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In a manner unique among activity-regulated immediate early genes (IEGs), mRNA encoded by Arc (also known as Arg3.1) undergoes rapid transport to dendrites and local synaptic translation. Despite this intrinsic appeal, relatively little is known about the neuronal and behavioral functions of Arc or its molecular mechanisms of action. Here, we attempt to distill recent advances on Arc spanning from its transcriptional and translational regulation, the functions of the Arc protein in multiple forms of neuronal plasticity (LTP, LTD and homeostatic plasticity), to its broader role in neural networks of behaving animals. Worley and colleagues show that Arc interacts with endophilin and dynamin, creating a post-synaptic trafficking endosome that selectively modifies the expression of AMPA type glutamate receptors at the excitatory synapses. Both LTD and homeostatic plasticity in the hippocampus are critically dependent on Arc-mediated endocytosis of AMPA receptors. LTD evoked by activation of metabotropic glutamate receptors depends on rapid Arc translation controlled by elongation factor 2. Bramham and colleagues show that sustained translation of newly induced Arc mRNA is necessary for cofilin phosphorylation and stable expansion of the F-actin cytoskeleton underlying LTP consolidation in the dentate gyrus of live rats. In addition to regulating F-actin, Arc synthesis maintains the activity of key translation factors during LTP consolidation. This process of Arc-dependent consolidation is activated by the secretory neurotrophin, BDNF. Moore and colleagues show that Arc mRNA is a natural target for nonsense-mediated mRNA decay (NMD) by virtue of its two conserved 3′-UTR introns. NMD and other related translation-dependent mRNA decay mechanisms may serve as critical brakes on protein expression that contribute to the fine spatial-temporal control of Arc synthesis. In studies in behaving rats, Guzowski and colleagues show that location-specific firing of CA3 and CA1 hippocampal neurons in the presence of theta rhythm provides the necessary stimuli for activation of Arc transcription. The impact of Arc transcription in memory processes may depend on the specific context of co-expressed IEGs, in addition to post-transcriptional regulation of Arc by neuromodulatory inputs from the amygdala and other brain regions. In sum, Arc is emerging as a versatile, finely-tuned system capable of coupling changes in neuronal activity patterns to diverse forms of synaptic plasticity, thereby optimizing information storage in active networks.
The immense capacity and specificity of memory storage in the mammalian central nervous system is thought to depend on the plasticity of neuronal communication at synapses. Dysfunction of synaptic plasticity is implicated in a range of disorders from Alzheimer’s disease to mental retardation and development of chronic pain states. Understanding how neural activity patterns are translated into lasting changes in synaptic connectivity that shape neural network functions and behavior is therefore a major goal. Glutamatergic synapses are capable of expressing diverse forms of activity-dependent potentiation and depression of synaptic efficacy. Persistent forms of synaptic change, as seen in long-term potentiation (LTP) and long-term depression (LTD), typically require rapid new gene expression. While these complex genomic responses are increasingly understood, it has proven difficult to show how changes in specific activity-regulated genes contribute to functional and structural modifications of synapses.
The mRNA encoded by the immediate early gene Arc (activity-regulated cytoskeleton-associated protein), also known as activity-regulated gene 3.1 (Arg3.1), traffics to dendrites and specifically accumulates at sites of synaptic activity (Steward et al., 1998;Lyford et al., 1995;Link et al., 1995). Arc protein also accumulates in dendrites and becomes enriched at the site of local synaptic activity suggesting that Arc protein is locally synthesized (Steward et al., 1998;Yin et al., 2002;Moga et al., 2004;Rodriguez et al., 2005). These unique features implied a function for Arc in coupling synaptic activity to protein synthesis-dependent synaptic plasticity. The goal of the symposium was to collate recent advances in this area, spanning from the transcriptional and translational regulation of Arc, its function in multiple forms of neuronal plasticity (LTP, LTD and homeostatic plasticity), to its role in neural networks and behavior. In writing this minireview, we have attempted to integrate the findings presented at the symposium with other recent advances in the field.
Arc is a single copy gene that encodes a single protein that is highly conserved in vertebrates and is induced in divergent behavioral paradigms in many species. Initial studies of predicted it to be a cytosolic protein, and noted a weak homology with spectrin (Lyford et al., 1995). Biochemical studies demonstrated that Arc protein co-precipitates with polymerized actin. Moreover, the intracellular distribution of Arc was noted to be co-extensive with polymerized actin in dendritic spines suggesting that it may contribute to actin-dependent cell biology. However, Arc did not co-precipitate with pure actin, and the relevant protein for this interaction remains unknown. Subsequent studies examined Arc protein interactions using the yeast two-hybrid system and identified two proteins, dynamin and endophilin, that are known to play a role in endocytosis (Chowdhury et al., 2006). The interaction between Arc and endophilin is notable in that it is selective for endophilin 2 and endophilin 3, which are enriched with Arc in the postsynaptic compartment. The Arc-endophilin interaction requires the C-terminus of the BAR domain of endophilin, which is thought to contact the curved lipid bilayer of the endosome. The Arc-dynamin interaction requires the PH domain of dynamin, which also is thought to be important for dynamin association with charged lipids. When co-expressed in heterologous cells, Arc, endophilin2/3 and dynamin co-associate on the surface of endosomes that traffic transferrin and EGFR. When co-expressed in neurons, Arc and endophilin associate with vesicles that selectively traffic AMPA-type glutamate receptors (AMPAR). Expression of Arc transgene increases the rate of surface AMPAR endocytosis and reduces the level of surface AMPAR. Arc similarly reduces synaptic AMPAR responses as revealed by analysis of spontaneous miniature excitatory synaptic currents (mEPSCs). In this process, Arc appears to be the rate-limiting molecule since overexpression of endophilin alone does not alter AMPAR trafficking. The molecular interactions that allow for selective trafficking of AMPAR remain unknown, but Arc, endophilin and dynamin do not directly bind AMPAR. These observations suggest that natural fluctuations of Arc protein that occur in neurons as a consequence of changes in neuronal activity would be linked to changes in surface/synaptic AMPAR. Consistent with this model, AMPAR levels at synapses of Arc KO neurons exhibit markedly reduced endocytosis, increased steady-state surface AMPAR levels and increased amplitude of spontaneously evoked minis.
The tight association between Arc expression and the strength of excitatory synapses suggested it plays a role in homeostatic plasticity (Shepherd et al., 2006;Rial Verde et al., 2006). Homeostatic plasticity is hypothesized to compensate for Hebbian forms of synaptic plasticity, such as LTP and LTD, by scaling neuronal output without changing the relative strength of individual synapses (Turrigiano, 2007). Homeostatic scaling can be modeled in neuronal cultures by changing the steady state level of network activity. Activity is reduced by addition of tetrodotoxin, and activity can be increased by addition of bicuculline to block GABA type A receptors. Under these conditions, the steady state level of Arc protein is decreased by tetrodotoxin and increased by bicuculline, consistent with a role in the reciprocal changes in surface AMPAR. Expression of Arc transgene blocks the homeostatic increases in AMPAR function induced by chronic neuronal inactivity. Conversely, loss of Arc results in increased AMPAR function and abolishes homeostatic scaling of AMPARs. These observations reveal the importance of Arc’s dynamic expression as it exerts continuous and precise control over synaptic strength and cellular excitability.
It was anticipated that Arc would contribute exclusively to transcription-dependent late-phase plasticity. However, studies that examined the mechanism of rapid, local translation of Arc mRNA reveal an essential role of Arc in a form of NMDA receptor-independent Hebbian plasticity that requires rapid, de novo protein synthesis in dendrites (Park et al., 2008;Waung et al., 2008). Arc is translationally induced within 5 min of activation of group 1 metabotropic glutamate receptors (mGluR), and this response is essential for mGluR-dependent LTD. The increase in Arc translation requires eukaryotic elongation factor 2 kinase (eEF2K), a Ca2+/calmodulin-dependent kinase that binds mGluR and dissociates upon mGluR activation, whereupon it phosphorylates eukaryotic elongation factor 2 (eEF2) (Park et al., 2008). Phospho-eEF2 acts to slow the elongation step of translation and inhibits general protein synthesis, but simultaneously increases Arc translation (Chotiner et al., 2003;Kanhema et al., 2006). In the report of Park and colleagues, genetic deletion of eEF2K resulted in a selective deficit in rapid mGluR-dependent Arc translation and mGluR-LTD. This rapid translational mechanism is disrupted in the Fragile X disease mouse (Fmr1 KO) in which mGluR-LTD does not require de novo protein synthesis, but does require Arc. These observations suggest that that eEF2K-eEF2 and FMRP coordinately control the dynamic translation of Arc mRNA in dendrites that is critical for synapse-specific LTD (Fig. 1).
Physiological studies in Arc KO mice have given important insights into the overall function of Arc in long-term synaptic plasticity. Early phase LTP is enhanced while late phase LTP is blocked in both the dentate gyrus in vivo and in the CA1 region of acute hippocampal slices (Plath et al., 2006). NMDA receptor-dependent LTD of the Schaffer collateral-CA1 synapse is also reduced in acute hippocampal slice recordings (Plath et al., 2006). Consistent with its role in plasticity, activity-dependent refinement of visual projections to the primary visual cortex is disrupted in Arc KO mice (Wang et al., 2006).
Messaoudi et al (2007) sought to resolve the dynamics function of Arc in LTP in vivo. Arc antisense (AS) oligodeoxynucleotides were transiently infused at various times after high-frequency stimulation (HFS) of the medial perforant path input to the dentate gyrus of anesthetized rats. The study revealed distinct temporal requirements for Arc synthesis. Surprisingly, early Arc synthesis is necessary for expression of LTP, while late synthesis is required for LTP consolidation. Infusion of Arc AS during early phase LTP (15 min post-HFS) resulted in transient inhibition of LTP, paralleled by inhibition and recovery of enhanced Arc synthesis. In contrast, application of AS at 2 hours (but not 4 hours) after HFS resulted in a rapid and permanent reversal of LTP and inhibition of dendritic Arc mRNA and protein expression. Taken together, the study suggests a critical role for sustained translation of newly induced, dendritically-transported Arc mRNA in LTP consolidation.
In addition to gene expression, formation of late phase LTP involves enduring structural changes including expansion of the postsynaptic density and enlargement of postsynaptic dendritic spines (Matsuzaki et al., 2004;Bourne and Harris, 2008). These structural changes depend on accumulation of F-actin as well as new protein synthesis (Fukazawa et al., 2003;Bramham, 2008;Tanaka et al., 2008). One of the major regulators of F-actin dynamics in spines is cofilin, which in its phosphorylated state promotes actin polymerization. In the study of Messaoudi and colleagues (2007), inhibition of LTP consolidation was associated with rapid dephosphorylation of hyperphosphorylated cofilin and corresponding loss of nascent F-actin at medial perforant path synapses. Importantly, the ability of Arc AS to reverse LTP was blocked by the F-actin stabilizing drug, jasplakinolide. Thus, Arc effectively couples new gene expression to F-actin expansion underlying stable LTP. Interestingly, local F-actin formation is necessary for selective localization of Arc mRNA to activated synapses (Huang et al., 2007), raising the possibility of a two-way interaction between Arc synthesis and actin polymerization. Although it is not known how Arc synthesis regulates cofilin phosphorylation, a recent study employing yeast two-hybrid and protein pulldown strategies has identified the actin-binding protein WAVE3 as an Arc binding-partner (Peebles et al., Soc Neurosci. Abstr., 2008).
LTP consolidation is strongly modulated by signaling events initiated after LTP induction. One of the major regulators of this process is the secretory peptide brain-derived neurotrophic factor (BDNF) (Bramham and Messaoudi, 2005;Lynch et al., 2007). HFS of excitatory input triggers release of BDNF leading to activation of postsynaptic TrkB receptors which can mobilize further BDNF secretion. Stimulus protocols generating late phase LTP are associated with a period of sustained BDNF release, and disruption of the BDNF-TrkB interaction blocks late phase LTP (Aicardi et al., 2004;Santi et al., 2006;Kang et al., 1997). Exogenous application of BDNF induces a lasting potentiation of excitatory synaptic transmission (BDNF-LTP) in several brain structures. In the dentate gyrus, BDNF-LTP is transcription-dependent, occluded by prior expression of late phase LTP, and associated with dendritic transport of Arc mRNA (Messaoudi et al., 2002;Wibrand et al., 2006;Ying et al., 2002). BDNF-LTP induction is completely abolished by prior treatment with Arc AS (Messaoudi et al., 2007). The maintenance of BDNF-LTP and the associated phosphorylation of cofilin is also rapidly reserved by Arc AS application during a critical time window, as seen for HFS-LTP. Together, the evidence suggests that BDNF is capable of directly activating Arc-dependent LTP consolidation. Furthermore, BDNF signaling phosphorylates cofilin in dendritic spines and is necessary for expansion of dendritic spines during LTP (Rex et al., 2007;Tanaka et al., 2008). A model of Arc-dependent LTP consolidation is shown in Fig. 2.
Protein synthesis-dependent modification of synaptic strength is tightly controlled at the post-transcriptional level, and local regulation in dendrites provides a compelling mechanism for coupling synaptic input patterns to local changes in protein composition (Sutton and Schuman, 2006;Bramham and Wells, 2007). Modulation of translation factor activity through regulated phosphorylation is a major means of adjusting translation rates. LTP in the dentate gyrus is associated with rapid, ERK-dependent phosphorylation of eukaryotic initiation factor 4E (eIF4E) and eEF2 along with enhanced Arc protein synthesis (Kanhema et al., 2006). However, the mechanisms that sustain eIF4E and eEF2 phosphorylation during LTP maintenance are unknown. F-actin formation has long been implicated in the fine-localization of the translational machinery and the activity of certain F-actin associated translation factors (Smart et al., 2003;Gross and Kinzy, 2007). This prompted investigation of whether Arc synthesis contributes to the regulation of translation factor activity (Tiron and Bramham, Soc Neurosci Abstr., 2008). Infusion of Arc AS reversed ongoing LTP and abolished the persistent hyperphosphorylation of the eIF4E and eEF2, without affecting LTP-evoked hyperphosphorylation of ribosomal protein S6. Thus, in addition to regulating F-actin dynamics, Arc appears to play a prominent role in regulating the activity of the translational machinery during LTP consolidation.
Whereas the mechanisms involved in localization and translational repression/activation of Arc and other dendritic mRNAs have received considerable attention in recent years (Czaplinski and Singer, 2006;Bramham and Wells, 2007), how their expression is kept in check once it is activated is much less understood. One means for limiting synaptic protein abundance is through activity-dependent recruitment and sequestration of proteasomes at activated synapses (Bingol and Schuman, 2005;Bingol and Schuman, 2006). On-site ubiquitin-dependent degradation by such proteasomes has been proposed to play a key role in sculpting local protein composition in response to synaptic activity. Another potential means for achieving tight control of gene expression at synapses is through the regulation of transcript abundance via translation-dependent mRNA decay. Because such decay pathways are thought to work exclusively on translationally active mRNAs, they would not be expected to interfere with the transport and local accumulation of translationally repressed mRNPs at distal reaches of the cytoplasm such as dendritic spines. Once translation is activated, however, the mRNA can be quickly degraded, resulting in a tightly controlled burst of protein synthesis
One well known translation-dependent mRNA degradation pathway is nonsense-mediated mRNA decay (NMD). Traditionally, NMD has been viewed as a quality control mechanism for the rapid elimination of mRNAs with premature stop codons. Prevailing wisdom holds that such aberrant mRNAs, the products of either genetic mutation or faulty pre-mRNA processing, must be destroyed because of their potential for encoding deleterious truncated proteins (Isken and Maquat, 2007). Recently, however, it has become apparent that numerous wild type mRNAs, including Arc, are also subject to this pathway (Wittmann et al., 2006;Ni et al., 2007;Giorgi and Moore, 2007) .
NMD of mammalian transcripts is thought to occur upon the first or "pioneer" round of translation (Maquat, 2004). Thus natural substrates for NMD potentially give rise to just a single molecule of protein prior to their demise. One trigger for NMD in mammalian cells is the presence of an exon junction complex (EJC) in the 3′-UTR. EJCs are stably deposited on mRNAs upstream of exon-exon junctions as a consequence of pre-mRNA splicing. Structurally, the EJC consists of a stably-bound tetrameric core that serves as a binding platform for other more transiently associated factors. Within this core, the DEAD-box protein eIF4AIII serves as the main RNA-binding constituent.
Recent work of Giorgi and colleagues (Giorgi et al., 2007) has shown that Arc is a natural target for nonsense-mediated mRNA decay (NMD) by virtue of two conserved introns in its 3′-UTR. In cortical neuronal cultures, eIF4AIII colocalizes with Arc as well as the RNA-binding proteins FMRP and STAU1 in dendritic RNA granules. This colocalization strongly suggests that a major fraction of Arc mRNA in dendrites has not yet undergone a pioneer round of translation. Inhibition of NMD by knockdown of eIF4AIII resulted in elevated expression of Arc mRNA and protein combined with an increase in excitatory synaptic transmission (selective increase in mEPSC amplitude). Bioinformatic analysis also revealed a number of other potential synaptic NMD targets, including two cadherins, a neurexin, a neuregulin, a synaptotagmin and several ion channels.
Functionally, translation-dependent decay of Arc and other dendritic mRNAs could act as a crucial brake to create a strictly limited burst of protein synthesis. In the case of Arc, NMD could allow a time-limited, quantity-limited, and mRNA-specific translational response. This has several important implications for synaptic function and plasticity. For example, sustained translation of Arc during LTP consolidation may be strictly limited by the delivery of newly-induced Arc mRNA to synapses.
The regulation of Arc gene expression by behavioral experience has received considerable attention, due, in large part, to the dendritic localization of Arc mRNA / protein and its requirement in synaptic plasticity and long-term memory (Guzowski et al., 2000;Plath et al., 2006;Messaoudi et al., 2007;Steward and Worley, 2001) These behavioral studies show Arc to be dynamically regulated in many regions of the brain critical for learning and memory (reviewed in (Miyashita et al., 2008a). The most compelling evidence that Arc transcription is tightly and specifically linked to neural activity associated with information processing (Kubik et al., 2007;Guzowski et al., 2005;Guzowski et al., 2004;Vazdarjanova and Guzowski, 2004), and not a nonspecific response to stress or behavioral arousal, was provided by the “catFISH” approach (cellular compartment analysis of temporal activity by fluorescence in situ hybridization; (Guzowski et al., 1999). By exploiting the precise timing of immediate-early gene (IEG) transcription and mRNA processing, catFISH allows cellular activity maps, for two or three discrete behavioral experiences, to be compared within the same brain (Guzowski et al., 1999;Marrone et al., 2008;Guzowski et al., 2005). Using fluorescence in situ hybridization (FISH) and confocal microscopy, the subcellular distribution of Arc alone, or Arc and another IEG, Homer 1a, provides activity history “time stamps” for individual neurons throughout the forebrain (Guzowski et al., 2005) (Fig. 4a). catFISH studies show that Arc transcription is activated in neural ensembles of hippocampal subfields in a highly context-specific fashion, in a manner that is both quantitatively and qualitatively similar to hippocampal cell firing activity recorded in electrophysiological studies (Kubik et al., 2007;Guzowski et al., 2006;Guzowski et al., 2004).
A recent study has more precisely defined the relationship between neural activity and Arc transcriptional activation. Using a closed track paradigm, as used in electrophysiological studies of hippocampal “place cells” (Miyashita et al., 2008b) showed that behavioral experience as minimal as walking a single initial lap around the track was sufficient to activate Arc transcription in the full CA3 ensemble, consistent with a role for CA3 in rapid encoding of episodic information. By contrast, activation of Arc transcription in a complete CA1 ensemble required additional experience on the track. To determine whether location-specific place cell activity is sufficient for activating Arc transcription, rats were tested on the track following infusion of tetracaine or vehicle into the medial septum. Previous studies have demonstrated that intra-septal tetracaine infusions block hippocampal theta rhythm, impair LTP, and disrupt hippocampal-dependent learning, without affecting place specific firing of CA1 neurons. Behaviorally-driven Arc induction was abolished in both CA3 and CA1 neurons of the tetracaine-treated rats (Miyashita et al., 2008b). Together with past catFISH studies, these data support the hypothesis that location-specific firing of CA3 and CA1 neurons in the presence of theta rhythm provide the necessary stimuli for activation of Arc transcription.
The tight coupling of Arc transcription to hippocampal place cell activity, however, is not static, but instead plastic. Guzowski et al. (2006) showed that recent, but not remote, firing history can strongly inhibit further Arc transcriptional activation. This “electro-transcriptional coupling” represents a form of metaplasticity and may function to maintain proper levels of Arc for regulating synaptic homeostasis or plasticity. In addition, the coupling of Arc mRNA expression to functional protein expression can also be subject to modulation (McIntyre et al., 2005). In rats trained in inhibitory avoidance and given post-training intra-amygdala infusions of drugs that either enhanced or impaired long-term memory, hippocampal Arc protein levels were increased or decreased (respectively) relative to controls. These changes in Arc protein expression were not accompanied by similar drug-dependent changes in mRNA expression, suggesting post-transcriptional regulation of Arc protein expression by amygdala-dependent neuromodulatory processes. Thus, functional Arc gene expression enables the integration of multiple neural signals to optimize information storage in active networks. Although Arc is amongst the most dynamically regulated genes in the forebrain, other IEGs share similar robust transcriptional regulation by behavioral stimuli. In a recent study, hippocampal expression of Arc and other known IEGs (e.g., zif268, c-fos, and Homer 1a) remained strongly coupled to ongoing neural activity in rats trained in a spatial water maze task, irrespective of the stage of learning (Fig. 4b;(Miyashita et al., 2008a)). However, distinct and overlapping patterns of experience-dependent gene expression were seen during initial learning, in overtraining, and in reversal learning. Thus, the contribution of Arc to specific neuroplastic mechanisms may be dependent on other activity-regulated genes present in a neuron, at a given time. In this view, the function of Arc or any other IEG to behavioral plasticity can only be fully understood in the “molecular context” of other co-expressed genes.
Arc is emerging as a versatile, finely-tuned system capable of translating neural activity patterns into various forms of protein synthesis-dependent synaptic plasticity. Current evidence suggests a critical role for Arc in AMPAR endocytosis underlying LTD and homeostatic plasticity as well as in F-actin formation underlying LTP consolidation. These new insights have spawned a host of questions that are likely to fuel further discovery. For one, what are the mechanisms that selectively engage newly synthesized Arc in LTP or LTD? Do the various forms of Arc-dependent plasticity co-exist in the same neuron and experimental preparation? For example, local Arc translation underlying F-actin expansion and LTP consolidation could be followed by a delayed, dendrite-wide homeostatic plasticity that globally resets synaptic excitability while preserving the relative differences in synaptic strength. Recent evidence also suggests that Arc RNA synthesis, localization, translation and metabolism are all highly regulated. Differential regulation of these processes, for example through activation of TrkB and mGluR receptors, might provide the requisite cellular contexts for Arc-dependent LTP or LTD. Surprisingly, post-translational regulation of Arc has so far received little attention. Post-translational modification of Arc or one of its binding-partners might dictate the functional mode of Arc at synapses. Another looming question, not addressed at the symposium, is the possible role of Arc in the nucleus, where it accumulates and binds to promyelocytic leukemia nuclear bodies (Bloomer et al., 2007).
The versatile role of Arc in synaptic plasticity coupled with behavioral studies of Arc transcription in physiologically defined neural networks suggests a dedicated role for this immediate early gene in optimizing information storage. A major challenge of future research is to ascribe specific neural network functions to Arc-dependent regulation of LTP, LTD, and homeostatic synaptic plasticity.
C.B was supported by the Norwegian Research Council and European Union RTN grant GENE-MEMORY (504321). P.W. was supported by MH053608 and DA266. M.M. was supported by the Howard Hughes Medical Institute. J.G. was supported by NIH RO1 MH060123