Synaptic protein synthesis associated with memory is regulated by the RISC pathway in Drosophila

Long-lasting forms of memory require protein synthesis, but how the pattern of synthesis is related to the storage of a memory has not been determined. Here we show that neural activity directs the mRNA of the Drosophila Ca(2+), Calcium/Calmodulin-dependent Kinase II (CaMKII), to postsynaptic sites, where it is rapidly translated. These features of CaMKII synthesis are recapitulated during the induction of a long-term memory and produce patterns of local protein synthesis specific to the memory. We show that mRNA transport and synaptic protein synthesis are regulated by components of the RISC pathway, including the SDE3 helicase Armitage, which is specifically required for long-lasting memory. Armitage is localized to synapses and lost in a memory-specific pattern that is inversely related to the pattern of synaptic protein synthesis. Therefore, we propose that degradative control of the RISC pathway underlies the pattern of synaptic protein synthesis associated with a stable memory.     

RISC-y Memories

Local protein synthesis in the synapse is required for synaptic plasticity and has been implicated in learning and memory. However, direct evidence that behavioral training induces local protein synthesis has been lacking. In this issue of Cell, Ashraf et al. (2006) observe persistent local protein synthesis in the antennal lobe synapses of the fruit fly following olfactory-avoidance learning. This protein synthesis is regulated by the RNA-induced silencing complex (RISC).     

Protein synthesis in the dendrite

In neurons, many proteins that are involved in the transduction of synaptic activity and the expression of neural plasticity are specifically localized at synapses. How these proteins are targeted is not clearly understood. One mechanism is synaptic protein synthesis. According to this idea, messenger RNA (mRNA) translation from the polyribosomes that are observed at the synaptic regions provides a local source of synaptic proteins. Although an increasing number of mRNA species has been detected in the dendrite, information about the synaptic synthesis of specific proteins in a physiological context is still limited. The physiological function of synaptic synthesis of specific proteins in synaptogenesis and neural plasticity expression remains to be shown. Experiments aimed at understanding the mechanisms and functions f synaptic protein synthesis might provide important information about the molecular nature of neural plasticity.     

NMDAR‐dependent Argonaute 2 phosphorylation regulates miRNA activity and dendritic spine plasticity

MicroRNAs (miRNAs) repress translation of target mRNAs by associating with Argonaute (Ago) proteins to form the RNA‐induced silencing complex (RISC), underpinning a powerful mechanism for fine‐tuning protein expression. Specific miRNAs are required for NMDA receptor (NMDAR)‐dependent synaptic plasticity by modulating the translation of proteins involved in dendritic spine morphogenesis or synaptic transmission. However, it is unknown how NMDAR stimulation stimulates RISC activity to rapidly repress translation of synaptic proteins. We show that NMDAR stimulation transiently increases Akt‐dependent phosphorylation of Ago2 at S387, which causes an increase in binding to GW182 and a rapid increase in translational repression of LIMK1 via miR‐134. Furthermore, NMDAR‐dependent down‐regulation of endogenous LIMK1 translation in dendrites and dendritic spine shrinkage requires phospho‐regulation of Ago2 at S387. AMPAR trafficking and hippocampal LTD do not involve S387 phosphorylation, defining this mechanism as a specific pathway for structural plasticity. This work defines a novel mechanism for the rapid transduction of NMDAR stimulation into miRNA‐mediated translational repression to control dendritic spine morphology.     

Cells Lacking the Fragile X Mental Retardation Protein (FMRP) have Normal RISC Activity but Exhibit Altered Stress Granule Assembly

The fragile X mental retardation protein (FMRP) is an RNA-binding protein involved in the mRNA metabolism. The absence of FMRP in neurons leads to alterations of the synaptic plasticity, probably as a result of translation regulation defects. The exact molecular mechanisms by which FMRP plays a role in translation regulation have remained elusive. The finding of an interaction between FMRP and the RNA interference silencing complex (RISC), a master of translation regulation, has suggested that both regulators could be functionally linked. We investigated here this link, and we show that FMRP exhibits little overlap both physically and functionally with the RISC machinery, excluding a direct impact of FMRP on RISC function. Our data indicate that FMRP and RISC are associated to distinct pools of mRNAs. FMRP, unlike RISC machinery, associates with the pool of mRNAs that eventually goes into stress granules upon cellular stress. Furthermore, we show that FMRP plays a positive role in this process as the lack of FMRP or a point mutant causing a severe fragile X alter stress granule formation. Our data support the proposal that FMRP plays a role in controlling the fate of mRNAs after translation arrest.     

Regulation and function of local protein synthesis in neuronal dendrites

It has long been shown that protein synthesis can occur in neuronal dendrites, but its significance remained unclear until relatively recently. Studies suggest that local protein synthesis has crucial roles in synaptic plasticity, the change in neuronal communication efficiency that is probably a cellular basis of learning and memory. Induced by neuronal activity, local protein synthesis provides key factors for the modification of activated synapses. In this review, we summarize the evidence for local protein synthesis and its functions in synaptic plasticity. We also discuss the molecular mechanisms by which neuronal activity induces the synthesis of proteins that allow for changes in synaptic function.     

Protein homeostasis and synaptic plasticity

It is clear that de novo protein synthesis has an important function in synaptic transmission and plasticity. A substantial amount of work has shown that mRNA translation in the hippocampus is spatially controlled and that dendritic protein synthesis is required for different forms of long‐term synaptic plasticity. More recently, several studies have highlighted a function for protein degradation by the ubiquitin proteasome system in synaptic plasticity. These observations suggest that changes in synaptic transmission involve extensive regulation of the synaptic proteome. Here, we review experimental data supporting the idea that protein homeostasis is a regulatory motif for synaptic plasticity.     

Rapid ocular dominance plasticity requires cortical but not geniculate protein synthesis

Synaptic plasticity is a multistep process in which rapid, early phases eventually give way to slower, more enduring stages. Diverse forms of synaptic change share a common requirement for protein synthesis in the late stages of plasticity, which are often associated with structural rearrangements. Ocular dominance plasticity in the primary visual cortex (V1) is a long-lasting form of activity-dependent plasticity comprised of well-defined physiological and anatomical stages. The molecular events underlying these stages remain poorly understood. Using the protein synthesis inhibitor cycloheximide, we investigated a role for protein synthesis in ocular dominance plasticity. Suppression of cortical, but not geniculate, protein synthesis impaired rapid ocular dominance plasticity, while leaving neuronal responsiveness intact. These findings suggest that structural changes underlying ocular dominance plasticity occur rapidly following monocular occlusion, and cortical changes guide subsequent alterations in thalamocortical afferents.     

Biochemical mechanisms for translational regulation in synaptic plasticity

Changes in gene expression are required for long-lasting synaptic plasticity and long-term memory in both invertebrates and vertebrates. Regulation of local protein synthesis allows synapses to control synaptic strength independently of messenger RNA synthesis in the cell body. Recent reports indicate that several biochemical signalling cascades couple neurotransmitter and neurotrophin receptors to translational regulatory factors in protein synthesis-dependent forms of synaptic plasticity and memory. In this review, we highlight these translational regulatory mechanisms and the signalling pathways that govern the expression of synaptic plasticity in response to specific types of neuronal stimulation.     

Single-cell optogenetic excitation drives homeostatic synaptic depression

Homeostatic processes have been proposed to explain the discrepancy between the dynamics of synaptic plasticity and the stability of brain function. Forms of synaptic plasticity such as long-term potentiation alter synaptic activity in a synapse- and cell-specific fashion. Although network-wide excitation triggers compensatory homeostatic changes, it is unknown whether neurons initiate homeostatic synaptic changes in response to cell-autonomous increases in excitation. Here we employ optogenetic tools to cell-autonomously excite CA1 pyramidal neurons and find that a compensatory postsynaptic depression of both AMPAR and NMDAR function results. Elevated calcium influx through L-type calcium channels leads to activation of a pathway involving CaM kinase kinase and CaM kinase 4 that induces synaptic depression of AMPAR and NMDAR responses. The synaptic depression of AMPARs but not of NMDARs requires protein synthesis and the GluA2 AMPAR subunit, indicating that downstream of CaM kinase activation divergent pathways regulate homeostatic AMPAR and NMDAR depression.     

Arc/Arg3.1 interacts with the endocytic machinery to regulate AMPA receptor trafficking

Arc/Arg3.1 is an immediate-early gene whose mRNA is rapidly transcribed and targeted to dendrites of neurons as they engage in information processing and storage. Moreover, Arc/Arg3.1 is known to be required for durable forms of synaptic plasticity and learning. Despite these intriguing links to plasticity, Arc/Arg3.1's molecular function remains enigmatic. Here, we demonstrate that Arc/Arg3.1 protein interacts with dynamin and specific isoforms of endophilin to enhance receptor endocytosis. Arc/Arg3.1 selectively modulates trafficking of AMPA-type glutamate receptors (AMPARs) in neurons by accelerating endocytosis and reducing surface expression. The Arc/Arg3.1-endocytosis pathway appears to regulate basal AMPAR levels since Arc/Arg3.1 KO neurons exhibit markedly reduced endocytosis and increased steady-state surface levels. These findings reveal a novel molecular pathway that is regulated by Arc/Arg3.1 and likely contributes to late-phase synaptic plasticity and memory consolidation.     

The autistic neuron: troubled translation?

Autism is a complex genetic disorder, but single-gene disorders with a high prevalence of autism offer insight into its pathogenesis. Recent evidence suggests that some molecular defects in autism may interfere with the mechanisms of synaptic protein synthesis. We propose that aberrant synaptic protein synthesis may represent one possible pathway leading to autistic phenotypes, including cognitive impairment and savant abilities.     

Dendritic mRNA: transport, translation and function

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

Removing pathogenic memories

Experimental research examining the neural bases of nondeclarative memory has offered intriguing insight into how functional and dysfunctional implicit learning affects the brain. Long-term modifications of synaptic transmission, in particular, are currently considered the most plausible mechanism underlying memory trace encoding and compulsions, addiction, anxiety, and phobias. Therefore, an effective psychotherapy must be directed to erase maladaptive implicit memories and aberrant synaptic plasticity. This article describes the neurobiological bases of pathogenic memory disruption to provide some insight into how psychotherapy works. At least two mechanisms of unwanted memory erasing appear to be implicated in the effects of psychotherapy: inhibition of memory consolidation/reconsolidation and extinction. Behavioral evidence demonstrated that these two ways to forget are profoundly distinct in nature, and it is increasingly clear that their cellular, synaptic, and molecular underpinnings are different. Accordingly, the blockade of consolidation/reconsolidation erases memories by reversing the plasticity associated with memory maintenance, whereas extinction is a totally new form of plasticity that, similar to the plasticity underlying the old memory, requires protein synthesis-dependent synaptic remodeling.     

Dopaminergic stimulation of local protein synthesis enhances surface expression of GluR1 and synaptic transmission in hippocampal neurons

The use-dependent modification of synapses is strongly influenced by dopamine, a transmitter that participates in both the physiology and pathophysiology of animal behavior. In the hippocampus, dopaminergic signaling is thought to play a key role in protein synthesis-dependent forms of synaptic plasticity. The molecular mechanisms by which dopamine influences synaptic function, however, are not well understood. Using a GFP-based reporter, as well as a small-molecule reporter of endogenous protein synthesis, we show that dopamine D1/D5 receptor activation stimulates local protein synthesis in the dendrites of hippocampal neurons. We also identify the GluR1 subunit of AMPA receptors as one protein upregulated by dopamine receptor activation, with increased incorporation of surface GluR1 at synaptic sites. The insertion of new GluRs is accompanied by an increase in the frequency of miniature synaptic events. Together, these data suggest a local protein synthesis-dependent activation of previously silent synapses as a result of dopamine receptor stimulation.     

Dynamic visualization of local protein synthesis in hippocampal neurons

Using pharmacological approaches, several recent studies suggest that local protein synthesis is required for synaptic plasticity. Convincing demonstrations of bona fide dendritic protein synthesis in mammalian neurons are rare, however. We developed a protein synthesis reporter in which the coding sequence of green fluorescent protein is flanked by the 5' and 3' untranslated regions from CAMKII-alpha, conferring both dendritic mRNA localization and translational regulation. In cultured hippocampal neurons, we show that BDNF, a growth factor involved in synaptic plasticity, stimulates protein synthesis of the reporter in intact, mechanically, or "optically" isolated dendrites. The stimulation of protein synthesis is blocked by anisomycin and not observed in untreated neurons. In addition, dendrites appear to possess translational hot spots, regions near synapses where protein synthesis consistently occurs over time.     

Critical role of CDK5 and Polo-like kinase 2 in homeostatic synaptic plasticity during elevated activity

Homeostatic plasticity keeps neuronal spiking output within an optimal range in the face of chronically altered levels of network activity. Little is known about the underlying molecular mechanisms, particularly in response to elevated activity. We report that, in hippocampal neurons experiencing heightened activity, the activity-inducible protein kinase Polo-like kinase 2 (Plk2, also known as SNK) was required for synaptic scaling-a principal mechanism underlying homeostatic plasticity. Synaptic scaling also required CDK5, which acted as a "priming" kinase for the phospho-dependent binding of Plk2 to its substrate SPAR, a postsynaptic RapGAP and scaffolding molecule that is degraded following phosphorylation by Plk2. RNAi knockdown of SPAR weakened synapses, and overexpression of a SPAR mutant resistant to Plk2-dependent degradation prevented synaptic scaling. Thus, priming phosphorylation of the Plk2 binding site in SPAR by CDK5, followed by Plk2 recruitment and SPAR phosphorylation-degradation, constitutes a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity.     

Tag-Trigger-Consolidation: A Model of Early and Late Long-Term-Potentiation and Depression

Changes in synaptic efficacies need to be long-lasting in order to serve as a substrate for memory. Experimentally, synaptic plasticity exhibits phases covering the induction of long-term potentiation and depression (LTP/LTD) during the early phase of synaptic plasticity, the setting of synaptic tags, a trigger process for protein synthesis, and a slow transition leading to synaptic consolidation during the late phase of synaptic plasticity. We present a mathematical model that describes these different phases of synaptic plasticity. The model explains a large body of experimental data on synaptic tagging and capture, cross-tagging, and the late phases of LTP and LTD. Moreover, the model accounts for the dependence of LTP and LTD induction on voltage and presynaptic stimulation frequency. The stabilization of potentiated synapses during the transition from early to late LTP occurs by protein synthesis dynamics that are shared by groups of synapses. The functional consequence of this shared process is that previously stabilized patterns of strong or weak synapses onto the same postsynaptic neuron are well protected against later changes induced by LTP/LTD protocols at individual synapses.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.