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Local protein synthesis during axon guidance and synaptic plasticity
Local protein synthesis during axon guidance and synaptic plasticity Kelsey C Martin mRNA localization and regulated translation take central roles in axon guidance and synaptic plasticity. By spatially restricting gene expression within neurons, local protein synthesis provides growth cones and synapses with the capacity to autonomously regulate their structure and function. Studies in a variety of systems have provided insight into the specific roles of local protein synthesis during axonal navigation and during synaptic plasticity, and have begun to delineate the mechanisms underlying mRNA localization and regulated translation. Several powerful new tools have recently been developed to visualize each of these processes. Addresses Department of Psychiatry and Biobehavioral Sciences, Department of Biological Chemistry, Neuropsychiatric Institute, University of California, Los Angeles, Gonda Research Building 3506C, 695 Charles Young Drive South, Los Angeles, California 90095-1761, USA e-mail: [email protected]
Current Opinion in Neurobiology 2004, 14:305–310 This review comes from a themed issue on Signalling mechanisms Edited by Richard L Huganir and S Lawrence Zipursky Available online 20th May 2004 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.05.009 Abbreviations BDNF brain derived neurotrophic factor CaMKII calcium/calmodulin kinase II CPE cytoplasmic polyadenylation element CPEB cytoplasmic polyadenylation element binding protein ELH egg laying hormone FMRP fragile X mental retardation protein GFP green fluorescent protein IRES internal ribosomal entry site LTP long-term potentiation MAP mitogen activated protein miRNAs micro RNAs mRNA messenger RNA PSD postsynaptic density UTR untranslated region ZBP zipcode-binding protein
Introduction The concept of messenger RNA (mRNA) localization and regulated translation at synapses in neurons has gained increasing acceptance in recent years. The discovery of polyribosomes at the base of spines in hippocampal neurons [1] first gave rise to the possibility that protein synthesis could be regulated at a synaptic, as opposed to a www.sciencedirect.com
cell-wide level. This idea was appealing because it provided the neuron with a means of rapidly altering protein composition in a spatially restricted manner. During the past decade, local translation has been found to underlie plasticity at several synapses, including brain derived neurotrophic factor (BDNF)-induced potentiation of hippocampal Schaeffer collateral synapses [2], serotonininduced facilitation of Aplysia californica sensory–motor synapses [3,4], and metabotropic glutamate receptor (mGluR) dependent long-term depression of hippocampal neurons [5]. Several mRNAs and components of the translational machinery have been detected in post-synaptic compartments of mature vertebrate neurons and in preand post-synaptic compartments of mature invertebrate neurons. The mechanisms underlying both the localization of these transcripts and the regulation of their translation are beginning to be delineated in a variety of systems. Although the existence of local translation in dendrites has been widely accepted, the question of whether or not translation occurs in axons has remained more controversial. While several mRNAs have been detected in specific axonal compartments, ribosomes have generally not been detected in these same preparations. This has led many to believe that protein synthesis never occurs in mature axons. During the past several years, however, several studies have challenged this view and have sparked new interest in the role of axonal presynaptic protein synthesis (for a recent review, see Giuditta et al. [6]). Together, these studies clearly indicate that presynaptic protein synthesis plays a part in the navigation of axonal growth cones in developing neurons, that presynaptic translation plays a part in invertebrate neurons and in specific classes of vertebrate neurons whose axonal/dendritic polarity might not be not fully established, and finally, that axonal protein synthesis is recruited during regeneration of injured axons [7]. In this review, I discuss recent studies defining potential roles for local translation during both axon guidance and synaptic plasticity. I also discuss several new tools that have been developed to study mRNA localization and local translation, and to identify the population of mRNAs present in synapses. I then focus on studies that aim to elucidate the mechanisms whereby mRNAs are localized in neurons and the mechanisms whereby stimulation regulates translation of these mRNAs.
A role for local translation during axon guidance During development axons travel long distances to reach their targets, and along this pathway their growth cones Current Opinion in Neurobiology 2004, 14:305–310
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are capable of responding rapidly to local cues. Studies from the 1980s [8] had shown that guidance of retinal ganglion cells could occur in the absence of soma, which indicates that steering decisions could be made locally. In studying this phenomenon, Campbell and Holt [9] found that the application of either netrin-1 or semaphorin 3A, both of which are secreted protein guidance cues, to cultured Xenopus laevis retinal axons induced local protein synthesis within the growth cone, and, furthermore, that blocking translation inhibited the turning but not the growth of these axons. Induction of translation in these growth cones was later shown to depend on activation of the p42/44 and p38 mitogen activated protein (MAP) kinase pathways [10]. Subsequent investigation of the role of protein synthesis in the turning of Xenopus spinal axons revealed a subtler role for translation during growth cone navigation. In these studies, Poo and co-workers [11] found that local translation was required to preserve the sensitivity of a growth cone as it traveled through a gradient of chemical guidance cues. Specifically, they found that cultured spinal growth cones underwent cycles of desensitization and resensitization to netrin-1, and that resensitization, as opposed to the initial turning response, required local translation of new proteins. A role for local translation in switching the growth cone’s responsiveness to cues has emerged from studies of commissural axon targeting in chicks, where the EphA2 receptor is only expressed at high levels in axonal segments that have crossed the midline. Brittis et al. [12] discovered a sequence in the 30 untranslated region (UTR) of EphA2 that allowed the mRNA to be translated only after it had crossed the midline. Thus, when the 30 UTR sequence was fused to a reporter construct, the reporter gene was expressed in the same pattern as EphA2. To demonstrate that translation occurred specifically in the process, the authors infected axons lacking cell bodies with recombinant Sindbis virus. Local translation in axons was also demonstrated to underlie the ability of the neurotrophin BDNF to potentiate neurotransmitter release in growing Xenopus nerve-muscle cultures [13]. In these studies, Zhang and Poo [13] found that local exposure to a bead coated with BDNF led to an increase in transmitter release from the developing synapse, and that this potentiation of release required protein synthesis in the axon. These findings indicate that axonal translation might play a part not only in axon guidance but also in synapse formation.
A role for local translation during synaptic plasticity A general requirement for localized translation has been demonstrated in several model systems of learningCurrent Opinion in Neurobiology 2004, 14:305–310
related synaptic plasticity. Recent studies have begun to uncover more defined roles for local translation during synaptic plasticity and memory. In a very elegant experiment, Miller, Mayford, and co-workers [14] tested the function of one of the best characterized dendritically localized mRNAs, that encoding calcium/calmodulin kinase II (CaMKII) a, by generating a mouse in which the protein-coding region of CaMKII a was intact but its mRNA was restricted to the soma. In the absence of dendritically localized CaMKII a mRNA, mice demonstrated a reduction in late-phase long-term potentiation (LTP), as well as impairments in long-term spatial memory, associative fear conditioning, and object recognition memory. Their studies also indicated that most of the CaMKII a protein present in the post-synaptic density (PSD) derives from locally synthesized protein, as removal of dendritic mRNA produced a dramatic reduction of CaMKII a in the PSD. Ultrastructural studies performed by Ostroff et al. [15] also supported a role for protein synthesis in altering the structure of the PSD in the rat hippocampus. These investigators found that LTP induction greatly increased the percentage of CA1 spines containing polyribosomes. Notably, the postsynaptic densities on spines containing polyribosomes were larger after LTP stimulation, which suggests that local translation served to promote growth of the PSD. Si, Lindquist, and Kandel [16] have discovered a remarkable and novel potential function for local translation in neurons — generation of a prion-like switch at the synapse. In characterizing the Aplysia cytoplasmic polyadenylation element binding protein (CPEB), whose mRNA is localized to distal sensory neurites and is translationally upregulated by serotonin [17], these investigators found that CPEB has a highly glutaminerich amino-terminal sequence and that it functions as a prion-like protein when grown in yeast. Specifically, it was capable of conversion into an altered conformational state that was heritable and transmissible. Taken together, their data suggest that the increased translation of CPEB at the synapse triggers a conformational change to the prion-like state, and that this long-lasting transmissible conformation of the protein serves as a tag or local ‘memory’ of the synaptic stimulation. Interestingly, in its prion-like aggregated state CPEB appears to function more effectively in cytoplasmic polyadenylation, which indicates that one function of this tag might be to promote local translation of mRNAs containing a cytoplasmic polyadenylation element (CPE). That this could be a general mechanism in memory is suggested from the finding that glutamine-rich isoforms of CPEB are also present in the Drosophila, mouse, and human genomes [16]. www.sciencedirect.com
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New tools for visualizing local protein synthesis Recently developed constructs for visualizing local translation have provided powerful tools to study local regulation of translation in neurons. Aakalu et al. [18] fused the 30 and 50 UTRs of CaMKII a to green fluorescent protein (GFP) and transfected this reporter construct into cultured hippocampal neurons. Imaging GFP fluorescence using confocal microscopy, the authors found that BDNF application led to an increase in translation of the GFP reporter in dendrites, including in dendrites that had been severed from the cell body. Job and Eberwine [19] transfected GFP mRNA into severed hippocampal dendrites and, using two photon microscopy imaging, found that mGluR agonists stimulated translation of the GFP construct at ‘hotspots’ within the isolated dendrite. To uncouple mRNA localization from translation, Kiebler and co-workers [20] developed a GFP reporter construct containing the CaMKII a 30 UTR, which localized the mRNA to dendrites, and an iron responsive element (IRE) in its 50 UTR, rendering its translation dependent on the presence of iron in the medium. Using this construct, the authors found that KCl depolarization, glutamate application, and BDNF application all specifically increased translation as opposed to localization of the GFP reporter. A recent study by Ju et al. [21] has utilized a new method to visualize dendritic translation, based upon membranepermeant biarsenical dyes that fluoresce only after binding to short sequences containing four cysteine residues. By transfecting hippocampal cells with recombinant a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor subunits GluR1 and GluR2 that contain the tetracysteine motif in their intracellular carboxy domains, the authors demonstrated that translation of both gluR1 and gluR2 in transected dendrites was regulated by activity. Recently developed methods have also allowed visualization of mRNA localization in neurons in real-time. One of these techniques involves the use of a fusion protein consisting of GFP linked to the RNA binding coat protein of the phage MS2 and a transgene encoding the RNA of interest, to which binding sites for the fusion protein have been inserted [22]. The GFP–MS2 also has a nuclear localization signal (NLS), so it accumulates in the nucleus, and is only transported out into the cytoplasm after it has bound to an mRNA. Using this technique to follow the movement of CaMKII a mRNA in cultured hippocampal neurons, Rook et al. [23] showed that depolarization led to a change from oscillatory to unidirectional movements of CaMKII a mRNA into the dendrite. Bassell and co-workers [24] used GFP-tagged zipcodebinding protein 1 (ZBP1) to follow mRNA localization in www.sciencedirect.com
living cultured neurons. ZBP1 is an RNA binding protein shown to bind to sequences in the mRNA (termed ‘zip codes’), which direct the localization of the mRNA to dendrites. Combining live imaging with immunocytochemistry and in situ hybridization, they also found that depolarization led to a net movement of ZBP1 from the cell soma into dendrites.
Identification of localized transcripts To identify the population of mRNAs that are localized in dendrites, Eberwine et al. [25] developed methods for extracting RNA from isolated dendrites and amplifying the mRNA to generate probes for microarray analysis. Using this strategy, they have identified approximately 400 mRNAs that are localized to dendrites of cultured hippocampal neurons, which represents 5% of expressed genes. To identify localized mRNAs involved in longterm facilitation of Aplysia sensory–motor synapses, Moccia et al. [26] used a preparation of isolated sensory neurites as the starting material for a complementary DNA (cDNA) library. Sequencing of this library indicated that it contained approximately 250 distinct transcripts. The library was enriched for cytoskeletal elements and molecules involved in translation. The latter finding led to the hypothesis that local translation might function to set up a ‘sink’ for translation, such that transcriptionally induced genes are preferentially translated at previously stimulated synapses. These studies, together with earlier studies using synaptosome preparations as the starting material for identification of localized mRNAs [27], indicate that a surprisingly large number of mRNAs are present in neuronal processes. This endows the synapse with the capacity to alter its protein composition in complex and subtle ways in response to distinct stimuli.
Mechanisms underlying mRNA localization As has been found to be the case in non-neuronal cells, mRNA localization in neurons depends on cis-acting sequences, usually in the 30 UTR, and on trans-acting factors that bind to these sequences. Several groups have demonstrated that the RNA binding protein Staufen is present in adult neurons and that it participates in the trafficking of mRNAs into dendrites [28,29]. Staufen is proposed to bind to RNA-containing granules and to mediate transport of these granules along microtubules into dendrites. By isolating Staufen-containing ribonucleoprotein particles, Kiebler and co-workers [30] have also identified the mammalian homolog of Barentz, an RNA-binding protein involved in mRNA localization in Drosophila oocytes, as a candidate protein that mediates mRNA localization in neurons. Bassell, Singer, and co-workers have demonstrated that the mRNA encoding b-actin is localized to neurites and growth cones of immature neurons, and have identified Current Opinion in Neurobiology 2004, 14:305–310
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the cis-acting sequences, termed the ‘zip codes’ that mediate this localization [31]. Several RNA binding proteins bind this sequence, including ZBP1 and ZBP2, which is a homolog of KSRP (KH-type splicing regulatory protein), an RNA binding protein involved in pre-mRNA splicing [32]. Studies in which ZBP1 was downregulated by morpholino antisense oligonucleotides, or in which ZBP1 was upregulated by overexpression, indicated that b-actin mRNA localization by ZBP1 regulates filopodial dynamics in neurons [33]. Studies of translational regulation in neurons had previously shown that the CPE, a cis element in the 30 UTRs of specific dendritic mRNAs, promotes cytoplasmic polyadenylation-induced translation in response to synaptic stimulation [34]. More recently, Huang et al. [35] found that the CPE and its binding protein CPEB function to facilitate mRNA transport to dendrites. Thus, addition of a CPE was sufficient to target a reporter mRNA into dendrites of cultured hippocampal neurons, overexpression of CPEB promoted the dendritic targeting of the CPE-containing mRNA encoding MAP2, and overexpression of a mutant CPEB (which is incapable of interacting with molecular motors) inhibited the dendritic localization of MAP2. The fragile X mental retardation protein (FMRP) gene is mutated in the most common heritable form of mental retardation (fragile X mental retardation, FXMR). FMRP encodes a protein that is localized to synapses, and is believed to be involved both in the targeting of dendritic mRNAs and in their translational regulation [36]. Darnell et al. [37] used RNA selection to identify the RNA motifs recognized by FMRP, finding that they bound to an element called a ‘G-quartet’, and used this motif to identify synaptic protein containing G-quartets and showed that many of these had altered polysome distribution in patients with FXMR [38]. In a separate set of studies, Eberwine and co-workers [39] developed a novel approach, antibody-positioned RNA amplification, to identify mRNAs associated with FMRP, and found that many of the proteins encoded by these mRNAs showed altered abundance or localization in FMRP null mice. FMRP also interacts with a noncoding, dendritically localized RNA polymerase III transcript, BC1 RNA, and this interaction is essential for the translational repression of FMRP-bound mRNAs at the synapse [40].
Mechanisms underlying translational regulation of synaptically localized transcripts The translation of dendritically localized mRNAs appears to be regulated by many mechanisms. One such mechanism is rapamycin-dependent translational regulation, which involves regulation of translational initiation through the 50 UTR of mRNAs. Components of rapamycin-sensitive translation have been detected in the denCurrent Opinion in Neurobiology 2004, 14:305–310
drites of hippocampal neurons [41] and in Aplysia synaptosomes [42]. A role for rapamycin dependent translation has been described in the stabilization of long-lasting synapse-specific plasticity of Aplysia sensory–motor synapses [17,43]. Stimulation-induced translation of localized mRNAs can also be mediated by cytoplasmic polyadenylation [34,44]. Specifically, NMDA receptor stimulation leads to phosphorylation and activation of CPEB, which results in the length of the polyA tail of CaMKII a being increased, and increased translation of the message. Translation of mRNAs using internal ribosomal entry sites (IRESs) has also been proposed to provide a mechanism for specifically regulating translation of several dendritically localized messages [45]. Dyer et al. [46] have shown, using a reporter construct, that the mRNA encoding egg-laying hormone (ELH) in Aplysia bag cells contains a functional IRES, and furthermore, that events known to stimulate ELH translation trigger translation of a reporter gene construct downstream from the ELH IRES. Finally, microRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to take a special role in translational regulation in neurons. Many miRNAs have been identified in mammalian neurons, have been shown to be developmentally regulated in their expression patterns, and, furthermore, have been shown to associate with polyribosomes [47]. These studies raise the possibility that miRNAs at the synapse might function to maintain mRNAs in a translationally dormant state, and to thereby negatively regulate dendritic translation. Interestingly, mammalian FMRP has recently been shown to interact with miRNAs and with components of the miRNA pathways, including Dicer and the mammalian ortholog of Argonaute 1 [48]. This interaction was further shown to be essential to FMRP function in neural development and synaptogenesis, which suggests that FMRP might regulate neuronal translation through miRNAs.
Conclusions The concept of local translation at growth cones and at synapses in neurons has gained increasing acceptance during the past decade. Recent studies have outlined specific roles for local translation in growth cones during axon navigation and synapse formation, and for synaptic protein synthesis during long-lasting synaptic plasticity and memory formation in the adult brain. Novel tools have been used to identify localized transcripts, to elucidate the mechanisms whereby mRNAs are localized and whereby their translation is regulated by neuronal activity. Taken together with studies pointing to a role for local degradation of proteins in growth cones and at synapses [49], these investigations indicate that neurons are endowed with a remarkable capacity for rapidly www.sciencedirect.com
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regulating gene expression in spatially restricted subcellular compartments. Present challenges in the field include characterizing the entire population of localized transcripts in neurons, understanding how neuronal activity regulates their mRNA localization and translation, and ultimately elucidating the specific roles the locally translated proteins play in synaptic function. As we understand more about these local events, it will become particularly interesting to address the question of how local processes at the growth cone or synapse are integrated, temporally and spatially, with changes in gene expression occurring at the level of the nucleus.
Acknowledgements I would like to thank the members of my laboratory for stimulating discussions.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
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Current Opinion in Neurobiology 2004, 14:305–310 This review comes from a themed issue on Signalling mechanisms Edited by Richard L Huganir and S Lawrence Zipursky Available online 20th May 2004 0959-4388/$ – see front matter ß 2004 Elsevier Ltd. All rights reserved. DOI 10.1016/j.conb.2004.05.009 Abbreviations BDNF brain derived neurotrophic factor CaMKII calcium/calmodulin kinase II CPE cytoplasmic polyadenylation element CPEB cytoplasmic polyadenylation element binding protein ELH egg laying hormone FMRP fragile X mental retardation protein GFP green fluorescent protein IRES internal ribosomal entry site LTP long-term potentiation MAP mitogen activated protein miRNAs micro RNAs mRNA messenger RNA PSD postsynaptic density UTR untranslated region ZBP zipcode-binding protein
Introduction The concept of messenger RNA (mRNA) localization and regulated translation at synapses in neurons has gained increasing acceptance in recent years. The discovery of polyribosomes at the base of spines in hippocampal neurons [1] first gave rise to the possibility that protein synthesis could be regulated at a synaptic, as opposed to a www.sciencedirect.com
cell-wide level. This idea was appealing because it provided the neuron with a means of rapidly altering protein composition in a spatially restricted manner. During the past decade, local translation has been found to underlie plasticity at several synapses, including brain derived neurotrophic factor (BDNF)-induced potentiation of hippocampal Schaeffer collateral synapses [2], serotonininduced facilitation of Aplysia californica sensory–motor synapses [3,4], and metabotropic glutamate receptor (mGluR) dependent long-term depression of hippocampal neurons [5]. Several mRNAs and components of the translational machinery have been detected in post-synaptic compartments of mature vertebrate neurons and in preand post-synaptic compartments of mature invertebrate neurons. The mechanisms underlying both the localization of these transcripts and the regulation of their translation are beginning to be delineated in a variety of systems. Although the existence of local translation in dendrites has been widely accepted, the question of whether or not translation occurs in axons has remained more controversial. While several mRNAs have been detected in specific axonal compartments, ribosomes have generally not been detected in these same preparations. This has led many to believe that protein synthesis never occurs in mature axons. During the past several years, however, several studies have challenged this view and have sparked new interest in the role of axonal presynaptic protein synthesis (for a recent review, see Giuditta et al. [6]). Together, these studies clearly indicate that presynaptic protein synthesis plays a part in the navigation of axonal growth cones in developing neurons, that presynaptic translation plays a part in invertebrate neurons and in specific classes of vertebrate neurons whose axonal/dendritic polarity might not be not fully established, and finally, that axonal protein synthesis is recruited during regeneration of injured axons [7]. In this review, I discuss recent studies defining potential roles for local translation during both axon guidance and synaptic plasticity. I also discuss several new tools that have been developed to study mRNA localization and local translation, and to identify the population of mRNAs present in synapses. I then focus on studies that aim to elucidate the mechanisms whereby mRNAs are localized in neurons and the mechanisms whereby stimulation regulates translation of these mRNAs.
A role for local translation during axon guidance During development axons travel long distances to reach their targets, and along this pathway their growth cones Current Opinion in Neurobiology 2004, 14:305–310
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are capable of responding rapidly to local cues. Studies from the 1980s [8] had shown that guidance of retinal ganglion cells could occur in the absence of soma, which indicates that steering decisions could be made locally. In studying this phenomenon, Campbell and Holt [9] found that the application of either netrin-1 or semaphorin 3A, both of which are secreted protein guidance cues, to cultured Xenopus laevis retinal axons induced local protein synthesis within the growth cone, and, furthermore, that blocking translation inhibited the turning but not the growth of these axons. Induction of translation in these growth cones was later shown to depend on activation of the p42/44 and p38 mitogen activated protein (MAP) kinase pathways [10]. Subsequent investigation of the role of protein synthesis in the turning of Xenopus spinal axons revealed a subtler role for translation during growth cone navigation. In these studies, Poo and co-workers [11] found that local translation was required to preserve the sensitivity of a growth cone as it traveled through a gradient of chemical guidance cues. Specifically, they found that cultured spinal growth cones underwent cycles of desensitization and resensitization to netrin-1, and that resensitization, as opposed to the initial turning response, required local translation of new proteins. A role for local translation in switching the growth cone’s responsiveness to cues has emerged from studies of commissural axon targeting in chicks, where the EphA2 receptor is only expressed at high levels in axonal segments that have crossed the midline. Brittis et al. [12] discovered a sequence in the 30 untranslated region (UTR) of EphA2 that allowed the mRNA to be translated only after it had crossed the midline. Thus, when the 30 UTR sequence was fused to a reporter construct, the reporter gene was expressed in the same pattern as EphA2. To demonstrate that translation occurred specifically in the process, the authors infected axons lacking cell bodies with recombinant Sindbis virus. Local translation in axons was also demonstrated to underlie the ability of the neurotrophin BDNF to potentiate neurotransmitter release in growing Xenopus nerve-muscle cultures [13]. In these studies, Zhang and Poo [13] found that local exposure to a bead coated with BDNF led to an increase in transmitter release from the developing synapse, and that this potentiation of release required protein synthesis in the axon. These findings indicate that axonal translation might play a part not only in axon guidance but also in synapse formation.
A role for local translation during synaptic plasticity A general requirement for localized translation has been demonstrated in several model systems of learningCurrent Opinion in Neurobiology 2004, 14:305–310
related synaptic plasticity. Recent studies have begun to uncover more defined roles for local translation during synaptic plasticity and memory. In a very elegant experiment, Miller, Mayford, and co-workers [14] tested the function of one of the best characterized dendritically localized mRNAs, that encoding calcium/calmodulin kinase II (CaMKII) a, by generating a mouse in which the protein-coding region of CaMKII a was intact but its mRNA was restricted to the soma. In the absence of dendritically localized CaMKII a mRNA, mice demonstrated a reduction in late-phase long-term potentiation (LTP), as well as impairments in long-term spatial memory, associative fear conditioning, and object recognition memory. Their studies also indicated that most of the CaMKII a protein present in the post-synaptic density (PSD) derives from locally synthesized protein, as removal of dendritic mRNA produced a dramatic reduction of CaMKII a in the PSD. Ultrastructural studies performed by Ostroff et al. [15] also supported a role for protein synthesis in altering the structure of the PSD in the rat hippocampus. These investigators found that LTP induction greatly increased the percentage of CA1 spines containing polyribosomes. Notably, the postsynaptic densities on spines containing polyribosomes were larger after LTP stimulation, which suggests that local translation served to promote growth of the PSD. Si, Lindquist, and Kandel [16] have discovered a remarkable and novel potential function for local translation in neurons — generation of a prion-like switch at the synapse. In characterizing the Aplysia cytoplasmic polyadenylation element binding protein (CPEB), whose mRNA is localized to distal sensory neurites and is translationally upregulated by serotonin [17], these investigators found that CPEB has a highly glutaminerich amino-terminal sequence and that it functions as a prion-like protein when grown in yeast. Specifically, it was capable of conversion into an altered conformational state that was heritable and transmissible. Taken together, their data suggest that the increased translation of CPEB at the synapse triggers a conformational change to the prion-like state, and that this long-lasting transmissible conformation of the protein serves as a tag or local ‘memory’ of the synaptic stimulation. Interestingly, in its prion-like aggregated state CPEB appears to function more effectively in cytoplasmic polyadenylation, which indicates that one function of this tag might be to promote local translation of mRNAs containing a cytoplasmic polyadenylation element (CPE). That this could be a general mechanism in memory is suggested from the finding that glutamine-rich isoforms of CPEB are also present in the Drosophila, mouse, and human genomes [16]. www.sciencedirect.com
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New tools for visualizing local protein synthesis Recently developed constructs for visualizing local translation have provided powerful tools to study local regulation of translation in neurons. Aakalu et al. [18] fused the 30 and 50 UTRs of CaMKII a to green fluorescent protein (GFP) and transfected this reporter construct into cultured hippocampal neurons. Imaging GFP fluorescence using confocal microscopy, the authors found that BDNF application led to an increase in translation of the GFP reporter in dendrites, including in dendrites that had been severed from the cell body. Job and Eberwine [19] transfected GFP mRNA into severed hippocampal dendrites and, using two photon microscopy imaging, found that mGluR agonists stimulated translation of the GFP construct at ‘hotspots’ within the isolated dendrite. To uncouple mRNA localization from translation, Kiebler and co-workers [20] developed a GFP reporter construct containing the CaMKII a 30 UTR, which localized the mRNA to dendrites, and an iron responsive element (IRE) in its 50 UTR, rendering its translation dependent on the presence of iron in the medium. Using this construct, the authors found that KCl depolarization, glutamate application, and BDNF application all specifically increased translation as opposed to localization of the GFP reporter. A recent study by Ju et al. [21] has utilized a new method to visualize dendritic translation, based upon membranepermeant biarsenical dyes that fluoresce only after binding to short sequences containing four cysteine residues. By transfecting hippocampal cells with recombinant a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) glutamate receptor subunits GluR1 and GluR2 that contain the tetracysteine motif in their intracellular carboxy domains, the authors demonstrated that translation of both gluR1 and gluR2 in transected dendrites was regulated by activity. Recently developed methods have also allowed visualization of mRNA localization in neurons in real-time. One of these techniques involves the use of a fusion protein consisting of GFP linked to the RNA binding coat protein of the phage MS2 and a transgene encoding the RNA of interest, to which binding sites for the fusion protein have been inserted [22]. The GFP–MS2 also has a nuclear localization signal (NLS), so it accumulates in the nucleus, and is only transported out into the cytoplasm after it has bound to an mRNA. Using this technique to follow the movement of CaMKII a mRNA in cultured hippocampal neurons, Rook et al. [23] showed that depolarization led to a change from oscillatory to unidirectional movements of CaMKII a mRNA into the dendrite. Bassell and co-workers [24] used GFP-tagged zipcodebinding protein 1 (ZBP1) to follow mRNA localization in www.sciencedirect.com
living cultured neurons. ZBP1 is an RNA binding protein shown to bind to sequences in the mRNA (termed ‘zip codes’), which direct the localization of the mRNA to dendrites. Combining live imaging with immunocytochemistry and in situ hybridization, they also found that depolarization led to a net movement of ZBP1 from the cell soma into dendrites.
Identification of localized transcripts To identify the population of mRNAs that are localized in dendrites, Eberwine et al. [25] developed methods for extracting RNA from isolated dendrites and amplifying the mRNA to generate probes for microarray analysis. Using this strategy, they have identified approximately 400 mRNAs that are localized to dendrites of cultured hippocampal neurons, which represents 5% of expressed genes. To identify localized mRNAs involved in longterm facilitation of Aplysia sensory–motor synapses, Moccia et al. [26] used a preparation of isolated sensory neurites as the starting material for a complementary DNA (cDNA) library. Sequencing of this library indicated that it contained approximately 250 distinct transcripts. The library was enriched for cytoskeletal elements and molecules involved in translation. The latter finding led to the hypothesis that local translation might function to set up a ‘sink’ for translation, such that transcriptionally induced genes are preferentially translated at previously stimulated synapses. These studies, together with earlier studies using synaptosome preparations as the starting material for identification of localized mRNAs [27], indicate that a surprisingly large number of mRNAs are present in neuronal processes. This endows the synapse with the capacity to alter its protein composition in complex and subtle ways in response to distinct stimuli.
Mechanisms underlying mRNA localization As has been found to be the case in non-neuronal cells, mRNA localization in neurons depends on cis-acting sequences, usually in the 30 UTR, and on trans-acting factors that bind to these sequences. Several groups have demonstrated that the RNA binding protein Staufen is present in adult neurons and that it participates in the trafficking of mRNAs into dendrites [28,29]. Staufen is proposed to bind to RNA-containing granules and to mediate transport of these granules along microtubules into dendrites. By isolating Staufen-containing ribonucleoprotein particles, Kiebler and co-workers [30] have also identified the mammalian homolog of Barentz, an RNA-binding protein involved in mRNA localization in Drosophila oocytes, as a candidate protein that mediates mRNA localization in neurons. Bassell, Singer, and co-workers have demonstrated that the mRNA encoding b-actin is localized to neurites and growth cones of immature neurons, and have identified Current Opinion in Neurobiology 2004, 14:305–310
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the cis-acting sequences, termed the ‘zip codes’ that mediate this localization [31]. Several RNA binding proteins bind this sequence, including ZBP1 and ZBP2, which is a homolog of KSRP (KH-type splicing regulatory protein), an RNA binding protein involved in pre-mRNA splicing [32]. Studies in which ZBP1 was downregulated by morpholino antisense oligonucleotides, or in which ZBP1 was upregulated by overexpression, indicated that b-actin mRNA localization by ZBP1 regulates filopodial dynamics in neurons [33]. Studies of translational regulation in neurons had previously shown that the CPE, a cis element in the 30 UTRs of specific dendritic mRNAs, promotes cytoplasmic polyadenylation-induced translation in response to synaptic stimulation [34]. More recently, Huang et al. [35] found that the CPE and its binding protein CPEB function to facilitate mRNA transport to dendrites. Thus, addition of a CPE was sufficient to target a reporter mRNA into dendrites of cultured hippocampal neurons, overexpression of CPEB promoted the dendritic targeting of the CPE-containing mRNA encoding MAP2, and overexpression of a mutant CPEB (which is incapable of interacting with molecular motors) inhibited the dendritic localization of MAP2. The fragile X mental retardation protein (FMRP) gene is mutated in the most common heritable form of mental retardation (fragile X mental retardation, FXMR). FMRP encodes a protein that is localized to synapses, and is believed to be involved both in the targeting of dendritic mRNAs and in their translational regulation [36]. Darnell et al. [37] used RNA selection to identify the RNA motifs recognized by FMRP, finding that they bound to an element called a ‘G-quartet’, and used this motif to identify synaptic protein containing G-quartets and showed that many of these had altered polysome distribution in patients with FXMR [38]. In a separate set of studies, Eberwine and co-workers [39] developed a novel approach, antibody-positioned RNA amplification, to identify mRNAs associated with FMRP, and found that many of the proteins encoded by these mRNAs showed altered abundance or localization in FMRP null mice. FMRP also interacts with a noncoding, dendritically localized RNA polymerase III transcript, BC1 RNA, and this interaction is essential for the translational repression of FMRP-bound mRNAs at the synapse [40].
Mechanisms underlying translational regulation of synaptically localized transcripts The translation of dendritically localized mRNAs appears to be regulated by many mechanisms. One such mechanism is rapamycin-dependent translational regulation, which involves regulation of translational initiation through the 50 UTR of mRNAs. Components of rapamycin-sensitive translation have been detected in the denCurrent Opinion in Neurobiology 2004, 14:305–310
drites of hippocampal neurons [41] and in Aplysia synaptosomes [42]. A role for rapamycin dependent translation has been described in the stabilization of long-lasting synapse-specific plasticity of Aplysia sensory–motor synapses [17,43]. Stimulation-induced translation of localized mRNAs can also be mediated by cytoplasmic polyadenylation [34,44]. Specifically, NMDA receptor stimulation leads to phosphorylation and activation of CPEB, which results in the length of the polyA tail of CaMKII a being increased, and increased translation of the message. Translation of mRNAs using internal ribosomal entry sites (IRESs) has also been proposed to provide a mechanism for specifically regulating translation of several dendritically localized messages [45]. Dyer et al. [46] have shown, using a reporter construct, that the mRNA encoding egg-laying hormone (ELH) in Aplysia bag cells contains a functional IRES, and furthermore, that events known to stimulate ELH translation trigger translation of a reporter gene construct downstream from the ELH IRES. Finally, microRNAs (miRNAs), a class of small noncoding RNAs, have been postulated to take a special role in translational regulation in neurons. Many miRNAs have been identified in mammalian neurons, have been shown to be developmentally regulated in their expression patterns, and, furthermore, have been shown to associate with polyribosomes [47]. These studies raise the possibility that miRNAs at the synapse might function to maintain mRNAs in a translationally dormant state, and to thereby negatively regulate dendritic translation. Interestingly, mammalian FMRP has recently been shown to interact with miRNAs and with components of the miRNA pathways, including Dicer and the mammalian ortholog of Argonaute 1 [48]. This interaction was further shown to be essential to FMRP function in neural development and synaptogenesis, which suggests that FMRP might regulate neuronal translation through miRNAs.
Conclusions The concept of local translation at growth cones and at synapses in neurons has gained increasing acceptance during the past decade. Recent studies have outlined specific roles for local translation in growth cones during axon navigation and synapse formation, and for synaptic protein synthesis during long-lasting synaptic plasticity and memory formation in the adult brain. Novel tools have been used to identify localized transcripts, to elucidate the mechanisms whereby mRNAs are localized and whereby their translation is regulated by neuronal activity. Taken together with studies pointing to a role for local degradation of proteins in growth cones and at synapses [49], these investigations indicate that neurons are endowed with a remarkable capacity for rapidly www.sciencedirect.com
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regulating gene expression in spatially restricted subcellular compartments. Present challenges in the field include characterizing the entire population of localized transcripts in neurons, understanding how neuronal activity regulates their mRNA localization and translation, and ultimately elucidating the specific roles the locally translated proteins play in synaptic function. As we understand more about these local events, it will become particularly interesting to address the question of how local processes at the growth cone or synapse are integrated, temporally and spatially, with changes in gene expression occurring at the level of the nucleus.
Acknowledgements I would like to thank the members of my laboratory for stimulating discussions.
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