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1.  GKAP/SAPAP orchestrates activity-dependent postsynaptic protein remodeling and homeostatic scaling 
Nature neuroscience  2012;15(12):1655-1666.
How does chronic activity modulation lead to global remodeling of proteins at synapses and synaptic scaling? Here we report a role of guanylate-kinase-associated-protein (GKAP; also known as SAPAP), a scaffolding molecule linking NMDA receptor-PSD-95 to Shank-Homer complexes, in these processes. Over-excitation removes GKAP from synapses via ubiquitin-proteasome system, while inactivity induces synaptic accumulation of GKAP in rat hippocampal neurons. The bi-directional changes of synaptic GKAP levels are controlled by specific CaMKII isoforms coupled to different Ca2+ channels. α-CaMKII activated by NMDA receptor phosphorylates Serine-54 of GKAP to induce poly-ubiquitination of GKAP. In contrast, β-CaMKII activation via L-type voltage-dependent calcium channel promotes GKAP recruitment by phosphorylating Serine-340 and Serine-384 residues, which uncouples GKAP from MyoVa motor complex. Remarkably, overexpressing GKAP turnover mutants not only hampers activity-dependent remodeling of PSD-95 and Shank but also blocks bi-directional synaptic scaling. Therefore, activity-dependent turnover of PSD proteins orchestrated by GKAP is critical for homeostatic plasticity.
doi:10.1038/nn.3259
PMCID: PMC3804128  PMID: 23143515
2.  Dynamic remodeling of scaffold interactions in dendritic spines controls synaptic excitability 
The Journal of Cell Biology  2012;198(2):251-263.
Synaptic activity–dependent remodeling of the glutamate receptor scaffold complex generates a negative feedback loop that limits further NMDA receptor activation.
Scaffolding proteins interact with membrane receptors to control signaling pathways and cellular functions. However, the dynamics and specific roles of interactions between different components of scaffold complexes are poorly understood because of the dearth of methods available to monitor binding interactions. Using a unique combination of single-cell bioluminescence resonance energy transfer imaging in living neurons and electrophysiological recordings, in this paper, we depict the role of glutamate receptor scaffold complex remodeling in space and time to control synaptic transmission. Despite a broad colocalization of the proteins in neurons, we show that spine-confined assembly/disassembly of this scaffold complex, physiologically triggered by sustained activation of synaptic NMDA (N-methyl-d-aspartate) receptors, induces physical association between ionotropic (NMDA) and metabotropic (mGlu5a) synaptic glutamate receptors. This physical interaction results in an mGlu5a receptor–mediated inhibition of NMDA currents, providing an activity-dependent negative feedback loop on NMDA receptor activity. Such protein scaffold remodeling represents a form of homeostatic control of synaptic excitability.
doi:10.1083/jcb.201110101
PMCID: PMC3410417  PMID: 22801779
3.  Degradation of Postsynaptic Scaffold GKAP and Regulation of Dendritic Spine Morphology by the TRIM3 Ubiquitin Ligase in Rat Hippocampal Neurons 
PLoS ONE  2010;5(3):e9842.
Changes in neuronal activity modify the structure of dendritic spines and alter the function and protein composition of synapses. Regulated degradation of postsynaptic density (PSD) proteins by the ubiquitin-proteasome system is believed to play an important role in activity-dependent synaptic remodeling. Stimulating neuronal activity in vitro and in vivo induces the ubiquitination and degradation of GKAP/SAPAP and Shank, major scaffold proteins of the PSD. However, the specific ubiquitin ligases that regulate postsynaptic protein composition have not been identified. Here we identify the RING finger-containing protein TRIM3 as a specific E3 ubiquitin ligase for the PSD scaffold GKAP/SAPAP1. Present in PSD fractions from rat brain, TRIM3 stimulates ubiquitination and proteasome-dependent degradation of GKAP, and induces the loss of GKAP and associated scaffold Shank1 from postsynaptic sites. Suppression of endogenous TRIM3 by RNA interference (RNAi) results in increased accumulation of GKAP and Shank1 at synapses, as well as enlargement of dendritic spine heads. RNAi of TRIM3 also prevented the loss of GKAP induced by synaptic activity. Thus, TRIM3 is a novel E3 ligase that mediates activity-dependent turnover of PSD scaffold proteins and is a negative regulator of dendritic spine morphology.
doi:10.1371/journal.pone.0009842
PMCID: PMC2844417  PMID: 20352094
4.  Regulated RalBP1 Binding to RalA and PSD-95 Controls AMPA Receptor Endocytosis and LTD 
PLoS Biology  2009;7(9):e1000187.
A two step mechanism was identified that regulates receptor endocytosis during the development of long-term depression (LTD), a long-lasting decrease in synaptic transmission.
Long-term depression (LTD) is a long-lasting activity-dependent decrease in synaptic strength. NMDA receptor (NMDAR)–dependent LTD, an extensively studied form of LTD, involves the endocytosis of AMPA receptors (AMPARs) via protein dephosphorylation, but the underlying mechanism has remained unclear. We show here that a regulated interaction of the endocytic adaptor RalBP1 with two synaptic proteins, the small GTPase RalA and the postsynaptic scaffolding protein PSD-95, controls NMDAR-dependent AMPAR endocytosis during LTD. NMDAR activation stimulates RalA, which binds and translocates widespread RalBP1 to synapses. In addition, NMDAR activation dephosphorylates RalBP1, promoting the interaction of RalBP1 with PSD-95. These two regulated interactions are required for NMDAR-dependent AMPAR endocytosis and LTD and are sufficient to induce AMPAR endocytosis in the absence of NMDAR activation. RalA in the basal state, however, maintains surface AMPARs. We propose that NMDAR activation brings RalBP1 close to PSD-95 to promote the interaction of RalBP1-associated endocytic proteins with PSD-95-associated AMPARs. This suggests that scaffolding proteins at specialized cellular junctions can switch their function from maintenance to endocytosis of interacting membrane proteins in a regulated manner.
Author Summary
Neurons adapt over time in order to dampen their response to prolonged or particularly strong stimuli. This process, termed long-term depression (LTD), results in a long-lasting decrease in the efficiency of synaptic transmission. One extensively studied form of LTD requires the activation of a specific class of receptors known as NMDA glutamate receptors (NMDARs). A key molecular event initiated by NMDA receptor activation is the stimulation of protein phosphatases. Another key event is internalization via endocytosis of synaptic AMPA glutamate receptors (AMPARs). However, the mechanism by which protein dephosphorylation is coupled to AMPAR endocytosis has remained unclear. Here, we help to define this mechanism. We show that endocytic proteins, including RalBP1, are widely distributed in neurons under normal conditions. Upon NMDAR activation, the small GTPase RalA becomes activated and binds to RalBP1, resulting in the translocation of RalBP1 and RalBP1-associated endocytic proteins to synapses. At the same time, RalBP1 becomes dephosphorylated, which promotes its binding to the postsynaptic scaffold protein PSD-95, a protein that itself associates with AMPARs. This concerted recruitment of endocytic proteins to the vicinity of AMPARs results in AMPAR endocytosis. On the basis of our data, we propose a model in which dual binding of RalBP1 to both RalA and PSD-95 following RalBP1 dephosphorylation is essential for NMDAR-dependent AMPAR endocytosis during LTD.
doi:10.1371/journal.pbio.1000187
PMCID: PMC2730530  PMID: 19823667
5.  Synaptic dysfunction and abnormal behaviors in mice lacking major isoforms of Shank3 
Human Molecular Genetics  2011;20(15):3093-3108.
SHANK3 is a synaptic scaffolding protein enriched in the postsynaptic density (PSD) of excitatory synapses. Small microdeletions and point mutations in SHANK3 have been identified in a small subgroup of individuals with autism spectrum disorder (ASD) and intellectual disability. SHANK3 also plays a key role in the chromosome 22q13.3 microdeletion syndrome (Phelan–McDermid syndrome), which includes ASD and cognitive dysfunction as major clinical features. To evaluate the role of Shank3 in vivo, we disrupted major isoforms of the gene in mice by deleting exons 4–9. Isoform-specific Shank3e4–9 homozygous mutant mice display abnormal social behaviors, communication patterns, repetitive behaviors and learning and memory. Shank3e4–9 male mice display more severe impairments than females in motor coordination. Shank3e4–9 mice have reduced levels of Homer1b/c, GKAP and GluA1 at the PSD, and show attenuated activity-dependent redistribution of GluA1-containing AMPA receptors. Subtle morphological alterations in dendritic spines are also observed. Although synaptic transmission is normal in CA1 hippocampus, long-term potentiation is deficient in Shank3e4–9 mice. We conclude that loss of major Shank3 species produces biochemical, cellular and morphological changes, leading to behavioral abnormalities in mice that bear similarities to human ASD patients with SHANK3 mutations.
doi:10.1093/hmg/ddr212
PMCID: PMC3131048  PMID: 21558424
6.  S-SCAM/MAGI-2 is an essential synaptic scaffolding molecule for the GluA2-containing maintenance pool of AMPA receptors 
The Journal of Neuroscience  2012;32(20):6967-6980.
Synaptic plasticity, the cellular basis of learning and memory, involves the dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses. One of the remaining key unanswered aspects of AMPAR trafficking is the mechanism by which synaptic strength is preserved in spite of protein turnover. In particular, the identity of AMPAR scaffolding molecule(s) involved in the maintenance of GluA2-containing AMPARs is completely unknown. Here we report that Synaptic scaffolding molecule (S-SCAM, also called membrane-associated guanylate kinase inverted-2 and atrophin interacting protein-1) plays the critical role of maintaining synaptic strength. Increasing S-SCAM levels in rat hippocampal neurons led to specific increases in the surface AMPAR levels, enhanced AMPAR-mediated synaptic transmission, and enlargement of dendritic spines, without significantly effecting GluN levels or NMDAR EPSC. Conversely, decreasing S-SCAM levels by RNA interference-mediated knockdown caused the loss of synaptic AMPARs, which was followed by a severe reduction in the dendritic spine density. Importantly, S-SCAM regulated synaptic AMPAR levels in a manner, dependent on GluA2 not GluA1, sensitive to NSF interaction, and independent of activity. Further, S-SCAM increased surface AMPAR levels in the absence of PSD-95, while PSD-95 was dependent on S-SCAM to increase surface AMPAR levels. Finally, S-SCAM overexpression hampered NMDA-induced internalization of AMPARs and prevented the induction of long term depression, while S-SCAM knockdown did not affect long term depression. Together, these results suggest that S-SCAM is an essential AMPAR scaffolding molecule for the GluA2-containing pool of AMPARs, which are involved in the constitutive pathway of maintaining synaptic strength.
doi:10.1523/JNEUROSCI.0025-12.2012
PMCID: PMC3365591  PMID: 22593065
7.  A Myosin Va Mutant Mouse with Disruptions in Glutamate Synaptic Development and Mature Plasticity in Visual Cortex 
MyosinVa (MyoVa) mediates F-actin-based vesicular transport toward the plasma membrane and is found at neuronal postsynaptic densities (PSDs), but the role of MyoVa in synaptic development and function is largely unknown. Here, in studies using the dominant negative MyoVa neurological mutant mouse Flailer, we find that MyoVa plays an essential role in activity-dependent delivery of PSD-95 and other critical PSD molecules to synapses and in endocytosis of AMPA-type glutamate receptors (AMPAR) in the dendrites of CNS neurons. MyoVa is known to carry a complex containing the major scaffolding proteins of the mature PSD, PSD-95, SAPAP1/GKAP, Shank and Homer, to dendritic spine synapses. In Flailer, neurons show abnormal dendritic shaft localization of PSD-95, stargazin, dynamin3, AMPA glutamate receptors (AMPARs) and abnormal spine morphology. Flailer neurons also have abnormally high AMPAR miniature current frequencies and spontaneous AMPAR currents that are more frequent and larger than in WT while numbers of NMDAR containing synapses remain normal. The AMPAR abnormalities are consistent with a severely disrupted developmental regulation of long-term depression that we find in cortical Flailer neurons. Thus MyoVa plays a fundamentally important role both in localizing mature glutamate synapses to spines and in organizing the synapse for normal function. For this reason Flailer mice will be valuable in further dissecting the role of MyoVa in normal synaptic and circuit refinement and also in studies of neurological and neuropsychiatric diseases where disruptions of normal glutamate synapses are frequently observed.
doi:10.1523/JNEUROSCI.4585-12.2013
PMCID: PMC3699337  PMID: 23658184
8.  CDK5 Is Essential for Soluble Amyloid β-Induced Degradation of GKAP and Remodeling of the Synaptic Actin Cytoskeleton 
PLoS ONE  2011;6(7):e23097.
The early stages of Alzheimer's disease are marked by synaptic dysfunction and loss. This process results from the disassembly and degradation of synaptic components, in particular of scaffolding proteins that compose the post-synaptic density (PSD), namely PSD95, Homer and Shank. Here we investigated in rat frontal cortex dissociated culture the mechanisms involved in the downregulation of GKAP (SAPAP1), which links the PSD95 complex to the Shank complex and cytoskeletal structures within the PSD. We show that Aβ causes the rapid loss of GKAP from synapses through a pathway that critically requires cdk5 activity, and is set in motion by NMDAR activity and Ca2+ influx. We show that GKAP is a direct substrate of cdk5 and that its phosphorylation results in polyubiquitination and proteasomal degradation of GKAP and remodeling (collapse) of the synaptic actin cytoskeleton; the latter effect is abolished in neurons expressing GKAP mutants that are resistant to phosphorylation by cdk5. Given that cdk5 also regulates degradation of PSD95, these results underscore the central position of cdk5 in mediating Aβ-induced PSD disassembly and synapse loss.
doi:10.1371/journal.pone.0023097
PMCID: PMC3146526  PMID: 21829588
9.  Dlg1 binds GKAP to control dynein association with microtubules, centrosome positioning, and cell polarity 
The Journal of Cell Biology  2010;191(3):585-598.
The small GTPase Cdc42 regulates interactions of dynein with microtubules through the polarity protein Dlg1 and the scaffolding protein GKAP.
Centrosome positioning is crucial during cell division, cell differentiation, and for a wide range of cell-polarized functions including migration. In multicellular organisms, centrosome movement across the cytoplasm is thought to result from a balance of forces exerted by the microtubule-associated motor dynein. However, the mechanisms regulating dynein-mediated forces are still unknown. We show here that during wound-induced cell migration, the small G protein Cdc42 acts through the polarity protein Dlg1 to regulate the interaction of dynein with microtubules of the cell front. Dlg1 interacts with dynein via the scaffolding protein GKAP and together, Dlg1, GKAP, and dynein control microtubule dynamics and organization near the cell cortex and promote centrosome positioning. Our results suggest that, by modulating dynein interaction with leading edge microtubules, the evolutionary conserved proteins Dlg1 and GKAP control the forces operating on microtubules and play a fundamental role in centrosome positioning and cell polarity.
doi:10.1083/jcb.201002151
PMCID: PMC3003329  PMID: 21041448
10.  Synaptic State-Dependent Functional Interplay between Postsynaptic Density-95 and Synapse-Associated Protein 102 
The Journal of Neuroscience  2013;33(33):13398-13409.
Activity-dependent regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is the basis for establishing differences in synaptic weights among individual synapses during developmental and experience-dependent synaptic plasticity. Synaptic signaling scaffolds of the Discs large (DLG)–membrane-associated guanylate kinase (MAGUK) protein family regulate these processes by tethering signaling proteins to receptor complexes. Using a molecular replacement strategy with RNAi-mediated knockdown in rat and mouse hippocampal organotypic slice cultures, a postsynaptic density-95 (PSD-95) knock-out mouse line and electrophysiological analysis, our current study identified a functional interplay between two paralogs, PSD-95 and synapse-associated protein 102 (SAP102) to regulate synaptic AMPARs. During synaptic development, the SAP102 protein levels normally plateau but double if PSD-95 expression is prevented during synaptogenesis. For an autonomous function of PSD-95 in regulating synaptic AMPARs, in addition to the previously demonstrated N-terminal multimerization and the first two PDZ (PSD-95, Dlg1, zona occludens-1) domains, the PDZ3 and guanylate kinase domains were required. The Src homology 3 domain was dispensable for the PSD-95-autonomous regulation of basal synaptic transmission. However, it mediated the functional interaction with SAP102 of PSD-95 mutants to enhance AMPARs. These results depict a protein domain-based multifunctional aspect of PSD-95 in regulating excitatory synaptic transmission and unveil a novel form of domain-based interplay between signaling scaffolds of the DLG–MAGUK family.
doi:10.1523/JNEUROSCI.6255-11.2013
PMCID: PMC3742927  PMID: 23946397
11.  GKAP, a Novel Synaptic Protein That Interacts with the Guanylate Kinase-like Domain of the PSD-95/SAP90 Family of Channel Clustering Molecules 
The Journal of Cell Biology  1997;136(3):669-678.
The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH2-terminal PDZ domains, with certain ion channels (NMDA receptors and K+ channels), thereby promoting the clustering of these proteins. Although the function of the NH2-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K+ channels/ NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein– protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).
PMCID: PMC2134290  PMID: 9024696
12.  Brownian diffusion of AMPA receptors is sufficient to explain fast onset of LTP 
BMC Systems Biology  2010;4:25.
Background
Long-Term Potentiation (LTP) of synapses is thought to be due in part to a change in AMPA Receptor trafficking leading to an increase in the number of AMPA Receptors at the synapse. LTP onset occurs within seconds after the induction signal. A particle-based stochastic simulation software is used to investigate the effect of Brownian diffusion of glutamate receptors on receptor incorporation into the synaptic specialisation and the time-course of LTP expression. The model of the dendritic spine includes receptors diffusing within the membrane, scaffold molecules within the synaptic specialisation capable of binding receptors and a molecular picket-fence surrounding the synaptic membrane area, all features found within the biological system.
Results
During simulations, receptors accumulate rapidly at the post-synaptic density (PSD) from the extra-synaptic membrane under a number of biologically observed conditions. The time of half-saturation, t1/2, defined as the time-point at which half the available scaffold proteins are occupied with receptors, is found to be 710 ms. Different scaffold distributions are shown to have little effect on this time-course. Decreasing the probability of escape of receptors from the PSD domain, thus localising receptors closer to the scaffold proteins, substantially decreases t1/2. A decrease of escape probability from 1 to 0 brings about a non-linear decrease in t1/2 from 710 ms to 390 ms. Release-location of receptors within the spine is found to affect the initial rate of receptor incorporation. We simulate three possible sources of receptors: (i) receptors distributed within the spine extra-synaptic membrane; (ii) receptors from exocytotic vesicles released to the synaptic spine; and (iii) receptors entering the spine from the dendritic shaft through the spine neck. Receptors released from exocytotic vesicles initially accumulate faster than receptors released from the other two sources. A model of glutamate release and glutamate-receptor interaction shows that newly inserted receptors make a substantial contribution to a glutamate evoked response within the observed time-frame.
Conclusions
Fast accumulation of AMPA Receptors is consistent with experimentally observed fast onset of LTP expression.
doi:10.1186/1752-0509-4-25
PMCID: PMC2847995  PMID: 20233407
13.  Coordination of synaptic adhesion with dendritic spine remodeling by AF-6 and kalirin-7 
Remodeling of central excitatory synapses is crucial for synapse maturation, plasticity, and contributes to neurodevelopmental and psychiatric disorders. Remodeling of dendritic spines and the associated synapses, has been postulated to require the coordination of adhesion with spine morphology and stability; however, the molecular mechanisms that functionally link adhesion molecules with regulators of dendritic spine morphology are largely unknown. Here we report that spine size and N-cadherin content are tightly coordinated. In rat mature cortical pyramidal neurons, N-cadherin-dependent adhesion modulates the morphology of existing spines by recruiting the Rac1 guanine-nucleotide exchange factor kalirin-7 to synapses through the scaffolding protein AF-6/afadin. In pyramidal neurons, N-cadherin, AF-6, and kalirin-7 colocalize at synapses and participate in the same multiprotein complexes. N-cadherin clustering promotes the reciprocal interaction and recruitment of N-cadherin, AF-6, and kalirin-7, increasing the content of Rac1 and in spines and PAK phosphorylation. N-cadherin-dependent spine enlargement requires AF-6 and kalirin-7 function. Conversely, disruption of N-cadherin leads to thin, long spines, with reduced Rac1 contact, caused by uncoupling of N-cadherin, AF-6, and kalirin-7 from each other. By dynamically linking N-cadherin with a regulator of spine plasticity, this pathway allows synaptic adhesion molecules to rapidly coordinate spine remodeling associated with synapse maturation and plasticity. This study hence identifies a novel mechanism whereby cadherins, a major class of synaptic adhesion molecules, signal to the actin cytoskeleton to control the morphology of dendritic spines, and outlines a mechanism that underlies the coordination of synaptic adhesion with spine morphology.
doi:10.1523/JNEUROSCI.1170-08.2008
PMCID: PMC2727754  PMID: 18550750
Rac1; GluR1; postsynaptic density; synaptic plasticity; cytoskeleton; synapse
14.  PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs) and Synaptic Plasticity 
Current opinion in neurobiology  2011;21(2):306-312.
Activity-dependent modification of excitatory synaptic transmission is fundamental for developmental plasticity of the neural circuits and experience-dependent plasticity. Synaptic glutamatergic receptors including AMPA receptors and NMDA receptors (AMPARs and NMDARs) are embedded in the highly organized protein network in the postsynaptic density. Overwhelming data have shown that PSD-95-like membrane associated guanylate kinases (PSD-MAGUKs), as a major family of scaffold proteins at glutamatergic synapses, regulate basal synaptic AMPAR function and trafficking. It is now clear that PSD-MAGUKs have multifaceted functions in terms of regulating synaptic transmission and plasticity. Here we discuss recent advancements in understanding the roles of PSD-95 and other family members of PSD-MAGUKs in synaptic plasticity, both as an anchoring protein for synaptic AMPARs and also as a signaling scaffold for mediating the interaction of the signaling complex and NMDARs.
doi:10.1016/j.conb.2011.03.001
PMCID: PMC3138136  PMID: 21450454
15.  Ephrin-A5 and EphA5 Interaction Induces Synaptogenesis during Early Hippocampal Development 
PLoS ONE  2010;5(8):e12486.
Background
Synaptogenesis is a fundamental step in neuronal development. For spiny glutamatergic synapses in hippocampus and cortex, synaptogenesis involves adhesion of pre and postsynaptic membranes, delivery and anchorage of pre and postsynaptic structures including scaffolds such as PSD-95 and NMDA and AMPA receptors, which are glutamate-gated ion channels, as well as the morphological maturation of spines. Although electrical activity-dependent mechanisms are established regulators of these processes, the mechanisms that function during early development, prior to the onset of electrical activity, are unclear. The Eph receptors and ephrins provide cell contact-dependent pathways that regulate axonal and dendritic development. Members of the ephrin-A family are glycosyl-phosphatidylinositol-anchored to the cell surface and activate EphA receptors, which are receptor tyrosine kinases.
Methodology/Principal Findings
Here we show that ephrin-A5 interaction with the EphA5 receptor following neuron-neuron contact during early development of hippocampus induces a complex program of synaptogenic events, including expression of functional synaptic NMDA receptor-PSD-95 complexes plus morphological spine maturation and the emergence of electrical activity. The program depends upon voltage-sensitive calcium channel Ca2+ fluxes that activate PKA, CaMKII and PI3 kinase, leading to CREB phosphorylation and a synaptogenic program of gene expression. AMPA receptor subunits, their scaffolds and electrical activity are not induced. Strikingly, in contrast to wild type, stimulation of hippocampal slices from P6 EphA5 receptor functional knockout mice yielded no NMDA receptor currents.
Conclusions/Significance
These studies suggest that ephrin-A5 and EphA5 signals play a necessary, activity-independent role in the initiation of the early phases of synaptogenesis. The coordinated expression of the NMDAR and PSD-95 induced by eprhin-A5 interaction with EphA5 receptors may be the developmental switch that induces expression of AMPAR and their interacting proteins and the transition to activity-dependent synaptic regulation.
doi:10.1371/journal.pone.0012486
PMCID: PMC2930854  PMID: 20824214
16.  Biphasic Synaptic Ca Influx Arising from Compartmentalized Electrical Signals in Dendritic Spines 
PLoS Biology  2009;7(9):e1000190.
Dendritic spines compartmentalize synaptically-evoked biochemical signals. The authors show that electrical compartmentalization provided by a spine endows the associated synapse with additional modes of calcium signaling by shaping the kinetics of synaptic calcium currents.
Excitatory synapses on mammalian principal neurons are typically formed onto dendritic spines, which consist of a bulbous head separated from the parent dendrite by a thin neck. Although activation of voltage-gated channels in the spine and stimulus-evoked constriction of the spine neck can influence synaptic signals, the contribution of electrical filtering by the spine neck to basal synaptic transmission is largely unknown. Here we use spine and dendrite calcium (Ca) imaging combined with 2-photon laser photolysis of caged glutamate to assess the impact of electrical filtering imposed by the spine morphology on synaptic Ca transients. We find that in apical spines of CA1 hippocampal neurons, the spine neck creates a barrier to the propagation of current, which causes a voltage drop and results in spatially inhomogeneous activation of voltage-gated Ca channels (VGCCs) on a micron length scale. Furthermore, AMPA and NMDA-type glutamate receptors (AMPARs and NMDARs, respectively) that are colocalized on individual spine heads interact to produce two kinetically and mechanistically distinct phases of synaptically evoked Ca influx. Rapid depolarization of the spine triggers a brief and large Ca current whose amplitude is regulated in a graded manner by the number of open AMPARs and whose duration is terminated by the opening of small conductance Ca-activated potassium (SK) channels. A slower phase of Ca influx is independent of AMPAR opening and is determined by the number of open NMDARs and the post-stimulus potential in the spine. Biphasic synaptic Ca influx only occurs when AMPARs and NMDARs are coactive within an individual spine. These results demonstrate that the morphology of dendritic spines endows associated synapses with specialized modes of signaling and permits the graded and independent control of multiple phases of synaptic Ca influx.
Author Summary
The vast majority of excitatory synapses in the mammalian central nervous system are made onto dendritic spines, small (< 1 fL) membranous structures stippled along the dendrite. The head of each spine is separated from its parent dendrite by a thin neck – a morphological feature that intuitively suggests it might function to limit the transmission of electrical and biochemical signals. Unfortunately, the extremely small size of spines has made direct measurements of their electrical properties difficult and, therefore, the functional implications of electrical compartmentalization have remained elusive. In this study, we use spatiotemporally controlled stimulation to generate calcium signals within the spine head and/or neighboring dendrite. By comparing these measurements we demonstrate that spines create specialized electrical signaling compartments, which has at least two functional consequences. First, synaptic stimulation, but not similar dendritic depolarization, can trigger the activation of voltage-gated calcium channels within the spine. Second, voltage changes in the spine head arising from compartmentalization shape the time course of synaptically evoked calcium influx such that it is biphasic. Thus, the electrical compartmentalization provided by spines allows for multiple modes of calcium signaling at excitatory synapses.
doi:10.1371/journal.pbio.1000190
PMCID: PMC2734993  PMID: 19753104
17.  Rapid Redistribution of Synaptic PSD-95 in the Neocortex In Vivo 
PLoS Biology  2006;4(11):e370.
Most excitatory synapses terminate on dendritic spines. Spines vary in size, and their volumes are proportional to the area of the postsynaptic density (PSD) and synaptic strength. PSD-95 is an abundant multi-domain postsynaptic scaffolding protein that clusters glutamate receptors and organizes the associated signaling complexes. PSD-95 is thought to determine the size and strength of synapses. Although spines and their synapses can persist for months in vivo, PSD-95 and other PSD proteins have shorter half-lives in vitro, on the order of hours. To probe the mechanisms underlying synapse stability, we measured the dynamics of synaptic PSD-95 clusters in vivo. Using two-photon microscopy, we imaged PSD-95 tagged with GFP in layer 2/3 dendrites in the developing (postnatal day 10–21) barrel cortex. A subset of PSD-95 clusters was stable for days. Using two-photon photoactivation of PSD-95 tagged with photoactivatable GFP (paGFP), we measured the time over which PSD-95 molecules were retained in individual spines. Synaptic PSD-95 turned over rapidly (median retention times τr ~ 22–63 min from P10–P21) and exchanged with PSD-95 in neighboring spines by diffusion. PSDs therefore share a dynamic pool of PSD-95. Large PSDs in large spines captured more diffusing PSD-95 and also retained PSD-95 longer than small PSDs. Changes in the sizes of individual PSDs over days were associated with concomitant changes in PSD-95 retention times. Furthermore, retention times increased with developmental age (τr ~ 100 min at postnatal day 70) and decreased dramatically following sensory deprivation. Our data suggest that individual PSDs compete for PSD-95 and that the kinetic interactions between PSD molecules and PSDs are tuned to regulate PSD size.
Using two-photon microscopy and photoactivation of a fluorescently tagged synaptic protein (PSD-95), the authors demonstrated rapid turnover of these molecules in dendritic spines of the mouse sensory cortex in vivo.
doi:10.1371/journal.pbio.0040370
PMCID: PMC1634879  PMID: 17090216
18.  Regulation of dendritic spine growth through activity-dependent recruitment of the brain-enriched Na+/H+ exchanger NHE5 
Molecular Biology of the Cell  2011;22(13):2246-2257.
pH homeostasis in neurons plays crucial roles in normal synaptic functions. It is found that the Na+/H+ exchanger NHE5 is targeted to the synapse on neuronal activation, regulates the synaptic pH, and controls the morphology of dendritic spines.
Subtle changes in cellular and extracellular pH within the physiological range have profound impacts on synaptic activities. However, the molecular mechanisms underlying local pH regulation at synapses and their influence on synaptic structures have not been elucidated. Dendritic spines undergo dynamic structural changes in response to neuronal activation, which contributes to induction and long-term maintenance of synaptic plasticity. Although previous studies have indicated the importance of cytoskeletal rearrangement, vesicular trafficking, cell signaling, and adhesion in this process, much less is known about the involvement of ion transporters. In this study we demonstrate that N-methyl-d-aspartate (NMDA) receptor activation causes recruitment of the brain-enriched Na+/H+ exchanger NHE5 from endosomes to the plasma membrane. Concomitantly, real-time imaging of green fluorescent protein–tagged NHE5 revealed that NMDA receptor activation triggers redistribution of NHE5 to the spine head. We further show that neuronal activation causes alkalinization of dendritic spines following the initial acidification, and suppression of NHE5 significantly retards the activity-induced alkalinization. Perturbation of NHE5 function induces spontaneous spine growth, which is reversed by inhibition of NMDA receptors. In contrast, overexpression of NHE5 inhibits spine growth in response to neuronal activity. We propose that NHE5 constrains activity-dependent dendritic spine growth via a novel, pH-based negative-feedback mechanism.
doi:10.1091/mbc.E11-01-0066
PMCID: PMC3128527  PMID: 21551074
19.  Ultrastructure of Synapses in the Mammalian Brain 
The morphology and molecular composition of synapses provide the structural basis for synaptic function. This article reviews the electron microscopy of excitatory synapses on dendritic spines, using data from rodent hippocampus, cerebral cortex, and cerebellar cortex. Excitatory synapses have a prominent postsynaptic density, in contrast with inhibitory synapses, which have less dense presynaptic or postsynaptic specializations and are usually found on the cell body or proximal dendritic shaft. Immunogold labeling shows that the presynaptic active zone provides a scaffold for key molecules involved in the release of neurotransmitter, whereas the postsynaptic density contains ligand-gated ionic channels, other receptors, and a complex network of signaling molecules. Delineating the structure and molecular organization of these axospinous synapses represents a crucial step toward understanding the mechanisms that underlie synaptic transmission and the dynamic modulation of neurotransmission associated with short- and long-term synaptic plasticity.
Direct visualization of the synaptic cleft was first made possible by electron microscopy (EM). High-resolution EM continues to define synaptic organization and unravel mechanisms of synaptic transmission.
doi:10.1101/cshperspect.a005587
PMCID: PMC3331701  PMID: 22357909
20.  In vivo composition of NMDA receptor signalling complexes differs between membrane subdomains and is modulated by PSD-95 and PSD-93 
Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids involved in the compartmentalization of signaling pathways, trafficking and sorting of proteins. At synapses, the glutamatergic NMDA receptor and its cytoplasmic scaffold protein PSD-95 move between postsynaptic density (PSD) and rafts following learning or ischemia. However it is not known if the signaling complexes formed by these proteins are different in rafts nor the molecular mechanisms that govern their localization. To examine these issues in vivo we used mice carrying genetically encoded tags for purification of protein complexes and specific mutations in NMDA receptors, PSD-95 and other postsynaptic scaffold proteins. Isolation of PSD-95 complexes from mice carrying tandem affinity purification tags showed differential composition in lipid rafts, postsynaptic density and detergent-soluble fractions. Raft PSD-95 complexes showed less CamKIIα and SynGAP and enrichment in Src and Arc/Arg3.1 compared with PSD complexes. Mice carrying knockouts of PSD-95 or PSD-93 show a key role for PSD-95 in localizing NR2A containing NMDA receptor complexes to rafts. Deletion of the NR2A carboxyl-terminus or the carboxyl-terminal valine residue of NR2B, which prevents all PDZ interactions, reduced the NR1 association with rafts. Interestingly, the deletion of the NR2B valine residue increased the total amount of lipid rafts. These data show critical roles for scaffold proteins and their interactions with NMDA receptor subunits in organizing the differential expression in rafts and postsynaptic densities of synaptic signaling complexes.
doi:10.1523/JNEUROSCI.1792-10.2010
PMCID: PMC2912510  PMID: 20554866
Lipid rafts; NMDAR; PSD-95; complexes; postsynaptic density; signal compartmentalization
21.  Facilitation of AMPA Receptor Synaptic Delivery as a Molecular Mechanism for Cognitive Enhancement 
PLoS Biology  2012;10(2):e1001262.
A small peptide from a neuronal cell adhesion molecule enhances synaptic plasticity in the hippocampus and results in improved cognitive performance in rats.
Cell adhesion molecules and downstream growth factor-dependent signaling are critical for brain development and synaptic plasticity, and they have been linked to cognitive function in adult animals. We have previously developed a mimetic peptide (FGL) from the neural cell adhesion molecule (NCAM) that enhances spatial learning and memory in rats. We have now investigated the cellular and molecular basis of this cognitive enhancement, using biochemical, morphological, electrophysiological, and behavioral analyses. We have found that FGL triggers a long-lasting enhancement of synaptic transmission in hippocampal CA1 neurons. This effect is mediated by a facilitated synaptic delivery of AMPA receptors, which is accompanied by enhanced NMDA receptor-dependent long-term potentiation (LTP). Both LTP and cognitive enhancement are mediated by an initial PKC activation, which is followed by persistent CaMKII activation. These results provide a mechanistic link between facilitation of AMPA receptor synaptic delivery and improved hippocampal-dependent learning, induced by a pharmacological cognitive enhancer.
Author Summary
The human brain contains trillions of neuronal connections, called synapses, whose pattern of activity controls all our cognitive functions. These synaptic connections are dynamic and constantly changing in their strength and properties, and this process of synaptic plasticity is essential for learning and memory. Alterations in synaptic plasticity mechanisms are thought to be responsible for multiple cognitive deficits, such as autism, Alzheimer's disease, and several forms of mental retardation. In this study, we show that synapses can be made more plastic using a small protein fragment (peptide) derived from a neuronal protein involved in cell-to-cell communication. This peptide (FGL) initiates a cascade of events inside the neuron that results in the facilitation of synaptic plasticity. Specifically, we find that FGL triggers delivery of a specific type of glutamate receptor (AMPA receptors) to synapses in a region of the brain called the hippocampus, which is known to be involved in multiple forms of learning and memory. Importantly, when this peptide was administered to rats, their ability to learn and retain spatial information was enhanced. Therefore, this work demonstrates that cognitive function can be improved pharmacologically in adult animals by enhancing the plasticity of synaptic connections in the brain.
doi:10.1371/journal.pbio.1001262
PMCID: PMC3283560  PMID: 22363206
22.  “The CUB domain protein Neto1 is an auxiliary protein of native synaptic kainate receptors” 
Ionotropic glutamate receptors of AMPA, NMDA and kainate receptor (KAR) subtypes mediate fast excitatory synaptic transmission in the vertebrate CNS. Auxiliary proteins have been identified for AMPA and NMDA receptor complexes, but little is known about KAR complex proteins. We previously identified the CUB-domain protein, Neto1, as an NMDA receptor-associated polypeptide. Here, we show that Neto1 is also an auxiliary subunit for endogenous synaptic KARs. We found that Neto1 and KARs co-immunoprecipitated from brain lysates, from post-synaptic densities (PSDs) and, in a manner dependent on Neto1 CUB domains, when co-expressed in heterologous cells. In Neto1-null mice, there was an ~50% reduction in the abundance of GluK2-KARs in hippocampal PSDs. Neto1 strongly localized to CA3 stratum lucidum and loss of Neto1 resulted in a selective deficit in KAR-mediated neurotransmission at mossy fiber-CA3 pyramidal cell synapses (MF-CA3): KAR-mediated EPSCs in Neto1-null mice were reduced in amplitude and decayed more rapidly than did those in wild-type mice. In contrast, the loss of Neto2, which also localizes to stratum lucidum and interacts with KARs, had no effect on KAR synaptic abundance or MF-CA3 transmission. Indeed MF-CA3 KAR deficits in Neto1/2 double null mutant mice were indistinguishable from Neto1 single null mice. Thus, our findings establish Neto1 as an auxiliary protein required for synaptic function of KARs. The ability of Neto1 to regulate both NMDARs and KARs reveals a unique dual role in controlling synaptic transmission by serving as an auxiliary protein for these two classes of ionotropic glutamate receptors in a synapse specific fashion.
doi:10.1523/JNEUROSCI.6617-10.2011
PMCID: PMC3148853  PMID: 21734292
23.  Spike-Driven Glutamate Electrodiffusion Triggers Synaptic Potentiation via a Homer-Dependent mGluR-NMDAR Link 
Neuron  2013;77(3):528-541.
Summary
Electric fields of synaptic currents can influence diffusion of charged neurotransmitters, such as glutamate, in the synaptic cleft. However, this phenomenon has hitherto been detected only through sustained depolarization of large principal neurons, and its adaptive significance remains unknown. Here, we find that in cerebellar synapses formed on electrically compact granule cells, a single postsynaptic action potential can retard escape of glutamate released into the cleft. This retardation boosts activation of perisynaptic group I metabotropic glutamate receptors (mGluRs), which in turn rapidly facilitates local NMDA receptor currents. The underlying mechanism relies on a Homer-containing protein scaffold, but not GPCR- or Ca2+-dependent signaling. Through the mGluR-NMDAR interaction, the coincidence between a postsynaptic spike and glutamate release triggers a lasting enhancement of synaptic transmission that alters the basic integrate-and-spike rule in the circuitry. Our results thus reveal an electrodiffusion-driven synaptic memory mechanism that requires high-precision coincidence detection suitable for high-fidelity circuitries.
Highlights
► A single action potential decelerates diffusion escape of released glutamate ► Slowing down glutamate diffusion enhances activation of perisynaptic group I mGluRs ► Activation of mGluRs boosts local NMDAR currents in a Homer1-dependent manner ► Postsynaptic spike-release pairing induces NMDAR- and mGluR-dependent LTP
Sylantyev et al. find that electric fields of postsynaptic spikes retard escape of glutamate from the synaptic cleft thus boosting activation of perisynaptic metabotropic receptors in cerebellar synapses. This leads to enhancement of synaptic NMDA receptor current leading to long-term potentiation of transmission.
doi:10.1016/j.neuron.2012.11.026
PMCID: PMC3568920  PMID: 23395378
24.  Glutamate Receptor Dynamics in Dendritic Microdomains 
Neuron  2008;58(4):472-497.
Among diverse factors regulating excitatory synaptic transmission, the abundance of postsynaptic glutamate receptors figures prominently in molecular memory and learning-related synaptic plasticity. To allow for both long-term maintenance of synaptic transmission and acute changes in synaptic strength, the relative rates of glutamate receptor insertion and removal must be tightly regulated. Interactions with scaffolding proteins control the targeting and signaling properties of glutamate receptors within the postsynaptic membrane. In addition, extrasynaptic receptor populations control the equilibrium of receptor exchange at synapses and activate distinct signaling pathways involved in plasticity. Here, we review recent findings that have shaped our current understanding of receptor mobility between synaptic and extrasynaptic compartments at glutamatergic synapses, focusing on AMPA and NMDA receptors. We also examine the cooperative relationship between intracellular trafficking and surface diffusion of glutamate receptors that underlies the expression of learning-related synaptic plasticity.
doi:10.1016/j.neuron.2008.04.030
PMCID: PMC2572138  PMID: 18498731
25.  Pin1-dependent signalling negatively affects GABAergic transmission by modulating neuroligin2/gephyrin interaction 
Nature Communications  2014;5:5066.
The cell adhesion molecule Neuroligin2 (NL2) is localized selectively at GABAergic synapses, where it interacts with the scaffolding protein gephyrin in the post-synaptic density. However, the role of this interaction for formation and plasticity of GABAergic synapses is unclear. Here, we demonstrate that endogenous NL2 undergoes proline-directed phosphorylation at its unique S714-P consensus site, leading to the recruitment of the peptidyl-prolyl cis–trans isomerase Pin1. This signalling cascade negatively regulates NL2’s ability to interact with gephyrin at GABAergic post-synaptic sites. As a consequence, enhanced accumulation of NL2, gephyrin and GABAA receptors was detected at GABAergic synapses in the hippocampus of Pin1-knockout mice (Pin1−/−) associated with an increase in amplitude of spontaneous GABAA-mediated post-synaptic currents. Our results suggest that Pin1-dependent signalling represents a mechanism to modulate GABAergic transmission by regulating NL2/gephyrin interaction.
The cell adhesion molecule Neuroligin2 (NL2) interacts with the scaffolding protein gephyrin to ensure the functional organization of GABAA receptors at inhibitory synapses. Here the authors show that NL2 binding of Pin1 negatively regulates its ability to complex with gephyrin at these synapses.
doi:10.1038/ncomms6066
PMCID: PMC4197815  PMID: 25297980

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