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• W1996198347 abstract "•The NR2B subunit of the NMDA receptor is phosphorylated at Ser1116 by Cdk5•Synaptic plasticity and memory formation alter NR2B Ser1116 phosphorylation•Disruption of NR2B-Cdk5 complex increases surface NR2B and synaptic transmission•Hippocampal infusion of NR2B-Cdk5 small interfering peptide improves memory Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers because of its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when overexpressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor’s cell surface expression. Disrupting NR2B-Cdk5 interaction via a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers.Video AbstracteyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJhMWEwN2UxODlmMWU5Yjc5ZmM0YjNiZDQ1ZDdhOWY0MCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4OTA4ODEyfQ.jp5loLuU1bt7TXajiAlzhgcesb2ZFBbi-9ccgITbPMcspbEnGmcYtwyaWrfa1PAxF7kG42mhbXiVYyjBgoWsuP6Tz-8jHYI9okH6q6at4ys2QEwNHZeZwqYrvMU7NmaUg7sxMj8y7PaRL9nniRrtGNjLOX3EJeWjvbD1bpTZZtNZVLwQumCTapxwrhleg2ay3jQpy-zq6rslIuydPeFi1UzKMAXzf5dbDSYrexBlRtU6I4i7P4MGRkS8qrkbsl_qLyImg-sn3n4crMPQbsX1QtBmCO2cQZpfX9cWwxwjeEf7na-Y1KvPtOSV3UvgOk7lDvw650e3zxIOIgw5smsqng(mp4, (50.8 MB) Download video Many psychiatric and neurological disorders are characterized by learning and memory deficits, for which cognitive enhancement is considered a valid treatment strategy. The N-methyl-D-aspartate receptor (NMDAR) is a prime target for the development of cognitive enhancers because of its fundamental role in learning and memory. In particular, the NMDAR subunit NR2B improves synaptic plasticity and memory when overexpressed in neurons. However, NR2B regulation is not well understood and no therapies potentiating NMDAR function have been developed. Here, we show that serine 1116 of NR2B is phosphorylated by cyclin-dependent kinase 5 (Cdk5). Cdk5-dependent NR2B phosphorylation is regulated by neuronal activity and controls the receptor’s cell surface expression. Disrupting NR2B-Cdk5 interaction via a small interfering peptide (siP) increases NR2B surface levels, facilitates synaptic transmission, and improves memory formation in vivo. Our results reveal a regulatory mechanism critical to NR2B function that can be targeted for the development of cognitive enhancers. N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that exhibit broad expression within the nervous system and are critically involved in neuronal processes such as synaptic plasticity and learning and memory (Bliss and Collingridge, 1993Bliss T.V. Collingridge G.L. A synaptic model of memory: long-term potentiation in the hippocampus.Nature. 1993; 361: 31-39Crossref PubMed Scopus (9558) Google Scholar, Martin et al., 2000Martin S.J. Grimwood P.D. Morris R.G. Synaptic plasticity and memory: an evaluation of the hypothesis.Annu. Rev. Neurosci. 2000; 23: 649-711Crossref PubMed Scopus (2084) Google Scholar). Functional NMDARs are obligate heterotetrameric complexes formed by two glycine-binding GluN1 subunits assembled with two of several isoforms of the glutamate-binding GluN2 (A, B, C, or D subtypes) or glycine-binding GluN3 (A or B subtypes) (Cull-Candy and Leszkiewicz, 2004Cull-Candy S.G. Leszkiewicz D.N. Role of distinct NMDA receptor subtypes at central synapses.Sci. STKE. 2004; 2004: re16Crossref PubMed Scopus (605) Google Scholar, Dingledine et al., 1999Dingledine R. Borges K. Bowie D. Traynelis S.F. The glutamate receptor ion channels.Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). The biophysical properties of NMDARs are dependent on their subunit composition (Morrisett, 1997Morrisett R. Electrophysiologic characteristics of heteromeric recombinant NMDA receptors.in: Monaghan D.T. Wenthold R.J. The Ionotropic Glutamate Receptors. Humana Press, Totowa1997Crossref Google Scholar). Indeed, NMDAR subunit composition varies greatly between different synapses and neurons, as well as during neuronal development (Monyer et al., 1994Monyer H. Burnashev N. Laurie D.J. Sakmann B. Seeburg P.H. Developmental and regional expression in the rat brain and functional properties of four NMDA receptors.Neuron. 1994; 12: 529-540Abstract Full Text PDF PubMed Scopus (2877) Google Scholar, Sheng et al., 1994Sheng M. Cummings J. Roldan L.A. Jan Y.N. Jan L.Y. Changing subunit composition of heteromeric NMDA receptors during development of rat cortex.Nature. 1994; 368: 144-147Crossref PubMed Scopus (1108) Google Scholar). Therefore, precise spatiotemporal regulation of NMDAR subunit expression, composition, trafficking, and localization is critical for proper neuronal function. Brain regions involved in mnemonic functions rely predominantly upon GluN2A (NR2A) and GluN2B (NR2B) subunit-containing NMDARs. During postnatal development, NR2B expression steadily decreases, whereas NR2A levels rise. Compared to NR2A-containing NMDARs, receptors that include NR2B inactivate more slowly and, consequently, have been associated with increased levels of synaptic plasticity (Cull-Candy and Leszkiewicz, 2004Cull-Candy S.G. Leszkiewicz D.N. Role of distinct NMDA receptor subtypes at central synapses.Sci. STKE. 2004; 2004: re16Crossref PubMed Scopus (605) Google Scholar, Lau and Zukin, 2007Lau C.G. Zukin R.S. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders.Nat. Rev. Neurosci. 2007; 8: 413-426Crossref PubMed Scopus (909) Google Scholar). Consistently, upregulation of NR2B expression in mice improves synaptic plasticity and memory formation (Crair and Malenka, 1995Crair M.C. Malenka R.C. A critical period for long-term potentiation at thalamocortical synapses.Nature. 1995; 375: 325-328Crossref PubMed Scopus (598) Google Scholar, Tang et al., 1999Tang Y.P. Shimizu E. Dube G.R. Rampon C. Kerchner G.A. Zhuo M. Liu G. Tsien J.Z. Genetic enhancement of learning and memory in mice.Nature. 1999; 401: 63-69Crossref PubMed Scopus (1564) Google Scholar). Numerous animal models that feature elevated NR2B levels via altered synthesis, transport, or degradation exhibit improved synaptic plasticity and memory (Lee and Silva, 2009Lee Y.S. Silva A.J. The molecular and cellular biology of enhanced cognition.Nat. Rev. Neurosci. 2009; 10: 126-140Crossref PubMed Scopus (267) Google Scholar). Hence, targeting NR2B and its regulatory machinery has been singled out as an attractive approach for cognitive enhancement (Bibb et al., 2010Bibb J.A. Mayford M.R. Tsien J.Z. Alberini C.M. Cognition enhancement strategies.J. Neurosci. 2010; 30: 14987-14992Crossref PubMed Scopus (38) Google Scholar, Collingridge et al., 2013Collingridge G.L. Volianskis A. Bannister N. France G. Hanna L. Mercier M. Tidball P. Fang G. Irvine M.W. Costa B.M. et al.The NMDA receptor as a target for cognitive enhancement.Neuropharmacology. 2013; 64: 13-26Crossref PubMed Scopus (184) Google Scholar). One possible strategy to increase NR2B levels involves induction of its de novo synthesis, for example, via cAMP response element (CRE)-mediated gene expression or chromatin remodeling (Fujita et al., 2012Fujita Y. Morinobu S. Takei S. Fuchikami M. Matsumoto T. Yamamoto S. Yamawaki S. Vorinostat, a histone deacetylase inhibitor, facilitates fear extinction and enhances expression of the hippocampal NR2B-containing NMDA receptor gene.J. Psychiatr. Res. 2012; 46: 635-643Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, Jiang et al., 2010Jiang Y. Jakovcevski M. Bharadwaj R. Connor C. Schroeder F.A. Lin C.L. Straubhaar J. Martin G. Akbarian S. Setdb1 histone methyltransferase regulates mood-related behaviors and expression of the NMDA receptor subunit NR2B.J. Neurosci. 2010; 30: 7152-7167Crossref PubMed Scopus (94) Google Scholar, Myers et al., 1999Myers S.J. Dingledine R. Borges K. Genetic regulation of glutamate receptor ion channels.Annu. Rev. Pharmacol. Toxicol. 1999; 39: 221-241Crossref PubMed Scopus (94) Google Scholar). Alternative targets include transport or degradation of NR2B via its coupling to distinct intracellular signaling pathways (Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar, Yin et al., 2011Yin X. Takei Y. Kido M.A. Hirokawa N. Molecular motor KIF17 is fundamental for memory and learning via differential support of synaptic NR2A/2B levels.Neuron. 2011; 70: 310-325Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). The molecular machinery regulating trafficking, subcellular localization, and degradation of NMDAR subunits is not yet well understood, but it has been recognized that phosphorylation of NMDAR subunits including NR2B is important for the regulation of such processes (Chen and Roche, 2007Chen B.S. Roche K.W. Regulation of NMDA receptors by phosphorylation.Neuropharmacology. 2007; 53: 362-368Crossref PubMed Scopus (304) Google Scholar, Ma and Jan, 2002Ma D. Jan L.Y. ER transport signals and trafficking of potassium channels and receptors.Curr. Opin. Neurobiol. 2002; 12: 287-292Crossref PubMed Scopus (152) Google Scholar, Wenthold et al., 2003Wenthold R.J. Prybylowski K. Standley S. Sans N. Petralia R.S. Trafficking of NMDA receptors.Annu. Rev. Pharmacol. Toxicol. 2003; 43: 335-358Crossref PubMed Scopus (292) Google Scholar). One protein kinase implicated in the metabolism of NR2B is Cdk5 (Cyclin-dependent kinase 5), although no direct phosphorylation of NR2B by Cdk5 has been reported (Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar). Cdk5 is a proline-directed serine/threonine kinase that is activated upon interaction with the neuron-specific cofactors p35 or p39 (Dhavan and Tsai, 2001Dhavan R. Tsai L.H. A decade of CDK5.Nat. Rev. Mol. Cell Biol. 2001; 2: 749-759Crossref PubMed Scopus (942) Google Scholar). Cdk5 has been implicated in numerous CNS processes, including cortical layer formation, neurotransmission, and mnemonic functions (Angelo et al., 2006Angelo M. Plattner F. Giese K.P. Cyclin-dependent kinase 5 in synaptic plasticity, learning and memory.J. Neurochem. 2006; 99: 353-370Crossref PubMed Scopus (110) Google Scholar). Accordingly, a previous study showed that Cdk5 conditional knockout (cKO) mice have improved synaptic plasticity and learning and memory via regulation of NR2B degradation by calpain (Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar). Interestingly, transgenic mice overexpressing the truncated Cdk5 activator p25 also exhibit enhanced plasticity and memory formation (Angelo et al., 2003Angelo M. Plattner F. Irvine E.E. Giese K.P. Improved reversal learning and altered fear conditioning in transgenic mice with regionally restricted p25 expression.Eur. J. Neurosci. 2003; 18: 423-431Crossref PubMed Scopus (80) Google Scholar, Angelo et al., 2006Angelo M. Plattner F. Giese K.P. Cyclin-dependent kinase 5 in synaptic plasticity, learning and memory.J. Neurochem. 2006; 99: 353-370Crossref PubMed Scopus (110) Google Scholar, Fischer et al., 2005Fischer A. Sananbenesi F. Pang P.T. Lu B. Tsai L.H. Opposing roles of transient and prolonged expression of p25 in synaptic plasticity and hippocampus-dependent memory.Neuron. 2005; 48: 825-838Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), suggesting that reduced Cdk5 expression or displacement from physiological substrates potentiates synaptic remodeling processes. Despite these results indicating a close mechanistic relationship between NR2B and Cdk5 in the control of mnemonic functions, the underlying molecular processes remain unknown. Here we show that Cdk5 directly phosphorylates NR2B at Ser1116 and that the phosphorylation state of this serine residue is specifically regulated by physiological neuronal activity. Cdk5-dependent Ser1116 phosphorylation prevents cell surface expression of NR2B-containing NMDAR and thus attenuates synaptic transmission. Disrupting the Cdk5-mediated regulation of NR2B via small interfering peptides (siPs) to block protein-protein interactions reduces this phosphorylation, increases NR2B surface levels, facilitates neurotransmission, and improves hippocampus-dependent learning and memory, suggesting that this regulatory mechanism may indeed be a suitable target for the development of cognitive enhancers. Cdk5 has been previously shown to contribute to synaptic plasticity and memory formation through its control of NR2B (GluR2B) levels via calpain-mediated degradation (Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar). Because Cdk5 directly associates with this receptor, we investigated whether it could phosphorylate NR2B. To identify novel Cdk5 phosphorylation sites, we employed a PhosphoScan approach utilizing antibody-based enrichment of phospho-peptides from mouse brain with a phospho-specific Cdk5 substrate antibody followed by tandem mass spectrometry (Rush et al., 2005Rush J. Moritz A. Lee K.A. Guo A. Goss V.L. Spek E.J. Zhang H. Zha X.M. Polakiewicz R.D. Comb M.J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells.Nat. Biotechnol. 2005; 23: 94-101Crossref PubMed Scopus (963) Google Scholar). This approach identified Ser1116 within the cytoplasmic carboxy-terminal of NR2B as a novel Cdk5 phosphorylation site (Figures 1A and 1B ). To monitor physiological changes in Ser1116 NR2B phosphorylation, a phosphorylation state-specific antibody was generated. The phospho-Ser1116 (P-S1116) NR2B antibody detected time-dependent in vitro phosphorylation of recombinant NR2B at Ser1116 by Cdk5 (Figure 1C). Analysis of various mouse brain regions, namely cortex, hippocampus, cerebellum, and striatum, further revealed that NR2B is phosphorylated at Ser1116 in vivo (Figures S1A and S1B available online), indicating that this NR2B phosphorylation functions broadly throughout the brain. Treatment of acute mouse hippocampal slices with the specific Cdk5 inhibitor CP681301 caused dose-dependent P-S1116 NR2B reduction (Figure 1D). Moreover, Cdk5 inhibitor-induced reduction of NR2B phosphorylation at Ser1116 followed a similar time course as did the reduction of phospho-Thr75 DARPP-32, a well-established Cdk5 substrate (Figure S1C; Bibb et al., 1999Bibb J.A. Snyder G.L. Nishi A. Yan Z. Meijer L. Fienberg A.A. Tsai L.-H. Kwon Y.T. Girault J.-A. Czernik A.J. et al.Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons.Nature. 1999; 402: 669-671Crossref PubMed Scopus (490) Google Scholar). Finally, P-S1116 NR2B levels were markedly reduced in hippocampal lysate of Cdk5 cKO mice (Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar) compared to wild-type (WT) controls (Figure 1E). Together, these results demonstrate that Ser1116 of NR2B is a physiological Cdk5 phosphorylation site and raise the question of how this posttranslational modification might affect NR2B function. NMDARs are assembled in the endoplasmic reticulum, trafficked along the secretory pathway, and delivered to the postsynaptic plasma membrane (Lau and Zukin, 2007Lau C.G. Zukin R.S. NMDA receptor trafficking in synaptic plasticity and neuropsychiatric disorders.Nat. Rev. Neurosci. 2007; 8: 413-426Crossref PubMed Scopus (909) Google Scholar, Wenthold et al., 2003Wenthold R.J. Prybylowski K. Standley S. Sans N. Petralia R.S. Trafficking of NMDA receptors.Annu. Rev. Pharmacol. Toxicol. 2003; 43: 335-358Crossref PubMed Scopus (292) Google Scholar). The molecular machinery underlying NMDAR trafficking and subcellular localization is not yet well characterized, but it has been recognized that phosphorylation of NMDAR subunits including NR2B is important for the regulation of such processes (Chen and Roche, 2007Chen B.S. Roche K.W. Regulation of NMDA receptors by phosphorylation.Neuropharmacology. 2007; 53: 362-368Crossref PubMed Scopus (304) Google Scholar, Ma and Jan, 2002Ma D. Jan L.Y. ER transport signals and trafficking of potassium channels and receptors.Curr. Opin. Neurobiol. 2002; 12: 287-292Crossref PubMed Scopus (152) Google Scholar, Wenthold et al., 2003Wenthold R.J. Prybylowski K. Standley S. Sans N. Petralia R.S. Trafficking of NMDA receptors.Annu. Rev. Pharmacol. Toxicol. 2003; 43: 335-358Crossref PubMed Scopus (292) Google Scholar). We noted that Ser1116 of NR2B occurs adjacent to a putative ER retention signal, RXR (Ma and Jan, 2002Ma D. Jan L.Y. ER transport signals and trafficking of potassium channels and receptors.Curr. Opin. Neurobiol. 2002; 12: 287-292Crossref PubMed Scopus (152) Google Scholar) (i.e., NH2-…RRRPPRS∗PDHK…COOH), and, therefore, investigated whether Cdk5-dependent phosphorylation of NR2B at Ser1116 affected its subcellular localization. The effect of Cdk5 inhibition on NR2B cell surface levels was assessed in cultured hippocampal neurons overexpressing NR2B tagged with extracellular N-terminal green-fluorescent protein (GFP-NR2B). Immunofluorescence staining via anti-GFP antibody thus enabled visualization of cell surface GFP-NR2B. Surface-stained GFP-NR2B exhibited a punctate pattern within dendritic processes (Figure 1F). Counterlabeling with Homer-1, a marker of postsynaptic densities (PSDs), demonstrated that the cell surface fraction of GFP-NR2B localized in proximity to PSDs. Treatment of cultured hippocampal neurons with the Cdk5 inhibitor CP681301 increased surface expression of NR2B in dendritic processes approximately 1.3-fold (Figures 1F and 1G), suggesting that NR2B phosphorylation by Cdk5 modulates NR2B surface levels. The abundance of postsynaptic NMDARs is a major determinant of synaptic plasticity and memory formation (Lee and Silva, 2009Lee Y.S. Silva A.J. The molecular and cellular biology of enhanced cognition.Nat. Rev. Neurosci. 2009; 10: 126-140Crossref PubMed Scopus (267) Google Scholar, Tang et al., 1999Tang Y.P. Shimizu E. Dube G.R. Rampon C. Kerchner G.A. Zhuo M. Liu G. Tsien J.Z. Genetic enhancement of learning and memory in mice.Nature. 1999; 401: 63-69Crossref PubMed Scopus (1564) Google Scholar). Thus, blocking Cdk5-dependent retention of NR2B from the cell surface could be important in regulating its contribution to NMDAR function. To assess this directly, mouse hippocampal brain slices were treated with either CP681301 or vehicle (Figure 2A). Cdk5 inhibition increased cell surface expression of NR2B 3.5-fold with concomitant reduction in P-S1116 levels to approximately 30% of untreated controls. The effect appeared specific for NR2B because no changes in surface levels of NR2A or the AMPA receptor subunit GluR1 were observed (Figures S2A and S2B). To evaluate the impact of Cdk5-dependent regulation of NR2B surface levels on physiological function, whole-cell voltage-clamp recording of synaptically evoked NMDAR-mediated excitatory postsynaptic currents (EPSCs) were conducted in CA1 pyramidal neurons of hippocampal slices treated with the Cdk5 inhibitor CP681301 (Figures 2B–2D). Administration of CP681301 increased the NMDAR-EPSC amplitude by approximately 1.5-fold. Furthermore, application of the NR2B-specific inhibitor ifenprodil had a greater attenuating effect on NMDAR-EPSCs in CP681301-treated slices (33% reduction) in comparison to controls (20% reduction) (Figure 2C), suggesting that the NR2B component of NMDAR-EPSCs was increased by Cdk5 inhibition. The deactivation kinetics of NMDAR EPSCs was increased in CP681301-treated slices (170.7 ± 19.4 ms) as compared to untreated slices (112.1 ± 10.5 ms) (Figure 2D). This result is consistent with increased NR2B function in response to CP681301 treatment, because NR2B-containing NMDARs inactivate more slowly than receptors composed of other subunits (Cull-Candy and Leszkiewicz, 2004Cull-Candy S.G. Leszkiewicz D.N. Role of distinct NMDA receptor subtypes at central synapses.Sci. STKE. 2004; 2004: re16Crossref PubMed Scopus (605) Google Scholar). Together, these results indicate that Cdk5 regulates the subcellular localization of NMDAR via NR2B phosphorylation at Ser1116, thereby controlling the level of functional NR2B-containing NMDARs within the synaptic membrane and modulating NMDAR-mediated synaptic currents. NMDARs are central to glutamatergic neurotransmission (Bliss and Collingridge, 1993Bliss T.V. Collingridge G.L. A synaptic model of memory: long-term potentiation in the hippocampus.Nature. 1993; 361: 31-39Crossref PubMed Scopus (9558) Google Scholar) and are controlled by complex molecular machinery integrating various postsynaptic signaling cascades (Husi et al., 2000Husi H. Ward M.A. Choudhary J.S. Blackstock W.P. Grant S.G. Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.Nat. Neurosci. 2000; 3: 661-669Crossref PubMed Scopus (1025) Google Scholar, Newpher and Ehlers, 2008Newpher T.M. Ehlers M.D. Glutamate receptor dynamics in dendritic microdomains.Neuron. 2008; 58: 472-497Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). To better understand how Cdk5-dependent NR2B phosphorylation might be regulated and contribute to NR2B function in plasticity and memory formation, the effect of glutamatergic neurotransmission on P-S1116 NR2B was assessed ex vivo and in vivo. Treatment of acute mouse hippocampus slices with NMDA dose-dependently attenuated P-S1116, when normalized to total NR2B levels (Figure 3A). NMDA treatment also reduced total NR2B levels, consistent with previous reports of NMDA-induced, calpain-mediated NR2B degradation (Guttmann et al., 2001Guttmann R.P. Baker D.L. Seifert K.M. Cohen A.S. Coulter D.A. Lynch D.R. Specific proteolysis of the NR2 subunit at multiple sites by calpain.J. Neurochem. 2001; 78: 1083-1093Crossref PubMed Scopus (97) Google Scholar, Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar). The ability of NMDA to reduce P-S1116 in hippocampal slices was attenuated by treatment with the PP2A/PP1 inhibitor okadaic acid (Figure S2C), indicating that some of the NMDAR-induced effect on P-S1116 is mediated via serine/threonine phosphatase activity. In agreement with the attenuating effect of NMDA on P-S1116, induction of in vivo long-term potentiation (LTP) in the hippocampal area CA1 also reduced P-S1116. Tetanic stimulation of mouse Schaffer commissural-CA1 pyramidal cell synapses in vivo caused a significant increase in fEPSP slope (1.4-fold as compared to baseline) that was maintained for 1 hr poststimulus (Figure 3B). One hour after LTP induction, P-S1116 was reduced to approximately 40% of the level in control CA1 lysates (Figure 3C), demonstrating the physiological regulation of P-S1116 in synaptic plasticity. In contrast, induction of long-term depression (LTD) did not affect P-S1116 levels (Figures 3D and 3E). Because excitatory activation of hippocampal NMDAR is integral to learning and memory (Nakazawa et al., 2004Nakazawa K. McHugh T.J. Wilson M.A. Tonegawa S. NMDA receptors, place cells and hippocampal spatial memory.Nat. Rev. Neurosci. 2004; 5: 361-372Crossref PubMed Scopus (472) Google Scholar), we also assessed the involvement of Cdk5-dependent NR2B phosphorylation in mnemonic processes by evaluating P-S1116 levels after contextual fear conditioning. One hour after training, P-S1116 was reduced in CA1 lysates of fear-conditioned mice to 80% of the levels in nonshocked context-exposed controls (Figure 3F). Foot shock in the absence of context had no effect on P-S1116. Taken together, these results show that NR2B phosphorylation at Ser1116 is regulated by glutamatergic neurotransmission, synaptic plasticity, and learning and memory, suggesting an important role for this signaling mechanism in cognition. NR2B and Cdk5 form a complex both in vitro and in vivo (Hawasli et al., 2007Hawasli A.H. Benavides D.R. Nguyen C. Kansy J.W. Hayashi K. Chambon P. Greengard P. Powell C.M. Cooper D.C. Bibb J.A. Cyclin-dependent kinase 5 governs learning and synaptic plasticity via control of NMDAR degradation.Nat. Neurosci. 2007; 10: 880-886Crossref PubMed Scopus (255) Google Scholar). To identify physiological protein-protein interaction sites between NR2B and Cdk5, overlapping eight amino acid peptide cassettes of the NR2B carboxy-terminal domain were synthesized on a peptide microarray microchip (Gao et al., 2004Gao X.B. Pellois J.P. Na Y. Kim Y. Gulari E. Zhou X. High density peptide microarrays. In situ synthesis and applications.Mol. Divers. 2004; 8: 177-187Crossref PubMed Scopus (34) Google Scholar) that was then probed for Cdk5 binding via recombinant Cdk5 followed by immunodetection with anti-Cdk5 antibody (Figure 4A). Four motifs exhibiting high Cdk5 binding were identified by this approach (Figure 4B). To determine whether these motifs were actual NR2B-Cdk5 interaction sites, peptides corresponding to these motifs were synthesized and tested for their ability to interfere with NR2B-Cdk5 binding. In this screen, the peptide corresponding to amino acid residues 1111–1127 (RRPPRSPDHKRYFRDKE; NR2B small interfering peptide [NR2B siP]) most potently blocked the pull-down of Cdk5 by recombinant GST-tagged NR2B in vitro (Figure 4C). Moreover, the NR2B siP dose-dependently inhibited in vitro phosphorylation of Ser1116 NR2B by Cdk5 (Figure 4D). In contrast, a scrambled control peptide (RRRSYFHKEDRPPRDK) did not attenuate Ser1116 phosphorylation in vitro. Interestingly, the NR2B siP did not inhibit the in vitro phosphorylation of inhibitor-1 (Figure S3A), a well-defined Cdk5 substrate (Nguyen et al., 2007Nguyen C. Nishi A. Kansy J.W. Fernandez J. Hayashi K. Gillardon F. Hemmings Jr., H.C. Nairn A.C. Bibb J.A. Regulation of protein phosphatase inhibitor-1 by cyclin-dependent kinase 5.J. Biol. Chem. 2007; 282: 16511-16520Crossref PubMed Scopus (24) Google Scholar), indicating that the inhibition of Ser1116 NR2B phosphorylation by NR2B siP was specific. To further assess the efficacy and consequences of targeting Cdk5-NR2B protein interactions by small interfering peptides ex vivo and in vivo, cell-permeabilizing polyarginine or penetratin N-terminal tags were included in the NR2B siP and control peptides. These tags are commonly used to deliver peptides across the cell membrane into the cytosol of neurons (Joliot and Prochiantz, 2004Joliot A. Prochiantz A. Transduction peptides: from technology to physiology.Nat. Cell Biol. 2004; 6: 189-196Crossref PubMed Scopus (448) Google Scholar). Consistent with the effects observed in vitro, application of the NR2B siP to hippocampal slices decreased P-S1116 in a time- and dose-dependent manner (Figures 4E and S3B). Treatment with the NR2B siP did not alter levels of NR2B or Cdk5 expression (Figures 4E and S3B). The specificity of NR2B siP observed in vitro was retained in vivo; it had no effect on the phosphorylation state of the well-characterized Cdk5 site Thr75 DARPP-32 (Bibb et al., 1999Bibb J.A. Snyder G.L. Nishi A. Yan Z. Meijer L. Fienberg A.A. Tsai L.-H. Kwon Y.T. Girault J.-A. Czernik A.J. et al.Phosphorylation of DARPP-32 by Cdk5 modulates dopamine signalling in neurons.Nature. 1999; 402: 669-671Crossref PubMed Scopus (490) Google Scholar), but it time-dependently reduced P-S1116 in acute mouse striatal slices (Figures S3C and S3D). These data show selective modulation of P-S1116 by siP and also that this mechanism may be targeted in multiple limbic structures mediating learning and memory. Furthermore, NR2B siP treatment of hippocampal slices effectively blocked subsequent coimmunoprecipitation of Cdk5 and NR2B (Figure 4F), indicating that siP disrupts Cdk5-NR2B protein-protein interactions. In contrast, treatment with scrambled control or the Cdk5 inhibitor CP681301 did not affect Cdk5-NR2B interactions. Consistent with our data that Cdk5 regulates NR2B cell surface expression, the siP induced a 3.3-fold increase in NR2B cell surface levels in hippocampal slices (Figure 4G). Finally, cell surface GFP-NR2B levels i" @default.
• W1996198347 created "2016-06-24" @default.
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• W1996198347 date "2014-03-01" @default.
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• W1996198347 title "Memory Enhancement by Targeting Cdk5 Regulation of NR2B" @default.
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• W1996198347 doi "https://doi.org/10.1016/j.neuron.2014.01.022" @default.
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