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.

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.

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 (). Alternative targets include transport or degradation of NR2B via its coupling to distinct intracellular signaling pathways (). 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 (). 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 (). Cdk5 is a proline-directed serine/threonine kinase that is activated upon interaction with the neuron-specific cofactors p35 or p39 (). Cdk5 has been implicated in numerous CNS processes, including cortical layer formation, neurotransmission, and mnemonic functions (). 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 (). Interestingly, transgenic mice overexpressing the truncated Cdk5 activator p25 also exhibit enhanced plasticity and memory formation (), 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.

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 (). Consistently, upregulation of NR2B expression in mice improves synaptic plasticity and memory formation (). Numerous animal models that feature elevated NR2B levels via altered synthesis, transport, or degradation exhibit improved synaptic plasticity and memory (). Hence, targeting NR2B and its regulatory machinery has been singled out as an attractive approach for cognitive enhancement ().

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 (). 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) (). The biophysical properties of NMDARs are dependent on their subunit composition (). Indeed, NMDAR subunit composition varies greatly between different synapses and neurons, as well as during neuronal development (). Therefore, precise spatiotemporal regulation of NMDAR subunit expression, composition, trafficking, and localization is critical for proper neuronal function.

Taken together, these findings show that selective disruption of NR2B-Cdk5 interactions via NR2B siP enhances hippocampus-dependent learning and memory through potentiation of a regulatory mechanism that is involved in memory formation. Thus, it is possible that this mechanism regulating NR2B function via Cdk5 may be a suitable target for the development of cognitive enhancers as therapeutics for memory impairment as well as age-dependent cognitive decline.

Previous studies found that NMDAR inhibition during the acquisition phase impaired fear memory (). Therefore, we evaluated whether an acute NR2B siP infusion shortly before the training session could also improve fear memory. For this purpose, 3-month-old mice were acutely surgerized under isofluorane anesthesia and NR2B siP was infused into dorsal hippocampus 90 min prior to fear conditioning. Acute NR2B siP infusion resulted in significantly elevated freezing behavior in the context test as compared to controls ( Figure 6 E). The freezing levels during the context test of the acute paradigm were, in fact, comparable to the levels observed with chronic infusion. The tone test did not reveal any significant changes between acute peptide-infused groups. Finally, to evaluate whether NR2B siP administration may be a valid treatment for age-related cognitive decline, 14-month-old mice were acutely infused with NR2B siP and tested for fear memory ( Figure S5 B). As observed for 3-month-old mice, acute NR2B siP infusion 90 min prior to training increased contextual fear memory 1.7-fold compared to control peptide or noninfused cannulated controls. Thus, increasing NR2B surface levels may be a suitable memory enhancement strategy even for later stages in life. These results are also consistent with studies showing that NR2B overexpression in forebrain neurons improves learning and memory in aged mice ().

Memory in aged mice is rescued by enhanced expression of the GluN2B subunit of the NMDA receptor.

Induction- and conditioning-protocol dependent involvement of NR2B-containing NMDA receptors in synaptic potentiation and contextual fear memory in the hippocampal CA1 region of rats.

The learning and memory improvements associated with NR2B siP infusion correlated directly with reduced P-S1116 NR2B. NR2B siP infusion and fear conditioning both decreased P-S1116 NR2B in CA1 1 hr after training ( Figure S5 A). Animals that had been infused with NR2B siP and undergone fear conditioning exhibited the lowest phosho-Ser1116 levels.

With these parameters established, the effect of NR2B siP infusion on fear memory was evaluated. Continuous bilateral NR2B siP infusion into rat dorsal hippocampus 24 hr prior to fear conditioning increased hippocampus-dependent contextual fear memory 1.7-fold compared to scrambled peptide controls ( Figure 6 D). In agreement with the hippocampal-independent nature of cue-associated fear learning (), no effect was observed on tone-induced freezing. Administration of neither NR2B siP nor scrambled control affected nociception, pain threshold, or motor reflexes ( Figures S4 B and S4C). During extinction trials, contextual memory enhancement persisted in NR2B siP-treated rats at 48 and 72 hr after initial training ( Figure S4 D and S4E). The conditioned fear response was extinguished by 96 hr for both groups ( Figure S4 E). After extinction and expiration of the mini-osmotic pump, rats were retrained. Twenty-four hours after retraining, the contextual fear memory was increased in rats previously infused with NR2B siP during the initial acquisition phase, but not scrambled controls ( Figure S4 F), suggesting that behavioral effects persist even after NR2B siP clearance. These data also indicate that the siP-improved memory may have been more retained or consolidated.

Within the FITC siP-infused region, P-S1116 was reduced by approximately 40% compared to FITC scrambled peptide-infused controls ( Figure 6 B). Infusion of FITC siP also caused a 3-fold increase in NR2B surface expression ( Figure 6 C). Together, these results validate this approach for the delivery of NR2B siP into pyramidal neurons of dorsal hippocampus and show that the NR2B siP induces biochemical and cellular changes in vivo comparable to our in vitro and ex vivo observations.

The data presented so far indicate that the subcellular localization of NR2B is regulated by Cdk5. Reduction in Cdk5 activity, as well as disruption of Cdk5-NR2B interaction, consistently increased NR2B surface levels and facilitated NMDA-mediated synaptic function. Because previous research has demonstrated that elevated NR2B function enhances cognitive performance (), we investigated whether administration of NR2B siP can improve mnemonic function. For this purpose, we first established robust in vivo protocols to deliver cell-penetrating NR2B siP to the appropriate brain regions and test their effect on learning and memory. NMDARs are required for hippocampus-dependent learning and memory, such as acquisition of contextual fear memory (). Accordingly, infusion of the selective NR2B inhibitors Ro25-6981 or ifenprodil into dorsal hippocampus was previously found to impair contextual fear learning, but not locomotor or anxiety-related behavior (). Hence, NR2B siP was infused bilaterally into the CA1 subfield of dorsal hippocampus continuously for 72 hr via mini-osmotic pumps. Because of the size of the osmotic pump and the robust behavioral performance, these initial experiments were performed in rat. Spatiotemporal tracking of fluorescein-tagged NR2B siP (FITC siP) confirmed correct targeting, continuity of the delivery, and neuronal uptake of the peptide. FITC siP was specifically detected in dorsal hippocampus at 12, 24, and 72 hr after initiation of pumping. The FITC siP was present in hippocampal area CA1 and labeled the cytoplasm of pyramidal neurons ( Figure 6 A). After pumping was stopped, intracellular FITC siP signal was markedly reduced by 6 hr and had largely disappeared by 24 hr ( Figure S4 A).

(F) Model of NR2B regulation via Ser1116 phosphorylation by Cdk5. At basal state, Cdk5 constitutively phosphorylates NR2B at Ser1116, withholding it from the cell surface. Glutamatergic neurotransmission reduces Cdk5-dependent NR2B phosphorylation, enabling translocation of NR2B to the cell surface and thereby contributing to memory formation. Addition of NR2B siP disrupts NR2B-Cdk5 interactions and reduces Ser1116 phosphorylation, and more NR2B translocates to the cell surface, thereby enhancing memory formation.

(E) Experimental design and quantitation of fear memory in acutely infused 3-month-old mice. Ninety minutes prior to training, mice were infused with NR2B siP or control peptide into dorsal hippocampus. Twenty-four hours later, NR2B siP-treated mice exhibited improved context freezing, but not freezing to tone (n = 6–10). Data are represented as mean ± SEM. See also Figures S4 and S5

(D) Experimental design and quantitation of context- and tone-elicited fear memory in 2-month-old rats chronically infused with scrambled or NR2B siP into dorsal hippocampus (n = 20) showing that NR2B siP improved contextual fear memory.

(B and C) Immunoblots showing infused NR2B siP versus scramble control reduces P-S1116 (n = 4) (B) and increases NR2B cell surface expression (4 pooled tissue punches for each condition) (C) in epifluorescent hippocampal area CA1.

Induction- and conditioning-protocol dependent involvement of NR2B-containing NMDA receptors in synaptic potentiation and contextual fear memory in the hippocampal CA1 region of rats.

To determine whether the NR2B siP-induced changes in hippocampal neurotransmission were due to NR2B, the effects of the NR2B siP on basal fEPSPs were assessed in slices coincubated with the specific NR2B antagonist Ro25-6981 ( Figures 5 F and 5G). Under these conditions, the NR2B siP-mediated increase was blocked. Cdk5 cKO mice lack approximately 70% of hippocampal Cdk5 and exhibit a reduced threshold for induction of LTP but have normal baselines for fEPSP fiber volley amplitude (). Interestingly, the ability of the NR2B siP to raise CA1-CA3 fEPSPs was almost absent in Cdk5 cKO mice with a small (1.2-fold) but still significant increase in fEPSP slopes ( Figures 5 F and 5G). Thus the effect of the peptide on synaptic transmission is mediated via NR2B and Cdk5. Together these results show that NR2B siP selectively disrupts NR2B-Cdk5 interaction, reduces P-S1116, increases cell surface levels of NR2B, and facilitates neurotransmission by enhancing NR2B function.

Given the importance of NR2B in mediating the potentiation of synaptic transmission during plasticity, we assessed the effects of NR2B siP on extracellularly recorded field excitatory postsynaptic potentials (fEPSP) in the hippocampal CA3-CA1 pathway. Treatment of hippocampal slices with NR2B siP rapidly induced a 1.9-fold increase in fEPSP slope compared to slices infused with a control peptide ( Figures 5 D and 5E). Upon washout of the NR2B siP, fEPSP slopes progressively decreased until they reached steady-state levels with fEPSP slopes that were 1.6-fold greater than control treated slices.

Given the efficacy of the NR2B siP in targeting Cdk5-NR2B interactions and increasing NR2B cell surface levels, its impact on NMDAR-mediated EPSCs was assessed. Treatment of cortical slices with the NR2B siP produced a 1.8-fold increase in the NMDA/AMPA EPSC ratio as compared to a scrambled control peptide ( Figure 5 A). Moreover, slices incubated with NR2B siP exhibited a 1.5-fold increase in the ifenprodil-sensitive NR2B component of NMDAR EPSCs ( Figures 5 B and 5C), suggesting that the NR2B siP-induced increase in NMDAR EPSCs is due to recruitment of additional NR2B-containing NMDAR to the cell surface.

(F and G) Extracellular field recordings in hippocampal slices incubated with the specific NR2B inhibitor Ro 25-6981 (3 μM) or from Cdk5 cKO were treated with NR2B siP (30 min, 2 μM) and quantification of fEPSP slope (n = 3) did not show a significant effect of NR2B siP with NR2B inhibition or in absence of Cdk5 (cKO).

(D and E) Extracellular CA3-CA1 field recordings in hippocampal slices treated with NR2B siP or control peptide (30 min, 2 μM). Sample traces of 2 min before (a) and at end (b) of siP application and Quantitation of fEPSP slope (n = 4).

(B and C) NMDAR EPSC sensitivity to the NR2B inhibitor, ifenprodil, in slices incubated with NR2B siP or scrambled control (n = 6–7).

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 4 G). Finally, cell surface GFP-NR2B levels increased 1.5-fold after 30 min treatment with NR2B siP as compared to control peptide (S1116A) in primary hippocampal cultures ( Figures 4 H and 4I). Thus, Cdk5-NR2B protein-protein interactions can be targeted in intact brain tissue with NR2B siP, resulting in reduced Cdk5-dependent phosphorylation of Ser1116 NR2B and appreciable increases in cell surface expression of the receptor.

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 4 E and S3 B). Treatment with the NR2B siP did not alter levels of NR2B or Cdk5 expression ( Figures 4 E and S3 B). 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 (), but it time-dependently reduced P-S1116 in acute mouse striatal slices ( Figures S3 C 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 4 F), 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.

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 ().

NR2B and Cdk5 form a complex both in vitro and in vivo (). 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 () that was then probed for Cdk5 binding via recombinant Cdk5 followed by immunodetection with anti-Cdk5 antibody ( Figure 4 A). Four motifs exhibiting high Cdk5 binding were identified by this approach ( Figure 4 B). 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 4 C). Moreover, the NR2B siP dose-dependently inhibited in vitro phosphorylation of Ser1116 NR2B by Cdk5 ( Figure 4 D). 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 S3 A), a well-defined Cdk5 substrate (), indicating that the inhibition of Ser1116 NR2B phosphorylation by NR2B siP was specific.

(G) NR2B cell surface levels are increased in hippocampal slices treated with NR2B siP (1 hr, 100 μM) or the Cdk5 inhibitor CP681301 (1 hr, 50 μM), but not with control peptide (Scramble; 1 hr, 100 μM) (n = 4; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ANOVA). NR2B Load is also indicated.

(F) NR2B siP, but not the scrambled peptide, nor CP681301 (CP) disrupts NR2B-Cdk5 interaction in hippocampal slices (four pooled slices for each sample) as evaluated by coimmunoprecipitation. Cdk5 Load is also shown.

(E) Time-dependent reduction of P-S1116 in hippocampal slices treated with NR2B siP (25 μM) or the Cdk5 inhibitor CP681301 (CP; 50 μM), but not scrambled (Scr) control peptide (n = 4; ∗ p < 0.05, ∗∗ p < 0.01; ANOVA).

(D) Immunoblots showing that NR2B siP dose dependently inhibits in vitro phosphorylation of Ser1116 NR2B by Cdk5, whereas the control peptide (scramble) has no effect.

(C) NR2B siP blocks pull-down of Cdk5 with recombinant NR2B immobilized on GST beads, demonstrating disruption of NR2B-Cdk5 interaction in vitro. Immunoblots show relative levels of NR2B and Cdk5 under conditions indicated. NR2B siP dose dependently disrupts NR2B-Cdk5 binding in vitro as depicted in plot (n = 3).

(B) The cytoplasmic C terminus amino acid sequence of NR2B with potential Cdk5 binding sites highlighted with their corresponding peptide array tiles.

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 3 B). One hour after LTP induction, P-S1116 was reduced to approximately 40% of the level in control CA1 lysates ( Figure 3 C), 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 3 D and 3E). Because excitatory activation of hippocampal NMDAR is integral to learning and memory (), 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 3 F). 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.

NMDARs are central to glutamatergic neurotransmission () and are controlled by complex molecular machinery integrating various postsynaptic signaling cascades (). 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 3 A). NMDA treatment also reduced total NR2B levels, consistent with previous reports of NMDA-induced, calpain-mediated NR2B degradation (). The ability of NMDA to reduce P-S1116 in hippocampal slices was attenuated by treatment with the PP2A/PP1 inhibitor okadaic acid ( Figure S2 C), indicating that some of the NMDAR-induced effect on P-S1116 is mediated via serine/threonine phosphatase activity.

(F) Quantitative immunoblots of CA1-specific lysates from contextual fear conditioned (FC) and control (Context and Foot Shock) mice obtained 1 hr after training for P-S1116 and total NR2B levels (n = 4–6).

(D) Induction of CA1 LTD in mouse hippocampal slices with sample traces of 5 min periods before (a) and after (b) low-frequency stimulation (LFS; 900 stimuli at 1 Hz).

(B) Induction of in vivo CA1 LTP in mouse. Sample traces of 5 min periods before (a) and after (b) high-frequency stimulation (HFS; two trains of 50 pulses at 100 Hz; 30 s intertrain interval) and LTP induction are shown.

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.

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 2 B–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 2 C), 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 2 D). 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 ().

The abundance of postsynaptic NMDARs is a major determinant of synaptic plasticity and memory formation (). 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 2 A). 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 S2 A and S2B).

(C and D) Quantitation of NMDAR EPSC recordings showing an increase of ifenprodil-sensitive NR2B component (C) and prolonged decay kinetics (D) of NMDAR EPSCs in cells incubated in the absence (-) or presence (+) of CP681301 (n = 6−7).

(B) Effect of Cdk5 inhibition on the NR2B component of NMDAR-EPSCs. Voltage-clamp EPSC recordings of total NMDAR-EPSCs (I NMDA ) in the absence or presence of the specific NR2B inhibitor ifenprodil from pyramidal neurons within the hippocampal area CA1 pretreated with vehicle (control) or the Cdk5 inhibitor CP681301 are shown.

(A) Increased cell surface NR2B correlates with reduced P-S1116 in hippocampal slices. Levels of cell surface-biotinylated NR2B pulled-down from lysates of hippocampal slices incubated in the absence (-) or presence (+) of the Cdk5 inhibitor CP681301 are shown with blots and quantitation (n = 4). Prior to pull-down, lysates were tested for NR2B Load and P-S1116 (bottom).

NMDARs are assembled in the endoplasmic reticulum, trafficked along the secretory pathway, and delivered to the postsynaptic plasma membrane (). 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 (). We noted that Ser1116 of NR2B occurs adjacent to a putative ER retention signal, RXR () (i.e., NH-…PPRSPDHK…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 1 F). 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 1 F and 1G), suggesting that NR2B phosphorylation by Cdk5 modulates NR2B surface levels.

Cdk5 has been previously shown to contribute to synaptic plasticity and memory formation through its control of NR2B (GluR2B) levels via calpain-mediated degradation (). 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 (). This approach identified Ser1116 within the cytoplasmic carboxy-terminal of NR2B as a novel Cdk5 phosphorylation site ( Figures 1 A 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 1 C). Analysis of various mouse brain regions, namely cortex, hippocampus, cerebellum, and striatum, further revealed that NR2B is phosphorylated at Ser1116 in vivo ( Figures S1 A 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 1 D). 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 S1 C;). Finally, P-S1116 NR2B levels were markedly reduced in hippocampal lysate of Cdk5 cKO mice () compared to wild-type (WT) controls ( Figure 1 E). 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.

(F) Inhibition of Cdk5 increases NR2B cell surface levels in hippocampal neurons. Top panels show immunofluorescence staining of cell surface GFP-NR2B (green) and postsynaptic, intraneuronal Homer-1 (red) in hippocampal neurons treated with vehicle (control) or the Cdk5 inhibitor CP681301. Bottom panels depict high magnification of stained dendrites.

(E) Quantitative immunoblot analysis of P-S1116 and total NR2B in hippocampal lysates from Cdk5 cKO and WT mice (n = 4). Loss of Cdk5 in cKO is also shown (bottom).

Discussion

In this study, we present a molecular mechanism regulating the subcellular localization of NR2B-containing NMDAR. This mechanism appears to be central to synaptic transmission and mnemonic functions and can be targeted to enhance memory. A model of this mechanism and the effects of targeting it may be suggested ( Figure 6 F). Upon glutamatergic neurotransmission, such as that occurring during synaptic plasticity induction or memory formation, Cdk5-dependent Ser1116 phosphorylation is reduced and subsequently leads to increased cell surface levels of NR2B-containing NMDAR. Consequently, selective disruption of NR2B-Cdk5 interactions via the NR2B siP increases NR2B surface levels, thereby facilitating synaptic transmission. Intrahippocampal infusion of the NR2B siP improved fear memory, suggesting that the regulation of NR2B by Cdk5 may be a suitable target for the development of cognitive enhancers.

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Teves L.

Tymianski M. Treatment of stroke with a PSD-95 inhibitor in the gyrencephalic primate brain. Peptides are now an integral component of CNS research, and protein-protein interactions and intracellular signaling mechanisms provide a very large base of new therapeutic targets (). Until recently, CNS drug discovery has focused on developing synthetic small molecules that act primarily upon targets involved in neurotransmission including receptors and ion channels. Drug target pool algorithms such as the “rule of five” () and “the druggable genome” () have been used in an effort to facilitate the discovery of small molecules of high specificity for oral delivery. However, these restrictions have often had the unintended consequences of producing molecules with poor selectivity and unwanted side effects. Larger molecules including peptides and peptidomemetics that interfere with protein-protein interactions provide more contact with targets and greater selectivity but have been limited by low bioavailability, poor membrane permeability, and metabolic instability. These obstacles are being circumvented by a number of creative means and protein- or peptide-based drugs are now roughly 10% of the pharmaceutical market and growing (). As an example of this potential, peptides that target NR2B interactions with the postsynaptic density have been shown to neuroprotect from ischemic injury in primates ().

Here we demonstrate memory enhancement by selectively targeting a single phosphorylation site and NR2B-Cdk5 protein-protein interactions with a peptide injected intracranially. It will be interesting to find out whether further optimization in the targeting of this and other synaptic mechanisms will lead to more practical delivery modes and clinically applicable therapies.