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1.  The X-Linked Intellectual Disability Protein TSPAN7 Regulates Excitatory Synapse Development and AMPAR Trafficking 
Neuron  2012;73(6):1143-1158.
Summary
Mutations in TSPAN7—a member of the tetraspanin protein superfamily—are implicated in some forms of X-linked intellectual disability. Here we show that TSPAN7 overexpression promotes the formation of filopodia and dendritic spines in cultured hippocampal neurons from embryonic rats, whereas TSPAN7 silencing reduces head size and stability of spines and AMPA receptor currents. Via its C terminus, TSPAN7 interacts with the PDZ domain of protein interacting with C kinase 1 (PICK1), to regulate PICK1 and GluR2/3 association and AMPA receptor trafficking. These findings indicate that, in hippocampal neurons, TSPAN7 regulates AMPA receptor trafficking by limiting PICK1 accessibility to AMPA receptors and suggest an additional mechanism for the functional maturation of glutamatergic synapses, whose impairment is implicated in intellectual disability.
Highlights
► TSPAN7 is required for spine maturation in hippocampal neurons ► TSPAN7 knockdown impairs AMPAR currents ► TSPAN7 binds PICK1 and through this interaction regulates AMPAR trafficking
Mutations in TSPAN7 protein cause human intellectual disability. Bassani et al. now find that TSPAN7 regulates trafficking of essential receptor proteins to neuron surfaces and that absence impairs neuronal maturation in young animals, potentially underlying this intellectual disability.
doi:10.1016/j.neuron.2012.01.021
PMCID: PMC3314997  PMID: 22445342
2.  TspanC8 tetraspanins regulate ADAM10/Kuzbanian trafficking and promote Notch activation in flies and mammals 
The Journal of Cell Biology  2012;199(3):481-496.
TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch activation.
The metalloprotease ADAM10/Kuzbanian catalyzes the ligand-dependent ectodomain shedding of Notch receptors and activates Notch. Here, we show that the human tetraspanins of the evolutionary conserved TspanC8 subfamily (Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33) directly interact with ADAM10, regulate its exit from the endoplasmic reticulum, and that four of them regulate ADAM10 surface expression levels. In an independent RNAi screen in Drosophila, two TspanC8 genes were identified as Notch regulators. Functional analysis of the three Drosophila TspanC8 genes (Tsp3A, Tsp86D, and Tsp26D) indicated that these genes act redundantly to promote Notch signaling. During oogenesis, TspanC8 genes were up-regulated in border cells and regulated Kuzbanian distribution, Notch activity, and cell migration. Furthermore, the human TspanC8 tetraspanins Tspan5 and Tspan14 positively regulated ligand-induced ADAM10-dependent Notch1 signaling. We conclude that TspanC8 tetraspanins have a conserved function in the regulation of ADAM10 trafficking and activity, thereby positively regulating Notch receptor activation.
doi:10.1083/jcb.201201133
PMCID: PMC3483123  PMID: 23091066
3.  An Essential Role for PICK1 in NMDA Receptor-Dependent Bidirectional Synaptic Plasticity 
Neuron  2008;57(6):872-882.
Summary
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.
doi:10.1016/j.neuron.2008.01.028
PMCID: PMC2336895  PMID: 18367088
4.  Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation 
Nature neuroscience  2010;13(10):1199-1207.
Trafficking of AMPA receptors is important for many forms of synaptic plasticity. However, the link between activity and resulting synaptic alterations is not fully understood. Here, we identified a direct interaction between NSF, an ATPase involved in membrane fusion events and stabilization of surface AMPARs, and Plk2, an activity-inducible kinase that homeostatically decreases excitatory synapse number and strength. Plk2 disrupted interaction of NSF with the GluA2 subunit of AMPARs, promoting extensive loss of surface GluA2 in rat hippocampal neurons, greater association of GluA2 with adapter proteins PICK1 and GRIP1, and decreased synaptic AMPAR current. Plk2 engagement of NSF, but not Plk2 kinase activity, was required for this mechanism and occurred through a novel motif within Plk2 independent from canonical polo box interaction sites. These data reveal that heightened synaptic activity, acting through Plk2, leads to homeostatic decreases in surface AMPAR expression via the direct dissociation of NSF from GluA2.
doi:10.1038/nn.2624
PMCID: PMC2947592  PMID: 20802490
Plk2; SNK; NSF; GluA2; GluR2; endocytosis; GluA1; GluR1; homeostatic plasticity; polo kinase
5.  Role of TARP interaction in S-SCAM-mediated regulation of AMPA receptors 
Channels  2012;6(5):393-397.
Scaffolding proteins are involved in the incorporation, anchoring, maintenance, and removal of AMPA receptors (AMPARs) at synapses, either through a direct interaction with AMPARs or via indirect association through auxiliary subunits of transmembrane AMPAR regulatory proteins (TARPs). Synaptic scaffolding molecule (S-SCAM) is a newly characterized member of the scaffolding proteins critical for the regulation and maintenance of AMPAR levels at synapses, and directly binds to TARPs through a PDZ interaction. However, the functional significance of S-SCAM–TARP interaction in the regulation of AMPARs has not been tested. Here we show that overexpression of the C-terminal peptide of TARP-γ2 fused to EGFP abolished the S-SCAM-mediated enhancement of surface GluA2 expression. Conversely, the deletion of the PDZ-5 domain of S-SCAM that binds TARPs greatly attenuated the S-SCAM-induced increase of surface GluA2 expression. In contrast, the deletion of the guanylate kinase domain of S-SCAM did not show a significant effect on the regulation of AMPARs. Together, these results suggest that S-SCAM is regulating AMPARs through TARPs.
doi:10.4161/chan.21301
PMCID: PMC3508780  PMID: 22878254
AMPA receptor; scaffolding proteins; TARP; S-SCAM; PDZ interaction
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.  Glutamate Receptor Subunit GluA1 is Necessary for Long-term Potentiation and Synapse Unsilencing, but not Long-term Depression in Mouse Hippocampus 
Brain Research  2011;1435:8-14.
Receptor subunit composition is believed to play a major role in the synaptic trafficking of AMPA receptors (AMPARs), and thus in activity-dependent synaptic plasticity. To isolate a physiological role of GluA1-containing AMPARs in area CA3 of the hippocampus, pair recordings were performed in organotypic hippocampal slices taken from genetically modified mice lacking the GluA1 subunit. We report here that long-term potentiation (LTP) is impaired not only at active but also at silent synapses when the GluA1 subunit is absent. The GluA1 knockout mice also exhibited reduced AMPAR-mediated evoked currents between pairs of CA3 pyramidal neurons under baseline conditions suggesting a significant role for GluA1-containing AMPARs in regulating basal synaptic transmission. In two independent measures, however, long-term depression (LTD) was unaffected in tissue from these mice. These data provide a further demonstration of the fundamental role that GluA1-containing AMPARs play in activity-dependent increases in synaptic strength but do not support a GluA1-dependent mechanism for reductions in synaptic strength.
doi:10.1016/j.brainres.2011.11.029
PMCID: PMC3268828  PMID: 22197030
Glutamate receptor; AMPA receptor; knockout; synaptic plasticity; silent synapse; long-term potentiation; long-term depression; hippocampus
8.  Calcium Binding to PICK1 is Essential for the Intracellular Retention of AMPA Receptors Underlying LTD 
NMDA receptor (NMDAR)-dependent LTD in the hippocampus is mediated primarily by the calcium-dependent removal of AMPA receptors (AMPARs) from the postsynaptic density. The AMPAR-binding, PDZ and BAR domain containing protein PICK1 has been implicated in the regulation of AMPAR trafficking underlying several forms of synaptic plasticity. Using a strategy involving shRNA-mediated knockdown of PICK1 and its replacement with recombinant PICK1, we performed a detailed structure-function analysis of the role of PICK1 in hippocampal synaptic plasticity and the underlying NMDAR-induced AMPAR trafficking. We found that PICK1 is not necessary for maintenance of the basal synaptic complement of AMPARs or expression of either mGluR-LTD or NMDAR-dependent LTP. Rather, PICK1 function is specific to NMDAR-dependent LTD and the underlying AMPAR trafficking. Furthermore, while PICK1 does not regulate the initial phase of NMDAR-induced AMPAR endocytosis, it is required for intracellular retention of internalized AMPARs. Detailed biophysical analysis of an N-terminal acidic motif indicated that it is involved in intramolecular electrostatic interactions that are disrupted by calcium. Mutations that interfered with the calcium-induced structural changes in PICK1 precluded LTD and the underlying NMDAR-induced intracellular retention of AMPARs. These findings support a model whereby calcium-induced modification of PICK1 structure is critical for its function in the retention of internalized AMPARs that underlies the expression of hippocampal NMDAR-dependent LTD.
doi:10.1523/JNEUROSCI.4478-10.2010
PMCID: PMC3004477  PMID: 21147983
Hippocampus; LTD; NMDA receptor; AMPA receptor; endocytic trafficking; plasticity
9.  CASK Regulates SAP97 Conformation and Its Interactions with AMPA and NMDA Receptors 
The Journal of Neuroscience  2013;33(29):12067-12076.
SAP97 interacts with AMPA receptors (AMPARs) and NMDA receptors (NMDARs) during sorting and trafficking to synapses. Here we addressed how SAP97 distinguishes between AMPARs and NMDARs and what role the adaptor/scaffold protein, CASK, plays in the process. Using intramolecular SAP97 Förster resonance energy transfer sensors, we demonstrated that SAP97 is in “extended” or “compact” conformations in vivo. SAP97 conformation was regulated by a direct interaction between SAP97 and CASK through L27 protein-interaction domains on each protein. Unbound SAP97 was mostly in the compact conformation, while CASK binding stabilized it in an extended conformation. In HEK cells and rat hippocampal neurons, SAP97 in the compact conformation preferentially associated and colocalized with GluA1-containing AMPARs, and in the extended conformation colocalized with GluN2B-containing NMDARs. Altogether, our findings suggest a molecular mechanism by which CASK binding regulates SAP97 conformation and its subsequent sorting and synaptic targeting of AMPARs and NMDARs during trafficking to synapses.
doi:10.1523/JNEUROSCI.0816-13.2013
PMCID: PMC3713737  PMID: 23864692
10.  Tetraspanin-13 modulates voltage-gated CaV2.2 Ca2+ channels 
Scientific Reports  2013;3:1777.
Integration of voltage-gated Ca2+ channels in a network of protein-interactions is a crucial requirement for proper regulation of channel activity. In this study, we took advantage of the specific properties of the yeast split-ubiquitin system to search for and characterize so far unknown interaction partners of CaV2 Ca2+ channels. We identified tetraspanin-13 (TSPAN-13) as an interaction partner of the α1 subunit of N-type CaV2.2, but not of P/Q-type CaV2.1 or L- and T-type Ca2+ channels. Interaction could be located between domain IV of CaV2.2 and transmembrane segments S1 and S2 of TSPAN-13. Electrophysiological analysis revealed that TSPAN-13 specifically modulates the efficiency of coupling between voltage sensor activation and pore opening of the channel and accelerates the voltage-dependent activation and inactivation of the Ba2+ current through CaV2.2. These data indicate that TSPAN-13 might regulate CaV2.2 Ca2+ channel activity in defined synaptic membrane compartments and thereby influences transmitter release.
doi:10.1038/srep01777
PMCID: PMC3646272  PMID: 23648579
11.  Regulation of Synaptic Strength and AMPA Receptor Subunit Composition by PICK1 
PICK1 (protein interacting with C kinase-1) regulates the surface expression of the AMPA receptor (AMPAR) GluR2 subunit, however, the functional consequences of this interaction are not well understood. Previous work has suggested that PICK1 promotes the internalization of AMPARs. However, we found that when PICK1 is virally expressed in the CA1 region of hippocampal slices, it causes an increase in AMPAR-mediated EPSC amplitude. This effect is associated with increased AMPAR rectification and sensitivity to polyamine toxin. These effects are blocked by PKC or calcium/calmodulin-dependent protein kinase II inhibitors, indicating that the virally expressed PICK1 signals through an endogenous kinase cascade. In contrast, blockade of interactions with GluR2 at the N-ethylmaleimide-sensitive factor site did not cause a change in subunit composition, suggesting that the effects of PICK1 are not simply a nonspecific consequence of removing AMPARs from the surface. Immunocytochemical and biochemical analyses in dissociated cultured hippocampal neurons show that PICK1 causes a decrease in endogenous GluR2 surface expression but no change in GluR1 surface levels. To address the physiological role of PICK1, we virally expressed C-terminal GluR2 peptides. Blockade of endogenous PICK1 PDZ (postsynaptic density-95/Discs large/zona occludens-1) domain interactions produced opposite effects on synaptic strength and AMPAR rectification to those observed with PICK1 expression. This demonstrates that AMPAR subunit composition is physiologically regulated through a mechanism involving PICK1 PDZ domain interactions. These findings suggest that PICK1 acts to downregulate the GluR2 content of AMPARs at hippocampal CA1 synapses, thereby increasing synaptic strength at resting membrane potentials.
doi:10.1523/JNEUROSCI.4378-03.2004
PMCID: PMC3310907  PMID: 15190111
glutamate; synaptic plasticity; hippocampus; interacting protein; AMPA receptor trafficking; protein interacting with C kinase
12.  A role for sorting nexin 27 in AMPA receptor trafficking 
Nature Communications  2014;5:3176.
Sorting nexin 27 (SNX27), a PDZ domain-containing endosomal protein, was recently shown to modulate glutamate receptor recycling in Down’s syndrome. However, the precise molecular role of SNX27 in GluA1 trafficking is unclear. Here we report that SNX27 is enriched in dendrites and spines, along with recycling endosomes. Significantly, the mobilization of SNX27 along with recycling endosomes into spines was observed. Mechanistically, SNX27 interacts with K-ras GTPase via the RA domain; and following chemical LTP stimuli, K-ras is recruited to SNX27-enriched endosomes through a Ca2+/CaM-dependent mechanism, which in turn drives the synaptic delivery of homomeric GluA1 receptors. Impairment of SNX27 prevents LTP and associated trafficking of AMPARs. These results demonstrate a role for SNX27 in neuronal plasticity, provide a molecular explanation for the K-ras signal during LTP and identify SNX27 as the PDZ-containing molecular linker that couples the plasticity stimuli to the delivery of postsynaptic cargo.
Sorting nexin 27 (SNX27) regulates endosomal sorting of glutamate receptors. Loo et al. show that SNX27 is localized to recycling endosomes within dendritic spines where it interacts with glutamate receptors, allowing them to be shuttled to the postsynaptic membrane.
doi:10.1038/ncomms4176
PMCID: PMC3921469  PMID: 24458027
13.  The Small GTPase Arf1 Modulates Arp2/3-Mediated Actin Polymerization via PICK1 to Regulate Synaptic Plasticity 
Neuron  2013;79(2):293-307.
Summary
Inhibition of Arp2/3-mediated actin polymerization by PICK1 is a central mechanism to AMPA receptor (AMPAR) internalization and long-term depression (LTD), although the signaling pathways that modulate this process in response to NMDA receptor (NMDAR) activation are unknown. Here, we define a function for the GTPase Arf1 in this process. We show that Arf1-GTP binds PICK1 to limit PICK1-mediated inhibition of Arp2/3 activity. Expression of mutant Arf1 that does not bind PICK1 leads to reduced surface levels of GluA2-containing AMPARs and smaller spines in hippocampal neurons, which occludes subsequent NMDA-induced AMPAR internalization and spine shrinkage. In organotypic slices, NMDAR-dependent LTD of AMPAR excitatory postsynaptic currents is abolished in neurons expressing mutant Arf1. Furthermore, NMDAR stimulation downregulates Arf1 activation and binding to PICK1 via the Arf-GAP GIT1. This study defines Arf1 as a critical regulator of actin dynamics and synaptic function via modulation of PICK1.
Highlights
•The Arf1-PICK1-Arp2/3 pathway regulates actin polymerization•NMDAR activation activates the Arf-GAP GIT1 to deactivate Arf1•Arf1 controls NMDAR-dependent, PICK1-mediated AMPAR trafficking and LTD•A noncanonical role is described for Arf1 in vesicle traffic, distinct from COPI regulation
Rocca et al. show that Arf1 regulates actin dynamics in dendritic spines by modulating PICK1-mediated Arp2/3 inhibition. This controls spine size, AMPAR trafficking, and hence synaptic transmission. This study defines Arf1 as a critical regulator of actin dynamics and synaptic plasticity via PICK1 modulation.
doi:10.1016/j.neuron.2013.05.003
PMCID: PMC3725416  PMID: 23889934
14.  Differential roles of GRIP1a and GRIP1b in AMPA receptor trafficking 
Neuroscience letters  2010;485(3):167-172.
Regulated trafficking controls AMPA receptor (AMPAR) number at the postsynaptic membrane to modify the efficiency of synaptic transmission. The PDZ proteins GRIP1 and the related ABP-L/GRIP2 bind AMPAR subunit GluA2, and have been proposed to play a role in AMPAR trafficking associated with Long Term Depression (LTD) of synaptic transmission. Both GRIP1 and ABP-L/GRIP2 exist in different splice isoforms, including alternative 18 amino acid domains at the extreme N-terminus, which determine whether the protein can be palmitoylated. The implications of this differential splicing for AMPAR trafficking is unknown. Here, we use surface biotinylation and quantitative Western blotting to show that the N-terminal splice variants GRIP1a and GRIP1b have differential effects in NMDA-induced AMPAR internalization in cultured hippocampal neurons. GRIP1a inhibits, but GRIP1b enhances this trafficking event. We further demonstrate that GRIP1a and GRIP1b have dramatically different subcellular distributions in cultured neurons and exhibit NMDA-dependent colocalisation with early endosomes. We propose that GRIP1 palmitoylation modulates NMDA-induced AMPAR internalisation by differential regulation of the early endosomal system.
doi:10.1016/j.neulet.2010.09.003
PMCID: PMC3310156  PMID: 20837103
Synaptic plasticity; LTD; Palmitoylation; Glutamate; PDZ domain
15.  Analysis of the Potential Role of GluA4 Carboxyl-Terminus in PDZ Interactions 
PLoS ONE  2010;5(1):e8715.
Background
Specific delivery to synapses of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.
Methodology/Principal Findings
Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.
Conclusion/Significance
We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect.
doi:10.1371/journal.pone.0008715
PMCID: PMC2806832  PMID: 20090852
16.  PICK1 loss of function occludes homeostatic synaptic scaling 
Homeostatic synaptic scaling calibrates neuronal excitability by adjusting synaptic strengths during prolonged changes in synaptic activity. The molecular mechanisms that regulate the trafficking of AMPA receptors (AMPARs) during synaptic scaling are largely unknown. Here we show that chronic activity blockade reduces PICK1 protein level on a time scale that coincides with the accumulation of surface AMPARs. PICK1 loss of function alters the subunit composition and the abundance of GluA2-containing AMPARs. Due to aberrant trafficking of these receptors, the increase in synaptic strength in response to synaptic inactivity is occluded in neurons generated from PICK1 knockout mouse. In agreement with electrophysiological recordings, no defect of AMPAR trafficking is observed in PICK1 knockout neurons in response to elevated neuronal activity. Overall, our data reveal an important role of PICK1 in inactivity-induced synaptic scaling by regulating the subunit composition, abundance and trafficking of GluA2-containing AMPARs.
doi:10.1523/JNEUROSCI.5633-10.2011
PMCID: PMC3071039  PMID: 21307255
AMPA receptors; PICK1; homeostatic plasticity; synaptic scaling
17.  Stress at Learning Facilitates Memory Formation by Regulating AMPA Receptor Trafficking Through a Glucocorticoid Action 
Neuropsychopharmacology  2009;35(3):674-685.
Stress and glucocorticoids (GCs) can facilitate memory formation. However, the molecular mechanisms mediating their effects are largely unknown. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR) trafficking has been implicated in the changes in synaptic strength at central glutamatergic synapses associated with memory formation. In cell cultures, corticosterone has been shown to condition the synaptic trafficking of the AMPAR GluA2 subunit. In this study, we investigated the involvement of GluA2 trafficking in the facilitation of learning by stress. Using the water maze spatial task involving different stress levels, mice trained under more stressful conditions (water at 22°C) showed better learning and memory, and higher post-training corticosterone levels, than mice trained under lower stress (water at 30°C). Strikingly, this facilitated learning by stress was accompanied by enhanced synaptic expression of GluA2 AMPARs that was not observed in mice trained under less stressful conditions. Interfering with GC actions by injecting the GC synthesis inhibitor, metyrapone, blocked both the memory facilitation and the enhanced GluA2 trafficking induced by stressful learning. Intracerebroventricular infusion of the peptide, pep2m, that blocks GluA2 synaptic trafficking by interfering with the interaction between N-ethylmaleimide-sensitive factor and GluA2, impaired immediate performance at learning as well as long-term memory retrieval, supporting a causal role for GluA2 trafficking in stress-induced facilitation of spatial learning and memory. Evidence for the involvement of the neural cell adhesion molecule N-cadherin in interaction with GluA2 is also provided. These findings underscore a new mechanism whereby stress can improve memory function.
doi:10.1038/npp.2009.172
PMCID: PMC3055605  PMID: 19890264
stress; corticosterone; learning; memory; GluA2; mice
18.  Two populations of neurokinin 1 receptor-expressing projection neurons in lamina I of the rat spinal cord that differ in AMPA receptor subunit composition and density of excitatory synaptic input 
Neuroscience  2010;167(4):1192-1204.
Lamina I of the spinal cord contains many projection neurons that express the neurokinin 1 receptor (NK1r). It has been reported that these cells can undergo long-term potentiation (LTP), which may result from insertion of AMPA-type glutamate receptors (AMPArs) containing GluA1 or GluA4 subunits. We therefore investigated synaptic AMPAr expression on these cells with immunocytochemistry following antigen-retrieval. We also examined their density of glutamatergic input (by analysing AMPAr synaptic puncta and contacts from glutamatergic boutons), and phosphorylation of extracellular signal-regulated kinases (pERKs) following noxious stimulation. Our results indicate that there are two populations of NK1r-expressing projection neurons: large GluA4+/GluA1− cells with a high density of glutamatergic input and small GluA1+/GluA4− cells with a much lower input density. Results from pERK experiments suggested that the two groups may not differ in the types of noxious stimulus that activate them. Glutamatergic synapses on distal dendrites of the large cells were significantly longer than those on proximal dendrites, which presumably compensates for the greater attenuation of distally-generated excitatory postsynaptic currents (EPSCs). Both types of cell received contacts from peptidergic primary afferents, however, on the large cells these appeared to constitute over half of the glutamatergic synapses, and were often associated with elongated AMPAr puncta. This suggests that these afferents, which probably contain substance P, provide a powerful, secure synaptic input to large NK1r-expressing projection neurons. These results demonstrate the importance of GluA4-containing AMPArs in nociceptive transmission and raise the possibility that different forms of LTP in lamina I projection neurons may be related to differential expression of GluA1/GluA4.
doi:10.1016/j.neuroscience.2010.03.028
PMCID: PMC3169794  PMID: 20303396
dorsal horn; pain; NK1 receptor; CGRP; VGLUT2; glutamatergic synapse; AMPAr, AMPA, receptor; CGRP, calcitonin gene-related peptide; LPb, lateral parabrachial area; LTP, long-term potentiation; NK1r, neurokinin 1 receptor; pERK, phosphorylated extracellular signal-regulated kinases; TSA, tyramide signal amplification; VGLUT, vesicular glutamate transporter
19.  Subunit composition of synaptic AMPA receptors revealed by a single-cell genetic approach 
Neuron  2009;62(2):254-268.
Summary
The precise subunit composition of synaptic ionotropic receptors in the brain is poorly understood. This information is of particular importance with regard to AMPA-type glutamate receptors, the multimeric complexes assembled from GluA1-A4 subunits, as the trafficking of these receptors into and out of synapses is proposed to depend upon the subunit composition of the receptor. We report a molecular quantification of synaptic AMPA receptors (AMPARs) by employing a single-cell genetic approach coupled with electrophysiology in hippocampal CA1 pyramidal neurons. In contrast to prevailing views, we find that GluA1A2 heteromers are the dominant AMPARs at CA1 cell synapses (~80%). In cells lacking GluA1, -A2 and -A3, synapses are devoid of AMPARs, yet synaptic NMDA receptors and dendritic morphology remain unchanged. These data demonstrate a functional dissociation of AMPARs from trafficking of NMDARs and neuronal morphogenesis. This study provides a functional quantification of the subunit composition of AMPARs in the CNS, and suggests novel roles for AMPAR subunits in receptor trafficking.
doi:10.1016/j.neuron.2009.02.027
PMCID: PMC3632349  PMID: 19409270
20.  Brain-derived neurotrophic factor signal enhances and maintains the expression of AMPA receptor-associated PDZ proteins in developing cortical neurons 
Developmental biology  2003;263(2):216-230.
Postsynaptic molecules with PDZ domains (PDZ proteins) interact with various glutamate receptors and regulate their subcellular trafficking and stability. In rat neocortical development, the protein expression of AMPA-type glutamate receptor GluR1 lagged behind its mRNA expression and rather paralleled an increase in PDZ protein levels. One of the neurotrophins, brain-derived neurotrophic factor (BDNF), appeared to contribute to this process, regulating the PDZ protein expression. In neocortical cultures, BDNF treatment upregulated SAP97, GRIP1, and Pick1 PDZ proteins. Conversely, BDNF gene targeting downregulated these same PDZ molecules. The BDNF-triggered increases in PDZ proteins resulted in the elevation of their total association with the AMPA receptors GluR1 and GluR2/3, which led to the increase in AMPA receptor proteins. When Sindbis viruses carrying GluR1 or GluR2 C-terminal decoys disrupted their interactions, GluR2 C-terminal decoys inhibited both BDNF-triggered GluR1 and GluR2/3 increases, whereas GluR1 C-terminal decoys blocked only the BDNF-triggered GluR1 increase. In agreement, coexpression of SAP97 and GluR1 in nonneuronal HEK293 cells increased both proteins compared with their single transfection, implying mutual stabilization. This work reveals a novel function of BDNF in postsynaptic development by regulating the PDZ protein expression.
PMCID: PMC3683555  PMID: 14597197
BDNF; GluR1; GluR2; GRIP1; Neurotrophin; Postsynaptic density; SAP97
21.  AKAP150-Anchored Calcineurin Regulates Synaptic Plasticity by Limiting Synaptic Incorporation of Ca2+-Permeable AMPA Receptors 
AMPA receptors (AMPARs) are tetrameric ion channels assembled from GluA1-GluA4 subunits that mediate the majority of fast excitatory synaptic transmission in the brain. In the hippocampus, most synaptic AMPARs are composed of GluA1/2 or GluA2/3 with the GluA2 subunit preventing Ca2+ influx. However, a small number of Ca2+-permeable GluA1 homomeric receptors reside in extrasynaptic locations where they can be rapidly recruited to synapses during synaptic plasticity. Phosphorylation of GluA1 S845 by the cAMP-dependent protein kinase (PKA) primes extrasynaptic receptors for synaptic insertion in response to NMDA receptor (NMDAR) Ca2+ signaling during long-term potentiation (LTP), while phosphatases dephosphorylate S845 and remove synaptic and extrasynaptic GluA1 during long-term depression (LTD). PKA and the Ca2+-activated phosphatase calcineurin (CaN) are targeted to GluA1 through binding to A-kinase anchoring protein (AKAP) 150 in a complex with PSD-95, but we do not understand how the opposing activities of these enzymes are balanced to control plasticity. Here, we generated AKAP150ΔPIX knock-in mice to selectively disrupt CaN anchoring in vivo. We found that AKAP150ΔPIX mice lack LTD but express enhanced LTP at CA1 synapses. Accordingly, basal GluA1 S845 phosphorylation is elevated in AKAP150ΔPIX hippocampus, and LTD-induced dephosphorylation and removal of GluA1, AKAP150, and PSD-95 from synapses is impaired. In addition, basal synaptic activity of GluA2-lacking AMPARs is increased in AKAP150ΔPIX mice and pharmacologic antagonism of these receptors restores normal LTD and inhibits the enhanced LTP. Thus, AKAP150-anchored CaN opposes PKA phosphorylation of GluA1 to restrict synaptic incorporation of Ca2+-permeable AMPARs both basally and during LTP and LTD.
doi:10.1523/JNEUROSCI.3326-12.2012
PMCID: PMC3504485  PMID: 23100425
22.  Cornichons modify channel properties of recombinant and glial AMPA receptors 
Ionotropic glutamate receptors, which underlie a majority of excitatory synaptic transmission in the CNS, associate with transmembrane proteins that modify their intracellular trafficking and channel gating. For AMPA-type glutamate receptors (AMPARs), significant advances have been made in our understanding of their regulation by transmembrane AMPAR regulatory proteins (TARPs). Less is known about the functional influence of cornichons – unrelated AMPAR-interacting proteins, identified by proteomic analysis. Here we confirm that cornichon homologs 2 and 3 (CNIH-2 and CNIH-3), but not CNIH-1, slow the deactivation and desensitization of both GluA2-containing calcium-impermeable (CI-) and GluA2-lacking calcium-permeable (CP-) AMPARs expressed in tsA201 cells. CNIH-2 and -3 also enhanced the glutamate sensitivity, single-channel conductance and calcium permeability of CP-AMPARs, while decreasing their block by intracellular polyamines. We examined the potential effects of CNIHs on native AMPARs by recording from rat optic nerve oligodendrocyte precursor cells (OPCs), known to express a significant population of CP-AMPARs. These glial cells exhibited surface labelling with an anti-CNIH-2/3 antibody. Two features of their AMPAR-mediated currents – the relative efficacy of the partial agonist kainate (IKA/IGlu ratio 0.4), and a greater than five-fold potentiation of kainate responses by cyclothiazide – suggest AMPAR association with CNIHs. Additionally, overexpression of CNIH-3 in OPCs markedly slowed AMPAR desensitization. Together, our experiments support the view that CNIHs are capable of altering key properties of AMPARs and suggest that they may do so in glia.
doi:10.1523/JNEUROSCI.0345-12.2012
PMCID: PMC3428874  PMID: 22815494
23.  The stoichiometry of AMPA receptors and TARPs varies by neuronal cell type 
Neuron  2009;62(5):633-640.
Summary
Fast synaptic transmission in the brain is mediated by activation of AMPA-type glutamate receptors (AMPARs). AMPARs are comprised of four pore forming subunits (GluAs) as well as auxiliary subunits referred to as transmembrane AMPA receptor regulatory proteins (TARPs). TARPs control the trafficking and gating of AMPARs. However, the number of TARP molecules that assemble within an individual AMPAR complex is unknown. Here, we covalently link GluAs to TARPs to investigate the properties of TARP/AMPAR complexes with known stoichiometry in HEK cells. We find that AMPARs are functional when associated with either four, two, or no TARPs, and that the efficacy of the partial agonist kainate varies across these conditions, providing a sensitive assay for TARP/AMPAR stoichiometry. By comparing these results with data obtained from hippocampal neurons, we show that native AMPARs are normally associated with multiple TARP molecules, and that native TARP/AMPAR stoichiometry varies with the expression level of endogenous and exogenous TARPs. Interestingly, AMPARs in hippocampal pyramidal cells contain more TARP molecules than those in dentate gyrus granule cells, suggesting a cell type-specific regulatory mechanism for TARP/AMPAR stoichiometry.
doi:10.1016/j.neuron.2009.05.016
PMCID: PMC3119531  PMID: 19524523
24.  Novel TSPAN12 mutations in patients with familial exudative vitreoretinopathy and their associated phenotypes 
Molecular Vision  2011;17:1128-1135.
Purpose
Mutations in tetraspanin 12 (TSPAN12) have recently been identified as a cause of autosomal dominant familial exudative vitreoretinopathy (FEVR). The purpose of this study was to detect TSPAN12 mutations in Chinese patients with FEVR and to describe the associated phenotypes.
Methods
Sanger sequencing was used to analyze the seven coding exons and their adjacent regions of TSPAN12 in 49 unrelated FEVR patients. Clinical phenotypes of the patients with TSPAN12 mutations were documented.
Results
Three novel heterozygous mutations in TSPAN12 were identified in three patients from unrelated families: c.146C>T (p.Thr49Met), c.313T>C (p.Cys105Arg), and c.601delC (p.Leu201PhefsX14). All three mutations involved highly conserved residues and were not present in 180 normal individuals. Ocular phenotypes included retinal folds, inferotemporal dragging of the optic disc and macula, increased vessels in the equatorial region, and a peripheral avascular zone. A father and his daughter had the same mutation but the father only had mild peripheral fundus changes while his daughter had obvious dragged disc and macular ectopia.
Conclusions
Our results suggest that TSPAN12 mutations are responsible for FEVR. Similar to patients with mutations in NDP, LRP5, or FZD4, the phenotypes associated with TSPAN12 mutations showed great variations between different individuals within a family and between the two eyes in individual patients.
PMCID: PMC3087453  PMID: 21552475
25.  Activity-dependent ubiquitination of the AMPA receptor subunit GluA2 
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.
doi:10.1523/JNEUROSCI.5944-10.2011
PMCID: PMC3081723  PMID: 21414928
Activity; glutamate receptor; AMPAR; ubiquitin; endocytosis

Results 1-25 (536419)