Traditionally, hippocampal long-term potentiation (LTP) of synaptic strength requires Ca2+/calmodulin(CaM)-dependent protein kinase II (CaMKII) and other kinases, while long-term depression (LTD) requires phosphatases. Here we found that LTD also requires CaMKII and its phospho-T286-induced “autonomous” (Ca2+-independent) activity. However, while LTP is known to induce phosphorylation of the AMPA-type glutamate receptor (AMPAR) subunit GluA1 at S831, LTD instead induced CaMKII-mediated phosphorylation at S567, a site known to reduce synaptic GluA1 localization. GluA1 S831 phosphorylation by “autonomous” CaMKII was further stimulated by Ca2+/CaM, as expected for traditional substrates. By contrast, GluA1 S567 represents a distinct substrate-class that is unaffected by such stimulation. This differential regulation caused GluA1 S831 to be favored by LTP-type stimuli (strong but brief), while GluA1 S567 was favored by LTD-type stimuli (weak but prolonged). Thus, requirement of autonomous CaMKII in opposing forms of plasticity involves distinct substrate classes that are differentially regulated to enable stimulus-dependent substrate-site preference.
In the mammalian central nervous system, the majority of fast excitatory synaptic transmission is mediated by glutamate acting on AMPA-type ionotropic glutamate receptors. The abundance of AMPA receptors at the synapse can be modulated through receptor trafficking, which dynamically regulates many fundamental brain functions, including learning and memory. Reversible posttranslational modifications, including phosphorylation, palmitoylation and ubiquitination of AMPA receptor subunits are important regulatory mechanisms for controlling synaptic AMPA receptor expression and function. In this review, we highlight recent advances in the study of AMPA receptor posttranslational modifications and discuss how these modifications regulate AMPA receptor trafficking and function at synapses.
Synaptic activity triggers a profound reorganization of the molecular composition of excitatory synapses. For example, NMDA receptors are removed from synapses in an activity- and calcium-dependent manner, via casein kinase 2 (CK2) phosphorylation of the PDZ-ligand of the GluN2B subunit (S1480). However, how synaptic activity drives this process remains unclear because CK2 is a constitutively active kinase, which is not directly regulated by calcium. We show here that activated CaMKII couples GluN2B and CK2 to form a tri-molecular complex and increase CK2-mediated phosphorylation of GluN2B S1480. In addition, a GluN2B mutant, which contains an insert to mimic the GluN2A sequence and cannot bind to CaMKII, displays reduced S1480 phosphorylation and increased surface-expression. Importantly, we find that although disrupting GluN2B/CaMKII binding reduces synapse number, it increases synaptic-GluN2B content. Therefore, the GluN2B/CaMKII association controls synapse density and PSD composition in an activity-dependent manner, including recruitment of CK2 to remove GluN2B from synapses.
N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptor, which play a central role in learning, memory, and synaptic development. NMDARs are assembled as tetramers composed of two GluN1 subunits and two GluN2 or GluN3 subunits. Although NMDARs are widely expressed throughout the central nervous system, their number, localization, and subunit composition are strictly regulated and differ in a cell- and synapse-specific manner. The brain area, developmental stage and level of synaptic activity are some of the factors that regulate NMDARs. Molecular mechanisms that control subunit-specific NMDAR function include developmental regulation of subunit transcription/translation, differential trafficking through the secretory pathway, post-transcriptional modifications such as phosphorylation, and protein-protein interactions. The GluN2A and GluN2B subunits are highly expressed in cortex and hippocampus and confer many of the distinct properties on endogenous NMDARs. Importantly, the synaptic NMDAR subunit composition changes from predominantly GluN2B-containing to GluN2A-containing NMDARs during synaptic maturation and in response to activity and experience. Some of the molecular mechanisms underlying this GluN2 subunit switch have been recently identified. In addition, the balance between synaptic and extrasynaptic NMDARs is altered in several neuronal disorders. Here, we summarize the recent advances in the identification of NMDAR subunit-specific regulatory mechanisms.
Membrane-associated guanylate kinases (MAGUKs) are the major family of scaffolding proteins at the postsynaptic density. The PSD-MAGUK subfamily, which includes PSD-95, PSD-93, SAP97 and SAP102, is well accepted to be primarily involved in the synaptic anchoring of numerous proteins, including N-methyl-D-aspartate receptors (NMDARs). Notably, the synaptic targeting of NMDARs depends on the binding of the PDZ ligand on the GluN2B subunit to MAGUK PDZ domains as disruption of this interaction dramatically decreases NMDAR surface and synaptic expression. We recently reported a secondary interaction between SAP102 and GluN2B, in addition to the PDZ interaction. Here, we identify two critical residues on GluN2B responsible for the non-PDZ binding to SAP102. Strikingly, either mutation of these critical residues or knock-down of endogenous SAP102 can rescue the defective surface expression and synaptic localization of PDZ binding-deficient GluN2B. These data reveal an unexpected, non-scaffolding role for SAP102 in the synaptic clearance of GluN2B-containing NMDARs.
Since the discovery of long-term potentiation (LTP), thousands of papers have been published on this phenomenon. With this massive amount of information, it is often difficult, especially for someone not directly involved in the field, not to be overwhelmed. The goal of this review is to peel away as many layers as possible, and probe the core properties of LTP. We would argue that the many dozens of proteins that have been implicated in the phenomenon are not essential, but rather modulate, often in indirect ways, the threshold and/or magnitude of LTP. What is required is NMDA receptor activation followed by CaMKII activation. The consequence of CaMKII activation is the rapid recruitment of AMPA receptors to the synapse. This recruitment is independent of AMPA receptor subunit type, but absolutely requires an adequate pool of surface receptors. An important unresolved issue is how exactly CaMKII activation leads to modifications in the PSD to allow rapid enrichment.
Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors that modulate excitatory neurotransmission and synaptic plasticity. The group I mGluRs (mGluR1 and mGluR5) have long intracellular C-terminal domains, which interact with many proteins. Our previous studies identified calmodulin (CaM) as a strong regulator of mGluR5 trafficking and mGluR5-induced calcium signaling. Although it has been accepted that both mGluR1 and mGluR5 interact with CaM, we now show that CaM specifically binds mGluR5 and not mGluR1. We have identified a single critical residue in mGluR5 (L896) that is required for CaM binding. In mGluR1, mutation of the corresponding residue, V909, to leucine is sufficient to confer CaM binding to mGluR1. To investigate the functional effects of CaM binding, we examined the surface expression of mGluR1 and mGluR5 in hippocampal neurons. The mutation in mGluR1 (V909L) that confers CaM binding dramatically increases mGluR1 surface expression, whereas the analogous mutation in mGluR5 that disrupts CaM binding (L896V) decreases mGluR5 surface expression. In addition, the critical residue that alters CaM binding regulates mGluR internalization. Furthermore, we find that mGluR-mediated AMPA receptor endocytosis is enhanced by CaM binding to group I mGluRs. Finally, we show that calcium responses evoked by group I mGluRs are modulated by these mutations, which regulate CaM binding. Our findings elucidate a critical mechanism that specifically affects mGluR5 trafficking and signaling, and distinguishes mGluR1 and mGluR5 regulation.
Calmodulin; metabotropic; glutamate; endocytosis; AMPA receptor; receptor trafficking
NMDA receptors (NMDARs) play a central role in development, synaptic plasticity and neurological disease. NMDAR subunit composition defines their biophysical properties and downstream signaling. Casein kinase 2 (CK2) phosphorylates the NR2B subunit within its PDZ-binding domain; however, the consequences for NMDAR localization and function are unclear. Here we show that CK2 phosphorylation of NR2B regulates synaptic NR2B and NR2A in response to activity. We find that CK2 phosphorylates NR2B, but not NR2A, to drive NR2B-endocytosis and remove NR2B from synapses resulting in an increase in synaptic NR2A expression. During development there is an activity-dependent switch from NR2B to NR2A at cortical synapses. We observe an increase in CK2 expression and NR2B phosphorylation over this same critical period, and show that the acute activity-dependent switch in NR2 subunit composition at developing hippocampal synapses requires CK2 activity. Thus CK2 plays a central role in determining the NR2 subunit content of synaptic NMDARs.
AMPA receptors (AMPARs) are postsynaptic glutamate-gated ion channels that mediate fast excitatory neurotransmission in the mammalian brain. Synaptic activity modulates the density of synaptic AMPARs, thereby affecting synaptic function, learning and memory. Consequently, there is intense interest in defining the molecular mechanisms regulating AMPAR trafficking. Protein expression in the postsynaptic density of excitatory synapses is tightly regulated by ubiquitination, a post-translational modification that dynamically regulates protein trafficking and degradation in response to synaptic activity. Surprisingly, the ubiquitination of mammalian AMPARs has not been reported. In this study, we demonstrate that increasing synaptic activity, via treatment with the GABA(A) receptor antagonist bicuculline, rapidly and robustly induces ubiquitination of the GluA2 AMPAR subunit. Similarly, treatment with AMPAR agonists results in GluA2 ubiquitination, which suggests that ligand-binding plays a critical role. Finally, we find that clathrin- and dynamin-dependent endocytosis of AMPARs is required for activity-dependent GluA2 ubiquitination. Our findings that GluA2 undergoes activity-dependent ubiquitination expand our understanding of how ubiquitination regulates synaptic plasticity.
Activity; glutamate receptor; AMPAR; ubiquitin; endocytosis
Regulated exocytosis is essential for many biological processes, and many components of the protein trafficking machinery are ubiquitous. However, there are also exceptions such as SNAP-25, a neuron-specific SNARE protein, which is essential for synaptic vesicle release from presynaptic nerve terminals. In contrast, SNAP-23 is the ubiquitously-expressed SNAP-25 homologue that is critical for regulated exocytosis in non-neuronal cells. However, the role of SNAP-23 in neurons has not been elucidated. We now find that SNAP-23 is enriched in dendritic spines and colocalizes with constituents of the postsynaptic density, whereas SNAP-25 is restricted to axons. In addition, loss of SNAP-23 using genetically-altered mice or shRNA targeted to SNAP-23 leads to a dramatic decrease in NMDA receptor surface expression and NMDA receptor currents, whereas loss of SNAP-25 does not. Therefore SNAP-23 plays a unique role in the functional regulation of postsynaptic glutamate receptors.
Abnormalities in NMDA receptor (NMDAR) function have been implicated in schizophrenia. Here we show that dysbindin, a schizophrenia-susceptibility gene widely expressed in the forebrain, controls the surface expression of NMDARs in a subunit-specific manner. Imaging analyses revealed a marked increase in surface NR2A, but not NR2B, in hippocampal neurons derived from dysbindin null mutant mice (Dys−/−). Exogenous expression of dysbindin reduced NR2A surface expression in both wild type and Dys−/− neurons. Biotinylation experiments also revealed an increase in surface expression of endogenous NR2A in Dys−/− neurons. Disruption of the dysbindin gene dramatically increased NR2A-mediated synaptic currents, without affecting AMPA receptor currents, in hippocampal CA1 neurons. The Dys−/− hippocampal slices exhibited an enhanced LTP, whereas basal synaptic transmission, presynaptic properties, and LTD were normal. Thus, dysbindin controls hippocampal LTP by selective regulation of the surface expression of NR2A. These results reveal subunit-specific regulation of NMDARs by dysbindin, providing an unexpected link between these two proteins implicated in schizophrenia.
NMDA receptor subunit composition varies throughout the brain, providing molecular diversity in NMDA receptor function. The NR2 subunits (NR2A-D) in large part dictate the distinct functional properties of NMDA receptors and differentially regulate receptor trafficking. Although the NR2C subunit is highly enriched in cerebellar granule cells and plays a unique role in cerebellar function, little is known about NR2C-specific regulation of NMDA receptors. Here we demonstrate that PKB/Akt directly phosphorylates NR2C on serine 1096 (S1096). In addition, we identify 14-3-3ε as a novel NR2C interactor, whose binding is dependent on S1096 phosphorylation. Both growth factor stimulation and NMDA receptor activity lead to a robust increase in both phosphorylation of NR2C on S1096 and surface expression of cerebellar NMDA receptors. Finally, we find that NR2C expression, unlike NR2A and NR2B, supports neuronal survival. Thus, our data provide a direct mechanistic link between growth factor stimulation and regulation of cerebellar NMDA receptors.
The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release, and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a novel form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo.
Cornichon-2 and -3 (CNIH-2/-3) are AMPA receptor (AMPAR) binding proteins that promote receptor trafficking, and markedly slow AMPAR deactivation in heterologous cells, but their role in neurons is unclear. Using CNIH-2 and -3 conditional knock-out mice, we find a profound reduction of AMPAR synaptic transmission in the hippocampus. This deficit is due to the selective loss of surface GluA1-containing AMPARs (GluA1A2 heteromers), leaving a small residual pool of synaptic GluA2A3 heteromers. The kinetics of AMPARs in neurons lacking CNIH2/3 are faster than those in WT neurons due to the fast kinetics of GluA2A3 heteromers. The remarkably selective effect of CNIHs on the GluA1 subunit, is likely mediated by TARP γ-8, which prevents a functional association of CNIHs with non-GluA1 subunits. These results point to a sophisticated interplay between CNIHs and γ-8 that dictates subunit-specific AMPAR trafficking and the strength and kinetics of synaptic AMPAR-mediated transmission.
Neuroligins are postsynaptic cell adhesion molecules that are important for synaptic function through their trans-synaptic interaction with neurexins (NRXNs). The localization and synaptic effects of neuroligin-1 (NL-1, also called NLGN1) are specific to excitatory synapses with the capacity to enhance excitatory synapses dependent on synaptic activity or Ca2+/calmodulin kinase II (CaMKII). Here we report that CaMKII robustly phosphorylates the intracellular domain of NL-1. We show that T739 is the dominant CaMKII site on NL-1 and is phosphorylated in response to synaptic activity in cultured rodent neurons and sensory experience in vivo. Furthermore, a phosphodeficient mutant (NL-1 T739A) reduces the basal and activity-driven surface expression of NL-1, leading to a reduction in neuroligin-mediated excitatory synaptic potentiation. To the best of our knowledge, our results are the first to demonstrate a direct functional interaction between CaMKII and NL-1, two primary components of excitatory synapses.
The extensive dendritic arbor of a pyramidal cell introduces considerable complexity to the integration of synaptic potentials. Propagation of dendritic potentials is largely passive, in contrast to regenerative axonal potentials that are maintained by voltage-gated sodium channels, leading to a declination in amplitude as dendritic potentials travel toward the soma in a manner that disproportionally affects distal synaptic inputs. To counteract this amplitude filtering, Schaffer collateral synapses onto CA1 pyramidal cells contain a varying number of AMPA receptors (AMPARs) per synapse that increases with distance from the soma, a phenomenon known as distance-dependent scaling. Here, we undertake an investigation into the molecular mechanisms of distance-dependent scaling. Using dendritic recordings from rat pyramidal neurons, we confirm the basic scaling phenomenon and find that it is expressed and can be manipulated cell autonomously. Finally, we show that it depends on the presence of both a reserve pool of AMPARs and the AMPAR subunit GluA2.
Glutamate is the major excitatory neurotransmitter in the mammalian CNS and acts on both ionotropic and metabotropic glutamate receptors (mGluRs). The mGluRs are widely distributed in the CNS and modulate a variety of neuronal processes including neurotransmitter release and ion channel function. In hippocampus and cortex, mGluR5 is highly expressed and plays an important role in the regulation of synaptic plasticity. CaM binding dynamically regulates mGluR5 surface expression; however, the mechanisms linking CaM to mGluR5 trafficking are not clear. Recent studies showed that CaM binding to mGluR7 regulates its trafficking in a phosphorylation-dependent manner by disrupting the binding of PICK1. The E3 ligase seven in absentia homolog (Siah)-1A binds to mGluR5 and competes with CaM binding making it an intriguing molecule to regulate phosphorylation-dependent trafficking of mGluR5. In the present study, we find that CaM competes with Siah-1A for mGluR5 binding in a phosphorylation-dependent manner in rat hippocampal neurons. Specifically, phosphorylation of mGluR5 S901 favors Siah-1A binding by displacing CaM. We identified critical residues regulating Siah-1A binding to mGluR5 and showed that binding is essential for the Siah-1A effects on mGluR5 trafficking. Siah-1A binding decreases mGluR5 surface expression and increases endosomal trafficking and lysosomal degradation of mGluR5. Thus CaM-regulated Siah-1A binding to mGluR5 dynamically regulates mGluR5 trafficking. These findings support a conserved role for CaM in regulating mGluR trafficking by PKC-dependent regulation of receptor binding proteins.
In cerebral cortex there is a developmental switch from NR2B- to NR2A-containing NMDA receptors (NMDARs) driven by activity and sensory experience. This subunit switch alters NMDAR function, influences synaptic plasticity, and its dysregulation is associated with neurological disorders. However, the mechanisms driving the subunit switch are not known. Here we show in hippocampal CA1 pyramidal neurons that the NR2B- to NR2A switch driven acutely by activity requires activation of NMDARs and mGluR5, involves PLC, Ca2+ release from IP3R-dependent stores and PKC activity. In mGluR5 knock-out mice the developmental NR2B-NR2A switch in CA1 is deficient. Moreover, in visual cortex of mGluR5 knock-out mice the NR2B-NR2A switch evoked in vivo by visual experience is absent. Thus we establish that mGluR5 and NMDARs are required for the activity-dependent NR2B-NR2A switch and play a critical role in experience-dependent regulation of NMDAR subunit composition in vivo.
Neuroligins, a family of postsynaptic adhesion molecules, are important in synaptogenesis through a well-characterized trans-synaptic interaction with neurexin. In addition, neuroligins are thought to drive postsynaptic assembly through binding of their intracellular domain to PSD-95. However, there is little direct evidence to support the functional necessity of the neuroligin intracellular domain in postsynaptic development. We found that presence of endogenous neuroligin obscured the study of exogenous mutated neuroligin. We therefore used chained microRNAs in rat organotypic hippocampal slices to generate a reduced background of endogenous neuroligin. On this reduced background, we found that neuroligin function was critically dependent on the cytoplasmic tail. However, this function required neither the PDZ ligand nor any other previously described cytoplasmic binding domain, but rather required a previously unknown conserved region. Mutation of a single critical residue in this region inhibited neuroligin-mediated excitatory synaptic potentiation. Finally, we found a functional distinction between neuroligins 1 and 3.
Membrane associated guanylate kinases (MAGUKs) are major components of the postsynaptic density and play important roles in synaptic organization and plasticity. Most excitatory synapses are located on dendritic spines, which are dynamic structures that undergo morphological changes during synapse formation and plasticity. Synapse-associated protein 102 (SAP102) is a MAGUK that is highly expressed early in development and mediates receptor trafficking during synaptogenesis. Mutations in human SAP102 cause mental retardation, which is often accompanied with abnormalities in dendritic spines. However, little is known about the role of SAP102 in regulating synapse formation or spine morphology. We now find that SAP102 contains a novel NMDA receptor binding site in the N-terminal domain, which is specific for the NR2B subunit. The interaction between SAP102 and NR2B is PDZ domain-independent and is regulated by alternative splicing of SAP102. We show that SAP102 that possesses an N-terminal insert is developmentally regulated at both mRNA and protein levels. In addition, expression of SAP102 increases synapse formation. Furthermore, the alternative splicing of SAP102 regulates dendritic spine morphology. SAP102 containing the N-terminal insert promotes lengthening of dendritic spines and preferentially promotes the formation of synapses at long spines, whereas an shRNA knockdown of the same SAP102 splice variant causes spine shrinkage. Finally, blocking NMDA receptor activity prevents the spine lengthening induced by the N-terminal splice variant of SAP102. Thus, our data provide the first evidence that SAP102 links NMDA receptor activation to alterations in spine morphology.
SAP102; NMDA; spine morphology; MAGUK; splice variants; PDZ proteins; glutamate receptors
SNARE-mediated membrane fusion is a pivotal event for a wide-variety of biological processes. SNAP-25, a neuron-specific SNARE protein, has been well-characterized and mouse embryos lacking Snap25 are viable. However, the phenotype of mice lacking SNAP-23, the ubiquitously expressed SNAP-25 homolog, remains unknown. To reveal the importance of SNAP-23 function in mouse development, we generated Snap23-null mice by homologous recombination. We were unable to obtain newborn SNAP-23-deficient mice, and analysis of pre-implantation embryos from Snap23Δ/wt matings revealed that Snap23-null blastocysts were dying prior to implantation at embryonic day E3.5. Thus these data reveal a critical role for SNAP-23 during embryogenesis.
PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 over expression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR-dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.
N-methyl-D-aspartate (NMDA) receptors are critical for neuronal development and synaptic plasticity. The molecular mechanisms underlying the synaptic localization and functional regulation of NMDA receptors have been the subject of extensive studies. In particular, phosphorylation has emerged as a fundamental mechanism that regulates NMDA receptor trafficking and can alter the channel properties of NMDA receptors. Here we summarize recent advances in the characterization of NMDA receptor phosphorylation, emphasizing subunit-specific phosphorylation, which differentially controls the trafficking and surface expression of NMDA receptors.
NMDA receptors; Phosphorylation; Kinase; Glutamate