this interaction is mediated by the C terminus of kalirin-7. Kalirin-7 and X11α co-localize at excitatory synapses in cultured cortical neurons. Using time-lapse imaging of fluorescence recovery after photobleaching, we show that X11α is present in a mobile fraction of the postsynaptic density. X11α also localizes to Golgi outposts in dendrites, and its overexpression induces the removal of kalirin-7 from spines and accumulation of kalirin-7 in Golgi outposts. In addition, neurons overexpressing X11α displayed thinner spines. These data support a novel mechanism of regulation of kalirin-7 localization and function in dendrites, providing insight into signaling pathways underlying neuronal plasticity. Dissecting the molecular mechanisms of synaptic structural plasticity will improve our understanding of neuropsychiatric and neurodegenerative disorders, as kalirin-7 has been associated with schizophrenia and Alzheimer disease.     

Selective regulation of neurite extension and synapse formation by the beta but not the alpha isoform of CaMKII

Neurite extension and branching are important neuronal plasticity mechanisms that can lead to the addition of synaptic contacts in developing neurons and changes in the number of synapses in mature neurons. Here we show that Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates movement, extension, and branching of filopodia and fine dendrites as well as the number of synapses in hippocampal neurons. Only CaMKIIbeta, which peaks in expression early in development, but not CaMKIIalpha, has this morphogenic activity. A small insert in CaMKIIbeta, which is absent in CaMKIIalpha, confers regulated F-actin localization to the enzyme and enables selective upregulation of dendritic motility. These results show that the two main neuronal CaMKII isoforms have markedly different roles in neuronal plasticity, with CaMKIIalpha regulating synaptic strength and CaMKIIbeta controlling the dendritic morphology and number of synapses.     

Statistical modeling of mRNP transport in dendrites: A comparative analysis of β-actin and Arc mRNP dynamics

Localization of messenger RNA (mRNA) in dendrites is crucial for regulating gene expression during long-term memory formation. mRNA binds to RNA-binding proteins (RBPs) to form messenger ribonucleoprotein (mRNP) complexes that are transported by motor proteins along microtubules to their target synapses. However, the dynamics by which mRNPs find their target locations in the dendrite have not been well understood. Here, we investigated the motion of endogenous β-actin and Arc mRNPs in dissociated mouse hippocampal neurons using the MS2 and PP7 stem-loop systems, respectively. By evaluating the statistical properties of mRNP movement, we found that the aging Lévy walk model effectively describes both β-actin and Arc mRNP transport in proximal dendrites. A critical difference between β-actin and Arc mRNPs was the aging time, the time lag between transport initiation and measurement initiation. The longer mean aging time of β-actin mRNP (~100 s) compared with that of Arc mRNP (~30 s) reflects the longer half-life of constitutively expressed β-actin mRNP. Furthermore, our model also permitted us to estimate the ratio of newly generated and pre-existing β-actin mRNPs in the dendrites. This study offers a robust theoretical framework for mRNP transport, which provides insight into how mRNPs locate their targets in neurons.     

Local protein synthesis, actin dynamics, and LTP consolidation

Modulation of local protein synthesis in neuronal dendrites plays a key role in the production of long-term, activity-dependent changes in synapse structure and functional efficacy. Such long-term changes also require regulation of actin dynamics in dendritic spines. Recent evidence couples local protein synthesis to regulation of actin dynamics in long-term synaptic plasticity. Translation of the dendritically localized mRNA, Arc, is required for consolidation of LTP and stabilization of nascent polymerized actin. BDNF signaling activates Arc-dependent LTP consolidation and is required for actin polymerization and stable expansion of dendritic spines during LTP. Regulation of actin pools within dendritic spines modulates spine size and enlargement, organization of the postsynaptic density, receptor trafficking, and localization of the translational machinery.     

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.     

Dendritic localization and a cis-acting dendritic targeting element of Kv4.2 mRNA

Kv4.2, a pore-forming α-subunit of voltage-gated A-type potassium channels, is expressed abundantly in the soma and dendrites of hippocampal neurons, and is responsible for somatodendritic I(A) current. Recent studies have suggested that changes in the surface levels of Kv4.2 potassium channels might be relevant to synaptic plasticity. Although the function and expression of Kv4.2 protein have been extensively studied, the dendritic localization of Kv4.2 mRNA is not well described. In this study, Kv4.2 mRNAs were shown to be localized in the dendrites near postsynaptic regions. The dendritic transport of Kv4.2 mRNAs were mediated by microtubule- based movement. The 500 nucleotides of specific regions within the 3'-untranslated region of Kv4.2 mRNA were found to be necessary and sufficient for its dendritic localization. Collectively, these results suggest that the dendritic localization of Kv4.2 mRNAs might regulate the dendritic surface level of Kv4.2 channels and synaptic plasticity.     

Subcellular Localization of Fragile X Mental Retardation Protein with the I304N Mutation in the RNA-Binding Domain in Cultured Hippocampal Neurons

1. Fragile X syndrome, the most common form of inherited mental retardation,iscaused by the lack or dysfunction of fragile X mental retardationprotein (FMRP). The I304N mutation in the RNA-binding domain of FMRP results in an exceptionally severe form of mental retardation.2. We have investigated the subcellular localization of FMRP and its I304N-mutated form in cultured hippocampal neurons and PC12 cells, using immunofluorescence microscopy. In PC12 cells, FMRP was predominantly localized to the cytoplasm and also to the processes after differentiation by NGF.3. In cultured hippocampal neurons, granular labeling was detected along the neuronal processes.4. Double-labeling with synaptophysin antibody revealed FMRP at synaptic sites in neurons.5. The I304N mutation did not appear to affect the transport of FMRP to dendrites or its localization at synaptic sites. Thus, FMRP is a synaptic protein and the severe phenotype observed in the patient with the I304N mutation is not produced by alterations in dendritic transport.     

The impacts of geometry and binding on CaMKII diffusion and retention in dendritic spines

We used a particle-based Monte Carlo simulation to dissect the regulatory mechanism of molecular translocation of CaMKII, a key regulator of neuronal synaptic function. Geometry was based upon measurements from EM reconstructions of dendrites in CA1 hippocampal pyramidal neurons. Three types of simulations were performed to investigate the effects of geometry and other mechanisms that control CaMKII translocation in and out of dendritic spines. First, the diffusional escape rate of CaMKII from model spines of varied morphologies was examined. Second, a postsynaptic density (PSD) was added to study the impact of binding sites on this escape rate. Third, translocation of CaMKII from dendrites and trapping in spines was investigated using a simulated dendrite. Based on diffusion alone, a spine of average dimensions had the ability to retain CaMKII for duration of ~4 s. However, binding sites mimicking those in the PSD controlled the residence time of CaMKII in a highly nonlinear manner. In addition, we observed that F-actin at the spine head/neck junction had a significant impact on CaMKII trapping in dendritic spines. We discuss these results in the context of possible mechanisms that may explain the experimental results that have shown extended accumulation of CaMKII in dendritic spines during synaptic plasticity and LTP induction.