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Sample records for psd-95 controls ampa

  1. Acute inactivation of PSD-95 destabilizes AMPA receptors at hippocampal synapses.

    PubMed

    Yudowski, Guillermo A; Olsen, Olav; Adesnik, Hillel; Marek, Kurt W; Bredt, David S

    2013-01-01

    Postsynatptic density protein (PSD-95) is a 95 kDa scaffolding protein that assembles signaling complexes at synapses. Over-expression of PSD-95 in primary hippocampal neurons selectively increases synaptic localization of AMPA receptors; however, mice lacking PSD-95 display grossly normal glutamatergic transmission in hippocampus. To further study the scaffolding role of PSD-95 at excitatory synapses, we generated a recombinant PSD-95-4c containing a tetracysteine motif, which specifically binds a fluorescein derivative and allows for acute and permanent inactivation of PSD-95. Interestingly, acute inactivation of PSD-95 in rat hippocampal cultures rapidly reduced surface AMPA receptor immunostaining, but did not affected NMDA or transferrin receptor localization. Acute photoinactivation of PSD-95 in dissociated neurons causes ∼80% decrease in GluR2 surface staining observed by live-cell microscopy within 15 minutes of PSD-95-4c ablation. These results confirm that PSD-95 stabilizes AMPA receptors at postsynaptic sites and provides insight into the dynamic interplay between PSD-95 and AMPA receptors in live neurons.

  2. A critical role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA receptors.

    PubMed

    Bhattacharyya, Samarjit; Biou, Virginie; Xu, Weifeng; Schlüter, Oliver; Malenka, Robert C

    2009-02-01

    The endocytosis of AMPA receptors (AMPARs) underlies several forms of synaptic plasticity, including NMDA receptor (NMDAR)-dependent long-term depression (LTD), but the molecular mechanisms responsible for this trafficking remain unknown. We found that PSD-95, a major postsynaptic density protein, is important for NMDAR-triggered endocytosis of synaptic AMPARs in rat neuron cultures because of its binding to A kinase-anchoring protein 150 (AKAP150), a scaffold for specific protein kinases and phosphatases. Knockdown of PSD-95 with shRNA blocked NMDAR-triggered, but not constitutive or mGluR-triggered, endocytosis of AMPARs. Deletion of PSD-95's Src homology 3 and guanylate kinase-like domains, as well as a point mutation (L460P), both of which inhibit binding of PSD-95 to AKAP150, also blocked NMDAR-triggered AMPAR endocytosis. Furthermore, expression of a mutant AKAP150 that does not bind calcineurin inhibited this NMDAR-triggered trafficking event. Our results suggest that PSD-95's interaction with AKAP150 is critical for NMDAR-triggered AMPAR endocytosis and LTD, possibly because these scaffolds position calcineurin in the appropriate subsynaptic domain.

  3. Palmitoylation regulates glutamate receptor distributions in postsynaptic densities through control of PSD95 conformation and orientation

    PubMed Central

    Jeyifous, Okunola; Lin, Eric I.; Chen, Xiaobing; Antinone, Sarah E.; Mastro, Ryan; Drisdel, Renaldo; Reese, Thomas S.; Green, William N.

    2016-01-01

    Postsynaptic density protein 95 (PSD95) and synapse-associated protein 97 (SAP97) are homologous scaffold proteins with different N-terminal domains, possessing either a palmitoylation site (PSD95) or an L27 domain (SAP97). Here, we measured PSD95 and SAP97 conformation in vitro and in postsynaptic densities (PSDs) using FRET and EM, and examined how conformation regulated interactions with AMPA-type and NMDA-type glutamate receptors (AMPARs/NMDARs). Palmitoylation of PSD95 changed its conformation from a compact to an extended configuration. PSD95 associated with AMPARs (via transmembrane AMPAR regulatory protein subunits) or NMDARs [via glutamate ionotropic receptor NMDA-type subunit 2B (GluN2B) subunits] only in its palmitoylated and extended conformation. In contrast, in its extended conformation, SAP97 associates with NMDARs, but not with AMPARs. Within PSDs, PSD95 and SAP97 were largely in the extended conformation, but had different orientations. PSD95 oriented perpendicular to the PSD membrane, with its palmitoylated, N-terminal domain at the membrane. SAP97 oriented parallel to the PSD membrane, likely as a dimer through interactions of its N-terminal L27 domain. Changing PSD95 palmitoylation in PSDs altered PSD95 and AMPAR levels but did not affect NMDAR levels. These results indicate that in PSDs, PSD95 palmitoylation, conformation, and its interactions are dynamic when associated with AMPARs and more stable when associated with NMDARs. Altogether, our results are consistent with differential regulation of PSD95 palmitoylation in PSDs resulting from the clustering of palmitoylating and depalmitoylating enzymes into AMPAR nanodomains segregated away from NMDAR nanodomains. PMID:27956638

  4. Bidirectional control of postsynaptic density-95 (PSD-95) clustering by Huntingtin.

    PubMed

    Parsons, Matthew P; Kang, Rujun; Buren, Caodu; Dau, Alejandro; Southwell, Amber L; Doty, Crystal N; Sanders, Shaun S; Hayden, Michael R; Raymond, Lynn A

    2014-02-07

    Huntington disease is associated with early alterations in corticostriatal synaptic function that precede cell death, and it is postulated that ameliorating such changes may delay clinical onset and/or prevent neurodegeneration. Although many of these synaptic alterations are thought to be attributable to a toxic gain of function of the mutant huntingtin protein, the role that nonpathogenic huntingtin (HTT) plays in synaptic function is relatively unexplored. Here, we compare the immunocytochemical localization of a major postsynaptic scaffolding protein, PSD-95, in striatal neurons from WT mice and mice overexpressing HTT with 18 glutamine repeats (YAC18, nonpathogenic). We found that HTT overexpression resulted in a palmitoylation- and BDNF-dependent increase in PSD-95 clustering at synaptic sites in striatal spiny projection neurons (SPNs) co-cultured with cortical neurons. Surprisingly, the latter effect was mediated presynaptically, as HTT overexpression in cortical neurons alone was sufficient to increase PSD-95 clustering in the postsynaptic SPNs. In contrast, antisense oligonucleotide knockdown of HTT in WT co-cultures resulted in a significant reduction of PSD-95 clustering in SPNs. Notably, despite these bidirectional changes in PSD-95 clustering, we did not observe an alteration in basal electrophysiological measures of AMPA and NMDA receptors. Thus, unlike in previous studies in the hippocampus, enhanced or decreased PSD-95 clustering alone was insufficient to drive AMPA or NMDA receptors into or out of SPN synapses. In all, our results demonstrate that nonpathogenic HTT can indeed influence synaptic protein localization and uncover a novel role of HTT in PSD-95 distribution.

  5. Bidirectional Control of Postsynaptic Density-95 (PSD-95) Clustering by Huntingtin*

    PubMed Central

    Parsons, Matthew P.; Kang, Rujun; Buren, Caodu; Dau, Alejandro; Southwell, Amber L.; Doty, Crystal N.; Sanders, Shaun S.; Hayden, Michael R.; Raymond, Lynn A.

    2014-01-01

    Huntington disease is associated with early alterations in corticostriatal synaptic function that precede cell death, and it is postulated that ameliorating such changes may delay clinical onset and/or prevent neurodegeneration. Although many of these synaptic alterations are thought to be attributable to a toxic gain of function of the mutant huntingtin protein, the role that nonpathogenic huntingtin (HTT) plays in synaptic function is relatively unexplored. Here, we compare the immunocytochemical localization of a major postsynaptic scaffolding protein, PSD-95, in striatal neurons from WT mice and mice overexpressing HTT with 18 glutamine repeats (YAC18, nonpathogenic). We found that HTT overexpression resulted in a palmitoylation- and BDNF-dependent increase in PSD-95 clustering at synaptic sites in striatal spiny projection neurons (SPNs) co-cultured with cortical neurons. Surprisingly, the latter effect was mediated presynaptically, as HTT overexpression in cortical neurons alone was sufficient to increase PSD-95 clustering in the postsynaptic SPNs. In contrast, antisense oligonucleotide knockdown of HTT in WT co-cultures resulted in a significant reduction of PSD-95 clustering in SPNs. Notably, despite these bidirectional changes in PSD-95 clustering, we did not observe an alteration in basal electrophysiological measures of AMPA and NMDA receptors. Thus, unlike in previous studies in the hippocampus, enhanced or decreased PSD-95 clustering alone was insufficient to drive AMPA or NMDA receptors into or out of SPN synapses. In all, our results demonstrate that nonpathogenic HTT can indeed influence synaptic protein localization and uncover a novel role of HTT in PSD-95 distribution. PMID:24347167

  6. Identification of PSD-95 Depalmitoylating Enzymes

    PubMed Central

    Yokoi, Norihiko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta

    2016-01-01

    Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. SIGNIFICANCE STATEMENT Protein palmitoylation, the most common lipid

  7. Identification of PSD-95 Depalmitoylating Enzymes.

    PubMed

    Yokoi, Norihiko; Fukata, Yuko; Sekiya, Atsushi; Murakami, Tatsuro; Kobayashi, Kenta; Fukata, Masaki

    2016-06-15

    Postsynaptic density (PSD)-95, the most abundant postsynaptic scaffolding protein, plays a pivotal role in synapse development and function. Continuous palmitoylation cycles on PSD-95 are essential for its synaptic clustering and regulation of AMPA receptor function. However, molecular mechanisms for palmitate cycling on PSD-95 remain incompletely understood, as PSD-95 depalmitoylating enzymes remain unknown. Here, we isolated 38 mouse or rat serine hydrolases and found that a subset specifically depalmitoylated PSD-95 in heterologous cells. These enzymes showed distinct substrate specificity. α/β-Hydrolase domain-containing protein 17 members (ABHD17A, 17B, and 17C), showing the strongest depalmitoylating activity to PSD-95, showed different localization from other candidates in rat hippocampal neurons, and were distributed to recycling endosomes, the dendritic plasma membrane, and the synaptic fraction. Expression of ABHD17 in neurons selectively reduced PSD-95 palmitoylation and synaptic clustering of PSD-95 and AMPA receptors. Furthermore, taking advantage of the acyl-PEGyl exchange gel shift (APEGS) method, we quantitatively monitored the palmitoylation stoichiometry and the depalmitoylation kinetics of representative synaptic proteins, PSD-95, GluA1, GluN2A, mGluR5, Gαq, and HRas. Unexpectedly, palmitate on all of them did not turn over in neurons. Uniquely, most of the PSD-95 population underwent rapid palmitoylation cycles, and palmitate cycling on PSD-95 decelerated accompanied by its increased stoichiometry as synapses developed, probably contributing to postsynaptic receptor consolidation. Finally, inhibition of ABHD17 expression dramatically delayed the kinetics of PSD-95 depalmitoylation. This study suggests that local palmitoylation machinery composed of synaptic DHHC palmitoylating enzymes and ABHD17 finely controls the amount of synaptic PSD-95 and synaptic function. Protein palmitoylation, the most common lipid modification, dynamically

  8. Interaction of nNOS with PSD-95 negatively controls regenerative repair after stroke.

    PubMed

    Luo, Chun-Xia; Lin, Yu-Hui; Qian, Xiao-Dan; Tang, Ying; Zhou, Hai-Hui; Jin, Xing; Ni, Huan-Yu; Zhang, Feng-Yun; Qin, Cheng; Li, Fei; Zhang, Yu; Wu, Hai-Yin; Chang, Lei; Zhu, Dong-Ya

    2014-10-01

    Stroke is a major public health concern. The lack of effective therapies heightens the need for new therapeutic targets. Mammalian brain has the ability to rewire itself to restore lost functionalities. Promoting regenerative repair, including neurogenesis and dendritic remodeling, may offer a new therapeutic strategy for the treatment of stroke. Here, we report that interaction of neuronal nitric oxide synthase (nNOS) with the protein postsynaptic density-95 (PSD-95) negatively controls regenerative repair after stroke in rats. Dissociating nNOS-PSD-95 coupling in neurons promotes neuronal differentiation of neural stem cells (NSCs), facilitates the migration of newborn cells into the injured area, and enhances neurite growth of newborn neurons and dendritic spine formation of mature neurons in the ischemic brain of rats. More importantly, blocking nNOS-PSD-95 binding during the recovery stage improves stroke outcome via the promotion of regenerative repair in rats. Histone deacetylase 2 in NSCs may mediate the role of nNOS-PSD-95 association. Thus, nNOS-PSD-95 can serve as a target for regenerative repair after stroke. Copyright © 2014 the authors 0270-6474/14/3413535-14$15.00/0.

  9. PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density.

    PubMed

    Chen, Xiaobing; Levy, Jonathan M; Hou, Austin; Winters, Christine; Azzam, Rita; Sousa, Alioscka A; Leapman, Richard D; Nicoll, Roger A; Reese, Thomas S

    2015-12-15

    The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95-like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD.

  10. PSD-95 family MAGUKs are essential for anchoring AMPA and NMDA receptor complexes at the postsynaptic density

    PubMed Central

    Chen, Xiaobing; Levy, Jonathan M.; Hou, Austin; Winters, Christine; Azzam, Rita; Sousa, Alioscka A.; Leapman, Richard D.; Nicoll, Roger A.; Reese, Thomas S.

    2015-01-01

    The postsynaptic density (PSD)-95 family of membrane-associated guanylate kinases (MAGUKs) are major scaffolding proteins at the PSD in glutamatergic excitatory synapses, where they maintain and modulate synaptic strength. How MAGUKs underlie synaptic strength at the molecular level is still not well understood. Here, we explore the structural and functional roles of MAGUKs at hippocampal excitatory synapses by simultaneous knocking down PSD-95, PSD-93, and synapse-associated protein (SAP)102 and combining electrophysiology and transmission electron microscopic (TEM) tomography imaging to analyze the resulting changes. Acute MAGUK knockdown greatly reduces synaptic transmission mediated by α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptors (AMPARs) and N-methyl-d-aspartate receptors (NMDARs). This knockdown leads to a significant rise in the number of silent synapses, diminishes the size of PSDs without changes in pre- or postsynaptic membrane, and depletes the number of membrane-associated PSD-95–like vertical filaments and transmembrane structures, identified as AMPARs and NMDARs by EM tomography. The differential distribution of these receptor-like structures and dependence of their abundance on PSD size matches that of AMPARs and NMDARs in the hippocampal synapses. The loss of these structures following MAGUK knockdown tracks the reduction in postsynaptic AMPAR and NMDAR transmission, confirming the structural identities of these two types of receptors. These results demonstrate that MAGUKs are required for anchoring both types of glutamate receptors at the PSD and are consistent with a structural model where MAGUKs, corresponding to membrane-associated vertical filaments, are the essential structural proteins that anchor and organize both types of glutamate receptors and govern the overall molecular organization of the PSD. PMID:26604311

  11. Effects of Dimeric PSD-95 Inhibition on Excitotoxic Cell Death and Outcome After Controlled Cortical Impact in Rats.

    PubMed

    Sommer, Jens Bak; Bach, Anders; Malá, Hana; Gynther, Mikko; Bjerre, Ann-Sofie; Gram, Marie Gajhede; Marschner, Linda; Strømgaard, Kristian; Mogensen, Jesper; Pickering, Darryl S

    2017-08-21

    Therapeutic effects of PSD-95 inhibition have been demonstrated in numerous studies of stroke; however only few studies have assessed the effects of PSD-95 inhibitors in traumatic brain injury (TBI). As the pathophysiology of TBI partially overlaps with that of stroke, PSD-95 inhibition may also be an effective therapeutic strategy in TBI. The objectives of the present study were to assess the effects of a dimeric inhibitor of PSD-95, UCCB01-144, on excitotoxic cell death in vitro and outcome after experimental TBI in rats in vivo. In addition, the pharmacokinetic parameters of UCCB01-144 were investigated in order to assess uptake of the drug into the central nervous system of rats. After a controlled cortical impact rats were randomized to receive a single injection of either saline or two different doses of UCCB01-144 (10 or 20 mg/kg IV) immediately after injury. Spatial learning and memory were assessed in a water maze at 2 weeks post-trauma, and at 4 weeks lesion volumes were estimated. Overall, UCCB01-144 did not protect against NMDA-toxicity in neuronal cultures or experimental TBI in rats. Important factors that should be investigated further in future studies assessing the effects of PSD-95 inhibitors in TBI are discussed.

  12. PSD-95 expression controls l-DOPA dyskinesia through dopamine D1 receptor trafficking

    PubMed Central

    Porras, Gregory; Berthet, Amandine; Dehay, Benjamin; Li, Qin; Ladepeche, Laurent; Normand, Elisabeth; Dovero, Sandra; Martinez, Audrey; Doudnikoff, Evelyne; Martin-Négrier, Marie-Laure; Chuan, Qin; Bloch, Bertrand; Choquet, Daniel; Boué-Grabot, Eric; Groc, Laurent; Bezard, Erwan

    2012-01-01

    l-DOPA–induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson’s disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson’s patients. PMID:23041629

  13. PMCA2 VIA PSD-95 CONTROLS CALCIUM SIGNALING BY α7-CONTAINING NICOTINIC ACETYLCHOLINE RECEPTORS ON ASPINY INTERNEURONS

    PubMed Central

    Gomez-Varela, David; Schmidt, Manuela; Schoellerman, Jeff; Peters, Eric C.; Berg, Darwin K.

    2012-01-01

    Local control of calcium concentration within neurons is critical for signaling and regulation of synaptic communication in neural circuits. How local control can be achieved in the absence of physical compartmentalization is poorly understood. Challenging examples are provided by nicotinic acetylcholine receptors that contain α7 nicotinic receptor subunits (α7-nAChRs). These receptors are highly permeable to calcium and are concentrated on aspiny dendrites of interneurons which lack obvious physical compartments for constraining calcium diffusion. Using functional proteomics on rat brain, we show that α7-nAChRs are associated with the membrane calcium pump PMCA2. Analysis of α7-nAChR function in hippocampal interneurons in culture shows that PMCA2 activity limits the duration of calcium elevations produced by the receptors. Unexpectedly, PMCA2 inhibition triggers rapid calcium-dependent loss of α7-nAChR clusters. This extreme regulatory response is mediated by CaMKII, involves proteasome activity, depends on the second intracellular loop of α7-nAChR subunits, and is specific in that it does not alter two other classes of calcium-permeable ionotropic receptors on the same neurons. A critical link is provided by the scaffold protein PSD-95, which is associated with α7-nAChRs and constrains their mobility as revealed by single-particle tracking on neurons. The PSD-95 link is required for PMCA2-mediated removal of α7-nAChR clusters. This three-component combination of PMCA2/PSD-95/α7-nAChR offers a novel mechanism for tight control of calcium dynamics in neurons. PMID:22593058

  14. Cdk5 regulates PSD-95 ubiquitination in neurons

    PubMed Central

    Bianchetta, Michael J.; Lam, TuKiet T.; Jones, Stephen N.; Morabito, Maria A.

    2011-01-01

    The kinase Cdk5 and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer’s, learning and memory, and synapse maturation and plasticity. However the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the Ubiquitin E3 Ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a non-proteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiqutination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis. PMID:21849563

  15. Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

    PubMed Central

    Zhang, Yonghong; Matt, Lucas; Patriarchi, Tommaso; Malik, Zulfiqar A; Chowdhury, Dhrubajyoti; Park, Deborah K; Renieri, Alessandra; Ames, James B; Hell, Johannes W

    2014-01-01

    Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca2+ influx via NMDA receptors. Here, we show that Ca2+/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1–16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca2+-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca2+-induced dissociation of PSD-95 from the postsynaptic membrane. PMID:24705785

  16. Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release.

    PubMed

    Zhang, Yonghong; Matt, Lucas; Patriarchi, Tommaso; Malik, Zulfiqar A; Chowdhury, Dhrubajyoti; Park, Deborah K; Renieri, Alessandra; Ames, James B; Hell, Johannes W

    2014-06-17

    Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane. © 2014 The Authors.

  17. PSD-95 is required for activity-driven synapse stabilization

    PubMed Central

    Ehrlich, Ingrid; Klein, Matthew; Rumpel, Simon; Malinow, Roberto

    2007-01-01

    The activity-dependent regulation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and the stabilization of synapses are critical to synaptic development and plasticity. One candidate molecule implicated in maturation, synaptic strengthening, and plasticity is PSD-95. Here we find that acute knockdown of PSD-95 in brain slice cultures by RNAi arrests the normal development of synaptic structure and function that is driven by spontaneous activity. Surprisingly, PSD-95 is not necessary for the induction and early expression of long-term potentiation (LTP). However, knockdown of PSD-95 leads to smaller increases in spine size after chemically induced LTP. Furthermore, although at this age spine turnover is normally low and LTP produces a transient increase, in cells with reduced PSD-95 spine turnover is high and remains increased after LTP. Taken together, our data support a model in which appropriate levels of PSD-95 are required for activity-dependent synapse stabilization after initial phases of synaptic potentiation. PMID:17360496

  18. Cyclin-dependent kinase 5 regulates PSD-95 ubiquitination in neurons.

    PubMed

    Bianchetta, Michael J; Lam, TuKiet T; Jones, Stephen N; Morabito, Maria A

    2011-08-17

    Cyclin-dependent kinase 5 (Cdk5) and its activator p35 have been implicated in drug addiction, neurodegenerative diseases such as Alzheimer's, learning and memory, and synapse maturation and plasticity. However, the molecular mechanisms by which Cdk5 regulates synaptic plasticity are still unclear. PSD-95 is a major postsynaptic scaffolding protein of glutamatergic synapses that regulates synaptic strength and plasticity. PSD-95 is ubiquitinated by the ubiquitin E3 ligase Mdm2, and rapid and transient PSD-95 ubiquitination has been implicated in NMDA receptor-induced AMPA receptor endocytosis. Here we demonstrate that genetic or pharmacological reduction of Cdk5 activity increases the interaction of Mdm2 with PSD-95 and enhances PSD-95 ubiquitination without affecting PSD-95 protein levels in vivo in mice, suggesting a nonproteolytic function of ubiquitinated PSD-95 at synapses. We show that PSD-95 ubiquitination correlates with increased interaction with β-adaptin, a subunit of the clathrin adaptor protein complex AP-2. This interaction is increased by genetic reduction of Cdk5 activity or NMDA receptor stimulation and is dependent on Mdm2. Together these results support a function for Cdk5 in regulating PSD-95 ubiquitination and its interaction with AP-2 and suggest a mechanism by which PSD-95 may regulate NMDA receptor-induced AMPA receptor endocytosis.

  19. Activity-dependent regulation of synaptic strength by PSD-95 in CA1 neurons.

    PubMed

    Zhang, Peng; Lisman, John E

    2012-02-01

    CaMKII and PSD-95 are the two most abundant postsynaptic proteins in the postsynaptic density (PSD). Overexpression of either can dramatically increase synaptic strength and saturate long-term potentiation (LTP). To do so, CaMKII must be activated, but the same is not true for PSD-95; expressing wild-type PSD-95 is sufficient. This raises the question of whether PSD-95's effects are simply an equilibrium process [increasing the number of AMPA receptor (AMPAR) slots] or whether activity is somehow involved. To examine this question, we blocked activity in cultured hippocampal slices with TTX and found that the effects of PSD-95 overexpression were greatly reduced. We next studied the type of receptors involved. The effects of PSD-95 were prevented by antagonists of group I metabotropic glutamate receptors (mGluRs) but not by antagonists of ionotropic glutamate receptors. The inhibition of PSD-95-induced strengthening was not simply a result of inhibition of PSD-95 synthesis. To understand the mechanisms involved, we tested the role of CaMKII. Overexpression of a CaMKII inhibitor, CN19, greatly reduced the effect of PSD-95. We conclude that PSD-95 cannot itself increase synaptic strength simply by increasing the number of AMPAR slots; rather, PSD-95's effects on synaptic strength require an activity-dependent process involving mGluR and CaMKII.

  20. Activity-dependent regulation of synaptic strength by PSD-95 in CA1 neurons

    PubMed Central

    Zhang, Peng

    2012-01-01

    CaMKII and PSD-95 are the two most abundant postsynaptic proteins in the postsynaptic density (PSD). Overexpression of either can dramatically increase synaptic strength and saturate long-term potentiation (LTP). To do so, CaMKII must be activated, but the same is not true for PSD-95; expressing wild-type PSD-95 is sufficient. This raises the question of whether PSD-95's effects are simply an equilibrium process [increasing the number of AMPA receptor (AMPAR) slots] or whether activity is somehow involved. To examine this question, we blocked activity in cultured hippocampal slices with TTX and found that the effects of PSD-95 overexpression were greatly reduced. We next studied the type of receptors involved. The effects of PSD-95 were prevented by antagonists of group I metabotropic glutamate receptors (mGluRs) but not by antagonists of ionotropic glutamate receptors. The inhibition of PSD-95-induced strengthening was not simply a result of inhibition of PSD-95 synthesis. To understand the mechanisms involved, we tested the role of CaMKII. Overexpression of a CaMKII inhibitor, CN19, greatly reduced the effect of PSD-95. We conclude that PSD-95 cannot itself increase synaptic strength simply by increasing the number of AMPAR slots; rather, PSD-95's effects on synaptic strength require an activity-dependent process involving mGluR and CaMKII. PMID:22114157

  1. PSD-95 is required to sustain the molecular organization of the postsynaptic density

    PubMed Central

    Chen, Xiaobing; Nelson, Christopher D; Li, Xiang; Winters, Christine A.; Azzam, Rita; Sousa, Alioscka A.; Leapman, Richard D.; Gainer, Harold; Sheng, Morgan; Reese, Thomas S.

    2011-01-01

    PSD-95, a membrane-associated guanylate kinase (MAGUK), is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNAi knockdown of PSD-95 leads to loss of entire patches of PSD material, and EM tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA, but not NMDA receptor type structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD. PMID:21525273

  2. Phosphorylation of threonine-19 of PSD-95 by GSK-3β is required for PSD-95 mobilization and long-term depression.

    PubMed

    Nelson, Christopher D; Kim, Myung Jong; Hsin, Honor; Chen, Yelin; Sheng, Morgan

    2013-07-17

    Activity of glycogen synthase kinase-3β (GSK-3β) is required for long-term depression (LTD) via molecular mechanisms that are incompletely understood. Here, we report that PSD-95, a major scaffold protein of the postsynaptic density (PSD) that promotes synaptic strength, is phosphorylated on threonine-19 (T19) by GSK-3β. In cultured rat hippocampal neurons, phosphorylation of T19 increases rapidly with chemical LTD and is attenuated by pharmacologic or genetic suppression of GSK-3β. In organotypic rat hippocampal slices, we find that a nonphosphorylatable PSD-95 mutant (T19A) tagged with photoactivatable green fluorescent protein (PAGFP) shows enhanced stability in dendritic spines versus wild-type PSD-95, whereas the phosphomimetic mutant (PSD-95-T19D) is more readily dispersed. Further, overexpression of PSD-95-T19A, but not WT-PSD-95, impairs AMPA receptor internalization and the induction of LTD. These data indicate that phosphorylation on T19 by GSK-3β destabilizes PSD-95 within the PSD and is a critical step for AMPA receptor mobilization and LTD.

  3. Phosphorylation of Threonine-19 of PSD-95 by GSK-3β is Required for PSD-95 Mobilization and Long-Term Depression

    PubMed Central

    Nelson, Christopher D.; Kim, Myung Jong; Hsin, Honor; Chen, Yelin

    2013-01-01

    Activity of glycogen synthase kinase-3β (GSK-3β) is required for long-term depression (LTD) via molecular mechanisms that are incompletely understood. Here, we report that PSD-95, a major scaffold protein of the postsynaptic density (PSD) that promotes synaptic strength, is phosphorylated on threonine-19 (T19) by GSK-3β. In cultured rat hippocampal neurons, phosphorylation of T19 increases rapidly with chemical LTD and is attenuated by pharmacologic or genetic suppression of GSK-3β. In organotypic rat hippocampal slices, we find that a nonphosphorylatable PSD-95 mutant (T19A) tagged with photoactivatable green fluorescent protein (PAGFP) shows enhanced stability in dendritic spines versus wild-type PSD-95, whereas the phosphomimetic mutant (PSD-95-T19D) is more readily dispersed. Further, overexpression of PSD-95-T19A, but not WT-PSD-95, impairs AMPA receptor internalization and the induction of LTD. These data indicate that phosphorylation on T19 by GSK-3β destabilizes PSD-95 within the PSD and is a critical step for AMPA receptor mobilization and LTD. PMID:23864697

  4. Durable fear memories require PSD-95

    PubMed Central

    Fitzgerald, Paul J.; Pinard, Courtney R.; Camp, Marguerite C.; Feyder, Michael; Sah, Anupam; Bergstrom, Hadley; Graybeal, Carolyn; Liu, Yan; Schlüter, Oliver; Grant, Seth G.N.; Singewald, Nicolas; Xu, Weifeng; Holmes, Andrew

    2014-01-01

    Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. While overly persistent fear memories underlie anxiety disorders such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Post-synaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Employing a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95GK), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95GK mice to retrieve remote cued fear memories was associated with hypoactivation of the infralimbic cortex (IL) (not anterior cingulate (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated PSD-95 virus-mediated knockdown in the IL, not ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories. PMID:25510511

  5. Durable fear memories require PSD-95.

    PubMed

    Fitzgerald, P J; Pinard, C R; Camp, M C; Feyder, M; Sah, A; Bergstrom, H C; Graybeal, C; Liu, Y; Schlüter, O M; Grant, S G; Singewald, N; Xu, W; Holmes, A

    2015-07-01

    Traumatic fear memories are highly durable but also dynamic, undergoing repeated reactivation and rehearsal over time. Although overly persistent fear memories underlie anxiety disorders, such as posttraumatic stress disorder, the key neural and molecular mechanisms underlying fear memory durability remain unclear. Postsynaptic density 95 (PSD-95) is a synaptic protein regulating glutamate receptor anchoring, synaptic stability and certain types of memory. Using a loss-of-function mutant mouse lacking the guanylate kinase domain of PSD-95 (PSD-95(GK)), we analyzed the contribution of PSD-95 to fear memory formation and retrieval, and sought to identify the neural basis of PSD-95-mediated memory maintenance using ex vivo immediate-early gene mapping, in vivo neuronal recordings and viral-mediated knockdown (KD) approaches. We show that PSD-95 is dispensable for the formation and expression of recent fear memories, but essential for the formation of precise and flexible fear memories and for the maintenance of memories at remote time points. The failure of PSD-95(GK) mice to retrieve remote cued fear memory was associated with hypoactivation of the infralimbic (IL) cortex (but not the anterior cingulate cortex (ACC) or prelimbic cortex), reduced IL single-unit firing and bursting, and attenuated IL gamma and theta oscillations. Adeno-associated virus-mediated PSD-95 KD in the IL, but not the ACC, was sufficient to impair recent fear extinction and remote fear memory, and remodel IL dendritic spines. Collectively, these data identify PSD-95 in the IL as a critical mechanism supporting the durability of fear memories over time. These preclinical findings have implications for developing novel approaches to treating trauma-based anxiety disorders that target the weakening of overly persistent fear memories.

  6. PSD-95 regulates synaptic kainate receptors at mouse hippocampal mossy fiber-CA3 synapses.

    PubMed

    Suzuki, Etsuko; Kamiya, Haruyuki

    2016-06-01

    Kainate-type glutamate receptors (KARs) are the third class of ionotropic glutamate receptors whose activation leads to the unique roles in regulating synaptic transmission and circuit functions. In contrast to AMPA receptors (AMPARs), little is known about the mechanism of synaptic localization of KARs. PSD-95, a major scaffold protein of the postsynaptic density, is a candidate molecule that regulates the synaptic KARs. Although PSD-95 was shown to bind directly to KARs subunits, it has not been tested whether PSD-95 regulates synaptic KARs in intact synapses. Using PSD-95 knockout mice, we directly investigated the role of PSD-95 in the KARs-mediated components of synaptic transmission at hippocampal mossy fiber-CA3 synapse, one of the synapses with the highest density of KARs. Mossy fiber EPSCs consist of AMPA receptor (AMPAR)-mediated fast component and KAR-mediated slower component, and the ratio was significantly reduced in PSD-95 knockout mice. The size of KARs-mediated field EPSP reduced in comparison with the size of the fiber volley. Analysis of KARs-mediated miniature EPSCs also suggested reduced synaptic KARs. All the evidence supports critical roles of PSD-95 in regulating synaptic KARs. Copyright © 2015 Elsevier Ireland Ltd and Japan Neuroscience Society. All rights reserved.

  7. PSD-95 and PSD-93 play critical but distinct roles in synaptic scaling up and down.

    PubMed

    Sun, Qian; Turrigiano, Gina G

    2011-05-04

    Synaptic scaling stabilizes neuronal firing through the homeostatic regulation of postsynaptic strength, but the mechanisms by which chronic changes in activity lead to bidirectional adjustments in synaptic AMPA receptor (AMPAR) abundance are incompletely understood. Furthermore, it remains unclear to what extent scaling up and scaling down use distinct molecular machinery. PSD-95 is a scaffold protein proposed to serve as a binding "slot" that determines synaptic AMPAR content, and synaptic PSD-95 abundance is regulated by activity, raising the possibility that activity-dependent changes in the synaptic abundance of PSD-95 or other membrane-associated guanylate kinases (MAGUKs) drives the bidirectional changes in AMPAR accumulation during synaptic scaling. We found that synaptic PSD-95 and SAP102 (but not PSD-93) abundance were bidirectionally regulated by activity, but these changes were not sufficient to drive homeostatic changes in synaptic strength. Although not sufficient, the PSD-95 MAGUKs were necessary for synaptic scaling, but scaling up and down were differentially dependent on PSD-95 and PSD-93. Scaling down was completely blocked by reduced or enhanced PSD-95, through a mechanism that depended on the PDZ1/2 domains. In contrast, scaling up could be supported by either PSD-95 or PSD-93 in a manner that depended on neuronal age and was unaffected by a superabundance of PSD-95. Together, our data suggest that scaling up and down of quantal amplitude is not driven by changes in synaptic abundance of PSD-95 MAGUKs, but rather that the PSD-95 MAGUKs serve as critical synaptic organizers that use distinct protein-protein interactions to mediate homeostatic accumulation and loss of synaptic AMPAR.

  8. Motor impairments, striatal degeneration, and altered dopamine-glutamate interplay in mice lacking PSD-95.

    PubMed

    Zhang, Jingping; Saur, Taixiang; Duke, Angela N; Grant, Seth G N; Platt, Donna M; Rowlett, James K; Isacson, Ole; Yao, Wei-Dong

    2014-01-01

    Excessive activation of the N-methyl-d-aspartate (NMDA) receptor and the neurotransmitter dopamine (DA) mediate neurotoxicity and neurodegeneration under many neurological conditions, including Huntington's disease (HD), an autosomal dominant neurodegenerative disease characterized by the preferential loss of medium spiny projection neurons (MSNs) in the striatum. PSD-95 is a major scaffolding protein in the postsynaptic density (PSD) of dendritic spines, where a classical role for PSD-95 is to stabilize glutamate receptors at sites of synaptic transmission. Our recent studies indicate that PSD-95 also interacts with the D1 DA receptor localized in spines and negatively regulates spine D1 signaling. Moreover, PSD-95 forms ternary protein complexes with D1 and NMDA receptors, and plays a role in limiting the reciprocal potentiation between both receptors from being escalated. These studies suggest a neuroprotective role for PSD-95. Here we show that mice lacking PSD-95, resulting from genetic deletion of the GK domain of PSD-95 (PSD-95-ΔGK mice), sporadically develop progressive neurological impairments characterized by hypolocomotion, limb clasping, and loss of DARPP-32-positive MSNs. Electrophysiological experiments indicated that NMDA receptors in mutant MSNs were overactive, suggested by larger, NMDA receptor-mediated miniature excitatory postsynaptic currents (EPSCs) and higher ratios of NMDA- to AMPA-mediated corticostriatal synaptic transmission. In addition, NMDA receptor currents in mutant cortical neurons were more sensitive to potentiation by the D1 receptor agonist SKF81297. Finally, repeated administration of the psychostimulant cocaine at a dose regimen not producing overt toxicity-related phenotypes in normal mice reliably converted asymptomatic mutant mice to clasping symptomatic mice. These results support the hypothesis that deletion of PSD-95 in mutant mice produces concomitant overactivation of both D1 and NMDA receptors that makes neurons more

  9. Serine Racemase Regulated by Binding to Stargazin and PSD-95

    PubMed Central

    Ma, Ting Martin; Paul, Bindu D.; Fu, Chenglai; Hu, Shaohui; Zhu, Heng; Blackshaw, Seth; Wolosker, Herman; Snyder, Solomon H.

    2014-01-01

    d-Serine, an endogenous co-agonist for the glycine site of the synaptic NMDA glutamate receptor, regulates synaptic plasticity and is implicated in schizophrenia. Serine racemase (SR) is the enzyme that converts l-serine to d-serine. In this study, we demonstrate that SR interacts with the synaptic proteins, postsynaptic density protein 95 (PSD-95) and stargazin, forming a ternary complex. SR binds to the PDZ3 domain of PSD-95 through the PDZ domain ligand at its C terminus. SR also binds to the C terminus of stargazin, which facilitates the cell membrane localization of SR and inhibits its activity. AMPA receptor activation internalizes SR and disrupts its interaction with stargazin, therefore derepressing SR activity, leading to more d-serine production and potentially facilitating NMDA receptor activation. These interactions regulate the enzymatic activity as well as the intracellular localization of SR, potentially coupling the activities of NMDA and AMPA receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation. PMID:25164819

  10. Hypersocial behavior and biological redundancy in mice with reduced expression of PSD95 or PSD93.

    PubMed

    Winkler, Daniela; Daher, Fernanda; Wüstefeld, Liane; Hammerschmidt, Kurt; Poggi, Giulia; Seelbach, Anna; Krueger-Burg, Dilja; Vafadari, Behnam; Ronnenberg, Anja; Liu, Yanling; Kaczmarek, Leszek; Schlüter, Oliver M; Ehrenreich, Hannelore; Dere, Ekrem

    2017-02-09

    increased PSD93 protein expression in hippocampal synaptic enrichment preparations of PSD95(+/-) mice. These data suggest that both PSD95 and PSD93 are involved in processing of social stimuli and control of social behavior. This important role may be partly assured by functional/behavioral and biological/biochemical redundancy.

  11. PSD-95 is required to sustain the molecular organization of the postsynaptic density.

    PubMed

    Chen, Xiaobing; Nelson, Christopher D; Li, Xiang; Winters, Christine A; Azzam, Rita; Sousa, Alioscka A; Leapman, Richard D; Gainer, Harold; Sheng, Morgan; Reese, Thomas S

    2011-04-27

    PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein in the excitatory postsynaptic density (PSD) and a potent regulator of synaptic strength. Here we show that PSD-95 is in an extended configuration and positioned into regular arrays of vertical filaments that contact both glutamate receptors and orthogonal horizontal elements layered deep inside the PSD in rat hippocampal spine synapses. RNA interference knockdown of PSD-95 leads to loss of entire patches of PSD material, and electron microscopy tomography shows that the patchy loss correlates with loss of PSD-95-containing vertical filaments, horizontal elements associated with the vertical filaments, and putative AMPA receptor-type, but not NMDA receptor-type, structures. These observations show that the orthogonal molecular scaffold constructed from PSD-95-containing vertical filaments and their associated horizontal elements is essential for sustaining the three-dimensional molecular organization of the PSD. Our findings provide a structural basis for understanding the functional role of PSD-95 at the PSD.

  12. Early Growth Response 1 (Egr-1) Regulates N-Methyl-d-aspartate Receptor (NMDAR)-dependent Transcription of PSD-95 and α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor (AMPAR) Trafficking in Hippocampal Primary Neurons*

    PubMed Central

    Qin, Xike; Jiang, Yongjun; Tse, Yiu Chung; Wang, Yunling; Wong, Tak Pan; Paudel, Hemant K.

    2015-01-01

    The N-methyl-d-aspartate receptor (NMDAR) controls synaptic plasticity and memory function and is one of the major inducers of transcription factor Egr-1 in the hippocampus. However, how Egr-1 mediates the NMDAR signal in neurons has remained unclear. Here, we show that the hippocampus of mice lacking Egr-1 displays electrophysiology properties and ultrastructure that are similar to mice overexpressing PSD-95, a major scaffolding protein of postsynaptic density involved in synapse formation, synaptic plasticity, and synaptic targeting of AMPA receptors (AMPARs), which mediate the vast majority of excitatory transmission in the CNS. We demonstrate that Egr-1 is a transcription repressor of the PSD-95 gene and is recruited to the PSD-95 promoter in response to NMDAR activation. Knockdown of Egr-1 in rat hippocampal primary neurons blocks NMDAR-induced PSD-95 down-regulation and AMPAR endocytosis. Likewise, overexpression of Egr-1 in rat hippocampal primary neurons causes reduction in PSD-95 protein level and promotes AMPAR endocytosis. Our data indicate that Egr-1 is involved in NMDAR-mediated PSD-95 down-regulation and AMPAR endocytosis, a process important in the expression of long term depression. PMID:26475861

  13. Early Growth Response 1 (Egr-1) Regulates N-Methyl-d-aspartate Receptor (NMDAR)-dependent Transcription of PSD-95 and α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor (AMPAR) Trafficking in Hippocampal Primary Neurons.

    PubMed

    Qin, Xike; Jiang, Yongjun; Tse, Yiu Chung; Wang, Yunling; Wong, Tak Pan; Paudel, Hemant K

    2015-12-04

    The N-methyl-d-aspartate receptor (NMDAR) controls synaptic plasticity and memory function and is one of the major inducers of transcription factor Egr-1 in the hippocampus. However, how Egr-1 mediates the NMDAR signal in neurons has remained unclear. Here, we show that the hippocampus of mice lacking Egr-1 displays electrophysiology properties and ultrastructure that are similar to mice overexpressing PSD-95, a major scaffolding protein of postsynaptic density involved in synapse formation, synaptic plasticity, and synaptic targeting of AMPA receptors (AMPARs), which mediate the vast majority of excitatory transmission in the CNS. We demonstrate that Egr-1 is a transcription repressor of the PSD-95 gene and is recruited to the PSD-95 promoter in response to NMDAR activation. Knockdown of Egr-1 in rat hippocampal primary neurons blocks NMDAR-induced PSD-95 down-regulation and AMPAR endocytosis. Likewise, overexpression of Egr-1 in rat hippocampal primary neurons causes reduction in PSD-95 protein level and promotes AMPAR endocytosis. Our data indicate that Egr-1 is involved in NMDAR-mediated PSD-95 down-regulation and AMPAR endocytosis, a process important in the expression of long term depression. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. A novel role for PSD-95 in mediating ethanol intoxication, drinking and place preference.

    PubMed

    Camp, Marguerite C; Feyder, Michael; Ihne, Jessica; Palachick, Benjamin; Hurd, Benita; Karlsson, Rose-Marie; Noronha, Bianca; Chen, Yi-Chyan; Coba, Marcelo P; Grant, Seth G N; Holmes, Andrew

    2011-07-01

    The synaptic signaling mechanisms mediating the behavioral effects of ethanol (EtOH) remain poorly understood. Post-synaptic density 95 (PSD-95, SAP-90, Dlg4) is a key orchestrator of N-methyl-D-aspartate receptors (NMDAR) and glutamatergic synapses, which are known to be major sites of EtOH's behavioral actions. However, the potential contribution of PSD-95 to EtOH-related behaviors has not been established. Here, we evaluated knockout (KO) mice lacking PSD-95 for multiple measures of sensitivity to the acute intoxicating effects of EtOH (ataxia, hypothermia, sedation/hypnosis), EtOH drinking under conditions of free access and following deprivation, acquisition and long-term retention of EtOH conditioned place preference (CPP) (and lithium chloride-induced conditioned taste aversion), and intoxication-potentiating responses to NMDAR antagonism. PSD-95 KO exhibited increased sensitivity to the sedative/hypnotic, but not ataxic or hypothermic, effects of acute EtOH relative to wild-type controls (WT). PSD-95 KO consumed less EtOH than WT, particularly at higher EtOH concentrations, although increases in KO drinking could be induced by concentration-fading and deprivation. PSD-95 KO showed normal EtOH CPP 1 day after conditioning, but showed significant aversion 2 weeks later. Lithium chloride-induced taste aversion was impaired in PSD-95 KO at both time points. Finally, the EtOH-potentiating effects of the NMDAR antagonist MK-801 were intact in PSD-95 KO at the dose tested. These data reveal a major, novel role for PSD-95 in mediating EtOH behaviors, and add to growing evidence that PSD-95 is a key mediator of the effects of multiple abused drugs.

  15. Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice

    PubMed Central

    2012-01-01

    Background Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. Results The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. Conclusions These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior. PMID:23268962

  16. Impaired synaptic clustering of postsynaptic density proteins and altered signal transmission in hippocampal neurons, and disrupted learning behavior in PDZ1 and PDZ2 ligand binding-deficient PSD-95 knockin mice.

    PubMed

    Nagura, Hitoshi; Ishikawa, Yasuyuki; Kobayashi, Katsunori; Takao, Keizo; Tanaka, Tomo; Nishikawa, Kouki; Tamura, Hideki; Shiosaka, Sadao; Suzuki, Hidenori; Miyakawa, Tsuyoshi; Fujiyoshi, Yoshinori; Doi, Tomoko

    2012-12-26

    Postsynaptic density (PSD)-95-like membrane-associated guanylate kinases (PSD-MAGUKs) are scaffold proteins in PSDs that cluster signaling molecules near NMDA receptors. PSD-MAGUKs share a common domain structure, including three PDZ (PDZ1/2/3) domains in their N-terminus. While multiple domains enable the PSD-MAGUKs to bind various ligands, the contribution of each PDZ domain to synaptic organization and function is not fully understood. Here, we focused on the PDZ1/2 domains of PSD-95 that bind NMDA-type receptors, and studied the specific roles of the ligand binding of these domains in the assembly of PSD proteins, synaptic properties of hippocampal neurons, and behavior, using ligand binding-deficient PSD-95 cDNA knockin (KI) mice. The KI mice showed decreased accumulation of mutant PSD-95, PSD-93 and AMPA receptor subunits in the PSD fraction of the hippocampus. In the hippocampal CA1 region of young KI mice, basal synaptic efficacy was reduced and long-term potentiation (LTP) was enhanced with intact long-term depression. In adult KI mice, there was no significant change in the magnitude of LTP in CA1, but robustly enhanced LTP was induced at the medial perforant path-dentate gyrus synapses, suggesting that PSD-95 has an age- and subregion-dependent role. In a battery of behavioral tests, KI mice showed markedly abnormal anxiety-like behavior, impaired spatial reference and working memory, and impaired remote memory and pattern separation in fear conditioning test. These findings reveal that PSD-95 including its ligand binding of the PDZ1/2 domains controls the synaptic clustering of PSD-MAGUKs and AMPA receptors, which may have an essential role in regulating hippocampal synaptic transmission, plasticity, and hippocampus-dependent behavior.

  17. Anchoring and synaptic stability of PSD-95 is driven by ephrin-B3.

    PubMed

    Hruska, Martin; Henderson, Nathan T; Xia, Nan L; Le Marchand, Sylvain J; Dalva, Matthew B

    2015-11-01

    Organization of signaling complexes at excitatory synapses by membrane-associated guanylate kinase (MAGUK) proteins regulates synapse development, plasticity, senescence and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches and in vivo models that the trans-synaptic organizing protein ephrin-B3 controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a newly identified mitogen-associated protein kinase (MAPK)-dependent phosphorylation site on ephrin-B3, Ser332. Unphosphorylated ephrin-B3 was enriched at synapses, and interacted directly with and stabilized PSD-95 at synapses. Activity-induced phosphorylation of Ser332 dispersed ephrin-B3 from synapses, prevented the interaction with PSD-95 and enhanced the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity.

  18. Ocular Dominance Plasticity after Stroke Was Preserved in PSD-95 Knockout Mice

    PubMed Central

    Greifzu, Franziska; Parthier, Daniel; Goetze, Bianka; Schlüter, Oliver M.; Löwel, Siegrid

    2016-01-01

    Neuronal plasticity is essential to enable rehabilitation when the brain suffers from injury, such as following a stroke. One of the most established models to study cortical plasticity is ocular dominance (OD) plasticity in the primary visual cortex (V1) of the mammalian brain induced by monocular deprivation (MD). We have previously shown that OD-plasticity in adult mouse V1 is absent after a photothrombotic (PT) stroke lesion in the adjacent primary somatosensory cortex (S1). Exposing lesioned mice to conditions which reduce the inhibitory tone in V1, such as raising animals in an enriched environment or short-term dark exposure, preserved OD-plasticity after an S1-lesion. Here we tested whether modification of excitatory circuits can also be beneficial for preserving V1-plasticity after stroke. Mice lacking postsynaptic density protein-95 (PSD-95), a signaling scaffold present at mature excitatory synapses, have lifelong juvenile-like OD-plasticity caused by an increased number of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) -silent synapses in V1 but unaltered inhibitory tone. In fact, using intrinsic signal optical imaging, we show here that OD-plasticity was preserved in V1 of adult PSD-95 KO mice after an S1-lesion but not in PSD-95 wildtype (WT)-mice. In addition, experience-enabled enhancement of the optomotor reflex of the open eye after MD was compromised in both lesioned PSD-95 KO and PSD-95 WT mice. Basic V1-activation and retinotopic map quality were, however, not different between lesioned PSD-95 KO mice and their WT littermates. The preserved OD-plasticity in the PSD-95 KO mice indicates that V1-plasticity after a distant stroke can be promoted by either changes in excitatory circuitry or by lowering the inhibitory tone in V1 as previously shown. Furthermore, the present data indicate that an increased number of AMPA-silent synapses preserves OD-plasticity not only in the healthy brain, but also in another experimental paradigm of

  19. Ocular Dominance Plasticity after Stroke Was Preserved in PSD-95 Knockout Mice.

    PubMed

    Greifzu, Franziska; Parthier, Daniel; Goetze, Bianka; Schlüter, Oliver M; Löwel, Siegrid

    2016-01-01

    Neuronal plasticity is essential to enable rehabilitation when the brain suffers from injury, such as following a stroke. One of the most established models to study cortical plasticity is ocular dominance (OD) plasticity in the primary visual cortex (V1) of the mammalian brain induced by monocular deprivation (MD). We have previously shown that OD-plasticity in adult mouse V1 is absent after a photothrombotic (PT) stroke lesion in the adjacent primary somatosensory cortex (S1). Exposing lesioned mice to conditions which reduce the inhibitory tone in V1, such as raising animals in an enriched environment or short-term dark exposure, preserved OD-plasticity after an S1-lesion. Here we tested whether modification of excitatory circuits can also be beneficial for preserving V1-plasticity after stroke. Mice lacking postsynaptic density protein-95 (PSD-95), a signaling scaffold present at mature excitatory synapses, have lifelong juvenile-like OD-plasticity caused by an increased number of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) -silent synapses in V1 but unaltered inhibitory tone. In fact, using intrinsic signal optical imaging, we show here that OD-plasticity was preserved in V1 of adult PSD-95 KO mice after an S1-lesion but not in PSD-95 wildtype (WT)-mice. In addition, experience-enabled enhancement of the optomotor reflex of the open eye after MD was compromised in both lesioned PSD-95 KO and PSD-95 WT mice. Basic V1-activation and retinotopic map quality were, however, not different between lesioned PSD-95 KO mice and their WT littermates. The preserved OD-plasticity in the PSD-95 KO mice indicates that V1-plasticity after a distant stroke can be promoted by either changes in excitatory circuitry or by lowering the inhibitory tone in V1 as previously shown. Furthermore, the present data indicate that an increased number of AMPA-silent synapses preserves OD-plasticity not only in the healthy brain, but also in another experimental paradigm of

  20. Pin1 Modulates the Synaptic Content of NMDA Receptors via Prolyl-Isomerization of PSD-95.

    PubMed

    Antonelli, Roberta; De Filippo, Roberto; Middei, Silvia; Stancheva, Stefka; Pastore, Beatrice; Ammassari-Teule, Martine; Barberis, Andrea; Cherubini, Enrico; Zacchi, Paola

    2016-05-18

    Phosphorylation of serine/threonine residues preceding a proline regulates the fate of its targets through postphosphorylation conformational changes catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. By flipping the substrate between two different functional conformations, this enzyme exerts a fine-tuning of phosphorylation signals. Pin1 has been detected in dendritic spines and shafts where it regulates protein synthesis required to sustain the late phase of long-term potentiation (LTP). Here, we demonstrate that Pin1 residing in postsynaptic structures can interact with postsynaptic density protein-95 (PSD-95), a key scaffold protein that anchors NMDA receptors (NMDARs) in PSD via GluN2-type receptor subunits. Pin1 recruitment by PSD-95 occurs at specific serine-threonine/proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. Upon binding, Pin1 triggers structural changes in PSD-95, thus negatively affecting its ability to interact with NMDARs. In electrophysiological experiments, larger NMDA-mediated synaptic currents, evoked in CA1 principal cells by Schaffer collateral stimulation, were detected in hippocampal slices obtained from Pin1(-/-) mice compared with controls. Similar results were obtained in cultured hippocampal cells expressing a PSD-95 mutant unable to undergo prolyl-isomerization, thus indicating that the action of Pin1 on PSD-95 is critical for this effect. In addition, an enhancement in spine density and size was detected in CA1 principal cells of Pin1(-/-) or in Thy-1GFP mice treated with the pharmacological inhibitor of Pin1 catalytic activity PiB.Our data indicate that Pin1 controls synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for LTP maintenance. PSD-95, a membrane-associated guanylate kinase, is the major scaffolding protein at excitatory postsynaptic densities and a potent regulator of synaptic strength and plasticity. The

  1. Posttranslational Modifications Regulate the Postsynaptic Localization of PSD-95.

    PubMed

    Vallejo, Daniela; Codocedo, Juan F; Inestrosa, Nibaldo C

    2017-04-01

    The postsynaptic density (PSD) consists of a lattice-like array of interacting proteins that organizes and stabilizes synaptic receptors, ion channels, structural proteins, and signaling molecules required for normal synaptic transmission and synaptic function. The scaffolding and hub protein postsynaptic density protein-95 (PSD-95) is a major element of central chemical synapses and interacts with glutamate receptors, cell adhesion molecules, and cytoskeletal elements. In fact, PSD-95 can regulate basal synaptic stability as well as the activity-dependent structural plasticity of the PSD and, therefore, of the excitatory chemical synapse. Several studies have shown that PSD-95 is highly enriched at excitatory synapses and have identified multiple protein structural domains and protein-protein interactions that mediate PSD-95 function and trafficking to the postsynaptic region. PSD-95 is also a target of several signaling pathways that induce posttranslational modifications, including palmitoylation, phosphorylation, ubiquitination, nitrosylation, and neddylation; these modifications determine the synaptic stability and function of PSD-95 and thus regulate the fates of individual dendritic spines in the nervous system. In the present work, we review the posttranslational modifications that regulate the synaptic localization of PSD-95 and describe their functional consequences. We also explore the signaling pathways that induce such changes.

  2. PSD-95 is post-transcriptionally repressed during early neural development by PTBP1 and PTBP2.

    PubMed

    Zheng, Sika; Gray, Erin E; Chawla, Geetanjali; Porse, Bo Torben; O'Dell, Thomas J; Black, Douglas L

    2012-01-15

    Postsynaptic density protein 95 (PSD-95) is essential for synaptic maturation and plasticity. Although its synaptic regulation has been widely studied, the control of PSD-95 cellular expression is not understood. We found that Psd-95 was controlled post-transcriptionally during neural development. Psd-95 was transcribed early in mouse embryonic brain, but most of its product transcripts were degraded. The polypyrimidine tract binding proteins PTBP1 and PTBP2 repressed Psd-95 (also known as Dlg4) exon 18 splicing, leading to premature translation termination and nonsense-mediated mRNA decay. The loss of first PTBP1 and then of PTBP2 during embryonic development allowed splicing of exon 18 and expression of PSD-95 late in neuronal maturation. Re-expression of PTBP1 or PTBP2 in differentiated neurons inhibited PSD-95 expression and impaired the development of glutamatergic synapses. Thus, expression of PSD-95 during early neural development is controlled at the RNA level by two PTB proteins whose sequential downregulation is necessary for synapse maturation.

  3. Anchoring and Synaptic stability of PSD-95 is driven by ephrin-B3

    PubMed Central

    Hruska, Martin; Henderson, Nathan T.; Xia, Nan L.; Le Marchand, Sylvain J.; Dalva, Matthew B.

    2015-01-01

    Summary Organization of signaling complexes at excitatory synapses by Membrane Associated Guanylate Kinase (MAGUK) proteins regulates synapse development, plasticity, senescence, and disease. Post-translational modification of MAGUK family proteins can drive their membrane localization, yet it is unclear how these intracellular proteins are targeted to sites of synaptic contact. Here we show using super-resolution imaging, biochemical approaches, and in vivo models that the trans-synaptic organizing protein, ephrin-B3, controls the synaptic localization and stability of PSD-95 and links these events to changes in neuronal activity via negative regulation of a novel MAPK-dependent phosphorylation site on ephrin-B3 (S332). Unphosphorylated ephrin-B3 is enriched at synapses, interacts directly with and stabilizes PSD-95 at synapses. Activity induced phosphorylation of S332 disperses ephrin-B3 from synapses, prevents the interaction with, and enhances the turnover of PSD-95. Thus, ephrin-B3 specifies the synaptic localization of PSD-95 and likely links the synaptic stability of PSD-95 to changes in neuronal activity. PMID:26479588

  4. Pathological reorganization of NMDA receptors subunits and postsynaptic protein PSD-95 distribution in Alzheimer's disease.

    PubMed

    Leuba, Genevieve; Vernay, Andre; Kraftsik, Rudolf; Tardif, Eric; Riederer, Beat Michel; Savioz, Armand

    2014-01-01

    In Alzheimer's disease (AD), synaptic alterations play a major role and are often correlated with cognitive changes. In order to better understand synaptic modifications, we compared alterations in NMDA receptors and postsynaptic protein PSD-95 expression in the entorhinal cortex (EC) and frontal cortex (FC; area 9) of AD and control brains. We combined immunohistochemical and image analysis methods to quantify on consecutive sections the distribution of PSD-95 and NMDA receptors GluN1, GluN2A and GluN2B in EC and FC from 25 AD and control cases. The density of stained receptors was analyzed using multivariate statistical methods to assess the effect of neurodegeneration. In both regions, the number of neuronal profiles immunostained for GluN1 receptors subunit and PSD-95 protein was significantly increased in AD compared to controls (3-6 fold), while the number of neuronal profiles stained for GluN2A and GluN2B receptors subunits was on the contrary decreased (3-4 fold). The increase in marked neuronal profiles was more prominent in a cortical band corresponding to layers 3 to 5 with large pyramidal cells. Neurons positive for GluN1 or PSD-95 staining were often found in the same localization on consecutive sections and they were also reactive for the anti-tau antibody AD2, indicating a neurodegenerative process. Differences in the density of immunoreactive puncta representing neuropile were not statistically significant. Altogether these data indicate that GluN1 and PSD-95 accumulate in the neuronal perikarya, but this is not the case for GluN2A and GluN2B, while the neuropile compartment is less subject to modifications. Thus, important variations in the pattern of distribution of the NMDA receptors subunits and PSD-95 represent a marker in AD and by impairing the neuronal network, contribute to functional deterioration.

  5. The LGI1-ADAM22 protein complex directs synapse maturation through regulation of PSD-95 function.

    PubMed

    Lovero, Kathryn L; Fukata, Yuko; Granger, Adam J; Fukata, Masaki; Nicoll, Roger A

    2015-07-28

    Synapse development is coordinated by a number of transmembrane and secreted proteins that come together to form synaptic organizing complexes. Whereas a variety of synaptogenic proteins have been characterized, much less is understood about the molecular networks that support the maintenance and functional maturation of nascent synapses. Here, we demonstrate that leucine-rich, glioma-inactivated protein 1 (LGI1), a secreted protein previously shown to modulate synaptic AMPA receptors, is a paracrine signal released from pre- and postsynaptic neurons that acts specifically through a disintegrin and metalloproteinase protein 22 (ADAM22) to set postsynaptic strength. We go on to describe a novel role for ADAM22 in maintaining excitatory synapses through PSD-95/Dlg1/zo-1 (PDZ) domain interactions. Finally, we show that in the absence of LGI1, the mature synapse scaffolding protein PSD-95, but not the immature synapse scaffolding protein SAP102, is unable to modulate synaptic transmission. These results indicate that LGI1 and ADAM22 form an essential synaptic organizing complex that coordinates the maturation of excitatory synapses by regulating the functional incorporation of PSD-95.

  6. PSD-95 promotes the stabilization of young synaptic contacts.

    PubMed

    Taft, Christine E; Turrigiano, Gina G

    2014-01-05

    Maintaining a population of stable synaptic connections is probably of critical importance for the preservation of memories and functional circuitry, but the molecular dynamics that underlie synapse stabilization is poorly understood. Here, we use simultaneous time-lapse imaging of post synaptic density-95 (PSD-95) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) to investigate the dynamics of protein composition at axodendritic (AD) contacts. Our data reveal that this composition is highly dynamic, with both proteins moving into and out of the same synapse independently, so that synapses cycle rapidly between states in which they are enriched for none, one or both proteins. We assessed how PSD-95 and CaMKII interact at stable and transient AD sites and found that both phospho-CaMKII and PSD-95 are present more often at stable than labile contacts. Finally, we found that synaptic contacts are more stable in older neurons, and this process can be mimicked in younger neurons by overexpression of PSD-95. Taken together, these data show that synaptic protein composition is highly variable over a time-scale of hours, and that PSD-95 is probably a key synaptic protein that promotes synapse stability.

  7. Impairment of TrkB-PSD-95 signaling in Angelman syndrome.

    PubMed

    Cao, Cong; Rioult-Pedotti, Mengia S; Migani, Paolo; Yu, Crystal J; Tiwari, Rakesh; Parang, Keykavous; Spaller, Mark R; Goebel, Dennis J; Marshall, John

    2013-01-01

    Angelman syndrome (AS) is a neurodevelopment disorder characterized by severe cognitive impairment and a high rate of autism. AS is caused by disrupted neuronal expression of the maternally inherited Ube3A ubiquitin protein ligase, required for the proteasomal degradation of proteins implicated in synaptic plasticity, such as the activity-regulated cytoskeletal-associated protein (Arc/Arg3.1). Mice deficient in maternal Ube3A express elevated levels of Arc in response to synaptic activity, which coincides with severely impaired long-term potentiation (LTP) in the hippocampus and deficits in learning behaviors. In this study, we sought to test whether elevated levels of Arc interfere with brain-derived neurotrophic factor (BDNF) TrkB receptor signaling, which is known to be essential for both the induction and maintenance of LTP. We report that TrkB signaling in the AS mouse is defective, and show that reduction of Arc expression to control levels rescues the signaling deficits. Moreover, the association of the postsynaptic density protein PSD-95 with TrkB is critical for intact BDNF signaling, and elevated levels of Arc were found to impede PSD-95/TrkB association. In Ube3A deficient mice, the BDNF-induced recruitment of PSD-95, as well as PLCγ and Grb2-associated binder 1 (Gab1) with TrkB receptors was attenuated, resulting in reduced activation of PLCγ-α-calcium/calmodulin-dependent protein kinase II (CaMKII) and PI3K-Akt, but leaving the extracellular signal-regulated kinase (Erk) pathway intact. A bridged cyclic peptide (CN2097), shown by nuclear magnetic resonance (NMR) studies to uniquely bind the PDZ1 domain of PSD-95 with high affinity, decreased the interaction of Arc with PSD-95 to restore BDNF-induced TrkB/PSD-95 complex formation, signaling, and facilitate the induction of LTP in AS mice. We propose that the failure of TrkB receptor signaling at synapses in AS is directly linked to elevated levels of Arc associated with PSD-95 and PSD-95 PDZ

  8. Impairment of TrkB-PSD-95 Signaling in Angelman Syndrome

    PubMed Central

    Cao, Cong; Rioult-Pedotti, Mengia S.; Migani, Paolo; Yu, Crystal J.; Tiwari, Rakesh; Parang, Keykavous; Spaller, Mark R.; Goebel, Dennis J.; Marshall, John

    2013-01-01

    Angelman syndrome (AS) is a neurodevelopment disorder characterized by severe cognitive impairment and a high rate of autism. AS is caused by disrupted neuronal expression of the maternally inherited Ube3A ubiquitin protein ligase, required for the proteasomal degradation of proteins implicated in synaptic plasticity, such as the activity-regulated cytoskeletal-associated protein (Arc/Arg3.1). Mice deficient in maternal Ube3A express elevated levels of Arc in response to synaptic activity, which coincides with severely impaired long-term potentiation (LTP) in the hippocampus and deficits in learning behaviors. In this study, we sought to test whether elevated levels of Arc interfere with brain-derived neurotrophic factor (BDNF) TrkB receptor signaling, which is known to be essential for both the induction and maintenance of LTP. We report that TrkB signaling in the AS mouse is defective, and show that reduction of Arc expression to control levels rescues the signaling deficits. Moreover, the association of the postsynaptic density protein PSD-95 with TrkB is critical for intact BDNF signaling, and elevated levels of Arc were found to impede PSD-95/TrkB association. In Ube3A deficient mice, the BDNF-induced recruitment of PSD-95, as well as PLCγ and Grb2-associated binder 1 (Gab1) with TrkB receptors was attenuated, resulting in reduced activation of PLCγ-α-calcium/calmodulin-dependent protein kinase II (CaMKII) and PI3K-Akt, but leaving the extracellular signal-regulated kinase (Erk) pathway intact. A bridged cyclic peptide (CN2097), shown by nuclear magnetic resonance (NMR) studies to uniquely bind the PDZ1 domain of PSD-95 with high affinity, decreased the interaction of Arc with PSD-95 to restore BDNF-induced TrkB/PSD-95 complex formation, signaling, and facilitate the induction of LTP in AS mice. We propose that the failure of TrkB receptor signaling at synapses in AS is directly linked to elevated levels of Arc associated with PSD-95 and PSD-95 PDZ

  9. Postsynaptic Clustering and Activation of Pyk2 by PSD-95

    PubMed Central

    Bartos, Jason A.; Ulrich, Jason D.; Li, Hongbin; Beazely, Michael A.; Chen, Yucui; MacDonald, John F.; Hell, Johannes W.

    2010-01-01

    The tyrosine kinase Pyk2 plays a unique role in intracellular signal transduction by linking Ca2+ influx to tyrosine phosphorylation, but the molecular mechanism of Pyk2 activation is unknown. We report that Pyk2 oligomerization by antibodies in vitro or overexpression of PSD-95 in PC6-3 cells induces trans-autophosphorylation of Tyr402, the first step in Pyk2 activation. In neurons, Ca2+ influx through NMDA-type glutamate receptors (NMDAR) causes postsynaptic clustering and autophosphorylation of endogenous Pyk2 via Ca2+- and calmodulin-stimulated binding to PSD-95. Accordingly, Ca2+ influx promotes oligomerization and thereby autoactivation of Pyk2 by stimulating its interaction with PSD-95. We show that this mechanism of Pyk2 activation is critical for LTP in the hippocampus CA1 region, which is thought to underlie learning and memory. PMID:20071509

  10. Postsynaptic degeneration as revealed by PSD-95 reduction occurs after advanced Aβ and tau pathology in transgenic mouse models of Alzheimer's disease.

    PubMed

    Shao, Charles Y; Mirra, Suzanne S; Sait, Hameetha B R; Sacktor, Todd C; Sigurdsson, Einar M

    2011-09-01

    Impairment of synaptic plasticity underlies memory dysfunction in Alzheimer's disease (AD). Molecules involved in this plasticity such as PSD-95, a major postsynaptic scaffold protein at excitatory synapses, may play an important role in AD pathogenesis. We examined the distribution of PSD-95 in transgenic mice of amyloidopathy (5XFAD) and tauopathy (JNPL3) as well as in AD brains using double-labeling immunofluorescence and confocal microscopy. In wild type control mice, PSD-95 primarily labeled neuropil with distinct distribution in hippocampal apical dendrites. In 3-month-old 5XFAD mice, PSD-95 distribution was similar to that of wild type mice despite significant Aβ deposition. However, in 6-month-old 5XFAD mice, PSD-95 immunoreactivity in apical dendrites markedly decreased and prominent immunoreactivity was noted in neuronal soma in CA1 neurons. Similarly, PSD-95 immunoreactivity disappeared from apical dendrites and accumulated in neuronal soma in 14-month-old, but not in 3-month-old, JNPL3 mice. In AD brains, PSD-95 accumulated in Hirano bodies in hippocampal neurons. Our findings support the notion that either Aβ or tau can induce reduction of PSD-95 in excitatory synapses in hippocampus. Furthermore, this PSD-95 reduction is not an early event but occurs as the pathologies advance. Thus, the time-dependent PSD-95 reduction from synapses and accumulation in neuronal soma in transgenic mice and Hirano bodies in AD may mark postsynaptic degeneration that underlies long-term functional deficits.

  11. Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling.

    PubMed

    Yoshii, Akira; Constantine-Paton, Martha

    2014-01-01

    Brain-derived neurotrophic factor (BDNF) and its receptor TrkB regulate synaptic plasticity. TrkB triggers three downstream signaling pathways; Phosphatidylinositol 3-kinase (PI3K), Phospholipase Cγ (PLCγ) and Mitogen activated protein kinases/Extracellular signal-regulated kinases (MAPK/ERK). We previously showed two distinct mechanisms whereby BDNF-TrkB pathway controls trafficking of PSD-95, which is the major scaffold at excitatory synapses and is critical for synapse maturation. BDNF activates the PI3K-Akt pathway and regulates synaptic delivery of PSD-95 via vesicular transport (Yoshii and Constantine-Paton, 2007). BDNF-TrkB signaling also triggers PSD-95 palmitoylation and its transport to synapses through the phosphorylation of the palmitoylation enzyme ZDHHC8 by a protein kinase C (PKC; Yoshii etal., 2011). The second study used PKC inhibitors chelerythrine as well as a synthetic zeta inhibitory peptide (ZIP) which was originally designed to block the brain-specific PKC isoform protein kinase Mϖ (PKMϖ). However, recent studies raise concerns about specificity of ZIP. Here, we assessed the contribution of TrkB and its three downstream pathways to the synaptic distribution of endogenous PSD-95 in cultured neurons using chemical and genetic interventions. We confirmed that TrkB, PLC, and PI3K were critical for the postsynaptic distribution of PSD-95. Furthermore, suppression of MAPK/ERK also disrupted PSD-95 expression. Next, we examined the contribution of PKC. While both chelerythrine and ZIP suppressed the postsynaptic localization of PSD-95, RNA interference for PKMϖ did not have a significant effect. This result suggests that the ZIP peptide, widely used as the "specific" PKMϖ antagonist by many investigators may block a PKC variant other than PKMϖ such as PKCλ/ι. Our results indicate that TrkB regulates postsynaptic localization of PSD-95 through all three downstream pathways, but also recommend further work to identify other PKC variants that

  12. PSD95 nanoclusters are postsynaptic building blocks in hippocampus circuits

    PubMed Central

    Broadhead, Matthew J.; Horrocks, Mathew H.; Zhu, Fei; Muresan, Leila; Benavides-Piccione, Ruth; DeFelipe, Javier; Fricker, David; Kopanitsa, Maksym V.; Duncan, Rory R.; Klenerman, David; Komiyama, Noboru H.; Lee, Steven F.; Grant, Seth G. N.

    2016-01-01

    The molecular features of synapses in the hippocampus underpin current models of learning and cognition. Although synapse ultra-structural diversity has been described in the canonical hippocampal circuitry, our knowledge of sub-synaptic organisation of synaptic molecules remains largely unknown. To address this, mice were engineered to express Post Synaptic Density 95 protein (PSD95) fused to either eGFP or mEos2 and imaged with two orthogonal super-resolution methods: gated stimulated emission depletion (g-STED) microscopy and photoactivated localisation microscopy (PALM). Large-scale analysis of ~100,000 synapses in 7 hippocampal sub-regions revealed they comprised discrete PSD95 nanoclusters that were spatially organised into single and multi-nanocluster PSDs. Synapses in different sub-regions, cell-types and locations along the dendritic tree of CA1 pyramidal neurons, showed diversity characterised by the number of nanoclusters per synapse. Multi-nanocluster synapses were frequently found in the CA3 and dentate gyrus sub-regions, corresponding to large thorny excrescence synapses. Although the structure of individual nanoclusters remained relatively conserved across all sub-regions, PSD95 packing into nanoclusters also varied between sub-regions determined from nanocluster fluorescence intensity. These data identify PSD95 nanoclusters as a basic structural unit, or building block, of excitatory synapses and their number characterizes synapse size and structural diversity. PMID:27109929

  13. Reduced SNAP-25 increases PSD-95 mobility and impairs spine morphogenesis

    PubMed Central

    Fossati, G; Morini, R; Corradini, I; Antonucci, F; Trepte, P; Edry, E; Sharma, V; Papale, A; Pozzi, D; Defilippi, P; Meier, J C; Brambilla, R; Turco, E; Rosenblum, K; Wanker, E E; Ziv, N E; Menna, E; Matteoli, M

    2015-01-01

    Impairment of synaptic function can lead to neuropsychiatric disorders collectively referred to as synaptopathies. The SNARE protein SNAP-25 is implicated in several brain pathologies and, indeed, brain areas of psychiatric patients often display reduced SNAP-25 expression. It has been recently found that acute downregulation of SNAP-25 in brain slices impairs long-term potentiation; however, the processes through which this occurs are still poorly defined. We show that in vivo acute downregulation of SNAP-25 in CA1 hippocampal region affects spine number. Consistently, hippocampal neurons from SNAP-25 heterozygous mice show reduced densities of dendritic spines and defective PSD-95 dynamics. Finally, we show that, in brain, SNAP-25 is part of a molecular complex including PSD-95 and p140Cap, with p140Cap being capable to bind to both SNAP-25 and PSD-95. These data demonstrate an unexpected role of SNAP-25 in controlling PSD-95 clustering and open the possibility that genetic reductions of the protein levels – as occurring in schizophrenia – may contribute to the pathology through an effect on postsynaptic function and plasticity. PMID:25678324

  14. A cyclic peptide targeted against PSD-95 blocks central sensitization and attenuates thermal hyperalgesia.

    PubMed

    LeBlanc, B W; Iwata, M; Mallon, A P; Rupasinghe, C N; Goebel, D J; Marshall, J; Spaller, M R; Saab, C Y

    2010-05-05

    Post-synaptic density protein PSD-95 is emerging as a valid target for modulating nociception in animal studies. Based on the key role of PSD-95 in neuronal plasticity and the maintenance of pain behavior, we predicted that CN2097, a peptide-based macrocycle of nine residues that binds to the PSD-95 Discs large, Zona occludens 1 (PDZ) domains of PSD-95, would interfere with physiologic phenomena in the spinal cord related to central sensitization. Furthermore, we tested whether spinal intrathecal injection of CN2097 attenuates thermal hyperalgesia in a rat model of sciatic neuropathy. Results demonstrate that spinal CN2097 reverses hyperexcitability of wide dynamic range (WDR) neurons in the dorsal horn of neuropathic rats and decreases their evoked responses to peripheral stimuli (brush, low caliber von Frey and pressure), whereas CN5125 ("negative control") has no effect. CN2097 also blocks C-fiber long-term potentiation (LTP) in the dorsal horn, which is linked to neuronal plasticity and central sensitization. At a molecular level, CN2097 attenuates the increase in phosphorylated p38 MAPK, a key intracellular signaling pathway in neuropathic pain. Moreover, spinal injection of CN2097 blocks thermal hyperalgesia in neuropathic rats. We conclude that CN2097 is a small molecule peptide with putative anti-nociceptive effects that modulates physiologic phenomena related to central sensitization under conditions of chronic pain.

  15. Iodine deficiency increases apoptosis and decreases synaptotagmin-1 and PSD-95 in rat hippocampus.

    PubMed

    Dong, Jing; Wang, Yuan; Wang, Yi; Wei, Wei; Min, Hui; Song, Binbin; Xi, Qi; Teng, Weiping; Chen, Jie

    2013-05-01

    Developmental iodine deficiency (ID) leads to inadequate thyroid hormone that impairs the development of the central nervous system with an unclear mechanism. Here, we show that hippocampal apoptosis, synaptotagmin-1, and PSD-95 are involved in the synaptic impairment following developmental ID. Two developmental rat models were created by administrating dam rats with either iodine-deficient diet or propylthiouracil (PTU, 15 ppm)-added drinking water from gestational day 6 till postnatal day (PND) 28. Then, the apoptosis in the hippocampus was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and the levels of synaptotagmin-1 and PSD-95 were detected with western blot on PND14, PND21, and PND28. The results showed that apoptosis cells and activity of caspase3 were increased in the iodine-deficient and PTU-treatment rats (P < 0.05, respectively). The iodine-deficient and PTU-treatment pups showed significantly lower level of synaptotagmin-1 and PSD-95 in hippocampus than that of controls (P < 0.05, respectively). Developmental ID resulted in the increase and delay of cell apoptosis and the decrease of synaptotagmin-1 and PSD-95 in the hippocampus, which were implicated in the impairment of brain development.

  16. Site-Specific Phosphorylation of PSD-95 PDZ Domains Reveals Fine-Tuned Regulation of Protein-Protein Interactions.

    PubMed

    Pedersen, Søren W; Albertsen, Louise; Moran, Griffin E; Levesque, Brié; Pedersen, Stine B; Bartels, Lina; Wapenaar, Hannah; Ye, Fei; Zhang, Mingjie; Bowen, Mark E; Strømgaard, Kristian

    2017-09-15

    The postsynaptic density protein of 95 kDa (PSD-95) is a key scaffolding protein that controls signaling at synapses in the brain through interactions of its PDZ domains with the C-termini of receptors, ion channels, and enzymes. PSD-95 is highly regulated by phosphorylation. To explore the effect of phosphorylation on PSD-95, we used semisynthetic strategies to introduce phosphorylated amino acids at four positions within the PDZ domains and examined the effects on interactions with a large set of binding partners. We observed complex effects on affinity. Most notably, phosphorylation at Y397 induced a significant increase in affinity for stargazin, as confirmed by NMR and single molecule FRET. Additionally, we compared the effects of phosphorylation to phosphomimetic mutations, which revealed that phosphomimetics are ineffective substitutes for tyrosine phosphorylation. Our strategy to generate site-specifically phosphorylated PDZ domains provides a detailed understanding of the role of phosphorylation in the regulation of PSD-95 interactions.

  17. A framework to understand the variations of PSD-95 expression in brain aging and in Alzheimer's disease.

    PubMed

    Savioz, Armand; Leuba, Geneviève; Vallet, Philippe G

    2014-11-01

    The postsynaptic density protein PSD-95 is a major element of synapses. PSD-95 is involved in aging, Alzheimer's disease (AD) and numerous psychiatric disorders. However, contradictory data about PSD-95 expression in aging and AD have been reported. Indeed in AD versus control brains PSD-95 varies according to regions, increasing in the frontal cortex, at least in a primary stage, and decreasing in the temporal cortex. In contrast, in transgenic mouse models of aging and AD PSD-95 expression is decreased, in behaviorally aged impaired versus unimpaired rodents it can decrease or increase and finally, it is increased in rodents grown in enriched environments. Different factors explain these contradictory results in both animals and humans, among others concomitant psychiatric endophenotypes, such as depression. The possible involvement of PSD-95 in reactive and/or compensatory mechanisms during AD progression is underscored, at least before the occurrence of important synaptic elimination. Thus, in AD but not in AD transgenic mice, enhanced expression might precede the diminution commonly observed in advanced aging. A two-compartments cell model, separating events taking place in cell bodies and synapses, is presented. Overall these data suggest that AD research will progress by untangling pathological from protective events, a prerequisite for effective therapeutic strategies. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Single-Molecule Imaging of PSD-95 mRNA Translation in Dendrites and Its Dysregulation in a Mouse Model of Fragile X Syndrome

    PubMed Central

    Ifrim, Marius F.; Williams, Kathryn R.

    2015-01-01

    Fragile X syndrome (FXS) is caused by the loss of the fragile X mental retardation protein (FMRP), an RNA binding protein that regulates translation of numerous target mRNAs, some of which are dendritically localized. Our previous biochemical studies using synaptoneurosomes demonstrate a role for FMRP and miR-125a in regulating the translation of PSD-95 mRNA. However, the local translation of PSD-95 mRNA within dendrites and spines, as well as the roles of FMRP or miR-125a, have not been directly studied. Herein, local synthesis of a Venus-PSD-95 fusion protein was directly visualized in dendrites and spines using single-molecule imaging of a diffusion-restricted Venus-PSD-95 reporter under control of the PSD-95 3′UTR. The basal translation rates of Venus-PSD-95 mRNA was increased in cultured hippocampal neurons from Fmr1 KO mice compared with WT neurons, which correlated with a transient elevation of endogenous PSD-95 within dendrites. Following mGluR stimulation with (S)-3,5-dihydroxyphenylglycine, the rate of Venus-PSD-95 mRNA translation increased rapidly in dendrites of WT hippocampal neurons, but not in those of Fmr1 KO neurons or when the binding site of miR125a, previously shown to bind PSD-95 3′UTR, was mutated. This study provides direct support for the hypothesis that local translation within dendrites and spines is dysregulated in FXS. Impairments in the regulated local synthesis of PSD-95, a critical regulator of synaptic structure and function, may affect the spatiotemporal control of PSD-95 levels and affect dendritic spine development and synaptic plasticity in FXS. PMID:25948262

  19. Single-Molecule Imaging of PSD-95 mRNA Translation in Dendrites and Its Dysregulation in a Mouse Model of Fragile X Syndrome.

    PubMed

    Ifrim, Marius F; Williams, Kathryn R; Bassell, Gary J

    2015-05-06

    Fragile X syndrome (FXS) is caused by the loss of the fragile X mental retardation protein (FMRP), an RNA binding protein that regulates translation of numerous target mRNAs, some of which are dendritically localized. Our previous biochemical studies using synaptoneurosomes demonstrate a role for FMRP and miR-125a in regulating the translation of PSD-95 mRNA. However, the local translation of PSD-95 mRNA within dendrites and spines, as well as the roles of FMRP or miR-125a, have not been directly studied. Herein, local synthesis of a Venus-PSD-95 fusion protein was directly visualized in dendrites and spines using single-molecule imaging of a diffusion-restricted Venus-PSD-95 reporter under control of the PSD-95 3'UTR. The basal translation rates of Venus-PSD-95 mRNA was increased in cultured hippocampal neurons from Fmr1 KO mice compared with WT neurons, which correlated with a transient elevation of endogenous PSD-95 within dendrites. Following mGluR stimulation with (S)-3,5-dihydroxyphenylglycine, the rate of Venus-PSD-95 mRNA translation increased rapidly in dendrites of WT hippocampal neurons, but not in those of Fmr1 KO neurons or when the binding site of miR125a, previously shown to bind PSD-95 3'UTR, was mutated. This study provides direct support for the hypothesis that local translation within dendrites and spines is dysregulated in FXS. Impairments in the regulated local synthesis of PSD-95, a critical regulator of synaptic structure and function, may affect the spatiotemporal control of PSD-95 levels and affect dendritic spine development and synaptic plasticity in FXS.

  20. Rapid Redistribution of Synaptic PSD-95 in the Neocortex In Vivo

    PubMed Central

    Bureau, Ingrid; Svoboda, Karel

    2006-01-01

    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 τ r ~ 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 (τ r ~ 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. PMID:17090216

  1. The role of PSD-95 in the rearrangement of Kv1.3 channels to the immunological synapse.

    PubMed

    Szilágyi, Orsolya; Boratkó, Anita; Panyi, György; Hajdu, Péter

    2013-09-01

    Establishment of the immunological synapse (IS) between T lymphocytes and antigen-presenting cells is a key step in the adaptive immune response. Several proteins accumulate in the IS, such as the Kv1.3 potassium channel; however, the mechanism of this translocation is unknown. PSD-95 and SAP97 are adaptor proteins that regulate the polarized cell surface expression and localization of Kv1 channels in neurons. We investigated whether these proteins affect the redistribution of Kv1.3 into the IS in non-excitable human T cells. We show here that PSD-95 and SAP97 are expressed in Jurkat and interact with the C terminus of Kv1.3. Disruption of the interaction between PSD-95 or SAP97 and Kv1.3 in Jurkat was realized by the expression of a C-terminal truncated Kv1.3, which lacks the binding domain for these proteins, or by the knockdown of the expression of PSD-95 or SAP97 using specific shRNA. Expression of the truncated Kv1.3 or knockdown of PSD-95, but not the knockdown of SAP97, inhibited the recruitment of Kv1.3 into the IS; the fraction of cells showing polarized Kv1.3 expression upon engagement in an IS was significantly lower than in control cells expressing the full-length Kv1.3, and the rearrangement of Kv1.3 did not show time dependence. In contrast, Jurkat cells expressing the full-length channel showed marked time dependence in the recruitment into the IS peaking at 1 min after the conjugation of the cells. These results demonstrate that PSD-95 participates in the targeting of Kv1.3 into the IS, implying its important role in human T-cell activation.

  2. Effect of complex aerobic physical exercise on PSD-95 in the hippocampus and on cognitive function in juvenile mice

    NASA Astrophysics Data System (ADS)

    Satriani, W. H.; Redjeki, S.; Kartinah, N. T.

    2017-08-01

    Increased neuroplasticity induced by complex aerobic physical exercise is associated with improved cognitive function in adult mice. Increased cognitive function is assumed to be based on increased synapse formation. One of the regions of the brain that is important in cognitive function is the hippocampus, which plays a role in memory formation. Post synaptic density-95 (PSD-95) is an adhesion protein of the post-synaptic density scaffolding that is essential to synaptic stabilization. As we age, the PSD-95 molecule matures the synapses needed for the formation of the basic circuitry of the nervous system in the brain. However, during the growth period, synapse elimination is higher than its formation. This study aims to determine whether complex aerobic exercise can improve cognitive function and PSD-95 levels in the hippocampus of juvenile mice during their growth stage. The mice performed complex aerobic exercise starting at five weeks of age and continuing for seven weeks with a gradual increase of 8 m/min. At eight weeks it was increased to 10 m/min. The exercise was done for five days of each week. The subjects of the study were tested for cognition one week before being sacrificed (at 12 weeks). The PSD-95 in the hippocampus was measured with ELISA. The results showed that there was a significant difference in cognitive function, where p < 0.05, between the group that was given complex aerobic exercise and a control group that did not. However, the PSD-95 levels did not differ significantly between the two groups. The results of this study indicate that early complex aerobic exercise can improve cognitive ability in adulthood but does not increase the levels of PSD-95 in adults.

  3. Synaptic Clustering of PSD-95 Is Regulated by c-Abl through Tyrosine Phosphorylation

    PubMed Central

    de Arce, Karen Perez; Varela-Nallar, Lorena; Farias, Olivia; Cifuentes, Alejandra; Bull, Paulina; Couch, Brian A.; Koleske, Anthony J.; Inestrosa, Nibaldo C.; Alvarez, Alejandra R.

    2010-01-01

    The c-Abl tyrosine kinase is present in mouse brain synapses, but its precise synaptic function is unknown. We found that c-Abl levels in the rat hippocampus increase postnatally, with expression peaking at the first postnatal week. In 14 d in vitro hippocampal neuron cultures, c-Abl localizes primarily to the postsynaptic compartment, in which it colocalizes with the postsynaptic scaffold protein postsynaptic density protein-95 (PSD-95) in apposition to presynaptic markers. c-Abl associates with PSD-95, and chemical or genetic inhibition of c-Abl kinase activity reduces PSD-95 tyrosine phosphorylation, leading to reduced PSD-95 clustering and reduced synapses in treated neurons. c-Abl can phosphorylate PSD-95 on tyrosine 533, and mutation of this residue reduces the ability of PSD-95 to cluster at postsynaptic sites. Our results indicate that c-Abl regulates synapse formation by mediating tyrosine phosphorylation and clustering of PSD-95. PMID:20220006

  4. Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95.

    PubMed

    Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J; Alkon, Daniel L

    2016-08-05

    Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95(S295)) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Protein Kinase Cϵ (PKCϵ) Promotes Synaptogenesis through Membrane Accumulation of the Postsynaptic Density Protein PSD-95*

    PubMed Central

    Sen, Abhik; Hongpaisan, Jarin; Wang, Desheng; Nelson, Thomas J.; Alkon, Daniel L.

    2016-01-01

    Protein kinase Cϵ (PKCϵ) promotes synaptic maturation and synaptogenesis via activation of synaptic growth factors such as BDNF, NGF, and IGF. However, many of the detailed mechanisms by which PKCϵ induces synaptogenesis are not fully understood. Accumulation of PSD-95 to the postsynaptic density (PSD) is known to lead to synaptic maturation and strengthening of excitatory synapses. Here we investigated the relationship between PKCϵ and PSD-95. We show that the PKCϵ activators dicyclopropanated linoleic acid methyl ester and bryostatin 1 induce phosphorylation of PSD-95 at the serine 295 residue, increase the levels of PSD-95, and enhance its membrane localization. Elimination of the serine 295 residue in PSD-95 abolished PKCϵ-induced membrane accumulation. Knockdown of either PKCϵ or JNK1 prevented PKCϵ activator-mediated membrane accumulation of PSD-95. PKCϵ directly phosphorylated PSD-95 and JNK1 in vitro. Inhibiting PKCϵ, JNK, or calcium/calmodulin-dependent kinase II activity prevented the effects of PKCϵ activators on PSD-95 phosphorylation. Increase in membrane accumulation of PKCϵ and phosphorylated PSD-95 (p-PSD-95S295) coincided with an increased number of synapses and increased amplitudes of excitatory post-synaptic potentials (EPSPs) in adult rat hippocampal slices. Knockdown of PKCϵ also reduced the synthesis of PSD-95 and the presynaptic protein synaptophysin by 30 and 44%, respectively. Prolonged activation of PKCϵ increased synapse number by 2-fold, increased presynaptic vesicle density, and greatly increased PSD-95 clustering. These results indicate that PKCϵ promotes synaptogenesis by activating PSD-95 phosphorylation directly through JNK1 and calcium/calmodulin-dependent kinase II and also by inducing expression of PSD-95 and synaptophysin. PMID:27330081

  6. Visual-spatial learning impairments are associated with hippocampal PSD-95 protein dysregulation in a mouse model of fragile X syndrome.

    PubMed

    Gandhi, Réno M; Kogan, Cary S; Messier, Claude; Macleod, Lindsey S

    2014-03-05

    Fragile X syndrome is the most common cause of inherited intellectual disability and is caused by the lack of fragile X mental retardation protein (FMRP) expression. In-vitro findings in mice and post-mortem autopsies in humans are characterized by dendritic spine abnormalities in the absence of Fmrp/FMRP. Biochemical and electrophysiological studies have identified postsynaptic density protein (PSD)-95 as having an established role in dendritic morphology as well as a molecular target of Fmrp. How Fmrp affects the expression of PSD-95 following behavioral learning is unknown. In the current study, wild type controls and Fmr1 knockout mice were trained in a subset of the Hebb-Williams (H-W) mazes. Dorsal hippocampal PSD-95 protein levels relative to a stable cytoskeleton protein (β-tubulin) were measured. We report a significant upregulation of PSD-95 protein levels in wild type mice, whereas training-related protein increases were blunted in Fmr1 knockout mice. In addition, there was a significant negative correlation between mean total errors on the mazes and PSD-95 protein levels. The coefficient of determination indicated that the mean total errors on the H-W mazes accounted for 35% of the variance in PSD-95 protein levels. These novel findings suggest that reduced PSD-95-associated postsynaptic plasticity may contribute to the learning and memory deficits observed in human fragile X syndrome patients.

  7. Loss of PSD-95 Enrichment is not a Prerequisite for Spine Retraction

    PubMed Central

    Woods, Georgia F.; Oh, Won Chan; Boudewyn, Lauren C.; Mikula, Sarah K.; Zito, Karen

    2011-01-01

    Changes in neuronal structure are thought to underlie long-term behavioral modifications associated with learning and memory. In particular, considerable evidence implicates the destabilization and retraction of dendritic spines along with the loss of spine synapses as an important cellular mechanism for refining brain circuits, yet the molecular mechanisms regulating spine elimination remain ill-defined. The postsynaptic density protein, PSD-95, is highly enriched in dendritic spines and has been associated with spine stability. Because spines with low levels of PSD-95 are more dynamic, and the recruitment of PSD-95 to nascent spines has been associated with spine stabilization, we hypothesized that loss of PSD-95 enrichment would be a prerequisite for spine retraction. To test this hypothesis, we used dual-color time-lapse two-photon microscopy to monitor rat hippocampal pyramidal neurons co-transfected with PSD-95-GFP and DsRed-Express, and we analyzed the relationship between PSD-95-GFP enrichment and spine morphological changes. Consistent with our hypothesis, we found that the majority of spines that retracted were relatively unenriched for PSD-95-GFP. However, in the subset of PSD-95-GFP-enriched spines that retracted, spine shrinkage and loss of PSD-95-GFP were tightly coupled, suggesting that loss of PSD-95-GFP enrichment did not precede spine retraction. Moreover, we found that in some instances spine retraction resulted in a significant enrichment of PSD-95-GFP on the dendritic shaft. Our data support a model of spine retraction in which loss of PSD-95 enrichment is not required prior to the destabilization of spines. PMID:21865455

  8. Loss of PSD-95 enrichment is not a prerequisite for spine retraction.

    PubMed

    Woods, Georgia F; Oh, Won Chan; Boudewyn, Lauren C; Mikula, Sarah K; Zito, Karen

    2011-08-24

    Changes in neuronal structure are thought to underlie long-term behavioral modifications associated with learning and memory. In particular, considerable evidence implicates the destabilization and retraction of dendritic spines along with the loss of spine synapses as an important cellular mechanism for refining brain circuits, yet the molecular mechanisms regulating spine elimination remain ill-defined. The postsynaptic density protein, PSD-95, is highly enriched in dendritic spines and has been associated with spine stability. Because spines with low levels of PSD-95 are more dynamic, and the recruitment of PSD-95 to nascent spines has been associated with spine stabilization, we hypothesized that loss of PSD-95 enrichment would be a prerequisite for spine retraction. To test this hypothesis, we used dual-color time-lapse two-photon microscopy to monitor rat hippocampal pyramidal neurons cotransfected with PSD-95-GFP and DsRed-Express, and we analyzed the relationship between PSD-95-GFP enrichment and spine morphological changes. Consistent with our hypothesis, we found that the majority of spines that retracted were relatively unenriched for PSD-95-GFP. However, in the subset of PSD-95-GFP-enriched spines that retracted, spine shrinkage and loss of PSD-95-GFP were tightly coupled, suggesting that loss of PSD-95-GFP enrichment did not precede spine retraction. Moreover, we found that, in some instances, spine retraction resulted in a significant enrichment of PSD-95-GFP on the dendritic shaft. Our data support a model of spine retraction in which loss of PSD-95 enrichment is not required prior to the destabilization of spines.

  9. PSD-95 stabilizes NMDA receptors by inducing the degradation of STEP61

    PubMed Central

    Won, Sehoon; Incontro, Salvatore; Nicoll, Roger A.; Roche, Katherine W.

    2016-01-01

    Phosphorylation regulates surface and synaptic expression of NMDA receptors (NMDARs). Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) are known to target the NMDA receptor subunit GluN2B on tyrosine 1472, which is a critical residue that mediates NMDAR endocytosis. STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs. However, nothing is known about a potential functional interaction between STEP and PSD-95. We now report that STEP61 binds to PSD-95 but not to other PSD-95 family members. We find that PSD-95 expression destabilizes STEP61 via ubiquitination and degradation by the proteasome. Using subcellular fractionation, we detect low amounts of STEP61 in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95–KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. Importantly, only extrasynaptic NMDAR expression and currents were increased upon STEP knockdown, as is consistent with low STEP61 localization in the PSD. Our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61. PMID:27457929

  10. PSD-95 stabilizes NMDA receptors by inducing the degradation of STEP61.

    PubMed

    Won, Sehoon; Incontro, Salvatore; Nicoll, Roger A; Roche, Katherine W

    2016-08-09

    Phosphorylation regulates surface and synaptic expression of NMDA receptors (NMDARs). Both the tyrosine kinase Fyn and the tyrosine phosphatase striatal-enriched protein tyrosine phosphatase (STEP) are known to target the NMDA receptor subunit GluN2B on tyrosine 1472, which is a critical residue that mediates NMDAR endocytosis. STEP reduces the surface expression of NMDARs by promoting dephosphorylation of GluN2B Y1472, whereas the synaptic scaffolding protein postsynaptic density protein 95 (PSD-95) stabilizes the surface expression of NMDARs. However, nothing is known about a potential functional interaction between STEP and PSD-95. We now report that STEP61 binds to PSD-95 but not to other PSD-95 family members. We find that PSD-95 expression destabilizes STEP61 via ubiquitination and degradation by the proteasome. Using subcellular fractionation, we detect low amounts of STEP61 in the PSD fraction. However, STEP61 expression in the PSD is increased upon knockdown of PSD-95 or in vivo as detected in PSD-95-KO mice, demonstrating that PSD-95 excludes STEP61 from the PSD. Importantly, only extrasynaptic NMDAR expression and currents were increased upon STEP knockdown, as is consistent with low STEP61 localization in the PSD. Our findings support a dual role for PSD-95 in stabilizing synaptic NMDARs by binding directly to GluN2B but also by promoting synaptic exclusion and degradation of the negative regulator STEP61.

  11. TrkB and PKMζ regulate synaptic localization of PSD-95 in developing cortex

    PubMed Central

    Yoshii, Akira; Murata, Yasunobu; Kim, Jihye; Zhang, Chao; Shokat, Kevan M.; Constantine-Paton, Martha

    2011-01-01

    Post-synaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We previously showed that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely signaling through PLCγ and the brain-specific PKC variant PKMζ. We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by over-expression ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as ageing brains. PMID:21849550

  12. TrkB and protein kinase Mζ regulate synaptic localization of PSD-95 in developing cortex.

    PubMed

    Yoshii, Akira; Murata, Yasunobu; Kim, Jihye; Zhang, Chao; Shokat, Kevan M; Constantine-Paton, Martha

    2011-08-17

    Postsynaptic density 95 (PSD-95), the major scaffold at excitatory synapses, is critical for synapse maturation and learning. In rodents, eye opening, the onset of pattern vision, triggers a rapid movement of PSD-95 from visual neuron somata to synapses. We showed previously that the PI3 kinase-Akt pathway downstream of BDNF/TrkB signaling stimulates synaptic delivery of PSD-95 via vesicular transport. However, vesicular transport requires PSD-95 palmitoylation to attach it to a lipid membrane. Also, PSD-95 insertion at synapses is known to require this lipid modification. Here, we show that BDNF/TrkB signaling is also necessary for PSD-95 palmitoylation and its transport to synapses in mouse visual cortical layer 2/3 neurons. However, palmitoylation of PSD-95 requires the activation of another pathway downstream of BDNF/TrkB, namely, signaling through phospholipase Cγ and the brain-specific PKC variant protein kinase M ζ (PKMζ). We find that PKMζ selectively regulates phosphorylation of the palmitoylation enzyme ZDHHC8. Inhibition of PKMζ results in a reduction of synaptic PSD-95 accumulation in vivo, which can be rescued by overexpressing ZDHHC8. Therefore, TrkB and PKMζ, two critical regulators of synaptic plasticity, facilitate PSD-95 targeting to synapses. These results also indicate that palmitoylation can be regulated by a trophic factor. Our findings have implications for neurodevelopmental disorders as well as aging brains.

  13. PSD-95 mediates membrane clustering of the human plasma membrane Ca2+ pump isoform 4b.

    PubMed

    Padányi, Rita; Pászty, Katalin; Strehler, Emanuel E; Enyedi, Agnes

    2009-06-01

    Besides the control of global calcium changes, specific plasma membrane calcium ATPase (PMCA) isoforms are involved in the regulation of local calcium signals. Although local calcium signaling requires the confinement of signaling molecules into microdomains, little is known about the specific organization of PMCA molecules within the plasma membrane. Here we show that co-expression with the postsynaptic density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its PDZ-binding sequence. Using the fluorescence recovery after photobleaching (FRAP) technique, we show that the lateral membrane mobility of the clustered PMCA4b is significantly lower than that of the non-clustered molecules. Disruption of the actin-based cytoskeleton by cytochalasin D resulted in increased cluster size. Our results suggest that PSD-95 promotes the formation of high-density PMCA4b microdomains in the plasma membrane and that the membrane cytoskeleton plays an important role in the regulation of this process.

  14. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility

    PubMed Central

    Stefanovic, Snezana; Bassell, Gary J.

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3′-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3′ UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson–Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. PMID:25406362

  15. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

    PubMed

    Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3' UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. © 2014 Stefanovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  16. PSD-95 and PSD-93 Play Critical but Distinct Roles in Synaptic Scaling Up and Down

    PubMed Central

    Sun, Qian; Turrigiano, Gina G.

    2011-01-01

    Synaptic scaling stabilizes neuronal firing through the homeostatic regulation of postsynaptic strength, but the mechanisms by which chronic changes in activity lead to bidirectional adjustments in synaptic AMPAR abundance are incompletely understood. Further, it remains unclear to what extent scaling up and scaling down utilize distinct molecular machinery. PSD-95 is a scaffold protein proposed to serve as a binding “slot” that determines synaptic AMPAR content, and synaptic PSD-95 abundance is regulated by activity, raising the possibility that activity-dependent changes in the synaptic abundance of PSD-95 or other MAGUKs drives the bidirectional changes in AMPAR accumulation during synaptic scaling. We found that synaptic PSD-95 and SAP102 (but not PSD-93) abundance were bidirectionally regulated by activity, but these changes were not sufficient to drive homeostatic changes in synaptic strength. Although not sufficient, the PSD-95-MAGUKs were necessary for synaptic scaling, but scaling up and down were differentially dependent on PSD-95 and PSD-93. Scaling down was completely blocked by reduced or enhanced PSD-95, through a mechanism that depended on the PDZ1/2 domains. In contrast scaling up could be supported by either PSD-95 or PSD-93 in a manner that depended on neuronal age, and was unaffected by a superabundance of PSD-95. Taken together, our data suggest that scaling up and down of quantal amplitude is not driven by changes in synaptic abundance of PSD-95-MAGUKs, but rather that the PSD-95 MAGUKs serve as critical synaptic organizers that utilize distinct protein-protein interactions to mediate homeostatic accumulation and loss of synaptic AMPAR. PMID:21543610

  17. PSD-95 is a negative regulator of the tyrosine kinase Src in the NMDA receptor complex

    PubMed Central

    Kalia, Lorraine V; Pitcher, Graham M; Pelkey, Kenneth A; Salter, Michael W

    2006-01-01

    The tyrosine kinase Src upregulates the activity of the N-methyl-D-aspartate subtype of glutamate receptor (NMDAR) and tyrosine phosphorylation of this receptor is critical for induction of NMDAR-dependent plasticity of synaptic transmission. A binding partner for Src within the NMDAR complex is the protein PSD-95. Here we demonstrate an interaction of PSD-95 with Src that does not require the well-characterized domains of PSD-95. Rather, we show binding to Src through a 12-amino-acid sequence in the N-terminal region of PSD-95, a region not previously known to participate in protein–protein interactions. This region interacts directly with the Src SH2 domain. Contrary to typical SH2 domain binding, the PSD-95–Src SH2 domain interaction is phosphotyrosine-independent. Binding of the Src-interacting region of PSD-95 inhibits Src kinase activity and reduces NMDAR phosphorylation. Intracellularly administering a peptide matching the Src SH2 domain-interacting region of PSD-95 depresses NMDAR currents in cultured neurons and inhibits induction of long-term potentiation in hippocampus. Thus, the PSD-95–Src SH2 domain interaction suppresses Src-mediated NMDAR upregulation, a finding that may be of broad importance for synaptic transmission and plasticity. PMID:16990796

  18. Ezh1 and Ezh2 differentially regulate PSD-95 gene transcription in developing hippocampal neurons.

    PubMed

    Henriquez, Berta; Bustos, Fernando J; Aguilar, Rodrigo; Becerra, Alvaro; Simon, Felipe; Montecino, Martin; van Zundert, Brigitte

    2013-11-01

    Polycomb Repressive Complex 2 (PRC2) mediates transcriptional silencing by catalyzing histone H3 lysine 27 trimethylation (H3K27me3), but its role in the maturation of postmitotic mammalian neurons remains largely unknown. We report that the PRC2 paralogs Ezh1 and Ezh2 are differentially expressed during hippocampal development. We show that depletion of Ezh2 leads to increased expression of PSD-95, a critical plasticity gene, and that reduced PSD-95 gene transcription is correlated with enrichment of Ezh2 at the PSD-95 gene promoter; however, the H3K27me3 epigenetic mark is not present at the PSD-95 gene promoter, likely due to the antagonizing effects of the H3S28P and H3K27Ac marks and the activity of the H3K27 demethylases JMJD3 and UTX. In contrast, increased PSD-95 gene transcription is accompanied by the presence of Ezh1 and elongation-engaged RNA Polymerase II complexes at the PSD-95 gene promoter, while knock-down of Ezh1 reduces PSD-95 transcription. These results indicate that Ezh1 and Ezh2 have antagonistic roles in regulating PSD-95 transcription. © 2013.

  19. PSD-95 regulates CRFR1 localization, trafficking and β-arrestin2 recruitment.

    PubMed

    Dunn, Henry A; Chahal, Harpreet S; Caetano, Fabiana A; Holmes, Kevin D; Yuan, George Y; Parikh, Ruchi; Heit, Bryan; Ferguson, Stephen S G

    2016-05-01

    Corticotropin-releasing factor (CRF) is a neuropeptide commonly associated with the hypothalamic-pituitary adrenal axis stress response. Upon release, CRF activates two G protein-coupled receptors (GPCRs): CRF receptor 1 (CRFR1) and CRF receptor 2 (CRFR2). Although both receptors contribute to mood regulation, CRFR1 antagonists have demonstrated anxiolytic and antidepressant-like properties that may be exploited in the generation of new pharmacological interventions for mental illnesses. Previous studies have demonstrated CRFR1 capable of heterologously sensitizing serotonin 2A receptor (5-HT2AR) signaling: another GPCR implicated in psychiatric disease. Interestingly, this phenomenon was dependent on Postsynaptic density 95 (PSD-95)/Disc Large/Zona Occludens (PDZ) interactions on the distal carboxyl termini of both receptors. In the current study, we demonstrate that endogenous PSD-95 can be co-immunoprecipitated with CRFR1 from cortical brain homogenate, and this interaction appears to be primarily via the PDZ-binding motif. Additionally, PSD-95 colocalizes with CRFR1 within the dendritic projections of cultured mouse neurons in a PDZ-binding motif-dependent manner. In HEK 293 cells, PSD-95 overexpression inhibited CRFR1 endocytosis, whereas PSD-95 shRNA knockdown enhanced CRFR1 endocytosis. Although PSD-95 does not appear to play a significant role in CRF-mediated cAMP or ERK1/2 signaling, PSD-95 was demonstrated to suppress β-arrestin2 recruitment: providing a potential mechanism for PSD-95's inhibition of endocytosis. In revisiting previously documented heterologous sensitization, PSD-95 shRNA knockdown did not prevent CRFR1-mediated enhancement of 5-HT2AR signaling. In conclusion, we have identified and characterized a novel functional relationship between CRFR1 and PSD-95 that may have implications in the design of new treatment strategies for mental illness. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. S-Nitrosylation and S-Palmitoylation Reciprocally Regulate Synaptic Targeting of PSD-95

    PubMed Central

    Ho, Gary P. H.; Selvakumar, Balakrishnan; Mukai, Jun; Hester, Lynda D.; Wang, Yuxuan; Gogos, Joseph A.; Snyder, Solomon H.

    2011-01-01

    PSD-95, a principal scaffolding component of the post-synaptic density, is targeted to synapses by palmitoylation where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction. PMID:21745643

  1. S-nitrosylation and S-palmitoylation reciprocally regulate synaptic targeting of PSD-95.

    PubMed

    Ho, Gary P H; Selvakumar, Balakrishnan; Mukai, Jun; Hester, Lynda D; Wang, Yuxuan; Gogos, Joseph A; Snyder, Solomon H

    2011-07-14

    PSD-95, a principal scaffolding component of the postsynaptic density, is targeted to synapses by palmitoylation, where it couples NMDA receptor stimulation to production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS). Here, we show that PSD-95 is physiologically S-nitrosylated. We identify cysteines 3 and 5, which are palmitoylated, as sites of nitrosylation, suggesting a competition between these two modifications. In support of this hypothesis, physiologically produced NO inhibits PSD-95 palmitoylation in granule cells of the cerebellum, decreasing the number of PSD-95 clusters at synaptic sites. Further, decreased palmitoylation, as seen in heterologous cells treated with 2-bromopalmitate or in ZDHHC8 knockout mice deficient in a PSD-95 palmitoyltransferase, results in increased PSD-95 nitrosylation. These data support a model in which NMDA-mediated production of NO regulates targeting of PSD-95 to synapses via mutually competitive cysteine modifications. Thus, differential modification of cysteines may represent a general paradigm in signal transduction. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. PKMζ is necessary and sufficient for synaptic clustering of PSD-95.

    PubMed

    Shao, Charles Y; Sondhi, Rachna; van de Nes, Paula S; Sacktor, Todd Charlton

    2012-07-01

    The persistent activity of protein kinase Mzeta (PKMζ), a brain-specific, constitutively active protein kinase C isoform, maintains synaptic long-term potentiation (LTP). Structural remodeling of the postsynaptic density is believed to contribute to the expression of LTP. We therefore examined the role of PKMζ in reconfiguring PSD-95, the major postsynaptic scaffolding protein at excitatory synapses. In primary cultures of hippocampal neurons, PKMζ activity was critical for increasing the size of PSD-95 clusters during chemical LTP (cLTP). Increasing PKMζ activity by overexpressing the kinase in hippocampal neurons was sufficient to increase PSD-95 cluster size, spine size, and postsynaptic AMPAR subunit GluA2. Overexpression of an inactive mutant of PKMζ did not increase PSD-95 clustering, and applications of the ζ-pseudosubstrate inhibitor ZIP reversed the PKMζ-mediated increases in PSD-95 clustering, indicating that the activity of PKMζ is necessary to induce and maintain the increased size of PSD-95 clusters. Thus the persistent activity of PKMζ is both necessary and sufficient for maintaining increases of PSD-95 clusters, providing a unified mechanism for long-term functional and structural modifications of synapses. Copyright © 2011 Wiley Periodicals, Inc.

  3. Spatiotemporal expression of PSD-95 in Fmr1 knockout mice brain.

    PubMed

    Zhu, Zhi-Wei; Xu, Qin; Zhao, Zheng-Yan; Gu, Wei-Zhong; Wu, Ding-Wen

    2011-06-01

    To investigate and compare the spatial and temporal expression of post-synaptic density-95 (PSD-95) in Fmr1 knockout mice (the animal model of fragile X syndrome, FXS) and wild-type mice brain, on postnatal day 7 (P7), P14, P21, P28 and P90, mice from each group were decapitated, and three principal brain regions (cerebral cortex, hippocampus and cerebellum) were obtained and stored for later experiments. PSD-95 mRNA in the three brain areas was analyzed with quantitative RT-PCR. PSD-95 protein was measured by immunohistochemical staining and Western blot. In the three principal brain areas of Fmr1 knockout mice and wild-type mice, the expression of PSD-95 mRNA and protein were detected at the lowest levels on P7, and then significantly increased on P14, reaching the peak levels in adolescents or adults. Moreover, it was found that PSD-95 mRNA and protein in the hippocampus were significantly decreased in Fmr1 knockout mice during the developmental period (P7, P14, P21 and P28) as well as at adulthood (P90) (P < 0.05, and P < 0.01, respectively). However, there was no significant difference of expression of PSD-95 in the cortex and cerebellum between Fmr1 knockout and wild mice. The expression of PSD-95 in the hippocampus might be regulated by fragile X mental retardation protein (FMRP) during mice early developmental and adult periods. It is suggested that impairment of PSD-95 is possibly involved in hippocampal-dependent learning defects, which are common in people with FXS. © 2010 Japanese Society of Neuropathology.

  4. Live imaging of endogenous PSD-95 using ENABLED: a conditional strategy to fluorescently label endogenous proteins.

    PubMed

    Fortin, Dale A; Tillo, Shane E; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V; Guo, Caiying; Mao, Tianyi; Zhong, Haining

    2014-12-10

    Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. Copyright © 2014 the authors 0270-6474/14/3416698-15$15.00/0.

  5. Stabilization of the angiotensin-(1-7) receptor Mas through interaction with PSD95.

    PubMed

    Bian, Weihua; Sun, Licui; Yang, Longyan; Li, Ji-Feng; Hu, Jia; Zheng, Shuai; Guo, Ruihan; Feng, Duiping; Ma, Qian; Shi, Xiaocui; Xiong, Ying; Yang, Xiaomei; Song, Ran; Xu, Jianguo; Wang, Songlin; He, Junqi

    2013-08-01

    The functions and signalling mechanisms of the Ang-(1-7) [angiotensin-(1-7)] receptor Mas have been studied extensively. However, less attention has been paid to the intracellular regulation of Mas protein. In the present study, PSD95 (postsynaptic density 95), a novel binding protein of Mas receptor, was identified, and their association was characterized further. Mas specifically interacts with PDZ1-2, but not the PDZ3, domain of PSD95 via Mas-CT (Mas C-terminus), and the last four amino acids [ETVV (Glu-Thr-Val-Val)] of Mas-CT were determined to be essential for this interaction, as shown by GST pull-down, co-immunoprecipitation and confocal co-localization experiments. Gain-of-function and loss-of-function studies indicated that PSD95 enhanced Mas protein expression by increasing the stabilization of the receptor. Mas degradation was robustly inhibited by the proteasome inhibitor MG132 in time- and dose-dependent manners, and the expression of PSD95 impaired Mas ubiquitination, indicating that the PSD95-Mas association inhibits Mas receptor degradation via the ubiquitin-proteasome proteolytic pathway. These findings reveal a novel mechanism of Mas receptor regulation by which its expression is modulated at the post-translational level by ubiquitination, and clarify the role of PSD95, which binds directly to Mas, blocking the ubiquitination and subsequent degradation of the receptor via the ubiquitin-proteasome proteolytic pathway.

  6. Live Imaging of Endogenous PSD-95 Using ENABLED: A Conditional Strategy to Fluorescently Label Endogenous Proteins

    PubMed Central

    Fortin, Dale A.; Tillo, Shane E.; Yang, Guang; Rah, Jong-Cheol; Melander, Joshua B.; Bai, Suxia; Soler-Cedeño, Omar; Qin, Maozhen; Zemelman, Boris V.; Guo, Caiying

    2014-01-01

    Stoichiometric labeling of endogenous synaptic proteins for high-contrast live-cell imaging in brain tissue remains challenging. Here, we describe a conditional mouse genetic strategy termed endogenous labeling via exon duplication (ENABLED), which can be used to fluorescently label endogenous proteins with near ideal properties in all neurons, a sparse subset of neurons, or specific neuronal subtypes. We used this method to label the postsynaptic density protein PSD-95 with mVenus without overexpression side effects. We demonstrated that mVenus-tagged PSD-95 is functionally equivalent to wild-type PSD-95 and that PSD-95 is present in nearly all dendritic spines in CA1 neurons. Within spines, while PSD-95 exhibited low mobility under basal conditions, its levels could be regulated by chronic changes in neuronal activity. Notably, labeled PSD-95 also allowed us to visualize and unambiguously examine otherwise-unidentifiable excitatory shaft synapses in aspiny neurons, such as parvalbumin-positive interneurons and dopaminergic neurons. Our results demonstrate that the ENABLED strategy provides a valuable new approach to study the dynamics of endogenous synaptic proteins in vivo. PMID:25505322

  7. PSD-95 alters microtubule dynamics via an association with EB3

    PubMed Central

    Sweet, Eric S.; Previtera, Michelle L.; Fernández, Jose R.; Charych, Erik I.; Tseng, Chia-Yi; Kwon, Munjin; Starovoytov, Valentin; Zheng, James Q.; Firestein, Bonnie L.

    2011-01-01

    Little is known about how the neuronal cytoskeleton is regulated when a dendrite decides whether to branch or not. Previously, we reported that postsynaptic density protein 95 (PSD-95) acts as a stop signal for dendrite branching. It is yet to be elucidated how PSD-95 affects the cytoskeleton and how this regulation relates to the dendritic arbor. Here, we show that the SH3 (src homology 3) domain of PSD-95 interacts with a proline-rich region within the microtubule end-binding protein EB3. Overexpression of PSD-95 or mutant EB3 results in a decreased lifetime of EB3 comets in dendrites. In line with these data, transfected rat neurons show that overexpression of PSD-95 results in less organized microtubules at dendritic branch points and decreased dendritogensis. The interaction between PSD-95 and EB3 elucidates a function for a novel region of EB3 and provides a new and important mechanism for the regulation of microtubules in determining dendritic morphology. PMID:21248129

  8. Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia

    PubMed Central

    Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

    2015-01-01

    Background: There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. Aims: To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Methods: Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. Results: We found a 20% decrease in NR1 protein (t(66)=−2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=−2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Conclusions: Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient. PMID:27336043

  9. Postsynaptic density levels of the NMDA receptor NR1 subunit and PSD-95 protein in prefrontal cortex from people with schizophrenia.

    PubMed

    Catts, Vibeke Sørensen; Derminio, Dominique Suzanne; Hahn, Chang-Gyu; Weickert, Cynthia Shannon

    2015-01-01

    There is converging evidence of involvement of N-methyl-d-aspartate (NMDA) receptor hypofunction in the pathophysiology of schizophrenia. Our group recently identified a decrease in total NR1 mRNA and protein expression in the dorsolateral prefrontal cortex in a case-control study of individuals with schizophrenia (n=37/group). The NR1 subunit is critical to NMDA receptor function at the postsynaptic density, a cellular structure rich in the scaffolding protein, PSD-95. The extent to which the NMDA receptor NR1 subunit is altered at the site of action, in the postsynaptic density, is not clear. To extend our previous results by measuring levels of NR1 and PSD-95 protein in postsynaptic density-enriched fractions of prefrontal cortex from the same individuals in the case-control study noted above. Postsynaptic density-enriched fractions were isolated from fresh-frozen prefrontal cortex (BA10) and subjected to western blot analysis for NR1 and PSD-95. We found a 20% decrease in NR1 protein (t(66)=-2.874, P=0.006) and a 30% decrease in PSD-95 protein (t(63)=-2.668, P=0.010) in postsynaptic density-enriched fractions from individuals with schizophrenia relative to unaffected controls. Individuals with schizophrenia have less NR1 protein, and therefore potentially fewer functional NMDA receptors, at the postsynaptic density. The associated decrease in PSD-95 protein at the postsynaptic density suggests that not only are glutamate receptors compromised in individuals with schizophrenia, but the overall spine architecture and downstream signaling supported by PSD-95 may also be deficient.

  10. Clustering of neuronal potassium channels is independent of their interaction with PSD-95

    PubMed Central

    Rasband, Matthew N.; Park, Eunice W.; Zhen, Dongkai; Arbuckle, Margaret I.; Poliak, Sebastian; Peles, Elior; Grant, Seth G.N.; Trimmer, James S.

    2002-01-01

    Voltage-dependent potassium channels regulate membrane excitability and cell–cell communication in the mammalian nervous system, and are found highly localized at distinct neuronal subcellular sites. Kv1 (mammalian Shaker family) potassium channels and the neurexin Caspr2, both of which contain COOH-terminal PDZ domain binding peptide motifs, are found colocalized at high density at juxtaparanodes flanking nodes of Ranvier of myelinated axons. The PDZ domain–containing protein PSD-95, which clusters Kv1 potassium channels in heterologous cells, has been proposed to play a major role in potassium channel clustering in mammalian neurons. Here, we show that PSD-95 colocalizes precisely with Kv1 potassium channels and Caspr2 at juxtaparanodes, and that a macromolecular complex of Kv1 channels and PSD-95 can be immunopurified from mammalian brain and spinal cord. Surprisingly, we find that the high density clustering of Kv1 channels and Caspr2 at juxtaparanodes is normal in a mutant mouse lacking juxtaparanodal PSD-95, and that the indirect interaction between Kv1 channels and Caspr2 is maintained in these mutant mice. These data suggest that the primary function of PSD-95 at juxtaparanodes lies outside of its accepted role in mediating the high density clustering of Kv1 potassium channels at these sites. PMID:12438413

  11. Wnt-5a/JNK Signaling Promotes the Clustering of PSD-95 in Hippocampal Neurons*

    PubMed Central

    Farías, Ginny G.; Alfaro, Iván E.; Cerpa, Waldo; Grabowski, Catalina P.; Godoy, Juan A.; Bonansco, Christian; Inestrosa, Nibaldo C.

    2009-01-01

    During the formation of synapses, specific regions of pre- and postsynaptic cells associate to form a single functional transmission unit. In this process, synaptogenic factors are necessary to modulate pre- and postsynaptic differentiation. In mammals, different Wnt ligands operate through canonical and non-canonical Wnt pathways, and their precise functions to coordinate synapse structure and function in the mature central nervous system are still largely unknown. Here, we studied the effect of different Wnt ligands on postsynaptic organization. We found that Wnt-5a induces short term changes in the clustering of PSD-95, without affecting its total levels. Wnt-5a promotes the recruitment of PSD-95 from a diffuse dendritic cytoplasmic pool to form new PSD-95 clusters in dendritic spines. Moreover, Wnt-5a acting as a non-canonical ligand regulates PSD-95 distribution through a JNK-dependent signaling pathway, as demonstrated by using the TAT-TI-JIP peptide in mature hippocampal neurons. Finally, using adult rat hippocampal slices, we found that Wnt-5a modulates glutamatergic synaptic transmission through a postsynaptic mechanism. Our studies indicate that the Wnt-5a/JNK pathway modulates the postsynaptic region of mammalian synapse directing the clustering and distribution of the physiologically relevant scaffold protein, PSD-95. PMID:19332546

  12. The relationship between PSD-95 clustering and spine stability in vivo.

    PubMed

    Cane, Michele; Maco, Bohumil; Knott, Graham; Holtmaat, Anthony

    2014-02-05

    The appearance and disappearance of dendritic spines, accompanied by synapse formation and elimination may underlie the experience-dependent reorganization of cortical circuits. The exact temporal relationship between spine and synapse formation in vivo remains unclear, as does the extent to which synapse formation enhances the stability of newly formed spines and whether transient spines produce synapses. We used in utero electroporation of DsRedExpress- and eGFP-tagged postsynaptic density protein 95 (PSD-95) to investigate the relationship between spine and PSD stability in mouse neocortical L2/3 pyramidal cells in vivo. Similar to previous studies, spines and synapses appeared and disappeared, even in naive animals. Cytosolic spine volumes and PSD-95-eGFP levels in spines covaried over time, suggesting that the strength of many individual synapses continuously changes in the adult neocortex. The minority of newly formed spines acquired PSD-95-eGFP puncta. Spines that failed to acquire a PSD rarely survived for more than a day. Although PSD-95-eGFP accumulation was associated with increased spine lifetimes, most new spines with a PSD did not convert into persistent spines. This indicates that transient spines may serve to produce short-lived synaptic contacts. Persistent spines that were destined to disappear showed, on average, reduced PSD-95-eGFP levels well before the actual pruning event. Altogether, our data indicate that the PSD size relates to spine stability in vivo.

  13. Ovarian Steroids Increase PSD-95 Expression and Dendritic Spines in the Dorsal Raphe of Ovariectomized Macaques

    PubMed Central

    Rivera, Heidi M.; Bethea, Cynthia L.

    2014-01-01

    Estradiol (E) and progesterone (P) promote spinogenesis in several brain areas. Intracellular signaling cascades that promote spinogenesis involve RhoGTPases, glutamate signaling and synapse assembly. We found that in serotonin neurons, E±P administration increases (a) gene and protein expression of RhoGTPases, (b) gene expression of glutamate receptors (c) gene expression of pivotal synapse assembly proteins. Therefore, in this study we determined whether structural changes in dendritic spines in the dorsal raphe follow the observed changes in gene and protein expression. Dendritic spines were examined with immunogold silver staining of a spine marker protein, postsynaptic density-95 (PSD-95) and with Golgi staining. In the PSD-95 study, adult Ovx monkeys received placebo, E, P, or E+P for 1 month (n=3/group). Sections were immunostained for PSD-95 and the number of PSD-95-positive puncta was determined with stereology. E, P and E+P treatment significantly increased the total number of PSD-95-positive puncta (ANOVA, P=0.04). In the Golgi study, adult Ovx monkeys received placebo, E or E+P for 1 month (n=3–4) and the midbrain was Golgi-stained. A total of 80 neurons were analyzed with Neurolucida software. There was a significant difference in spine density that depended on branch order (two-way ANOVA). E+P treatment significantly increased spine density in higher-order (3–5°) dendritic branches relative to Ovx group (Bonferroni, P<0.05). In summary, E+P leads to the elaboration of dendritic spines on dorsal raphe neurons. The ability of E to induce PSD-95, but not actual spines, suggests either a sampling or time lag issue. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and, in turn, increase serotonin neurotransmission throughout the brain. PMID:23959764

  14. Ovarian steroids increase PSD-95 expression and dendritic spines in the dorsal raphe of ovariectomized macaques.

    PubMed

    Rivera, Heidi M; Bethea, Cynthia L

    2013-12-01

    Estradiol (E) and progesterone (P) promote spinogenesis in several brain areas. Intracellular signaling cascades that promote spinogenesis involve RhoGTPases, glutamate signaling and synapse assembly. We found that in serotonin neurons, E ± P administration increases (a) gene and protein expression of RhoGTPases, (b) gene expression of glutamate receptors, and (c) gene expression of pivotal synapse assembly proteins. Therefore, in this study we determined whether structural changes in dendritic spines in the dorsal raphe follow the observed changes in gene and protein expression. Dendritic spines were examined with immunogold silver staining of a spine marker protein, postsynaptic density-95 (PSD-95) and with Golgi staining. In the PSD-95 study, adult Ovx monkeys received placebo, E, P, or E + P for 1 month (n = 3/group). Sections were immunostained for PSD-95 and the number of PSD-95-positive puncta was determined with stereology. E, P, and E + P treatment significantly increased the total number of PSD-95-positive puncta (ANOVA, P = 0.04). In the golgi study, adult Ovx monkeys received placebo, E or E + P for 1 month (n = 3-4) and the midbrain was golgi-stained. A total of 80 neurons were analyzed with Neurolucida software. There was a significant difference in spine density that depended on branch order (two-way ANOVA). E + P treatment significantly increased spine density in higher-order (3°-5°) dendritic branches relative to Ovx group (Bonferroni, P < 0.05). In summary, E + P leads to the elaboration of dendritic spines on dorsal raphe neurons. The ability of E to induce PSD-95, but not actual spines, suggests either a sampling or time lag issue. Increased spinogenesis on serotonin dendrites would facilitate excitatory glutamatergic input and, in turn, increase serotonin neurotransmission throughout the brain. Copyright © 2013 Wiley Periodicals, Inc.

  15. Small molecule inhibitors of PSD95-nNOS protein-protein interactions as novel analgesics

    PubMed Central

    Lee, Wan-Hung; Xu, Zhili; Ashpole, Nicole M.; Hudmon, Andy; Kulkarni, Pushkar M.; Thakur, Ganesh A.; Lai, Yvonne Y.; Hohmann, Andrea G.

    2015-01-01

    Aberrant increases in NMDA receptor (NMDAR) signaling contributes to central nervous system sensitization and chronic pain by activating neuronal nitric oxide synthase (nNOS) and generating nitric oxide (NO). Because the scaffolding protein postsynaptic density 95kDA (PSD95) tethers nNOS to NMDARs, the PSD95-nNOS complex represents a therapeutic target. Small molecule inhibitors IC87201 (EC5O: 23.94 µM) and ZL006 (EC50: 12.88 µM) directly inhibited binding of purified PSD95 and nNOS proteins in AlphaScreen without altering binding of PSD95 to ErbB4. Both PSD95-nNOS inhibitors suppressed glutamate-induced cell death with efficacy comparable to MK-801. IC87201 and ZL006 preferentially suppressed phase 2A pain behavior in the formalin test and suppressed allodynia induced by intraplantar complete Freund’s adjuvant administration. IC87201 and ZL006 suppressed mechanical and cold allodynia induced by the chemotherapeutic agent paclitaxel (ED50s: 2.47 and 0.93 mg/kg i.p. for IC87201 and ZL006, respectively). Efficacy of PSD95-nNOS disruptors was similar to MK-801. Motor ataxic effects were induced by MK-801 but not by ZL006 or IC87201. Finally, MK-801 produced hyperalgesia in the tail-flick test whereas IC87201 and ZL006 did not alter basal nociceptive thresholds. Our studies establish the utility of using AlphaScreen and purified protein pairs to establish and quantify disruption of protein-protein interactions. Our results demonstrate previously unrecognized antinociceptive efficacy of ZL006 and establish, using two small molecules, a broad application for PSD95-nNOS inhibitors in treating neuropathic and inflammatory pain. Collectively, our results demonstrate that disrupting PSD95-nNOS protein-protein interactions is effective in attenuating pathological pain without producing unwanted side effects (i.e. motor ataxia) associated with NMDAR antagonists. PMID:26071110

  16. Role of PSD95 in membrane association and catalytic activity of nNOSalpha in nitrergic varicosities in mice gut.

    PubMed

    Chaudhury, Arun; He, Xue-Dao; Goyal, Raj K

    2009-10-01

    We have recently shown that membrane association of neuronal nitric oxide synthase-alpha (nNOSalpha) is critical in the regulation of synthesis of NO during nitrergic neurotransmission. The purpose of this study was to examine the role of the synapse-associated proteins (SAPs) in membrane association of nNOSalpha. Varicosities (swellings on terminal axons) were isolated from mice gastrointestinal tract and examined for nNOSalpha, postsynaptic density protein 95 (PSD95), and membrane interactions by coimmunoprecipitation and SDS-PAGE. Our results show that PSD95 protein was present in the membrane fraction of the nerve varicosity, whereas both PSD95 and SAP97 were present in the cytosol. nNOSalpha was associated with PSD95 but not SAP97. nNOSalpha-PSD95 complex was bound to the membrane via palmitoylation of PSD95. Depalmitoylation of PSD95 with 2-bromopalmitate dislocates nNOSalpha and PSD95 from the varicosity membrane and abolishes NO production. These studies show that palmitoylation of PSD95 anchors nNOSalpha to the varicosity membrane and that it is obligatory for NO production by the enzyme. Palmitoylation of PSD95 may provide a novel target for regulation of nitrergic neurotransmission.

  17. Reversible inhibition of PSD-95 mRNA translation by miR-125a, FMRP phosphorylation and mGluR signaling

    PubMed Central

    Muddashetty, Ravi S.; Nalavadi, Vijayalaxmi C.; Gross, Christina; Yao, Xiaodi; Xing, Lei; Laur, Oskar; Warren, Stephen T.; Bassell, Gary J.

    2011-01-01

    Summary The molecular mechanism how RISC and microRNAs selectively and reversibly regulate mRNA translation in response to receptor signaling is unknown but could provide a means for temporal and spatial control of translation. Here we show that miR-125a targeting PSD-95 mRNA allows reversible inhibition of translation and regulation by mGluR signaling. Inhibition of miR-125a increased PSD-95 levels in dendrites and altered dendritic spine morphology. Bidirectional control of PSD-95 expression depends on miR-125a and FMRP phosphorylation status. miR-125a levels at synapses and its association with AGO2 is reduced in Fmr1 KO. FMRP phosphorylation promotes the formation of an AGO2-miR-125a inhibitory complex on PSD-95 mRNA, whereas mGluR signaling of translation requires FMRP dephosphorylation and release of AGO2 from the mRNA. These findings reveal a novel mechanism whereby FMRP phosphorylation provides a reversible switch for AGO2 and microRNA to selectively regulate mRNA translation at synapses in response to receptor activation. PMID:21658607

  18. Disrupting nNOS-PSD-95 coupling in the hippocampal dentate gyrus promotes extinction memory retrieval.

    PubMed

    Li, Jun; Han, Zhou; Cao, Bo; Cai, Cheng-Yun; Lin, Yu-Hui; Li, Fei; Wu, Hai-Ying; Chang, Lei; Luo, Chun-Xia; Zhu, Dong-Ya

    2017-09-06

    Granule cells in the dentate gyrus regenerate constantly in adult hippocampus and then integrate into neural circuits in the hippocampus thereby providing the neural basis for learning and memory. Promoting the neurogenesis in the hippocampus facilitates learning and memory such as spatial learning, object identification, and extinction learning. The interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) is reported to negatively regulate neurogenesis in brain, so we hypothesized that disrupting this interaction might facilitate the neurogenesis in the dentate gyrus (DG) and thus enhance the extinction memory retrieval of fear learning. We found that uncoupling the nNOS-PSD-95 complex in remote contextual fear condition promoted both neuronal proliferation and survival in the DG, contributing to an enhanced retrieval of the extinction memory. Moreover, the nNOS-PSD-95 uncoupling-induced neurogenesis may be mediated by the extracellular signal-regulated kinase (ERK) as the phosphorylation level of ERK1/2 was increased after uncoupling. These findings suggest that the nNOS-PSD-95 complex may serve as a novel target for the treatment of post-traumatic stress disorder (PTSD). Copyright © 2017 Elsevier Inc. All rights reserved.

  19. D-Serine and Serine Racemase Are Associated with PSD-95 and Glutamatergic Synapse Stability.

    PubMed

    Lin, Hong; Jacobi, Ariel A; Anderson, Stewart A; Lynch, David R

    2016-01-01

    D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating

  20. D-Serine and Serine Racemase Are Associated with PSD-95 and Glutamatergic Synapse Stability

    PubMed Central

    Lin, Hong; Jacobi, Ariel A.; Anderson, Stewart A.; Lynch, David R.

    2016-01-01

    D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs), synthesized by serine racemase (SR) through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking α7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR) are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1), in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) and 7-chlorokynurenic acid (7-CK), a specific antagonist at the glycine site of NMDARs, demonstrating

  1. Structural Modeling of Protein Interactions by Analogy: Application to PSD-95

    PubMed Central

    Korkin, Dmitry; Davis, Fred P; Alber, Frank; Luong, Tinh; Shen, Min-Yi; Lucic, Vladan; Kennedy, Mary B; Sali, Andrej

    2006-01-01

    We describe comparative patch analysis for modeling the structures of multidomain proteins and protein complexes, and apply it to the PSD-95 protein. Comparative patch analysis is a hybrid of comparative modeling based on a template complex and protein docking, with a greater applicability than comparative modeling and a higher accuracy than docking. It relies on structurally defined interactions of each of the complex components, or their homologs, with any other protein, irrespective of its fold. For each component, its known binding modes with other proteins of any fold are collected and expanded by the known binding modes of its homologs. These modes are then used to restrain conventional molecular docking, resulting in a set of binary domain complexes that are subsequently ranked by geometric complementarity and a statistical potential. The method is evaluated by predicting 20 binary complexes of known structure. It is able to correctly identify the binding mode in 70% of the benchmark complexes compared with 30% for protein docking. We applied comparative patch analysis to model the complex of the third PSD-95, DLG, and ZO-1 (PDZ) domain and the SH3-GK domains in the PSD-95 protein, whose structure is unknown. In the first predicted configuration of the domains, PDZ interacts with SH3, leaving both the GMP-binding site of guanylate kinase (GK) and the C-terminus binding cleft of PDZ accessible, while in the second configuration PDZ interacts with GK, burying both binding sites. We suggest that the two alternate configurations correspond to the different functional forms of PSD-95 and provide a possible structural description for the experimentally observed cooperative folding transitions in PSD-95 and its homologs. More generally, we expect that comparative patch analysis will provide useful spatial restraints for the structural characterization of an increasing number of binary and higher-order protein complexes. PMID:17096593

  2. Assessment of ZnT3 and PSD95 protein levels in Lewy body dementias and Alzheimer's disease: association with cognitive impairment.

    PubMed

    Whitfield, David R; Vallortigara, Julie; Alghamdi, Amani; Howlett, David; Hortobágyi, Tibor; Johnson, Mary; Attems, Johannes; Newhouse, Stephen; Ballard, Clive; Thomas, Alan J; O'Brien, John T; Aarsland, Dag; Francis, Paul T

    2014-12-01

    The loss of zinc transporter 3 (ZnT3) has been implicated in age-related cognitive decline in mice, and the protein has been associated with plaques. We investigated the levels of ZnT3 and postsynaptic density protein 95 (PSD95), a marker of the postsynaptic terminal, in people with Parkinson's disease dementia (PDD, n = 31), dementia with Lewy bodies (DLB, n = 44), Alzheimer's disease (AD, n = 16), and controls (n = 24), using semiquantitative western blotting and immunohistochemistry in 3 cortical regions. Standardized cognitive assessments during life and semiquantitative scoring of amyloid β (Aβ), tau, and α-synuclein at postmortem were used to investigate the relationship between ZnT3 and PSD95, cognition and pathology. Associations were observed between ZnT3 and PSD95 levels in prefrontal cortex and cognitive impairment (p = 0.001 and p = 0.002, respectively) and between ZnT3 levels in the parietal cortex and cognitive impairment (p = 0.036). Associations were also seen between ZnT3 levels in cingulate cortex and severity of Aβ (p = 0.003) and tau (p = 0.011) pathologies. DLB and PDD were characterized by significant reductions of PSD95 (p < 0.05) and ZnT3 (p < 0.001) in prefrontal cortex compared with controls and AD. PSD95 levels in the parietal cortex were found to be decreased in AD cases compared with controls (p = 0.02) and PDD (p = 0.005). This study has identified Zn(2+) modulation as a possible novel target for the treatment of cognitive impairment in DLB and PDD and the potential for synaptic proteins to be used as a biomarker for the differentiation of DLB and PDD from AD.

  3. Effects of the dimeric PSD-95 inhibitor UCCB01-144 on functional recovery after fimbria-fornix transection in rats.

    PubMed

    Sommer, Jens Bak; Bach, Anders; Malá, Hana; Strømgaard, Kristian; Mogensen, Jesper; Pickering, Darryl S

    2017-10-01

    Pharmacological inhibition of PSD-95 is a promising therapeutic strategy in the treatment of stroke, and positive effects of monomeric and dimeric PSD-95 inhibitors have been reported in numerous studies. However, whether therapeutic effects will generalize to other types of acute brain injury such as traumatic brain injury (TBI), which has pathophysiological mechanisms in common with stroke, is currently uncertain. We have previously found a lack of neuroprotective effects of dimeric PSD-95 inhibitors in the controlled cortical impact model of TBI in rats. However, as no single animal model is currently able to mimic the complex and heterogeneous pathophysiology of TBI, it is necessary to assess treatment effects across a range of models. In this preliminary study we investigated the neuroprotective abilities of the dimeric PSD-95 inhibitor UCCB01-144 after fimbria-fornix (FF) transection in rats. UCCB01-144 or saline was injected into the lateral tail vein of rats immediately after sham surgery or FF-transection, and effects on spatial delayed alternation in a T-maze were assessed over a 28-day period. Task acquisition was significantly impaired in FF-transected animals, but there were no significant effects of UCCB01-144 on spatial delayed alternation after FF-transection or sham surgery, although decelerated learning curves were seen after treatment with UCCB01-144 in FF-transected animals. The results of the present study are consistent with previous research showing a lack of neuroprotective effects of PSD-95 inhibition in experimental models of TBI. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Perturbing NR2B-PSD-95 interaction relieves neuropathic pain by inactivating CaMKII-CREB signaling.

    PubMed

    Xu, Fangxia; Zhao, Xin; Liu, Lin; Song, Jia; Zhu, Yingjun; Chu, Shuaishuai; Shao, Xueming; Li, Xiuxiu; Ma, Zhengliang; Gu, Xiaoping

    2017-09-06

    Neuropathic pain is characterized by central sensitization. The interaction between N-methyl-D-aspartate receptors (NMDARs) and postsynaptic density protein-95 (PSD-95) plays a major role in central sensitization. Here, we aimed to investigate the analgesic effect of disruption of the interaction between NMDAR and PSD-95. Chronic dorsal root ganglia compression model rats were used to mimic sciatica. Thermal hyperalgesia and mechanical allodynia were evaluated. The expression of spinal phospho-NR2B, PSD-95, calcium/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element binding protein (CREB) was measured using western blotting. A mimetic peptide Myr-NR2B9c was injected intrathecally to disrupt the interaction between PSD-95 and NR2B and detected by coimmunoprecipitation. Chronic dorsal root ganglia compression surgery induced thermal hyperalgesia and mechanical allodynia, and upregulated pain-related proteins such as phospho-NR2B, PSD-95, CaMKII, and CREB expressions in the spinal cord. Myr-NR2B9c disrupted the interaction between NR2B-containing NMDARs and PSD-95 in the spinal cord. Intrathecal administration of Myr-NR2B9c attenuated neuropathic pain behaviors and downregulated the expressions of phospho-NR2B, PSD-95, CaMKII, and CREB in the spinal cord. The present study indicates that dissociation of NR2B-containing NMDARs from PSD-95 inactivates CaMKII and CREB signaling and relieves pain.

  5. A Cyclic Peptide Targeted against PSD-95 Blocks Central Sensitization and Attenuates Thermal Hyperalgesia

    PubMed Central

    LeBlanc, Brian W.; Iwata, Masashi; Mallon, Andrew P.; Rupasinghe, Chamila N.; Goebel, Dennis J.; Marshall, John; Spaller, Mark R.; Saab, Carl Y.

    2010-01-01

    Post-synaptic density protein PSD-95 is emerging as a valid target for modulating nociception in animal studies. Based on the key role of PSD-95 in neuronal plasticity and the maintenance of pain behavior, we predicted that CN2097, a peptide-based macrocycle of 9 residues that binds to the PDZ domains of PSD-95, would interfere with physiologic phenomena in the spinal cord related to central sensitization. Furthermore, we tested whether spinal intrathecal injection of CN2097 attenuates thermal hyperalgesia in a rat model of sciatic neuropathy. Results demonstrate that spinal CN2097 reverses hyperexcitability of WDR neurons in the dorsal horn of neuropathic rats and decreases their evoked responses to peripheral stimuli (brush, low caliber von Frey and pressure), whereas CN5125 (‘negative control’) has no effect. CN2097 also blocks C-fiber LTP in the dorsal horn, which is linked to neuronal plasticity and central sensitization. At a molecular level, CN2097 attenuates the increase in phosphorylated p38 MAPK, a key intracellular signaling pathway in neuropathic pain. Moreover, spinal injection of CN2097 blocks thermal hyperalgesia in neuropathic rats. We conclude that CN2097 is a small molecule peptide with putative anti-nociceptive effects that modulates physiologic phenomena related to central sensitization under conditions of chronic pain. PMID:20167266

  6. Coxsackievirus and adenovirus receptor (CAR) mediates trafficking of acid sensing ion channel 3 (ASIC3) via PSD-95.

    PubMed

    Excoffon, Katherine J D A; Kolawole, Abimbola O; Kusama, Nobuyoshi; Gansemer, Nicholas D; Sharma, Priyanka; Hruska-Hageman, Alesia M; Petroff, Elena; Benson, Christopher J

    2012-08-17

    We have previously shown that the Coxsackievirus and adenovirus receptor (CAR) can interact with post-synaptic density 95 (PSD-95) and localize PSD-95 to cell-cell junctions. We have also shown that activity of the acid sensing ion channel (ASIC3), a H(+)-gated cation channel that plays a role in mechanosensation and pain signaling, is negatively modulated by PSD-95 through a PDZ-based interaction. We asked whether CAR and ASIC3 simultaneously interact with PSD-95, and if so, whether co-expression of these proteins alters their cellular distribution and localization. Results indicate that CAR and ASIC3 co-immunoprecipitate only when co-expressed with PSD-95. CAR also brings both PSD-95 and ASIC3 to the junctions of heterologous cells. Moreover, CAR rescues PSD-95-mediated inhibition of ASIC3 currents. These data suggest that, in addition to activity as a viral receptor and adhesion molecule, CAR can play a role in trafficking proteins, including ion channels, in a PDZ-based scaffolding complex. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Lengthening of the Stargazin Cytoplasmic Tail Increases Synaptic Transmission by Promoting Interaction to Deeper Domains of PSD-95.

    PubMed

    Hafner, Anne-Sophie; Penn, Andrew C; Grillo-Bosch, Dolors; Retailleau, Natacha; Poujol, Christel; Philippat, Amandine; Coussen, Françoise; Sainlos, Matthieu; Opazo, Patricio; Choquet, Daniel

    2015-04-22

    PSD-95 is a prominent organizer of the postsynaptic density (PSD) that can present a filamentous orientation perpendicular to the plasma membrane. Interactions between PSD-95 and transmembrane proteins might be particularly sensitive to this orientation, as "long" cytoplasmic tails might be required to reach deeper PSD-95 domains. Extension/retraction of transmembrane protein C-tails offer a new way of regulating binding to PSD-95. Using stargazin as a model, we found that enhancing the apparent length of stargazin C-tail through phosphorylation or by an artificial linker was sufficient to potentiate binding to PSD-95, AMPAR anchoring, and synaptic transmission. A linear extension of stargazin C-tail facilitates binding to PSD-95 by preferentially engaging interaction with the farthest located PDZ domains regarding to the plasma membrane, which present a greater affinity for the stargazin PDZ-domain-binding motif. Our study reveals that the concerted orientation of the stargazin C-tail and PSD-95 is a major determinant of synaptic strength. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity.

    PubMed

    Wu, Qian; Sun, Miao; Bernard, Laura P; Zhang, Huaye

    2017-09-29

    Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. A Specific Nutrient Combination Attenuates the Reduced Expression of PSD-95 in the Proximal Dendrites of Hippocampal Cell Body Layers in a Mouse Model of Phenylketonuria

    PubMed Central

    Bruinenberg, Vibeke M.; van Vliet, Danique; Attali, Amos; de Wilde, Martijn C.; Kuhn, Mirjam; van Spronsen, Francjan J.; van der Zee, Eddy A.

    2016-01-01

    The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice. PMID:27102170

  10. A Specific Nutrient Combination Attenuates the Reduced Expression of PSD-95 in the Proximal Dendrites of Hippocampal Cell Body Layers in a Mouse Model of Phenylketonuria.

    PubMed

    Bruinenberg, Vibeke M; van Vliet, Danique; Attali, Amos; de Wilde, Martijn C; Kuhn, Mirjam; van Spronsen, Francjan J; van der Zee, Eddy A

    2016-03-26

    The inherited metabolic disease phenylketonuria (PKU) is characterized by increased concentrations of phenylalanine in the blood and brain, and as a consequence neurotransmitter metabolism, white matter, and synapse functioning are affected. A specific nutrient combination (SNC) has been shown to improve synapse formation, morphology and function. This could become an interesting new nutritional approach for PKU. To assess whether treatment with SNC can affect synapses, we treated PKU mice with SNC or an isocaloric control diet and wild-type (WT) mice with an isocaloric control for 12 weeks, starting at postnatal day 31. Immunostaining for post-synaptic density protein 95 (PSD-95), a post-synaptic density marker, was carried out in the hippocampus, striatum and prefrontal cortex. Compared to WT mice on normal chow without SNC, PKU mice on the isocaloric control showed a significant reduction in PSD-95 expression in the hippocampus, specifically in the granular cell layer of the dentate gyrus, with a similar trend seen in the cornus ammonis 1 (CA1) and cornus ammonis 3 (CA3) pyramidal cell layer. No differences were found in the striatum or prefrontal cortex. PKU mice on a diet supplemented with SNC showed improved expression of PSD-95 in the hippocampus. This study gives the first indication that SNC supplementation has a positive effect on hippocampal synaptic deficits in PKU mice.

  11. Immunolocalization of NR1, NR2A, and PSD-95 in rat hippocampal subregions during postnatal development.

    PubMed

    Ling, Wei; Chang, Lirong; Song, Yizhi; Lu, Tao; Jiang, Yuhua; Li, Youxiang; Wu, Yan

    2012-05-01

    Although the expression of NMDARs and synaptic-associated proteins has been widely studied, the temporospatial distribution of NMDAR subunits and synaptic proteins in different hippocampal subregions during postnatal development still lacks detailed information, and the relationship between NR1 or NR2 subunits and PSD-95 family proteins is controversial. In this study, we used immunofluorescent staining to assess NR1 or NR2A and PSD-95 expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on postnatal (P) days: P0, P4, P7, P10, P14, P21, P28, P56. The results showed that from P0 to P56, NR1, NR2A, and PSD-95 expressions increased gradually, and the time points of their expression peak differed in CA1, CA3, and DG during postnatal development. Interestingly, although the expression of PSD-95 was positively correlated to both NR1 and NR2A, the NR1 and PSD-95 coexpressed puncta were greatest in CA3, while NR2A and PSD-95 coexpressed puncta were greatest in CA1, compared to other subregions. Surprisingly, at P21, among different strata of CA1, the area of highest expression of NR2A was dramatically changed from stratum pyramidale to stratum polymorphum and stratum moleculare, and returned to stratum pyramidale gradually on the later observed days again, indicating that P21 may be one critical timepoint during postnatal development in CA1. The specific temporospatial distribution pattern of NR1, NR2A, and PSD-95 might be related to the different physiological functions during postnatal development. Discovering the alteration of the relationship between PSD-95 and NMDAR subunits expression may be helpful for understanding mechanisms and therapy of neurodegenerative diseases. Copyright © 2011 Elsevier GmbH. All rights reserved.

  12. PSD-95 interacts with NBCn1 and enhances channel-like activity without affecting Na/HCO(3) cotransport.

    PubMed

    Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R Kyle; Smith, Yoland; Yun, C Chris; Choi, Inyeong

    2012-01-01

    The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO(3)(-) movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO(3) cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO(3) cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. Copyright © 2012 S. Karger AG, Basel.

  13. PSD-95 Interacts with NBCn1 and Enhances Channel-like Activity without Affecting Na/HCO3 Cotransport

    PubMed Central

    Lee, Soojung; Yang, Han Soo; Kim, Eunjin; Ju, Eun Ji; Kwon, Min Hyung; Dudley, R. Kyle; Smith, Yoland; Yun, C. Chris; Choi, Inyeong

    2013-01-01

    Background/Aims The sodium/bicarbonate transporter NBCn1 plays an essential role in intracellular pH regulation and transepithelial HCO3− movement in the body. NBCn1 also has sodium channel-like activity uncoupled to Na/HCO3 cotransport. We previously reported that NBCn1 interacts with the postsynaptic density protein PSD-95 in the brain. Here, we elucidated the structural determinant and functional consequence of NBCn1/PSD-95 interaction. Methods: Results In rat hippocampal CA3 neurons, NBCn1 was localized to the postsynaptic membranes of both dendritic shafts and spines and occasionally to the presynaptic membranes. A GST/NBCn1 fusion protein containing the C-terminal 131 amino acids of NBCn1 pulled down PSD-95 from rat brain lysates, whereas GST/NBCn1-ΔETSL (deletion of the last four amino acids) and GST/NBCn2 (NCBE) lacking the same ETSL did not. NBCn1 and PSD-95 were coimmunoprecipitated in HEK 293 cells, and their interaction did not affect the efficacy of PSD-95 to bind to the NMDA receptor NR2A. PSD-95 has negligible effects on intracellular pH changes mediated by NBCn1 in HEK 293 cells and Xenopus oocytes. However, PSD-95 increased an ionic conductance produced by NBCn1 channel-like activity. This increase was abolished by NBCn1-ΔETSL or by the peptide containing the last 15 amino acids of NBCn1. Conclusion Our data suggest that PSD-95 interacts with NBCn1 and increases its channel-like activity while negligibly affecting Na/HCO3 cotransport. The possibility that the channel-like activity occurs via an intermolecular cavity of multimeric NBCn1 proteins is discussed. PMID:23183381

  14. Destabilization of the Postsynaptic Density by PSD-95 Serine 73 Phosphorylation Inhibits Spine Growth and Synaptic Plasticity

    PubMed Central

    Steiner, Pascal; Higley, Michael J.; Xu, Weifeng; Czervionke, Brian L.; Malenka, Robert C.; Sabatini, Bernardo L.

    2009-01-01

    SUMMARY Long-term potentiation (LTP) is accompanied by dendritic spine growth and changes in the composition of the postsynaptic density (PSD). We find that activity-dependent growth of apical spines of CA1 pyramidal neurons is accompanied by destabilization of the PSD that results in transient loss and rapid replacement of PSD-95 and SHANK2. Signaling through PSD-95 is required for activity-dependent spine growth and trafficking of SHANK2. N-terminal PDZ and C-terminal guanylate kinase domains of PSD-95 are required for both processes, indicating that PSD-95 coordinates multiple signals to regulate morphological plasticity. Activity-dependent trafficking of PSD-95 is triggered by phosphorylation at serine 73, a conserved calcium/calmodulin-dependent protein kinase II (CaMKII) consensus phosphorylation site, which negatively regulates spine growth and potentiation of synaptic currents. We propose that PSD-95 and CaMKII act at multiple steps during plasticity induction to initially trigger and later terminate spine growth by trafficking growth-promoting PSD proteins out of the active spine. PMID:19081375

  15. Differential localization of SAP102 and PSD-95 is revealed in hippocampal spines using super-resolution light microscopy.

    PubMed

    Zheng, Chan-Ying; Wang, Ya-Xia; Kachar, Bechara; Petralia, Ronald S

    2011-01-01

    Synapse-associated protein 102 (SAP102) and postsynaptic density 95 (PSD-95) are two major cytoskeleton proteins in the postsynaptic density (PSD). Both of them belong to the membrane-associated guanylate kinase (MAGUK) family, which clusters and anchors glutamate receptors and other proteins at synapses. In our previous study, we found that SAP102 and PSD-95 have different distributions, using combined light/electron microscopy (LM/EM) methods.1 Here, we double labeled endogenous SAP102 and PSD-95 in mature hippocampal neurons, and then took images by two different kinds of super resolution microscopy-Stimulated Emission Depletion microscopy (STED) and DeltaVision OMX 3D super resolution microscopy. We found that our 2D and 3D super resolution data were consistent with our previous LM/EM data, showing significant differences in the localization of SAP102 and PSD-95 in spines: SAP102 is distributed in both the PSD and cytoplasm of spines, while PSD-95 is concentrated only in the PSD area. These results indicate functional differences between SAP102 and PSD-95 in synaptic organization and plasticity.

  16. Transferrin Receptor Controls AMPA Receptor Trafficking Efficiency and Synaptic Plasticity

    PubMed Central

    Liu, Ke; Lei, Run; Li, Qiong; Wang, Xin-Xin; Wu, Qian; An, Peng; Zhang, Jianchao; Zhu, Minyan; Xu, Zhiheng; Hong, Yang; Wang, Fudi; Shen, Ying; Li, Hongchang; Li, Huashun

    2016-01-01

    Transferrin receptor (TFR) is an important iron transporter regulating iron homeostasis and has long been used as a marker for clathrin mediated endocytosis. However, little is known about its additional function other than iron transport in the development of central nervous system (CNS). Here we demonstrate that TFR functions as a regulator to control AMPA receptor trafficking efficiency and synaptic plasticity. The conditional knockout (KO) of TFR in neural progenitor cells causes mice to develop progressive epileptic seizure, and dramatically reduces basal synaptic transmission and long-term potentiation (LTP). We further demonstrate that TFR KO remarkably reduces the binding efficiency of GluR2 to AP2 and subsequently decreases AMPA receptor endocytosis and recycling. Thus, our study reveals that TFR functions as a novel regulator to control AMPA trafficking efficiency and synaptic plasticity. PMID:26880306

  17. Palmitoylation-dependent CDKL5–PSD-95 interaction regulates synaptic targeting of CDKL5 and dendritic spine development

    PubMed Central

    Zhu, Yong-Chuan; Li, Dan; Wang, Lu; Lu, Bin; Zheng, Jing; Zhao, Shi-Lin; Zeng, Rong; Xiong, Zhi-Qi

    2013-01-01

    The X-linked gene cyclin-dependent kinase-like 5 (CDKL5) is mutated in severe neurodevelopmental disorders, including some forms of atypical Rett syndrome, but the function and regulation of CDKL5 protein in neurons remain to be elucidated. Here, we show that CDKL5 binds to the scaffolding protein postsynaptic density (PSD)-95, and that this binding promotes the targeting of CDKL5 to excitatory synapses. Interestingly, this binding is not constitutive, but governed by palmitate cycling on PSD-95. Furthermore, pathogenic mutations that truncate the C-terminal tail of CDKL5 diminish its binding to PSD-95 and synaptic accumulation. Importantly, down-regulation of CDKL5 by RNA interference (RNAi) or interference with the CDKL5–PSD-95 interaction inhibits dendritic spine formation and growth. These results demonstrate a critical role of the palmitoylation-dependent CDKL5–PSD-95 interaction in localizing CDKL5 to synapses for normal spine development and suggest that disruption of this interaction by pathogenic mutations may be implicated in the pathogenesis of CDKL5-related disorders. PMID:23671101

  18. Resequencing and Association Analysis of Six PSD-95-Related Genes as Possible Susceptibility Genes for Schizophrenia and Autism Spectrum Disorders

    PubMed Central

    Xing, Jingrui; Kimura, Hiroki; Wang, Chenyao; Ishizuka, Kanako; Kushima, Itaru; Arioka, Yuko; Yoshimi, Akira; Nakamura, Yukako; Shiino, Tomoko; Oya-Ito, Tomoko; Takasaki, Yuto; Uno, Yota; Okada, Takashi; Iidaka, Tetsuya; Aleksic, Branko; Mori, Daisuke; Ozaki, Norio

    2016-01-01

    PSD-95 associated PSD proteins play a critical role in regulating the density and activity of glutamate receptors. Numerous previous studies have shown an association between the genes that encode these proteins and schizophrenia (SZ) and autism spectrum disorders (ASD), which share a substantial portion of genetic risks. We sequenced the protein-encoding regions of DLG1, DLG2, DLG4, DLGAP1, DLGAP2, and SynGAP in 562 cases (370 SZ and 192 ASD patients) on the Ion PGM platform. We detected 26 rare (minor allele frequency <1%), non-synonymous mutations, and conducted silico functional analysis and pedigree analysis when possible. Three variants, G344R in DLG1, G241S in DLG4, and R604C in DLGAP2, were selected for association analysis in an independent sample set of 1315 SZ patients, 382 ASD patients, and 1793 healthy controls. Neither DLG4-G241S nor DLGAP2-R604C was detected in any samples in case or control sets, whereas one additional SZ patient was found that carried DLG1-G344R. Our results suggest that rare missense mutations in the candidate PSD genes may increase susceptibility to SZ and/or ASD. These findings may strengthen the theory that rare, non-synonymous variants confer substantial genetic risks for these disorders. PMID:27271353

  19. Structure of the PSD-95/MAP1A complex reveals a unique target recognition mode of the MAGUK GK domain.

    PubMed

    Xia, Yitian; Shang, Yuan; Zhang, Rongguang; Zhu, Jinwei

    2017-08-10

    The PSD-95 family of membrane-associated guanylate kinases (MAGUKs) are major synaptic scaffold proteins and play crucial roles in the dynamic regulation of dendritic remodelling, which is understood to be the foundation of synaptogenesis and synaptic plasticity. The guanylate kinase (GK) domain of MAGUK family proteins functions as a phosphor-peptide binding module. However, the GK domain of PSD-95 has been found to directly bind to a peptide sequence within the C-terminal region of neuronal-specific microtubule-associated protein 1A (MAP1A), although the detailed molecular mechanism governing this phosphorylation-independent interaction at the atomic level is missing. In the present study, we determine the crystal structure of PSD-95 GK in complex with the MAP1A peptide at 2.6-Å resolution. The complex structure reveals that, unlike a linear and elongated conformation in the phosphor-peptide/GK complexes, the MAP1A peptide adopts a unique conformation with a stretch of hydrophobic residues far from each other in the primary sequence clustering and interacting with the 'hydrophobic site' of PSD-95 GK and a highly conserved aspartic acid of MAP1A (D2117) mimicking the phosphor-serine/threonine in binding to the 'phosphor-site' of PSD-95 GK. We demonstrate that the MAP1A peptide may undergo a conformational transition upon binding to PSD-95 GK. Further structural comparison of known DLG GK-mediated complexes reveals the target recognition specificity and versatility of DLG GKs. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  20. Functional Uncoupling NMDAR NR2A Subunit from PSD-95 in the Prefrontal Cortex: Effects on Behavioral Dysfunction and Parvalbumin Loss after Early-Life Stress.

    PubMed

    Ganguly, Prabarna; Holland, Freedom H; Brenhouse, Heather C

    2015-11-01

    Exposure to early-life stress increases vulnerability to psychiatric disorders, including depression, schizophrenia, and anxiety. Growing evidence implicates aberrant development of the prefrontal cortex (PFC) in the effects of early-life stress, which often emerge in adolescence or young adulthood. Specifically, early-life stress in the form of maternal separation (MS) in rodents has been shown to decrease parvalbumin (PVB)-positive interneurons in the adolescent PFC; however, the mechanism underpinning behavioral dysfunction and PVB loss is not yet known. We recently reported that MS causes overexpression of the NMDA subunit NR2A in the PFC of adolescent rats. Elevated PFC NR2A is also found in developmental models of schizophrenia and is correlated with behavioral deficits, acting largely through its association with the postsynaptic protein PSD-95. In addition, adolescent maturation of PVB-positive interneurons relies on NR2A-driven NMDA activity. Therefore, it is possible that the NR2A/PSD-95 signaling complex has a role in adolescent MS effects. Here, we aimed to determine whether a discrete manipulation of PFC NR2A could prevent MS effects on PFC-controlled behaviors, including cognition, anxiety, and novelty-induced hyperlocomotion, as well as PVB loss in adolescence. We intracranially infused the NR2A-specific blocking peptide TAT2A in order to uncouple NR2A from PSD-95 in the early-adolescent PFC, without antagonizing the NMDA receptor. We demonstrated that MS rats treated with TAT2A during early adolescence were protected from MS-induced PVB loss and exhibited less anxious behavior than those infused with control peptide. These data implicate NR2A-related N-methyl-D-aspartate receptor development in adolescent behavioral and neural consequences of early-life stress.

  1. In vitro and in vivo effects of a novel dimeric inhibitor of PSD-95 on excitotoxicity and functional recovery after experimental traumatic brain injury.

    PubMed

    Sommer, Jens Bak; Bach, Anders; Malá, Hana; Strømgaard, Kristian; Mogensen, Jesper; Pickering, Darryl S

    2017-01-01

    PSD-95 inhibitors have been shown to be neuroprotective in stroke, but have only to a very limited extent been evaluated in the treatment of traumatic brain injury (TBI) that has pathophysiological mechanisms in common with stroke. The aims of the current study were to assess the effects of a novel dimeric inhibitor of PSD-95, UCCB01-147, on histopathology and long-term cognitive outcome after controlled cortical impact (CCI) in rats. As excitotoxic cell death is thought to be a prominent part of the pathophysiology of TBI, we also investigated the neuroprotective effects of UCCB01-147 and related compounds on NMDA-induced cell death in cultured cortical neurons. Anesthetized rats were given a CCI or sham injury, and were randomized to receive an injection of either UCCB01-147 (10 mg/kg), the non-competitive NMDAR-receptor antagonist MK-801 (1 mg/kg) or saline immediately after injury. At 2 and 4 weeks post-trauma, spatial learning and memory were assessed in a water maze, and at 3 months, brains were removed for estimation of lesion volumes. Overall, neither treatment with UCCB01-147 nor MK-801 resulted in significant improvements of cognition and histopathology after CCI. Although MK-801 provided robust neuroprotection against NMDA-induced toxicity in cultured cortical neurons, UCCB01-147 failed to reduce cell death and became neurotoxic at high doses. The data suggest potential differential effects of PSD-95 inhibition in stroke and TBI that should be investigated further in future studies taking important experimental factors such as timing of treatment, dosage, and anesthesia into consideration.

  2. TARP gamma-8 controls hippocampal AMPA receptor number, distribution and synaptic plasticity.

    PubMed

    Rouach, Nathalie; Byrd, Keith; Petralia, Ronald S; Elias, Guillermo M; Adesnik, Hillel; Tomita, Susumu; Karimzadegan, Siavash; Kealey, Colin; Bredt, David S; Nicoll, Roger A

    2005-11-01

    Synaptic plasticity involves activity-dependent trafficking of AMPA-type glutamate receptors. Numerous cytoplasmic scaffolding proteins are postulated to control AMPA receptor trafficking, but the detailed mechanisms remain unclear. Here, we show that the transmembrane AMPA receptor regulatory protein (TARP) gamma-8, which is preferentially expressed in the mouse hippocampus, is important for AMPA receptor protein levels and extrasynaptic surface expression. By controlling the number of AMPA receptors, gamma-8 is also important in long-term potentiation, but not long-term depression. This study establishes gamma-8 as a critical protein for basal AMPA receptor expression and localization at extrasynaptic sites in the hippocampus and raises the possibility that TARP-dependent control of AMPA receptors during synapse development and plasticity may be widespread.

  3. Neonatal sevoflurane anesthesia induces long-term memory impairment and decreases hippocampal PSD-95 expression without neuronal loss.

    PubMed

    Wang, S-Q; Fang, F; Xue, Z-G; Cang, J; Zhang, X-G

    2013-04-01

    Volatile anesthetics are widely used in the clinic, and sevoflurane is the most prevalent volatile anesthetic in pediatric anesthesia. Recent findings question the potential risks of volatile anesthetics on brain development. Evidence suggests that sevoflurane may cause neuronal deficiency. This study investigates the long-term effect of sevoflurane in the developing brain. We anesthetized 7 day-old rats for 4 h with 2.5% sevoflurane. A Morris water maze was used to evaluate hippocampal function 7 weeks after sevoflurane exposure. Nissl staining was performed to analyze neuronal loss. PSD-95 (postsynaptic density protein-95) expression in the hippocampus was measured using a western blot. The exposure to 2.5% sevoflurane caused long-term deficits in hippocampal function and decreased hippocampal PSD-95 expression without neuronal loss. This study demonstrates that P7 rats exposed for 4 h to 2.5% sevoflurane have significant spatial learning and memory impairment 7 weeks after anesthesia. In addition, PSD-95 expression in the hippocampus decreased at P56 without neuronal loss. These data suggest that sevoflurane causes neurotoxicity in the developing brain, which may be attributed to decreased PSD-95 in the hippocampus.

  4. Prickle2 is localized in the postsynaptic density and interacts with PSD-95 and NMDA receptors in the brain.

    PubMed

    Hida, Yamato; Fukaya, Masahiro; Hagiwara, Akari; Deguchi-Tawarada, Maki; Yoshioka, Toshinori; Kitajima, Isao; Inoue, Eiji; Watanabe, Masahiko; Ohtsuka, Toshihisa

    2011-06-01

    The planar cell polarity (PCP) protein, Prickle (Pk), is conserved in invertebrates and vertebrates, and regulates cellular morphogenesis and movement. Vertebrate Pk consists of at least two family members, Pk1 and Pk2, both of which are expressed in the brain; however, their localization and function at synapses remain elusive. Here, we show that Pk2 is expressed mainly in the adult brain and is tightly associated with the postsynaptic density (PSD) fraction obtained by subcellular fractionation. In primary cultured rat hippocampal neurons, Pk2 is colocalized with PSD-95 and synaptophysin at synapses. Moreover, immunoelectron microcopy shows that Pk2 is localized at the PSD of asymmetric synapses in the hippocampal CA1 region. Biochemical assays identified that Pk2 forms a complex with PSD proteins including PSD-95 and NMDA receptor subunits via the direct binding to the C-terminal guanylate kinase domain of PSD-95. These results indicate that Pk2 is a novel PSD protein that interacts with PSD-95 and NMDA receptors through complex formations in the brain.

  5. Interfering of the Reelin/ApoER2/PSD95 Signaling Axis Reactivates Dendritogenesis of Mature Hippocampal Neurons.

    PubMed

    Ampuero, Estibaliz; Jury, Nur; Härtel, Steffen; Marzolo, María-Paz; van Zundert, Brigitte

    2017-05-01

    Reelin, an extracellular glycoprotein secreted in embryonic and adult brain, participates in neuronal migration and neuronal plasticity. Extensive evidence shows that reelin via activation of the ApoER2 and VLDLR receptors promotes dendrite and spine formation during early development. Further evidence suggests that reelin signaling is needed to maintain a stable architecture in mature neurons, but, direct evidence is lacking. During activity-dependent maturation of the neuronal circuitry, the synaptic protein PSD95 is inserted into the postsynaptic membrane to induce structural refinement and stability of spines and dendrites. Given that ApoER2 interacts with PSD95, we tested if reelin signaling interference in adult neurons reactivates the dendritic architecture. Unlike findings in developing cultures, the presently obtained in vitro and in vivo data show, for the first time, that reelin signaling interference robustly increase dendritogenesis and reduce spine density in mature hippocampal neurons. In particular, the expression of a mutant ApoER2 form (ApoER2-tailless), which is unable to interact with PSD95 and hence cannot transduce reelin signaling, resulted in robust dendritogenesis in mature hippocampal neurons in vitro. These results indicate that reelin/ApoER2/PSD95 signaling is important for neuronal structure maintenance in mature neurons. Mechanistically, obtained immunofluorescent data indicate that reelin signaling impairment reduced synaptic PSD95 levels, consequently leading to synaptic re-insertion of NR2B-NMDARs. Our findings underscore the importance of reelin in maintaining adult network stability and reveal a new mode for reactivating dendritogenesis in neurological disorders where dendritic arbor complexity is limited, such as in depression, Alzheimer's disease, and stroke. J. Cell. Physiol. 232: 1187-1199, 2017. © 2016 Wiley Periodicals, Inc.

  6. Effects of the dimeric PSD-95 inhibitor UCCB01-144 in mouse models of pain, cognition and motor function.

    PubMed

    Andreasen, Jesper T; Nasser, Arafat; Caballero-Puntiverio, Maitane; Sahlholt, Maj; Bach, Anders; Gynther, Mikko; Strømgaard, Kristian; Pickering, Darryl S

    2016-06-05

    NMDAR antagonism shows analgesic action in humans and animal pain models, but disrupts cognitive and motor functions. NMDAR-dependent NO production requires tethering of the NMDAR to neuronal NO synthase (nNOS) by the postsynaptic density protein-95 (PSD-95). Perturbing the NMDAR/PSD-95/nNOS interaction has therefore been proposed as an alternative analgesic mechanism. We recently reported that UCCB01-125, a dimeric PSD-95 inhibitor with limited blood-brain-barrier permeability, reduced mechanical hypersensitivity in the complete Freund's adjuvant (CFA) inflammatory pain model, without disrupting cognitive or motor functions. Here, we investigated the analgesic efficacy in the CFA model of UCCB01-144, a PSD-95 inhibitor with improved blood-brain-barrier permeability. To extend the comparison of UCCB01-125 and UCCB01-144, we also tested both compounds in the spared nerve injury (SNI) model of neuropathic pain. Potential cognitive effects of UCCB01-144 were examined using the social transmission of food preference (STFP) test and the V-maze test, and motor coordination was assessed with the rotarod test. UCCB01-144 (10mg/kg) reversed CFA-induced mechanical hypersensitivity after 1h, and completely normalised sensitivity after 24h. In the SNI model, UCCB01-144 (30mg/kg) partially reversed hypersensitivity after 1h, but no effect was observed after 24h. UCCB01-125 did not affect SNI-induced hypersensitivity. Rotarod performance was unaffected by UCCB01-144, but 30mg/kg UCCB01-144 impaired performance in the STFP test. Collectively, UCCB01-144 reversed both CFA and SNI-induced hypersensitivity, but the efficacy in the SNI model was only transient. This suggests that enhanced BBB permeability of PSD-95 inhibitors improves the analgesic action in neuropathic pain states.

  7. Steroid receptor coactivator-1 mediates letrozole induced downregulation of postsynaptic protein PSD-95 in the hippocampus of adult female rats.

    PubMed

    Liu, Mengying; Huangfu, Xuhong; Zhao, Yangang; Zhang, Dongmei; Zhang, Jiqiang

    2015-11-01

    Hippocampus local estrogen which is converted from androgen that catalyzed by aromatase has been shown to play important roles in the regulation of learning and memory as well as cognition through action on synaptic plasticity, but the underlying mechanisms are poorly understood. Steroid receptor coactivator-1 (SRC-1) is one of the coactivators of steroid nuclear receptors; it is widely distributed in brain areas that related to learning and memory, reproductive regulation, sensory and motor information integration. Previous studies have revealed high levels of SRC-1 immunoreactivities in the hippocampus; it is closely related to the levels of synaptic proteins such as PSD-95 under normal development or gonadectomy, but its exact roles in the regulation of these proteins remains unclear. In this study, we used aromatase inhibitor letrozole in vivo and SRC-1 RNA interference in vitro to investigate whether SRC-1 mediated endogenous estrogen regulation of hippocampal PSD-95. The results revealed that letrozole injection synchronously decreased hippocampal SRC-1 and PSD-95 in a dose-dependant manner. Furthermore, when SRC-1 specific shRNA pool was applied to block the expression of SRC-1 in the primary hippocampal neuron culture, both immunocytochemistry and Western blot revealed that levels of PSD-95 were also decreased significantly. Taking together, these results provided the first evidence that SRC-1 mediated endogenous estrogen regulation of hippocampal synaptic plasticity by targeting the expression of synaptic protein PSD-95. Additionally, since letrozole is frequently used to treat estrogen-sensitive breast cancer, the above results also indicate its potential side effects in clinical administration. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Integrin-Linked Kinase Is Involved in Cocaine Sensitization by Regulating PSD-95 and Synapsin I Expression and GluR1 Ser845 Phosphorylation

    PubMed Central

    Chen, Qiang; Zhu, Xiongzhao; Zhang, Yu; Wetsel, William C.; Lee, Tong H.

    2013-01-01

    Our recent studies have demonstrated that integrin-linked kinase (ILK) is involved in the induction and maintenance of cocaine behavioral sensitization and chronic cocaine-induced neural plasticity in the nucleus accumbens (NAc) core. In the present study, we used ILK silencing to investigate how ILK may influence cocaine-induced neural plasticity. Adeno-associated virus carrying a small interfering RNA-ILK cassette under the control of an inducible Tet-On system was injected into the NAc core of Sprague–Dawley rats. Induced silencing was established during repeated cocaine injections (sensitization induction period) or between withdrawal days 9 and 22 (sensitization maintenance period). Under both paradigms, established cocaine sensitization under non-silenced conditions was associated with enhanced PSD-95 and synapsin I protein expression as well as enhanced Ser845 phosphorylation of the GluR1 subunit on withdrawal day. Silencing ILK expression under both paradigms prevented or reversed these changes. Importantly, ILK appears to form a complex with PSD-95 and synapsin I because it co-immunoprecipitated with each of these proteins. Together, these data suggest that ILK exerts pleiotropic actions by regulating pre- and postsynaptic neural plasticities within the NAc core in response to repeated cocaine exposure. PMID:19629758

  9. Integrin-linked kinase is involved in cocaine sensitization by regulating PSD-95 and synapsin I expression and GluR1 Ser845 phosphorylation.

    PubMed

    Chen, Qiang; Zhu, Xiongzhao; Zhang, Yu; Wetsel, William C; Lee, Tong H; Zhang, Xiuwu

    2010-03-01

    Our recent studies have demonstrated that integrin-linked kinase (ILK) is involved in the induction and maintenance of cocaine behavioral sensitization and chronic cocaine-induced neural plasticity in the nucleus accumbens (NAc) core. In the present study, we used ILK silencing to investigate how ILK may influence cocaine-induced neural plasticity. Adeno-associated virus carrying a small interfering RNA-ILK cassette under the control of an inducible Tet-On system was injected into the NAc core of Sprague-Dawley rats. Induced silencing was established during repeated cocaine injections (sensitization induction period) or between withdrawal days 9 and 22 (sensitization maintenance period). Under both paradigms, established cocaine sensitization under non-silenced conditions was associated with enhanced PSD-95 and synapsin I protein expression as well as enhanced Ser(845) phosphorylation of the GluR1 subunit on withdrawal day. Silencing ILK expression under both paradigms prevented or reversed these changes. Importantly, ILK appears to form a complex with PSD-95 and synapsin I because it co-immunoprecipitated with each of these proteins. Together, these data suggest that ILK exerts pleiotropic actions by regulating pre- and postsynaptic neural plasticities within the NAc core in response to repeated cocaine exposure.

  10. Drug Transporters and Na+/H+ Exchange Regulatory Factor PSD-95/Drosophila Discs Large/ZO-1 Proteins

    PubMed Central

    Walsh, Dustin R.; Nolin, Thomas D.

    2015-01-01

    Drug transporters govern the absorption, distribution, and elimination of pharmacologically active compounds. Members of the solute carrier and ATP binding-cassette drug transporter family mediate cellular drug uptake and efflux processes, thereby coordinating the vectorial movement of drugs across epithelial barriers. To exert their physiologic and pharmacological function in polarized epithelia, drug transporters must be targeted and stabilized to appropriate regions of the cell membrane (i.e., apical versus basolateral). Despite the critical importance of drug transporter membrane targeting, the mechanisms that underlie these processes are largely unknown. Several clinically significant drug transporters possess a recognition sequence that binds to PSD-95/Drosophila discs large/ZO-1 (PDZ) proteins. PDZ proteins, such as the Na+/H+ exchanger regulatory factor (NHERF) family, act to stabilize and organize membrane targeting of multiple transmembrane proteins, including many clinically relevant drug transporters. These PDZ proteins are normally abundant at apical membranes, where they tether membrane-delimited transporters. NHERF expression is particularly high at the apical membrane in polarized tissue such as intestinal, hepatic, and renal epithelia, tissues important to drug disposition. Several recent studies have highlighted NHERF proteins as determinants of drug transporter function secondary to their role in controlling membrane abundance and localization. Mounting evidence strongly suggests that NHERF proteins may have clinically significant roles in pharmacokinetics and pharmacodynamics of several pharmacologically active compounds and may affect drug action in cancer and chronic kidney disease. For these reasons, NHERF proteins represent a novel class of post-translational mediators of drug transport and novel targets for new drug development. PMID:26092975

  11. Activation of PERK Elicits Memory Impairment through Inactivation of CREB and Downregulation of PSD95 After Traumatic Brain Injury.

    PubMed

    Sen, Tanusree; Gupta, Rajaneesh; Kaiser, Helen; Sen, Nilkantha

    2017-06-14

    The PKR-like ER kinase (PERK), a transmembrane protein, resides in the endoplasmic reticulum (ER). Its activation serves as a key sensor of ER stress, which has been implicated in traumatic brain injury (TBI). The loss of memory is one of the most common symptoms after TBI, but the precise role of PERK activation in memory impairment after TBI has not been well elucidated. Here, we have shown that blocking the activation of PERK using GSK2656157 prevents the loss of dendritic spines and rescues memory deficits after TBI. To elucidate the molecular mechanism, we found that activated PERK phosphorylates CAMP response element binding protein (CREB) and PSD95 directly at the S129 and T19 residues, respectively. Phosphorylation of CREB protein prevents its interaction with a coactivator, CREB-binding protein, and subsequently reduces the BDNF level after TBI. Conversely, phosphorylation of PSD95 leads to its downregulation in pericontusional cortex after TBI in male mice. Treatment with either GSK2656157 or overexpression of a kinase-dead mutant of PERK (PERK-K618A) rescues BDNF and PSD95 levels in the pericontusional cortex by reducing phosphorylation of CREB and PSD95 proteins after TBI. Similarly, administration of either GSK2656157 or overexpression of PERK-K618A in primary neurons rescues the loss of dendritic outgrowth and number of synapses after treatment with a PERK activator, tunicamycin. Therefore, our study suggests that inhibition of PERK phosphorylation could be a potential therapeutic target to restore memory deficits after TBI.SIGNIFICANCE STATEMENT Traumatic brain injury (TBI) is the leading cause of death and disability around the world and affects 1.7 million Americans each year. Here, we have shown that TBI-activated PKR-like ER kinase (PERK) is responsible for memory deficiency, which is the most common problem in TBI patients. A majority of PERK's biological activities have been attributed to its function as an eIF2α kinase. However, our study

  12. The association of GPR85 with PSD-95-neuroligin complex and autism spectrum disorder: a molecular analysis.

    PubMed

    Fujita-Jimbo, Eriko; Tanabe, Yuko; Yu, Zhiling; Kojima, Karin; Mori, Masato; Li, Hong; Iwamoto, Sadahiko; Yamagata, Takanori; Momoi, Mariko Y; Momoi, Takashi

    2015-01-01

    Autism spectrum disorder (ASD) has a complex genetic etiology. Some symptoms and mutated genes, including neuroligin (NLGN), neurexin (NRXN), and SH3 and multiple ankyrin repeat domains protein (SHANK), are shared by schizophrenia and ASD. Little is known about the molecular pathogenesis of ASD. One of the possible molecular pathogenesis is an imbalance of excitatory and inhibitory receptors linked with the NLGN-PSD-95-SHANK complex via postsynaptic density protein/Drosophila disc large tumor suppressor/zonula occludens-1 protein (PDZ) binding. In the present study, we focused on GPR85 as a candidate gene for ASD because the C-terminal amino acid sequence of GPR85 [Thr-Cys-Val-Ile (YCVI)] is classified as a type II PDZ-binding motif, and GPR85 is a risk factor for schizophrenia. GPR85 is an orphan receptor that regulates neural and synaptic plasticity and modulates diverse behaviors, including learning and memory. While searching for molecules that associate with GPR85, we found that GPR85 was associated with postsynaptic density protein (PSD)-95 linked with NLGN in the brain. We examined the proteins that associate with the C-terminal sequence of GPR85 by pull-down assay and immunoblot analysis and searched for a mutation of the GPR85 gene in patients with ASD. We used immunostaining to examine the intracellular localization of mutated GPR85 and its influence on the morphology of cells and neurons. The C-terminal sequence of GPR85 interacted with PSD-95 at PDZ1, while NLGN interacted with PSD-95 at PDZ3. Two male patients with ASD from independent Japanese families possessed inherited missense mutations at conserved sites in GPR85: one had T1033C (M152T) and the other had G1239T (V221L). These mutations were located in a domain related to G protein interaction and signal transduction. In contrast to wild-type GPR85, mutated GPR85 was more preferentially accumulated, causing endoplasmic reticulum stress, and disturbed the dendrite formation of hippocampal neurons

  13. PSD-95 uncoupling from NMDA receptors by Tat- N-dimer ameliorates neuronal depolarization in cortical spreading depression.

    PubMed

    Kucharz, Krzysztof; Søndergaard Rasmussen, Ida; Bach, Anders; Strømgaard, Kristian; Lauritzen, Martin

    2017-05-01

    Cortical spreading depression is associated with activation of NMDA receptors, which interact with the postsynaptic density protein 95 (PSD-95) that binds to nitric oxide synthase (nNOS). Here, we tested whether inhibition of the nNOS/PSD-95/NMDA receptor complex formation by anti-ischemic compound, UCCB01-144 (Tat- N-dimer) ameliorates the persistent effects of cortical spreading depression on cortical function. Using in vivo two-photon microscopy in somatosensory cortex in mice, we show that fluorescently labelled Tat- N-dimer readily crosses blood-brain barrier and accumulates in nerve cells during the first hour after i.v. injection. The Tat- N-dimer suppressed stimulation-evoked synaptic activity by 2-20%, while cortical blood flow and cerebral oxygen metabolic (CMRO2) responses were preserved. During cortical spreading depression, the Tat- N-dimer reduced the average amplitude of the negative shift in direct current potential by 33% (4.1 mV). Furthermore, the compound diminished the average depression of spontaneous electrocorticographic activity by 11% during first 40 min of post-cortical spreading depression recovery, but did not mitigate the suppressing effect of cortical spreading depression on cortical blood flow and CMRO2. We suggest that uncoupling of PSD-95 from NMDA receptors reduces overall neuronal excitability and the amplitude of the spreading depolarization wave. These findings may be of interest for understanding the neuroprotective effects of the nNOS/PSD-95 uncoupling in stroke.

  14. ATP from synaptic terminals and astrocytes regulates NMDA receptors and synaptic plasticity through PSD-95 multi-protein complex

    PubMed Central

    Lalo, U.; Palygin, O.; Verkhratsky, A.; Grant, S. G. N.; Pankratov, Y.

    2016-01-01

    Recent studies highlighted the importance of astrocyte-secreted molecules, such as ATP, for the slow modulation of synaptic transmission in central neurones. Biophysical mechanisms underlying the impact of gliotransmitters on the strength of individual synapse remain, however, unclear. Here we show that purinergic P2X receptors can bring significant contribution to the signalling in the individual synaptic boutons. ATP released from astrocytes facilitates a recruitment of P2X receptors into excitatory synapses by Ca2+-dependent mechanism. P2X receptors, co-localized with NMDA receptors in the excitatory synapses, can be activated by ATP co-released with glutamate from pre-synaptic terminals and by glia-derived ATP. An activation of P2X receptors in turn leads to down-regulation of postsynaptic NMDA receptors via Ca2+-dependent de-phosphorylation and interaction with PSD-95 multi-protein complex. Genetic deletion of the PSD-95 or P2X4 receptors obliterated ATP-mediated down-regulation of NMDA receptors. Impairment of purinergic modulation of NMDA receptors in the PSD-95 mutants dramatically decreased the threshold of LTP induction and increased the net magnitude of LTP. Our findings show that synergistic action of glia- and neurone-derived ATP can pre-modulate efficacy of excitatory synapses and thereby can have an important role in the glia-neuron communications and brain meta-plasticity. PMID:27640997

  15. Post-translational modifications modulate ligand recognition by the third PDZ domain of the MAGUK protein PSD-95.

    PubMed

    Murciano-Calles, Javier; Corbi-Verge, Carles; Candel, Adela M; Luque, Irene; Martinez, Jose C

    2014-01-01

    The relative promiscuity of hub proteins such as postsynaptic density protein-95 (PSD-95) can be achieved by alternative splicing, allosteric regulation, and post-translational modifications, the latter of which is the most efficient method of accelerating cellular responses to environmental changes in vivo. Here, a mutational approach was used to determine the impact of phosphorylation and succinimidation post-translational modifications on the binding affinity of the postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ3) domain of PSD-95. Molecular dynamics simulations revealed that the binding affinity of this domain is influenced by an interplay between salt-bridges linking the α3 helix, the β2-β3 loop and the positively charged Lys residues in its high-affinity hexapeptide ligand KKETAV. The α3 helix is an extra structural element that is not present in other PDZ domains, which links PDZ3 with the following SH3 domain in the PSD-95 protein. This regulatory mechanism was confirmed experimentally via thermodynamic and NMR chemical shift perturbation analyses, discarding intra-domain long-range effects. Taken together, the results presented here reveal the molecular basis of the regulatory role of the α3 extra-element and the effects of post-translational modifications of PDZ3 on its binding affinity, both energetically and dynamically.

  16. ASIC2 Subunits Target Acid-Sensing Ion Channels to the Synapse via an Association with PSD-95

    PubMed Central

    Zha, Xiang-ming; Costa, Vivian; Harding, Anne Marie S.; Reznikov, Leah; Benson, Christopher J.; Welsh, Michael J.

    2009-01-01

    Acid-sensing ion channel-1a (ASIC1a) mediates H+-gated current to influence normal brain physiology and impact several models of disease. Although ASIC2 subunits are widely expressed in brain and modulate ASIC1a current, their function remains poorly understood. We identified ASIC2a in dendrites, dendritic spines, and brain synaptosomes. This localization largely relied on ASIC2a binding to PSD-95 and matched that of ASIC1a, which does not co-immunoprecipitate with PSD-95. We found that ASIC2 and ASIC1a associated in brain, and through its interaction with PSD-95, ASIC2 increased ASIC1a localization in dendritic spines. Consistent with earlier work showing that acidic pH elevated spine [Ca2+]i by activating ASIC1a, loss of ASIC2 decreased the percentage of spines responding to acid. Moreover, like a reduction of ASIC1a, the number of spine synapses fell in ASIC2-/- neurons. These results indicate that ASIC2 facilitates ASIC1a localization and function in dendritic spines and suggest that the two subunits work in concert to regulate neuronal function. PMID:19571134

  17. 2-Methyl-6-(phenylethynyl) pyridine (MPEP) reverses maze learning and PSD-95 deficits in Fmr1 knock-out mice

    PubMed Central

    Gandhi, Réno M.; Kogan, Cary S.; Messier, Claude

    2014-01-01

    Fragile X Syndrome (FXS) is caused by the lack of expression of the fragile X mental retardation protein (FMRP), which results in intellectual disability and other debilitating symptoms including impairment of visual-spatial functioning. FXS is the only single-gene disorder that is highly co-morbid with autism spectrum disorder and can therefore provide insight into its pathophysiology. Lack of FMRP results in altered group I metabotropic glutamate receptor (mGluR) signaling, which is a target for putative treatments. The Hebb-Williams (H-W) mazes are a set of increasingly complex spatial navigation problems that depend on intact hippocampal and thus mGluR-5 functioning. In the present investigation, we examined whether an antagonist of mGluR-5 would reverse previously described behavioral deficits in fragile X mental retardation 1 knock-out (Fmr1 KO) mice. Mice were trained on a subset of the H-W mazes and then treated with either 20 mg/kg of an mGluR-5 antagonist, 2-Methyl-6-(phenylethynyl) pyridine (MPEP; n = 11) or an equivalent dose of saline (n = 11) prior to running test mazes. Latency and errors were dependent variables recorded during the test phase. Immediately after completing each test, marble-burying behavior was assessed, which confirmed that the drug treatment was pharmacologically active during maze learning. Although latency was not statistically different between the groups, MPEP treated Fmr1 KO mice made significantly fewer errors on mazes deemed more difficult suggesting a reversal of the behavioral deficit. MPEP treated mice were also less perseverative and impulsive when navigating mazes. Furthermore, MPEP treatment reversed post-synaptic density-95 (PSD-95) protein deficits in Fmr1 KO treated mice, whereas levels of a control protein (β-tubulin) remained unchanged. These data further validate MPEP as a potentially beneficial treatment for FXS. Our findings also suggest that adapted H-W mazes may be a useful tool to document alterations in

  18. Colocalization of APC and PSD-95 in the nerve fiber as well as in the post-synapse of matured neurons.

    PubMed

    Onouchi, Takanori; Takamori, Nobutaka; Senda, Takao

    2012-06-01

    Adenomatous polyposis coli protein (APC) is highly expressed in the nervous tissue, but its function there is not yet known. We previously found that the microtubule-bundling activity of APC is stimulated by its interaction with PSD-95, a neuronal scaffolding protein, in cultured COS-7 cells. In the present study, we investigated the distribution and localization of the intrinsic APC and PSD-95 in both cultured rat hippocampal neurons and in the rat cerebellum by immunofluorescence and immunoelectron microscopy. In cultured neurons, most of the PSD-95 immunofluorescence puncta were colocalized with APC, and the APC and PSD-95 immunogolds were colocalized in the nerve fibers as well as in the postsynaptic site, but not in the presynaptic site. In the molecular layer of the rat cerebellum, colocalization of APC and PSD-95 was also detected in the nerve fibers and the postsynaptic site, but not in the presynaptic site. Based on these results, we conclude that APC and PSD-95 colocalize and bind to form a protein complex in nerve fibers as well as in postsynaptic sites in matured neurons, suggesting the involvement of the APC/PSD-95 complex in the microtubule functions within the nerve fibers and the synapse functions at the postsynaptic site.

  19. Postsynaptic density protein (PSD)-95 expression is markedly decreased in the hippocampal CA1 region after experimental ischemia-reperfusion injury.

    PubMed

    Yan, Bing Chun; Park, Joon Ha; Ahn, Ji Hyeon; Lee, Jae-Chul; Won, Moo-Ho; Kang, Il-Jun

    2013-07-15

    Synaptic plasticity is important for functional recovery after cerebral ischemic injury. In the present study, we investigated chronological change in the immunoreactivity of PSD-95, a kind of postsynaptic density protein, in the hippocampus proper (CA1-3 regions) after 5 min of transient cerebral ischemia in gerbils. PSD-95 immunoreactivity was observed in MAP-2-immunoreactive dendrites in the CA1-3 regions of the sham group. The PSD-95 immunoreactivity was shown as beaded structure in the MAP-2-immunoreactive dendrites. However, PSD-95 immunoreactivity began to be dramatically decreased in MAP-2-immunoreactive dendrites in the CA1 region, not CA2-3 region, at early time after ischemia-reperfusion. At 5 days after ischemia-reperfusion, MAP-2 immunoreactivity almost disappeared in the ischemic CA1 region, and PSD-95 immunoreactivity was much lower than that in the sham group. In brief, PSD-95 immunoreactivity in the CA1 dendrites was markedly decreased at early time after ischemia-reperfusion. We suggest that decreased PSD-95 immunoreactivity in the ischemic CA1 region may lead to a deficit of postsynaptic plasticity in the brain. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Structural analysis of the PSD-95 cluster by electron tomography and CEMOVIS: a proposal for the application of the genetically encoded metallothionein tag.

    PubMed

    Hirabayashi, Ai; Fukunaga, Yuko; Miyazawa, Atsuo

    2014-06-01

    Postsynaptic density-95 (PSD-95) accumulates at excitatory postsynapses and plays important roles in the clustering and anchoring of numerous proteins at the PSD. However, a detailed ultrastructural analysis of clusters exclusively consisting of PSD-95 has never been performed. Here, we employed a genetically encoded tag, three tandem repeats of metallothionein (3MT), to study the structure of PSD-95 clusters in cells by electron tomography and cryo-electron microscopy of vitreous sections. We also performed conventional transmission electron microscopy (TEM). Cultured hippocampal neurons expressing a fusion protein of PSD-95 coupled to 3MT (PDS-95-3MT) were incubated with CdCl2 to result in the formation of Cd-bound PSD-95-3MT. Two types of electron-dense deposits composed of Cd-bound PSD-95-3MT were observed in these cells by TEM, as reported previously. Electron tomography revealed the presence of membrane-shaped structures representing PSD-95 clusters at the PSD and an ellipsoidal structure located in the non-synaptic cytoplasm. By TEM, the PSD-95 clusters appeared to be composed of a number of dense cores. In frozen hydrated sections, these dense cores were also found beneath the postsynaptic membrane. Taken together, our findings suggest that dense cores of PSD-95 aggregate to form the larger clusters present in the PSD and the non-synaptic cytoplasm. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  1. Investigation into the effects of prenatal alcohol exposure on postnatal spine development and expression of synaptophysin and PSD95 in rat hippocampus.

    PubMed

    Elibol-Can, Birsen; Kilic, Ertugrul; Yuruker, Sinan; Jakubowska-Dogru, Ewa

    2014-04-01

    Ethanol is known as a potent teratogen responsible for the fetal alcohol syndrome characterized by cognitive deficits especially pronounced in juveniles but ameliorating in adults. Since the mechanisms of these deficits and following partial recovery are not fully elucidated, the aim of the present study was to investigate the process of synaptogenesis in the hippocampus over the first two months of life in control and fetal-alcohol rats. Ethanol was delivered to the pregnant dams by intragastric intubation throughout 7-21 gestation days at the daily dose of 6g/kg generating a mean blood alcohol level of 246.6±40.9mg/dl on gestation day 20. The spine densities as well as the expression of pre- and postsynaptic proteins, synaptophysin (SYP) and PSD-95 protein, were evaluated for three distinct hippocampal regions: CA1, CA2+3, and DG and four postnatal days: PD1, PD10, PD30 and PD60, independently. Our results confirmed an intensive synaptogenesis within the brain spurt period (first 10 postnatal days), however, the temporal pattern of changes in the SYP and PSD-95 expression was different. The ethanol exposure during half of the 1st and the whole 2nd human trimester equivalent resulted in an overall trend toward lower values of synaptic indices at PD1 with a fast recovery from these deficits observed already at PD10. At PD30, around the age when the most pronounced behavioral deficits have been previously reported in juvenile fetal-alcohol subjects, no significant changes were found in either the hippocampal levels of synaptic proteins or in the spine density in principal hippocampal neurons.

  2. Identification of N-methyl-D-aspartic acid (NMDA) receptor subtype-specific binding sites that mediate direct interactions with scaffold protein PSD-95.

    PubMed

    Cousins, Sarah L; Stephenson, F Anne

    2012-04-13

    N-methyl-D-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149-1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins.

  3. Identification of N-Methyl-d-aspartic Acid (NMDA) Receptor Subtype-specific Binding Sites That Mediate Direct Interactions with Scaffold Protein PSD-95*

    PubMed Central

    Cousins, Sarah L.; Stephenson, F. Anne

    2012-01-01

    N-methyl-d-aspartate (NMDA) neurotransmitter receptors and the postsynaptic density-95 (PSD-95) membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins are integral components of post-synaptic macromolecular signaling complexes that serve to propagate glutamate responses intracellularly. Classically, NMDA receptor NR2 subunits associate with PSD-95 MAGUKs via a conserved ES(E/D)V amino acid sequence located at their C termini. We previously challenged this dogma to demonstrate a second non-ES(E/D)V PSD-95-binding site in both NMDA receptor NR2A and NR2B subunits. Here, using a combination of co-immunoprecipitations from transfected mammalian cells, yeast two-hybrid interaction assays, and glutathione S-transferase (GST) pulldown assays, we show that NR2A subunits interact directly with PSD-95 via the C-terminal ESDV motif and additionally via an Src homology 3 domain-binding motif that associates with the Src homology 3 domain of PSD-95. Peptide inhibition of co-immunoprecipitations of NR2A and PSD-95 demonstrates that both the ESDV and non-ESDV sites are required for association in native brain tissue. Furthermore, we refine the non-ESDV site within NR2B to residues 1149–1157. These findings provide a molecular basis for the differential association of NMDA receptor subtypes with PSD-95 MAGUK scaffold proteins. These selective interactions may contribute to the organization, lateral mobility, and ultimately the function of NMDA receptor subtypes at synapses. Furthermore, they provide a more general molecular mechanism by which the scaffold, PSD-95, may discriminate between potential interacting partner proteins. PMID:22375001

  4. DA Negatively Regulates IGF-I Actions Implicated in Cognitive Function via Interaction of PSD95 and nNOS in Minimal Hepatic Encephalopathy

    PubMed Central

    Ding, Saidan; Zhuge, Weishan; Wang, Xuebao; Yang, Jianjing; Lin, Yuanshao; Wang, Chengde; Hu, Jiangnan; Zhuge, Qichuan

    2017-01-01

    Insulin-like growth factor I (IGF-I) has been positively correlated with cognitive ability. Cognitive decline in minimal hepatic encephalopathy (MHE) was shown to be induced by elevated intracranial dopamine (DA). The beneficial effect of IGF-I signaling in MHE remains unknown. In this study, we found that IGF-I content was reduced in MHE rats and that IGF-I administration mitigated cognitive decline of MHE rats. A protective effect of IGF-I on the DA-induced interaction between postsynaptic density protein 95 (PSD95) and neuronal nitric oxide synthase (nNOS) was found in neurons. Ribosomal S6 protein kinase (RSK) phosphorylated nNOS in response to IGF-I by recruiting extracellular signal-regulated kinase (ERK1/2). In turn, DA inactivated the ERK1/2/RSK pathway and stimulated the PSD95–nNOS interaction by downregulating IGF-I. Inhibition of the interaction between PSD95 and nNOS ameliorated DA-induced memory impairment. As DA induced deficits in the ERK1/2/RSK pathway and the interaction between PSD95 and nNOS in MHE brains, IGF-I administration exerted a protective effect via interruption of the interaction between PSD95 and nNOS. These results suggest that IGF-I antagonizes DA-induced cognitive loss by disrupting PSD95–nNOS interactions in MHE. PMID:28932186

  5. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers

    PubMed Central

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-01-01

    Summary Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like MAGUKs. Among the three classes of ionotropic glutamate receptors (AMPA-, NMDA, kainate-type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively-charged lipid bilayers in its phosphorylation dependent manner, and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction. PMID:20547132

  6. TARP phosphorylation regulates synaptic AMPA receptors through lipid bilayers.

    PubMed

    Sumioka, Akio; Yan, Dan; Tomita, Susumu

    2010-06-10

    Neurons use neurotransmitters to communicate across synapses, constructing neural circuits in the brain. AMPA-type glutamate receptors are the predominant excitatory neurotransmitter receptors mediating fast synaptic transmission. AMPA receptors localize at synapses by forming protein complexes with transmembrane AMPA receptor regulatory proteins (TARPs) and PSD-95-like membrane-associated guanylate kinases. Among the three classes of ionotropic glutamate receptors (AMPA, NMDA, and kainate type), AMPA receptor activity is most regulatable by neuronal activity to adjust synaptic strength. Here, we mutated the prototypical TARP, stargazin, and found that TARP phosphorylation regulates synaptic AMPA receptor activity in vivo. We also found that stargazin interacts with negatively charged lipid bilayers in a phosphorylation-dependent manner and that the lipid interaction inhibited stargazin binding to PSD-95. Cationic lipids dissociated stargazin from lipid bilayers and enhanced synaptic AMPA receptor activity in a stargazin phosphorylation-dependent manner. Thus, TARP phosphorylation plays a critical role in regulating AMPA receptor-mediated synaptic transmission via a lipid bilayer interaction.

  7. Spinal SIRPα1-SHP2 interaction regulates spinal nerve ligation-induced neuropathic pain via PSD-95-dependent NR2B activation in rats.

    PubMed

    Peng, Hsien-Yu; Chen, Gin-Den; Lai, Cheng-Yuang; Hsieh, Ming-Chun; Lin, Tzer-Bin

    2012-05-01

    The fact that neuropathic pain mechanisms are not well understood is a major impediment in the development of effective clinical treatments. We examined whether the interaction between signal regulatory protein alpha 1 (SIRPα1) and Src homology-2 domain-containing protein tyrosine phosphatase 2 (SHP2), and the downstream spinal SHP2/postsynaptic density 95 (PSD-95)/N-methyl-d-aspartate receptor NR2B subunit signaling cascade play a role in neuropathic pain. Following spinal nerve ligation (L5), we assessed tactile allodynia using the von Frey filament test and analyzed dorsal horn samples (L4-5) by Western blotting, reverse transcription polymerase chain reaction, coimmunoprecipitation, and immunofluorescence. Nerve ligation induced allodynia, SIRPα1, SHP2, phosphorylated SHP2 (pSHP2), and phosphorylated NR2B (pNR2B) expression, and SHP2-PSD-95, pSHP2-PSD-95, PSD-95-NR2B, and PSD-95-pNR2B coimmunoprecipitation in the ipsilateral dorsal horn. In allodynic rats, injury-induced SHP2 immunoreactivity was localized in the ipsilateral dorsal horn neurons and coincident with PSD-95 and NR2B immunoreactivity. SIRPα1 silencing using small interfering RNA (siRNA; 1, 3, or 5μg/rat for 7days) prevented injury-induced allodynia and the associated changes in protein expression, phosphorylation, and coimmunoprecipitation. Intrathecal administration of NSC-87877 (an SHP2 antagonist; 1, 10, or 100μM/rat) and SIRPα1-neutralizing antibodies (1, 10, or 30μg/rat) suppressed spinal nerve ligation-induced allodynia, spinal SHP2 and NR2B phosphorylation, and SHP2/phosphorylated SHP2-PSD-95 and PSD-95-NR2B/phosphorylated NR2B coprecipitation. SHP2 siRNA led to similar effects as the NSC-87877 and SIRPα1 antibody treatments, except it prevented the allodynia-associated spinal SHP2 expression. In conclusion, our results suggest that a spinal SIRPα1-SHP2 interaction exists that subsequently triggers SHP2/PSD-95/NR2B signaling, thereby playing a role in neuropathic pain development.

  8. Melatonin improves neuroplasticity by upregulating the growth-associated protein-43 (GAP-43) and NMDAR postsynaptic density-95 (PSD-95) proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to transient focal cerebral ischemia even during a long-term recovery period.

    PubMed

    Juan, Wei-Sheng; Huang, Sheng-Yang; Chang, Che-Chao; Hung, Yu-Chang; Lin, Yu-Wen; Chen, Tsung-Ying; Lee, Ai-Hua; Lee, Ai-Chiang; Wu, Tian-Shung; Lee, E-Jian

    2014-03-01

    Recent evidence shows that the NMDAR postsynaptic density-95 (PSD-95), growth-associated protein-43 (GAP-43), and matrix metalloproteinase-9 (MMP-9) protein enhance neuroplasticity at the subacute stage of stroke. Here, we evaluated whether melatonin would modulate the PSD-95, GAP-43, and MMP-9 proteins in cultured neurons exposed to glutamate excitotoxicity and in rats subjected to experimental stroke. Adult male Sprague-Dawley rats were treated with melatonin (5 mg/kg) or vehicle at reperfusion onset after transient occlusion of the right middle cerebral artery (tMCAO) for 90 min. Animals were euthanized for Western immunoblot analyses for the PSD-95 and GAP-43 proteins and gelatin zymography for the MMP-9 activity at 7 days postinsult. Another set of animals was sacrificed for histologic and Golgi-Cox-impregnated sections at 28 days postinsult. In cultured neurons exposed to glutamate excitotoxicity, melatonin significantly upregulated the GAP-43 and PSD-95 expressions and improved dendritic aborizations (P<0.05, respectively). Relative to controls, melatonin-treated stroke animals caused a significant improvement in GAP-43 and PSD-95 expressions as well as the MMP-9 activity in the ischemic brain (P<0.05). Consequently, melatonin also significantly promoted the dendritic spine density and reduced infarction in the ischemic brain, and improved neurobehaviors as well at 28 days postinsult (P<0.05, respectively). Together, melatonin upregulates GAP-43, PSD-95, and MMP-9 proteins, which likely accounts for its actions to improve neuroplasticity in cultured neurons exposed to glutamate excitotoxicity and to enhance long-term neuroprotection, neuroplasticity, and brain remodeling in stroke rats. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Structure-based design of PDZ ligands as inhibitors of 5-HT(2A) receptor/PSD-95 PDZ1 domain interaction possessing anti-hyperalgesic activity.

    PubMed

    Vogrig, Alexandre; Dorr, Liam; Bouzidi, Naoual; Boucherle, Benjamin; Wattiez, Anne-Sophie; Cassier, Elisabeth; Vallon, Gary; Ripoche, Isabelle; Abrunhosa-Thomas, Isabelle; Marin, Philippe; Nauton, Lionel; Thery, Vincent; Courteix, Christine; Lian, Lu-Yun; Ducki, Sylvie

    2013-10-18

    Disrupting the interaction between the PDZ protein PSD-95 and the C-terminal domain of the 5-HT2A serotonin receptor has been shown to reduce hyperalgesia in a rodent model of neuropathic pain. Here, we designed and synthesized PDZ ligands capable of binding to the first PDZ domain (PDZ1) of the PSD-95 protein and evaluated their biological activity in vitro and in vivo. A series of substituted indoles was identified by docking simulations, and six novel analogues were synthesized. Three analogues displayed strong interactions with the first PDZ domain (PDZ1) of PDZ-95 in (1)H-(15)N heteronuclear single-quantum coherence (HSQC) experiments and two of them were able to inhibit the interaction between PSD-95 and the 5-HT2A receptor in vitro. We identified compound 8b as the analogue able to significantly suppress mechanical hyperalgesia in an experimental model of traumatic neuropathic pain in the rat. This effect was suppressed by the coadministration of the 5-HT2A receptor antagonist M100907, consistent with an inhibitory effect upon 5-HT2A receptor/PSD-95 interaction. Finally, we determined an NMR-restraint driven model structure for the PSD95 PDZ1/8b complex, which confirms that indole 8b binds to the putative PDZ-ligand binding site.

  10. Biochemical investigations of the mechanism of action of small molecules ZL006 and IC87201 as potential inhibitors of the nNOS-PDZ/PSD-95-PDZ interactions

    PubMed Central

    Bach, Anders; Pedersen, Søren W.; Dorr, Liam A.; Vallon, Gary; Ripoche, Isabelle; Ducki, Sylvie; Lian, Lu-Yun

    2015-01-01

    ZL006 and IC87201 have been presented as efficient inhibitors of the nNOS/PSD-95 protein-protein interaction and shown great promise in cellular experiments and animal models of ischemic stroke and pain. Here, we investigate the proposed mechanism of action of ZL006 and IC87201 using biochemical and biophysical methods, such as fluorescence polarization (FP), isothermal titration calorimetry (ITC), and 1H-15N HSQC NMR. Our data show that under the applied in vitro conditions, ZL006 and IC87201 do not interact with the PDZ domains of nNOS or PSD-95, nor inhibit the nNOS-PDZ/PSD-95-PDZ interface by interacting with the β-finger of nNOS-PDZ. Our findings have implications for further medicinal chemistry efforts of ZL006, IC87201 and analogues, and challenge the general and widespread view on their mechanism of action. PMID:26177569

  11. PDZ1 inhibitor peptide protects neurons against ischemia via inhibiting GluK2-PSD-95-module-mediated Fas signaling pathway.

    PubMed

    Yin, Xiao-Hui; Yan, Jing-Zhi; Yang, Guo; Chen, Li; Xu, Xiao-Feng; Hong, Xi-Ping; Wu, Shi-Liang; Hou, Xiao-Yu; Zhang, GuangYi

    2016-04-15

    Respecting the selective inhibition of peptides on protein-protein interactions, they might become potent methods in ischemic stroke therapy. In this study, we investigated the effect of PDZ1 inhibitor peptide on ischemic neuron apoptosis and the relative mechanism. Results showed that PDZ1 inhibitor peptide, which significantly disrupted GluK2-PSD-95 interaction, efficiently protected neuron from ischemia/reperfusion-induced apoptosis. Further, PDZ1 inhibited FasL expression, DISC assembly and activation of Caspase 8, Bid, Caspase 9 and Caspase 3 after global brain ischemia. Based on our previous report that GluK2-PSD-95 pathway increased FasL expression after global brain ischemia, the neuron protection effect of PDZ1 inhibitor peptide was considered to be achieved by disrupting GluK2-PSD-95 interaction and subsequently inhibiting FasL expression and Fas apoptosis pathway. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. PSD95 suppresses dendritic arbor development in mature hippocampal neurons by occluding the clustering of NR2B-NMDA receptors.

    PubMed

    Bustos, Fernando J; Varela-Nallar, Lorena; Campos, Matias; Henriquez, Berta; Phillips, Marnie; Opazo, Carlos; Aguayo, Luis G; Montecino, Martin; Constantine-Paton, Martha; Inestrosa, Nibaldo C; van Zundert, Brigitte

    2014-01-01

    Considerable evidence indicates that the NMDA receptor (NMDAR) subunits NR2A and NR2B are critical mediators of synaptic plasticity and dendritogenesis; however, how they differentially regulate these processes is unclear. Here we investigate the roles of the NR2A and NR2B subunits, and of their scaffolding proteins PSD-95 and SAP102, in remodeling the dendritic architecture of developing hippocampal neurons (2-25 DIV). Analysis of the dendritic architecture and the temporal and spatial expression patterns of the NMDARs and anchoring proteins in immature cultures revealed a strong positive correlation between synaptic expression of the NR2B subunit and dendritogenesis. With maturation, the pruning of dendritic branches was paralleled by a strong reduction in overall and synaptic expression of NR2B, and a significant elevation in synaptic expression of NR2A and PSD95. Using constructs that alter the synaptic composition, we found that either over-expression of NR2B or knock-down of PSD95 by shRNA-PSD95 augmented dendritogenesis in immature neurons. Reactivation of dendritogenesis could also be achieved in mature cultured neurons, but required both manipulations simultaneously, and was accompanied by increased dendritic clustering of NR2B. Our results indicate that the developmental increase in synaptic expression of PSD95 obstructs the synaptic clustering of NR2B-NMDARs, and thereby restricts reactivation of dendritic branching. Experiments with shRNA-PSD95 and chimeric NR2A/NR2B constructs further revealed that C-terminus of the NR2B subunit (tail) was sufficient to induce robust dendritic branching in mature hippocampal neurons, and suggest that the NR2B tail is important in recruiting calcium-dependent signaling proteins and scaffolding proteins necessary for dendritogenesis.

  13. Expression of the postsynaptic scaffold PSD-95 and development of synaptic physiology during giant terminal formation in the auditory brainstem of the chicken.

    PubMed

    Goyer, David; Fensky, Luisa; Hilverling, Anna Maria; Kurth, Stefanie; Kuenzel, Thomas

    2015-05-01

    In the avian nucleus magnocellularis (NM) endbulb of Held giant synapses develop from temporary bouton terminals. The molecular regulation of this process is not well understood. Furthermore, it is unknown how the postsynaptic specialization of the endbulb synapses develops. We therefore analysed expression of the postsynaptic scaffold protein PSD-95 during the transition from bouton-to-endbulb synapses. PSD-95 has been implicated in the regulation of the strength of glutamatergic synapses and could accordingly be of functional relevance for giant synapse formation. PSD-95 protein was expressed at synaptic sites in embryonic chicken auditory brainstem and upregulated between embryonic days (E)12 and E16. We applied immunofluorescence staining and confocal microscopy to quantify pre-and postsynaptic protein signals during bouton-to-endbulb transition. Giant terminal formation progressed along the tonotopic axis in NM, but was absent in low-frequency NM. We found a tonotopic gradient of postsynaptic PSD-95 signals in NM. Furthermore, PSD-95 immunosignals showed the greatest increase between E12 and E15, temporally preceding the bouton-to-endbulb transition. We then applied whole-cell electrophysiology to measure synaptic currents elicited by synaptic terminals during bouton-to-endbulb transition. With progressing endbulb formation postsynaptic currents rose more rapidly and synapses were less susceptible to short-term depression, but currents were not different in amplitude or decay-time constant. We conclude that development of presynaptic specializations follows postsynaptic development and speculate that the early PSD-95 increase could play a functional role in endbulb formation. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  14. Spinal serum-inducible and glucocorticoid-inducible kinase 1 mediates neuropathic pain via kalirin and downstream PSD-95-dependent NR2B phosphorylation in rats.

    PubMed

    Peng, Hsien-Yu; Chen, Gin-Den; Lai, Cheng-Yuan; Hsieh, Ming-Chun; Lin, Tzer-Bin

    2013-03-20

    The coupling of the spinal postsynaptic density-95 (PSD-95) with the glutamatergic N-methyl-d-aspartate receptor NR2B subunit and the subsequent NR2B phosphorylation contribute to pain-related plasticity. Increasing evidence reveals that kalirin, a Rho-guanine nucleotide exchange factor, modulates PSD-95-NR2B-dependent neuroplasticity. Our laboratory recently demonstrated that serum-inducible and glucocorticoid-inducible kinase 1 (SGK1) participates in inflammation-associated pain hypersensitivity by modulating spinal glutamatergic neurotransmission. Because kalirin is one of the proteins in PSD that is highly phosphorylated by various kinases, we tested whether kalirin could be a downstream target of spinal SGK1 that participates in neuropathic pain development via regulation of the PSD-95-NR2B coupling-dependent phosphorylation of NR2B. We observed that spinal nerve ligation (SNL, L5) in male Sprague Dawley rats resulted in behavioral allodynia, which was associated with phosphorylated SGK1 (pSGK1), kalirin, and phosphorylated NR2B (pNR2B) expression and an increase in pSGK1-kalirin-PSD-95-pNR2B coprecipitation in the ipsilateral dorsal horn (L4-L5). SNL-enhanced kalirin immunofluorescence was coincident with pSGK1, PSD-95, and pNR2B immunoreactivity. Small-interfering RNA (siRNA) that targeted spinal kalirin mRNA expression (10 μg, 10 μl; i.t.) reduced SNL-induced allodynia, kalirin and pNR2B expression, as well as kalirin-PSD-95 and PSD-95-pNR2B coupling and costaining without affecting SGK1 phosphorylation. Daily administration of GSK-650394 (an SGK1 antagonist; 100 nm, 10 μl, i.t.) not only exhibited effects similar to the kalirin mRNA-targeting siRNA but also attenuated pSGK1-kalirin costaining and SGK1-kalirin coupling. We suggest that nerve injury could induce spinal SGK1 phosphorylation that subsequently interacts with and upregulates kalirin to participate in neuropathic pain development via PSD-95-NR2B coupling-dependent NR2B phosphorylation.

  15. The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions*

    PubMed Central

    Belotti, Edwige; Polanowska, Jolanta; Daulat, Avais M.; Audebert, Stéphane; Thomé, Virginie; Lissitzky, Jean-Claude; Lembo, Frédérique; Blibek, Karim; Omi, Shizue; Lenfant, Nicolas; Gangar, Akanksha; Montcouquiol, Mireille; Santoni, Marie-Josée; Sebbagh, Michael; Aurrand-Lions, Michel; Angers, Stéphane; Kodjabachian, Laurent; Reboul, Jérome; Borg, Jean-Paul

    2013-01-01

    Protein–protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRβ, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions. PMID:23722234

  16. DHA-PC and PSD-95 decrease after loss of synaptophysin and before neuronal loss in patients with Alzheimer's disease.

    PubMed

    Yuki, Dai; Sugiura, Yuki; Zaima, Nobuhiro; Akatsu, Hiroyasu; Takei, Shiro; Yao, Ikuko; Maesako, Masato; Kinoshita, Ayae; Yamamoto, Takayuki; Kon, Ryo; Sugiyama, Keikichi; Setou, Mitsutoshi

    2014-11-20

    Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by senile plaques, neurofibrillary tangles, synaptic disruption, and neuronal loss. Several studies have demonstrated decreases of docosahexaenoic acid-containing phosphatidylcholines (DHA-PCs) in the AD brain. In this study, we used matrix-assisted laser desorption/ionization imaging mass spectrometry in postmortem AD brain to show that PC molecular species containing stearate and DHA, namely PC(18:0/22:6), was selectively depleted in the gray matter of patients with AD. Moreover, in the brain regions with marked amyloid β (Aβ) deposition, the magnitude of the PC(18:0/22:6) reduction significantly correlated with disease duration. Furthermore, at the molecular level, this depletion was associated with reduced levels of the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin. Interestingly, this reduction in PC(18:0/22:6) levels did not correlate with the degrees of Aβ deposition and neuronal loss in AD. The analysis of the correlations of key factors and disease duration showed that their effects on the disease time course were arranged in order as Aβ deposition, presynaptic disruption, postsynaptic disruption coupled with PC(18:0/22:6) reduction, and neuronal loss.

  17. DHA-PC and PSD-95 decrease after loss of synaptophysin and before neuronal loss in patients with Alzheimer's disease

    PubMed Central

    Yuki, Dai; Sugiura, Yuki; Zaima, Nobuhiro; Akatsu, Hiroyasu; Takei, Shiro; Yao, Ikuko; Maesako, Masato; Kinoshita, Ayae; Yamamoto, Takayuki; Kon, Ryo; Sugiyama, Keikichi; Setou, Mitsutoshi

    2014-01-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disease that is characterized by senile plaques, neurofibrillary tangles, synaptic disruption, and neuronal loss. Several studies have demonstrated decreases of docosahexaenoic acid-containing phosphatidylcholines (DHA-PCs) in the AD brain. In this study, we used matrix-assisted laser desorption/ionization imaging mass spectrometry in postmortem AD brain to show that PC molecular species containing stearate and DHA, namely PC(18:0/22:6), was selectively depleted in the gray matter of patients with AD. Moreover, in the brain regions with marked amyloid β (Aβ) deposition, the magnitude of the PC(18:0/22:6) reduction significantly correlated with disease duration. Furthermore, at the molecular level, this depletion was associated with reduced levels of the postsynaptic protein PSD-95 but not the presynaptic protein synaptophysin. Interestingly, this reduction in PC(18:0/22:6) levels did not correlate with the degrees of Aβ deposition and neuronal loss in AD. The analysis of the correlations of key factors and disease duration showed that their effects on the disease time course were arranged in order as Aβ deposition, presynaptic disruption, postsynaptic disruption coupled with PC(18:0/22:6) reduction, and neuronal loss. PMID:25410733

  18. Characterization of physiological phenotypes of dentate gyrus synapses of PDZ1/2 domain-deficient PSD-95-knockin mice.

    PubMed

    Nagura, Hitoshi; Doi, Tomoko; Fujiyoshi, Yoshinori

    2016-03-01

    The hippocampal formation is involved in several important brain functions of animals, such as memory formation and pattern separation, and the synapses in the dentate gyrus (DG) play critical roles as the first step in the hippocampal circuit. Previous studies have reported that mice with genetic modifications of the PDZ1/2 domains of postsynaptic density (PSD)-95 exhibit altered synaptic properties in the DG and impaired hippocampus-dependent behaviors. Based on the involvement of the DG in the regulation of behaviors, these data suggest that the abnormal behavior of these knockin (KI) mice is due partly to altered DG function. Precise understanding of the phenotypes of these mutant mice requires characterization of the synaptic properties of the DG, and here we provide detailed studies of DG synapses. We have demonstrated global changes in the PSD membrane-associated guanylate kinase expression pattern in the DG of mutant mice, and DG synapses in these mice exhibited increased long-term potentiation under a wide range of stimulus intensities, although the N-methyl-d-aspartic acid receptor dependence of the long-term potentiation was unchanged. Furthermore, our data also indicate increased silent synapses in the DG of the KI mice. These findings suggest that abnormal protein expression and physiological properties disrupt the function of DG neurons in these KI mice.

  19. Source memory in rats is impaired by an NMDA receptor antagonist but not by PSD95-nNOS protein-protein interaction inhibitors

    PubMed Central

    Smith, Alexandra E.; Xu, Zhili; Lai, Yvonne Y.; Kulkarni, Pushkar M.; Thakur, Ganesh A.; Hohmann, Andrea G.; Crystal, Jonathon D.

    2016-01-01

    Limitations of preclinical models of human memory contribute to the pervasive view that rodent models do not adequately predict therapeutic efficacy in producing cognitive impairments or improvements in humans. We used a source-memory model (i.e. a representation of the origin of information) we developed for use in rats to evaluate possible drug-induced impairments of both spatial memory and higher order memory functions in the same task. Memory impairment represents a major barrier to use of NMDAR antagonists as pharmacotherapies. The scaffolding protein postsynaptic density 95kDa (PSD95) links NMDARs to the neuronal enzyme nitric oxide synthase (nNOS), which catalyzes production of the signaling molecule nitric oxide (NO). Therefore, interrupting PSD95-nNOS protein-protein interactions downstream of NMDARs represents a novel therapeutic strategy to interrupt NMDAR-dependent NO signaling while bypassing unwanted side effects of NMDAR antagonists. We hypothesized that the NMDAR antagonist MK-801 would impair source memory. We also hypothesized that PSD95-nNOS inhibitors (IC87201 and ZL006) would lack the profile of cognitive impairment associated with global NMDAR antagonists. IC87201 and ZL006 suppressed NMDA-stimulated formation of cGMP, a marker of NO production, in cultured hippocampal neurons. MK-801, at doses that did not impair motor function, impaired source memory under conditions in which spatial memory was spared. Thus, source memory was more vulnerable than spatial memory to impairment. By contrast, PSD95-nNOS inhibitors, IC87201 and ZL006, administered at doses that are behaviorally effective in rats, spared source memory, spatial memory, and motor function. Thus, PSD95-nNOS inhibitors are likely to exhibit favorable therapeutic ratios compared to NMDAR antagonists. PMID:26909849

  20. AMPA receptor regulation mechanisms: future target for safer neuroprotective drugs.

    PubMed

    Jayakar, Selwyn S; Dikshit, Madhu

    2004-06-01

    The post-synaptic AMPA receptors play an important role in mediating fast excitatory transmission in the mammalian brain. Over-activated AMPA receptors induce excitotoxicity, implicated in a number of Chronic neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and AIDS encephalitis. AMPA receptor antagonists offer protection against neurodegeneration in the experimental models even if they are given 24 h after the injury. Because AMPA receptors seem to be involved in the neurodegenerative diseases, modulating the activity of the AMPA receptors could be an attractive approach to reduce or prevent excitotoxicity. Studies conducted recently have exhibited a number of new mechanisms for AMPA receptor regulation. Modulations of these were found to have protective implications. AMPA receptor depolarization and desensitization are protective to the neurons. Receptor desensitization depends on the receptor subunit composition. The R/G editing site and the flip/flop cassettes in AMPA receptor subunits contribute to a great extent in receptor desensitization and recovery rates. Molecules that could quicken receptor desensitization or delay recovery could be of use. AMPA receptors limit neuronal entry of Ca2+ ions by regulating Ca2+-permeability. Ca2+-permeable receptor channels are made up of GluR1, GluR3, or GluR4 subunits, whereas presence of the GluR2 subunit restricts Ca2+ entry and renders the receptor Ca2+-impermeable. GluR2 levels, however, experience a fall after neuronal insult rendering the AMPA receptors Ca2+-permeable, thus factors that could interfere with this event might prove to be very beneficial against excitotoxicity. AMPA receptor clusters are stabilized by PSD-95, which requires palmitoylation at two sites. Targeting palmitoylation of the PSD-95 can also be a useful approach to disperse AMPA clusters at the synapse. In the perisynaptic region, mGluRs are present a little away from the synapse and are among the glutamate

  1. CDKL5 ensures excitatory synapse stability by reinforcing NGL-1-PSD95 interaction in the postsynaptic compartment and is impaired in patient iPSC-derived neurons.

    PubMed

    Ricciardi, Sara; Ungaro, Federica; Hambrock, Melanie; Rademacher, Nils; Stefanelli, Gilda; Brambilla, Dario; Sessa, Alessandro; Magagnotti, Cinzia; Bachi, Angela; Giarda, Elisa; Verpelli, Chiara; Kilstrup-Nielsen, Charlotte; Sala, Carlo; Kalscheuer, Vera M; Broccoli, Vania

    2012-09-01

    Mutations of the cyclin-dependent kinase-like 5 (CDKL5) and netrin-G1 (NTNG1) genes cause a severe neurodevelopmental disorder with clinical features that are closely related to Rett syndrome, including intellectual disability, early-onset intractable epilepsy and autism. We report here that CDKL5 is localized at excitatory synapses and contributes to correct dendritic spine structure and synapse activity. To exert this role, CDKL5 binds and phosphorylates the cell adhesion molecule NGL-1. This phosphorylation event ensures a stable association between NGL-1 and PSD95. Accordingly, phospho-mutant NGL-1 is unable to induce synaptic contacts whereas its phospho-mimetic form binds PSD95 more efficiently and partially rescues the CDKL5-specific spine defects. Interestingly, similarly to rodent neurons, iPSC-derived neurons from patients with CDKL5 mutations exhibit aberrant dendritic spines, thus suggesting a common function of CDKL5 in mice and humans.

  2. [Effects of electroacupuncture at different acupoints on learning and memory ability and PSD-95 protein expression on hippocampus CA1 in rats with autism].

    PubMed

    Zhang, Xue-Jun; Wu, Qiang

    2013-07-01

    To explore the effect mechanism of electroacupuncture (EA) at Changqiang (GV 1) or Baihui (GV 20) on autism based on molecular biology. The autism model was established by intraperitoneal injection of sodium valproate (VPA) in Wistar pregnant rats. Forty young rats with autism were selected and randomly divided into a model group, a non-acupoint group, an electroacupuncture at "Changqiang" (GV 1) (EAGV 1 for short) group and an electroacupuncture at "Baihui" (GV 20) (EAGV 20 for short) group. Another 10 normal young rats were selected as a blank group. In the EAGV 1 group, acupuncture was applied at Houhai [as Changqiang (GV 1)], then EA apparatus was connected with continuous wave, 2 Hz, 20 min, once a day for consecutive 20 days. The same EA manipulation as EAGV 1 group was used in the EAGV 20 group where "Baihui" (GV 20) was selected and non-acupoint group where non-acupoint in the right rib was selected. Blank group and model group were reared under the same conditions without any intervention. The escape latency and the ratio of swimming distance in platform quadrant to total swimming distance in each group were observed by using Morris water maze, and the PSD-95 protein expression in hippocampal CA 1 was measured by immunohistochemical techniques. Compared with the blank group, the escape latency in the model group and the non-acupoint group lengthened (both P < 0.05), the ratio of swimming distance in platform quadrant to total swimming distance was decreased (both P < 0.05), the PSD-95 protein expression was decreased (P < 0.05). Compared with the model group, the escape latency in the EAGV 1 group and the EAGV 20 group were decreased (both P < 0.05), the ratio of swimming distance in platform quadrant to total swimming distance was increased, the PSD-95 protein expression was increased (both P < 0.05). But the escape latency, the ratio of swimming distance in platform quadrant to total swimming distance and the PSD-95 protein expression had no significant

  3. Small-molecule inhibitors at the PSD-95/nNOS interface protect against glutamate-induced neuronal atrophy in primary cortical neurons.

    PubMed

    Doucet, M V; O'Toole, E; Connor, T; Harkin, A

    2015-08-20

    Glutamate and nitric oxide (NO) are important regulators of dendrite and axon development in the central nervous system. Excess glutamatergic stimulation is a feature of many pathological conditions and manifests in neuronal atrophy and shrinkage with eventual neurodegeneration and cell death. Here we demonstrate that treatment of cultured primary cortical rat neurons for 24h with glutamate (500μM) or N-methyl-d-aspartate (NMDA) (100-500μM) combined with glycine suppresses neurite outgrowth. A similar reduction of neurite outgrowth was observed with the NO precursor l-arginine and NO donor sodium nitroprusside (SNP) (100 and 300μM). The NMDA-receptor (NMDA-R) antagonists ketamine and MK-801 (10nM) counteracted the NMDA/glycine-induced reduction in neurite outgrowth and the neuronal NO synthase (nNOS) inhibitor 1-[2-(trifluoromethyl)phenyl] imidazole (TRIM) (100nM) counteracted both the NMDA/glycine and l-arginine-induced decreases in neurite outgrowth. Furthermore, targeting soluble guanylate cyclase (sGC), a downstream target of NO, with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10μM) also protected against l-arginine-induced decreases in neurite outgrowth. Since the NMDA-R is functionally coupled to nNOS via the postsynaptic protein 95kDa (PSD-95), inhibitors of the PSD-95/nNOS interaction were tested for their ability to protect against glutamate-induced suppression in neurite outgrowth. Treatment with the small-molecule inhibitors of the PSD-95/nNOS interface 2-((1H-benzo[d] [1,2,3]triazol-5-ylamino) methyl)-4,6-dichlorophenol (IC87201) (10 and 100nM) and 4-(3,5-dichloro-2-hydroxy-benzylamino)-2-hydroxybenzoic acid (ZL-006) (10 and 100nM) attenuated NMDA/glycine-induced decreases in neurite outgrowth. These data support the hypothesis that targeting the NMDA-R/PSD-95/nNOS interaction downstream of NMDA-R promotes neurotrophic effects by preventing neurite shrinkage in response to excess glutamatergic stimulation. The PSD-95/n

  4. Binding of amyloid beta peptide to beta2 adrenergic receptor induces PKA-dependent AMPA receptor hyperactivity.

    PubMed

    Wang, Dayong; Govindaiah, G; Liu, Ruijie; De Arcangelis, Vania; Cox, Charles L; Xiang, Yang K

    2010-09-01

    Progressive decrease in neuronal function is an established feature of Alzheimer's disease (AD). Previous studies have shown that amyloid beta (Abeta) peptide induces acute increase in spontaneous synaptic activity accompanied by neurotoxicity, and Abeta induces excitotoxic neuronal death by increasing calcium influx mediated by hyperactive alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. An in vivo study has revealed subpopulations of hyperactive neurons near Abeta plaques in mutant amyloid precursor protein (APP)-transgenic animal model of Alzheimer's disease (AD) that can be normalized by an AMPA receptor antagonist. In the present study, we aim to determine whether soluble Abeta acutely induces hyperactivity of AMPA receptors by a mechanism involving beta(2) adrenergic receptor (beta(2)AR). We found that the soluble Abeta binds to beta(2)AR, and the extracellular N terminus of beta(2)AR is critical for the binding. The binding is required to induce G-protein/cAMP/protein kinase A (PKA) signaling, which controls PKA-dependent phosphorylation of GluR1 and beta(2)AR, and AMPA receptor-mediated excitatory postsynaptic currents (EPSCs). beta(2)AR and GluR1 also form a complex comprising postsynaptic density protein 95 (PSD95), PKA and its anchor AKAP150, and protein phosphotase 2A (PP2A). Both the third intracellular (i3) loop and C terminus of beta(2)AR are required for the beta(2)AR/AMPA receptor complex. Abeta acutely induces PKA phosphorylation of GluR1 in the complex without affecting the association between two receptors. The present study reveals that non-neurotransmitter Abeta has a binding capacity to beta(2)AR and induces PKA-dependent hyperactivity in AMPA receptors.

  5. TARP subtypes differentially and dose-dependently control synaptic AMPA receptor gating.

    PubMed

    Milstein, Aaron D; Zhou, Wei; Karimzadegan, Siavash; Bredt, David S; Nicoll, Roger A

    2007-09-20

    A family of transmembrane AMPA receptor regulatory proteins (TARPs) profoundly affects the trafficking and gating of AMPA receptors (AMPARs). Although TARP subtypes are differentially expressed throughout the CNS, it is unclear whether this imparts functional diversity to AMPARs in distinct neuronal populations. Here, we examine the effects of each TARP subtype on the kinetics of AMPAR gating in heterologous cells and in neurons. We report a striking heterogeneity in the effects of TARP subtypes on AMPAR deactivation and desensitization, which we demonstrate controls the time course of synaptic transmission. In addition, we find that some TARP subtypes dramatically slow AMPAR activation kinetics. Synaptic AMPAR kinetics also depend on TARP expression level, suggesting a variable TARP/AMPAR stoichiometry. Analysis of quantal synaptic transmission in a TARP gamma-4 knockout (KO) mouse corroborates our expression data and demonstrates that TARP subtype-specific gating of AMPARs contributes to the kinetics of native AMPARs at central synapses.

  6. Employment of Molecularly Imprinted Polymers to High-Throughput Screen nNOS-PSD-95 Interruptions: Structure and Dynamics Investigations on Monomer-Template Complexation.

    PubMed

    Wang, Yongwei; Zhao, Ting; Dai, Peng; Jiang, Nan; Li, Fei

    2016-03-16

    Molecularly imprinted polymers (MIPs) are employed to screen nNOS-PSD-95 (neuronal nitric oxide synthase post-synaptic density protein-95) interruptions. 5-(3,5-Dichloro-2-hydroxybenzylamino)-2-hydroxybenzoic acid (ZL006; a potential drug candidate for the treatment of stroke, depression, and pain) is employed as a template. Four kinds of functional monomers (2-VP: 2-vinylpyridine; 4-VP: 4-vinylpyridine; MMA: methyl methacrylate; and MAAM: methacrylamide) are designed, and their complexation with ZL006 in various solvents (methanol, acetonitrile, toluene, chloroform) is investigated by molecular dynamics simulations and quantum mechanics calculations. Both 4-VP and MAAM have stronger interactions with ZL006 than those of 2-VP and MMA. The appropriate ratio of monomer to template is 3:1. Intermolecular hydrogen bonds play a dominant role in monomer-template complexation. Ideal solvents are toluene and chloroform, and the solvation effect on monomer-template complexation is revealed. Both molecular modeling and adsorption experiments demonstrate that as-synthesized ZL006-MIP with 4-VP as a monomer has better selectivity than that employing MAAM to screen for nNOS-PSD-95 interruptions. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Depalmitoylation preferentially downregulates AMPA induced Ca2+ signaling and neurotoxicity in motor neurons.

    PubMed

    Krishnamurthy, Karthik; Mehta, Bhupesh; Singh, Mahendra; Tewari, Bhanu P; Joshi, Preeti G; Joshi, Nanda B

    2013-09-05

    Excessive activation of AMPA receptor has been implicated in motor neuron degeneration in amyotrophic lateral sclerosis (ALS). However, it is not clear why motor neurons are preferentially sensitive to AMPA receptor mediated excessive [Ca(2+)]i rise and excitotoxicity. In the present study we examined whether palmitoylation regulates Ca(2+) permeability of AMPA receptor and excitotoxicity in cultured spinal cord neurons. We adapted chronic 2-bromopalmitate (2-BrP) treatment to achieve depalmitoylation and examined its effect on the cytotoxicity in spinal cord neurons exposed to AMPA. The change in AMPA induced signaling and cytotoxicity in motor neurons and other spinal neurons under identical conditions of exposure to AMPA was studied. 2-BrP treatment inhibited AMPA induced rise in [Ca(2+)]i and cytotoxicity in both types of neurons but the degree of inhibition was significantly higher in motor neurons as compared to other spinal neurons. The AMPA induced [Na(+)]i rise was moderately affected in both type of neurons on depalmitoylation. Depalmitoylation reduced the expression levels of AMPA receptor subunits (GluR1 and GluR2) and also PSD-95 but stargazin levels remained unaffected. Our results demonstrate that 2-BrP attenuates AMPA receptor activated Ca(2+) signaling and cytotoxicity preferentially in motor neurons and suggest that AMPA receptor modulation by depalmitoylation could play a significant role in preventing motor neuron degeneration. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. A thermodynamic study of the third PDZ domain of MAGUK neuronal protein PSD-95 reveals a complex three-state folding behavior.

    PubMed

    Murciano-Calles, Javier; Martinez, Jose C; Marin-Argany, Marta; Villegas, Sandra; Cobos, Eva S

    2014-01-01

    The relevance of the C-terminal α helix of the PDZ3 domain of PSD95 in its unfolding process has been explored by achieving the thermodynamic characterization of a construct where the sequence of the nine residues corresponding to such motif has been deleted. Calorimetric traces at neutral pH require the application of a three-state model displaying three different equilibrium processes in which the intermediate state self-associates upon heating, being stable and populated in a wide temperature range. Temperature scans followed by circular dichroism, Fourier transform infrared spectroscopy and dynamic light scattering support the presence of such oligomeric-partially folded species. This study reveals that the deletion of the α3-helix sequence results in a more complex description of the domain unfolding.

  9. Association of mouse Dlg4 (PSD-95) gene deletion and human DLG4 gene variation with phenotypes relevant to autism spectrum disorders and Williams' syndrome.

    PubMed

    Feyder, Michael; Karlsson, Rose-Marie; Mathur, Poonam; Lyman, Matthew; Bock, Roland; Momenan, Reza; Munasinghe, Jeeva; Scattoni, Maria Luisa; Ihne, Jessica; Camp, Marguerite; Graybeal, Carolyn; Strathdee, Douglas; Begg, Alison; Alvarez, Veronica A; Kirsch, Peter; Rietschel, Marcella; Cichon, Sven; Walter, Henrik; Meyer-Lindenberg, Andreas; Grant, Seth G N; Holmes, Andrew

    2010-12-01

    Research is increasingly linking autism spectrum disorders and other neurodevelopmental disorders to synaptic abnormalities ("synaptopathies"). PSD-95 (postsynaptic density-95, DLG4) orchestrates protein-protein interactions at excitatory synapses and is a major functional bridge interconnecting a neurexinneuroligin-SHANK pathway implicated in autism spectrum disorders. The authors characterized behavioral, dendritic, and molecular phenotypic abnormalities relevant to autism spectrum disorders in mice with PSD-95 deletion (Dlg4⁻(/)⁻). The data from mice led to the identification of single-nucleotide polymorphisms (SNPs) in human DLG4 and the examination of associations between these variants and neural signatures of Williams' syndrome in a normal population, using functional and structural neuroimaging. Dlg4⁻(/)⁻ showed increased repetitive behaviors, abnormal communication and social behaviors, impaired motor coordination, and increased stress reactivity and anxiety-related responses. Dlg4⁻(/)⁻ had subtle dysmorphology of amygdala dendritic spines and altered forebrain expression of various synaptic genes, including Cyln2, which regulates cytoskeletal dynamics and is a candidate gene for Williams' syndrome. A signifi-cant association was observed between variations in two human DLG4 SNPs and reduced intraparietal sulcus volume and abnormal cortico-amygdala coupling, both of which characterize Williams' syndrome. These findings demonstrate that DLG4 gene disruption in mice produces a complex range of behavioral and molecular abnormalities relevant to autism spectrum disorders and Williams' syndrome. The study provides an initial link between human DLG4 gene variation and key neural endophenotypes of Williams' syndrome and perhaps corticoamygdala regulation of emotional and social processes more generally.

  10. Association of Mouse Dlg4 (PSD-95) Gene Deletion and Human DLG4 Gene Variation With Phenotypes Relevant to Autism Spectrum Disorders and Williams’ Syndrome

    PubMed Central

    Feyder, Michael; Karlsson, Rose-Marie; Mathur, Poonam; Lyman, Matthew; Bock, Roland; Momenan, Reza; Munasinghe, Jeeva; Scattoni, Maria Luisa; Ihne, Jessica; Camp, Marguerite; Graybeal, Carolyn; Strathdee, Douglas; Begg, Alison; Alvarez, Veronica A.; Kirsch, Peter; Rietschel, Marcella; Cichon, Sven; Walter, Henrik; Meyer-Lindenberg, Andreas; Grant, Seth G.N.; Holmes, Andrew

    2011-01-01

    Objective Research is increasingly linking autism spectrum disorders and other neurodevelopmental disorders to synaptic abnormalities (“synaptopathies”). PSD-95 (postsynaptic density-95, DLG4) orchestrates protein-protein interactions at excitatory synapses and is a major functional bridge interconnecting a neurexin-neuroligin-SHANK pathway implicated in autism spectrum disorders. Method The authors characterized behavioral, dendritic, and molecular phenotypic abnormalities relevant to autism spectrum disorders in mice with PSD-95 deletion (Dlg4−/−). The data from mice led to the identification of single-nucleotide polymorphisms (SNPs) in human DLG4 and the examination of associations between these variants and neural signatures of Williams’ syndrome in a normal population, using functional and structural neuroimaging. Results Dlg4−/− showed increased repetitive behaviors, abnormal communication and social behaviors, impaired motor coordination, and increased stress reactivity and anxiety-related responses. Dlg4−/− had subtle dysmorphology of amygdala dendritic spines and altered forebrain expression of various synaptic genes, including Cyln2, which regulates cytoskeletal dynamics and is a candidate gene for Williams’ syndrome. A significant association was observed between variations in two human DLG4 SNPs and reduced intraparietal sulcus volume and abnormal cortico-amygdala coupling, both of which characterize Williams’ syndrome. Conclusions These findings demonstrate that Dlg4 gene disruption in mice produces a complex range of behavioral and molecular abnormalities relevant to autism spectrum disorders and Williams’ syndrome. The study provides an initial link between human DLG4 gene variation and key neural endophenotypes of Williams’ syndrome and perhaps cortico-amygdala regulation of emotional and social processes more generally. PMID:20952458

  11. Increased Excitatory Synaptic Transmission of Dentate Granule Neurons in Mice Lacking PSD-95-Interacting Adhesion Molecule Neph2/Kirrel3 during the Early Postnatal Period.

    PubMed

    Roh, Junyeop D; Choi, Su-Yeon; Cho, Yi Sul; Choi, Tae-Yong; Park, Jong-Sil; Cutforth, Tyler; Chung, Woosuk; Park, Hanwool; Lee, Dongsoo; Kim, Myeong-Heui; Lee, Yeunkum; Mo, Seojung; Rhee, Jeong-Seop; Kim, Hyun; Ko, Jaewon; Choi, Se-Young; Bae, Yong Chul; Shen, Kang; Kim, Eunjoon; Han, Kihoon

    2017-01-01

    Copy number variants and point mutations of NEPH2 (also called KIRREL3) gene encoding an immunoglobulin (Ig) superfamily adhesion molecule have been linked to autism spectrum disorders, intellectual disability and neurocognitive delay associated with Jacobsen syndrome, but the physiological roles of Neph2 in the mammalian brain remain largely unknown. Neph2 is highly expressed in the dentate granule (DG) neurons of the hippocampus and is localized in both dendrites and axons. It was recently shown that Neph2 is required for the formation of mossy fiber filopodia, the axon terminal structure of DG neurons forming synapses with GABAergic neurons of CA3. In contrast, however, it is unknown whether Neph2 also has any roles in the postsynaptic compartments of DG neurons. We here report that, through its C-terminal PDZ domain-binding motif, Neph2 directly interacts with postsynaptic density (PSD)-95, an abundant excitatory postsynaptic scaffolding protein. Moreover, Neph2 protein is detected in the brain PSD fraction and interacts with PSD-95 in synaptosomal lysates. Functionally, loss of Neph2 in mice leads to age-specific defects in the synaptic connectivity of DG neurons. Specifically, Neph2(-/-) mice show significantly increased spontaneous excitatory synaptic events in DG neurons at postnatal week 2 when the endogenous Neph2 protein expression peaks, but show normal excitatory synaptic transmission at postnatal week 3. The evoked excitatory synaptic transmission and synaptic plasticity of medial perforant pathway (MPP)-DG synapses are also normal in Neph2(-/-) mice at postnatal week 3, further confirming the age-specific synaptic defects. Together, our results provide some evidence for the postsynaptic function of Neph2 in DG neurons during the early postnatal period, which might be implicated in neurodevelopmental and cognitive disorders caused by NEPH2 mutations.

  12. Increased Excitatory Synaptic Transmission of Dentate Granule Neurons in Mice Lacking PSD-95-Interacting Adhesion Molecule Neph2/Kirrel3 during the Early Postnatal Period

    PubMed Central

    Roh, Junyeop D.; Choi, Su-Yeon; Cho, Yi Sul; Choi, Tae-Yong; Park, Jong-Sil; Cutforth, Tyler; Chung, Woosuk; Park, Hanwool; Lee, Dongsoo; Kim, Myeong-Heui; Lee, Yeunkum; Mo, Seojung; Rhee, Jeong-Seop; Kim, Hyun; Ko, Jaewon; Choi, Se-Young; Bae, Yong Chul; Shen, Kang; Kim, Eunjoon; Han, Kihoon

    2017-01-01

    Copy number variants and point mutations of NEPH2 (also called KIRREL3) gene encoding an immunoglobulin (Ig) superfamily adhesion molecule have been linked to autism spectrum disorders, intellectual disability and neurocognitive delay associated with Jacobsen syndrome, but the physiological roles of Neph2 in the mammalian brain remain largely unknown. Neph2 is highly expressed in the dentate granule (DG) neurons of the hippocampus and is localized in both dendrites and axons. It was recently shown that Neph2 is required for the formation of mossy fiber filopodia, the axon terminal structure of DG neurons forming synapses with GABAergic neurons of CA3. In contrast, however, it is unknown whether Neph2 also has any roles in the postsynaptic compartments of DG neurons. We here report that, through its C-terminal PDZ domain-binding motif, Neph2 directly interacts with postsynaptic density (PSD)-95, an abundant excitatory postsynaptic scaffolding protein. Moreover, Neph2 protein is detected in the brain PSD fraction and interacts with PSD-95 in synaptosomal lysates. Functionally, loss of Neph2 in mice leads to age-specific defects in the synaptic connectivity of DG neurons. Specifically, Neph2−/− mice show significantly increased spontaneous excitatory synaptic events in DG neurons at postnatal week 2 when the endogenous Neph2 protein expression peaks, but show normal excitatory synaptic transmission at postnatal week 3. The evoked excitatory synaptic transmission and synaptic plasticity of medial perforant pathway (MPP)-DG synapses are also normal in Neph2−/− mice at postnatal week 3, further confirming the age-specific synaptic defects. Together, our results provide some evidence for the postsynaptic function of Neph2 in DG neurons during the early postnatal period, which might be implicated in neurodevelopmental and cognitive disorders caused by NEPH2 mutations. PMID:28381988

  13. N-terminal SAP97 isoforms differentially regulate synaptic structure and postsynaptic surface pools of AMPA receptors.

    PubMed

    Goodman, Lucy; Baddeley, David; Ambroziak, Wojciech; Waites, Clarissa L; Garner, Craig C; Soeller, Christian; Montgomery, Johanna M

    2017-02-28

    The location and density of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is controlled by scaffolding proteins within the postsynaptic density (PSD). SAP97 is a PSD protein with two N-terminal isoforms, α and β, that have opposing effects on synaptic strength thought to result from differential targeting of AMPA receptors into distinct synaptic versus extrasynaptic locations, respectively. In this study, we have applied dSTORM super resolution imaging in order to localize the synaptic and extrasynaptic pools of AMPA receptors in neurons expressing α or βSAP97. Unexpectedly, we observed that both α and βSAP97 enhanced the localization of AMPA receptors at synapses. However, this occurred via different mechanisms: αSAP97 increased PSD size and consequently the number of receptor binding sites, whilst βSAP97 increased synaptic receptor cluster size and surface AMPA receptor density at the PSD edge and surrounding perisynaptic sites without changing PSD size. αSAP97 also strongly enlarged presynaptic active zone protein clusters, consistent with both presynaptic and postsynaptic enhancement underlying the previously observed αSAP97-induced increase in AMPA receptor-mediated currents. In contrast, βSAP97-expressing neurons increased the proportion of immature filopodia that express higher levels of AMPA receptors, decreased the number of functional presynaptic terminals, and also reduced the size of the dendritic tree and delayed the maturation of mushroom spines. Our data reveal that SAP97 isoforms can specifically regulate surface AMPA receptor nanodomain clusters, with βSAP97 increasing extrasynaptic receptor domains at peri-synaptic and filopodial sites. Moreover, βSAP97 negatively regulates synaptic maturation both structurally and functionally. These data support diverging presynaptic and postsynaptic roles of SAP97 N-terminal isoforms in synapse maturation and plasticity. As numerous splice isoforms exist in

  14. Post-synaptic density-95 (PSD-95) binding capacity of G-protein-coupled receptor 30 (GPR30), an estrogen receptor that can be identified in hippocampal dendritic spines.

    PubMed

    Akama, Keith T; Thompson, Louisa I; Milner, Teresa A; McEwen, Bruce S

    2013-03-01

    The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.

  15. Post-synaptic Density-95 (PSD-95) Binding Capacity of G-protein-coupled Receptor 30 (GPR30), an Estrogen Receptor That Can Be Identified in Hippocampal Dendritic Spines*

    PubMed Central

    Akama, Keith T.; Thompson, Louisa I.; Milner, Teresa A.; McEwen, Bruce S.

    2013-01-01

    The estrogen 17β-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity. PMID:23300088

  16. Homeostatic regulation of AMPA receptor trafficking and degradation by light-controlled single synaptic activation

    PubMed Central

    Hou, Qingming; Gilbert, James; Man, Heng-Ye

    2011-01-01

    During homeostatic adjustment in response to alterations in neuronal activity, synaptic expression of AMPA receptors (AMPARs) is globally tuned up- or down so that the neuronal activity is restored to a physiological range. Given that a central neuron receives multiple presynaptic inputs, whether and how AMPAR synaptic expression is homeostatically regulated at individual synapses remains unclear. In cultured hippocampal neurons, we report that when activity of an individual presynaptic terminal is selectively elevated by light-controlled excitation, AMPAR abundance at the excited synapses is selectively down-regulated in an NMDAR-dependent manner. The reduction in surface AMPARs is accompanied by enhanced receptor endocytosis and dependent on proteasomal activity. Synaptic activation also leads to a site-specific increase in the ubiquitin ligase Nedd4 and polyubiquitination levels, consistent with AMPAR ubiquitination and degradation in the spine. These results indicate that AMPAR accumulation at individual synapses is subject to autonomous homeostatic regulation in response to synaptic activity. PMID:22153376

  17. Intrathecal injection of the peptide myr-NR2B9c attenuates bone cancer pain via perturbing N-methyl-D-aspartate receptor-PSD-95 protein interactions in mice.

    PubMed

    Liu, Yue; Cui, Xinlong; Sun, Yu-E; Yang, Xuli; Ni, Kun; Zhou, Yu; Ma, Zhengliang; Gu, Xiaoping

    2014-06-01

    N-methyl-D-aspartate receptor (NMDARs)-dependent central sensitization plays an important role in cancer pain. Binding of NMDAR subunit 2B (NR2B) by postsynaptic density protein-95 (PSD-95) can couple NMDAR activity to intracellular enzymes, such as neuronal nitric oxide synthase (nNOS), facilitate downstream signaling pathways, and modulate NMDAR stability, contributing to synaptic plasticity. In this study, we investigated whether perturbing the specific interaction between spinal NR2B-containing NMDAR and PSD-95, using a peptide-mimetic strategy, could attenuate bone cancer-related pain behaviors. Osteosarcoma cells were implanted into the intramedullary space of the right femurs of C3H/HeJ mice to induce progressive bone cancer-related pain behaviors. Western blotting was applied to examine the expression of spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95. We further investigated the effects of intrathecal injection of the mimetic peptide Myr-NR2B9c, which competitively disrupts the interaction between PSD-95 and NR2B, on nociceptive behaviors and on the upregulation of phospho-Tyr1472 NR2B, nNOS, and PSD-95 associated with bone cancer pain in the spinal cord. Inoculation of osteosarcoma cells induced progressive bone cancer pain and resulted in a significant upregulation of phospho-Tyr1472 NR2B, nNOS, and PSD-95. Intrathecal administration of Myr-NR2B9c attenuated bone cancer-evoked mechanical allodynia, thermal hyperalgesia, and reduced spinal phospho-Tyr1472 NR2B, nNOS, and PSD-95 expression. Intrathecal administration of Myr-NR2B9c reduced bone cancer pain. Internalization of spinal NR2B and dissociation NR2B-containing NMDARs activation from downstream nNOS signaling may contribute to the analgesic effects of Myr-NR2B9c. This approach may circumvent the negative consequences associated with blocking NMDARs, and may be a novel strategy for the treatment of bone cancer pain.

  18. Hippocampal AMPA receptor gating controlled by both TARP and cornichon proteins.

    PubMed

    Kato, Akihiko S; Gill, Martin B; Ho, Michelle T; Yu, Hong; Tu, Yuan; Siuda, Edward R; Wang, He; Qian, Yue-Wei; Nisenbaum, Eric S; Tomita, Susumu; Bredt, David S

    2010-12-22

    Transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon proteins (CNIH-2/3) independently modulate AMPA receptor trafficking and gating. However, the potential for interactions of these subunits within an AMPA receptor complex is unknown. Here, we find that TARPs γ-4, γ-7, and γ-8, but not γ-2, γ-3, or γ-5, cause AMPA receptors to "resensitize" upon continued glutamate application. With γ-8, resensitization occurs with all GluA subunit combinations; however, γ-8-containing hippocampal neurons do not display resensitization. In recombinant systems, CNIH-2 abrogates γ-8-mediated resensitization and modifies AMPA receptor pharmacology and gating to match that of hippocampal neurons. In hippocampus, γ-8 and CNIH-2 associate in postsynaptic densities and CNIH-2 protein levels are markedly diminished in γ-8 knockout mice. Manipulating neuronal CNIH-2 levels modulates the electrophysiological properties of extrasynaptic and synaptic γ-8-containing AMPA receptors. Thus, γ-8 and CNIH-2 functionally interact with common hippocampal AMPA receptor complexes to modulate synergistically kinetics and pharmacology.

  19. Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices.

    PubMed

    Bolea, S; Avignone, E; Berretta, N; Sanchez-Andres, J V; Cherubini, E

    1999-05-01

    Glutamate controls the induction of GABA-mediated giant depolarizing potentials through AMPA receptors in neonatal rat hippocampal slices. Giant depolarizing potentials (GDPs) are generated by the interplay of the depolarizing action of GABA and glutamate. In this study, single and dual whole cell recordings (in current-clamp configuration) were performed from CA3 pyramidal cells in hippocampal slices obtained from postnatal (P) days P1- to P6-old rats to evaluate the role of ionotropic glutamate receptors in GDP generation. Superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (10-40 microM) completely blocked GDPs. However, in the presence of CNQX, it was still possible to re-induce the appearance of GDPs with GABA (20 microM) or (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxadepropionate (AMPA) (5 microM). This effect was prevented by the more potent and selective AMPA receptor antagonist GYKI 53655 (50-100 microM). In the presence of GYKI 53655, both kainic or domoic acid (0.1-1 microM) were unable to induce GDPs. In contrast, bath application of D-(-)-2-amino-5-phosphonopentanoic acid (50 microM) or (+)-3-(2carboxy-piperazin-4-yl)-propyl-L-phosphonic acid (20 microM) produced only a 37 +/- 9% (SE) and 36 +/- 11% reduction in GDPs frequency, respectively. Cyclothiazide, a selective blocker of AMPA receptor desensitization, increased GDP frequency by 76 +/- 14%. Experiments were also performed with an intracellular solution containing KF to block GABAA receptor-mediated responses. In these conditions, a glutamatergic component of GDP was revealed. GDPs could still be recorded synchronous with those detected simultaneously with KCl-filled electrodes, although their amplitude was smaller. Similar results were found in pair recordings obtained from minislices containing only a small portion of the CA3 area. These data suggest that GDP generation requires activation of AMPA receptors by local release of glutamate from recurrent collaterals.

  20. Effects of the AMPA Antagonist ZK 200775 on Visual Function: A Randomized Controlled Trial

    PubMed Central

    Bergholz, Richard; Staks, Thomas; Rüther, Klaus

    2010-01-01

    Background ZK 200775 is an antagonist at the α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor and had earned attention as a possible neuroprotective agent in cerebral ischemia. Probands receiving the agent within phase I trials reported on an alteration of visual perception. In this trial, the effects of ZK 200775 on the visual system were analyzed in detail. Methodology In a randomised controlled trial we examined eyes and vision before and after the intravenous administration of two different doses of ZK 200775 and placebo. There were 3 groups of 6 probands each: Group 1 recieved 0.03 mg/kg/h, group 2 0.75 mg/kg/h of ZK 200775, the control group received 0.9% sodium chloride solution. Probands were healthy males aged between 57 and 69 years. The following methods were applied: clinical examination, visual acuity, ophthalmoscopy, colour vision, rod absolute threshold, central visual field, pattern-reversal visual evoked potentials (pVEP), ON-OFF and full-field electroretinogram (ERG). Principal Findings No effect of ZK 200775 was seen on eye position or motility, stereopsis, pupillary function or central visual field testing. Visual acuity and dark vision deteriorated significantly in both treated groups. Color vision was most remarkably impaired. The dark-adapted ERG revealed a reduction of oscillatory potentials (OP) and partly of the a- and b-wave, furthermore an alteration of b-wave morphology and an insignificantly elevated b/a-ratio. Cone-ERG modalities showed decreased amplitudes and delayed implicit times. In the ON-OFF ERG the ON-answer amplitudes increased whereas the peak times of the OFF-answer were reduced. The pattern VEP exhibited lower amplitudes and prolonged peak times. Conclusions The AMPA receptor blockade led to a strong impairment of typical OFF-pathway functions like color vision and the cone ERG. On the other hand the ON-pathway as measured by dark vision and the scotopic ERG was affected as well. This further elucidates

  1. Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover

    PubMed Central

    Szíber, Zsófia; Liliom, Hanna; Morales, Carlos O. Oueslati; Ignácz, Attila; Rátkai, Anikó Erika; Ellwanger, Kornelia; Link, Gisela; Szűcs, Attila; Hausser, Angelika; Schlett, Katalin

    2017-01-01

    Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase–dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity. PMID:27852895

  2. Ras and Rab interactor 1 controls neuronal plasticity by coordinating dendritic filopodial motility and AMPA receptor turnover.

    PubMed

    Szíber, Zsófia; Liliom, Hanna; Morales, Carlos O Oueslati; Ignácz, Attila; Rátkai, Anikó Erika; Ellwanger, Kornelia; Link, Gisela; Szűcs, Attila; Hausser, Angelika; Schlett, Katalin

    2017-01-15

    Ras and Rab interactor 1 (RIN1) is predominantly expressed in the nervous system. RIN1-knockout animals have deficits in latent inhibition and fear extinction in the amygdala, suggesting a critical role for RIN1 in preventing the persistence of unpleasant memories. At the molecular level, RIN1 signals through Rab5 GTPases that control endocytosis of cell-surface receptors and Abl nonreceptor tyrosine kinases that participate in actin cytoskeleton remodeling. Here we report that RIN1 controls the plasticity of cultured mouse hippocampal neurons. Our results show that RIN1 affects the morphology of dendritic protrusions and accelerates dendritic filopodial motility through an Abl kinase-dependent pathway. Lack of RIN1 results in enhanced mEPSC amplitudes, indicating an increase in surface AMPA receptor levels compared with wild-type neurons. We further provide evidence that the Rab5 GEF activity of RIN1 regulates surface GluA1 subunit endocytosis. Consequently loss of RIN1 blocks surface AMPA receptor down-regulation evoked by chemically induced long-term depression. Our findings indicate that RIN1 destabilizes synaptic connections and is a key player in postsynaptic AMPA receptor endocytosis, providing multiple ways of negatively regulating memory stabilization during neuronal plasticity.

  3. AMPA Receptors Control Fear Extinction through an Arc-Dependent Mechanism

    ERIC Educational Resources Information Center

    Trent, Simon; Barnes, Philip; Hall, Jeremy; Thomas, Kerrie L.

    2017-01-01

    Activity-regulated cytoskeleton-associated protein (Arc) supports fear memory through synaptic plasticity events requiring actin cytoskeleton rearrangements. We have previously shown that reducing hippocampal Arc levels through antisense knockdown leads to the premature extinction of contextual fear. Here we show that the AMPA receptor antagonist…

  4. GRIP1 interlinks N-cadherin and AMPA receptors at vesicles to promote combined cargo transport into dendrites

    PubMed Central

    Heisler, Frank F.; Lee, Han Kyu; Gromova, Kira V.; Pechmann, Yvonne; Schurek, Beate; Ruschkies, Laura; Schroeder, Markus; Schweizer, Michaela; Kneussel, Matthias

    2014-01-01

    The GluA2 subunit of AMPA-type glutamate receptors (AMPARs) regulates excitatory synaptic transmission in neurons. In addition, the transsynaptic cell adhesion molecule N-cadherin controls excitatory synapse function and stabilizes dendritic spine structures. At postsynaptic membranes, GluA2 physically binds N-cadherin, underlying spine growth and synaptic modulation. We report that N-cadherin binds to PSD-95/SAP90/DLG/ZO-1 (PDZ) domain 2 of the glutamate receptor interacting protein 1 (GRIP1) through its intracellular C terminus. N-cadherin and GluA2-containing AMPARs are presorted to identical transport vesicles for dendrite delivery, and live imaging reveals cotransport of both proteins. The kinesin KIF5 powers GluA2/N-cadherin codelivery by using GRIP1 as a multilink interface. Notably, GluA2 and N-cadherin use different PDZ domains on GRIP1 to simultaneously bind the transport complex, and interference with either binding motif impairs the turnover of both synaptic cargoes. Depolymerization of microtubules, deletion of the KIF5 motor domain, or specific blockade of AMPAR exocytosis affects delivery of GluA2/N-cadherin vesicles. At the functional level, interference with this cotransport reduces the number of spine protrusions and excitatory synapses. Our data suggest the concept that the multi-PDZ-domain adaptor protein GRIP1 can act as a scaffold at trafficking vesicles in the combined delivery of AMPARs and N-cadherin into dendrites. PMID:24639525

  5. Involvement of neuronal and glial activities in control of the extracellular d-serine concentrations by the AMPA glutamate receptor in the mouse medial prefrontal cortex.

    PubMed

    Ishiwata, Sayuri; Umino, Asami; Nishikawa, Toru

    2017-09-28

    It has been well accepted that d-serine may be an exclusive endogenous coagonist for the N-methyl-d-aspartate (NMDA)-type glutamate receptor in mammalian forebrain regions. We have recently found by using an in vivo dialysis method that an intra-medial prefrontal cortex infusion of S-α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (S-AMPA), a selective AMPA-type glutamate receptor agonist, causes a reduction in the extracellular levels of d-serine in a calcium-permeable AMPA receptor antagonist-sensitive manner. The inhibitory influence by the AMPA receptor on the extracellular d-serine, however, contradicts the data obtained from in vitro experiments that the AMPA receptor stimulation leads to facilitation of the d-serine liberation. This discrepancy appears to be due to the different cell setups between the in vivo and in vitro preparations. From the viewpoints of the previous reports indicating (1) the neuronal presence of d-serine synthesizing enzyme, serine racemase, and d-serine-like immunoreactivity and (2) the same high tissue concentrations of d-serine in the glia-enriched white matter and in the neuron-enriched gray matter of the mammalian neocortex, we have now investigated in the mouse medial prefrontal cortex, the effects of attenuation of neuronal and glial activities, by tetrodotoxin or fluorocitrate, respectively, on the S-AMPA-induced downregulation of the extracellular d-serine contents. In vivo dialysis studies revealed that a local infusion of tetrodotoxin or fluorocitrate eliminated the ability of S-AMPA given intra-cortically to cause a significant decrease in the dialysate concentrations of d-serine without affecting the elevating effects of S-AMPA on those of glycine, another intrinsic coagonist for the NMDA receptor. These findings suggest that the control by the AMPA receptor of the extracellular d-serine levels could be modulated by the neuronal and glial activities in the prefrontal cortex. It cannot be excluded that

  6. The impact of extra-domain structures and post-translational modifications in the folding/misfolding behaviour of the third PDZ domain of MAGUK neuronal protein PSD-95.

    PubMed

    Murciano-Calles, Javier; Marin-Argany, Marta; Cobos, Eva S; Villegas, Sandra; Martinez, Jose C

    2014-01-01

    The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2-β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2-β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post

  7. The Impact of Extra-Domain Structures and Post-Translational Modifications in the Folding/Misfolding Behaviour of the Third PDZ Domain of MAGUK Neuronal Protein PSD-95

    PubMed Central

    Cobos, Eva S.; Villegas, Sandra; Martinez, Jose C.

    2014-01-01

    The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post

  8. AMPA-receptor specific biogenesis complexes control synaptic transmission and intellectual ability

    PubMed Central

    Brechet, Aline; Buchert, Rebecca; Schwenk, Jochen; Boudkkazi, Sami; Zolles, Gerd; Siquier-Pernet, Karine; Schaber, Irene; Bildl, Wolfgang; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Reis, Andre; Sticht, Heinrich; Al-Sanna’a, Nouriya; Rolfs, Arndt; Kulik, Akos; Schulte, Uwe; Colleaux, Laurence; Abou Jamra, Rami; Fakler, Bernd

    2017-01-01

    AMPA-type glutamate receptors (AMPARs), key elements in excitatory neurotransmission in the brain, are macromolecular complexes whose properties and cellular functions are determined by the co-assembled constituents of their proteome. Here we identify AMPAR complexes that transiently form in the endoplasmic reticulum (ER) and lack the core-subunits typical for AMPARs in the plasma membrane. Central components of these ER AMPARs are the proteome constituents FRRS1l (C9orf4) and CPT1c that specifically and cooperatively bind to the pore-forming GluA1-4 proteins of AMPARs. Bi-allelic mutations in the human FRRS1L gene are shown to cause severe intellectual disability with cognitive impairment, speech delay and epileptic activity. Virus-directed deletion or overexpression of FRRS1l strongly impact synaptic transmission in adult rat brain by decreasing or increasing the number of AMPARs in synapses and extra-synaptic sites. Our results provide insight into the early biogenesis of AMPARs and demonstrate its pronounced impact on synaptic transmission and brain function. PMID:28675162

  9. Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats

    PubMed Central

    Pacheco, Anibal; Aguayo, Felipe I.; Aliaga, Esteban; Muñoz, Mauricio; García-Rojo, Gonzalo; Olave, Felipe A.; Parra-Fiedler, Nicolas A.; García-Pérez, Alexandra; Tejos-Bravo, Macarena; Rojas, Paulina S.; Parra, Claudio S.; Fiedler, Jenny L.

    2017-01-01

    Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in dorsal and ventral hippocampus (VH). Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day) during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH). By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR) subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR) subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area. PMID:28848384

  10. Chronic Stress Triggers Expression of Immediate Early Genes and Differentially Affects the Expression of AMPA and NMDA Subunits in Dorsal and Ventral Hippocampus of Rats.

    PubMed

    Pacheco, Anibal; Aguayo, Felipe I; Aliaga, Esteban; Muñoz, Mauricio; García-Rojo, Gonzalo; Olave, Felipe A; Parra-Fiedler, Nicolas A; García-Pérez, Alexandra; Tejos-Bravo, Macarena; Rojas, Paulina S; Parra, Claudio S; Fiedler, Jenny L

    2017-01-01

    Previous studies in rats have demonstrated that chronic restraint stress triggers anhedonia, depressive-like behaviors, anxiety and a reduction in dendritic spine density in hippocampal neurons. In this study, we compared the effect of repeated stress on the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptor subunits in dorsal and ventral hippocampus (VH). Adult male Sprague-Dawley rats were randomly divided into control and stressed groups, and were daily restrained in their motion (2.5 h/day) during 14 days. We found that chronic stress promotes an increase in c-Fos mRNA levels in both hippocampal areas, although it was observed a reduction in the immunoreactivity at pyramidal cell layer. Furthermore, Arc mRNAs levels were increased in both dorsal and VH, accompanied by an increase in Arc immunoreactivity in dendritic hippocampal layers. Furthermore, stress triggered a reduction in PSD-95 and NR1 protein levels in whole extract of dorsal and VH. Moreover, a reduction in NR2A/NR2B ratio was observed only in dorsal pole. In synaptosomal fractions, we detected a rise in NR1 in dorsal hippocampus (DH). By indirect immunofluorescence we found that NR1 subunits rise, especially in neuropil areas of dorsal, but not VH. In relation to AMPA receptor (AMPAR) subunits, chronic stress did not trigger any change, either in dorsal or ventral hippocampal areas. These data suggest that DH is more sensitive than VH to chronic stress exposure, mainly altering the expression of NMDA receptor (NMDAR) subunits, and probably favors changes in the configuration of this receptor that may influence the function of this area.

  11. Mice lacking the PSD-95–interacting E3 ligase, Dorfin/Rnf19a, display reduced adult neurogenesis, enhanced long-term potentiation, and impaired contextual fear conditioning

    PubMed Central

    Park, Hanwool; Yang, Jinhee; Kim, Ryunhee; Li, Yan; Lee, Yeunkum; Lee, Chungwoo; Park, Jongil; Lee, Dongmin; Kim, Hyun; Kim, Eunjoon

    2015-01-01

    Protein ubiquitination has a significant influence on diverse aspects of neuronal development and function. Dorfin, also known as Rnf19a, is a RING finger E3 ubiquitin ligase implicated in amyotrophic lateral sclerosis and Parkinson’s disease, but its in vivo functions have not been explored. We report here that Dorfin is a novel binding partner of the excitatory postsynaptic scaffolding protein PSD-95. Dorfin-mutant (Dorfin−/−) mice show reduced adult neurogenesis and enhanced long-term potentiation in the hippocampal dentate gyrus, but normal long-term potentiation in the CA1 region. Behaviorally, Dorfin−/− mice show impaired contextual fear conditioning, but normal levels of cued fear conditioning, fear extinction, spatial learning and memory, object recognition memory, spatial working memory, and pattern separation. Using a proteomic approach, we also identify a number of proteins whose ubiquitination levels are decreased in the Dorfin−/− brain. These results suggest that Dorfin may regulate adult neurogenesis, synaptic plasticity, and contextual fear memory. PMID:26553645

  12. Regulation of AMPA receptor surface trafficking and synaptic plasticity by a cognitive enhancer and antidepressant molecule.

    PubMed

    Zhang, H; Etherington, L-A; Hafner, A-S; Belelli, D; Coussen, F; Delagrange, P; Chaouloff, F; Spedding, M; Lambert, J J; Choquet, D; Groc, L

    2013-04-01

    The plasticity of excitatory synapses is an essential brain process involved in cognitive functions, and dysfunctions of such adaptations have been linked to psychiatric disorders such as depression. Although the intracellular cascades that are altered in models of depression and stress-related disorders have been under considerable scrutiny, the molecular interplay between antidepressants and glutamatergic signaling remains elusive. Using a combination of electrophysiological and single nanoparticle tracking approaches, we here report that the cognitive enhancer and antidepressant tianeptine (S 1574, [3-chloro-6-methyl-5,5-dioxo-6,11-dihydro-(c,f)-dibenzo-(1,2-thiazepine)-11-yl) amino]-7 heptanoic acid, sodium salt) favors synaptic plasticity in hippocampal neurons both under basal conditions and after acute stress. Strikingly, tianeptine rapidly reduces the surface diffusion of AMPA receptor (AMPAR) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism that enhances the binding of AMPAR auxiliary subunit stargazin with PSD-95. This prevents corticosterone-induced AMPAR surface dispersal and restores long-term potentiation of acutely stressed mice. Collectively, these data provide the first evidence that a therapeutically used drug targets the surface diffusion of AMPAR through a CaMKII-stargazin-PSD-95 pathway, to promote long-term synaptic plasticity.

  13. Stress- and mitogen-induced phosphorylation of the synapse-associated protein SAP90/PSD-95 by activation of SAPK3/p38gamma and ERK1/ERK2.

    PubMed Central

    Sabio, Guadalupe; Reuver, Suzana; Feijoo, Carmen; Hasegawa, Masato; Thomas, Gareth M; Centeno, Francisco; Kuhlendahl, Sven; Leal-Ortiz, Sergio; Goedert, Michel; Garner, Craig; Cuenda, Ana

    2004-01-01

    SAPK3 (stress-activated protein kinase-3, also known as p38gamma) is a member of the mitogen-activated protein kinase family; it phosphorylates substrates in response to cellular stress, and has been shown to bind through its C-terminal sequence to the PDZ domain of alpha1-syntrophin. In the present study, we show that SAP90 [(synapse-associated protein 90; also known as PSD-95 (postsynaptic density-95)] is a novel physiological substrate for both SAPK3/p38gamma and the ERK (extracellular-signal-regulated protein kinase). SAPK3/p38gamma binds preferentially to the third PDZ domain of SAP90 and phosphorylates residues Thr287 and Ser290 in vitro, and Ser290 in cells in response to cellular stresses. Phosphorylation of SAP90 is dependent on the binding of SAPK3/p38gamma to the PDZ domain of SAP90. It is not blocked by SB 203580, which inhibits SAPK2a/p38alpha and SAPK2b/p38beta but not SAPK3/p38gamma, or by the ERK pathway inhibitor PD 184352. However, phosphorylation is abolished when cells are treated with a cell-permeant Tat fusion peptide that disrupts the interaction of SAPK3/p38gamma with SAP90. ERK2 also phosphorylates SAP90 at Thr287 and Ser290 in vitro, but this does not require PDZ-dependent binding. SAP90 also becomes phosphorylated in response to mitogens, and this phosphorylation is prevented by pretreatment of the cells with PD 184352, but not with SB 203580. In neurons, SAP90 and SAPK3/p38gamma co-localize and they are co-immunoprecipitated from brain synaptic junctional preparations. These results demonstrate that SAP90 is a novel binding partner for SAPK3/p38gamma, a first physiological substrate described for SAPK3/p38gamma and a novel substrate for ERK1/ERK2, and that phosphorylation of SAP90 may play a role in regulating protein-protein interactions at the synapse in response to adverse stress- or mitogen-related stimuli. PMID:14741046

  14. Delayed reduction in hippocampal post-synaptic density protein-95 expression temporally correlates with cognitive dysfunction following controlled cortical impact in mice

    PubMed Central

    Wakade, Chandramohan; Sangeetha, S.R.; Laird, Melissa D.; Dhandapani, Krishnan M.; Vender, John R.

    2011-01-01

    Objective Traumatic brain injury (TBI) induces significant neurological damage, including deficits in learning and memory which contribute to a poor clinical prognosis. Treatment options to limit cognitive decline and promote neurological recovery are lacking, in part, due to a poor understanding of the secondary/delayed processes which contribute to brain injury. In the present study, we characterized the temporal and spatial changes in the expression of PSD-95, a key scaffolding protein implicated in excitatory synaptic signaling, following controlled cortical impact in mice. Neurological injury, as assessed by the open field activity test and the novel object recognition test, were compared with changes in PSD-95 expression. Methods Adult male CD-1 mice were subjected to controlled cortical impact to simulate a moderate traumatic brain injury in humans. The spatial and temporal expression of PSD-95 was analyzed in the cerebral cortex and hippocampus at various time points following injury. Neurological assessments were performed to compare changes in PSD-95 with cognitive deficits. Results A significant decrease in PSD-95 expression was observed in the ipsilateral hippocampus beginning at day 7 post-injury. The loss of PSD-95 corresponded with a concomitant reduction in immunoreactivity for NeuN, a neuronal-specific marker. Aside from the contused cortex, significant loss of PSD-95 immunoreactivity was not observed in the cerebral cortex. The delayed loss of hippocampal PSD-95 directly correlated with the onset of behavioral deficits, suggesting a possible causative role for PSD-95 in behavioral abnormalities following a head trauma. Conclusion Delayed loss of hippocampal synapses was observed following head trauma in mice. These data may suggest a cellular mechanism to explain the delayed learning and memory deficits in humans and provide a potential framework for further testing to implicate PSD-95 as a clinically-relevant therapeutic target. PMID:20397893

  15. AMPA Receptors as Therapeutic Targets for Neurological Disorders.

    PubMed

    Lee, Kevin; Goodman, Lucy; Fourie, Chantelle; Schenk, Susan; Leitch, Beulah; Montgomery, Johanna M

    2016-01-01

    Almost every neurological disease directly or indirectly affects synapse function in the brain. However, these diseases alter synapses through different mechanisms, ultimately resulting in altered synaptic transmission and/or plasticity. Glutamate is the major neurotransmitter that mediates excitatory synaptic transmission in the brain through activation of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors. These receptors have therefore been identified as a target for the development of therapeutic treatments for neurological disorders including epilepsy, neurodegenerative diseases, autism, and drug addiction. The fact that AMPA receptors play a dominant role throughout the brain raises the significant challenge of selectively targeting only those regions affected by disease, and clinical trials have raised doubt regarding the feasibility of specifically targeting AMPA receptors for new therapeutic options. Benzamide compounds that act as positive allosteric AMPA receptor modulators, known as AMPAkines, can act on specific brain regions and were initially proposed to revolutionize the treatment of cognitive deficits associated with neurological disorders. Their therapeutic potential has since declined due to inconsistent results in clinical trials. However, recent advances in basic biomedical research are significantly increasing our knowledge of AMPA receptor structure, binding sites, and interactions with auxiliary proteins. In particular, the large complex of postsynaptic proteins that interact with AMPA receptor subunits have been shown to control AMPA receptor insertion, location, pharmacology, synaptic transmission, and plasticity. These proteins are now being considered as alternative therapeutic target sites for modulating AMPA receptors in neurological disorders.

  16. Development- and age-related alterations in the expression of AMPA receptor subunit GluR2 and its trafficking proteins in the hippocampus of male mouse brain.

    PubMed

    Pandey, Surya P; Rai, Rakesh; Gaur, Pankaj; Prasad, S

    2015-06-01

    AMPA type glutamate receptor (AMPAR) on the post synaptic membrane plays important role in the process of synaptic plasticity involving various scaffolding and trafficking proteins. However, their alterations during development- and aging are not well understood. Here, we report that the expression of AMPAR-GluR2 subunit is gradually up regulated in the hippocampus from 0 day to adult (20 week) and down regulated thereafter in 70 week old male mice. This pattern of GluR2 during development (0-, 7- and 15 day), maturation (45 day) and adult age resembles with similar expression pattern of the scaffolding protein PSD95. Expression pattern of Stargazin (TARPγ-2) largely follows almost similar pattern up to adult age but is up regulated in old age. Pattern of PICK1 expression, however, is opposite to our GluR2 data till adult age but its expression is significantly down regulated in old age. Our data on alterations in the expression of GluR2 in the hippocampus during development and aging indicates a high- and low positive correlations with PSD95 and Stargazin, respectively whereas negative correlation with PICK1 except in old age where expression of Stargazin is higher and that of PICK1 is lower. Our findings suggest that increasing expression pattern of GluR2 during developmental periods and at adult age may be associated with achieving cognitive abilities whereas its low expression in old age may be linked with cognitive decline and proteins like PSD95, Stargazin and PICK1 might be differentially associated with development- and age-dependent alterations in AMPAR-dependent synaptic plasticity and hence learning and memory.

  17. Postsynaptic density-95 mimics and occludes hippocampal long-term potentiation and enhances long-term depression.

    PubMed

    Stein, Valentin; House, David R C; Bredt, David S; Nicoll, Roger A

    2003-07-02

    Previous studies have shown that overexpression of the protein PSD-95 (postsynaptic density-95) selectively enhances AMPA receptor-mediated synaptic responses in hippocampal pyramidal cells. To determine whether this effect is related to synaptic plasticity at these synapses, we examined whether PSD-95 expression mimics long-term potentiation (LTP), and also whether it influences LTP and long-term depression (LTD) in hippocampal slice cultures. Using simultaneous recording from transfected or infected cells and control pyramidal cells, we found that PSD-95, similar to LTP, increases the amplitude and frequency of miniature EPSCs. It also converts silent synapses to functional synapses, as does LTP. In addition, LTP is completely occluded in cells expressing PSD-95, whereas LTD is greatly enhanced. These results suggest that common mechanisms are involved in controlling synaptic AMPA receptors by PSD-95 and synaptic plasticity.

  18. Superactivation of AMPA receptors by auxiliary proteins

    PubMed Central

    Carbone, Anna L.; Plested, Andrew J. R.

    2016-01-01

    Glutamate receptors form complexes in the brain with auxiliary proteins, which control their activity during fast synaptic transmission through a seemingly bewildering array of effects. Here we devise a way to isolate the activation of complexes using polyamines, which enables us to show that transmembrane AMPA receptor regulatory proteins (TARPs) exert their effects principally on the channel opening reaction. A thermodynamic argument suggests that because TARPs promote channel opening, receptor activation promotes AMPAR-TARP complexes into a superactive state with high open probability. A simple model based on this idea predicts all known effects of TARPs on AMPA receptor function. This model also predicts unexpected phenomena including massive potentiation in the absence of desensitization and supramaximal recovery that we subsequently detected in electrophysiological recordings. This transient positive feedback mechanism has implications for information processing in the brain, because it should allow activity-dependent facilitation of excitatory synaptic transmission through a postsynaptic mechanism. PMID:26744192

  19. AMPA experimental communications systems

    NASA Technical Reports Server (NTRS)

    Beckerman, D.; Fass, S.; Keon, T.; Sielman, P.

    1982-01-01

    The program was conducted to demonstrate the satellite communication advantages of Adaptive Phased Array Technology. A laboratory based experiment was designed and implemented to demonstrate a low earth orbit satellite communications system. Using a 32 element, L-band phased array augmented with 4 sets of weights (2 for reception and 2 for transmission) a high speed digital processing system and operating against multiple user terminals and interferers, the AMPA system demonstrated: communications with austere user terminals, frequency reuse, communications in the face of interference, and geolocation. The program and experiment objectives are described, the system hardware and software/firmware are defined, and the test performed and the resultant test data are presented.

  20. GluA2-Containing AMPA Receptors Distinguish Ribbon-Associated from Ribbonless Afferent Contacts on Rat Cochlear Hair Cells123

    PubMed Central

    Martinez-Monedero, Rodrigo

    2016-01-01

    Abstract Mechanosensory hair cells release glutamate at ribbon synapses to excite postsynaptic afferent neurons, via AMPA-type ionotropic glutamate receptors (AMPARs). However, type II afferent neurons contacting outer hair cells in the mammalian cochlea were thought to differ in this respect, failing to show GluA immunolabeling and with many “ribbonless” afferent contacts. Here it is shown that antibodies to the AMPAR subunit GluA2 labeled afferent contacts below inner and outer hair cells in the rat cochlea, and that synaptic currents in type II afferents had AMPAR-specific pharmacology. Only half the postsynaptic densities of type II afferents that labeled for PSD-95, Shank, or Homer were associated with GluA2 immunopuncta or presynaptic ribbons, the “empty slots” corresponding to ribbonless contacts described previously. These results extend the universality of AMPAergic transmission by hair cells, and support the existence of silent afferent contacts. PMID:27257620

  1. Lamina-specific abnormalities of AMPA receptor trafficking and signaling molecule transcripts in the prefrontal cortex in schizophrenia.

    PubMed

    Beneyto, Monica; Meador-Woodruff, James H

    2006-12-15

    Ampakines, positive AMPA receptor modulators, can improve cognitive function in schizophrenia, and enhancement of AMPA receptor-mediated currents by them potentiates the activity of antipsychotics. In vitro studies have revealed that trafficking of AMPA receptors is mediated by specific interactions of a complex network of proteins that also target and anchor them at the postsynaptic density (PSD). The aim of this study was to determine whether there are abnormalities of the molecules associated with trafficking and localization of AMPA receptors at the PSD in the dorsolateral prefrontal cortex (DLPFC) in schizophrenia. We analyzed AMPA receptor expression in DLPFC in schizophrenia, major depression, bipolar disorder, and a control group, by examining transcript levels of all four AMPA receptor subunits by in situ hybridization. We found decreased GluR2 subunit expression in all three illnesses, decreased GluR3 in major depression, and decreased GluR4 in schizophrenia. However, autoradiography experiments showed no changes in AMPA receptor binding; thus, we hypothesized that these changes in receptor subunit stoichiometry do not alter binding to the assembled receptor, but rather intracellular processing. In situ hybridization for AMPA-trafficking molecules showed decreased expression of PICK1 and increased expression of stargazin in DLPFC in schizophrenia, both restricted to large cells of cortical layer III. These data suggest that AMPA-mediated glutamatergic neurotransmission is compromised in schizophrenia, particularly at the level of AMPA-related PSD proteins that mediate AMPA receptor trafficking, synaptic surface expression, and intracellular signaling.

  2. Calcium-permeable AMPA receptors and silent synapses in cocaine-conditioned place preference.

    PubMed

    Shukla, Avani; Beroun, Anna; Panopoulou, Myrto; Neumann, Peter A; Grant, Seth Gn; Olive, M Foster; Dong, Yan; Schlüter, Oliver M

    2017-02-15

    Exposure to cocaine generates silent synapses in the nucleus accumbens (NAc), whose eventual unsilencing/maturation by recruitment of calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) after drug withdrawal results in profound remodeling of NAc neuro-circuits. Silent synapse-based NAc remodeling was shown to be critical for several drug-induced behaviors, but its role in acquisition and retention of the association between drug rewarding effects and drug-associated contexts has remained unclear. Here, we find that the postsynaptic proteins PSD-93, PSD-95, and SAP102 differentially regulate excitatory synapse properties in the NAc. Mice deficient for either of these scaffold proteins exhibit distinct maturation patterns of silent synapses and thus provided instructive animal models to examine the role of NAc silent synapse maturation in cocaine-conditioned place preference (CPP). Wild-type and knockout mice alike all acquired cocaine-CPP and exhibited increased levels of silent synapses after drug-context conditioning. However, the mice differed in CPP retention and CP-AMPAR incorporation. Collectively, our results indicate that CP-AMPAR-mediated maturation of silent synapses in the NAc is a signature of drug-context association, but this maturation is not required for establishing or retaining cocaine-CPP. © 2017 The Authors.

  3. Shisa6 traps AMPA receptors at postsynaptic sites and prevents their desensitization during synaptic activity

    PubMed Central

    Klaassen, Remco V.; Stroeder, Jasper; Coussen, Françoise; Hafner, Anne-Sophie; Petersen, Jennifer D.; Renancio, Cedric; Schmitz, Leanne J. M.; Normand, Elisabeth; Lodder, Johannes C.; Rotaru, Diana C.; Rao-Ruiz, Priyanka; Spijker, Sabine; Mansvelder, Huibert D.; Choquet, Daniel; Smit, August B.

    2016-01-01

    Trafficking and biophysical properties of AMPA receptors (AMPARs) in the brain depend on interactions with associated proteins. We identify Shisa6, a single transmembrane protein, as a stable and directly interacting bona fide AMPAR auxiliary subunit. Shisa6 is enriched at hippocampal postsynaptic membranes and co-localizes with AMPARs. The Shisa6 C-terminus harbours a PDZ domain ligand that binds to PSD-95, constraining mobility of AMPARs in the plasma membrane and confining them to postsynaptic densities. Shisa6 expressed in HEK293 cells alters GluA1- and GluA2-mediated currents by prolonging decay times and decreasing the extent of AMPAR desensitization, while slowing the rate of recovery from desensitization. Using gene deletion, we show that Shisa6 increases rise and decay times of hippocampal CA1 miniature excitatory postsynaptic currents (mEPSCs). Shisa6-containing AMPARs show prominent sustained currents, indicating protection from full desensitization. Accordingly, Shisa6 prevents synaptically trapped AMPARs from depression at high-frequency synaptic transmission. PMID:26931375

  4. Protein Interacting with C-kinase 1 (PICK1) Binding Promiscuity Relies on Unconventional PSD-95/Discs-Large/ZO-1 Homology (PDZ) Binding Modes for Nonclass II PDZ Ligands*

    PubMed Central

    Erlendsson, Simon; Rathje, Mette; Heidarsson, Pétur O.; Poulsen, Flemming M.; Madsen, Kenneth L.; Teilum, Kaare; Gether, Ulrik

    2014-01-01

    PDZ domain proteins control multiple cellular functions by governing assembly of protein complexes. It remains unknown why individual PDZ domains can bind the extreme C terminus of very diverse binding partners and maintain selectivity. By employing NMR spectroscopy, together with molecular modeling, mutational analysis, and fluorescent polarization binding experiments, we identify here three structural mechanisms explaining why the PDZ domain of PICK1 selectively binds >30 receptors, transporters, and kinases. Class II ligands, including the dopamine transporter, adopt a canonical binding mode with promiscuity obtained via differential packing in the binding groove. Class I ligands, such as protein kinase Cα, depend on residues upstream from the canonical binding sequence that are likely to interact with flexible loop residues of the PDZ domain. Finally, we obtain evidence that the unconventional ligand ASIC1a has a dual binding mode involving a canonical insertion and a noncanonical internal insertion with the two C-terminal residues forming interactions outside the groove. Together with an evolutionary analysis, the data show how unconventional binding modes might evolve for a protein recognition domain to expand the repertoire of functionally important interactions. PMID:25023278

  5. Recycling Endosomes Supply AMPA Receptors for LTP

    NASA Astrophysics Data System (ADS)

    Park, Mikyoung; Penick, Esther C.; Edwards, Jeffrey G.; Kauer, Julie A.; Ehlers, Michael D.

    2004-09-01

    Long-term potentiation (LTP) of synaptic strength, the most established cellular model of information storage in the brain, is expressed by an increase in the number of postsynaptic AMPA receptors. However, the source of AMPA receptors mobilized during LTP is unknown. We report that AMPA receptors are transported from recycling endosomes to the plasma membrane for LTP. Stimuli that triggered LTP promoted not only AMPA receptor insertion but also generalized recycling of cargo and membrane from endocytic compartments. Thus, recycling endosomes supply AMPA receptors for LTP and provide a mechanistic link between synaptic potentiation and membrane remodeling during synapse modification.

  6. Gestational nicotine exposure regulates expression of AMPA and NMDA receptors and their signaling apparatus in developing and adult rat hippocampus

    PubMed Central

    Wang, Hong; Dávila-García, Martha I.; Yarl, Weonpo; Gondré-Lewis, Marjorie C.

    2011-01-01

    Untimely activation of nicotinic acetylcholine receptor (nAChR) by nicotine results in short- and long-term consequences on learning and behavior. In this study, the aim was to determine how prenatal nicotine exposure affects components of glutamatergic signaling in the hippocampus during postnatal development. We investigated regulation of both nAChRs and glutamate receptors for α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA), from postnatal day (P) 1 to P63 after a temporally restricted exposure to saline or nicotine for 14 days in utero. We analyzed postsynaptic density components associated with AMPAR and NMDAR signaling: Calcium/calmodulin-dependent protein kinase II α (CaMKIIα), Calmodulin (CaM), and postsynaptic density-95 (PSD95), as well as presynaptically localized synaptosomal-associated protein 25 (SNAP25). At P1, there was significantly heightened expression of AMPAR subunit GluR1 but not GluR2, and of NMDAR subunits NR1, NR2a and NR2d but not NR2b. NR2c was not detectable. At P1, the postsynaptic proteins CaMKIIα, CaM, and PSD95 were also significantly upregulated, together with presynaptic SNAP25. This enhanced expression of glutamate receptors and signaling proteins was concomitant with elevated levels of [3H] Epibatidine (EB) binding in prenatal nicotine-exposed hippocampus, indicating that α4β2 nAChR may influence glutamatergic function in the hippocampus at P1. By P14, neither [3H]EB binding nor the expression levels of subunits GluR1, GluR2, NR1, NR2a, NR2b, NR2c, or NR2d seemed changed with prenatal nicotine. However, CaMKIIα was significantly upregulated with nicotine treatment while CaM showed downregulation at P14. The effects of nicotine persisted in young adult brains at P63. They exhibited significantly downregulated GluR2, NR1, and NR2c expression levels in hippocampal homogenates and a considerably muted overall distribution of [3H]AMPA binding in areas CA1, CA2, CA3, and the dentate

  7. TARP γ-2 and γ-8 Differentially Control AMPAR Density Across Schaffer Collateral/Commissural Synapses in the Hippocampal CA1 Area.

    PubMed

    Yamasaki, Miwako; Fukaya, Masahiro; Yamazaki, Maya; Azechi, Hirotsugu; Natsume, Rie; Abe, Manabu; Sakimura, Kenji; Watanabe, Masahiko

    2016-04-13

    The number of AMPA-type glutamate receptors (AMPARs) at synapses is the major determinant of synaptic strength and varies from synapse to synapse. To clarify the underlying molecular mechanisms, the density of AMPARs, PSD-95, and transmembrane AMPAR regulatory proteins (TARPs) were compared at Schaffer collateral/commissural (SCC) synapses in the adult mouse hippocampal CA1 by quantitative immunogold electron microscopy using serial sections. We examined four types of SCC synapses: perforated and nonperforated synapses on pyramidal cells and axodendritic synapses on parvalbumin-positive (PV synapse) and pravalbumin-negative interneurons (non-PV synapse). SCC synapses were categorized into those expressing high-density (perforated and PV synapses) or low-density (nonperforated and non-PV synapses) AMPARs. Although the density of PSD-95 labeling was fairly constant, the density and composition of TARP isoforms was highly variable depending on the synapse type. Of the three TARPs expressed in hippocampal neurons, the disparity in TARP γ-2 labeling was closely related to that of AMPAR labeling. Importantly, AMPAR density was significantly reduced at perforated and PV synapses in TARP γ-2-knock-out (KO) mice, resulting in a virtual loss of AMPAR disparity among SCC synapses. In comparison, TARP γ-8 was the only TARP expressed at nonperforated synapses, where AMPAR labeling further decreased to a background level in TARP γ-8-KO mice. These results show that synaptic inclusion of TARP γ-2 potently increases AMPAR expression and transforms low-density synapses into high-density ones, whereas TARP γ-8 is essential for low-density or basal expression of AMPARs at nonperforated synapses. Therefore, these TARPs are critically involved in AMPAR density control at SCC synapses. Although converging evidence implicates the importance of transmembrane AMPA-type glutamate receptor (AMPAR) regulatory proteins (TARPs) in AMPAR stabilization during basal transmission and synaptic

  8. Electrophysiological properties of AMPA receptors are differentially modulated depending on the associated member of the TARP family.

    PubMed

    Kott, Sabine; Werner, Markus; Körber, Christoph; Hollmann, Michael

    2007-04-04

    The family of AMPA receptors is encoded by four genes that are differentially spliced to result in the flip or flop versions of the four subunits GluR1 to GluR4. GluR2 is further modified at the so-called Q/R site by posttranscriptional RNA editing. Delivery of AMPA receptors to the plasma membrane and synaptic trafficking are controlled by transmembrane AMPA receptor regulatory proteins (TARPs). Additionally, TARPs influence essential electrophysiological properties of AMPA receptor channels such as desensitization and agonist efficacies. Here, we compare the influence of all known TARPs (gamma2, gamma3, gamma4, and gamma8) on agonist-induced currents of the four AMPA receptor subunits, including flip and flop splice variants and editing variants. We show that, although agonist-induced currents of all homomeric AMPA receptor subunits as well as all heteromeric combinations tested are significantly potentiated when coexpressed with members of the TARP family in Xenopus laevis oocytes, the extent of TARP-mediated increase in agonist-induced responses is highly dependent on both the AMPA receptor subunit and the coexpressed TARP. Moreover, we demonstrate that the splice variant of the AMPA receptor plays a key role in determining the modulation of electrophysiological properties by associated TARPs. We furthermore present evidence that individual TARP-AMPA receptor interactions control the degree of desensitization of AMPA receptors. Consequently, because of their subunit-specific impact on the electrophysiological properties, TARPs play a major role as modulatory subunits of AMPA receptors and thus contribute to the functional diversity of AMPA receptors encountered in the CNS.

  9. Urban contributions of glyphosate and its degradate AMPA to streams in the United States

    USGS Publications Warehouse

    Kolpin, D.W.; Thurman, E.M.; Lee, E.A.; Meyer, M.T.; Furlong, E.T.; Glassmeyer, S.T.

    2006-01-01

    Glyphosate is the most widely used herbicide in the world, being routinely applied to control weeds in both agricultural and urban settings. Microbial degradation of glyphosate produces aminomethyl phosphonic acid (AMPA). The high polarity and water-solubility of glyphosate and AMPA has, until recently, made their analysis in water samples problematic. Thus, compared to other herbicides (e.g. atrazine) there are relatively few studies on the environmental occurrence of glyphosate and AMPA. In 2002, treated effluent samples were collected from 10 wastewater treatment plants (WWTPs) to study the occurrence of glyphosate and AMPA. Stream samples were collected upstream and downstream of the 10 WWTPs. Two reference streams were also sampled. The results document the apparent contribution of WWTP effluent to stream concentrations of glyphosate and AMPA, with roughly a two-fold increase in their frequencies of detection between stream samples collected upstream and those collected downstream of the WWTPs. Thus, urban use of glyphosate contributes to glyphosate and AMPA concentrations in streams in the United States. Overall, AMPA was detected much more frequently (67.5%) compared to glyphosate (17.5%).

  10. In search of novel AMPA potentiators.

    PubMed

    Francotte, Pierre; de Tullio, Pascal; Fraikin, Pierre; Counerotte, Stéphane; Goffin, Eric; Pirotte, Bernard

    2006-11-01

    Glutamate is the major excitatory neurotransmitter in the brain. Amongst ionotropic receptors responding to glutamate, the AMPA subtype has been considered as essential for the fast excitatory neurotransmission in the central nervous system and the expression and maintenance of long-term potentiation. As glutamate is known to be involved in many neurological and psychiatric disorders, AMPA receptors seem to represent interesting targets to develop therapeutic drugs. Hence, the enhancement of AMPA signals is an approach currently investigated for the management of Alzheimer's disease, schizophrenia or mood disorders. In particular, many efforts are being conducted in the development of AMPA positive allosteric modulators ("potentiators"), which alter the rate of receptor desensitization. The major chemical families developed as AMPA potentiators are aniracetam derivatives, cyclothiazide derivatives and biarylpropylsulfonamides derivatives.

  11. Toxicity of AMPA to the earthworm Eisenia andrei Bouché, 1972 in tropical artificial soil

    NASA Astrophysics Data System (ADS)

    Domínguez, Anahí; Brown, George Gardner; Sautter, Klaus Dieter; Ribas de Oliveira, Cintia Mara; de Vasconcelos, Eliane Carvalho; Niva, Cintia Carla; Bartz, Marie Luise Carolina; Bedano, José Camilo

    2016-01-01

    Aminomethylphosphonic acid (AMPA) - one of glyphosate’s main metabolites - has been classified as persistent in soils, raising concern regarding the widespread use of glyphosate in agriculture and forestry. Glyphosate may have negative or neutral effects on soil biota, but no information is available on the toxicity of AMPA to soil invertebrates. Therefore our aim was to study the effect of AMPA on mortality and reproduction of the earthworm species Eisenia andrei using standard soil ecotoxicological methods (ISO). Field-relevant concentrations of AMPA had no significant effects on mortality in acute or chronic assays. Except at the highest concentration tested, a significant biomass loss was observed compared to controls in the chronic assay. The number of juveniles and cocoons increased with higher concentrations of AMPA applied, but their mean weights decreased. This mass loss indicates higher sensitivity of juveniles than adults to AMPA. Our results suggest that earthworms coming from parents grown in contaminated soils may have reduced growth, limiting their beneficial roles in key soil ecosystem functions. Nevertheless, further research is needed to better understand the mechanisms underlying the sublethal effects observed here.

  12. Mannose-specific lectins modulate ligand binding to AMPA-type glutamate receptors.

    PubMed

    Hoffman, K B; Kessler, M; Ta, J; Lam, L; Lynch, G

    1998-06-08

    Binding of [3H]AMPA was increased above control levels in rat brain membranes that had been incubated with concanavalin A (Con A) or a lectin from Lens culinaris (LC), both of which bind mannose residues. This did not occur with any of six lectins with other specificities. The magnitude of the increased binding varied from 15% in cortex to 70% in hippocampus and decreased significantly between 3 weeks and 6 months of age. Succinylated Con A was without effect and neither Con A nor LC increased binding to solubilized AMPA receptors. Increases in binding were not obtained in membranes purified from HEK293 cell lines expressing homomeric AMPA receptors. This indicates that mannose specific lectins may enhance binding by cross-linking AMPA receptors to each other or to proteins that are specific to brain. Con A has been reported to reduce glutamate receptor desensitization with higher efficacy at kainate than at AMPA receptors; the increase in binding reported here appears to be unrelated to such effects because (1) it was not affected by drugs that block desensitization and (2) [3H]kainate binding was reduced rather than increased by Con A. These observations suggest that AMPA receptor kinetic properties not involving desensitization are influenced by extracellular interactions between the receptors and other transmembrane proteins. Copyright 1998 Elsevier Science B.V. All rights reserved.

  13. Toxicity of AMPA to the earthworm Eisenia andrei Bouché, 1972 in tropical artificial soil

    PubMed Central

    Domínguez, Anahí; Brown, George Gardner; Sautter, Klaus Dieter; Ribas de Oliveira, Cintia Mara; de Vasconcelos, Eliane Carvalho; Niva, Cintia Carla; Bartz, Marie Luise Carolina; Bedano, José Camilo

    2016-01-01

    Aminomethylphosphonic acid (AMPA) - one of glyphosate’s main metabolites - has been classified as persistent in soils, raising concern regarding the widespread use of glyphosate in agriculture and forestry. Glyphosate may have negative or neutral effects on soil biota, but no information is available on the toxicity of AMPA to soil invertebrates. Therefore our aim was to study the effect of AMPA on mortality and reproduction of the earthworm species Eisenia andrei using standard soil ecotoxicological methods (ISO). Field-relevant concentrations of AMPA had no significant effects on mortality in acute or chronic assays. Except at the highest concentration tested, a significant biomass loss was observed compared to controls in the chronic assay. The number of juveniles and cocoons increased with higher concentrations of AMPA applied, but their mean weights decreased. This mass loss indicates higher sensitivity of juveniles than adults to AMPA. Our results suggest that earthworms coming from parents grown in contaminated soils may have reduced growth, limiting their beneficial roles in key soil ecosystem functions. Nevertheless, further research is needed to better understand the mechanisms underlying the sublethal effects observed here. PMID:26792548

  14. AMPA-silent synapses in brain development and pathology.

    PubMed

    Hanse, Eric; Seth, Henrik; Riebe, Ilse

    2013-12-01

    Synapses are constantly generated at a high rate in the developing, prepubescent brain. Newly generated glutamatergic synapses lack functional AMPA receptor-mediated transmission. Most of these 'AMPA-silent' synapses are eliminated during the developmental period, but some are specifically selected for AMPA unsilencing by correlated pre-and postsynaptic activity as the first step in a process that leads to stabilization of the synapse. Premature, or delayed, unsilencing of AMPA-silent synapses has been implicated in neurodevelopmental disorders, and abnormal generation of AMPA-silent synapses is associated with brain trauma, addiction and neurodegenerative disorders, further highlighting the importance of AMPA-silent synapses in brain pathology.

  15. Impaired motor learning attributed to altered AMPA receptor function in the cerebellum of rats with temporal lobe epilepsy: ameliorating effects of Withania somnifera and withanolide A.

    PubMed

    Soman, Smijin; Anju, T R; Jayanarayanan, S; Antony, Sherin; Paulose, C S

    2013-06-01

    The aim of this study was to investigate the effect of Withania somnifera (WS) extract, withanolide A (WA), and carbamazepine (CBZ) on cerebellar AMPA receptor function in pilocarpine-induced temporal lobe epilepsy (TLE). In the present study, motor learning deficit was studied by rotarod test, grid walk test, and narrow beam test. Motor learning was significantly impaired in rats with epilepsy. The treatment with WS and WA significantly reversed the motor learning deficit in rats with epilepsy when compared with control rats. There was an increase in glutamate content and IP3 content observed in rats with epilepsy which was reversed in WS- and WA-treated rats with epilepsy. alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor dysfunction was analyzed using radiolabeled AMPA receptor binding assay, AMPA receptor mRNA expression, and immunohistochemistry using anti-AMPA receptor antibody. Our results suggest that there was a decrease in Bmax, mRNA expression, and AMPA receptor expression indicating AMPA receptor dysfunction, which is suggested to have contributed to the motor learning deficit observed in rats with epilepsy. Moreover, treatment with WS and WA resulted in physiological expression of AMPA receptors. There was also alteration in GAD and GLAST expression which supplemented the increase in extracellular glutamate. The treatment with WS and WA reversed the GAD and GLAST expression. These findings suggest that WS and WA regulate AMPA receptor function in the cerebellum of rats with TLE, which has therapeutic application in epilepsy.

  16. Quantitative Analysis of the Feedback of the Robust Signaling Pathway Network of Myosin V Molecular Motors on GluR1 of AMPA in Neurons: A Networking Approach for Controlling Nanobiomachines

    NASA Astrophysics Data System (ADS)

    Liu, Jian-Qin; Nakano, Tadashi

    Acting as nanobiomachines within the cell, myosin V molecular motors contribute greatly to the LTP (Long Term Potentiation) in neural signaling, which transport the recycling endosomes from the dendrite to the spine of neurons and the GluR1 in AMPA receptors lead to the activities of memorization in brains. However it is unknown that how the restriction of GluR1 at the spine of neuron is caused by the signaling cascade of myosin V and Rab11/Rab11-FIP2 during the myosin V centered signaling process in neurons. Here we report that the feedback of the biochemical reaction for binding Myosin V and Rab11/Rab11-FIP2 plays a pivotal role to restrict the accumulation of GluR1 at the spine. We have investigated the feedback of myosin V and Rab11/Rab11-FIP2 on the convergence of GluR1 by using the computational model of intracellular signaling pathway networks we designed and the simulation software Cell Illustrator Professional Version 3.0 ®. The obtained results show that controllability of molecular motor based nanobiomachines is inevitable for exploring the molecular mechanism of neuroscience at the nanoscale.

  17. Wind erosion as an environmental transport pathway of glyphosate and AMPA

    NASA Astrophysics Data System (ADS)

    Bento, Célia P. M.; Goossens, Dirk; Rezaei, Mahrooz; Riksen, Michel; Mol, Hans G. J.; Ritsema, Coen J.; Geissen, Violette

    2017-04-01

    Glyphosate is the active ingredient of many commercial formulations of herbicides extensively used worldwide for weed control. Because glyphosate and its main metabolite aminomethylphosphonic acid (AMPA) are considered non-volatile, their loss to the atmosphere is considered negligible. Both compounds strongly adsorb to soil particles and wind-eroded sediment and dust are thus a possible environmental transport pathway. This can result in environmental and human exposure far beyond the agricultural areas where it has been applied. Therefore, special attention is required to the airborne transport of glyphosate and AMPA. In this study, we investigated the behavior of glyphosate and AMPA in wind-eroded sediment by measuring their content in different size fractions (median diameters between 715 and 8 µm) of a loess soil, during a period of 28 days after glyphosate application. Granulometrical extraction was done using a wind tunnel and a Soil Fine Particle Extractor. Extractions were conducted on days 0, 3, 7, 14, 21 and 28 after glyphosate application. Results indicated that glyphosate and AMPA contents were significantly higher in the finest particle fractions (median diameters between 8 and 18 µm), and lowered significantly with the increase in particle size. Glyphosate and AMPA contents correlated positively with clay, organic matter, and silt content. The dissipation of glyphosate over time was very low, which was associated to the low soil moisture content of the sediment. Consequently, the formation of AMPA was also very low. The low dissipation of glyphosate in our study indicates that the risk of glyphosate transport in dry sediment to off-target areas by wind can be very high. The highest glyphosate and AMPA contents were found in the smallest soil fractions (PM10 and less), which are easily inhaled. This contributes to the risk of human and animal exposure and, therefore, more attention should be paid to this route of exposure in environmental and human

  18. NASA Adaptive Multibeam Phased Array (AMPA): An application study

    NASA Technical Reports Server (NTRS)

    Mittra, R.; Lee, S. W.; Gee, W.

    1982-01-01

    The proposed orbital geometry for the adaptive multibeam phased array (AMPA) communication system is reviewed and some of the system's capabilities and preliminary specifications are highlighted. Typical AMPA user link models and calculations are presented, the principal AMPA features are described, and the implementation of the system is demonstrated. System tradeoffs and requirements are discussed. Recommendations are included.

  19. AMPA receptor inhibition by synaptically released zinc

    PubMed Central

    Kalappa, Bopanna I.; Anderson, Charles T.; Lippard, Stephen J.; Tzounopoulos, Thanos

    2015-01-01

    The vast amount of fast excitatory neurotransmission in the mammalian central nervous system is mediated by AMPA-subtype glutamate receptors (AMPARs). As a result, AMPAR-mediated synaptic transmission is implicated in nearly all aspects of brain development, function, and plasticity. Despite the central role of AMPARs in neurobiology, the fine-tuning of synaptic AMPA responses by endogenous modulators remains poorly understood. Here we provide evidence that endogenous zinc, released by single presynaptic action potentials, inhibits synaptic AMPA currents in the dorsal cochlear nucleus (DCN) and hippocampus. Exposure to loud sound reduces presynaptic zinc levels in the DCN and abolishes zinc inhibition, implicating zinc in experience-dependent AMPAR synaptic plasticity. Our results establish zinc as an activity-dependent, endogenous modulator of AMPARs that tunes fast excitatory neurotransmission and plasticity in glutamatergic synapses. PMID:26647187

  20. Activity-Dependent Palmitoylation Controls SynDIG1 Stability, Localization, and Function

    PubMed Central

    Kaur, Inderpreet; Yarov-Yarovoy, Vladimir; Kirk, Lyndsey M.; Plambeck, Kristopher E.; Barragan, Eden V.; Ontiveros, Eric S.

    2016-01-01

    Synapses are specialized contacts between neurons. Synapse differentiation-induced gene I (SynDIG1) plays a critical role during synapse development to regulate AMPA receptor (AMPAR) and PSD-95 content at excitatory synapses. Palmitoylation regulates the localization and function of many synaptic proteins, including AMPARs and PSD-95. Here we show that SynDIG1 is palmitoylated, and investigate the effects of palmitoylation on SynDIG1 stability and localization. Structural modeling of SynDIG1 suggests that the membrane-associated region forms a three-helical bundle with two cysteine residues located at positions 191 and 192 in the juxta-transmembrane region exposed to the cytoplasm. Site-directed mutagenesis reveals that C191 and C192 are palmitoylated in heterologous cells and positively regulates dendritic targeting in neurons. Like PSD-95, activity blockade in a rat hippocampal slice culture increases SynDIG1 palmitoylation, which is consistent with our prior demonstration that SynDIG1 localization at synapses increases upon activity blockade. These data demonstrate that palmitoylation of SynDIG1 is regulated by neuronal activity, and plays a critical role in regulating its stability and subcellular localization, and thereby its function. SIGNIFICANCE STATEMENT Palmitoylation is a reversible post-translation modification that has recently been recognized as playing a critical role in the localization and function of many synaptic proteins. Here we show that activity-dependent palmitoylation of the atypical AMPA receptor auxiliary transmembrane protein SynDIG1 regulates its stability and localization at synapses to regulate function and synaptic strength. PMID:27445135

  1. S-palmitoylation regulates AMPA receptors trafficking and function: a novel insight into synaptic regulation and therapeutics

    PubMed Central

    Han, Jun; Wu, Pengfei; Wang, Fang; Chen, Jianguo

    2014-01-01

    Glutamate acting on AMPA-type ionotropic glutamate receptor (AMPAR) mediates the majority of fast excitatory synaptic transmission in the mammalian central nervous system. Dynamic regulation of AMPAR by post-translational modifications is one of the key elements that allow the nervous system to adapt to environment stimulations. S-palmitoylation, an important lipid modification by post-translational addition of a long-chain fatty acid to a cysteine residue, regulates AMPA receptor trafficking, which dynamically affects multiple fundamental brain functions, such as learning and memory. In vivo, S-palmitoylation is controlled by palmitoyl acyl transferases and palmitoyl thioesterases. In this review, we highlight advances in the mechanisms for dynamic AMPA receptors palmitoylation, and discuss how palmitoylation affects AMPA receptors function at synapses in recent years. Pharmacological regulation of S-palmitoylation may serve as a novel therapeutic strategy for neurobiological diseases. PMID:26579419

  2. Mechanism-based design of 2,3-benzodiazepine inhibitors for AMPA receptors

    PubMed Central

    Niu, Li

    2015-01-01

    2,3-Benzodiazepine (2,3-BDZ) compounds represent a group of structurally diverse, small-molecule antagonists of (R, S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors. Antagonists of AMPA receptors are drug candidates for potential treatment of a number of neurological disorders such as epilepsy, stroke and amyotrophic lateral sclerosis (ALS). How to make better inhibitors, such as 2,3-BDZs, has been an enduring quest in drug discovery. Among a few available tools to address this specific question for making better 2,3-BDZs, perhaps the best one is to use mechanistic clues from studies of the existing antagonists to design and discover more selective and more potent antagonists. Here I review recent work in this area, and propose some ideas in the continuing effort of developing newer 2,3-BDZs for tighter control of AMPA receptor activities in vivo. PMID:26713266

  3. Exendin-4 promotes the membrane trafficking of the AMPA receptor GluR1 subunit and ADAM10 in the mouse neocortex.

    PubMed

    Ohtake, Nobuaki; Saito, Mieko; Eto, Masaaki; Seki, Kenjiro

    2014-05-01

    Glucagon-like peptide-1 (GLP-1) is a novel treatment modality for type 2 diabetes mellitus. However, GLP-1 has been suggested as a therapeutic target for Alzheimer's disease (AD). In rodent studies, GLP-1 reduces amyloid beta (Aβ) and facilitates synaptic plasticity. Therefore, in the present study, we investigated how GLP-1 facilitates synaptic plasticity and reduces the Aβ in vivo. Exendin-4, a GLP-1 receptor agonist that can cross the blood brain barrier, was subcutaneously administered to adult mice. We then extracted the total and the plasma membrane proteins from the mouse neocortex. Exendin-4 significantly increased the phosphorylation level of cAMP response element-binding protein (CREB). Consistently, the expression level of brain-derived neurotrophic factor (BDNF), a transcriptional target of CREB, was increased. Furthermore, exendin-4 increased the membrane protein level of the AMPA receptor GluR1 subunit and postsynaptic density protein-95 (PSD-95), whereas GluR2 was unaffected. These exendin-4-dependent increases in membrane GluR1, total PSD-95 and BDNF were abrogated by pretreatment with temozolomide (TMZ), a DNA-alkylating agent, indicating that these alterations were dependent on exendin-4-induced transcriptional activity. In addition, we found that exendin-4 increased the level of the α-C terminal fragment (α-CTF) of amyloid precursor protein (APP). Furthermore, protein levels of both mature and immature ADAM10, the α-secretase of APP in the plasma membrane, were increased, whereas the total mature and immature ADAM10 levels were unchanged. These exendin-4-dependent increases in α-CTF and ADAM10 were not affected by TMZ. These findings suggested that GLP-1 facilitates the GluR1 membrane insertion through CREB activation and increases α-secretase activity through ADAM10 membrane trafficking. Upregulation of GluR1 and ADAM10 at the plasma membrane were also observed in mice with intracerebroventricular administration of Aβ oligomer

  4. TARP redundancy is critical for maintaining AMPA receptor function.

    PubMed

    Menuz, Karen; O'Brien, Jessica L; Karmizadegan, Siavash; Bredt, David S; Nicoll, Roger A

    2008-08-27

    Transmembrane AMPA receptor regulatory proteins (TARPs) are AMPA receptor auxiliary subunits that influence diverse aspects of receptor function. However, the full complement of physiological roles for TARPs in vivo remains poorly understood. Here we find that double knock-out mice lacking TARPs gamma-2 and gamma-3 are profoundly ataxic and fail to thrive. We demonstrate that these TARPs are critical for the synaptic targeting and kinetics of AMPA receptors in cerebellar Golgi cells, but that either alone is sufficient to fully preserve function. By analyzing the few remaining synaptic AMPA receptors in the gamma-2, gamma-3 double knock-out mice, we unexpectedly find that these TARPs specify AMPA receptor subunit composition. This study establishes a new role for TARPs in regulating AMPA receptor assembly and suggests that TARPs are necessary for proper AMPA receptor localization and function in most, if not all, neurons of the CNS.

  5. AMPA receptors in epilepsy and as targets for antiepileptic drugs.

    PubMed

    Rogawski, M A; Donevan, S D

    1999-01-01

    alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are key mediators of seizure spread in the nervous system and represent promising targets for antiepileptic drugs. There is emerging evidence that AMPA receptors may play a role in epileptogenesis and in seizure-induced brain damage. This evidence suggests that AMPA receptor antagonists could have broad utility in epilepsy therapy. Regional, developmental, and disease-associated variations in AMPA receptors produced by differential expression of AMPA receptor subunits and variations in posttranscriptional processing, including alternative splicing and pre-mRNA editing, provide a diversity of functionally distinct AMPA receptor isoforms that allow opportunities for selective drug targeting. Four types of AMPA receptor antagonist are discussed in this chapter: (a) competitive AMPA recognition site antagonists, including those of the quinoxalinedione and newer nonquinoxalinedione classes, (b) 2,3-benzodiazepine noncompetitive (allosteric) antagonists, (c) desensitization enhancing antagonists, exemplified by SCN-, and (d) antagonists of Ca(2+)-permeable AMPA receptors, including polyamine amide arthropod toxins and their synthetic analogues. Competitive and noncompetitive AMPA receptor antagonists are broad-spectrum anticonvulsants in animal seizure models. Their effectiveness and safety for humans remain to be determined. There is evidence that these antagonists can potentiate the antiseizure activity of N-methyl-D-aspartate (NMDA) receptor antagonists and conventional antiepileptic drugs. This evidence suggests that the preferred use of AMPA receptor antagonists may be in combination therapies. Agents that enhance desensitization may have advantages in comparison with other AMPA receptor antagonists to the extent that they preferentially block high-frequency synaptic signaling and avoid depressing AMPA receptors on interneurons, which would lead to disinhibition and enhanced excitability

  6. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain

    PubMed Central

    Wright, Amanda; Vissel, Bryce

    2012-01-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are comprised of different combinations of GluA1–GluA4 (also known asGluR1–GluR4 and GluR-A to GluR-D) subunits. The GluA2 subunit is subject to RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Gln; Q), present in the GluA2 gene, to a codon for arginine (Arg; R) found in the mRNA. AMPA receptors are calcium (Ca2+)-permeable if they contain the unedited GluA2(Q) subunit or if they lack the GluA2 subunit. While most AMPA receptors in the brain contain the edited GluA2(R) subunit and are therefore Ca2+-impermeable, recent evidence suggests that Ca2+-permeable AMPA receptors are important in synaptic plasticity, learning, and disease. Strong evidence supports the notion that Ca2+-permeable AMPA receptors are usually GluA2-lacking AMPA receptors, with little evidence to date for a significant role of unedited GluA2 in normal brain function. However, recent detailed studies suggest that Ca2+-permeable AMPA receptors containing unedited GluA2 do in fact occur in neurons and can contribute to excitotoxic cell loss, even where it was previously thought that there was no unedited GluA2.This review provides an update on the role of GluA2 RNA editing in the healthy and diseased brain and summarizes recent insights into the mechanisms that control this process. We suggest that further studies of the role of unedited GluA2 in normal brain function and disease are warranted, and that GluA2 editing should be considered as a possible contributing factor when Ca2+-permeable AMPA receptors are observed. PMID:22514516

  7. Elevated glucose concentration changes the content and cellular localization of AMPA receptors in the retina but not in the hippocampus.

    PubMed

    Castilho, A F; Liberal, J T; Baptista, F I; Gaspar, J M; Carvalho, A L; Ambrósio, A F

    2012-09-06

    Diabetic retinopathy and diabetic encephalopathy are two common complications of diabetes mellitus. The impairment of glutamatergic neurotransmission in the retina and hippocampus has been suggested to be involved in the pathogenesis of these diabetic complications. In this study, we investigated the effect of elevated glucose concentration and diabetes on the protein content and surface expression of AMPA receptor subunits in the rat retina and hippocampus. We have used two models, cultured retinal and hippocampal cells exposed to elevated glucose concentration and an animal model of streptozotocin-induced type 1 diabetes. The immunoreactivity of GluA1, GluA2 and GluA4 was evaluated by Western blot and immunocytochemistry. The levels of these subunits at the plasma membrane were evaluated by biotinylation and purification of plasma membrane-associated proteins. Elevated glucose concentration increased the total levels of GluA2 subunit of AMPA receptors in retinal neural cells, but not of the subunits GluA1 or GluA4. However, at the plasma membrane, elevated glucose concentration induced an increase of all AMPA receptor subunits. In cultured hippocampal neurons, elevated glucose concentration did not induce significant alterations in the levels of AMPA receptor subunits. In the retinas of diabetic rats there were no persistent changes in the levels of AMPA receptor subunits comparing to aged-matched control retinas. Also, no consistent changes were detected in the levels of GluA1, GluA2 or GluA4 in the hippocampus of diabetic rats. We demonstrate that elevated glucose concentration induces early changes in AMPA receptor subunits, mainly in GluA2 subunit, in retinal neural cells. Conversely, hippocampal neurons seem to remain unaffected by elevated glucose concentration, concerning the expression of AMPA receptors, suggesting that AMPA receptors are more susceptible to the stress caused by elevated glucose concentration in retinal cells than in hippocampal neurons.

  8. Desensitization of AMPA receptors and AMPA-NMDA receptor interaction: an in vivo cyclic GMP microdialysis study in rat cerebellum.

    PubMed Central

    Fedele, E.; Raiteri, M.

    1996-01-01

    1. Desensitization is an important characteristic of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type. 2. Stimulation of N-methyl-D-aspartate (NMDA) or AMPA receptors in cerebellum results in increased production of cyclic GMP. We have investigated AMPA receptor desensitization in vivo by monitoring extracellular cyclic GMP during intracerebellar microdialysis in conscious unrestrained adult rats. 3. Local infusion of AMPA (10 to 100 microM) caused dose-related elevations of cyclic GMP levels. The effect of AMPA was prevented by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX) and by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NOARG). 4. In the absence of AMPA, DNQX lowered the basal levels of cyclic GMP whereas the NMDA receptor channel antagonist dizocilpine (MK-801) was ineffective. 5. Cyclothiazide, a blocker of AMPA receptor desensitization, potentiated the cyclic GMP response to exogenous AMPA. Moreover, cyclothiazide (100-300 microM) produced on its own dose-dependent elevations of extracellular cyclic GMP. The cyclothiazide-induced response was prevented not only by DNQX but also by MK-801. 6. While the cyclic GMP response elicited by AMPA was totally insensitive to MK-801, the response produced by AMPA (10 microM) plus cyclothiazide (30 microM) was strongly attenuated by the NMDA receptor antagonist (30 microM). 7. The results suggest that (a) AMPA receptors linked to the NO-cyclic GMP pathway in the cerebellum can undergo desensitization in vivo during exposure to exogenous AMPA; cyclothiazide inhibits such desensitization; (b) AMPA receptors (but not NMDA receptors) are 'tonically' activated and kept in a partly desensitized state by endogenous glutamate; (c) if cyclothiazide is present, activation of AMPA receptors may permit endogenous activation of NMDA receptors. PMID:8882607

  9. Glyphosate and AMPA passive sampling in freshwater using a microporous polyethylene diffusion sampler.

    PubMed

    Fauvelle, Vincent; Montero, Natalia; Mueller, Jochen F; Banks, Andrew; Mazzella, Nicolas; Kaserzon, Sarit L

    2017-12-01

    Glyphosate (PMG) is one of the most widely used herbicides with a reported 8.6 million tons applied globally in 2016. Due to widespread use and limited understanding of long-term environmental impacts, it is expected that future monitoring requirements for PMG and its primary metabolite aminomethyl phosphonic acid (AMPA) in aquatic environments will increase, along with the need for low cost monitoring and risk assessment strategies. The aim of this study was to investigate a microporous polyethylene tube (MPT; 2-mm thickness, 17.6 cm(2) surface area, 35% porosity, 2.5 μm pore size) as a diffusive layer for the passive sampling of PMG and AMPA. Levels of PMG and AMPA sorbed to MPT were low (Kmw close to 1 mL g(-1)), validating MPT as a diffusive layer. Uptake experiments were conducted first under controlled laboratory conditions (pH = 6.8, 6 days), followed by an in situ freshwater lake system deployment (pH = 7.3, 11 days). PMG and AMPA accumulated linearly (slope relative standard deviation < 6%) under laboratory conditions with sampling rates (Rs) of 18 and 25 mL d(-1), respectively. PMG in situ Rs was 28 mL d(-1), and was not different from the one found in the laboratory. AMPA was below the limit of quantification (LOQ, 1 ng mL(-1)) in grab water samples, but was detected (>LOQ) in all passive samplers. Results illustrate the gain in sensitivity provided by the passive sampling technique, and the applicability of the device developed for the passive sampling of PMG and AMPA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. URBAN CONTRIBUTIONS OF GLYSPHOSATE AND ITS DEGRADATE AMPA TO STREAMS IN THE UNITED STATES

    EPA Science Inventory

    Glyphosate is the most widely used herbicide in the world, being routinely applied to control weeds in both agricultural and urban settings. Microbial degradation of glyphosate produces aminomethyl phosphonic acid (AMPA). The high polarity and water-solubility of glyphosate and A...

  11. URBAN CONTRIBUTIONS OF GLYSPHOSATE AND ITS DEGRADATE AMPA TO STREAMS IN THE UNITED STATES

    EPA Science Inventory

    Glyphosate is the most widely used herbicide in the world, being routinely applied to control weeds in both agricultural and urban settings. Microbial degradation of glyphosate produces aminomethyl phosphonic acid (AMPA). The high polarity and water-solubility of glyphosate and A...

  12. Revisiting AMPA receptors as an antiepileptic drug target.

    PubMed

    Rogawski, Michael A

    2011-03-01

    In the 1990s there was intense interest in ionotropic glutamate receptors as therapeutic targets for diverse neurological disorders, including epilepsy. NMDA receptors were thought to play a key role in the generation of seizures, leading to clinical studies of NMDA receptor blocking drugs in epilepsy. Disappointing results dampened enthusiasm for ionotropic glutamate receptors as a therapeutic target. Eventually it became appreciated that another type of ionotropic glutamate receptor, the AMPA receptor, is actually the predominant mediator of excitatory neurotransmission in the central nervous system and moreover that AMPA receptors are critical to the generation and spread of epileptic activity. As drugs became available that selectively target AMPA receptors, it was possible to demonstrate that AMPA receptor antagonists have powerful antiseizure activity in in vitro and in vivo models. A decade later, promising clinical studies with AMPA receptor antagonists, including the potent noncompetitive antagonist perampanel, are once again focusing attention on AMPA receptors as a drug target for epilepsy therapy.

  13. AMPA receptor trafficking in recent vs. remote memory.

    PubMed

    Ghazal, Pasha

    2016-11-01

    It is now established that iteration of memory circuits takes place from hippocampus to cortical regions. The recall of recent event is largely dependent on the hippocampus networks, however, with passage of time, the cortical regions become largely involved in the recall of remote events. Molecular events, specifically, the AMPA receptor regulation underlying this iteration remains largely elusive. Separate groups of mice were fear conditioned using contextual fear conditioning paradigm. Memory retrieval test was performed 1 day post-training, for the recent memory group, together with respective controls. Where as, in case of remote group retrieval was performed 30 days post training. One hour post retrieval session, hippocampus and anterior cingulate regions were harvested after decapitation from all the groups, which were processed for synaptic membrane isolation and quantitative western blotting. We observed endocytosis of GluA1 and 2 exclusively in the anterior cingulate regions in the remote memory group, one hour post retrieval session, whereas in recent group, endocytosis of AMPA receptor units was only observed in the hippocampal regions. The endocytosis of GluA1-2 containing AMPARs upon retrieval, showed the weakened state of synapse. At this time point modification in content and strength of memory is possible for treatment of traumatic memories. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Sucrose Ingestion Induces Rapid AMPA Receptor Trafficking

    PubMed Central

    Tukey, David S.; Ferreira, Jainne M.; Antoine, Shannon O.; D’amour, James A.; Ninan, Ipe; de Vaca, Soledad Cabeza; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K.; Hartner, Diana T.; Guarini, Carlo B.; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F.; Khatri, Megna; Marzan, Dave S.; Mahajan, Shahana S.; Wang, Jing; Froemke, Robert C.; Carr, Kenneth D.; Aoki, Chiye; Ziff, Edward B.

    2013-01-01

    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPA receptors containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca2+-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPA receptors. Electrophysiological, biochemical and quantitative electron microscopy studies revealed that sucrose training (7 days) induced a stable (>24 hr) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 hr) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7-day protocol of daily ingestion of a 3% solution of saccharin, a non-caloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multi-step GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose. PMID:23554493

  15. AMPA receptors in the therapeutic management of depression.

    PubMed

    Bleakman, D; Alt, A; Witkin, J M

    2007-04-01

    There is an increasing body of evidence implicating a role for alpha-amino-3-hydroxy-5-methyl-4 isoxazoleproprionic acid (AMPA) receptors in major depression and in the actions of antidepressant drugs. Alterations in AMPA receptors and other ionotropic glutamate receptors have been reported in depression, and following antidepressant treatment. Compounds which augment signaling through AMPA receptors (AMPA receptor potentiators) exhibit antidepressant-like behavioral effects in animal models, and produce neuronal effects similar to those produced by currently available antidepressants, including neurotrophin induction and increases in hippocampal progenitor cell proliferation. Additionally, the antidepressant fluoxetine has been found to alter AMPA receptor phosphorylation in a manner that is expected to increase AMPA receptor signaling. Data from mutant mice suggest that AMPA receptors may regulate the expression of brain-derived neurotrophic factor, a neurotrophin which has been implicated in the actions of antidepressant therapies. Combined, these data suggest that AMPA receptors may be in a key position to regulate mood disorders, and that compounds which target AMPA receptors may prove useful in the clinical management of depression.

  16. Blockade of calcium-permeable AMPA receptors protects hippocampal neurons against global ischemia-induced death

    PubMed Central

    Noh, Kyung-Min; Yokota, Hidenori; Mashiko, Toshihiro; Castillo, Pablo E.; Zukin, R. Suzanne; Bennett, Michael V. L.

    2005-01-01

    Transient global or forebrain ischemia induced experimentally in animals can cause selective, delayed neuronal death of hippocampal CA1 pyramidal neurons. A striking feature is a delayed rise in intracellular free Zn2+ in CA1 neurons just before the onset of histologically detectable cell death. Here we show that α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) at Schaffer collateral to CA1 synapses in postischemic hippocampus exhibit properties of Ca2+/Zn2+-permeable, Glu receptor 2 (GluR2)-lacking AMPARs before the rise in Zn2+ and cell death. At 42 h after ischemia, AMPA excitatory postsynaptic currents exhibited pronounced inward rectification and marked sensitivity to 1-naphthyl acetyl spermine (Naspm), a selective channel blocker of GluR2-lacking AMPARs. In control hippocampus, AMPA excitatory postsynaptic currents were electrically linear and relatively insensitive to Naspm. Naspm injected intrahippocampally at 9-40 h after insult greatly reduced the late rise in intracellular free Zn2+ in postischemic CA1 neurons and afforded partial protection against ischemia-induced cell death. These results implicate GluR2-lacking AMPA receptors in the ischemia-induced rise in free Zn2+ and death of CA1 neurons, although a direct action at the time of the rise in Zn2+ is unproven. This receptor subtype appears to be an important therapeutic target for intervention in ischemia-induced neuronal death in humans. PMID:16093311

  17. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    PubMed

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  18. Occurrence of glyphosate and AMPA in an agricultural watershed from the southeastern region of Argentina.

    PubMed

    Lupi, Leonardo; Miglioranza, Karina S B; Aparicio, Virginia C; Marino, Damian; Bedmar, Francisco; Wunderlin, Daniel A

    2015-12-01

    Glyphosate (GLY) and AMPA concentrations were determined in sandy soil profiles, during pre- and post-application events in two agricultural soybean fields (S1 and S2). Streamwater and sediment samples were also analyzed. Post-application sampling was carried out one month later from the event. Concentrations of GLY+AMPA in surface soils (0-5 cm depth) during pre-application period showed values 20-fold higher (0.093-0.163 μg/g d.w.) than control area (0.005 μg/g d.w.). After application event soils showed markedly higher pesticide concentrations. A predominance of AMPA (80%) was observed in S1 (early application), while 34% in S2 for surface soils. GLY+AMPA concentrations decreased with depth and correlated strongly with organic carbon (r between 0.74 and 0.88, p<0.05) and pH (r between -0.81 and -0.76, p<0.001). The slight enrichment of pesticides observed from 25 cm depth to deeper layer, in addition to the alkaline pH along the profile, is of high concern about groundwater contamination. Sediments from pre-application period showed relatively lower pesticide levels (0.0053-0.0263 μg/g d.w.) than surface soil with a predominance of glyphosate, indicating a limited degradation. Levels of contaminants (mainly AMPA) in streamwater (ND-0.5 ng/mL) denote the low persistence of these compounds. However, a direct relationship in AMPA concentration was observed between sediment and streamwater. Despite the known relatively short half-life of glyphosate in soils, GLY+AMPA occurrence is registered in almost all matrices at different sampling times (pre- and post-application events). The physicochemical characteristics (organic carbon, texture, pH) and structure of soils and sediment in addition to the time elapsed from application determined the behavior of these contaminants in the environment. As a whole, the results warn us about vertical transport trough soil profile with the possibility of reaching groundwater.

  19. Benzoxazinones as potent positive allosteric AMPA receptor modulators: part I.

    PubMed

    Mueller, Rudolf; Li, Yong-Xin; Hampson, Aidan; Zhong, Sheng; Harris, Clayton; Marrs, Christopher; Rachwal, Stanislaw; Ulas, Jolanta; Nielsson, Lena; Rogers, Gary

    2011-07-01

    AMPA receptors (AMPARs) are an increasingly important therapeutic target in the CNS. Aniracetam, the first identified potentiator of AMPARs, led to the rigid and more potent CX614. This lead molecule was optimized in order to increase affinity towards the AMPA receptor. The substitution of the dioxine with a benzoxazinone ring system increased the activity and allowed further investigation of the sidechain SAR.

  20. The Biochemistry, Ultrastructure, and Subunit Assembly Mechanism of AMPA Receptors

    PubMed Central

    2010-01-01

    The AMPA-type ionotropic glutamate receptors (AMPA-Rs) are tetrameric ligand-gated ion channels that play crucial roles in synaptic transmission and plasticity. Our knowledge about the ultrastructure and subunit assembly mechanisms of intact AMPA-Rs was very limited. However, the new studies using single particle EM and X-ray crystallography are revealing important insights. For example, the tetrameric crystal structure of the GluA2cryst construct provided the atomic view of the intact receptor. In addition, the single particle EM structures of the subunit assembly intermediates revealed the conformational requirement for the dimer-to-tetramer transition during the maturation of AMPA-Rs. These new data in the field provide new models and interpretations. In the brain, the native AMPA-R complexes contain auxiliary subunits that influence subunit assembly, gating, and trafficking of the AMPA-Rs. Understanding the mechanisms of the auxiliary subunits will become increasingly important to precisely describe the function of AMPA-Rs in the brain. The AMPA-R proteomics studies continuously reveal a previously unexpected degree of molecular heterogeneity of the complex. Because the AMPA-Rs are important drug targets for treating various neurological and psychiatric diseases, it is likely that these new native complexes will require detailed mechanistic analysis in the future. The current ultrastructural data on the receptors and the receptor-expressing stable cell lines that were developed during the course of these studies are useful resources for high throughput drug screening and further drug designing. Moreover, we are getting closer to understanding the precise mechanisms of AMPA-R-mediated synaptic plasticity. PMID:21080238

  1. CP-465,022, a selective noncompetitive AMPA receptor antagonist, blocks AMPA receptors but is not neuroprotective in vivo.

    PubMed

    Menniti, Frank S; Buchan, Alistair M; Chenard, Bertrand L; Critchett, Donald J; Ganong, Alan H; Guanowsky, Victor; Seymour, Patricia A; Welch, Willard M

    2003-01-01

    Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibition has been hypothesized to provide neuroprotective efficacy after cerebral ischemia on the basis of the activity in experimental ischemia models of a variety of compounds with varying selectivity for AMPA over other glutamate receptor subtypes. CP-465,022 is a new, potent, and selective noncompetitive AMPA receptor antagonist. The present study investigated the ability of this compound to reduce neuronal loss after experimental cerebral ischemia to probe the neuroprotective potential of AMPA receptor inhibition. To demonstrate that CP-465,022 gains access to the brain, the effects of systemic administration of CP-465,022 were investigated on AMPA receptor-mediated electrophysiological responses in hippocampus and on chemically induced seizures in rats. The compound was then investigated for neuroprotective efficacy in rat global and focal ischemia models at doses demonstrated to be maximally effective in the electrophysiology and seizure models. CP-465,022 potently and efficaciously inhibited AMPA receptor-mediated hippocampal synaptic transmission and the induction of seizures. However, at comparable doses, CP-465,022 failed to prevent CA1 neuron loss after brief global ischemia or to reduce infarct volume after temporary middle cerebral artery occlusion. Given the high selectivity of CP-465,022 for AMPA over kainate and N-methyl-D-aspartate subtypes of glutamate receptors, the lack of neuroprotective efficacy of the compound calls into question the neuroprotective efficacy of AMPA receptor inhibition after ischemia.

  2. AMPA receptor competitive antagonism reduces halothane MAC in rats.

    PubMed

    McFarlane, C; Warner, D S; Todd, M M; Nordholm, L

    1992-12-01

    Various subtypes of receptors have been identified for glutamate, an excitatory neurotransmitter. Previous studies have shown that antagonism of glutamate at the NMDA receptors reduces minimum alveolar concentration (MAC) for volatile anesthetics. NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline) is a selective antagonist at the glutamatergic AMPA receptor. The purpose of this experiment was to determine whether AMPA receptor antagonism influences halothane MAC in the rat. Sprague-Dawley rats were anesthetized with halothane in 50% O2/balance N2, tracheally intubated and the lungs were mechanically ventilated. Increasing doses of NBQX were intravenously infused in three groups while the control group was infused with vehicle (D5W). Halothane MAC was then determined by the tail-clamp method. Halothane MAC was log-linearly related to plasma NBQX concentrations (MAC = 0.125 (In plasma concentration NBQX) + 1.035, r2 = 0.77). A maximal 58% reduction of halothane MAC was achieved with an NBQX loading dose of 42 mg/kg followed by a continuous infusion rate of 36 mg x kg-1 x h-1 (control = 1.02 +/- 0.07%; NBQX = 0.43 +/- 0.12%; P < .01). Larger doses of NBQX were not possible because of the poor aqueous solubility of this compound. In a separate experiment, awake rats were randomly assigned to groups based on the dose of NBQX infused. Pa(CO2) and mean arterial pressure were measured at time 0 and at 5 and 30 min after start of NBQX infusion. The infusion was then stopped. Time until recovery of the righting reflex was recorded.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. AMPA receptor potentiators for the treatment of CNS disorders.

    PubMed

    O'Neill, Michael J; Bleakman, David; Zimmerman, Dennis M; Nisenbaum, Eric S

    2004-06-01

    Glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors mediate most of the excitatory neurotransmission in the mammalian central nervous system and also participate in forms of synaptic plasticity thought to underlie memory and learning, and the formation of neural networks during development. Molecular cloning techniques have shown that the AMPA receptor family is composed of four different subunits named GluR1-4 or GluRA-D (newly termed as Glu(A1)-Glu(A4)) and native AMPA receptors are most likely tetramers generated by the assembly of one or more of these subunits, yielding homomeric or heteromeric receptors. Additional complexity among AMPA receptors is conferred by alternative splicing of RNA for each subunit giving rise to flip and flop variants. Clinical and experimental data have suggested that positive modulation of AMPA receptors may be therapeutically effective in the treatment of cognitive deficits. Several classes of AMPA receptor potentiators have been reported, including pyrroliddones (piracetam, aniracetam), benzothiazides (cyclothiazide), benzylpiperidines (CX-516, CX-546) and more recently biarylpropylsulfonamides (LY392098, LY404187 and LY503430). These molecules enhance cognitive function in rodents, which appears to correlate with increased hippocampal activity. In addition, clinical studies have suggested that AMPA receptor modulators enhance cognitive function in elderly subjects, as well as patients suffering from neurological and psychiatric disorders. Several independent studies have suggested that AMPA receptors can increase BDNF expression by both calcium-dependent and independent pathways. For example, recent studies have shown that AMPA receptors interact with the protein tyrosine kinase, Lyn. Activation of Lyn can recruit the mitogen-activated protein kinase (MAPK) signalling pathway and increase the expression of BDNF. Therefore, in addition to directly enhancing glutamatergic synaptic transmission, AMPA

  4. Autoimmune encephalomyelitis ameliorated by AMPA antagonists.

    PubMed

    Smith, T; Groom, A; Zhu, B; Turski, L

    2000-01-01

    Multiple sclerosis is an immune-mediated disorder of the central nervous system leading to progressive decline of motor and sensory functions and permanent disability. The therapy of multiple sclerosis is only partially effective, despite anti-inflammatory, immunosuppresive and immunomodulatory measures. White matter inflammation and loss of myelin, the pathological hallmarks of multiple sclerosis, are thought to determine disease severity. Experimental autoimmune encephalomyelitis reproduces the features of multiple sclerosis in rodents and in nonhuman primates. The dominant early clinical symptom of acute autoimmune encephalomyelitis is progressive ascending muscle weakness. However, demyelination may not be profound and its extent may not correlate with severity of neurological decline, indicating that targets unrelated to myelin or oligodendrocytes may contribute to the pathogenesis of acute autoimmune encephalomyelitis. Here we report that within the spinal cord in the course of autoimmune encephalomyelitis not only myelin but also neurons are subject to lymphocyte attack and may degenerate. Blockade of glutamate AMPA receptors ameliorated the neurological sequelae of autoimmune encephalomyelitis, indicating the potential for AMPA antagonists in the therapy of multiple sclerosis.

  5. Sucrose ingestion induces rapid AMPA receptor trafficking.

    PubMed

    Tukey, David S; Ferreira, Jainne M; Antoine, Shannon O; D'amour, James A; Ninan, Ipe; Cabeza de Vaca, Soledad; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K; Hartner, Diana T; Guarini, Carlo B; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F; Khatri, Megna; Marzan, Dave S; Mahajan, Shahana S; Wang, Jing; Froemke, Robert C; Carr, Kenneth D; Aoki, Chiye; Ziff, Edward B

    2013-04-03

    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

  6. Regulation of AMPA receptor gating and pharmacology by TARP auxiliary subunits.

    PubMed

    Milstein, Aaron D; Nicoll, Roger A

    2008-07-01

    Presynaptic glutamate release elicits brief waves of membrane depolarization in neurons by activating AMPA receptors. Depending on its precise size and shape, current through AMPA receptors gates downstream processes like NMDA receptor activation and action potential generation. Over a decade of research on AMPA receptor structure and function has identified binding sites on AMPA receptors for agonists, antagonists and allosteric modulators as well as key residues underlying differences in the gating behavior of various AMPA receptor subtypes. However, the recent discovery that AMPA receptors are accompanied in the synaptic membrane by a family of auxiliary subunits known as transmembrane AMPA receptor regulatory proteins (TARPs) has revealed that the kinetics and pharmacology of neuronal AMPA receptors differ in many respects from those predicted by classical studies of AMPA receptors in heterologous systems. Here, we summarize recent work and discuss remaining questions concerning the structure and function of native TARP-AMPA receptor complexes.

  7. Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP.

    PubMed

    Wu, Dick; Bacaj, Taulant; Morishita, Wade; Goswami, Debanjan; Arendt, Kristin L; Xu, Wei; Chen, Lu; Malenka, Robert C; Südhof, Thomas C

    2017-04-20

    Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca(2+) influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca(2+) induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca(2+)-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca(2+)-dependent presynaptic vesicle exocytosis, also blocked Ca(2+)-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca(2+)-sensors for Ca(2+)-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.

  8. Differences of AMPA and kainate receptor interactomes identify a novel AMPA receptor auxiliary subunit, GSG1L

    PubMed Central

    Shanks, Natalie F.; Savas, Jeffrey N.; Maruo, Tomohiko; Cais, Ondrej; Hirao, Atsushi; Oe, Souichi; Ghosh, Anirvan; Noda, Yasuko; Greger, Ingo H.; Yates, John R.; Nakagawa, Terunaga

    2012-01-01

    AMPA receptor (AMPA-R) complexes consist of channel forming subunits, GluA1–4 and auxiliary proteins including TARPs, CNIHs, synDIG1, and CKAMP44, which can modulate AMPA-R function in specific ways. Combinatorial effects of four GluA subunits binding to various auxiliary subunits amplify the functional diversity of AMPA-Rs. The significance and magnitude of molecular diversity, however, remain elusive. To gain insight into the molecular complexity of AMPA and kainate receptors (KA-Rs), we compared the proteins that co-purify with each receptor type in rat brain. This interactome study identified the majority of known interacting proteins and more importantly, provides novel candidates for further studies. We validate the claudin homologue GSG1L as a novel binding protein and unique modulator of AMPA-R gating, as determined by detailed molecular, cellular, electrophysiological, and biochemical experiments. GSG1L extends the functional variety of AMPA-R complexes and further investigation of other candidates may reveal additional complexity of ionotropic glutamate receptor function. PMID:22813734

  9. Activation of somatostatin receptor (sst 5) protects the rat retina from AMPA-induced neurotoxicity.

    PubMed

    Kiagiadaki, Foteini; Savvaki, Maria; Thermos, Kyriaki

    2010-01-01

    In a recent study, we employed an in vivo model of retinal excitotoxicity to investigate the neuroprotective effect of somatostatinergic agents. Intravitreal administration of somatostatin and sst(2) selective agonists protected the retina from (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) induced excitotoxicity. The sst(1) and sst(4) selective ligands had no effect (Kiagiadaki and Thermos, 2008). The presence of sst(5) receptors in rat retina was only recently reported (Ke and Zhong, 2007). Synthetic agonists that activate sst(2) receptors also bind with high affinity to the sst(5) subtype. In the present study the putative neuroprotective effects of sst(5) receptor activation were investigated. Adult female and male Sprague-Dawley (250-350g) rats were employed. Groups of animals received intravitreally PBS (50mM) or AMPA (42 nmol/eye) alone or in combination with L-817,818 (sst(5), 10(-5), 10(-4)M). To exclude neuroprotective effects via the activation of sst(2) receptors, L-817,818 (10(-4)M) was coinjected with the sst(2) antagonist CYN-154806 (10(-4)M). Immunohistochemistry (IHC) studies using the anti-retinal marker choline acetyltransferase (ChAT) and TUNEL staining were employed to examine retinal cell loss and protection. IHC and Western blot analysis were also employed to assess whether the sst(5) receptors are viable in the AMPA treated tissue as compared to control retina. sst(5) receptors were not affected by AMPA. L-817,818 protected the retina from the AMPA insult in the dose of 10(-4)M, while CYN-154806 (10(-4)M) had no effect on the sst(5) neuroprotection. TUNEL staining confirmed the AMPA-induced retinal toxicity and the L-817,818 neuroprotection. These results demonstrate for the first time that sst(5) receptors are functional in the retina, and that sst(5) analogs administered intravitreally protect the retina from excitotoxicity. Further studies are essential to ascertain the therapeutic relevance of these

  10. TARP modulation of synaptic AMPA receptor trafficking and gating depends on multiple intracellular domains.

    PubMed

    Milstein, Aaron D; Nicoll, Roger A

    2009-07-07

    Previous work has established stargazin and its related family of transmembrane AMPA receptor regulatory proteins (TARPs) as auxiliary subunits of AMPA receptors (AMPARs) that control synaptic strength both by targeting AMPARs to synapses through an intracellular PDZ-binding motif and by modulating their gating through an extracellular domain. However, TARPs gamma-2 and gamma-8 differentially regulate the synaptic targeting of AMPARs, despite having identical PDZ-binding motifs. Here, we investigate the structural elements that contribute to this functional difference between TARP subtypes by using domain transplantation and truncation. We identify a component of synaptic AMPAR trafficking that is independent of the TARP C-terminal PDZ-binding motif, and we establish previously uncharacterized roles for the TARP intracellular N terminus, loop, and C terminus in modulating both the trafficking and gating of synaptic AMPARs.

  11. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  12. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    ERIC Educational Resources Information Center

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  13. AMPA receptor subunits expression and phosphorylation in cingulate cortex in rats following esophageal acid exposure

    PubMed Central

    BANERJEE, B.; MEDDA, B. K.; POCHIRAJU, S.; KANNAMPALLI, P.; LANG, I. M.; SENGUPTA, J. N.; SHAKER, R.

    2014-01-01

    Background We recently reported an increase in N-methyl-d-aspartate (NMDA) receptor subunit expression and CaMKII-dependent phosphorylation of NR2B in the rostral cingulate cortical (rCC) neurons following esophageal acid exposure in rats. As α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors mediate the fast excitatory transmission and play a critical role in synaptic plasticity, in this study, we investigated the effect of esophageal acid exposure in rats on the expression of AMPA receptor subunits and the involvement of these molecular alterations in acid-induced sensitization of neurons in the anterior cingulate (ACC) and midcingulate (MCC) cortices. Methods In molecular study, we examined GluA1 and GluA2 expression and phosphorylation in membrane preparations and in the isolated postsynaptic densities (PSDs) from rats receiving acute esophageal exposure of either saline (control group) or 0.1 NHCl (experimental group). In electrophysiological study, the effect of selective AMPA receptor (Ca2+ permeable) antagonist IEM-1460 and CaMKII inhibitor KN-93 was tested on responses of cortical neurons during acid infusion to address the underlying molecular mechanism of acid-induced sensitization. Key Results The acid exposure significantly increased expression of GluA1, pGluA1Ser831, and phosphorylated CaMKIIThr286, in the cortical membrane preparations. In isolated PSDs, a significant increase in pGluA1Ser831 was observed in acid-treated rats compared with controls. Microinjection of IEM-1460 or KN-93 near the recording site significantly attenuated acid-induced sensitization of cortical neurons. Conclusions & Inferences The underlying mechanism of acid-induced cortical sensitization involves upregulation and CaMKII-mediated phosphorylation of GluA1. These molecular changes of AMPA receptors subunit GluA1 in the cortical neurons might play an important role in acid-induced esophageal hypersensitivity. PMID:24118589

  14. Xenon reduces glutamate-, AMPA-, and kainate-induced membrane currents in cortical neurones.

    PubMed

    Dinse, A; Föhr, K J; Georgieff, M; Beyer, C; Bulling, A; Weigt, H U

    2005-04-01

    The anaesthetic, analgesic, and neuroprotective effects of xenon (Xe) are believed to be mediated by a block of the NMDA (N-methyl-D-aspartate) receptor channel. Interestingly, the clinical profile of the noble gas differs markedly from that of specific NMDA receptor antagonists. The aim of this study was, therefore, to investigate whether Xe might be less specific, also inhibiting the two other subtypes of glutamate receptor channels, such as the alpha-amino-3-hydroxy-5-methyl-4-isoxazolole propionate (AMPA) and kainate receptors. The study was performed on voltage-clamped cortical neurones from embryonic mice and SH-SY5Y cells expressing GluR6 kainate receptors. Drugs were applied by a multi-barreled fast perfusion system. Xe, dissolved at approximately 3.45 mM in aqueous solution, diminished the peak and even more the plateau of AMPA and glutamate induced currents. At the control EC(50) value for AMPA (29 microM) these reductions were by about 40 and 56% and at 3 mM glutamate the reductions were by 45 and 66%, respectively. Currents activated at the control EC(50) value for kainate (57 microM) were inhibited by 42%. Likewise, Xe showed an inhibitory effect on kainate-induced membrane currents of SH-SY5Y cells transfected with the GluR6 subunit of the kainate receptor. Xe reduced kainate-induced currents by between 35 and 60%, depending on the kainate concentration. Xe blocks not only NMDA receptors, but also AMPA and kainate receptors in cortical neurones as well as GluR6-type receptors expressed in SH-SY5Y cells. Thus, Xe seems to be rather non-specific as a channel blocker and this may contribute to the analgesic and anaesthetic potency of Xe.

  15. Novel bivalent positive allosteric modulators of AMPA receptor.

    PubMed

    Lavrov, M I; Grigor'ev, V V; Bachurin, S O; Palyulin, V A; Zefirov, N S

    2015-01-01

    A positive allosteric modulator of AMPA receptors has been designed using computer-aided molecular modeling techniques. It possessed a record high experimentally confirmed potency in the picomolar concentration range and belongs to a new type of bivalent AMPA receptor ligands containing bicyclo[3.3.1]nonane scaffold. The suggested structure could serve as a basis for further optimization and development of drugs for the treatment of neurodegenerative diseases, cognition enhancement, and improvement of memory.

  16. AMPA Receptors as a Molecular Target in Epilepsy Therapy

    PubMed Central

    Rogawski, Michael A.

    2013-01-01

    Epileptic seizures occur as a result of episodic abnormal synchronous discharges in cerebral neuronal networks. Although a variety of nonconventional mechanisms may play a role in epileptic synchronization, cascading excitation within networks of synaptically connected excitatory glutamatergic neurons is a classical mechanism. As is the case throughout the central nervous system, fast synaptic excitation within and between brain regions relevant to epilepsy is mediated predominantly by AMPA receptors. By inhibiting glutamate-mediated excitation, AMPA receptor antagonists markedly reduce or abolish epileptiform activity in in vitro preparations and confer seizure protection in a broad range of animal seizure models. NMDA receptors may also contribute to epileptiform activity, but NMDA receptor blockade is not sufficient to eliminate epileptiform discharges. AMPA receptors move into and out of the synapse in a dynamic fashion in forms of synaptic plasticity, underlying learning and memory. Often the trigger for these dynamic movements is activation of NMDA receptors. While NMDA receptor antagonists inhibit these forms of synaptic plasticity, AMPA receptor antagonists do not impair synaptic plasticity and do not inhibit memory formation or retrieval. The demonstrated clinical efficacy of perampanel, a high-potency, orally active noncompetitive AMPA receptor antagonist, supports the concept that AMPA receptors are critical to epileptic synchronization and the generation and spread of epileptic discharges in human epilepsy. PMID:23480151

  17. Regulation of postsynaptic AMPA responses by synaptojanin 1.

    PubMed

    Gong, Liang-Wei; De Camilli, Pietro

    2008-11-11

    Endocytosis of postsynaptic AMPA receptors is a mechanism through which efficiency of neurotransmission is regulated. We have genetically tested the hypothesis that synaptojanin 1, a phosphoinositide phosphatase implicated in the endocytosis of synaptic vesicles presynaptically, may also function in the endocytosis of AMPA receptors postsynaptically. Electrophysiological recordings of cultured hippocampal neurons showed that miniature excitatory postsynaptic current amplitudes were larger in synaptojanin 1 knockout (KO) neurons because of an increase of surface-exposed AMPA receptors. This change did not represent an adaptive response to decreased presynaptic release in KO cultures and was rescued by the expression of wild type, but not catalytically inactive synaptojanin 1, in the postsynaptic neuron. NMDA-induced internalization of pHluorin-tagged AMPA receptors (GluR2) was impaired in KO neurons. These results reveal a function of synaptojanin 1 in constitutive and triggered internalization of AMPA receptors and thus indicate a role for phosphatidylinositol(4,5)-bisphosphate metabolism in the regulation of postsynaptic AMPA responses.

  18. AMPA receptors as a molecular target in epilepsy therapy.

    PubMed

    Rogawski, M A

    2013-01-01

    Epileptic seizures occur as a result of episodic abnormal synchronous discharges in cerebral neuronal networks. Although a variety of non-conventional mechanisms may play a role in epileptic synchronization, cascading excitation within networks of synaptically connected excitatory glutamatergic neurons is a classical mechanism. As is the case throughout the central nervous system, fast synaptic excitation within and between brain regions relevant to epilepsy is mediated predominantly by AMPA receptors. By inhibiting glutamate-mediated excitation, AMPA receptor antagonists markedly reduce or abolish epileptiform activity in in vitro preparations and confer seizure protection in a broad range of animal seizure models. NMDA receptors may also contribute to epileptiform activity, but NMDA receptor blockade is not sufficient to eliminate epileptiform discharges. AMPA receptors move into and out of the synapse in a dynamic fashion in forms of synaptic plasticity, underlying learning and memory. Often, the trigger for these dynamic movements is the activation of NMDA receptors. While NMDA receptor antagonists inhibit these forms of synaptic plasticity, AMPA receptor antagonists do not impair synaptic plasticity and do not inhibit memory formation or retrieval. The demonstrated clinical efficacy of perampanel, a high-potency, orally active non-competitive AMPA receptor antagonist, supports the concept that AMPA receptors are critical to epileptic synchronization and the generation and spread of epileptic discharges in human epilepsy.

  19. Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes

    PubMed Central

    Droste, Damian; Seifert, Gerald; Seddar, Laura; Jädtke, Oliver; Steinhäuser, Christian; Lohr, Christian

    2017-01-01

    Ca2+ signaling in astrocytes is considered to be mainly mediated by metabotropic receptors linked to intracellular Ca2+ release. However, recent studies demonstrate a significant contribution of Ca2+ influx to spontaneous and evoked Ca2+ signaling in astrocytes, suggesting that Ca2+ influx might account for astrocytic Ca2+ signaling to a greater extent than previously thought. Here, we investigated AMPA-evoked Ca2+ influx into olfactory bulb astrocytes in mouse brain slices using Fluo-4 and GCaMP6s, respectively. Bath application of AMPA evoked Ca2+ transients in periglomerular astrocytes that persisted after neuronal transmitter release was inhibited by tetrodotoxin and bafilomycin A1. Withdrawal of external Ca2+ suppressed AMPA-evoked Ca2+ transients, whereas depletion of Ca2+ stores had no effect. Both Ca2+ transients and inward currents induced by AMPA receptor activation were partly reduced by Naspm, a blocker of Ca2+-permeable AMPA receptors lacking the GluA2 subunit. Antibody staining revealed a strong expression of GluA1 and GluA4 and a weak expression of GluA2 in periglomerular astrocytes. Our results indicate that Naspm-sensitive, Ca2+-permeable AMPA receptors contribute to Ca2+ signaling in periglomerular astrocytes in the olfactory bulb. PMID:28322255

  20. KIS, a kinase associated with microtubule regulators, enhances translation of AMPA receptors and stimulates dendritic spine remodeling.

    PubMed

    Pedraza, Neus; Ortiz, Raúl; Cornadó, Alba; Llobet, Artur; Aldea, Martí; Gallego, Carme

    2014-10-15

    Local regulation of protein synthesis allows a neuron to rapidly alter the proteome in response to synaptic signals, an essential mechanism in synaptic plasticity that is altered in many neurological diseases. Synthesis of many synaptic proteins is under local control and much of this regulation occurs through structures termed RNA granules. KIS is a protein kinase that associates with stathmin, a modulator of the tubulin cytoskeleton. Furthermore, KIS is found in RNA granules and stimulates translation driven by the β-actin 3'UTR in neurites. Here we explore the physiological and molecular mechanisms underlying the action of KIS on hippocampal synaptic plasticity in mice. KIS downregulation compromises spine development, alters actin dynamics, and reduces postsynaptic responsiveness. The absence of KIS results in a significant decrease of protein levels of PSD-95, a postsynaptic scaffolding protein, and the AMPAR subunits GluR1 and GluR2 in a CPEB3-dependent manner. Underlying its role in spine maturation, KIS is able to suppress the spine developmental defects caused by CPEB3 overexpression. Moreover, either by direct or indirect mechanisms, KIS counteracts the inhibitory activity of CPEB3 on the GluR2 3'UTR at both mRNA translation and polyadenylation levels. Our study provides insights into the mechanisms that mediate dendritic spine morphogenesis and functional synaptic maturation, and suggests KIS as a link regulating spine cytoskeleton and postsynaptic activity in memory formation.

  1. Glyphosate and AMPA contents in sediments produced by wind erosion of agricultural soils in Argentina

    NASA Astrophysics Data System (ADS)

    Aparicio, Virginia; Aimar, Silvia; De Gerónimo, Eduardo; Buschiazzo, Daniel; Mendez, Mariano; Costa, José Luis

    2014-05-01

    Wind erosion of soils is an important event in arid and semiarid regions of Argentina. The magnitude of wind erosion occurring under different management practices is relatively well known in this region but less information is available on the quality of the eroded material. Considering that the intensification of agriculture may increase the concentrations of substances in the eroded material, producing potential negative effects on the environment, we analyzed the amount of glyphosate and AMPA in sediments produced by wind erosion of agricultural soils of Argentina. Wind eroded materials were collected by means of BSNE samplers in two loess sites of the semiarid region of Argentina: Chaco and La Pampa. Samples were collected from 1 ha square fields at 13.5, 50 and 150 cm height. Results showed that at higher heights the concentrations of glyphosate and AMPA were mostly higher. The glyphosate concentration was more variable and higher in Chaco (0.66 to 313 µg kg-1) than in La Pampa (4.17 to 114 µg kg-1). These results may be due to the higher use of herbicides in Chaco, where the predominant crops are soybeans and corn, produced under no-tillage. Under these conditions the use of glyphosate for weeds control is a common practice. Conversely, AMPA concentrations were higher in La Pampa (13.1 to 101.3 µg kg-1) than in Chaco (1.3 to 83 µg kg-1). These preliminary results show high concentrations of glyphosate and AMPA in wind eroded materials of agricultural soils of Argentina. More research is needed to confirm these high concentrations in other conditions in order to detect the temporal and spatial distribution patterns of the herbicide.

  2. PACSIN1 regulates the dynamics of AMPA receptor trafficking.

    PubMed

    Widagdo, Jocelyn; Fang, Huaqiang; Jang, Se Eun; Anggono, Victor

    2016-08-04

    Dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses plays an important role in synaptic plasticity. We previously reported that the protein kinase C and casein kinase II substrate in neurons (PACSIN) forms a complex with AMPARs through its interaction with the protein interacting with C-kinase 1 (PICK1) to regulate NMDA receptor (NMDAR)-induced AMPAR endocytosis and cerebellar long-term depression. However, the molecular mechanism by which PACSIN regulates the dynamics of AMPAR trafficking remains unclear. Using a pH-sensitive green fluorescent protein, pHluorin, tagged to the extracellular domain of the GluA2 subunit of AMPARs, we demonstrate dual roles for PACSIN1 in controlling the internalization and recycling of GluA2 after NMDAR activation. Structure and function analysis reveals a requirement for the PACSIN1 F-BAR and SH3 domains in controlling these NMDAR-dependent processes. Interestingly, the variable region, which binds to PICK1, is not essential for NMDAR-dependent GluA2 internalization and is required only for the correct recycling of AMPARs. These results indicate that PACSIN is a versatile membrane deformation protein that links the endocytic and recycling machineries essential for dynamic AMPAR trafficking in neurons.

  3. PACSIN1 regulates the dynamics of AMPA receptor trafficking

    PubMed Central

    Widagdo, Jocelyn; Fang, Huaqiang; Jang, Se Eun; Anggono, Victor

    2016-01-01

    Dynamic trafficking of AMPA receptors (AMPARs) into and out of synapses plays an important role in synaptic plasticity. We previously reported that the protein kinase C and casein kinase II substrate in neurons (PACSIN) forms a complex with AMPARs through its interaction with the protein interacting with C-kinase 1 (PICK1) to regulate NMDA receptor (NMDAR)-induced AMPAR endocytosis and cerebellar long-term depression. However, the molecular mechanism by which PACSIN regulates the dynamics of AMPAR trafficking remains unclear. Using a pH-sensitive green fluorescent protein, pHluorin, tagged to the extracellular domain of the GluA2 subunit of AMPARs, we demonstrate dual roles for PACSIN1 in controlling the internalization and recycling of GluA2 after NMDAR activation. Structure and function analysis reveals a requirement for the PACSIN1 F-BAR and SH3 domains in controlling these NMDAR-dependent processes. Interestingly, the variable region, which binds to PICK1, is not essential for NMDAR-dependent GluA2 internalization and is required only for the correct recycling of AMPARs. These results indicate that PACSIN is a versatile membrane deformation protein that links the endocytic and recycling machineries essential for dynamic AMPAR trafficking in neurons. PMID:27488904

  4. Unified quantitative model of AMPA receptor trafficking at synapses.

    PubMed

    Czöndör, Katalin; Mondin, Magali; Garcia, Mikael; Heine, Martin; Frischknecht, Renato; Choquet, Daniel; Sibarita, Jean-Baptiste; Thoumine, Olivier R

    2012-02-28

    Trafficking of AMPA receptors (AMPARs) plays a key role in synaptic transmission. However, a general framework integrating the two major mechanisms regulating AMPAR delivery at postsynapses (i.e., surface diffusion and internal recycling) is lacking. To this aim, we built a model based on numerical trajectories of individual AMPARs, including free diffusion in the extrasynaptic space, confinement in the synapse, and trapping at the postsynaptic density (PSD) through reversible interactions with scaffold proteins. The AMPAR/scaffold kinetic rates were adjusted by comparing computer simulations to single-particle tracking and fluorescence recovery after photobleaching experiments in primary neurons, in different conditions of synapse density and maturation. The model predicts that the steady-state AMPAR number at synapses is bidirectionally controlled by AMPAR/scaffold binding affinity and PSD size. To reveal the impact of recycling processes in basal conditions and upon synaptic potentiation or depression, spatially and temporally defined exocytic and endocytic events were introduced. The model predicts that local recycling of AMPARs close to the PSD, coupled to short-range surface diffusion, provides rapid control of AMPAR number at synapses. In contrast, because of long-range diffusion limitations, extrasynaptic recycling is intrinsically slower and less synapse-specific. Thus, by discriminating the relative contributions of AMPAR diffusion, trapping, and recycling events on spatial and temporal bases, this model provides unique insights on the dynamic regulation of synaptic strength.

  5. Soil concentration of glyphosate and AMPA under rice cultivation with contrasting levels of fertilization

    NASA Astrophysics Data System (ADS)

    Rey Montoya, Tania; Micaela Biassoni, María; Graciela Herber, Luciana; De Geronimo, Eduardo; Aparicio, Virginia

    2017-04-01

    Rice (Oryza sativa) is the world's most important crop species and occupies c. 150 mill ha. The province of Corrientes in Argentina leads the national production of rice cultivation. Glyphosate is a non-selective herbicide commonly used to control weeds. The molecule is inactivated once applied due to its adsorption in the soil, and once desorbed is degraded by soil microflora resulting in sarcosine and aminomethylphosphoric acid (AMPA) molecules. The objective of this investigation was to compare glyphosate and AMPA concentration in soil under different levels of fertilization along the growth season of the rice crop. A field experiment following a completely randomized design was carried out with four replicates. We evaluated four levels of fertilization (0-18-40): Control: 0 kg ha-1, Dose 1: 120 kg ha-1, Dose 2: 150 kg ha-1, Dose 3: 180 kg ha-1; and two levels of Glyphosate: with (Gly) or without (No) application. Four sampling moments were defined: pre-sowing (taken as reference), vegetative stage (V4, 30 days after application), in floral primordial differentiation-DPF (80 days post-application), and at physiological maturity-MF (125 days after application). Flooding was applied in V4 after sampling. The method used for determination and quantification was by ultra high-pressure liquid chromatography coupled to ESI UHPLC-MS / MS tandem mass spectrometer (+/-) (Acquit-Quattro Premier). We found that glyphosate and AMPA varied their concentration in soil according to the time of sampling. Detected levels of both molecules at pre-sowing indicate the persistence of this herbicide from earlier crop seasons. The highest concentration was measured in MF followed by V4. Interestingly, AMPA concentration showed higher values in V4 without application compared to the treatment with glyphosate application. On the other hand, in flooded soil both molecules presented a decrease in their concentration probably because of their dilution in water, increasing it again after

  6. Hypoxia-induced retinal ganglion cell damage through activation of AMPA receptors and the neuroprotective effects of DNQX.

    PubMed

    Sivakumar, V; Foulds, W S; Luu, C D; Ling, E A; Kaur, C

    2013-04-01

    Hypoxia-induced glutamate accumulation in neural tissues results in damage to neurons through excitotoxic mechanisms via activation of glutamate receptors (GluRs). Here we examine whether hypoxia in the developing retina would cause activation of the ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) GluRs and increase in Ca(2+) influx into retinal ganglion cells (RGCs) that might ultimately lead to their death. Neonatal Wistar rats were subjected to hypoxia for 2h and then sacrificed at various time points after the exposure together with normal age matched control rats. Primary cultures of RGCs were also prepared and subjected to hypoxia. Expression of AMPA glutamate receptor (GluR) 1-4 was examined in the retina. Additionally, expression of GluRs, intracellular Ca(2+) influx, reactive oxygen species (ROS) generation and cell death were investigated in cultured RGCs. GluR1-4 mRNA and protein expression showed a significant increase (P < 0.01) over control values after the hypoxic exposure both in vivo and in vitro. Cells expressing GluR1-4 in the retina were identified as RGCs by double immunofluorescence labeling with Thy1.1. Increased intracellular Ca(2+) in cultured RGCs following hypoxic exposure was reduced (P < 0.01) by 10 μM AMPA antagonist 6, 7-dinitroquinoxaline-2,3-dione (DNQX). Our results suggest that following a hypoxic insult, an increased amount of glutamate accumulates in the neonatal retina. This would then activate AMPA receptors which may damage RGCs through increased Ca(2+) accumulation and ROS generation. The involvement of AMPA receptors in damaging the RGCs is evidenced by suppression of intracellular Ca(2+) influx by DNQX which also decreased ROS generation and cell death by 50%. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Interactions between recording technique and AMPA receptor modulators.

    PubMed

    Lin, Bin; Colgin, Laura Lee; Brücher, Fernando Andres; Arai, Amy Christine; Lynch, Gary

    2002-11-15

    Whole cell recording (EPSCs) and extracellular recording (field EPSPs) were compared in hippocampal field CA1 with regard to the effects of experimental treatments that increase AMPA receptor gated currents. Cyclothiazide, which maintains AMPA receptors in the sensitized state, caused a rapid and pronounced increase in EPSCs but only minor changes in field EPSPs. This difference was evident in recordings carried out at 22 and 32 degrees C and with different solutions in the clamp pipette. The larger effect of cyclothiazide on EPSCs was unaffected by blockade of GABA and NMDA receptors. Two-dimensional current source density analyses derived from 64 recording sites were used to provide extracellular estimates of AMPA receptor mediated synaptic currents. With this method, cyclothiazide again had much smaller effects than were obtained with whole cell clamp. Differences between whole cell and extracellular recordings were present, although not as pronounced, for the ampakines, a class of drugs that slow both deactivation and desensitization of AMPA receptors. Additionally, increases in synaptic responses produced by frequency facilitation, a manipulation that enhances the number of bound receptors, were not qualitatively different between recording techniques. These results support the conclusion that the whole cell clamp technique may alter AMPA receptors in such a way as to increase the relative importance of desensitization.

  8. Are AMPA Receptor Positive Allosteric Modulators Potential Pharmacotherapeutics for Addiction?

    PubMed Central

    Watterson, Lucas R.; Olive, M. Foster

    2013-01-01

    Positive allosteric modulators (PAMs) of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a diverse class of compounds that increase fast excitatory transmission in the brain. AMPA PAMs have been shown to facilitate long-term potentiation, strengthen communication between various cortical and subcortical regions, and some of these compounds increase the production and release of brain-derived neurotrophic factor (BDNF) in an activity-dependent manner. Through these mechanisms, AMPA PAMs have shown promise as broad spectrum pharmacotherapeutics in preclinical and clinical studies for various neurodegenerative and psychiatric disorders. In recent years, a small collection of preclinical animal studies has also shown that AMPA PAMs may have potential as pharmacotherapeutic adjuncts to extinction-based or cue-exposure therapies for the treatment of drug addiction. The present paper will review this preclinical literature, discuss novel data collected in our laboratory, and recommend future research directions for the possible development of AMPA PAMs as anti-addiction medications. PMID:24380895

  9. Mechanism of Positive Allosteric Modulators Acting on AMPA Receptors

    SciTech Connect

    Jin,R.; Clark, S.; Weeks, A.; Dudman, J.; Gouaux, E.; Partin, K.

    2005-01-01

    Ligand-gated ion channels involved in the modulation of synaptic strength are the AMPA, kainate, and NMDA glutamate receptors. Small molecules that potentiate AMPA receptor currents relieve cognitive deficits caused by neurodegenerative diseases such as Alzheimer's disease and show promise in the treatment of depression. Previously, there has been limited understanding of the molecular mechanism of action for AMPA receptor potentiators. Here we present cocrystal structures of the glutamate receptor GluR2 S1S2 ligand-binding domain in complex with aniracetam [1-(4-methoxybenzoyl)-2-pyrrolidinone] or CX614 (pyrrolidino-1, 3-oxazino benzo-1, 4-dioxan-10-one), two AMPA receptor potentiators that preferentially slow AMPA receptor deactivation. Both potentiators bind within the dimer interface of the nondesensitized receptor at a common site located on the twofold axis of molecular symmetry. Importantly, the potentiator binding site is adjacent to the 'hinge' in the ligand-binding core 'clamshell' that undergoes conformational rearrangement after glutamate binding. Using rapid solution exchange, patch-clamp electrophysiology experiments, we show that point mutations of residues that interact with potentiators in the cocrystal disrupt potentiator function. We suggest that the potentiators slow deactivation by stabilizing the clamshell in its closed-cleft, glutamate-bound conformation.

  10. Molecular Mechanism of AMPA Receptor Modulation by TARP/Stargazin.

    PubMed

    Ben-Yaacov, Anat; Gillor, Moshe; Haham, Tomer; Parsai, Alon; Qneibi, Mohammad; Stern-Bach, Yael

    2017-03-08

    AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain and critically contribute to synaptic plasticity and pathology. AMPAR trafficking and gating are tightly controlled by auxiliary transmembrane AMPAR regulatory proteins (TARPs). Here, using systematic domain swaps with the TARP-insensitive kainate receptor GluK2, we show that AMPAR interaction with the prototypical TARP stargazin/γ2 primarily involves the AMPAR membrane domains M1 and M4 of neighboring subunits, initiated or stabilized by the AMPAR C-tail, and that these interactions are sufficient to enable full receptor modulation. Moreover, employing TARP chimeras disclosed a key role in this process also for the TARP transmembrane domains TM3 and TM4 and extracellular loop 2. Mechanistically, our data support a two-step action in which binding of TARP to the AMPAR membrane domains destabilizes the channel closed state, thereby enabling an efficient opening upon agonist binding, which then stabilizes the open state via subsequent interactions.

  11. Glyphosate and AMPA, "pseudo-persistent" pollutants under real-world agricultural management practices in the Mesopotamic Pampas agroecosystem, Argentina.

    PubMed

    Primost, Jezabel E; Marino, Damián J G; Aparicio, Virginia C; Costa, José Luis; Carriquiriborde, Pedro

    2017-10-01

    In the Pampas, public concern has strongly risen because of the intensive use of glyphosate for weed control and fallow associated with biotech crops. The present study was aimed to evaluate the occurrence and concentration of the herbicide and its main metabolite (AMPA) in soil and other environmental compartments of the mentioned agroecosystem, including groundwater, in relation to real-world agricultural management practices in the region. Occurrence was almost ubiquitous in solid matrices (83-100%) with maximum concentrations among the higher reported in the world (soil: 8105 and 38939; sediment: 3294 and 7219; suspended particulate matter (SPM): 584 and 475 μg/kg of glyphosate and AMPA). Lower detection frequency was observed in surface water (27-55%) with maximum concentrations in whole water of 1.80 and 1.90 μg/L of glyphosate and AMPA, indicating that SPM analysis would be more sensitive for detection in the aquatic ecosystem. No detectable concentrations of glyphosate or AMPA were observed in groundwater. Glyphosate soil concentrations were better correlated with the total cumulative dose and total number of applications than the last spraying event dose, and an increment of 1 mg glyphosate/kg soil every 5 spraying events was estimated. Findings allow to infer that, under current practices, application rates are higher than dissipation rates. Hence, glyphosate and AMPA should be considered "pseudo-persistent" pollutants and a revisions of management procedures, monitoring programs, and ecological risk for soil and sediments should be also recommended. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. The PARP inhibitor benzamide protects against kainate and NMDA but not AMPA lesioning of the mouse striatum in vivo.

    PubMed

    Cosi, Cristina; Guerin, Karen; Marien, Marc; Koek, Wouter; Rollet, Karin

    2004-01-16

    Overactivation of poly(ADP-ribose) polymerase (PARP) in response to genotoxic insults can cause cell death by energy deprivation. We previously reported that neurotoxic amounts of kainic acid (KA) injected into the rat striatum produce time-dependent changes in striatal PARP activity in vivo. Here, we have investigated the time-course of KA-induced toxicity and the effects of the PARP inhibitor benzamide on KA, AMPA and NMDA neurotoxicities in vivo, by measuring changes in the volume of the lesion and in NAD+ and ATP levels induced by the intra-striatal injection of these excitotoxins in C57Bl/6N mice. The KA-induced lesion volume was dependent on the amount of toxin injected and the survival time. The lesion was well developed at 48 h and was almost undetectable after one week. KA produced an extensive astrogliosis at one week. Benzamide partially prevented both KA- and NMDA- but not AMPA-induced lesions when measured at 48 h after the treatment. The effects of benzamide appeared to be in part related to changes in energy metabolism, since KA produced decreases in striatal levels of NAD+ and ATP that were partially prevented by benzamide at 48 h and which returned to control levels at one week. NMDA did not affect NAD+ and induced little alteration in ATP levels. Benzamide had no effect on AMPA-induced decreases in either NAD+ or ATP levels at 48 h. These results (1) indicate that PARP overactivation and energy depletion could be responsible in part for the cellular demise during the development of the lesion induced by KA; (2) confirm that PARP is involved in NMDA but not AMPA toxicities; (3) suggest the existence of differences between KA and AMPA-mediated toxicities; and (4) provide further evidence supporting PARP as a novel target for new drug treatments against neurodegenerative disorders.

  13. The neurosteroid dehydroepiandrosterone (DHEA) protects the retina from AMPA-induced excitotoxicity: NGF TrkA receptor involvement.

    PubMed

    Kokona, Despina; Charalampopoulos, Ioannis; Pediaditakis, Iosif; Gravanis, Achille; Thermos, Kyriaki

    2012-04-01

    The aim of the present study was to investigate the neuroprotective properties of the endogenous neurosteroid dehydroepiandrosterone (DHEA) in an in vivo model of retinal excitotoxicity, and the involvement of Nerve Growth Factor (NGF) in its actions. Adult Sprague-Dawley rats (250-300 g) received intravitreally (RS)-alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA; 42 nmol/eye) alone or in combination with DHEA (10(-8), 10(-7), 10(-6) M), or PBS (50 mM, control group). To examine the involvement of NGF and its TrkA receptor in the pharmacological effects of DHEA, animals received AMPA and NGF (60 pg/eye) in the absence or presence of a TrkA receptor inhibitor (Calbiochem 648450, 10(-6) M) or AMPA, DHEA (10(-6) M) and TrkA receptor inhibitor (10(-6), 10(-5) M). Immunohistochemistry studies [choline acetyltransferase (ChAT), brain nitric oxide synthetase (bNOS), calbindin, and TUNEL] and fluorescence-activated cell sorting (FACS) were used to examine retinal cell loss and protection. TrkA receptor immunoreactivity (-IR) and colocalization studies with relevant markers were also performed. AMPA (42 nmol) treatment resulted in a loss of bNOS, ChAT and calbindin immunoreactivities 24 h after its administration. DHEA, administered intravitreally, protected the retina from excitotoxicity in a dose-dependent manner. This effect was mimicked by NGF, and reversed by the NGF TrkA receptor inhibitor. The TrkA receptor is expressed in ganglion cells of rat retina. TUNEL staining and FACS analysis substantiated the neuroprotective actions of DHEA. These results demonstrate for the first time that the neurosteroid DHEA, administered intravitreally, protects the retina from AMPA excitotoxicity. An NGF TrkA receptor mechanism appears to be involved in this neuroprotection. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. AMPA receptors as drug targets in neurological disease--advantages, caveats, and future outlook.

    PubMed

    Chang, Philip K-Y; Verbich, David; McKinney, R Anne

    2012-06-01

    Most excitatory transmission in the brain is mediated by the AMPA receptor subtype of the ionotropic glutamate receptors. In many neurological diseases, synapse structure and AMPA receptor function are altered, thus making AMPA receptors potential therapeutic targets for clinical intervention. The work summarized in this review suggests a link between AMPA receptor function and debilitating neuropathologies, and discusses the current state of therapies targeting AMPA receptors in four diseases. In amyotrophic lateral sclerosis, AMPA receptors allow cytotoxic levels of calcium into neurons, leading to motor neuron death. Likewise, in some epilepsies, overactivation of AMPA receptors leads to neuron damage. The same is true for ischemia, where oxygen deprivation leads to excitotoxicity. Conversely, Alzheimer's disease is characterized by decreased AMPA activation and synapse loss. Unfortunately, many clinical studies have had limited success by directly targeting AMPA receptors in these diseases. We also discuss how the use of AMPA receptor modulators, commonly known as ampakines, in neurological diseases initially seemed promising in animal studies, but mostly ineffective in clinical trials. We propose that indirectly affecting AMPA receptors, such as by modulating transmembrane AMPA receptor regulatory proteins or, more generally, by regulating glutamatergic transmission, may provide new therapeutic potential for neurological disorders.

  15. Ganglioside Regulation of AMPA Receptor Trafficking

    PubMed Central

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  16. Ganglioside regulation of AMPA receptor trafficking.

    PubMed

    Prendergast, Jillian; Umanah, George K E; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G; Cole, Robert N; Huganir, Richard L; Dawson, Ted M; Dawson, Valina L; Schnaar, Ronald L

    2014-09-24

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10(-4)), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.

  17. Roles of fragile X mental retardation protein in dopaminergic stimulation-induced synapse-associated protein synthesis and subsequent alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) receptor internalization.

    PubMed

    Wang, Hansen; Kim, Susan S; Zhuo, Min

    2010-07-09

    Fragile X syndrome, the most common form of inherited mental retardation, is caused by the absence of the RNA-binding protein fragile X mental retardation protein (FMRP). FMRP regulates local protein synthesis in dendritic spines. Dopamine (DA) is involved in the modulation of synaptic plasticity. Activation of DA receptors can regulate higher brain functions in a protein synthesis-dependent manner. Our recent study has shown that FMRP acts as a key messenger for DA modulation in forebrain neurons. Here, we demonstrate that FMRP is critical for DA D1 receptor-mediated synthesis of synapse-associated protein 90/PSD-95-associated protein 3 (SAPAP3) in the prefrontal cortex (PFC). DA D1 receptor stimulation induced dynamic changes of FMRP phosphorylation. The changes in FMRP phosphorylation temporally correspond with the expression of SAPAP3 after D1 receptor stimulation. Protein phosphatase 2A, ribosomal protein S6 kinase, and mammalian target of rapamycin are the key signaling molecules for FMRP linking DA D1 receptors to SAPAP3. Knockdown of SAPAP3 did not affect surface expression of alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-4-propionate (AMPA) GluR1 receptors induced by D1 receptor activation but impaired their subsequent internalization in cultured PFC neurons; the subsequent internalization of GluR1 was also impaired in Fmr1 knock-out PFC neurons, suggesting that FMRP may be involved in subsequent internalization of GluR1 through regulating the abundance of SAPAP3 after DA D1 receptor stimulation. Our study thus provides further insights into FMRP involvement in DA modulation and may help to reveal the molecular mechanisms underlying impaired learning and memory in fragile X syndrome.

  18. AMPA receptor/TARP stoichiometry visualized by single-molecule subunit counting.

    PubMed

    Hastie, Peter; Ulbrich, Maximilian H; Wang, Hui-Li; Arant, Ryan J; Lau, Anthony G; Zhang, Zhenjie; Isacoff, Ehud Y; Chen, Lu

    2013-03-26

    Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R-mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R-mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.

  19. SynDIG1 promotes excitatory synaptogenesis independent of AMPA receptor trafficking and biophysical regulation.

    PubMed

    Lovero, Kathryn L; Blankenship, Sabine M; Shi, Yun; Nicoll, Roger A

    2013-01-01

    AMPA receptors-mediators of fast, excitatory transmission and synaptic plasticity in the brain-achieve great functional diversity through interaction with different auxiliary subunits, which alter both the trafficking and biophysical properties of these receptors. In the past several years an abundance of new AMPA receptor auxiliary subunits have been identified, adding astounding variety to the proteins known to directly bind and modulate AMPA receptors. SynDIG1 was recently identified as a novel AMPA receptor interacting protein that directly binds to the AMPA receptor subunit GluA2 in heterologous cells. Functionally, SynDIG1 was found to regulate the strength and density of AMPA receptor containing synapses in hippocampal neurons, though the way in which SynDIG1 exerts these effects remains unknown. Here, we aimed to determine if SynDIG1 acts as a traditional auxiliary subunit, directly regulating the function and localization of AMPA receptors in the rat hippocampus. We find that, unlike any of the previously characterized AMPA receptor auxiliary subunits, SynDIG1 expression does not impact AMPA receptor gating, pharmacology, or surface trafficking. Rather, we show that SynDIG1 regulates the number of functional excitatory synapses, altering both AMPA and NMDA receptor mediated transmission. Our findings suggest that SynDIG1 is not a typical auxiliary subunit to AMPA receptors, but instead is a protein critical to excitatory synaptogenesis.

  20. Effects of cyclothiazide on GluR1/AMPA receptors

    PubMed Central

    Fucile, Sergio; Miledi, Ricardo; Eusebi, Fabrizio

    2006-01-01

    Cyclothiazide (CTZ), a positive allosteric modulator of ionotropic α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors, is used frequently to block the desensitization of both native and heterologously expressed AMPA receptors. Specifically, CTZ is known to produce a fast inhibition of AMPA receptor desensitization and a much slower potentiation of the AMPA current. By using patch-clamp techniques, the effects of CTZ were studied in HEK 293 cells stably transfected with the rat flip GluR1 subunit. Upon CTZ treatment, we found an increased apparent affinity for the agonist, a slow whole-cell current potentiation, a fast inhibition of desensitization, and a lengthening of single-channel openings. Furthermore, we show that CTZ alters the channel gating events modifying the relative contribution of different single-channel classes of conductance (γ), increasing and decreasing, respectively, the contributions of γM (medium) and γL (low) without altering that of the γH (high) conductance channels. We also present a kinetic model that predicts well all of the experimental findings of CTZ action. Finally, we suggest a protocol for standard cell treatment with CTZ to attain maximal efficacy of CTZ on GluR1 receptors. PMID:16473938

  1. Cognitive improvement by acute growth hormone is mediated by NMDA and AMPA receptors and MEK pathway.

    PubMed

    Ramis, Margarita; Sarubbo, Fiorella; Sola, Jessica; Aparicio, Sara; Garau, Celia; Miralles, Antonio; Esteban, Susana

    2013-08-01

    It has been reported that Growth hormone (GH) has an immediate effect enhancing excitatory postsynaptic potentials mediated by AMPA and NMDA receptors in hippocampal area CA1. As GH plays a role in adult memory processing, this work aims to study the acute effects of GH on working memory tasks in rodents and the possible involvement of NMDA and AMPA receptors and also the MEK/ERK signalling pathway. To evaluate memory processes, two different tests were used, the spatial working memory 8-arm radial maze, and the novel object recognition as a form of non-spatial working memory test. Acute GH treatment (1mg/kg i.p., 1h) improved spatial learning in the radial maze respect to the control group either in young rats (reduction of 46% in the performance trial time and 61% in the number of errors), old rats (reduction of 38% in trial time and 48% in the number of errors), and adult mice (reduction of 32% in the performance time and 34% in the number of errors). GH treatment also increased the time spent exploring the novel object respect to the familiar object compared to the control group in young rats (from 63% to 79%), old rats (from 53% to 70%), and adult mice (from 61 to 68%). The improving effects of GH on working memory tests were blocked by the NMDA antagonist MK801 dizocilpine (0.025 mg/kg i.p.) injected 10 min before the administration of GH, in both young and old rats. In addition, the AMPA antagonist DNQX (1mg/kg i.p.) injected 10 min before the administration of GH to young rats, blocked the positive effect of GH. Moreover, in mice, the MEK inhibitor SL 327 (20mg/kg i.p.) injected 30 min before the administration of GH, blocked the positive effect of GH on radial maze and the novel object recognition. In conclusion, GH improved working memory processes through both glutamatergic receptors NMDA and AMPA and it required the activation of extracellular MEK/ERK signalling pathway. These effects could be related to the enhancement of excitatory synaptic

  2. Redefining the classification of AMPA-selective ionotropic glutamate receptors

    PubMed Central

    Bowie, Derek

    2012-01-01

    Abstract AMPA-type ionotropic glutamate receptors (iGluRs) represent the major excitatory neurotransmitter receptor in the developing and adult vertebrate CNS. They are crucial for the normal hardwiring of glutamatergic circuits but also fine tune synaptic strength by cycling into and out of synapses during periods of sustained patterned activity or altered homeostasis. AMPARs are grouped into two functionally distinct tetrameric assemblies based on the inclusion or exclusion of the GluA2 receptor subunit. GluA2-containing receptors are thought to be the most abundant AMPAR in the CNS, typified by their small unitary events, Ca2+ impermeability and insensitivity to polyamine block. In contrast, GluA2-lacking AMPARs exhibit large unitary conductance, marked divalent permeability and nano- to micromolar polyamine affinity. Here, I review evidence for the existence of a third class of AMPAR which, though similarly Ca2+ permeable, is characterized by its near-insensitivity to internal and external channel block by polyamines. This novel class of AMPAR is most notably found at multivesicular release synapses found in the avian auditory brainstem and mammalian retina. Curiously, these synapses lack NMDA-type iGluRs, which are conventionally associated with controlling AMPAR insertion. The lack of NMDARs suggests that a different set of rules may govern AMPAR cycling at these synapses. AMPARs with similar functional profiles are also found on some glial cells suggesting they may have a more widespread distribution in the mammalian CNS. I conclude by noting that modest changes to the ion-permeation pathway might be sufficient to retain divalent permeability whilst eliminating polyamine sensitivity. Consequently, this emerging AMPAR subclass need not be assembled from novel subunits, yet to be cloned, but could simply occur by varying the stoichiometry of existing proteins. PMID:22106175

  3. Positive modulation of AMPA receptors increases neurotrophin expression by hippocampal and cortical neurons.

    PubMed

    Lauterborn, J C; Lynch, G; Vanderklish, P; Arai, A; Gall, C M

    2000-01-01

    This study investigated whether positive modulators of AMPA-type glutamate receptors influence neurotrophin expression by forebrain neurons. Treatments with the ampakine CX614 markedly and reversibly increased brain-derived neurotrophic factor (BDNF) mRNA and protein levels in cultured rat entorhinal/hippocampal slices. Acute effects of CX614 were dose dependent over the range in which the drug increased synchronous neuronal discharges; threshold concentrations for acute responses had large effects on mRNA content when applied for 3 d. Comparable results were obtained with a second, structurally distinct ampakine CX546. Ampakine-induced upregulation was broadly suppressed by AMPA, but not NMDA, receptor antagonists and by reducing transmitter release. Antagonism of L-type voltage-sensitive calcium channels blocked induction in entorhinal cortex but not hippocampus. Prolonged infusions of suprathreshold ampakine concentrations produced peak BDNF mRNA levels at 12 hr and a return to baseline levels by 48 hr. In contrast, BDNF protein remained elevated throughout a 48 hr incubation with the drug. Nerve growth factor mRNA levels also were increased by ampakines but with a much more rapid return to control levels during chronic administration. Finally, intraperitoneal injections of CX546 increased hippocampal BDNF mRNA levels in aged rats and middle-aged mice. The present results provide evidence of regional differences in mechanisms via which activity regulates neurotrophin expression. Moreover, these data establish that changes in synaptic potency produce sufficient network level physiological effects for inducing neurotrophin genes, indicate that the response becomes refractory during prolonged ampakine exposure, and raise the possibility of using positive AMPA modulators to regulate neurotrophin levels in aged brain.

  4. Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation.

    PubMed

    Bonini, Daniela; Mora, Cristina; Tornese, Paolo; Sala, Nathalie; Filippini, Alice; La Via, Luca; Milanese, Marco; Calza, Stefano; Bonanno, Gianbattista; Racagni, Giorgio; Gennarelli, Massimo; Popoli, Maurizio; Musazzi, Laura; Barbon, Alessandro

    2016-01-01

    Clinical studies on patients with stress-related neuropsychiatric disorders reported functional and morphological changes in brain areas where glutamatergic transmission is predominant, including frontal and prefrontal areas. In line with this evidence, several preclinical works suggest that glutamate receptors are targets of both rapid and long-lasting effects of stress. Here we found that acute footshock- (FS-) stress, although inducing no transcriptional and RNA editing alterations of ionotropic AMPA and NMDA glutamate receptor subunits, rapidly and transiently modulates their protein expression, phosphorylation, and localization at postsynaptic spines in prefrontal and frontal cortex. In total extract, FS-stress increased the phosphorylation levels of GluA1 AMPA subunit at Ser(845) immediately after stress and of GluA2 Ser(880) 2 h after start of stress. At postsynaptic spines, stress induced a rapid decrease of GluA2 expression, together with an increase of its phosphorylation at Ser(880), suggesting internalization of GluA2 AMPA containing receptors. GluN1 and GluN2A NMDA receptor subunits were found markedly upregulated in postsynaptic spines, 2 h after start of stress. These results suggest selected time-dependent changes in glutamatergic receptor subunits induced by acute stress, which may suggest early and transient enhancement of AMPA-mediated currents, followed by a transient activation of NMDA receptors.

  5. Acute Footshock Stress Induces Time-Dependent Modifications of AMPA/NMDA Protein Expression and AMPA Phosphorylation

    PubMed Central

    Bonini, Daniela; Mora, Cristina; Tornese, Paolo; Sala, Nathalie; Filippini, Alice; La Via, Luca; Milanese, Marco; Calza, Stefano; Bonanno, Gianbattista; Racagni, Giorgio; Gennarelli, Massimo; Popoli, Maurizio; Musazzi, Laura; Barbon, Alessandro

    2016-01-01

    Clinical studies on patients with stress-related neuropsychiatric disorders reported functional and morphological changes in brain areas where glutamatergic transmission is predominant, including frontal and prefrontal areas. In line with this evidence, several preclinical works suggest that glutamate receptors are targets of both rapid and long-lasting effects of stress. Here we found that acute footshock- (FS-) stress, although inducing no transcriptional and RNA editing alterations of ionotropic AMPA and NMDA glutamate receptor subunits, rapidly and transiently modulates their protein expression, phosphorylation, and localization at postsynaptic spines in prefrontal and frontal cortex. In total extract, FS-stress increased the phosphorylation levels of GluA1 AMPA subunit at Ser845 immediately after stress and of GluA2 Ser880 2 h after start of stress. At postsynaptic spines, stress induced a rapid decrease of GluA2 expression, together with an increase of its phosphorylation at Ser880, suggesting internalization of GluA2 AMPA containing receptors. GluN1 and GluN2A NMDA receptor subunits were found markedly upregulated in postsynaptic spines, 2 h after start of stress. These results suggest selected time-dependent changes in glutamatergic receptor subunits induced by acute stress, which may suggest early and transient enhancement of AMPA-mediated currents, followed by a transient activation of NMDA receptors. PMID:26966584

  6. Agricultural non-point source pollution of glyphosate and AMPA at a catchment scale

    NASA Astrophysics Data System (ADS)

    Okada, Elena; Perez, Debora; De Geronimo, Eduardo; Aparicio, Virginia; Costa, Jose Luis

    2017-04-01

    can be attributed to deposition of soil particles that are washed from the field. On the other hand, more than 90 % of the soil samples had glyphosate and AMPA. The highest concentrations were found in the month of June, corresponding to the fallow period were glyphosate is applied as a chemical weed controller in no-till systems. Glyphosate and AMPA detection in groundwater samples was 24% and 35%, respectively. The highest glyphosate levels in groundwater also corresponded to the month of June. Glyphosate occurrence in groundwater was transient, that is, in most of the cases glyphosate was not detected in the subsequent sampling months. The contamination of shallow groundwater (<20 m) by glyphosate is likely relatively short-term in duration due to its fast degradation. These results indicate that there is a higher risk of agricultural non-point source pollution in the months of herbicide application, that may contribute to groundwater contamination. This is of extreme relevance in the southeast of the Buenos Aires Province, since the main source of drinking water and irrigation is from groundwater resources

  7. AMPA receptor trafficking and the mechanisms underlying synaptic plasticity and cognitive aging

    PubMed Central

    Henley, Jeremy M.; Wilkinson, Kevin A.

    2013-01-01

    Even in healthy individuals there is an inexorable agerelated decline in cognitive function. This is due, in large part, to reduced synaptic plasticity caused by changes in the molecular composition of the postsynaptic membrane. AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the overwhelming majority of fast excitatory transmission in the brain. Changes in AMPAR number and/or function are a core feature of synaptic plasticity and age-related cognitive decline, AMPARs are highly dynamic proteins that are subject to highly controlled trafficking, recycling, and/or degradation and replacement. This active regulation of AMPAR synthesis, targeting, synaptic dwell time, and degradation is fundamentally important for memory formation and storage. Further, aberrant AMPAR trafficking and consequent detrimental changes in synapses are strongly implicated in many brain diseases, which represent a vast social and economic burden. The purpose of this article is to provide an overview of the molecular and cellular AMPA receptor trafficking events that control synaptic responsiveness and plasticity, and highlight what is known currently known about how these processes change with age and disease. PMID:23576886

  8. AMPA receptor trafficking and the mechanisms underlying synaptic plasticity and cognitive aging.

    PubMed

    Henley, Jeremy M; Wilkinson, Kevin A

    2013-03-01

    Even in healthy individuals there is an inexorable agerelated decline in cognitive function. This is due, in large part, to reduced synaptic plasticity caused by changes in the molecular composition of the postsynaptic membrane. AMPA receptors (AMPARs) are glutamate-gated cation channels that mediate the overwhelming majority of fast excitatory transmission in the brain. Changes in AMPAR number and/or function are a core feature of synaptic plasticity and age-related cognitive decline, AMPARs are highly dynamic proteins that are subject to highly controlled trafficking, recycling, and/or degradation and replacement. This active regulation of AMPAR synthesis, targeting, synaptic dwell time, and degradation is fundamentally important for memory formation and storage. Further, aberrant AMPAR trafficking and consequent detrimental changes in synapses are strongly implicated in many brain diseases, which represent a vast social and economic burden. The purpose of this article is to provide an overview of the molecular and cellular AMPA receptor trafficking events that control synaptic responsiveness and plasticity, and highlight what is known currently known about how these processes change with age and disease.

  9. AMPA receptors in post-mortem brains of Cloninger type 1 and 2 alcoholics: a whole-hemisphere autoradiography study.

    PubMed

    Kärkkäinen, Olli; Kupila, Jukka; Häkkinen, Merja; Laukkanen, Virpi; Tupala, Erkki; Kautiainen, Hannu; Tiihonen, Jari; Storvik, Markus

    2013-12-30

    Dysfunction of the brain glutamate system has been associated with alcoholism. Ionotropic glutamatergic alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) play an important role in both neurotransmission and post-synaptic plasticity. Alterations in AMPAR densities may also play a role in the neurobiological changes associated with alcoholism. In the present study, [(3)H] AMPA binding density was evaluated in the nucleus accumbens (NAc), frontal cortex, anterior cingulate cortex (ACC), dentate gyrus and hippocampus of Cloninger type 1 (n=9) and 2 (n=8) alcoholics, and compared with non-alcoholic control subjects (n=10) by post-mortem whole-hemisphere autoradiography. The [(3)H] AMPA binding density was significantly higher in the ACC of early onset type 2 alcoholics when compared with controls (p=0.011). There was also a significant negative correlation between [(3)H] AMPA binding and previously published results of dopamine transporter (DAT) density in the ACC in these same brain samples (R=-0.95, p=0.001). Although preliminary, and from a relatively small diagnostic group, the present results help to further explain the pathology of alcohol dependence and impulsive behaviour in type 2 alcoholics.

  10. [Reciprocal suppression of the AMPA and NMDA components of the excitatory postsynaptic potentials in the CA1 area of the rat hippocampus in vitro].

    PubMed

    Bazhenov, A V; Kleshchevnikov, A M

    1998-01-01

    The interaction between N-methyl-d-aspartate (NMDA)- and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-dependent components of excitatory postsynaptic potentials (EPSP) was studied in rat hippocampal slices. Responses evoked by stimulation of the collateral commissural fibers were recorded in the radial layer of the CA1 area. Contribution of the NMDA component was changed by application of solutions with different concentrations of magnesium. In solutions with low magnesium concentration, when both AMPA and NMDA components contribute significantly to EPSP, suppression of one of the components by application of selective antagonist resulted in increase in the area of another component. Thus, the sum of pharmacologically isolated AMPA and NMDA components was significantly higher than the control EPSP. For example, at 0.1 mM of magnesium in the extracellular solution the sum of the components was 340 +/- 120% of the control EPSP (p < 0.01, N = 6). The data imply that under the control conditions the EPSP components suppress each other. The mutual suppression of the AMPA and NMDA component of the EPSP can be an important factor which influences the conductivity and plastic properties of central glutamatergic synaptic pathways.

  11. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons

    NASA Technical Reports Server (NTRS)

    Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.

    2002-01-01

    The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

  12. The AMPA receptor subunit GluR1 regulates dendritic architecture of motor neurons

    NASA Technical Reports Server (NTRS)

    Inglis, Fiona M.; Crockett, Richard; Korada, Sailaja; Abraham, Wickliffe C.; Hollmann, Michael; Kalb, Robert G.

    2002-01-01

    The morphology of the mature motor neuron dendritic arbor is determined by activity-dependent processes occurring during a critical period in early postnatal life. The abundance of the AMPA receptor subunit GluR1 in motor neurons is very high during this period and subsequently falls to a negligible level. To test the role of GluR1 in dendrite morphogenesis, we reintroduced GluR1 into rat motor neurons at the end of the critical period and quantitatively studied the effects on dendrite architecture. Two versions of GluR1 were studied that differed by the amino acid in the "Q/R" editing site. The amino acid occupying this site determines single-channel conductance, ionic permeability, and other essential electrophysiologic properties of the resulting receptor channels. We found large-scale remodeling of dendritic architectures in a manner depending on the amino acid occupying the Q/R editing site. Alterations in the distribution of dendritic arbor were not prevented by blocking NMDA receptors. These observations suggest that the expression of GluR1 in motor neurons modulates a component of the molecular substrate of activity-dependent dendrite morphogenesis. The control of these events relies on subunit-specific properties of AMPA receptors.

  13. AMPA receptor potentiation can prevent ethanol-induced intoxication.

    PubMed

    Jones, Nicholas; Messenger, Marcus J; O'Neill, Michael J; Oldershaw, Anna; Gilmour, Gary; Simmons, Rosa M A; Iyengar, Smriti; Libri, Vincenzo; Tricklebank, Mark; Williams, Steve C R

    2008-06-01

    We present a substantial series of behavioral and imaging experiments, which demonstrate, for the first time, that increasing AMPA receptor-mediated neurotransmission via administration of potent and selective biarylsulfonamide AMPA potentiators LY404187 and LY451395 reverses the central effects of an acutely intoxicating dose of ethanol in the rat. Using pharmacological magnetic resonance imaging (phMRI), we observed that LY404187 attenuated ethanol-induced reductions in blood oxygenation level dependent (BOLD) in the anesthetized rat brain. A similar attenuation was apparent when measuring local cerebral glucose utilization (LCGU) via C14-2-deoxyglucose autoradiography in freely moving conscious rats. Both LY404187 and LY451395 significantly and dose-dependently reversed ethanol-induced deficits in both motor coordination and disruptions in an operant task where animals were trained to press a lever for food reward. Both prophylactic and acute intervention treatment with LY404187 reversed ethanol-induced deficits in motor coordination. Given that LY451395 and related AMPA receptor potentiators/ampakines are tolerated in both healthy volunteers and elderly patients, these data suggest that such compounds may form a potential management strategy for acute alcohol intoxication.

  14. Glutamate mediates platelet activation through the AMPA receptor

    PubMed Central

    Morrell, Craig N.; Sun, Henry; Ikeda, Masahiro; Beique, Jean-Claude; Swaim, Anne Marie; Mason, Emily; Martin, Tanika V.; Thompson, Laura E.; Gozen, Oguz; Ampagoomian, David; Sprengel, Rolf; Rothstein, Jeffrey; Faraday, Nauder; Huganir, Richard; Lowenstein, Charles J.

    2008-01-01

    Glutamate is an excitatory neurotransmitter that binds to the kainate receptor, the N-methyl-D-aspartate (NMDA) receptor, and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAR). Each receptor was first characterized and cloned in the central nervous system (CNS). Glutamate is also present in the periphery, and glutamate receptors have been identified in nonneuronal tissues, including bone, heart, kidney, pancreas, and platelets. Platelets play a central role in normal thrombosis and hemostasis, as well as contributing greatly to diseases such as stroke and myocardial infarction. Despite the presence of glutamate in platelet granules, the role of glutamate during hemostasis is unknown. We now show that activated platelets release glutamate, that platelets express AMPAR subunits, and that glutamate increases agonist-induced platelet activation. Furthermore, we demonstrate that glutamate binding to the AMPAR increases intracellular sodium concentration and depolarizes platelets, which are important steps in platelet activation. In contrast, platelets treated with the AMPAR antagonist CNQX or platelets derived from GluR1 knockout mice are resistant to AMPA effects. Importantly, mice lacking GluR1 have a prolonged time to thrombosis in vivo. Our data identify glutamate as a regulator of platelet activation, and suggest that the AMPA receptor is a novel antithrombotic target. PMID:18283118

  15. Amyloid-β-Induced Dysregulation of AMPA Receptor Trafficking

    PubMed Central

    Guntupalli, Sumasri; Widagdo, Jocelyn; Anggono, Victor

    2016-01-01

    Evidence from neuropathological, genetic, animal model, and biochemical studies has indicated that the accumulation of amyloid-beta (Aβ) is associated with, and probably induces, profound neuronal changes in brain regions critical for memory and cognition in the development of Alzheimer's disease (AD). There is considerable evidence that synapses are particularly vulnerable to AD, establishing synaptic dysfunction as one of the earliest events in pathogenesis, prior to neuronal loss. It is clear that excessive Aβ levels can disrupt excitatory synaptic transmission and plasticity, mainly due to dysregulation of the AMPA and NMDA glutamate receptors in the brain. Importantly, AMPA receptors are the principal glutamate receptors that mediate fast excitatory neurotransmission. This is essential for synaptic plasticity, a cellular correlate of learning and memory, which are the cognitive functions that are most disrupted in AD. Here we review recent advances in the field and provide insights into the molecular mechanisms that underlie Aβ-induced dysfunction of AMPA receptor trafficking. This review focuses primarily on NMDA receptor- and metabotropic glutamate receptor-mediated signaling. In particular, we highlight several mechanisms that underlie synaptic long-term depression as common signaling pathways that are hijacked by the neurotoxic effects of Aβ. PMID:27073700

  16. Activity Level-Dependent Synapse-Specific AMPA Receptor Trafficking Regulates Transmission Kinetics

    PubMed Central

    Zhu, J. Julius

    2009-01-01

    Central glutamatergic synapses may express AMPA-sensitive glutamate receptors (AMPA-Rs) with distinct gating properties and exhibit different transmission dynamics, which are important for computing various synaptic inputs received at different populations of synapses. However, how glutamatergic synapses acquire AMPA-Rs with distinct kinetics to influence synaptic integration remains poorly understood. Here I report synapse-specific trafficking of distinct AMPA-Rs in rat cortical layer 4 stellate and layer 5 pyramidal neurons. The analysis indicates that in single layer 4 stellate neurons thalamocortical synapses generate faster synaptic responses than intracortical synapses. Moreover, GluR1-containing AMPA-Rs traffic selectively into intracortical synapses, and this process requires sensory experience-dependent activity and slows down transmission kinetics. GluR4-containing AMPA-Rs traffic more heavily into thalamocortical synapses than intracortical synapses, and this process requires spontaneous synaptic activity and speeds up transmission kinetics. GluR2-containing AMPA-Rs traffic equally into both thalamocortical and intracortical synapses, and this process requires no synaptic activity and resets transmission kinetics. Notably, synaptic trafficking of distinct AMPA-Rs differentially regulates synaptic integration. Thus, synapse-specific AMPA-R trafficking coarsely sets and synaptic activity finely tunes transmission kinetics and integration properties at different synapses in central neurons. PMID:19439609

  17. TARP γ-8 glycosylation regulates the surface expression of AMPA receptors.

    PubMed

    Zheng, Chan-Ying; Chang, Kai; Suh, Young Ho; Roche, Katherine W

    2015-02-01

    TARP [transmembrane AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor regulatory protein] γ-8 is an auxiliary subunit of AMPA receptors that is widely distributed in the hippocampus. It has been shown that TARP γ-8 promotes surface expression of AMPA receptors; however, how TARP γ-8 regulates the expression of AMPA receptors remains unclear. In the present study, we examined the effect of TARP glycosylation on AMPA receptor trafficking. We first showed that TARP γ-8 is an N-glycosylated protein, which contains two glycosylation sites, Asn53 and Asn56, and compared this with the glycosylation of TARP γ-2 and the AMPA receptor auxiliary protein CNIH-2 (cornichon homologue 2). We next examine the effect of TARP glycosylation on TARP trafficking and also on AMPA receptor surface expression. We find that TARP γ-8 glycosylation is critical for surface expression of both TARP γ-8 and GluA1 in heterologous cells and neurons. Specifically, knockdown of TARP γ-8 causes a decrease in both total and surface AMPA receptors. We find that the expression of unglycosylated TARP γ-8 in cultured neurons is unable to restore GluA1 expression fully. Furthermore, when the maturation of TARP γ-8 is impaired, a large pool of immature GluA1 is retained intracellularly. Taken together, our data reveal an important role for the maturation of TARP γ-8 in the trafficking and function of the AMPA receptor complex.

  18. One aptamer, two functions: the full-length aptamer inhibits AMPA receptors, while the short one inhibits both AMPA and kainate receptors

    PubMed Central

    Jaremko, William J.; Huang, Zhen; Wen, Wei; Wu, Andrew; Karl, Nicholas; Niu, Li

    2017-01-01

    AMPA and kainate receptors, along with NMDA receptors, are distinct subtypes of glutamate ion channels. Excessive activity of AMPA and kainate receptors has been implicated in neurological diseases, such as epilepsy and neuropathic pain. Antagonists that block their activities are therefore potential drug candidates. In a recent article in the Journal of Biological Chemistry by Jaremko et al. 2017, we have reported on the discovery and molecular characterization of an RNA aptamer of a dual functionality: the full-length RNA (101 nucleotide) inhibits AMPA receptors while the truncated or the short (55 nucleotide) RNA inhibits both the AMPA and kainate receptors. The full-length RNA aptamer was isolated through a specially designed, systematic evolution of ligands by exponential enrichment (SELEX) using only a single type of AMPA receptors expressed in HEK-293 cells. The design feature and the results of our recent article are highlighted here, as they demonstrate the utility of the SELEX approach and the potential of using a single AMPA receptor type to develop potent, novel RNA aptamers targeting multiple subunits and AMPA/kainate receptor subtypes with length-dependent functionalities. PMID:28804757

  19. Discovery of the First α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Antagonist Dependent upon Transmembrane AMPA Receptor Regulatory Protein (TARP) γ-8.

    PubMed

    Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon K; Ornstein, Paul L; Spinazze, Patrick; Stevens, F Craig; Hahn, Patric; Hollinshead, Sean P; Mayhugh, Daniel; Schkeryantz, Jeff; Khilevich, Albert; De Frutos, Oscar; Gleason, Scott D; Kato, Akihiko S; Luffer-Atlas, Debra; Desai, Prashant V; Swanson, Steven; Burris, Kevin D; Ding, Chunjin; Heinz, Beverly A; Need, Anne B; Barth, Vanessa N; Stephenson, Gregory A; Diseroad, Benjamin A; Woods, Tim A; Yu, Hong; Bredt, David; Witkin, Jeffrey M

    2016-05-26

    Transmembrane AMPA receptor regulatory proteins (TARPs) are a family of scaffolding proteins that regulate AMPA receptor trafficking and function. TARP γ-8 is one member of this family and is highly expressed within the hippocampus relative to the cerebellum. A selective TARP γ-8-dependent AMPA receptor antagonist (TDAA) is an innovative approach to modulate AMPA receptors in specific brain regions to potentially increase the therapeutic index relative to known non-TARP-dependent AMPA antagonists. We describe here, for the first time, the discovery of a noncompetitive AMPA receptor antagonist that is dependent on the presence of TARP γ-8. Three major iteration cycles were employed to improve upon potency, CYP1A2-dependent challenges, and in vivo clearance. An optimized molecule, compound (-)-25 (LY3130481), was fully protective against pentylenetetrazole-induced convulsions in rats without the motor impairment associated with non-TARP-dependent AMPA receptor antagonists. Compound (-)-25 could be utilized to provide proof of concept for antiepileptic efficacy with reduced motor side effects in patients.

  20. Identification and Characterization of RNA Aptamers: A Long Aptamer Blocks the AMPA Receptor and a Short Aptamer Blocks Both AMPA and Kainate Receptors.

    PubMed

    Jaremko, William J; Huang, Zhen; Wen, Wei; Wu, Andrew; Karl, Nicholas; Niu, Li

    2017-03-21

    AMPA and kainate receptors, along with NMDA receptors, represent different subtypes of glutamate ion channels. AMPA and kainate receptors share a high degree of sequence and structural similarities, and excessive activity of these receptors has been implicated in neurological diseases such as epilepsy. Therefore, blocking detrimental activity of both receptor types could be therapeutically beneficial. Here, we report the use of an in vitro evolution approach involving systematic evolution of ligands by exponential enrichment with a single AMPA receptor target (i.e. GluA1/2R) to isolate RNA aptamers that can potentially inhibit both AMPA and kainate receptors. A full-length or 101-nucleotide (nt) aptamer selectively inhibited GluA1/2R with a KI of ~5 µM, along with GluA1 and GluA2 AMPA receptor subunits. Of note, its shorter version (55 nt) inhibited both AMPA and kainate receptors. In particular, this shorter aptamer blocked equally potently the activity of both the GluK1 and GluK2 kainate receptors. Using homologous binding and whole-cell recording assays, we found that an RNA aptamer most likely binds to the receptor's regulatory site and inhibits it noncompetitively. Our results suggest the potential of using a single receptor target to develop RNA aptamers with dual activity for effectively blocking both AMPA and kainate receptors.

  1. Learning, AMPA receptor mobility and synaptic plasticity depend on n-cofilin-mediated actin dynamics

    PubMed Central

    Rust, Marco B; Gurniak, Christine B; Renner, Marianne; Vara, Hugo; Morando, Laura; Görlich, Andreas; Sassoè-Pognetto, Marco; Banchaabouchi, Mumna Al; Giustetto, Maurizio; Triller, Antoine; Choquet, Daniel; Witke, Walter

    2010-01-01

    Neuronal plasticity is an important process for learning, memory and complex behaviour. Rapid remodelling of the actin cytoskeleton in the postsynaptic compartment is thought to have an important function for synaptic plasticity. However, the actin-binding proteins involved and the molecular mechanisms that in vivo link actin dynamics to postsynaptic physiology are not well understood. Here, we show that the actin filament depolymerizing protein n-cofilin is controlling dendritic spine morphology and postsynaptic parameters such as late long-term potentiation and long-term depression. Loss of n-cofilin-mediated synaptic actin dynamics in the forebrain specifically leads to impairment of all types of associative learning, whereas exploratory learning is not affected. We provide evidence for a novel function of n-cofilin function in synaptic plasticity and in the control of extrasynaptic excitatory AMPA receptors diffusion. These results suggest a critical function of actin dynamics in associative learning and postsynaptic receptor availability. PMID:20407421

  2. Presence of glyphosate and AMPA in orchard soils and water in the upper Río Negro and Neuquén valley

    NASA Astrophysics Data System (ADS)

    Holzmann, Rosa; Sheridan, Miguel; De Geronimo, Eduardo; Aparicio, Virginia; Costa, Jose Luis

    2017-04-01

    The Upper Valley of Río Negro and Neuquén provinces is the most important region of Argentina for pear and apple production. The local climate is arid, with deficits of plant available water of 1,200 mm per year with soils classified as Entisols and Aridisols. Flooding irrigation provides approximately 2,000 mm yearly. The weeds control consists on the application of glyphosate along the planting row 0.5 m each at both sides of the trees. The aim of this work was to detect the presence of glyphosate and AMPA (aminomethylphosphonic acid) remaining in water and soil. Some orchards were monitored one year after the herbicide application. Soil composed samples were taken at the 0 to 10 cm depth and also in the canals. Percolation water was taken from drainage canals until its final destination. Irrigation water before entering the orchards were also sampled. The presence of glyphosate and AMPA was detected in all samples. The soil in the canals had 1,098 and 340.5 µg.kg-1 of glyphosate and AMPA respectively. 934 and 1,864.5 µg.kg-1 of glyphosate and AMPA respectively on a land where the herbicide was recently applied; figures from 11 y 208µg.kg-1 (minimun) to 149.5 y 583 µg.kg-1 (maximum) of glyphosate and AMPA respectively in orchards on which the herbicide was applied one year before; finally, 13 and 17.5 µg.kg-1 minimun, and 32 and 30.5 µg.kg-1 maximun of glyphosate and AMPA respectively in draining channel sediments. As regards waters, and according to the quantity of molecules and the level allowed by the EU of 0.5 µg.l-1, the water source contained 0.56 µg.l-1, while, in the draining waters, we found concentrations between 1.5 and 12.21µg.l-1 right after soil percolation and between 0.49 and 5.0 µg.l-1 in secondary drainage canals and finally, between 0.5 and 1.4 µg.l-1 in the main canal. Glyphosate and AMPA comprised between 73% and 99.9% of the sum of total molecules in all cases.

  3. Insulin inhibits AMPA-induced neuronal damage via stimulation of protein kinase B (Akt).

    PubMed

    Kim, S-J; Han, Y

    2005-02-01

    We designed a series of experiments to explore the neuroprotective effects of insulin. Insulin significantly inhibited the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced neuronal cell damage as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay. However, insulin had little affect on the AMPA-induced glial cell damage. To determine whether insulin inhibits AMPA-induced excitotoxicity, we performed grease-gap recording assays using rat brain slices. In these experiments, insulin also significantly inhibited AMPA-induced depolarization. Flow cytometry and DNA fragmentation assays showed that insulin inhibits AMPA-induced apoptosis and DNA fragmentation, respectively. Insulin stimulated protein kinase B (Akt) activity, whereas AMPA pretreatment did not alter the insulin-stimulated Akt activity. On the contrary, insulin blocked induction of SAPK/JNK, which AMPA stimulated. Taken together, these results suggest that insulin exerts neuroprotective effects by inhibiting AMPA-induced excitotoxicity and apoptosis, possibly by activating Akt and blocking SAPK/JNK.

  4. Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism.

    PubMed

    Kato, Akihiko S; Burris, Kevin D; Gardinier, Kevin M; Gernert, Douglas L; Porter, Warren J; Reel, Jon; Ding, Chunjin; Tu, Yuan; Schober, Douglas A; Lee, Matthew R; Heinz, Beverly A; Fitch, Thomas E; Gleason, Scott D; Catlow, John T; Yu, Hong; Fitzjohn, Stephen M; Pasqui, Francesca; Wang, He; Qian, Yuewei; Sher, Emanuele; Zwart, Ruud; Wafford, Keith A; Rasmussen, Kurt; Ornstein, Paul L; Isaac, John T R; Nisenbaum, Eric S; Bredt, David S; Witkin, Jeffrey M

    2016-12-01

    Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

  5. Afferent-specific innervation of two distinct AMPA receptor subtypes on single hippocampal interneurons.

    PubMed

    Tóth, K; McBain, C J

    1998-11-01

    Using the polyamine toxin philanthotoxin, which selectively blocks calcium-permeable AMPA receptors, we show that synaptic transmission onto single hippocampal interneurons occurs by afferent-specific activation of philanthotoxin-sensitive and -insensitive AMPA receptors. Calcium-permeable AMPA receptors are found exclusively at synapses from mossy fibers. In contrast, synaptic responses evoked by stimulation of CA3 pyramidal neurons are mediated by calcium-impermeable AMPA receptors. Both pathways converge onto single interneurons and can be discriminated with Group II mGluR agonists. Thus, single interneurons target AMPA receptors of different subunit composition to specific postsynaptic sites, providing a mechanism to increase the synapse-specific computational properties of hippocampal interneurons.

  6. Glyphosate and AMPA inhibit cancer cell growth through inhibiting intracellular glycine synthesis

    PubMed Central

    Li, Qingli; Lambrechts, Mark J; Zhang, Qiuyang; Liu, Sen; Ge, Dongxia; Yin, Rutie; Xi, Mingrong; You, Zongbing

    2013-01-01

    Glycine is a nonessential amino acid that is reversibly converted from serine intracellularly by serine hydroxymethyltransferase. Glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), are analogs to glycine, thus they may inhibit serine hydroxymethyltransferase to decrease intracellular glycine synthesis. In this study, we found that glyphosate and AMPA inhibited cell growth in eight human cancer cell lines but not in two immortalized human normal prostatic epithelial cell lines. AMPA arrested C4-2B and PC-3 cancer cells in the G1/G0 phase and inhibited entry into the S phase of the cell cycle. AMPA also promoted apoptosis in C4-2B and PC-3 cancer cell lines. AMPA upregulated p53 and p21 protein levels as well as procaspase 9 protein levels in C4-2B cells, whereas it downregulated cyclin D3 protein levels. AMPA also activated caspase 3 and induced cleavage of poly (adenosine diphosphate [ADP]-ribose) polymerase. This study provides the first evidence that glyphosate and AMPA can inhibit proliferation and promote apoptosis of cancer cells but not normal cells, suggesting that they have potentials to be developed into a new anticancer therapy. PMID:23983455

  7. Spinal nociceptin inhibits AMPA-induced nociceptive behavior and Fos expression in rat spinal cord.

    PubMed

    Menéndez, Luis; Lastra, Ana; Villanueva, Noemí; Hidalgo, Agustín; Baamonde, Ana

    2003-02-01

    The effects of intrathecal nociceptin (NOCI) on the nociceptive behavior (biting, scratching and licking; BSL) and the spinal Fos expression induced by intrathecal administration of N-methyl-D-aspartate (NMDA, 4 microg/rat) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA, 2 microg/rat) were studied. Coadministration of NOCI (3 and 10 nmol/rat) with NMDA did not modify the NMDA-induced BSL or Fos expression. In contrast, NOCI (0.1-3 nmol/rat) dose-dependently inhibited the BSL behavior induced by AMPA. Furthermore, coadministration of NOCI (3 and 10 nmol/rat) significantly reduced the AMPA-induced expression of Fos protein in the superficial layers of the spinal dorsal horn. In order to test whether classical or opioid receptor-like type 1 (ORL1) receptors are involved in the inhibitions by NOCI of AMPA-evoked BSL, the corresponding antagonists were assayed. The administration of the nonselective opioid receptor antagonist, naloxone (10 mg/kg i.p.), did not modify the NOCI-induced inhibition of AMPA-evoked BSL. However, the selective ORL1 receptor antagonist, [N-Phe(1)]nociceptin-(1-13)-NH(2) (90 nmol/rat i.t.), completely prevented the NOCI-mediated inhibition of the nociceptive responses evoked by AMPA. In conclusion, NOCI, acting at ORL1 receptors can, at least in part, induce spinal analgesia by blocking the nociceptive responses produced through the stimulation of AMPA receptors.

  8. Phosphorylation of the AMPA receptor GluA1 subunit regulates memory load capacity.

    PubMed

    Olivito, Laura; Saccone, Paola; Perri, Valentina; Bachman, Julia L; Fragapane, Paola; Mele, Andrea; Huganir, Richard L; De Leonibus, Elvira

    2016-01-01

    Memory capacity (MC) refers to the number of elements one can maintain for a short retention interval. The molecular mechanisms underlying MC are unexplored. We have recently reported that mice as well as humans have a limited MC, which is reduced by hippocampal lesions. Here, we addressed the molecular mechanisms supporting MC. GluA1 AMPA-receptors (AMPA-R) mediate the majority of fast excitatory synaptic transmission in the brain and are critically involved in memory. Phosphorylation of GluA1 at serine residues S831 and S845 is promoted by CaMKII and PKA, respectively, and regulates AMPA-R function in memory duration. We hypothesized that AMPA-R phosphorylation may also be a key plastic process for supporting MC because it occurs in a few minutes, and potentiates AMPA-R ion channel function. Here, we show that knock-in mutant mice that specifically lack both of S845 and S831 phosphorylation sites on the GluA1 subunit had reduced MC in two different behavioral tasks specifically designed to assess MC in mice. This demonstrated a causal link between AMPA-R phosphorylation and MC. We then showed that information load regulates AMPA-R phosphorylation within the hippocampus, and that an overload condition associated with impaired memory is paralleled by a lack of AMPA-R phosphorylation. Accordingly, we showed that in conditions of high load, but not of low load, the pharmacological inhibition of the NMDA-CaMKII-PKA pathways within the hippocampus prevents memory as well as associated AMPA-R phosphorylation. These data provide the first identified molecular mechanism that regulates MC.

  9. Urban and agricultural contribution of annual loads of glyphosate and AMPA towards surface waters at the Orge River catchment scale (France)

    NASA Astrophysics Data System (ADS)

    Botta, Fabrizio; Chevreuil, Marc; Blanchoud, Hélène

    2010-05-01

    The general use of pesticides in the Orge Basin, located in the southern part of the Paris suburb (France), is damaging surface water quality. Consequently, an increase in the water supply costs is registered by the water supply agencies that are situated downstream the Orge confluence with the Seine River. In this catchment, high uses of glyphosate are registered for fallow fields (upstream part) and for roadway weed control (downstream part). The proportion of glyphosate coming from these two zones was not well known, along with the double source of its metabolite AMPA originated from the degradation of some detergent phosphonates. The aim of this work was firstly to identify the potential sources of glyphosate and AMPA in urban sectors (such as sewerage system inputs) and in agricultural areas and to quantify the origins of urban pesticides pathways towards surface waters at the basin scale. The new approach of this project was to collect information at three different scales to establish a first step of modeling. At the basin scale, 1 year of surface water monitoring at the outlet of the Orge River was useful to establish the inputs towards the Seine River. At the urban catchment scale, the investigations have permitted to record glyphosate and AMPA loads transferred by storm waters and by wastewaters. Loads were estimated during and out of application calendar, in different hydrological conditions such as rainfall with high intensity or dry conditions. Impact of WWTP on surface water was also demonstrated. The third phase of this work was the interpretation of agricultural inputs from two different agricultural catchments of the Orge River. The results showed the impact of urban uses of glyphosate upon the Orge River contamination with annual loads from 100 times higher from the urban zone than from the agricultural one. Storm sewers were recognized to be the main way for glyphosate transfer towards surface waters. A budget of glyphosate and AMPA inputs and

  10. Modern approaches to the design of memory and cognitive function stimulants based on AMPA receptor ligands

    NASA Astrophysics Data System (ADS)

    Grigoriev, V. V.; Proshin, A. N.; Kinzirsky, A. S.; Bachurin, Sergey O.

    2009-05-01

    Data on the structure and properties of compounds acting on AMPA receptors, the key subtype of ionotropic glutamate receptors of the mammalian central nervous system, are analyzed. Data on the role of these receptors in provision of memory and cognitive function formation and impairment processes are presented. The attention is focused on the modern views on the mechanisms of AMPA receptor desensitization and deactivation and action of substances affecting these processes. The structures of key positive modulators of AMPA receptors are given. The problems of application of these substances as therapeutic means for preventing and treating neurodegenerative and psychoneurological diseases are discussed. Bibliography — 121 references.

  11. AMPA Receptors Are Involved in Store-Operated Calcium Entry and Interact with STIM Proteins in Rat Primary Cortical Neurons

    PubMed Central

    Gruszczynska-Biegala, Joanna; Sladowska, Maria; Kuznicki, Jacek

    2016-01-01

    The process of store-operated calcium entry (SOCE) leads to refilling the endoplasmic reticulum (ER) with calcium ions (Ca2+) after their release into the cytoplasm. Interactions between (ER)-located Ca2+ sensors (stromal interaction molecule 1 [STIM1] and STIM2) and plasma membrane-located Ca2+ channel-forming protein (Orai1) underlie SOCE and are well described in non-excitable cells. In neurons, however, SOCE appears to be more complex because of the importance of Ca2+ influx via voltage-gated or ionotropic receptor-operated Ca2+ channels. We found that the SOCE inhibitors ML-9 and SKF96365 reduced α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-induced [Ca2+]i amplitude by 80% and 53%, respectively. To assess the possible involvement of AMPA receptors (AMPARs) in SOCE, we used their specific inhibitors. As estimated by Fura-2 acetoxymethyl (AM) single-cell Ca2+ measurements in the presence of CNQX or NBQX, thapsigargin (TG)-induced Ca2+ influx decreased 2.2 or 3.7 times, respectively. These results suggest that under experimental conditions of SOCE when Ca2+ stores are depleted, Ca2+ can enter neurons also through AMPARs. Using specific antibodies against STIM proteins or GluA1/GluA2 AMPAR subunits, co-immunoprecipitation assays indicated that when Ca2+ levels are low in the neuronal ER, a physical association occurs between endogenous STIM proteins and endogenous AMPAR receptors. Altogether, our data suggest that STIM proteins in neurons can control AMPA-induced Ca2+ entry as a part of the mechanism of SOCE. PMID:27826230

  12. Alternative Splicing of AMPA Subunits in Prefrontal Cortical Fields of Cynomolgus Monkeys Following Chronic Ethanol Self-Administration

    PubMed Central

    Acosta, Glen; Freidman, David P.; Grant, Kathleen A.; Hemby, Scott E.

    2012-01-01

    Functional impairment of the orbital and medial prefrontal cortex underlies deficits in executive control that characterize addictive disorders, including alcohol addiction. Previous studies indicate that alcohol alters glutamate neurotransmission and one substrate of these effects may be through the reconfiguration of the subunits constituting ionotropic glutamate receptor (iGluR) complexes. Glutamatergic transmission is integral to cortico-cortical and cortico-subcortical communication and alcohol-induced changes in the abundance of the receptor subunits and/or their splice variants may result in critical functional impairments of prefrontal cortex in alcohol dependence. To this end, the effects of chronic ethanol self-administration on glutamate receptor ionotropic AMPA (GRIA) subunit variant and kainate (GRIK) subunit mRNA expression were studied in the orbitofrontal cortex (OFC), dorsolateral prefrontal cortex (DLPFC), and anterior cingulate cortex (ACC) of male cynomolgus monkeys. In DLPFC, total AMPA splice variant expression and total kainate receptor subunit expression were significantly decreased in alcohol drinking monkeys. Expression levels of GRIA3 flip and flop and GRIA4 flop mRNAs in this region were positively correlated with daily ethanol intake and blood ethanol concentrations (BEC) averaged over the 6 months prior to necropsy. In OFC, AMPA subunit splice variant expression was reduced in the alcohol treated group. GRIA2 flop mRNA levels in this region were positively correlated with daily ethanol intake and BEC averaged over the 6 months prior to necropsy. Results from these studies provide further evidence of transcriptional regulation of iGluR subunits in the primate brain following chronic alcohol self-administration. Additional studies examining the cellular localization of such effects in the framework of primate prefrontal cortical circuitry are warranted. PMID:22291662

  13. AMPA RECEPTOR POTENTIATORS: FROM DRUG DESIGN TO COGNITIVE ENHANCEMENT

    PubMed Central

    PARTIN, KATHRYN M.

    2014-01-01

    Positive allosteric modulators of ionotropic glutamate receptors have emerged as a target for treating cognitive impairment and neurodegeneration, but also mental illnesses such as major depressive disorder. The possibility of creating a new class of pharmaceutical agent to treat refractive mental health issues has compelled researchers to redouble their efforts to develop a safe, effective treatment for memory and cognition impairments. Coupled with the more robust research methodologies that have emerged, including more sophisticated high-throughput-screens, higher resolution structural biology techniques, and more focused assessment on pharmacokinetics, the development of positive modulators of AMPA receptors holds great promise. We describe recent approaches that improve our understanding of the basic physiology underlying memory and cognition, and their application towards promoting human health. PMID:25462292

  14. AMPA receptor potentiators: from drug design to cognitive enhancement.

    PubMed

    Partin, Kathryn M

    2015-02-01

    Positive allosteric modulators of ionotropic glutamate receptors have emerged as a target for treating cognitive impairment and neurodegeneration, but also mental illnesses such as major depressive disorder. The possibility of creating a new class of pharmaceutical agent to treat refractive mental health issues has compelled researchers to redouble their efforts to develop a safe, effective treatment for memory and cognition impairments. Coupled with the more robust research methodologies that have emerged, including more sophisticated high-throughput-screens, higher resolution structural biology techniques, and more focused assessment on pharmacokinetics, the development of positive modulators of AMPA receptors holds great promise. We describe recent approaches that improve our understanding of the basic physiology underlying memory and cognition, and their application toward promoting human health.

  15. Chemical labelling for visualizing native AMPA receptors in live neurons

    NASA Astrophysics Data System (ADS)

    Wakayama, Sho; Kiyonaka, Shigeki; Arai, Itaru; Kakegawa, Wataru; Matsuda, Shinji; Ibata, Keiji; Nemoto, Yuri L.; Kusumi, Akihiro; Yuzaki, Michisuke; Hamachi, Itaru

    2017-04-01

    The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

  16. Direct imaging of lateral movements of AMPA receptors inside synapses

    PubMed Central

    Tardin, Catherine; Cognet, Laurent; Bats, Cécile; Lounis, Brahim; Choquet, Daniel

    2003-01-01

    Trafficking of AMPA receptors in and out of synapses is crucial for synaptic plasticity. Previous studies have focused on the role of endo/exocytosis processes or that of lateral diffusion of extra-synaptic receptors. We have now directly imaged AMPAR movements inside and outside synapses of live neurons using single- molecule fluorescence microscopy. Inside individual synapses, we found immobile and mobile receptors, which display restricted diffusion. Extra-synaptic receptors display free diffusion. Receptors could also exchange between these membrane compartments through lateral diffusion. Glutamate application increased both receptor mobility inside synapses and the fraction of mobile receptors present in a juxtasynaptic region. Block of inhibitory transmission to favor excitatory synaptic activity induced a transient increase in the fraction of mobile receptors and a decrease in the proportion of juxtasynaptic receptors. Altogether, our data show that rapid exchange of receptors between a synaptic and extra-synaptic localization occurs through regulation of receptor diffusion inside synapses. PMID:12970178

  17. Chemical labelling for visualizing native AMPA receptors in live neurons.

    PubMed

    Wakayama, Sho; Kiyonaka, Shigeki; Arai, Itaru; Kakegawa, Wataru; Matsuda, Shinji; Ibata, Keiji; Nemoto, Yuri L; Kusumi, Akihiro; Yuzaki, Michisuke; Hamachi, Itaru

    2017-04-07

    The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

  18. 3’-Deoxyadenosine (Cordycepin) Produces a Rapid and Robust Antidepressant Effect via Enhancing Prefrontal AMPA Receptor Signaling Pathway

    PubMed Central

    Li, Bai; Hou, Yangyang; Zhu, Ming; Bao, Hongkun; Nie, Jun; Zhang, Grace Y.; Shan, Liping; Yao, Yao; Du, Kai; Yang, Hongju; Li, Meizhang; Zheng, Bingrong; Xu, Xiufeng; Xiao, Chunjie; Du, Jing

    2016-01-01

    Background: The development of rapid and safe antidepressants for the treatment of major depression is in urgent demand. Converging evidence suggests that glutamatergic signaling seems to play important roles in the pathophysiology of depression. Methods: We studied the antidepressant effects of 3’-deoxyadenosine (3’-dA, Cordycepin) and the critical role of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor in male CD-1 mice via behavioral and biochemical experiments. After 3’-dA treatment, the phosphorylation and synaptic localization of the AMPA receptors GluR1 and GluR2 were determined in the prefrontal cortex (PFC) and hippocampus (HIP). The traditional antidepressant imipramine was applied as a positive control. Results: We found that an injection of 3’-dA led to a rapid and robust antidepressant effect, which was significantly faster and stronger than imipramine, after 45min in tail suspension and forced swim tests. This antidepressant effect remained after 5 days of treatment with 3’-dA. Unlike the psycho-stimulants, 3’-dA did not show a hyperactive effect in the open field test. After 45min or 5 days of treatment, 3’-dA enhanced GluR1 S845 phosphorylation in both the PFC and HIP. In addition, after 45min of treatment, 3’-dA significantly up-regulated GluR1 S845 phosphorylation and GluR1, but not GluR2 levels, at the synapses in the PFC. After 5 days of treatment, 3’-dA significantly enhanced GluR1 S845 phosphorylation and GluR1, but not GluR2, at the synapses in the PFC and HIP. Moreover, the AMPA-specific antagonist GYKI 52466 was able to block the rapid antidepressant effects of 3’-dA. Conclusion: This study identified 3’-dA as a novel rapid antidepressant with clinical potential and multiple beneficial mechanisms, particularly in regulating the prefrontal AMPA receptor signaling pathway. PMID:26443809

  19. Dynamic imaging of AMPA receptor trafficking in vitro and in vivo.

    PubMed

    Roth, Richard H; Zhang, Yong; Huganir, Richard L

    2017-04-12

    Modulation of synaptic strength through trafficking of AMPA receptors is a fundamental mechanism underlying synaptic plasticity and has been shown to be an important process in higher brain functions such as learning and memory. Many studies have used live time-lapse imaging of fluorescently tagged AMPA receptors to directly monitor their membrane trafficking in the basal state as well as during synaptic plasticity. While most of these studies are performed in vitro using neuronal cell cultures, in the past years technological advances have enabled the imaging of synaptic proteins in vivo in intact organisms. This has allowed for visualization of synaptic plasticity on a molecular level in living and behaving animals. Here, we discuss key studies and approaches using dynamic imaging to visualize AMPA receptor trafficking in vitro as well as imaging synaptic proteins, including AMPA receptors, in vivo.

  20. Mechanisms for Antagonistic Regulation of AMPA and NMDA-D1 Receptor Complexes at Postsynaptic Sites

    NASA Technical Reports Server (NTRS)

    Schumann, Johann; Scheler, Gabriele

    2004-01-01

    From the analysis of these pathways we conclude that postsynaptic processes that regulate synaptic transmission undergo significant cross-talk with respect to glutamatergic and neuromodulatory (dopamine) signals. The main hypothesis is that of a compensatory regulation, a competitive switch between the induction of increased AMPA conductance by CaMKII-dependent phosphorylation and reduced expression of PP2A, and increased D1 receptor sensitivity and expression by increased PKA, PP2A and decreased PP-1/calcineurin expression. Both types of plasticity are induced by NMDA receptor activation and increased internal calcium, they require different internal conditions to become expressed. Specifically we propose that AMPA regulation and D1 regulation are inversely coupled;The net result may be a bifurcation of synaptic state into predominantly AMPA or NMDA-D1 synapses. This could have functional consequences: stable connections for AMPA and conditional gating for NMDA-D1 synapses.

  1. Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

    PubMed

    McNair, L F; Kohlmeier, K A

    2015-06-01

    Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the

  2. Calcium permeable AMPA receptors and autoreceptors in external tufted cells of rat olfactory bulb

    PubMed Central

    Ma, Jie; Lowe, Graeme

    2007-01-01

    Glomeruli are functional units of the olfactory bulb responsible for early processing of odor information encoded by single olfactory receptor genes. Glomerular neural circuitry includes numerous external tufted (ET) cells whose rhythmic burst firing may mediate synchronization of bulbar activity with the inhalation cycle. Bursting is entrained by glutamatergic input from olfactory nerve terminals, so specific properties of ionotropic glutamate receptors on ET cells are likely to be important determinants of olfactory processing. Particularly intriguing is recent evidence that α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of juxta-glomerular neurons may permeate calcium. This could provide a novel pathway for regulating ET cell signaling. We tested the hypothesis that ET cells express functional calcium-permeable AMPA receptors. In rat olfactory bulb slices, excitatory postsynaptic currents (EPSCs) in ET cells were evoked by olfactory nerve shock, and by uncaging glutamate. We found attenuation of AMPA/kainate EPSCs by 1-naphthyl acetyl-spermine (NAS), an open-channel blocker specific for calcium permeable AMPA receptors. Cyclothiazide strongly potentiated EPSCs, indicating a major contribution from AMPA receptors. The current-voltage (I-V) relation of uncaging EPSCs showed weak inward rectification which was lost after > ~ 10 min of whole-cell dialysis, and was absent in NAS. In kainate-stimulated slices, Co2+ ions permeated cells of the glomerular layer. Large AMPA EPSCs were accompanied by fluorescence signals in fluo-4 loaded cells, suggesting calcium permeation. Depolarizing pulses evoked slow tail currents with pharmacology consistent with involvement of calcium permeable AMPA autoreceptors. Tail currents were abolished by Cd2+ and NBQX, and were sensitive to NAS block. Glutamate autoreceptors were confirmed by uncaging intracellular calcium to evoke a large inward current. Our results provide evidence that calcium permeable AMPA

  3. Rational Design of a Novel AMPA Receptor Modulator through a Hybridization Approach.

    PubMed

    Caldwell, Nicola; Harms, Jonathan E; Partin, Kathryn M; Jamieson, Craig

    2015-04-09

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a family of glutamate ion channels of considerable interest in excitatory neurotransmission and associated disease processes. Here, we demonstrate how exploitation of the available X-ray crystal structure of the receptor ligand binding domain enabled the development of a new class of AMPA receptor positive allosteric modulators (7) through hybridization of known ligands (5 and 6), leading to a novel chemotype with promising pharmacological properties.

  4. Rational Design of a Novel AMPA Receptor Modulator through a Hybridization Approach

    PubMed Central

    2015-01-01

    The α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are a family of glutamate ion channels of considerable interest in excitatory neurotransmission and associated disease processes. Here, we demonstrate how exploitation of the available X-ray crystal structure of the receptor ligand binding domain enabled the development of a new class of AMPA receptor positive allosteric modulators (7) through hybridization of known ligands (5 and 6), leading to a novel chemotype with promising pharmacological properties. PMID:25893038

  5. Differential effects of glyphosate and aminomethylphosphonic acid (AMPA) on photosynthesis and chlorophyll metabolism in willow plants.

    PubMed

    Gomes, Marcelo Pedrosa; Le Manac'h, Sarah Gingras; Maccario, Sophie; Labrecque, Michel; Lucotte, Marc; Juneau, Philippe

    2016-06-01

    We used a willow species (Salix miyabeana cultivar SX64) to examine the differential secondary-effects of glyphosate and aminomethylphosphonic acid (AMPA), the principal glyphosate by-product, on chlorophyll metabolism and photosynthesis. Willow plants were treated with different concentrations of glyphosate (equivalent to 0, 1.4, 2.1 and 2.8kgha(-1)) and AMPA (equivalent to 0, 0.28, 1.4 and 2.8kgha(-1)) and evaluations of pigment contents, chlorophyll fluorescence, and oxidative stress markers (hydrogen peroxide content and antioxidant enzyme activities) in leaves were performed after 12h of exposure. We observed that AMPA and glyphosate trigger different mechanisms leading to decreases in chlorophyll content and photosynthesis rates in willow plants. Both chemicals induced ROS accumulation in willow leaves although only glyphosate-induced oxidative damage through lipid peroxidation. By disturbing chlorophyll biosynthesis, AMPA induced decreases in chlorophyll contents, with consequent effects on photosynthesis. With glyphosate, ROS increases were higher than the ROS-sensitive threshold, provoking chlorophyll degradation (as seen by pheophytin accumulation) and invariable decreases in photosynthesis. Peroxide accumulation in both AMPA and glyphosate-treated plants was due to the inhibition of antioxidant enzyme activities. The different effects of glyphosate on chlorophyll contents and photosynthesis as described in the literature may be due to various glyphosate:AMPA ratios in those plants.

  6. Incorporation of inwardly rectifying AMPA receptors at silent synapses during hippocampal long-term potentiation.

    PubMed

    Morita, Daiju; Rah, Jong Cheol; Isaac, John T R

    2014-01-05

    Despite decades of study, the mechanisms by which synapses express the increase in strength during long-term potentiation (LTP) remain an area of intense interest. Here, we have studied how AMPA receptor subunit composition changes during the early phases of hippocampal LTP in CA1 pyramidal neurons. We studied LTP at silent synapses that initially lack AMPA receptors, but contain NMDA receptors. We show that strongly inwardly rectifying AMPA receptors are initially incorporated at silent synapses during LTP and are then subsequently replaced by non-rectifying AMPA receptors. These findings suggest that silent synapses initially incorporate GluA2-lacking, calcium-permeable AMPA receptors during LTP that are then replaced by GluA2-containing calcium-impermeable receptors. We also show that LTP consolidation at CA1 synapses requires a rise in intracellular calcium concentration during the early phase of expression, indicating that calcium influx through the GluA2-lacking AMPA receptors drives their replacement by GluA2-containing receptors during LTP consolidation. Taken together with previous studies in hippocampus and in other brain regions, these findings suggest that a common mechanism for the expression of activity-dependent glutamatergic synaptic plasticity involves the regulation of GluA2-subunit composition and highlights a critical role for silent synapses in this process.

  7. Chronic Intermittent Ethanol and Withdrawal Differentially Modulate Basolateral Amygdala AMPA-type Glutamate Receptor Function and Trafficking

    PubMed Central

    Christian, Daniel T; Alexander, Nancy J; Diaz, Marvin R; Robinson, Stacey; McCool, Brian A

    2012-01-01

    The amygdala plays a critical role in the generation and expression of anxiety-like behaviors including those expressed following withdrawal (WD) from chronic intermittent ethanol (CIE) exposure. In particular, the BLA glutamatergic system controls the expression of both innate and pathological anxiety. Recent data suggests that CIE and WD may functionally alter this system in a manner that closely parallels memory-related phenomena like long term potentiation (LTP). We therefore specifically dissected CIE/WD-induced changes in glutamatergic signaling using electrophysiological and biochemical approaches with a particular focus on the plasticity-related components of this neurotransmitter system. Our results indicate that cortical glutamatergic inputs arriving at BLA principal via the external capsule undergo predominantly post-synaptic alterations in AMPA receptor function following CIE and WD. Biochemical analysis revealed treatment-dependent changes in AMPA receptor surface expression and subunit phosphorylation that are complemented by changes in total protein levels and/or phosphorylation status of several key, plasticity-associated protein kinases such as calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). Together, these data show that CIE- and WD-induced changes in BLA glutamatergic function both functionally and biochemically mimic plasticity-related states. These mechanisms likely contribute to long-term increases in anxiety-like behavior following chronic ethanol exposure. PMID:22387532

  8. Chronic intermittent ethanol and withdrawal differentially modulate basolateral amygdala AMPA-type glutamate receptor function and trafficking.

    PubMed

    Christian, Daniel T; Alexander, Nancy J; Diaz, Marvin R; Robinson, Stacey; McCool, Brian A

    2012-06-01

    The amygdala plays a critical role in the generation and expression of anxiety-like behaviors including those expressed following withdrawal (WD) from chronic intermittent ethanol (CIE) exposure. In particular, the BLA glutamatergic system controls the expression of both innate and pathological anxiety. Recent data suggests that CIE and WD may functionally alter this system in a manner that closely parallels memory-related phenomena like long-term potentiation (LTP). We therefore specifically dissected CIE/WD-induced changes in glutamatergic signaling using electrophysiological and biochemical approaches with a particular focus on the plasticity-related components of this neurotransmitter system. Our results indicate that cortical glutamatergic inputs arriving at BLA principal via the external capsule undergo predominantly post-synaptic alterations in AMPA receptor function following CIE and WD. Biochemical analysis revealed treatment-dependent changes in AMPA receptor surface expression and subunit phosphorylation that are complemented by changes in total protein levels and/or phosphorylation status of several key, plasticity-associated protein kinases such as calcium/calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). Together, these data show that CIE- and WD-induced changes in BLA glutamatergic function both functionally and biochemically mimic plasticity-related states. These mechanisms likely contribute to long-term increases in anxiety-like behavior following chronic ethanol exposure. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Glyphosate-Resistant and Conventional Canola (Brassica napus L.) Responses to Glyphosate and Aminomethylphosphonic Acid (AMPA) Treatment.

    PubMed

    Corrêa, Elza Alves; Dayan, Franck E; Owens, Daniel K; Rimando, Agnes M; Duke, Stephen O

    2016-05-11

    Glyphosate-resistant (GR) canola contains two transgenes that impart resistance to the herbicide glyphosate: (1) the microbial glyphosate oxidase gene (gox) encoding the glyphosate oxidase enzyme (GOX) that metabolizes glyphosate to aminomethylphosphonic acid (AMPA) and (2) cp4 that encodes a GR form of the glyphosate target enzyme 5-enolpyruvylshikimic acid-3-phosphate synthase. The objectives of this research were to determine the phytotoxicity of AMPA to canola, the relative metabolism of glyphosate to AMPA in GR and conventional non-GR (NGR) canola, and AMPA pool sizes in glyphosate-treated GR canola. AMPA applied at 1.0 kg ha(-1) was not phytotoxic to GR or NGR. At this AMPA application rate, NGR canola accumulated a higher concentration of AMPA in its tissues than GR canola. At rates of 1 and 3.33 kg ae ha(-1) of glyphosate, GR canola growth was stimulated. This stimulatory effect is similar to that of much lower doses of glyphosate on NGR canola. Both shikimate and AMPA accumulated in tissues of these glyphosate-treated plants. In a separate experiment in which young GR and NGR canola plants were treated with non-phytotoxic levels of [(14)C]-glyphosate, very little glyphosate was metabolized in NGR plants, whereas most of the glyphosate was metabolized to AMPA in GR plants at 7 days after application. Untreated leaves of GR plants accumulated only metabolites (mostly AMPA) of glyphosate, indicating that GOX activity is very high in the youngest leaves. These data indicate that more glyphosate is transformed to AMPA rapidly in GR canola and that the accumulated AMPA is not toxic to the canola plant.

  10. AMPA-Kainate Receptor Inhibition Promotes Neurologic Recovery in Premature Rabbits with Intraventricular Hemorrhage

    PubMed Central

    Dohare, Preeti; Zia, Muhammad T.; Ahmed, Ehsan; Ahmed, Asad; Yadala, Vivek; Schober, Alexandra L.; Ortega, Juan Alberto; Kayton, Robert; Ungvari, Zoltan; Mongin, Alexander A.

    2016-01-01

    Intraventricular hemorrhage (IVH) in preterm infants leads to cerebral inflammation, reduced myelination of the white matter, and neurological deficits. No therapeutic strategy exists against the IVH-induced white matter injury. AMPA-kainate receptor induced excitotoxicity contributes to oligodendrocyte precursor cell (OPC) damage and hypomyelination in both neonatal and adult models of brain injury. Here, we hypothesized that IVH damages white matter via AMPA receptor activation, and that AMPA-kainate receptor inhibition suppresses inflammation and restores OPC maturation, myelination, and neurologic recovery in preterm newborns with IVH. We tested these hypotheses in a rabbit model of glycerol-induced IVH and evaluated the expression of AMPA receptors in autopsy samples from human preterm infants. GluR1-GluR4 expressions were comparable between preterm humans and rabbits with and without IVH. However, GluR1 and GluR2 levels were significantly lower in the embryonic white matter and germinal matrix relative to the neocortex in both infants with and without IVH. Pharmacological blockade of AMPA-kainate receptors with systemic NBQX, or selective AMPA receptor inhibition by intramuscular perampanel restored myelination and neurologic recovery in rabbits with IVH. NBQX administration also reduced the population of apoptotic OPCs, levels of several cytokines (TNFα, IL-β, IL-6, LIF), and the density of Iba1+ microglia in pups with IVH. Additionally, NBQX treatment inhibited STAT-3 phosphorylation, but not astrogliosis or transcription factors regulating gliosis. Our data suggest that AMPA-kainate receptor inhibition alleviates OPC loss and IVH-induced inflammation and restores myelination and neurologic recovery in preterm rabbits with IVH. Therapeutic use of FDA-approved perampanel treatment might enhance neurologic outcome in premature infants with IVH. SIGNIFICANCE STATEMENT Intraventricular hemorrhage (IVH) is a major complication of prematurity and a large number

  11. Excitatory Mechanisms in the Suprachiasmatic Nucleus: The Role of AMPA/KA Glutamate Receptors

    PubMed Central

    Michel, Stephan; Itri, Jason; Colwell, Christopher S.

    2008-01-01

    A variety of evidence suggests that the effects of light on the mammalian circadian system are mediated by direct retinal ganglion cell projection to the suprachiasmatic nucleus (SCN). This synaptic connection is glutamatergic and the release of glutamate is detected by both N-methyl-d-asparate (NMDA) and amino-methyl proprionic acid/kainate (AMPA/KA) iontotropic glutamate receptors (GluRs). It is well established that NMDA GluRs play a critical role in mediating the effects of light on the circadian system; however, the role of AMPA/KA GluRs has received less attention. In the present study, we sought to better understand the contribution of AMPA/KA-mediated currents in the circadian system based in the SCN. First, whole cell patch-clamp electrophysiological techniques were utilized to measure spontaneous excitatory postsynaptic currents (sEPSCs) from SCN neurons. These currents were widespread in the SCN and not just restricted to the retino-recipient region. The sEPSC frequency and amplitude did not vary with the daily cycle. Similarly, currents evoked by the exogenous application of AMPA onto SCN neurons were widespread within the SCN and did not exhibit a diurnal rhythm in their magnitude. Fluorometric techniques were utilized to estimate AMPA-induced calcium (Ca2+) concentration changes in SCN neurons. The resulting data indicate that AMPA-evoked Ca2+ transients were widespread in the SCN and that there was a daily rhythm in the magnitude of AMPA-induced Ca2+ transients that peaked during the night. By itself, blocking AMPA/KA GluRs with a receptor blocker decreased the spontaneous firing of some SCN neurons as well as reduced resting Ca2+ levels, suggesting tonic glutamatergic excitation. Finally, immunohistochemical techniques were used to describe expression of the AMPA-preferring GluR subunits GluR1 and GluR2/3s within the SCN. Overall, our data suggest that glutamatergic synaptic transmission mediated by AMPA/KA GluRs play an important role throughout

  12. AMPA-Kainate Receptor Inhibition Promotes Neurologic Recovery in Premature Rabbits with Intraventricular Hemorrhage.

    PubMed

    Dohare, Preeti; Zia, Muhammad T; Ahmed, Ehsan; Ahmed, Asad; Yadala, Vivek; Schober, Alexandra L; Ortega, Juan Alberto; Kayton, Robert; Ungvari, Zoltan; Mongin, Alexander A; Ballabh, Praveen

    2016-03-16

    Intraventricular hemorrhage (IVH) in preterm infants leads to cerebral inflammation, reduced myelination of the white matter, and neurological deficits. No therapeutic strategy exists against the IVH-induced white matter injury. AMPA-kainate receptor induced excitotoxicity contributes to oligodendrocyte precursor cell (OPC) damage and hypomyelination in both neonatal and adult models of brain injury. Here, we hypothesized that IVH damages white matter via AMPA receptor activation, and that AMPA-kainate receptor inhibition suppresses inflammation and restores OPC maturation, myelination, and neurologic recovery in preterm newborns with IVH. We tested these hypotheses in a rabbit model of glycerol-induced IVH and evaluated the expression of AMPA receptors in autopsy samples from human preterm infants. GluR1-GluR4 expressions were comparable between preterm humans and rabbits with and without IVH. However, GluR1 and GluR2 levels were significantly lower in the embryonic white matter and germinal matrix relative to the neocortex in both infants with and without IVH. Pharmacological blockade of AMPA-kainate receptors with systemic NBQX, or selective AMPA receptor inhibition by intramuscular perampanel restored myelination and neurologic recovery in rabbits with IVH. NBQX administration also reduced the population of apoptotic OPCs, levels of several cytokines (TNFα, IL-β, IL-6, LIF), and the density of Iba1(+) microglia in pups with IVH. Additionally, NBQX treatment inhibited STAT-3 phosphorylation, but not astrogliosis or transcription factors regulating gliosis. Our data suggest that AMPA-kainate receptor inhibition alleviates OPC loss and IVH-induced inflammation and restores myelination and neurologic recovery in preterm rabbits with IVH. Therapeutic use of FDA-approved perampanel treatment might enhance neurologic outcome in premature infants with IVH. Intraventricular hemorrhage (IVH) is a major complication of prematurity and a large number of survivors with

  13. Losses of glyphosate and AMPA via drainflow in a typical Belgian residential area

    NASA Astrophysics Data System (ADS)

    Tang, Ting; Boënne, Wesley; van Griensven, Ann; Seuntjens, Piet; Bronders, Jan; Desmet, Nele

    2014-05-01

    Urban hard surfaces are considered as important facilitators for pesticide transport into urban streams. To obtain concurrent high-resolution data for a detailed investigation on the losses of pesticide runoff from hard surfaces, a monitoring campaign was performed in a typical Belgian residential area (9.5 ha) between 7 May and 7 August, 2013. The campaign yielded a concurrent dataset of rainfall (1-mm rainfall interval), discharge (1-min interval), glyphosate application by the residents and the occurrences of glyphosate and its major degradation product - aminomethylphosphonic acid (AMPA) in the separated storm drainage outflow during 12 rainfall events. In addition, detailed information was obtained on the spatial characteristics of the study area. The resulting dataset allows us to investigate the relevance of catchment hydrology, urban surface properties and pesticide application to the transport and losses of glyphosate in a residential environment. During the campaign, glyphosate was only applied by local residents, mainly on their private driveways. As a result of their continuous use, both glyphosate and AMPA were detected in all analysed outflow samples, with maximum concentrations of 6.1 μg/L and 5.8 μg/L, respectively. Overall, the storm drainage system collected 0.43% of the applied amount of glyphosate. However, this loss rate varied considerably among rainfall events, ranging from 0.04% to 23.36%. According to statistical analysis of the 12 rainfall events, the loss rate was significantly correlated with three factors: the application amount prior to a rainfall event (p < 0.005), rainfall amount during the event (p < 0.02) and the weighted lag time between glyphosate application and the start of the rainfall event (negatively, p < 0.05). A regression analysis showed that these three factors can explain more than 85% of the variation in the loss rate of glyphosate. Furthermore, three types of glyphosate runoff were classified by a clustering

  14. Individual stress vulnerability is predicted by short-term memory and AMPA receptor subunit ratio in the hippocampus.

    PubMed

    Schmidt, Mathias V; Trümbach, Dietrich; Weber, Peter; Wagner, Klaus; Scharf, Sebastian H; Liebl, Claudia; Datson, Nicole; Namendorf, Christian; Gerlach, Tamara; Kühne, Claudia; Uhr, Manfred; Deussing, Jan M; Wurst, Wolfgang; Binder, Elisabeth B; Holsboer, Florian; Müller, Marianne B

    2010-12-15

    Increased vulnerability to aversive experiences is one of the main risk factors for stress-related psychiatric disorders as major depression. However, the molecular bases of vulnerability, on the one hand, and stress resilience, on the other hand, are still not understood. Increasing clinical and preclinical evidence suggests a central involvement of the glutamatergic system in the pathogenesis of major depression. Using a mouse paradigm, modeling increased stress vulnerability and depression-like symptoms in a genetically diverse outbred strain, and we tested the hypothesis that differences in AMPA receptor function may be linked to individual variations in stress vulnerability. Vulnerable and resilient animals differed significantly in their dorsal hippocampal AMPA receptor expression and AMPA receptor binding. Treatment with an AMPA receptor potentiator during the stress exposure prevented the lasting effects of chronic social stress exposure on physiological, neuroendocrine, and behavioral parameters. In addition, spatial short-term memory, an AMPA receptor-dependent behavior, was found to be predictive of individual stress vulnerability and response to AMPA potentiator treatment. Finally, we provide evidence that genetic variations in the AMPA receptor subunit GluR1 are linked to the vulnerable phenotype. Therefore, we propose genetic variations in the AMPA receptor system to shape individual stress vulnerability. Those individual differences can be predicted by the assessment of short-term memory, thereby opening up the possibility for a specific treatment by enhancing AMPA receptor function.

  15. AMPA receptor-induced local brain-derived neurotrophic factor signaling mediates motor recovery after stroke.

    PubMed

    Clarkson, Andrew N; Overman, Justine J; Zhong, Sheng; Mueller, Rudolf; Lynch, Gary; Carmichael, S Thomas

    2011-03-09

    Stroke is the leading cause of adult disability. Recovery after stroke shares similar molecular and cellular properties with learning and memory. A main component of learning-induced plasticity involves signaling through AMPA receptors (AMPARs). We systematically tested the role of AMPAR function in motor recovery in a mouse model of focal stroke. AMPAR function controls functional recovery beginning 5 d after the stroke. Positive allosteric modulators of AMPARs enhance recovery of limb control when administered after a delay from the stroke. Conversely, AMPAR antagonists impair motor recovery. The contributions of AMPARs to recovery are mediated by release of brain-derived neurotrophic factor (BDNF) in periinfarct cortex, as blocking local BDNF function in periinfarct cortex blocks AMPAR-mediated recovery and prevents the normal pattern of motor recovery. In contrast to a delayed AMPAR role in motor recovery, early administration of AMPAR agonists after stroke increases stroke damage. These findings indicate that the role of glutamate signaling through the AMPAR changes over time in stroke: early potentiation of AMPAR signaling worsens stroke damage, whereas later potentiation of the same signaling system improves functional recovery.

  16. Glyphosate and AMPA distribution in wind-eroded sediment derived from loess soil.

    PubMed

    Bento, Célia P M; Goossens, Dirk; Rezaei, Mahrooz; Riksen, Michel; Mol, Hans G J; Ritsema, Coen J; Geissen, Violette

    2017-01-01

    Glyphosate is one of the most used herbicides in agricultural lands worldwide. Wind-eroded sediment and dust, as an environmental transport pathway of glyphosate and of its main metabolite aminomethylphosphonic acid (AMPA), can result in environmental- and human exposure far beyond the agricultural areas where it has been applied. Therefore, special attention is required to the airborne transport of glyphosate and AMPA. In this study, we investigated the behavior of glyphosate and AMPA in wind-eroded sediment by measuring their content in different size fractions (median diameters between 715 and 8 μm) of a loess soil, during a period of 28 days after glyphosate application. Granulometrical extraction was done using a wind tunnel and a Soil Fine Particle Extractor. Extractions were conducted on days 0, 3, 7, 14, 21 and 28 after glyphosate application. Results indicated that glyphosate and AMPA contents were significantly higher in the finest particle fractions (median diameters between 8 and 18 μm), and lowered significantly with the increase in particle size. However, their content remained constant when aggregates were present in the sample. Glyphosate and AMPA contents correlated positively with clay, organic matter, and silt content. The dissipation of glyphosate over time was very low, which was most probably due to the low soil moisture content of the sediment. Consequently, the formation of AMPA was also very low. The low dissipation of glyphosate in our study indicates that the risk of glyphosate transport in dry sediment to off-target areas by wind can be very high. The highest glyphosate and AMPA contents were found in the smallest soil fractions (PM10 and less), which are easily inhaled and, therefore, contribute to human exposure.

  17. Enhanced AMPA Receptor Activity Increases Operant Alcohol Self-administration and Cue-Induced Reinstatement

    PubMed Central

    Cannady, Reginald; Fisher, Kristen R.; Durant, Brandon; Besheer, Joyce; Hodge, Clyde W.

    2012-01-01

    Long-term alcohol exposure produces neuroadaptations that contribute to the progression of alcohol abuse disorders. Chronic alcohol consumption results in strengthened excitatory neurotransmission and increased AMPA receptor signaling in animal models. However, the mechanistic role of enhanced AMPA receptor activity in alcohol reinforcement and alcohol-seeking behavior remains unclear. This study examined the role of enhanced AMPA receptor function using the selective positive allosteric modulator, aniracetam, in modulating operant alcohol self-administration and cue-induced reinstatement. Male alcohol-preferring (P-) rats, trained to self-administer alcohol (15%, v/v) versus water were pretreated with aniracetam to assess effects on maintenance of alcohol self-administration. To determine reinforcer specificity, P-rats were trained to self-administer sucrose (0.8%, w/v) versus water, and effects of aniracetam were tested. The role of aniracetam in modulating relapse of alcohol-seeking was assessed using a response-contingent cue-induced reinstatement procedure in P-rats trained to self-administer 15% alcohol. Aniracetam pretreatment significantly increased alcohol-reinforced responses relative to vehicle treatment. This increase was not attributed to aniracetam-induced hyperactivity as aniracetam pretreatment did not alter locomotor activity. AMPA receptor involvement was confirmed because DNQX (AMPA receptor antagonist) blocked the aniracetam-induced increase in alcohol self-administration. Aniracetam did not alter sucrose-reinforced responses in sucrose-trained P-rats, suggesting that enhanced AMPA receptor activity is selective in modulating the reinforcing function of alcohol. Finally, aniracetam pretreatment potentiated cue-induced reinstatement of alcohol-seeking behavior versus vehicle treated-P-rats. These data suggest that enhanced glutamate activity at AMPA receptors may be key in facilitating alcohol consumption and seeking behavior which could

  18. Natural Reward Experience Alters AMPA and NMDA Receptor Distribution and Function in the Nucleus Accumbens

    PubMed Central

    Pitchers, Kyle K.; Schmid, Susanne; Di Sebastiano, Andrea R.; Wang, Xu; Laviolette, Steven R.; Lehman, Michael N.; Coolen, Lique M.

    2012-01-01

    Natural reward and drugs of abuse converge upon the mesolimbic system which mediates motivation and reward behaviors. Drugs induce neural adaptations in this system, including transcriptional, morphological, and synaptic changes, which contribute to the development and expression of drug-related memories and addiction. Previously, it has been reported that sexual experience in male rats, a natural reward behavior, induces similar neuroplasticity in the mesolimbic system and affects natural reward and drug-related behavior. The current study determined whether sexual experience causes long-lasting changes in mating, or ionotropic glutamate receptor trafficking or function in the nucleus accumbens (NAc), following 3 different reward abstinence periods: 1 day, 1 week, or 1 month after final mating session. Male Sprague Dawley rats mated during 5 consecutive days (sexual experience) or remained sexually naïve to serve as controls. Sexually experienced males displayed facilitation of initiation and performance of mating at each time point. Next, intracellular and membrane surface expression of N-methyl-D-aspartate (NMDA: NR1 subunit) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA: GluA1, GluA2 subunits) receptors in the NAc was determined using a bis(sulfosuccinimidyl)suberate (BS3) protein cross-linking assay followed by Western Blot analysis. NR1 expression was increased at 1 day abstinence both at surface and intracellular, but decreased at surface at 1 week of abstinence. GluA2 was increased intracellularly at 1 week and increased at the surface after 1 month of abstinence. Finally, whole-cell patch clamp electrophysiological recordings determined reduced AMPA/NMDA ratio of synaptic currents in NAc shell neurons following stimulation of cortical afferents in sexually experienced males after all reward abstinence periods. Together, these data show that sexual experience causes long-term alterations in glutamate receptor expression and function in the

  19. AMPA receptor exchange underlies transient memory destabilization on retrieval.

    PubMed

    Hong, Ingie; Kim, Jeongyeon; Kim, Jihye; Lee, Sukwon; Ko, Hyoung-Gon; Nader, Karim; Kaang, Bong-Kiun; Tsien, Richard W; Choi, Sukwoo

    2013-05-14

    A consolidated memory can be transiently destabilized by memory retrieval, after which memories are reconsolidated within a few hours; however, the molecular substrates underlying this destabilization process remain essentially unknown. Here we show that at lateral amygdala synapses, fear memory consolidation correlates with increased surface expression of calcium-impermeable AMPA receptors (CI-AMPARs), which are known to be more stable at the synapse, whereas memory retrieval induces an abrupt exchange of CI-AMPARs to calcium-permeable AMPARs (CP-AMPARs), which are known to be less stable at the synapse. We found that blockade of either CI-AMPAR endocytosis or NMDA receptor activity during memory retrieval, both of which blocked the exchange to CP-AMPARs, prevented memory destabilization, indicating that this transient exchange of AMPARs may underlie the transformation of a stable memory into an unstable memory. These newly inserted CP-AMPARs gradually exchanged back to CI-AMPARs within hours, which coincided with the course of reconsolidation. Furthermore, blocking the activity of these newly inserted CP-AMPARs after retrieval impaired reconsolidation, suggesting that they serve as synaptic "tags" that support synapse-specific reconsolidation. Taken together, our results reveal unexpected physiological roles of CI-AMPARs and CP-AMPARs in transforming a consolidated memory into an unstable memory and subsequently guiding reconsolidation.

  20. Characterization of the AMPA-activated receptors present on motoneurons.

    PubMed

    Greig, A; Donevan, S D; Mujtaba, T J; Parks, T N; Rao, M S

    2000-01-01

    Motoneurons have been shown to be particularly sensitive to Ca2+-dependent glutamate excitotoxicity, mediated via AMPA receptors (AMPARs). To determine the molecular basis for this susceptibility we have used immunocytochemistry, RT-PCR, and electrophysiology to profile AMPARs on embryonic day 14.5 rat motoneurons. Motoneurons show detectable AMPAR-mediated calcium permeability in vitro and in vivo as determined by cobalt uptake and electrophysiology. Motoneurons express all four AMPAR subunit mRNAs, with glutamate receptor (GluR) 2 being the most abundant (63.9+/-4.8%). GluR2 is present almost exclusively in the edited form, and electrophysiology confirms that most AMPARs present are calcium-impermeant. However, the kainate current in motoneurons was blocked an average of 32.0% by Joro spider toxin, indicating that a subset of the AM PARs is Ca2+-permeable. Therefore, heterogeneity of AMPARs, rather than the absence of GluR2 or the presence of unedited GluR2, explains AMPAR-mediated Ca2+ permeability. The relative levels of flip/flop isoforms of each subunit were also examined by semiquantitative PCR. Both isoforms were present, but the relative proportion varied for each subunit, and the flip isoform predominated. Thus, our data show that despite high levels of edited GluR2 mRNA, some AMPARs are Ca2+-permeable, and this subset of AMPARs can account for the AMPAR-mediated Ca2+ inflow inferred from cobalt uptake and electrophysiology studies.

  1. Contextual learning requires synaptic AMPA receptor delivery in the hippocampus

    PubMed Central

    Mitsushima, Dai; Ishihara, Kouji; Sano, Akane; Kessels, Helmut W.; Takahashi, Takuya

    2011-01-01

    The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace. PMID:21746893

  2. Contextual learning requires synaptic AMPA receptor delivery in the hippocampus.

    PubMed

    Mitsushima, Dai; Ishihara, Kouji; Sano, Akane; Kessels, Helmut W; Takahashi, Takuya

    2011-07-26

    The hippocampus plays a central role in learning and memory. Although synaptic delivery of AMPA-type glutamate receptors (AMPARs) contributes to experience-dependent synaptic strengthening, its role in hippocampus-dependent learning remains elusive. By combining viral-mediated in vivo gene delivery with in vitro patch-clamp recordings, we found that the inhibitory avoidance task, a hippocampus-dependent contextual fear-learning paradigm, delivered GluR1-containing AMPARs into CA3-CA1 synapses of the dorsal hippocampus. To block the synaptic delivery of endogenous AMPARs, we expressed a fragment of the GluR1-cytoplasmic tail (the 14-aa GluR1 membrane-proximal region with two serines mutated to phospho-mimicking aspartates: MPR-DD). MPR-DD prevented learning-driven synaptic AMPAR delivery in CA1 neurons. Bilateral expression of MPR-DD in the CA1 region of the rat impaired inhibitory avoidance learning, indicating that synaptic GluR1 trafficking in the CA1 region of the hippocampus is required for encoding contextual fear memories. The fraction of CA1 neurons that underwent synaptic strengthening positively correlated with the performance in the inhibitory avoidance fear memory task. These data suggest that the robustness of a contextual memory depends on the number of hippocampal neurons that participate in the encoding of a memory trace.

  3. Synaptic activity regulates AMPA receptor trafficking through different recycling pathways

    PubMed Central

    Zheng, Ning; Jeyifous, Okunola; Munro, Charlotte; Montgomery, Johanna M; Green, William N

    2015-01-01

    Changes in glutamatergic synaptic strength in brain are dependent on AMPA-type glutamate receptor (AMPAR) recycling, which is assumed to occur through a single local pathway. In this study, we present evidence that AMPAR recycling occurs through different pathways regulated by synaptic activity. Without synaptic stimulation, most AMPARs recycled in dynamin-independent endosomes containing the GTPase, Arf6. Few AMPARs recycled in dynamin-dependent endosomes labeled by transferrin receptors (TfRs). AMPAR recycling was blocked by alterations in the GTPase, TC10, which co-localized with Arf6 endosomes. TC10 mutants that reduced AMPAR recycling had no effect on increased AMPAR levels with long-term potentiation (LTP) and little effect on decreased AMPAR levels with long-term depression. However, internalized AMPAR levels in TfR-containing recycling endosomes increased after LTP, indicating increased AMPAR recycling through the dynamin-dependent pathway with synaptic plasticity. LTP-induced AMPAR endocytosis is inconsistent with local recycling as a source of increased surface receptors, suggesting AMPARs are trafficked from other sites. DOI: http://dx.doi.org/10.7554/eLife.06878.001 PMID:25970033

  4. Cornichon proteins determine the subunit composition of synaptic AMPA receptors.

    PubMed

    Herring, Bruce E; Shi, Yun; Suh, Young Ho; Zheng, Chan-Ying; Blankenship, Sabine M; Roche, Katherine W; Nicoll, Roger A

    2013-03-20

    Cornichon-2 and cornichon-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 CNIH-3 conditional knockout 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 CNIH-2/-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 probably 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.

  5. AMPA receptor exchange underlies transient memory destabilization on retrieval

    PubMed Central

    Hong, Ingie; Kim, Jeongyeon; Kim, Jihye; Lee, Sukwon; Ko, Hyoung-Gon; Nader, Karim; Kaang, Bong-Kiun; Tsien, Richard W.; Choi, Sukwoo

    2013-01-01

    A consolidated memory can be transiently destabilized by memory retrieval, after which memories are reconsolidated within a few hours; however, the molecular substrates underlying this destabilization process remain essentially unknown. Here we show that at lateral amygdala synapses, fear memory consolidation correlates with increased surface expression of calcium-impermeable AMPA receptors (CI-AMPARs), which are known to be more stable at the synapse, whereas memory retrieval induces an abrupt exchange of CI-AMPARs to calcium-permeable AMPARs (CP-AMPARs), which are known to be less stable at the synapse. We found that blockade of either CI-AMPAR endocytosis or NMDA receptor activity during memory retrieval, both of which blocked the exchange to CP-AMPARs, prevented memory destabilization, indicating that this transient exchange of AMPARs may underlie the transformation of a stable memory into an unstable memory. These newly inserted CP-AMPARs gradually exchanged back to CI-AMPARs within hours, which coincided with the course of reconsolidation. Furthermore, blocking the activity of these newly inserted CP-AMPARs after retrieval impaired reconsolidation, suggesting that they serve as synaptic “tags” that support synapse-specific reconsolidation. Taken together, our results reveal unexpected physiological roles of CI-AMPARs and CP-AMPARs in transforming a consolidated memory into an unstable memory and subsequently guiding reconsolidation. PMID:23630279

  6. Pathogenic Mechanism of an Autism-Associated Neuroligin Mutation Involves Altered AMPA-Receptor Trafficking

    PubMed Central

    Chanda, Soham; Aoto, Jason; Lee, Sung-Jin; Wernig, Marius; Südhof, Thomas C.

    2015-01-01

    Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Although the general synaptic role of neuroligins is undisputed, their specific functions at a synapse remain unclear, even controversial. Moreover, many neuroligin gene mutations were associated with autism, but the pathophysiological relevance of these mutations is often unknown, and their mechanisms of action uninvestigated. Here, we examine the synaptic effects of an autism-associated neuroligin-4 substitution (called R704C) which mutates a cytoplasmic arginine residue that is conserved in all neuroligins. We show that the R704C mutation, when introduced into neuroligin-3, enhances the interaction between neuroligin-3 and AMPA-receptors, increases AMPA-receptor internalization, and decreases postsynaptic AMPA-receptor levels. When introduced into neuroligin-4, conversely, the R704C mutation unexpectedly elevated AMPA-receptor mediated synaptic responses. These results suggest a general functional link between neuroligins and AMPA-receptors, indicate that both neuroligin-3 and -4 act at excitatory synapses but perform surprisingly distinct functions, and demonstrate that the R704C mutation significantly impairs the normal function of neuroligin-4, thereby validating its pathogenicity. PMID:25778475

  7. Transfer of glyphosate and its degradate AMPA to surface waters through urban sewerage systems.

    PubMed

    Botta, Fabrizio; Lavison, Gwenaëlle; Couturier, Guillaume; Alliot, Fabrice; Moreau-Guigon, Elodie; Fauchon, Nils; Guery, Bénédicte; Chevreuil, Marc; Blanchoud, Hélène

    2009-09-01

    A study of glyphosate and aminomethyl phosphonic acid (AMPA) transfer in the Orge watershed (France) was carried out during 2007 and 2008. Water samples were collected in surface water, wastewater sewer, storm sewer and wastewater treatment plant (WWTP). These two molecules appeared to be the most frequently detected ones in the rivers and usually exceeded the European quality standard concentrations of 0.1microg L(-1) for drinking water. The annual glyphosate estimated load was 1.9 kg year(-1) upstream (agricultural zone) and 179.5 kg year(-1) at the catchment outlet (urban zone). This result suggests that the contamination of this basin by glyphosate is essentially from urban origin (road and railway applications). Glyphosate reached surface water prevalently through storm sewer during rainfall event. Maximum concentrations were detected in storm sewer just after a rainfall event (75-90 microg L(-1)). High concentrations of glyphosate in surface water during rainfall events reflected urban runoff impact. AMPA was always detected in the sewerage system. This molecule reached surface water mainly via WWTP effluent and also through storm sewer. Variations in concentrations of AMPA during hydrological episodes were minor compared to glyphosate variations. Our study highlights that AMPA and glyphosate origins in urban area are different. During dry period, detergent degradation seemed to be the major AMPA source in wastewater.

  8. DNQX-induced toxicity in cultured rat hippocampal neurons: an apparent AMPA receptor-independent effect?

    PubMed

    Martin, Alexandra; Récasens, Max; Guiramand, Janique

    2003-02-01

    To evaluate the involvement of AMPA receptor activation in neuronal cell death and survival, rat hippocampal neurons in culture were treated with AMPA receptor antagonists. A 46 h treatment with 6,7-dinitroquinoxaline-2,3-dione (DNQX), added 2 h after cell plating, induces a dose-dependent neurotoxicity. Similar effects are also observed in more mature hippocampal neurons (treatment at 14 days in vitro). DNQX toxic effect is neuron-specific since cultured hippocampal glial cells are unaffected. Attempts to characterise the site of action of DNQX suggest that ionotropic glutamate receptors would not be implicated. Indeed, (i) other AMPA receptor antagonists are either ineffective or only moderately efficient in mimicking DNQX effects; (ii) AMPA alone or in the presence of cyclothiazide, as well as, other AMPA receptor agonists, do not reverse DNQX action; (iii) DNQX neurotoxicity is not likely to involve blockade of NMDA receptor glycine site, since this effect is neither mimicked by 7-chlorokynurenate nor reversed by D-serine. Thus, DNQX toxicity in cultured hippocampal neurons is apparently mediated through an ionotropic glutamate receptor-independent way. Copyright 2003 Elsevier Science Ltd.

  9. The SOL-2/Neto auxiliary protein modulates the function of AMPA-subtype ionotropic glutamate receptors.

    PubMed

    Wang, Rui; Mellem, Jerry E; Jensen, Michael; Brockie, Penelope J; Walker, Craig S; Hoerndli, Frédéric J; Hauth, Linda; Madsen, David M; Maricq, Andres V

    2012-09-06

    The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning, and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants, and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. The SOL-2/Neto Auxiliary Protein Modulates the Function of AMPA-Subtype Ionotropic Glutamate Receptors

    PubMed Central

    Wang, Rui; Mellem, Jerry E.; Jensen, Michael; Brockie, Penelope J.; Walker, Craig S.; Hoerndli, Frédéric J.; Madsen, David M.; Maricq, Andres V.

    2012-01-01

    Summary The neurotransmitter glutamate mediates excitatory synaptic transmission by gating ionotropic glutamate receptors (iGluRs). AMPA receptors (AMPARs), a subtype of iGluR, are strongly implicated in synaptic plasticity, learning and memory. We previously discovered two classes of AMPAR auxiliary proteins in C. elegans that modify receptor kinetics and thus change synaptic transmission. Here, we have identified another auxiliary protein, SOL-2, a CUB-domain protein that associates with both the related auxiliary subunit SOL-1 and with the GLR-1 AMPAR. In sol-2 mutants, behaviors dependent on glutamatergic transmission are disrupted, GLR-1-mediated currents are diminished, and GLR-1 desensitization and pharmacology are modified. Remarkably, a secreted variant of SOL-1 delivered in trans can rescue sol-1 mutants and this rescue depends on in cis expression of SOL-2. Finally, we demonstrate that SOL-1 and SOL-2 have an ongoing role in the adult nervous system to control AMPAR-mediated currents. PMID:22958824

  11. Erbin interacts with TARP γ-2 for surface expression of AMPA receptors in cortical interneurons.

    PubMed

    Tao, Yanmei; Chen, Yong-Jun; Shen, Chengyong; Luo, Zhengyi; Bates, C Ryan; Lee, Daehoon; Marchetto, Sylvie; Gao, Tian-Ming; Borg, Jean-Paul; Xiong, Wen-Cheng; Mei, Lin

    2013-03-01

    Inhibitory neurons control the firing of glutamatergic neurons and synchronize brain activity. However, little is known about mechanisms of excitatory synapse formation in inhibitory neurons. Here we demonstrate that Erbin is specifically expressed in cortical inhibitory neurons. It localizes at excitatory synapses and regulates AMPA receptor (AMPAR) surface expression. Erbin mutation reduced mEPSCs and AMPAR currents specifically in parvalbumin (PV)-positive interneurons but not in pyramidal neurons. We found that the AMPAR auxiliary protein TARP γ-2 was specifically expressed in cortical interneurons. Erbin interacts with TARP γ-2 and is crucial for its stability. Deletion of the γ-2-interacting domain in Erbin attenuated surface AMPAR and excitatory transmission in PV-positive interneurons. Furthermore, we observed behavioral deficits in Erbin-null mice and in mice expressing an Erbin truncation mutant that is unable to interact with TARP γ-2. These observations demonstrate a crucial function for Erbin in AMPAR surface expression in cortical PV-positive interneurons and may contribute to a better understanding of psychiatric disorders.

  12. GluA1 signal peptide determines the spatial assembly of heteromeric AMPA receptors

    PubMed Central

    Li, Yan-Jun; Kalyanaraman, Chakrapani; Qiu, Li-Li; Chen, Chen; Xiao, Qi; Liu, Wen-Xue; Zhang, Wei; Yang, Jian-Jun; Chen, Guiquan; Jacobson, Matthew P.; Shi, Yun Stone

    2016-01-01

    AMPA-type glutamate receptors (AMPARs) mediate fast excitatory neurotransmission and predominantly assemble as heterotetramers in the brain. Recently, the crystal structures of homotetrameric GluA2 demonstrated that AMPARs are assembled with two pairs of conformationally distinct subunits, in a dimer of dimers formation. However, the structure of heteromeric AMPARs remains unclear. Guided by the GluA2 structure, we performed cysteine mutant cross-linking experiments in full-length GluA1/A2, aiming to draw the heteromeric AMPAR architecture. We found that the amino-terminal domains determine the first level of heterodimer formation. When the dimers further assemble into tetramers, GluA1 and GluA2 subunits have preferred positions, possessing a 1–2–1–2 spatial assembly. By swapping the critical sequences, we surprisingly found that the spatial assembly pattern is controlled by the excisable signal peptides. Replacements with an unrelated GluK2 signal peptide demonstrated that GluA1 signal peptide plays a critical role in determining the spatial priority. Our study thus uncovers the spatial assembly of an important type of glutamate receptors in the brain and reveals a novel function of signal peptides. PMID:27601647

  13. GSG1L suppresses AMPA receptor-mediated synaptic transmission and uniquely modulates AMPA receptor kinetics in hippocampal neurons

    PubMed Central

    Gu, Xinglong; Mao, Xia; Lussier, Marc P.; Hutchison, Mary Anne; Zhou, Liang; Hamra, F. Kent; Roche, Katherine W.; Lu, Wei

    2016-01-01

    Regulation of AMPA receptor (AMPAR)-mediated synaptic transmission is a key mechanism for synaptic plasticity. In the brain, AMPARs assemble with a number of auxiliary subunits, including TARPs, CNIHs and CKAMP44, which are important for AMPAR forward trafficking to synapses. Here we report that the membrane protein GSG1L negatively regulates AMPAR-mediated synaptic transmission. Overexpression of GSG1L strongly suppresses, and GSG1L knockout (KO) enhances, AMPAR-mediated synaptic transmission. GSG1L-dependent regulation of AMPAR synaptic transmission relies on the first extracellular loop domain and its carboxyl-terminus. GSG1L also speeds up AMPAR deactivation and desensitization in hippocampal CA1 neurons, in contrast to the effects of TARPs and CNIHs. Furthermore, GSG1L association with AMPARs inhibits CNIH2-induced slowing of the receptors in heterologous cells. Finally, GSG1L KO rats have deficits in LTP and show behavioural abnormalities in object recognition tests. These data demonstrate that GSG1L represents a new class of auxiliary subunit with distinct functional properties for AMPARs. PMID:26932439

  14. Mechanism of Inhibition of the GluA2 AMPA Receptor Channel Opening by Talampanel and Its Enantiomer: The Stereochemistry of the 4-Methyl Group on the Diazepine Ring of 2,3-Benzodiazepine Derivatives

    PubMed Central

    2013-01-01

    Stereoselectivity of 2,3-benzodiazepine compounds provides a unique way for the design of stereoisomers as more selective and more potent inhibitors as drug candidates for treatment of the neurological diseases involving excessive activity of AMPA receptors. Here we investigate a pair of enantiomers known as Talampanel and its (+) counterpart about their mechanism of inhibition and selectivity toward four AMPA receptor subunits or GluA1–4. We show that Talampanel is the eutomer with the endismic ratio being 14 for the closed-channel and 10 for the open-channel state of GluA2. Kinetic evidence supports that Talampanel is a noncompetitive inhibitor and it binds to the same site for those 2,3-benzodiazepine compounds with the C-4 methyl group on the diazepine ring. This site, which we term as the “M” site, recognizes preferentially those 2,3-benzodiazepine compounds with the C-4 methyl group being in the R configuration, as in the chemical structure of Talampanel. Given that Talampanel inhibits GluA1 and GluA2, but is virtually ineffective on the GluA3 and GluA4 AMPA receptor subunits, we hypothesize that the “M” site(s) on GluA1 and GluA2 to which Talampanel binds is different from that on GluA3 and GluA4. If the molecular properties of the AMPA receptors and Talampanel are used for selecting an inhibitor as a single drug candidate for controlling the activity of all AMPA receptors in vivo, Talampanel is not ideal. Our results further suggest that addition of longer acyl groups to the N-3 position should produce more potent 2,3-benzodiazepine inhibitors for the “M” site. PMID:23402301

  15. Quantal release of glutamate generates pure kainate and mixed AMPA/kainate EPSCs in hippocampal neurons.

    PubMed

    Cossart, Rosa; Epsztein, Jérôme; Tyzio, Roman; Becq, Hélène; Hirsch, June; Ben-Ari, Yehezkel; Crépel, Valérie

    2002-07-03

    The relative contribution of kainate receptors to ongoing glutamatergic activity is at present unknown. We report the presence of spontaneous, miniature, and minimal stimulation-evoked excitatory postsynaptic currents (EPSCs) that are mediated solely by kainate receptors (EPSC(kainate)) or by both AMPA and kainate receptors (EPSC(AMPA/kainate)). EPSC(kainate) and EPSC(AMPA/kainate) are selectively enriched in CA1 interneurons and mossy fibers synapses of CA3 pyramidal neurons, respectively. In CA1 interneurons, the decay time constant of EPSC(kainate) (circa 10 ms) is comparable to values obtained in heterologous expression systems. In both hippocampal neurons, the quantal release of glutamate generates kainate receptor-mediated EPSCs that provide as much as half of the total glutamatergic current. Kainate receptors are, therefore, key players of the ongoing glutamatergic transmission in the hippocampus.

  16. Screening for AMPA receptor auxiliary subunit specific modulators

    PubMed Central

    Azumaya, Caleigh M.; Days, Emily L.; Vinson, Paige N.; Stauffer, Shaun; Sulikowski, Gary; Weaver, C. David; Nakagawa, Terunaga

    2017-01-01

    AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes. PMID:28358902

  17. Probing TARP modulation of AMPA receptor conductance with polyamine toxins.

    PubMed

    Jackson, Alexander C; Milstein, Aaron D; Soto, David; Farrant, Mark; Cull-Candy, Stuart G; Nicoll, Roger A

    2011-05-18

    The properties of synaptic AMPA receptors (AMPARs) depend on their subunit composition and association with transmembrane AMPAR regulatory proteins (TARPs). Although both GluA2 incorporation and TARP association have been shown to influence AMPAR channel conductance, the manner in which different TARPs modulate the mean channel conductance of GluA2-containing AMPARs is unknown. Using ultrafast agonist application and nonstationary fluctuation analysis, we found that TARP subtypes differentially increase the mean channel conductance, but not the peak open probability, of recombinant GluA2-containing AMPARs. TARP γ-8, in particular, enhances mean channel conductance to a greater degree than γ-2, γ-3, or γ-4. We then examined the action of a use-dependent antagonist of GluA2-containing AMPARs, philanthotoxin-74 (PhTx-74), on recombinant AMPARs and on GluA2-containing AMPARs in cerebellar granule neurons from stargazer mice transfected with TARPs. We found that the rate and extent of channel block varies with TARP subtype, in a manner that correlates linearly with mean channel conductance. Furthermore, block of GluA2-containing AMPARs by polyamine toxins varied depending on whether channels were activated by the full agonist glutamate or the partial agonist kainate, consistent with conductance state-dependent block. Block of GluA2-lacking AMPARs by PhTx-433 is also modulated by TARP association and is a function of agonist efficacy. Our data indicate that channel block by polyamine toxins is sensitive to the mean channel conductance of AMPARs, which varies with TARP subtype and agonist efficacy. Furthermore, our results illustrate the utility of polyamine toxins as sensitive probes of AMPAR channel conductance and suggest the possibility that TARPs may influence their channel properties by selectively stabilizing specific channel conformations, rather than altering the pore structure.

  18. AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability

    PubMed Central

    Lykens, Nicole M.; Reddi, Jyoti M.

    2017-01-01

    Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability. PMID:28178321

  19. AMPA GluA1-flip targeted oligonucleotide therapy reduces neonatal seizures and hyperexcitability.

    PubMed

    Lykens, Nicole M; Coughlin, David J; Reddi, Jyoti M; Lutz, Gordon J; Tallent, Melanie K

    2017-01-01

    Glutamate-activated α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPA-Rs) mediate the majority of excitatory neurotransmission in brain and thus are major drug targets for diseases associated with hyperexcitability or neurotoxicity. Due to the critical nature of AMPA-Rs in normal brain function, typical AMPA-R antagonists have deleterious effects on cognition and motor function, highlighting the need for more precise modulators. A dramatic increase in the flip isoform of alternatively spliced AMPA-R GluA1 subunits occurs post-seizure in humans and animal models. GluA1-flip produces higher gain AMPA channels than GluA1-flop, increasing network excitability and seizure susceptibility. Splice modulating oligonucleotides (SMOs) bind to pre-mRNA to influence alternative splicing, a strategy that can be exploited to develop more selective drugs across therapeutic areas. We developed a novel SMO, GR1, which potently and specifically decreased GluA1-flip expression throughout the brain of neonatal mice lasting at least 60 days after single intracerebroventricular injection. GR1 treatment reduced AMPA-R mediated excitatory postsynaptic currents at hippocampal CA1 synapses, without affecting long-term potentiation or long-term depression, cellular models of memory, or impairing GluA1-dependent cognition or motor function in mice. Importantly, GR1 demonstrated anti-seizure properties and reduced post-seizure hyperexcitability in neonatal mice, highlighting its drug candidate potential for treating epilepsies and other neurological diseases involving network hyperexcitability.

  20. Exacerbation of NMDA, AMPA, and L-glutamate excitotoxicity by the succinate dehydrogenase inhibitor malonate.

    PubMed

    Greene, J G; Greenamyre, J T

    1995-05-01

    We report that a subtoxic dose of the succinate dehydrogenase (SDH) inhibitor malonate greatly enhances the neurotoxicity of three different excitatory amino acid agonists: N-methyl-D-aspartate (NMDA), S-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (S-AMPA), and L-glutamate. In male Sprague-Dawley rats, intrastriatal stereotaxic injection of malonate alone (0.6 mumol), NMDA alone (15 nmol), S-AMPA alone (1 nmol), or glutamate alone (0.6 mumol) produced negligible toxicity as assessed by measurement of lesion volume. Coinjection of subtoxic malonate with NMDA produced a large lesion (15.2 +/- 1.4 mm3), as did coinjection of malonate with S-AMPA (11.0 +/- 1.0 mm3) or glutamate (12.8 +/- 0.7 mm3). Administration of the noncompetitive NMDA antagonist MK-801 (5 mg/kg i.p.) completely blocked the toxicity of malonate plus NMDA (0.5 +/- 0.3 mm3). This dose of MK-801 had little effect on the lesion produced by malonate plus S-AMPA (9.0 +/- 0.7 mm3), but it attenuated the toxicity of malonate plus glutamate by approximately 40% (7.5 +/- 0.9 mm3). Coinjection of the AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)-quinoxaline (NBQX; 2 nmol) had no effect on malonate plus NMDA or malonate plus glutamate toxicity (12.3 +/- 1.8 and 14.0 +/- 0.9 mm3, respectively) but greatly attenuated malonate plus S-AMPA toxicity (1.5 +/- 0.9 mm3). Combination of the two antagonists conferred no additional neuroprotection in any paradigm. These results indicate that metabolic inhibition exacerbates both NMDA receptor- and non-NMDA receptor-mediated excitotoxicity.(ABSTRACT TRUNCATED AT 250 WORDS)

  1. CNQX and AMPA inhibit electrical synaptic transmission: a potential interaction between electrical and glutamatergic synapses

    PubMed Central

    Li, Qin; Burrell, Brian D.

    2008-01-01

    Electrical synapses play an important role in signaling between neurons and the synaptic connections between many neurons possess both electrical and chemical components. Although modulation of electrical synapses is frequently observed, the cellular processes that mediate such changes have not been studied as thoroughly as plasticity in chemical synapses. In the leech (Hirudo sp), the competitive AMPA receptor antagonist CNQX inhibited transmission at the rectifying electrical synapse of a mixed glutamatergic/electrical synaptic connection. This CNQX-mediated inhibition of the electrical synapse was blocked by concanavalin A (Con A) and dynamin inhibitory peptide (DIP), both of which are known to inhibit endocytosis of neurotransmitter receptors. CNQX-mediated inhibition was also blocked by pep2-SVKI (SVKI), a synthetic peptide that prevents internalization of AMPA-type glutamate receptor. AMPA itself also inhibited electrical synaptic transmission and this AMPA-mediated inhibition was partially blocked by Con A, DIP and SVKI. Low frequency stimulation induced long-term depression (LTD) in both the electrical and chemical components of these synapses and this LTD was blocked by SVKI. GYKI 52466, a selective non-competitive antagonist of AMPA receptors, did not affect the electrical EPSP, although it did block the chemical component of these synapses. CNQX did not affect non-rectifying electrical synapses in two different pairs of neurons. These results suggest an interaction between AMPA-type glutamate receptors and the gap junction proteins that mediate electrical synaptic transmission. This putative interaction between glutamate receptors and gap junction proteins represents a novel mechanism for regulating the strength of synaptic transmission. PMID:18601913

  2. Involvement of prefrontal AMPA receptors in encounter stimulation-induced hyperactivity in isolation-reared mice.

    PubMed

    Araki, Ryota; Ago, Yukio; Hasebe, Shigeru; Nishiyama, Saki; Tanaka, Tatsunori; Oka, Satoshi; Takuma, Kazuhiro; Matsuda, Toshio

    2014-06-01

    We recently showed that social encounter stimulation induces hyperactivity in mice reared in social isolation from early life and this is associated with the transient activation of prefrontal dopaminergic and serotonergic systems. In the present study, we examined the effect of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist 2, 3-dioxo-6-nitro-1, 2, 3, 4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) on encounter-induced behavioural and neurochemical changes to study the role of the receptor in abnormal behaviours in isolation-reared mice. The encounter to an intruder mouse induced hyperactivity with transient increases in prefrontal dopamine and serotonin levels in isolation-reared mice. NBQX attenuated the encounter-induced hyperactivity and the associated neurochemical changes in isolation-reared mice. In addition, NBQX reduced aggressive behaviour and cognitive impairment in isolation-reared mice, but did not affect depressive-like behaviour or spontaneous hyper-locomotion in these animals. The AMPA receptor agonist (S)-AMPA increased prefrontal dopamine and serotonin release, and this effect was higher in isolation-reared mice than in the group-reared mice, suggesting higher prefrontal AMPA receptor activity in isolation-reared mice. Furthermore, isolation rearing increased the expression of AMPA receptor subunits (GluR1, GluR2 and GluR3) and GluR1 Ser845 phosphorylation in the prefrontal cortex, but not in the hippocampus or nucleus accumbens. Taken together, these results suggest that an increase in AMPA receptor activity in the prefrontal cortex contributes to some, but not all, abnormal behaviours in isolation-reared mice.

  3. The Fate and Transport of Glyphosate and its Degradation Product, Aminomethylphosphonic Acid (AMPA), in Water

    NASA Astrophysics Data System (ADS)

    Scribner, E.; Meyer, M. T.

    2006-05-01

    Since 2001, the U.S. Geological Survey (USGS) has investigated the fate and transport of glyphosate and its degradation product, aminomethylphosphonic acid (AMPA), in surface water, and more recently in tile-drain flow, soil, and wet deposition. According to U.S. Environmental Protection Agency sources, glyphosate is among the world's most widely used herbicides. In 2004, glyphosate usage estimates indicated that between 103 and 113 million pounds were applied annually to crops in the United States. The use of glyphosate over a wide geographic area suggests that this herbicide might be a potential concern for air, water, and soil quality as well as measured in high concentrations in streams; therefore, it is important to monitor its fate and transport in ground-water/surface-water systems. National, regional, and field-scale studies conducted by the USGS National Water-Quality Assessment and Toxic Substance Hydrology Programs have studied the fate and transport of glyphosate in overland flow, tile- drain flow, surface water, soil, and wet-deposition samples. The samples were analyzed for glyphosate and AMPA by using derivatization and online solid-phase extraction with liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS methods developed by the USGS Organic Geochemistry Research Laboratory in Lawrence, Kansas. During spring, summer, and fall 2002 runoff periods in 50 Midwestern streams, glyphosate was detected at or above the 0.10 micrograms per liter detection limit in 35, 41, and 31 percent of samples, respectively. AMPA was detected in 53, 82, and 75 percent of samples, respectively. Results of 128 samples from a field study showed that glyphosate was transported as a narrow high- concentration pulse during the first period of runoff after application and that the concentration of glyphosate in runoff was greater than the concentration of AMPA. In tile-drain flow, glyphosate and AMPA were transported in a broad low-concentration pulse during these same

  4. Involvement of AMPA receptors in the antidepressant-like effects of dextromethorphan in mice.

    PubMed

    Nguyen, Linda; Matsumoto, Rae R

    2015-12-15

    Dextromethorphan (DM) is an antitussive with rapid acting antidepressant potential based on pharmacodynamic similarities to ketamine. Building upon our previous finding that DM produces antidepressant-like effects in the mouse forced swim test (FST), the present study aimed to establish the antidepressant-like actions of DM in the tail suspension test (TST), another well-established model predictive of antidepressant efficacy. Additionally, using the TST and FST, we investigated the role of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors in the antidepressant-like properties of DM because accumulating evidence suggests that AMPA receptors play an important role in the pathophysiology of depression and may contribute to the efficacy of antidepressant medications, including that of ketamine. We found that DM displays antidepressant-like effects in the TST similar to the conventional and fast acting antidepressants characterized by imipramine and ketamine, respectively. Moreover, decreasing the first-pass metabolism of DM by concomitant administration of quinidine (CYP2D6 inhibitor) potentiated antidepressant-like actions, implying DM itself has antidepressant efficacy. Finally, in both the TST and FST, pretreatment with the AMPA receptor antagonist NBQX (2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide) significantly attenuated the antidepressant-like behavior elicited by DM. Together, the data show that DM exerts antidepressant-like actions through AMPA receptors, further suggesting DM may act as a safe and effective fast acting antidepressant drug.

  5. Activation of α7-containing nicotinic receptors on astrocytes triggers AMPA receptor recruitment to glutamateric synapses

    PubMed Central

    Wang, Xulong; Lippi, Giordano; Carlson, David M.; Berg, Darwin K.

    2014-01-01

    Astrocytes, an abundant form of glia, are known to promote and modulate synaptic signaling between neurons. They also express α7-containing nicotinic acetylcholine receptors (α7-nAChRs), but the functional relevance of these receptors is unknown. We show here that stimulation of α7-nAChRs on astrocytes releases components that induce hippocampal neurons to acquire more a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors post-synaptically at glutamatergic synapses. The increase is specific in that no change is seen in synaptic NMDA receptor clusters or other markers for glutamatergic synapses, or in markers for GABAergic synapses. Moreover, the increases in AMPA receptors on the neuron surface are accompanied by increases in the frequency of spontaneous miniature synaptic currents mediated by the receptors and increases in the ratio of evoked synaptic currents mediated by AMPA versus NMDA receptors. This suggests that stimulating α7-nAChRs on astrocytes can convert ‘silent’ glutamatergic synapses to functional status. Astrocyte-derived thrombospondin is necessary but not sufficient for the effect, while tumor necrosis factor-α is sufficient but not necessary. The results identify astrocyte α7-nAChRs as a novel pathway through which nicotinic cholinergic signaling can promote the development of glutamatergic networks, recruiting AMPA receptors to post-synaptic sites and rendering the synapses more functional. PMID:24032433

  6. mTOR Is Essential for Corticosteroid Effects on Hippocampal AMPA Receptor Function and Fear Memory

    ERIC Educational Resources Information Center

    Xiong, Hui; Casse, Frédéric; Zhou, Yang; Zhou, Ming; Xiong, Zhi-Qi; Joëls, Marian; Martin, Stéphane; Krugers, Harm J.

    2015-01-01

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptors (AMPARs), which are crucial for synaptic plasticity and memory formation. Combining a live imaging fluorescent recovery after photobleaching approach…

  7. Structural Determinants of the γ-8 TARP Dependent AMPA Receptor Antagonist.

    PubMed

    Lee, Matthew R; Gardinier, Kevin M; Gernert, Douglas L; Schober, Douglas A; Wright, Rebecca A; Wang, He; Qian, Yuewei; Witkin, Jeffrey M; Nisenbaum, Eric S; Kato, Akihiko S

    2017-09-05

    The forebrain specific AMPA receptor antagonist, LY3130481/CERC-611, which selectively antagonizes the AMPA receptors associated with TARP γ-8, an auxiliary subunit enriched in the forebrain, has potent antiepileptic activities without motor side effects. We designated the compounds with such activities as γ-8 TARP dependent AMPA receptor antagonists (γ-8 TDAAs). In this work, we further investigated the mechanisms of action using a radiolabeled γ-8 TDAA and ternary structural modeling with mutational validations to characterize the LY3130481 binding to γ-8. The radioligand binding to the cells heterologously expressing GluA1 and/or γ-8 revealed that γ-8 TDAAs binds to γ-8 alone without AMPA receptors. Homology modeling of γ-8, based on the crystal structures of a distant TARP homologue, murine claudin 19, in conjunction with knowledge of two γ-8 residues previously identified as critical for the LY3130481 TARP-dependent selectivity provided the basis for a binding mode prediction. This allowed further rational mutational studies for characterization of the structural determinants in TARP γ-8 for LY3130481 activities, both thermodynamically as well as kinetically.

  8. Peripheral AMPA receptors contribute to muscle nociception and c-fos activation

    PubMed Central

    Chun, Yang-Hyun; Frank, Dorie; Lee, Jong-Seok; Zhang, Youping; Auh, Q-Schick; Ro, Jin Y.

    2008-01-01

    In this study, involvement of peripheral AMPA receptors in mediating craniofacial muscle pain was investigated. AMPA receptor subunits, GluR1 and GluR2, were predominantly expressed in small to medium size neurons but more GluR2 positive labeling were encountered in trigeminal ganglia (TG) of male Sprague Dawley rats. A greater prevalence of GluR2 is reflected by the significantly higher percentage of GluR2 than GluR1 positive masseter afferents. Nocifensive behavior and c-fos immunoreactivity were assessed from the same animals that received intramuscular mustard oil (MO) with or without NBQX, a potent AMPA/KA receptor antagonist. Masseteric MO produced nocifensive hindpaw shaking responses that peaked in the first 30 seconds and gradually diminished over a few minutes. There was a significant difference in both peak and overall MO-induced nocifensive responses between NBQX and vehicle pre-treated rats. Subsequent Fos studies also showed that peripheral NBQX pre-treatment effectively reduced the MO-induced neuronal activation in the subnucleus caudalis of the trigeminal nerve (Vc). These combined results provide compelling evidence that acute muscle nociception is mediated, in part, by peripherally located AMPA/KA receptors, and that blockade of multiple peripheral glutamate receptor subtypes may provide a more effective means of reducing muscular pain and central neuronal activation. PMID:18655811

  9. A profile of the behavioral changes produced by facilitation of AMPA-type glutamate receptors.

    PubMed

    Davis, C M; Moskovitz, B; Nguyen, M A; Tran, B B; Arai, A; Lynch, G; Granger, R

    1997-09-01

    A newly developed group of benzoylpiperidine drugs that enhance AMPA-receptor-gated currents ("ampakines") has been shown to improve memory encoding in rats across a variety of experimental paradigms. The present experiments were intended to i) provide a partial profile of the behavioral changes produced by ampakines, ii) test if two ampakines (BDP-12 and BDP-20) that differ significantly in their effects on AMPA receptor kinetics produce similar behavioral profiles, and iii) determine if physiological potency is reflected in behavioral potency. BDP-20 reduced two measures of exploratory activity in aged rats but increased speed of performance in a radial maze; the drug also caused substantially improved retention of spatial information. These results are similar to those obtained with BDP-12, an analog that differs from BDP-20 in its effects on ligand binding to the AMPA receptor and on the physiological responses of the receptors to glutamate. BDP-20 was approximately ten-fold more potent in behavioral effects than BDP-12, which agrees with the relative potencies of the two drugs as assessed with excised patches and excitatory synaptic responses. These findings indicate that ampakines, though differing in their effects on AMPA-receptor-mediated responses, have similar effects at the behavioral level.

  10. Determination of glyphosate and AMPA on polyester-toner electrophoresis microchip with contactless conductivity detection.

    PubMed

    da Silva, Eduardo R; Segato, Thiago P; Coltro, Wendell K T; Lima, Renato S; Carrilho, Emanuel; Mazo, Luiz H

    2013-07-01

    This paper reports a method for rapid, simple, direct, and reproducible determination of glyphosate and its major metabolite aminomethylphosphonic acid (AMPA). The platform described herein uses polyester-toner microchips incorporating capacitively coupled contactless conductivity detection and electrophoresis separation of the analytes. The polyester-toner microchip presented 150 μm-wide and 12 μm-deep microchannels, with injection and separation lengths of 10 and 40 mm long, respectively. The best results were obtained with 320 kHz frequency, 4.5 Vpp excitation voltage, 80 mmol/L CHES/Tris buffer at pH 8.8, injection in -1.0 kV for 7 s, and separation in -1.5 kV. RSD values related to the peak areas for glyphosate and AMPA were 1.5 and 3.3% and 10.1 and 8.6% for intra- and interchip assays, respectively. The detection limits were 45.1 and 70.5 μmol/L, respectively, without any attempt of preconcentration of the analytes. Finally, the method was applied to river water samples in which glyphosate and AMPA (1.0 mmol/L each) were added. The recovery results were 87.4 and 83.7% for glyphosate and AMPA, respectively. The recovery percentages and LOD values obtained here were similar to others reported in the literature.

  11. Glyphosate and AMPA adsorption in soils: laboratory experiments and pedotransfer rules.

    PubMed

    Sidoli, Pauline; Baran, Nicole; Angulo-Jaramillo, Rafael

    2016-03-01

    Adsorption of the herbicide glyphosate and its main metabolite AMPA (aminomethylphosphonic acid) was investigated on 17 different agricultural soils. Batch equilibration adsorption data are shown by Freundlich adsorption isotherms. Glyphosate adsorption is clearly affected by equilibration concentrations, but the nonlinear AMPA adsorption isotherms indicate saturation of the adsorption sites with increasing equilibrium concentrations. pHCaCl2 (i.e. experimental pH) is the major parameter governing glyphosate and AMPA adsorption in soils. However, considering pHCaCl2 values, available phosphate amount, and amorphous iron and aluminium oxide contents by using a nonlinear multiple regression equation, obtains the most accurate and powerful pedotransfer rule for predicting the adsorption constants for these two molecules. As amorphous iron and aluminium oxide contents in soil are not systematically determined, we also propose a pedotransfer rule with two variables-pHCaCl2 values and available phosphate amount-that remains acceptable for both molecules. Moreover, the use of the commonly measured pHwater or pHKCl values gives less accurate results compared to pHCaCl2 measurements. To our knowledge, this study is the first AMPA adsorption characterization for a significant number of temperate climate soils.

  12. Selective antagonism of AMPA receptors unmasks kainate receptor-mediated responses in hippocampal neurons.

    PubMed

    Paternain, A V; Morales, M; Lerma, J

    1995-01-01

    Although both protein and mRNAs for kainate receptor subunits are abundant in several brain regions, the responsiveness of AMPA receptors to kainate has made it difficult to demonstrate the presence of functional kainate-type receptors in native cells. Recently, however, we have shown that many hippocampal neurons in culture express glutamate receptors of the kainate type. The large nondesensitizing response that kainate induces at AMPA receptors precludes detection and analysis of smaller, rapidly desensitizing currents induced by kainate at kainate receptors. Consequently, the functional significance of these strongly desensitizing glutamate receptors remains enigmatic. We report here that the family of new noncompetitive antagonists of AMPA receptors (GYKI 52466 and 53655) minimally affects kainate-induced responses at kainate receptors while completely blocking AMPA receptor-mediated currents, making it possible to separate the responses mediated by each receptor. These compounds will allow determination of the role played by kainate receptors in synaptic transmission and plasticity in the mammalian brain, as well as evaluation of their involvement in neurotoxicity.

  13. Disruption of the endocytic protein HIP1 results in neurological deficits and decreased AMPA receptor trafficking

    PubMed Central

    Metzler, Martina; Li, Bo; Gan, Lu; Georgiou, John; Gutekunst, Claire-Anne; Wang, Yushan; Torre, Enrique; Devon, Rebecca S.; Oh, Rosemary; Legendre-Guillemin, Valerie; Rich, Mark; Alvarez, Christine; Gertsenstein, Marina; McPherson, Peter S.; Nagy, Andras; Wang, Yu Tian; Roder, John C.; Raymond, Lynn A.; Hayden, Michael R.

    2003-01-01

    Huntingtin interacting protein 1 (HIP1) is a recently identified component of clathrin-coated vesicles that plays a role in clathrin-mediated endocytosis. To explore the normal function of HIP1 in vivo, we created mice with targeted mutation in the HIP1 gene (HIP1–/–). HIP1–/– mice develop a neurological phenotype by 3 months of age manifest with a failure to thrive, tremor and a gait ataxia secondary to a rigid thoracolumbar kyphosis accompanied by decreased assembly of endocytic protein complexes on liposomal membranes. In primary hippocampal neurons, HIP1 colocalizes with GluR1-containing AMPA receptors and becomes concentrated in cell bodies following AMPA stimulation. Moreover, a profound dose-dependent defect in clathrin-mediated internalization of GluR1-containing AMPA receptors was observed in neurons from HIP1–/– mice. Together, these data provide strong evidence that HIP1 regulates AMPA receptor trafficking in the central nervous system through its function in clathrin-mediated endocytosis. PMID:12839988

  14. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    ERIC Educational Resources Information Center

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  15. Enhanced AMPA Receptor Function Promotes Cerebellar Long-Term Depression Rather than Potentiation

    ERIC Educational Resources Information Center

    van Beugen, Boeke J.; Qiao, Xin; Simmons, Dana H.; De Zeeuw, Chris I.; Hansel, Christian

    2014-01-01

    Ampakines are allosteric modulators of AMPA receptors that facilitate hippocampal long-term potentiation (LTP) and learning, and have been considered for the treatment of cognition and memory deficits. Here, we show that the ampakine CX546 raises the amplitude and slows the decay time of excitatory postsynaptic currents (EPSCs) at cerebellar…

  16. mTOR Is Essential for Corticosteroid Effects on Hippocampal AMPA Receptor Function and Fear Memory

    ERIC Educational Resources Information Center

    Xiong, Hui; Casse, Frédéric; Zhou, Yang; Zhou, Ming; Xiong, Zhi-Qi; Joëls, Marian; Martin, Stéphane; Krugers, Harm J.

    2015-01-01

    Glucocorticoid hormones, via activation of their receptors, promote memory consolidation, but the exact underlying mechanisms remain elusive. We examined how corticosterone regulates AMPA receptors (AMPARs), which are crucial for synaptic plasticity and memory formation. Combining a live imaging fluorescent recovery after photobleaching approach…

  17. Topiramate antagonizes NMDA- and AMPA-induced seizure-like activity in planarians.

    PubMed

    Rawls, Scott M; Thomas, Timmy; Adeola, Mobilaji; Patil, Tanvi; Raymondi, Natalie; Poles, Asha; Loo, Michael; Raffa, Robert B

    2009-10-01

    The mechanism of anticonvulsant action of topiramate includes inhibition of glutamate-activated ion channels. The evidence is most convincing for direct inhibitory action at the ionotropic AMPA (alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid) and kainate ((2S,3S,4S)-3-(Carboxymethyl)-4-prop-1-en-2-ylpyrrolidine-2-carboxylic acid) glutamate receptor subtypes. Less direct connection has been made to the NMDA (N-Methyl-d-aspartate) subtype. In the present study, we demonstrate that NMDA and AMPA produce concentration-dependent seizure-like activity in planarians, a type of flatworm which possesses mammalian-like neurotransmitters. In contrast, planarians exposed to the inhibitory amino acid, glycine, did not display pSLA. For combination experiments, topiramate significantly reduced planarian seizure-like activity (pSLA) produced by NMDA or AMPA. Additionally, NMDA-induced pSLA was antagonized by either an NMDA receptor antagonist (MK-801) or AMPA receptor antagonist (DNQX), thus suggesting that NMDA-induced pSLA was mediated by NMDA and non-NMDA receptors. The present results provide pharmacologic evidence of a functional inhibitory action of topiramate on glutamate receptor activity in invertebrates and provide a sensitive, quantifiable end-point for studying anti-seizure pharmacology.

  18. Enhanced AMPA Receptor Function Promotes Cerebellar Long-Term Depression Rather than Potentiation

    ERIC Educational Resources Information Center

    van Beugen, Boeke J.; Qiao, Xin; Simmons, Dana H.; De Zeeuw, Chris I.; Hansel, Christian

    2014-01-01

    Ampakines are allosteric modulators of AMPA receptors that facilitate hippocampal long-term potentiation (LTP) and learning, and have been considered for the treatment of cognition and memory deficits. Here, we show that the ampakine CX546 raises the amplitude and slows the decay time of excitatory postsynaptic currents (EPSCs) at cerebellar…

  19. AMPA Receptor Endocytosis in Rat Perirhinal Cortex Underlies Retrieval of Object Memory

    ERIC Educational Resources Information Center

    Cazakoff, Brittany N.; Howland, John G.

    2011-01-01

    Mechanisms consistent with long-term depression in the perirhinal cortex (PRh) play a fundamental role in object recognition memory; however, whether AMPA receptor endocytosis is involved in distinct phases of recognition memory is not known. To address this question, we used local PRh infusions of the cell membrane-permeable Tat-GluA2[subscript…

  20. Glyphosate and AMPA in the estuaries of the Baltic Sea method optimization and field study.

    PubMed

    Skeff, Wael; Neumann, Christine; Schulz-Bull, Detlef E

    2015-11-15

    Water samples from ten German Baltic estuaries were collected in 2012 in order to study the presence of the herbicide glyphosate, its primary metabolite AMPA and their potential transport to the marine environment. For the analyses an LC-MS/MS based analytical method after derivatization with FMOC-Cl was optimized and validated for marine water samples. All investigated estuarine stations were contaminated with AMPA and nine of them also with glyphosate. Concentration ranges observed were 28 to 1690ng/L and 45 to 4156ng/L for glyphosate and AMPA, respectively with strong spatial and temporal fluctuations. Both contaminants were found at inbound sampling sites in the stream Muehlenfliess and concentrations decreased along the salinity gradient to the estuaries of the Baltic Sea. The data obtained in this study clearly depict the transport of glyphosate and AMPA to the Baltic Sea. Hence, detailed fate and risk assessment for both contaminants in marine environments are required. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum.

    PubMed

    Cost, Hoa N; Noratel, Elizabeth F; Blumberg, Daphne D

    2013-01-01

    The Dictyostelium discoideum ampA gene encodes a multifunctional regulator protein that modulates cell-cell and cell-substrate adhesions and actin polymerization during growth and is necessary for correct cell type specification and patterning during development. Insertional inactivation of the ampA gene results in defects that define two distinct roles for the ampA gene during development. AmpA is necessary in a non-cell autonomous manner to prevent premature expression of a prespore gene marker. It is also necessary in a cell autonomous manner for the anterior like cells, which express the ampA gene, to migrate to the upper cup during culmination. It is also necessary to prevent excessive cell-cell agglutination when cells are developed in a submerged suspension culture. Here, we demonstrate that a supernatant source of AmpA protein, added extracellularly, can prevent the premature mis-expression of the prespore marker. Synthetic oligopeptides are used to identify the domain of the AmpA protein that is important for preventing cells from mis-expressing the prespore gene. We further demonstrate that a factor capable of inducing additional cells to express the prespore gene marker accumulates extracellularly in the absence of AmpA protein. While the secreted AmpA acts extracellularly to suppress prespore gene expression, the effects of AmpA on cell agglutination and on actin polymerization in growing cells are not due to an extracellular role of secreted AmpA protein. Rather, these effects appear to reflect a distinct cell autonomous role of the ampA gene. Finally, we show that secretion of AmpA protein is brought about by elevating the levels of expression of ampA so that the protein accumulates to an excessive level.

  2. Mathematical modelling of non-stationary fluctuation analysis for studying channel properties of synaptic AMPA receptors.

    PubMed

    Benke, T A; Lüthi, A; Palmer, M J; Wikström, M A; Anderson, W W; Isaac, J T; Collingridge, G L

    2001-12-01

    1. The molecular properties of synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors are an important factor determining excitatory synaptic transmission in the brain. Changes in the number (N) or single-channel conductance (gamma) of functional AMPA receptors may underlie synaptic plasticity, such as long-term potentiation (LTP) and long-term depression (LTD). These parameters have been estimated using non-stationary fluctuation analysis (NSFA). 2. The validity of NSFA for studying the channel properties of synaptic AMPA receptors was assessed using a cable model with dendritic spines and a microscopic kinetic description of AMPA receptors. Electrotonic, geometric and kinetic parameters were altered in order to determine their effects on estimates of the underlying gamma. 3. Estimates of gamma were very sensitive to the access resistance of the recording (R(A)) and the mean open time of AMPA channels. Estimates of gamma were less sensitive to the distance between the electrode and the synaptic site, the electrotonic properties of dendritic structures, recording electrode capacitance and background noise. Estimates of gamma were insensitive to changes in spine morphology, synaptic glutamate concentration and the peak open probability (P(o)) of AMPA receptors. 4. The results obtained using the model agree with biological data, obtained from 91 dendritic recordings from rat CA1 pyramidal cells. A correlation analysis showed that R(A) resulted in a slowing of the decay time constant of excitatory postsynaptic currents (EPSCs) by approximately 150 %, from an estimated value of 3.1 ms. R(A) also greatly attenuated the absolute estimate of gamma by approximately 50-70 %. 5. When other parameters remain constant, the model demonstrates that NSFA of dendritic recordings can readily discriminate between changes in gamma vs. changes in N or P(o). Neither background noise nor asynchronous activation of multiple synapses prevented reliable

  3. Developmental changes in AMPA and kainate receptor-mediated quantal transmission at thalamocortical synapses in the barrel cortex.

    PubMed

    Bannister, Neil J; Benke, Timothy A; Mellor, Jack; Scott, Helen; Gürdal, Esra; Crabtree, John W; Isaac, John T R

    2005-05-25

    During the first week of life, there is a shift from kainate to AMPA receptor-mediated thalamocortical transmission in layer IV barrel cortex. However, the mechanisms underlying this change and the differential properties of AMPA and kainate receptor-mediated transmission remain essentially unexplored. To investigate this, we studied the quantal properties of AMPA and kainate receptor-mediated transmission using strontium-evoked miniature EPSCs. AMPA and kainate receptor-mediated transmission exhibited very different quantal properties but were never coactivated by a single quantum of transmitter, indicating complete segregation to different synapses within the thalamocortical input. Nonstationary fluctuation analysis showed that synaptic AMPA receptors exhibited a range of single-channel conductance (gamma) and a strong negative correlation between gamma and functional channel number, indicating that these two parameters are reciprocally regulated at thalamocortical synapses. We obtained the first estimate of gamma for synaptic kainate receptors (<2 pS), and this primarily accounted for the small quantal size of kainate receptor-mediated transmission. Developmentally, the quantal contribution to transmission of AMPA receptors increased and that of kainate receptors decreased. No changes in AMPA or kainate quantal amplitude or in AMPA receptor gamma were observed, demonstrating that the developmental change was attributable to a decrease in the number of kainate synapses and an increase in the number of AMPA synapses contributing to transmission. Therefore, we demonstrate fundamental differences in the quantal properties for these two types of synapse. Thus, the developmental switch in transmission will dramatically alter information transfer at thalamocortical inputs to layer IV.

  4. Griflola frondosa (GF) produces significant antidepressant effects involving AMPA receptor activation in mice.

    PubMed

    Bao, Hongkun; Ran, Pengzhan; Sun, Lijuan; Hu, Weihong; Li, Hongliang; Xiao, Chunjie; Zhu, Keming; Du, Jing

    2017-12-01

    Griflola frondosa (Fr) S.F. Gray (Meripilaceae) (GF) is a medical mushroom, and its regulation of the immune system is of interest for the treatment of mood disorders. α-Amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are the central mediator for the treatment of depression. This study examines the antidepressant effects of GF and the role of AMPA in these antidepressant effects. The CD-1 mice were fed with GF- or Pleurotus ostreatus [(Jacq.: Fr) Kumm (Pleurotaceae)] (PO)-containing food for 1 day or 5 days. The antidepressant effects was determined in the tail suspension test (TST), forced swim test (FST), and open field test (OFT). The involvement of AMPA receptors was determined by the application of the AMPA-specific blocker GYKI 52466. Treatments with 20%, 33% or 50% of GF-containing food significantly decreased the immobility time (63.6, 56.9, and 52.0% in TST; and 50.8, 43.2, and 38.2% in FST) after 1 day and (62.3, 51.8, and 52.8% in TST; and 49.5, 45.1, and 40.3% in FST) after 5 days. GF-containing food did not cause hyperactive effects in the OFT. The antidepressant effects of the 33% of GF-containing food (down-to 51.3% in 1-day TST and 46.8% in 5-day FST) were significantly stronger than that of the 33% of PO-containing food (down-to 85.5% in 1-day TST and 82.0% in 5-day FST). AMPA-specific blocker GYKI 52466 was able to block the antidepressant effects of the GF-containing food. GF demonstrated the potential as a safe medical food supplement for the patient with depression.

  5. The effects of AMPA blockade on the spectral profile of human early visual cortex recordings studied with non-invasive MEG.

    PubMed

    Muthukumaraswamy, Suresh D; Routley, Bethany; Droog, Wouter; Singh, Krish D; Hamandi, Khalid

    2016-08-01

    The generation of gamma-band (>30 Hz) cortical activity is thought to depend on the reciprocal connections of excitatory glutamatergic principal cells with inhibitory GABAergic interneurons. Both in vitro and in vivo animal studies have shown that blockade of glutamatergic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors reduces the amplitude of gamma-band activity. In this registered report, we hypothesised that similar effects would be observed in humans following administration of perampanel, a first in class AMPA antagonist, used in the treatment of epilepsy. In a single-blind placebo-controlled crossover study, 20 healthy male participants completed two study days. On one day participants were given a 6 mg dose of perampanel and on the other an inactive placebo. magnetoencephalography (MEG) recordings of brain activity were taken before and two hours after drug administration, with activity in the visual cortex probed using a stimulation protocol known to induce gamma-band activity in the primary visual cortex. As hypothesised, our results indicated a decrease in gamma-band amplitudes following perampanel administration. The decreases in gamma-band amplitudes observed were temporally restricted to the early time-period of stimulus presentation (up to 400 msec) with no significant effects observed on early evoked responses or alpha rhythms. This suggests that the early time-window of induced visual gamma-band activity, thought to reflect input to the visual cortex from the lateral geniculate nucleus, is most sensitive to AMPA blocking drugs.

  6. Reciprocal inhibition of the AMPA and NMDA components of excitatory postsynaptic potentials in field CA1 of the rat hippocampus in vitro.

    PubMed

    Bazhenov, A V; Kleshchevnikov, A M

    1999-01-01

    The mutual effects of components of excitatory postsynaptic potentials (EPSP) induced by activation of glutamate receptors sensitive to alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) were studied on living slices of rat hippocampus. Evoked responses were recorded in the radial layer (stratum radialis) in field CA1 after stimulation of collateral-commissural fibers. The contribution of the NMDA component to the total EPSP was altered by extracellular application of solutions containing different concentrations of magnesium. At low magnesium concentrations, when both components made significant contributions to EPSP, inhibition of one of the components by application of antagonists of the appropriate receptors led to increases in the area of the other component. Thus, the total magnitude of pharmacologically isolated components were significantly greater than the control response (for example, at 0.1 mM magnesium, the sum of the components was 340 +/- 120% of the control two-component EPSP (p < 0.01; N = 6). These results suggest that in controls, the AMPA and NMDA components of EPSP inhibit each other. The mutual inhibition of components may be an important factor affecting the conductivity and plastic properties of central glutamatergic synaptic pathways.

  7. The food intake-suppressive effects of glucagon-like peptide-1 receptor signaling in the ventral tegmental area are mediated by AMPA/kainate receptors

    PubMed Central

    Mietlicki-Baase, Elizabeth G.; Ortinski, Pavel I.; Rupprecht, Laura E.; Olivos, Diana R.; Alhadeff, Amber L.; Pierce, R. Christopher

    2013-01-01

    Glucagon-like peptide-1 receptor (GLP-1R) activation in the ventral tegmental area (VTA) is physiologically relevant for the control of palatable food intake. Here, we tested whether the food intake-suppressive effects of VTA GLP-1R activation are mediated by glutamatergic signaling within the VTA. Intra-VTA injections of the GLP-1R agonist exendin-4 (Ex-4) reduced palatable high-fat food intake in rats primarily by reducing meal size; these effects were mediated in part via glutamatergic AMPA/kainate but not NMDA receptor signaling. Additional behavioral data indicated that GLP-1R expressed specifically within the VTA can partially mediate the intake- and body weight-suppressive effects of systemically administered Ex-4, offering the intriguing possibility that this receptor population may be clinically relevant for food intake control. Intra-VTA Ex-4 rapidly increased tyrosine hydroxylase levels within the VTA, suggesting that GLP-1R activation modulates VTA dopaminergic signaling. Further evidence for this hypothesis was provided by electrophysiological data showing that Ex-4 increased the frequency of AMPA-mediated currents and reduced the paired/pulse ratio in VTA dopamine neurons. Together, these data provide novel mechanisms by which GLP-1R agonists in the mesolimbic reward system control for palatable food intake. PMID:24105414

  8. Variation in glyphosate and AMPA concentrations of surface water and groundwater

    NASA Astrophysics Data System (ADS)

    Caprile, Ana Clara; Aparicio, Virginia; Sasal, Carolina; Andriulo, Enrique

    2017-04-01

    The presence of pesticides in various environmental matrices indicate that the soil's ability to function as a bio-physical-chemical reactor is declining. As it operates as an interface between air and water, it causes a negative impact on these two vital resources. Currently, the pampa agriculture is simplified with a marked tendency towards spring-summer crops, where the main crops are RR soybean and corn. Herbicides are neither retained nor degraded in the soil, which results in polluted groundwater and surface waters. The objectives of this study were: a) to verify the presence of glyphosate and aminomethylphosphonic acid (AMPA) in Pergamino stream (a typical representative of the most productive agricultural region of Argentina) under different land use and to detect if in the detections there was a space-time pattern, and b) to verify the detection of these molecules in groundwater of the upper same basin under exclusively rural land use. Surface stream was sampling in six sites (five under rural land use and one under urban-industrial land use) at a rate of one sample by spring, summer and winter seasons (2010-2013, 54 total samples). Groundwater glyphosate and AMPA concentrations were determined in 24 piezometers constructed at two positions of the landscape, across the groundwater flow direction, sampled at two sampling dates (2010 and 2012, 45 total samples). In surface water, glyphosate and AMPA were detected in 54 and 69% of the samples analyzed, respectively. The median concentrations were 0.9 and 0.8 µg L-1 for glyphosate and AMPA and maximal concentrations 258 and 5865 µg L-1, respectively. The sampling site under urban-industrial land use had abnormally high concentrations of glyphosate in the spring (attributed to point pollution), a fact that not allowed to see differences in the remaining sampling times under different land uses. AMPA concentrations under urban-industrial land use were high and higher than rural land use in 3 studied seasons

  9. Potent and Selective Inhibition of the Open-Channel Conformation of AMPA Receptors by an RNA Aptamer†

    PubMed Central

    Huang, Zhen; Han, Yan; Wang, Congzhou; Niu, Li

    2010-01-01

    Inhibitors of AMPA receptors are useful as biochemical probes for structure-function studies and as drug candidates for a number of neurological disorders and diseases. Here we report the identification of an RNA inhibitor or aptamer by an in vitro evolution approach. Using a laser-pulse photolysis technique, we further characterized the mechanism of inhibition of this aptamer on the AMPA receptor channel-opening rate process in the microsecond-to-millisecond time domain. Our results show that the aptamer we isolated is a noncompetitive inhibitor that selectively inhibits the open-channel conformation of AMPA receptors with nanomolar affinity. The potency and the selectivity of this noncompetitive aptamer rival those of small molecule inhibitors. Our results therefore demonstrate the utility of this approach to develop water-soluble, highly potent and conformation-selective noncompetitive inhibitors of AMPA receptors. PMID:20518485

  10. Member of the Ampakine class of memory enhancers prolongs the single channel open time of reconstituted AMPA receptors.

    PubMed

    Suppiramaniam, V; Bahr, B A; Sinnarajah, S; Owens, K; Rogers, G; Yilma, S; Vodyanoy, V

    2001-05-01

    Ampakines are small benzamide compounds that allosterically produce the positive modulation of AMPA receptors and improve performance on a variety of behavioral tasks. To test if the native synaptic membrane is necessary for the effects of such positive modulators, the mechanism of action of the Ampakine 1-(1,3-benzodioxol-5-ylcarbonyl)-1,2,3,6-tetrahydropyridine (CX509) was investigated in isolated rat brain AMPA receptors reconstituted in lipid bilayers. The drug increased the open time of AMPA-induced single channel current fluctuations with an EC(50) of 4 microM. The action of CX509 was highly selective since it had no effect on the amplitude or close time of channel events. The open time effect had a maximum enhancement of 70-fold and the modulated currents were blocked by CNQX. It is concluded that the synaptic membrane environment is not necessary for Ampakine effects. In fact, CX509 was about 100 times more potent on the reconstituted AMPA receptors than on receptors in their native membrane. These findings indicate that centrally active Ampakines modulate specific kinetic properties of AMPA currents. They also raise the possibility that AMPA receptors are regulated by factors present in situ, thus explaining the more efficient modulatory effects of CX509 when acting on receptors removed from their synaptic location.

  11. Role of AMPA and NMDA receptors and back-propagating action potentials in spike timing-dependent plasticity.

    PubMed

    Fuenzalida, Marco; Fernández de Sevilla, David; Couve, Alejandro; Buño, Washington

    2010-01-01

    The cellular mechanisms that mediate spike timing-dependent plasticity (STDP) are largely unknown. We studied in vitro in CA1 pyramidal neurons the contribution of AMPA and N-methyl-d-aspartate (NMDA) components of Schaffer collateral (SC) excitatory postsynaptic potentials (EPSPs; EPSP(AMPA) and EPSP(NMDA)) and of the back-propagating action potential (BAP) to the long-term potentiation (LTP) induced by a STDP protocol that consisted in pairing an EPSP and a BAP. Transient blockade of EPSP(AMPA) with 7-nitro-2,3-dioxo-1,4-dihydroquinoxaline-6-carbonitrile (CNQX) during the STDP protocol prevented LTP. Contrastingly LTP was induced under transient inhibition of EPSP(AMPA) by combining SC stimulation, an imposed EPSP(AMPA)-like depolarization, and BAP or by coupling the EPSP(NMDA) evoked under sustained depolarization (approximately -40 mV) and BAP. In Mg(2+)-free solution EPSP(NMDA) and BAP also produced LTP. Suppression of EPSP(NMDA) or BAP always prevented LTP. Thus activation of NMDA receptors and BAPs are needed but not sufficient because AMPA receptor activation is also obligatory for STDP. However, a transient depolarization of another origin that unblocks NMDA receptors and a BAP may also trigger LTP.

  12. Abnormally increased surface expression of AMPA receptors in the cerebellum, cortex and striatum of Cln3(-/-) mice.

    PubMed

    Kovács, Attila D; Hof, Caitlin; Pearce, David A

    2015-10-21

    Mutations in the CLN3 gene cause a fatal neurodegenerative disorder, juvenile CLN3 disease. Exploring the cause of the motor coordination deficit in the Cln3(-/-) mouse model of the disease we have previously found that attenuation of AMPA receptor activity in 1-month-old Cln3(-/-) mice significantly improves their motor coordination [20]. To elucidate the mechanism of the abnormally increased AMPA receptor function in Cln3(-/-) mice, we examined the surface expression of AMPA receptors using surface cross-linking in brain slices from 1-month-old wild type (WT) and Cln3(-/-) mice. In surface cross-linked brain samples, Western blotting for AMPA receptor subunits revealed significantly increased surface levels of GluA1 and GluA2 in the cerebellum, and of GluA2 in the cortex and striatum of Cln3(-/-) mice as compared to WT mice. Expression levels of the GluA4 subunit were similar in the cerebellum of WT and Cln3(-/-) mice. While intracellular GluA1 levels in the WT and Cln3(-/-) cerebellum or cortex were similar, the intracellular expression of GluA1 in the Cln3(-/-) striatum was decreased to 56% of the WT level. Our results show a prominent increase in AMPA receptor surface expression in the brain of Cln3(-/-) mice and suggest that CLN3 is involved in the regulation of AMPA receptor surface expression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. Age-dependent requirement of AKAP150-anchored PKA and GluR2-lacking AMPA receptors in LTP

    PubMed Central

    Lu, Yuan; Allen, Margaret; Halt, Amy R; Weisenhaus, Michael; Dallapiazza, Robert F; Hall, Duane D; Usachev, Yuriy M; McKnight, G Stanley; Hell, Johannes W

    2007-01-01

    Association of PKA with the AMPA receptor GluR1 subunit via the A kinase anchor protein AKAP150 is crucial for GluR1 phosphorylation. Mutating the AKAP150 gene to specifically prevent PKA binding reduced PKA within postsynaptic densities (>70%). It abolished hippocampal LTP in 7–12 but not 4-week-old mice. Inhibitors of PKA and of GluR2-lacking AMPA receptors blocked single tetanus LTP in hippocampal slices of 8 but not 4-week-old WT mice. Inhibitors of GluR2-lacking AMPA receptors also prevented LTP in 2 but not 3-week-old mice. Other studies demonstrate that GluR1 homomeric AMPA receptors are the main GluR2-lacking AMPA receptors in adult hippocampus and require PKA for their functional postsynaptic expression during potentiation. AKAP150-anchored PKA might thus critically contribute to LTP in adult hippocampus in part by phosphorylating GluR1 to foster postsynaptic accumulation of homomeric GluR1 AMPA receptors during initial LTP in 8-week-old mice. PMID:17972919

  14. Myelin Proteolipid Protein Complexes with αv Integrin and AMPA Receptors In Vivo and Regulates AMPA-Dependent Oligodendrocyte Progenitor Cell Migration through the Modulation of Cell-Surface GluR2 Expression

    PubMed Central

    Harlow, Danielle E.; Saul, Katherine E.; Komuro, Hitoshi

    2015-01-01

    In previous studies, stimulation of ionotropic AMPA/kainate glutamate receptors on cultured oligodendrocyte cells induced the formation of a signaling complex that includes the AMPA receptor, integrins, calcium-binding proteins, and, surprisingly, the myelin proteolipid protein (PLP). AMPA stimulation of cultured oligodendrocyte progenitor cells (OPCs) also caused an increase in OPC migration. The current studies focused primarily on the formation of the PLP–αv integrin–AMPA receptor complex in vivo and whether complex formation impacts OPC migration in the brain. We found that in wild-type cerebellum, PLP associates with αv integrin and the calcium-impermeable GluR2 subunit of the AMPA receptor, but in mice lacking PLP, αv integrin did not associate with GluR2. Live imaging studies of OPC migration in ex vivo cerebellar slices demonstrated altered OPC migratory responses to neurotransmitter stimulation in the absence of PLP and GluR2 or when αv integrin levels were reduced. Chemotaxis assays of purified OPCs revealed that AMPA stimulation was neither attractive nor repulsive but clearly increased the migration rate of wild-type but not PLP null OPCs. AMPA receptor stimulation of wild-type OPCs caused decreased cell-surface expression of the GluR2 AMPA receptor subunit and increased intracellular Ca2+ signaling, whereas PLP null OPCs did not reduce GluR2 at the cell surface or increase Ca2+ signaling in response to AMPA treatment. Together, these studies demonstrate that PLP is critical for OPC responses to glutamate signaling and has important implications for OPC responses when levels of glutamate are high in the extracellular space, such as following demyelination. SIGNIFICANCE STATEMENT After demyelination, such as occurs in multiple sclerosis, remyelination of axons is often incomplete, leading to loss of neuronal function and clinical disability. Remyelination may fail because oligodendrocyte precursor cells (OPCs) do not completely migrate into

  15. Odor preference learning and memory modify GluA1 phosphorylation and GluA1 distribution in the neonate rat olfactory bulb: testing the AMPA receptor hypothesis in an appetitive learning model.

    PubMed

    Cui, Wen; Darby-King, Andrea; Grimes, Matthew T; Howland, John G; Wang, Yu Tian; McLean, John H; Harley, Carolyn W

    2011-01-01

    An increase in synaptic AMPA receptors is hypothesized to mediate learning and memory. AMPA receptor increases have been reported in aversive learning models, although it is not clear if they are seen with memory maintenance. Here we examine AMPA receptor changes in a cAMP/PKA/CREB-dependent appetitive learning model: odor preference learning in the neonate rat. Rat pups were given a single pairing of peppermint and 2 mg/kg isoproterenol, which produces a 24-h, but not a 48-h, peppermint preference in the 7-d-old rat pup. GluA1 PKA-dependent phosphorylation peaked 10 min after the 10-min training trial and returned to baseline within 90 min. At 24 h, GluA1 subunits did not change overall but were significantly increased in synaptoneurosomes, consistent with increased membrane insertion. Immunohistochemistry revealed a significant increase in GluA1 subunits in olfactory bulb glomeruli, the targets of olfactory nerve axons. Glomerular increases were seen at 3 and 24 h after odor exposure in trained pups, but not in control pups. GluA1 increases were not seen as early as 10 min after training and were no longer observed 48 h after training when odor preference is no longer expressed behaviorally. Thus, the pattern of increased GluA1 membrane expression closely follows the memory timeline. Further, blocking GluA1 insertion using an interference peptide derived from the carboxyl tail of the GluA1 subunit inhibited 24 h odor preference memory providing causative support for our hypothesis. PKA-mediated GluA1 phosphorylation and later GluA1 insertion could, conjointly, provide increased AMPA function to support both short-term and long-term appetitive memory.

  16. Reconsolidation of Reminder-Induced Amnesia: Role of NMDA and AMPA Glutamate Receptors.

    PubMed

    Nikitin, V P; Kozyrev, S A; Solntseva, S V

    2015-11-01

    We studied the role of glutamate receptors and reminder in the mechanisms of amnesia maintenance caused by disruption of conditioned food aversion reconsolidation with an antagonist of NMDA glutamate receptor in snails. At the early stage of amnesia (day 3 after induction), injection or NMDA of AMPA glutamate receptor antagonists prior to reminder (presentation of the conditioned food stimulus) led to memory recovery. Reminder alone or injection of antagonists without reminder or after reminder was ineffective. At the late stage of amnesia (day 10), antagonists/reminder had no effect on amnesia maintenance. It was hypothesized that reminder at the early stage of amnesia led to reactivation and reconsolidation of the molecular processes of amnesia including activation NMDA and AMPA glutamate receptors. Injection of antagonists of these receptors prior to reminder led to disruption of reactivation/reconsolidation of amnesia and recovery of the conditioned food aversion memory.

  17. Ionotropic AMPA-type glutamate and metabotropic GABAB receptors: determining cellular physiology by proteomes.

    PubMed

    Bettler, Bernhard; Fakler, Bernd

    2017-03-07

    Ionotropic AMPA-type glutamate receptors and G-protein-coupled metabotropic GABAB receptors are key elements of neurotransmission whose cellular functions are determined by their protein constituents. Over the past couple of years unbiased proteomic approaches identified comprehensive sets of protein building blocks of these two types of neurotransmitter receptors in the brain (termed receptor proteomes). This provided the opportunity to match receptor proteomes with receptor physiology and to study the structural organization, regulation and function of native receptor complexes in an unprecedented manner. In this review we discuss the principles of receptor architecture and regulation emerging from the functional characterization of the proteomes of AMPA and GABAB receptors. We also highlight progress in unraveling the role of unexpected protein components for receptor physiology.

  18. Habitual responding for alcohol depends upon both AMPA and D2 receptor signaling in the dorsolateral striatum

    PubMed Central

    Corbit, Laura H.; Nie, Hong; Janak, Patricia H.

    2014-01-01

    Chronic alcohol self-administration leads to alcohol-seeking behavior that is habitual and insensitive to changes in the value of the earned alcohol. Such behavior has been shown to rely on the dorsolateral region of the striatum in rats but the specific pharmacological control of output from this region is not yet understood. In the following experiments rats were trained to self-administer unsweetened 10% (v/v) ethanol in daily sessions for 8 weeks prior to testing for sensitivity to outcome devaluation. We examined the role of glutamatergic AMPA-receptor activation by testing the effects of the antagonist NBQX (0.3 and 1.0 μg/μl) infused specifically into the dorsolateral striatum (DLS) before devaluation testing. In a separate group of rats we examined the role of dopaminergic D2-receptor activation using the D2-receptor antagonist raclopride (0.2 and 1.0 μg/μl) infused into the DLS before devaluation testing. Following control (saline) infusions rats’ lever-press performance was insensitive to devaluation of ethanol thus showing evidence of habitual responding. NBQX and racolpride each restored goal-directed control of responding at doses that did not impair overall lever-press rates. These data demonstrate that expression of habitual alcohol seeking relies on glutamatergic inputs to the DLS and D2 receptors within the DLS. PMID:25228865

  19. EM colocalization of AMPA and NMDA receptor subunits at synapses in rat cerebral cortex.

    PubMed

    Kharazia, V N; Phend, K D; Rustioni, A; Weinberg, R J

    1996-05-24

    Electrophysiology and light microscopy suggest that a single excitatory synapse may use both amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors. Using immunogold electron microscopy, we here provide direct evidence for colocalization at individual synapses in sensorimotor cortex of adult rats. Colocalization was most commonly observed on dendritic spines; subunits of the two classes of receptors seemed to be independently distributed within the synaptic active zone.

  20. Glutamate receptors on myelinated spinal cord axons: II)AMPA and GluR5 receptors

    PubMed Central

    Ouardouz, M.; Coderre, E.; Zamponi, G. W.; Hameed, S.; Yin, X.; Trapp, B.D.; Stys, P.K.

    2010-01-01

    Objective Glutamate receptors, which play a major role in the physiology and pathology of CNS gray matter, are also involved in the pathophysiology of white matter. However the cellular and molecular mechanisms responsible for excitotoxic damage to white matter elements are not fully understood. We explored the roles of AMPA and GluR5 kainate receptors in axonal Ca2+ deregulation. Methods Dorsal column axons were loaded with a Ca2+ indicator and imaged in vitro using confocal microscopy. Results Both AMPA and a GluR5 kainate receptor agonists increased intra-axonal Ca2+ in myelinated rat dorsal column fibers. These responses were inhibited by selective antagonists of these glutamate receptors. The GluR5-mediated Ca2+ rise was mediated by both canonical (i.e. ionotropic) and non-canonical (metabotropic) signalling, dependent on a pertussis toxin-sensitive G protein and a phospholipase C-dependent pathway, promoting Ca2+ release from IP3-dependent stores. Additionally, the GluR5 response was significantly reduced by intra-axonal NO scavengers. In contrast, GluR4 AMPA receptors operated via Ca2+ induced Ca2+ release, dependent on ryanodine receptors, and unaffected by NO scavengers. Neither pathway depended on L-type Ca2+ channels, in contrast to GlurR6 kainate receptor action 1. Immunohistochemistry confirmed the presence of GluR4 and GluR5 clustered at the surface of myelinated axons; GluR5 co-immunoprecipitated with nNOS and often co-localized with nNOS clusters on the internodal axon. Interpretation Central myelinated axons express functional AMPA and GluR5 kainate receptors, and can directly respond to glutamate receptor agonists. These glutamate receptor-dependent signalling pathways promote an increase in intra-axonal Ca2+ levels potentially contributing to axonal degeneration. PMID:19224531

  1. Differential trafficking of AMPA receptors following activation of NMDA receptors and mGluRs.

    PubMed

    Sanderson, Thomas M; Collingridge, Graham L; Fitzjohn, Stephen M

    2011-07-27

    The removal of AMPA receptors from synapses is a major component of long-term depression (LTD). How this occurs, however, is still only partially understood. To investigate the trafficking of AMPA receptors in real-time we previously tagged the GluA2 subunit of AMPA receptors with ecliptic pHluorin and studied the effects of NMDA receptor activation. In the present study we have compared the effect of NMDA receptor and group I mGluR activation, using GluA2 tagged with super ecliptic pHluorin (SEP-GluA2) expressed in cultured hippocampal neurons. Surprisingly, agonists of the two receptors, which are both able to induce chemical forms of LTD, had clearly distinct effects on AMPA receptor trafficking. In agreement with our previous work we found that transient NMDA receptor activation results in an initial decrease in surface GluA2 from extrasynaptic sites followed by a delayed reduction in GluA2 from puncta (putative synapses). In contrast, transient activation of group I mGluRs, using DHPG, led to a pronounced but more delayed decrease in GluA2 from the dendritic shafts. Surprisingly, there was no average change in the fluorescence of the puncta. Examination of fluorescence at individual puncta, however, indicated that alterations did take place, with some puncta showing an increase and others a decrease in fluorescence. The effects of DHPG were, like DHPG-induced LTD, prevented by treatment with a protein tyrosine phosphatase (PTP) inhibitor. The electrophysiological correlate of the effects of DHPG in the SEP-GluA2 infected cultures was a reduction in mEPSC frequency with no change in amplitude. The implications of these findings for the initial mechanisms of expression of both NMDA receptor- and mGluR-induced LTD are discussed.

  2. Discovery and Characterization of AMPA Receptor Modulators Selective for TARP-γ8.

    PubMed

    Maher, Michael P; Wu, Nyantsz; Ravula, Suchitra; Ameriks, Michael K; Savall, Brad M; Liu, Changlu; Lord, Brian; Wyatt, Ryan M; Matta, Jose A; Dugovic, Christine; Yun, Sujin; Ver Donck, Luc; Steckler, Thomas; Wickenden, Alan D; Carruthers, Nicholas I; Lovenberg, Timothy W

    2016-05-01

    Members of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediate the majority of fast synaptic transmission within the mammalian brain and spinal cord, representing attractive targets for therapeutic intervention. Here, we describe novel AMPA receptor modulators that require the presence of the accessory protein CACNG8, also known as transmembrane AMPA receptor regulatory protein γ8 (TARP-γ8). Using calcium flux, radioligand binding, and electrophysiological assays of wild-type and mutant forms of TARP-γ8, we demonstrate that these compounds possess a novel mechanism of action consistent with a partial disruption of the interaction between the TARP and the pore-forming subunit of the channel. One of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118), had excellent pharmacokinetic properties and achieved high receptor occupancy following oral administration. This molecule showed strong, dose-dependent inhibition of neurotransmission within the hippocampus, and a strong anticonvulsant effect. At high levels of receptor occupancy in rodent in vivo models, JNJ-55511118 showed a strong reduction in certain bands on electroencephalogram, transient hyperlocomotion, no motor impairment on rotarod, and a mild impairment in learning and memory. JNJ-55511118 is a novel tool for reversible AMPA receptor inhibition, particularly within the hippocampus, with potential therapeutic utility as an anticonvulsant or neuroprotectant. The existence of a molecule with this mechanism of action demonstrates the possibility of pharmacological targeting of accessory proteins, increasing the potential number of druggable targets.

  3. Distribution of transmembrane AMPA receptor regulatory protein (TARP) isoforms in the rat spinal cord.

    PubMed

    Larsson, M; Agalave, N; Watanabe, M; Svensson, C I

    2013-09-17

    The transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor regulatory proteins (TARPs) are a family of auxiliary AMPA receptor subunits that differentially modulate trafficking and many functional properties of the receptor. To investigate which TARP isoforms may be involved in AMPA receptor-mediated spinal synaptic transmission, we have mapped the localization of five of the known TARP isoforms, namely γ-2 (also known as stargazin), γ-3, γ-4, γ-7 and γ-8, in the rat spinal cord. Immunoblotting showed expression of all isoforms in the spinal cord to varying degrees. At the light microscopic level, immunoperoxidase labeling of γ-4, γ-7 and γ-8 was found throughout spinal gray matter. In white matter, γ-4 and γ-7 immunolabeling was observed in astrocytic processes and in mature oligodendrocytes. In pepsin-treated spinal cord, γ-7 often colocalized with GluA2 immunopositive puncta in the deep dorsal horn as well as in the ventral horn, but not in the superficial dorsal horn. Postembedding immunogold labeling was further used to assess the synaptic localization of γ-2, γ-7 and γ-8 in the dorsal horn. Synaptic immunogold labeling of γ-2 was sparse throughout the dorsal horn, with some primary afferent synapses weakly labeled, whereas relatively strong γ-7 immunogold labeling was found at deep dorsal horn synapses, including at synapses formed by low-threshold mechanosensitive primary afferent terminals. Prominent immunogold labeling of γ-8 was frequently detected at synapses established by primary afferent fibers. The spinal localization patterns of TARP isoforms reported here suggest that AMPA receptors at spinal synaptic populations and in glial cells may exhibit different functional characteristics owing to differences in auxiliary subunit composition.

  4. Precision therapy for a new disorder of AMPA receptor recycling due to mutations in ATAD1

    PubMed Central

    Ahrens-Nicklas, Rebecca C.; Umanah, George K.E.; Sondheimer, Neal; Deardorff, Matthew A.; Wilkens, Alisha B.; Conlin, Laura K.; Santani, Avni B.; Nesbitt, Addie; Juulsola, Jane; Ma, Erica; Dawson, Ted M.; Dawson, Valina L.

    2017-01-01

    Objective: ATAD1 encodes Thorase, a mediator of α-amino-3-hydroxy-5-methylisoxazole-4-proprionate (AMPA) receptor recycling; in this work, we characterized the phenotype resulting from ATAD1 mutations and developed a targeted therapy in both mice and humans. Methods: Using exome sequencing, we identified a novel ATAD1 mutation (p.E276X) as the etiology of a devastating neurologic disorder characterized by hypertonia, seizures, and death in a consanguineous family. We postulated that pathogenesis was a result of excessive AMPA receptor activity and designed a targeted therapeutic approach using perampanel, an AMPA-receptor antagonist. Results: Perampanel therapy in ATAD1 knockout mice reversed behavioral defects, normalized brain MRI abnormalities, prevented seizures, and prolonged survival. The ATAD1 patients treated with perampanel showed improvement in hypertonicity and resolution of seizures. Conclusions: This work demonstrates that identification of novel monogenic neurologic disorders and observation of response to targeted therapeutics can provide important insights into human nervous system functioning. PMID:28180185

  5. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives

    PubMed Central

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn

    2013-01-01

    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r2 = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders. PMID:24250113

  6. Inhibition of the NMDA and AMPA receptor channels by antidepressants and antipsychotics.

    PubMed

    Barygin, Oleg I; Nagaeva, Elina I; Tikhonov, Denis B; Belinskaya, Darya A; Vanchakova, Nina P; Shestakova, Natalia N

    2017-04-01

    It is known that some antidepressants and antipsychotics directly inhibit NMDA-type ionotropic glutamate receptors. In this study we systematically studied action of seven drugs (Fluoxetine, Citalopram, Desipramine, Amitriptyline, Atomoxetine, Chlorpromazine, and Clozapine) on NMDA receptors and Ca(2+)-permeable and -impermeable AMPA receptors in rat brain neurons by whole-cell patch-clamp technique. Except for weak effect of fluoxetine, all drugs were virtually inactive against Ca(2+)-impermeable AMPA receptors. Fluoxetine and desipramine significantly inhibited Ca(2+)-permeable AMPA receptors (IC50=43±7 and 105±12µM, respectively). Desipramine, atomoxetine and chlorpromazine inhibited NMDA receptors in clinically relevant low micromolar concentrations, while citalopram had only weak effect. All tested medicines have been clustered into two groups by their action on NMDA receptors: desipramine, amitriptyline, chlorpromazine, and atomoxetine display voltage- and magnesium-dependent open channel blocking mechanism. Action of fluoxetine and clozapine was found to be voltage- and magnesium-independent. All voltage-dependent compounds could be trapped in closed NMDA receptor channels. Possible contribution of NMDA receptor inhibition by certain antidepressants and antipsychotics to their analgesic effects in neuropathic pain is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. The AMPA modulator S 18986 improves declarative and working memory performances in aged mice.

    PubMed

    Marighetto, Aline; Valerio, Stephane; Jaffard, Robert; Mormede, Cecile; Muñoz, Carmen; Bernard, Katy; Morain, Philippe

    2008-05-01

    The aim of this study was to further characterize the memory-enhancing profile of S 18986 a positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. S 18986 was studied in two mouse models of age-related memory deficits, using radial maze paradigms involving long-term/declarative memory and short-term/working memory. Aged mice exhibited severe deficits when compared with their younger counterparts in the two behavioural tests. S 18986 at the dose of 0.1 mg/kg selectively improved aged mouse performance in the test of long-term/declarative memory flexibility and exerted a beneficial effect on short-term retention of successive arm-visits in the short-term/working memory test. This study confirms the memory-enhancing properties of S 18986 and, in line with emerging data on multiple AMPA modulators, highlights the relevance of targeting AMPA receptors in the development of new memory enhancers.

  8. Synergy of AMPA and NMDA Receptor Currents in Dopaminergic Neurons: A Modeling Study

    PubMed Central

    Zakharov, Denis; Lapish, Christopher; Gutkin, Boris; Kuznetsov, Alexey

    2016-01-01

    Dopaminergic (DA) neurons display two modes of firing: low-frequency tonic and high-frequency bursts. The high frequency firing within the bursts is attributed to NMDA, but not AMPA receptor activation. In our models of the DA neuron, both biophysical and abstract, the NMDA receptor current can significantly increase their firing frequency, whereas the AMPA receptor current is not able to evoke high-frequency activity and usually suppresses firing. However, both currents are produced by glutamate receptors and, consequently, are often co-activated. Here we consider combined influence of AMPA and NMDA synaptic input in the models of the DA neuron. Different types of neuronal activity (resting state, low frequency, or high frequency firing) are observed depending on the conductance of the AMPAR and NMDAR currents. In two models, biophysical and reduced, we show that the firing frequency increases more effectively if both receptors are co-activated for certain parameter values. In particular, in the more quantitative biophysical model, the maximal frequency is 40% greater than that with NMDAR alone. The dynamical mechanism of such frequency growth is explained in the framework of phase space evolution using the reduced model. In short, both the AMPAR and NMDAR currents flatten the voltage nullcline, providing the frequency increase, whereas only NMDA prevents complete unfolding of the nullcline, providing robust firing. Thus, we confirm a major role of the NMDAR in generating high-frequency firing and conclude that AMPAR activation further significantly increases the frequency. PMID:27252643

  9. Memory retrieval requires ongoing protein synthesis and NMDA receptor activity-mediated AMPA receptor trafficking.

    PubMed

    Lopez, Joëlle; Gamache, Karine; Schneider, Rilla; Nader, Karim

    2015-02-11

    Whereas consolidation and reconsolidation are considered dynamic processes requiring protein synthesis, memory retrieval has long been considered a passive readout of previously established plasticity. However, previous findings suggest that memory retrieval may be more dynamic than previously thought. This study therefore aimed at investigating the molecular mechanisms underlying memory retrieval in the rat. Infusion of protein synthesis inhibitors (rapamycin or anisomycin) in the amygdala 10 min before memory retrieval transiently impaired auditory fear memory expression, suggesting ongoing protein synthesis is required to enable memory retrieval. We then investigated the role of protein synthesis in NMDA receptor activity-mediated AMPA receptor trafficking. Coinfusion of an NMDA receptor antagonist (ifenprodil) or infusion of an AMPA receptor endocytosis inhibitor (GluA23Y) before rapamycin prevented this memory impairment. Furthermore, rapamycin transiently decreased GluA1 levels at the postsynaptic density (PSD), but did not affect extrasynaptic sites. This effect at the PSD was prevented by an infusion of GluA23Y before rapamycin. Together, these data show that ongoing protein synthesis is required before memory retrieval is engaged, and suggest that this protein synthesis may be involved in the NMDAR activity-mediated trafficking of AMPA receptors that takes place during memory retrieval.

  10. X-ray structures of AMPA receptor-cone snail toxin complexes illuminate activation mechanism.

    PubMed

    Chen, Lei; Dürr, Katharina L; Gouaux, Eric

    2014-08-29

    AMPA-sensitive glutamate receptors are crucial to the structural and dynamic properties of the brain, to the development and function of the central nervous system, and to the treatment of neurological conditions from depression to cognitive impairment. However, the molecular principles underlying AMPA receptor activation have remained elusive. We determined multiple x-ray crystal structures of the GluA2 AMPA receptor in complex with a Conus striatus cone snail toxin, a positive allosteric modulator, and orthosteric agonists, at 3.8 to 4.1 angstrom resolution. We show how the toxin acts like a straightjacket on the ligand-binding domain (LBD) "gating ring," restraining the domains via both intra- and interdimer cross-links such that agonist-induced closure of the LBD "clamshells" is transduced into an irislike expansion of the gating ring. By structural analysis of activation-enhancing mutants, we show how the expansion of the LBD gating ring results in pulling forces on the M3 helices that, in turn, are coupled to ion channel gating.

  11. The Influence of Synaptic Size on AMPA Receptor Activation: A Monte Carlo Model

    PubMed Central

    Montes, Jesus; Peña, Jose M.; DeFelipe, Javier; Herreras, Oscar; Merchan-Perez, Angel

    2015-01-01

    Physiological and electron microscope studies have shown that synapses are functionally and morphologically heterogeneous and that variations in size of synaptic junctions are related to characteristics such as release probability and density of postsynaptic AMPA receptors. The present article focuses on how these morphological variations impact synaptic transmission. We based our study on Monte Carlo computational simulations of simplified model synapses whose morphological features have been extracted from hundreds of actual synaptic junctions reconstructed by three-dimensional electron microscopy. We have examined the effects that parameters such as synaptic size or density of AMPA receptors have on the number of receptors that open after release of a single synaptic vesicle. Our results indicate that the maximum number of receptors that will open after the release of a single synaptic vesicle may show a ten-fold variation in the whole population of synapses. When individual synapses are considered, there is also a stochastical variability that is maximal in small synapses with low numbers of receptors. The number of postsynaptic receptors and the size of the synaptic junction are the most influential parameters, while the packing density of receptors or the concentration of extrasynaptic transporters have little or no influence on the opening of AMPA receptors. PMID:26107874

  12. AMPA receptor trafficking and synaptic plasticity require SQSTM1/p62.

    PubMed

    Jiang, Jianxiong; Parameshwaran, Kodeeswaran; Seibenhener, M Lamar; Kang, Myoung-Goo; Suppiramaniam, Vishnu; Huganir, Richard L; Diaz-Meco, Maria T; Wooten, Marie W

    2009-04-01

    SQSTM1/p62 is a multidomain/scaffold for the atypical protein kinase Cs (aPKC). Phosphorylation of AMPA receptors by PKC has been shown to regulate their insertion in the postsynaptic membrane. Here, we directly tested whether p62 could interact with AMPA receptor subunits and influence their trafficking and phosphorylation. GluR1 receptor intracellular loop L2-3 and the ZZ-type zinc finger domain of p62 are essential for the interaction between these two proteins. In this context, both p62 and aPKC-mediated phosphorylation were necessary for surface delivery of the receptor. Our findings reveal that p62 is the first protein identified that interacts with a region of the GluR receptor other than the C-terminal tail. Furthermore, mice deficient in p62 displayed impaired hippocampal CA1 long-term potentiation (LTP), along with diminished surface expression of GluR1 and phosphorylation of S818. Lastly, we identify a conserved sequence (ISExSL) shared by all p62 interacting-aPKC substrates. These findings support a model where p62 interaction and aPKC phosphorylation act together to mediate AMPA receptor trafficking and long-term synaptic plasticity in the hippocampus.

  13. The influence of synaptic size on AMPA receptor activation: a Monte Carlo model.

    PubMed

    Montes, Jesus; Peña, Jose M; DeFelipe, Javier; Herreras, Oscar; Merchan-Perez, Angel

    2015-01-01

    Physiological and electron microscope studies have shown that synapses are functionally and morphologically heterogeneous and that variations in size of synaptic junctions are related to characteristics such as release probability and density of postsynaptic AMPA receptors. The present article focuses on how these morphological variations impact synaptic transmission. We based our study on Monte Carlo computational simulations of simplified model synapses whose morphological features have been extracted from hundreds of actual synaptic junctions reconstructed by three-dimensional electron microscopy. We have examined the effects that parameters such as synaptic size or density of AMPA receptors have on the number of receptors that open after release of a single synaptic vesicle. Our results indicate that the maximum number of receptors that will open after the release of a single synaptic vesicle may show a ten-fold variation in the whole population of synapses. When individual synapses are considered, there is also a stochastical variability that is maximal in small synapses with low numbers of receptors. The number of postsynaptic receptors and the size of the synaptic junction are the most influential parameters, while the packing density of receptors or the concentration of extrasynaptic transporters have little or no influence on the opening of AMPA receptors.

  14. FUNCTIONAL INSIGHT INTO DEVELOPMENT OF POSITIVE ALLOSTERIC MODULATORS OF AMPA RECEPTORS

    PubMed Central

    Weeks, Autumn M.; Harms, Jonathan E.; Partin, Kathryn M.; Benveniste, Morris

    2014-01-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five “subsites” which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites “A” and “B” of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds. PMID:24878241

  15. Functional insight into development of positive allosteric modulators of AMPA receptors.

    PubMed

    Weeks, Autumn M; Harms, Jonathan E; Partin, Kathryn M; Benveniste, Morris

    2014-10-01

    Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five "subsites" which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites "A" and "B" of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds.

  16. in Silico investigation of the structural requirements for the AMPA receptor antagonism by quinoxaline derivatives.

    PubMed

    Azam, Faizul; Abugrain, Ismaiel Mohamed; Sanalla, Mohamed Hussin; Elnaas, Radwan Fatahalla; Rajab, Ibrahim Abdassalam Ibn

    2013-01-01

    Glutamate receptors have been implicated in various neurological disorders and their antagonism offers a suitable approach for the treatment of such disorders. The field of drug design and discovery aims to find best medicines to prevent, treat and cure diseases quickly and efficiently. In this regard, computational tools have helped medicinal chemists modify and optimize molecules to potent drug candidates with better pharmacokinetic profiles, and guiding biologists and pharmacologists to explore new disease genes as well as novel drug targets. In the present study, to understand the structural requirements for AMPA receptor antagonism, molecular docking study was performed on 41 structurally diverse antagonists based on quinoxaline nucleus. Lamarckian genetic algorithm methodology was employed for docking simulations using AutoDock 4.2 program. The results obtained signify that the molecular docking approach is reliable and produces a good correlation coefficient (r(2) = 0.6) between experimental and docking predicted AMPA receptor antagonistic activity. The aromatic moiety of quinoxaline core has been proved to be vital for hydrophobic contacts exhibiting - interactions in docked conformations. However, polar moieties such as carboxylic group and 1,2,4-triazole moieties were noted to be sites for hydrophilic interactions in terms of hydrogen bonding with the receptor. These analyses can be exploited to design and develop novel AMPA receptor antagonists for the treatment of different neurological disorders.

  17. Ca(2+) -permeable AMPA receptors associated with epileptogenesis of hypothalamic hamartoma.

    PubMed

    Kitaura, Hiroki; Sonoda, Masaki; Teramoto, Sayaka; Shirozu, Hiroshi; Shimizu, Hiroshi; Kimura, Tadashi; Masuda, Hiroshi; Ito, Yosuke; Takahashi, Hitoshi; Kwak, Shin; Kameyama, Shigeki; Kakita, Akiyoshi

    2017-04-01

    Hypothalamic hamartoma (HH), composed of neurons and glia without apparent cytologic abnormalities, is a rare developmental malformation in humans. Patients with HH often have characteristic medically refractory gelastic seizures, and intrinsic epileptogenesis within the lesions has been speculated. Herein we provide evidence to suggest that in HH neurons, Ca(2+) permeability through α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors is aberrantly elevated. In needle biopsy specimens of HH tissue, field potential recordings demonstrated spontaneous epileptiform activities similar to those observed in other etiologically distinct epileptogenic tissues. In HH, however, these activities were clearly abolished by application of Joro Spider Toxin (JSTX), a specific inhibitor of the Ca(2+) -permeable AMPA receptor. Consistent with these physiologic findings, the neuronal nuclei showed disappearance of adenosine deaminase acting on RNA 2 (ADAR2) immunoreactivity. Furthermore, examination of glutamate receptor 2 (GluA2) messenger RNA (mRNA) revealed that editing efficiency at the glutamine/arginine site was significantly low. These results suggest that neurons in HH may bear Ca(2+) -permeable AMPA receptors due to dislocation of ADAR2.

  18. Influence of AMPA/kainate receptors on extracellular 5-hydroxytryptamine in rat midbrain raphe and forebrain

    PubMed Central

    Tao, Rui; Ma, Zhiyuan; Auerbach, Sidney B

    1997-01-01

    The regulation of 5-hydroxytryptamine (5-HT) release by excitatory amino acid (EAA) receptors was examined by use of microdialysis in the CNS of freely behaving rats. Extracellular 5-HT was measured in the dorsal raphe nucleus (DRN), median raphe nucleus (MRN), nucleus accumbens, hypothalamus, frontal cortex, dorsal and ventral hippocampus. Local infusion of kainate produced increases in extracellular 5-HT in the DRN and MRN. Kainate infusion into forebrain sites had a less potent effect. In further studies of the DRN and nucleus accumbens, kainate-induced increases in extracellular 5-HT were blocked by the EAA receptor antagonists, kynurenate and 6,7-dinitroquinoxaline-2,3-dione (DNQX). The effect of infusing kainate into the DRN or nucleus accumbens was attenuated or abolished by tetrodotoxin (TTX), suggesting that the increase in extracellular 5-HT is dependent on 5-HT neuronal activity. In contrast, ibotenate-induced lesion of intrinsic neurones did not attenuate the effect of infusing kainate into the nucleus accumbens. Thus, the effect of kainate in the nucleus accumbens does not depend on intrinsic neurones. Infusion of α-amino-3-hydroxy-5-methyl-4-isoxazolaproprionate (AMPA) into the DRN and nucleus accumbens induced nonsignificant changes in extracellular 5-HT. Cyclothiazide and diazoxide, which attenuate receptor desensitization, greatly enhanced the effect of AMPA on 5-HT in the DRN, but not in the nucleus accumbens. In conclusion, AMPA/kainate receptors regulate 5-HT in the raphe and in forebrain sites. PMID:9283707

  19. Synaptic Consolidation Normalizes AMPAR Quantal Size following MAGUK Loss.

    PubMed

    Levy, Jonathan M; Chen, Xiaobing; Reese, Thomas S; Nicoll, Roger A

    2015-08-05

    The mechanisms controlling synapse growth and maintenance are of critical importance for learning and memory. The MAGUK family of synaptic scaffolding proteins is abundantly expressed at glutamatergic central synapses, but their importance in controlling the synaptic content of glutamate receptors is poorly understood. Here, we use a chained RNAi-mediated knockdown approach to simultaneously remove PSD-93, PSD-95, and SAP102, the MAGUKs previously shown to be responsible for synaptic local