GluN2B Subunit Research Articles

Developmental light deprivation transiently reduces the expression of vGluT2 and GluN2B in the rat ventral suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.

GluN2B inhibition confers resilience against long-term cocaine-induced neurocognitive sequelae.

Cocaine self-administration can disrupt the capacity of humans and rodents to flexibly modify familiar behavioral routines, even when they become maladaptive or unbeneficial. However, mechanistic factors, particularly those driving long-term behavioral changes, are still being determined. Here, we capitalized on individual differences in oral cocaine self-administration patterns in adolescent mice and revealed that the post-synaptic protein PSD-95 was reduced in the orbitofrontal cortex (OFC) of escalating, but not stable, responders, which corresponded with later deficits in flexible decision-making behavior. Meanwhile, NMDA receptor GluN2B subunit content was lower in the OFC of mice that were resilient to escalatory oral cocaine seeking. This discovery led us to next co-administer the GluN2B-selective antagonist ifenprodil with cocaine, blocking the later emergence of cocaine-induced decision-making abnormalities. GluN2B inhibition also prevented cocaine-induced dysregulation of neuronal structure and function in the OFC, preserving mature, mushroom-shaped dendritic spine densities on deep-layer pyramidal neurons, which were otherwise lower with cocaine, and safeguarding functional BLA→OFC connections necessary for action flexibility. We posit that cocaine potentiates GluN2B-dependent signaling, which triggers a series of durable adaptations that result in the dysregulation of post-synaptic neuronal structure in the OFC and disruption of BLA→OFC connections, ultimately weakening the capacity for flexible choice. And thus, inhibiting GluN2B-NMDARs promotes resilience to long-term cocaine-related sequelae.

Changes in NMDA Receptor Function in Rapid Ischemic Tolerance: A Potential Role for Tri-Heteromeric NMDA Receptors.

In this study, we characterize biophysical changes in NMDA receptor function in response to brief non-injurious ischemic stress (ischemic preconditioning). Electrophysiological studies show NMDA receptor function is reduced following preconditioning in cultured rat cortical neurons. This functional change is not due to changes in the reversal potential of the receptor, but an increase in desensitization. We performed concentration–response analysis of NMDA-evoked currents, and demonstrate that preconditioned neurons show a reduced potency of NMDA to evoke currents, an increase in Mg2+ sensitivity, but no change in glycine sensitivity. Antagonists studies show a reduced inhibition of GluN2B antagonists that have an allosteric mode of action (ifenprodil and R-25-6981), but competitive antagonists at the GluR2A and 2B receptor (NVP-AMM077 and conantokin-G) appear to have similar potency to block currents. Biochemical studies show a reduction in membrane surface GluN2B subunits, and an increased co-immunoprecipitation of GluN2A with GluN2B subunits, suggestive of tri-heteromeric receptor formation. Finally, we show that blocking actin remodeling with jasplakinolide, a mechanism of rapid ischemic tolerance, prevents NMDA receptor functional changes and co-immunoprecipitation of GluN2A and 2B subunits. Together, this study shows that alterations in NMDA receptor function following preconditioning ischemia are associated with neuroprotection in rapid ischemic tolerance.

Region-Specific Enhancement of c-fos Expression by Combined Treatment With NMDA Receptor Agonists and Antagonists With Antidepressant Potential.

Rapastinel, formerly Glyx-13, is a novel positive allosteric modulator of the N-methyl-D-aspartate-receptor (NMDAR) that counteracts psychotomimetic actions of NMDAR antagonists. We set out to evaluate the effect of rapastinel alone or in combination with the global and GluN2B subunit-specific NMDAR antagonists MK-801 and Ro25-6981, respectively, on neuronal activation in relevant regions using c-fos brain mapping. Whereas rapastinel alone did not trigger significant c-fos expression beyond the prelimbic cortex, it strongly increased the c-fos expression induced by MK-801 in hippocampal, cingulate, and retrosplenial areas. Similar results were obtained when rapastinel was replaced by D-cycloserine. Our results reveal new interactions at network level between NMDAR modulators with possible implications regarding their therapeutic effects.

Assays for L-type voltage gated calcium channels

Voltage gated calcium channels (VGCCs) are pursued as drug targets for neurodegenerative and cardiovascular diseases. High throughput drug screening targeting VGCCs depends on patch-clamp electrophysiology or fluorophore-based calcium imaging that requires powerful equipment and specialized expertise thus leading to cost escalation. Moreover, VGCC needs to be transfected into cell lines such as HEK-293. We report the presence of L-type VGCC (L-VGCC) subunit proteins, Cav1.2, α2δ and β in HEK-293 cells and the application of simple methods for its assay. Endogenous expression of the channel in HEK-293 cells overcomes the need for transfection. L-VGCC in HEK-293 cells was activated either by the agonist, BayK8644 or by KCl-mediated depolarization. Activity was detected using the calcium sensing probe, GCaMP6m by live imaging. L-VGCC activity induced enhancement in GCaMP6m fluorescence returned to baseline corresponding to channel-closure. Activity was also shown using a methodology involving end-point detection of the calcium dependent interaction of α-CaMKII with NMDA receptor subunit GluN2B sequence. This methodology further simplifies the assay as it eliminates the need for real time imaging. Activation was blocked by the specific L-type VGCC antagonist, nifedipine. Finding the protein and activity of L-VGCC in HEK-293 cells offers commercially viable assays for drug screening.

Phenol-benzoxazolone bioisosteres of GluN2B-NMDA receptor antagonists: Unexpected rearrangement during reductive alkylation with phenylcyclohexanone.

Negative allosteric modulators of N-methyl- d-aspartatereceptors containing the GluN2B subunit represent promising drug candidates for the treatment of various neurological disorders including stroke, epilepsy, and Parkinson's disease. To increase the bioavailability and GluN2B affinity, the phenol of the potent benzazepine-based inhibitor, WMS-1410 (3), was replaced bioisosterically by a benzoxazolone moiety and the phenylbutyl side chain was conformationally restricted in a phenylcyclohexyl substituent. A four-step, one-pot procedure transformed the oxazolo-benzazepine 7 into the phenylcyclohexyl derivative 11. The same protocol was applied to the methylated analog 12, which unexpectedly led to ring-contracted oxazolo-isoquinolines 18. This rearrangement was explained by the additional methyl moiety in the 8-position inhibiting the formation of the planar intermediate iminium ion with phenylcyclohexanone. The allyl protective group of 11 and 18 was removed with RhCl3 and HCl to obtain the tricyclic compounds 5 and 19 without substituent at the oxazolone ring. The structures of the rearranged products 18 and 19 were elucidated by X-ray crystal structure analysis. The oxazolo-isoquinoline trans-18 with allyl moiety (Ki = 89 nM) and the oxazolo-benzazepine 5 without substituent at the oxazolone ring (Ki = 114 nM) showed GluN2B affinity in the same range as the lead compound 3. In two-electrode voltage clamp measurements, 5 displayed only weak inhibitory activity.

Molecular Insights into the Role of Pathogenic nsSNPs in GRIN2B Gene Provoking Neurodevelopmental Disorders.

The GluN2B subunit of N-methyl-D-aspartate receptors plays an important role in the physiology of different neurodevelopmental diseases. Genetic variations in the GluN2B coding gene (GRIN2B) have consistently been linked to West syndrome, intellectual impairment with focal epilepsy, developmental delay, macrocephaly, corticogenesis, brain plasticity, as well as infantile spasms and Lennox–Gastaut syndrome. It is unknown, however, how GRIN2B genetic variation impacts protein function. We determined the cumulative pathogenic impact of GRIN2B variations on healthy participants using a computational approach. We looked at all of the known mutations and calculated the impact of single nucleotide polymorphisms on GRIN2B, which encodes the GluN2B protein. The pathogenic effect, functional impact, conservation analysis, post-translation alterations, their driving residues, and dynamic behaviors of deleterious nsSNPs on protein models were then examined. Four polymorphisms were identified as phylogenetically conserved PTM drivers and were related to structural and functional impact: rs869312669 (p.Thr685Pro), rs387906636 (p.Arg682Cys), rs672601377 (p.Asn615Ile), and rs1131691702 (p.Ser526Pro). The combined impact of protein function is accounted for by the calculated stability, compactness, and total globularity score. GluN2B hydrogen occupancy was positively associated with protein stability, and solvent-accessible surface area was positively related to globularity. Furthermore, there was a link between GluN2B protein folding, movement, and function, indicating that both putative high and low local movements were linked to protein function. Multiple GRIN2B genetic variations are linked to gene expression, phylogenetic conservation, PTMs, and protein instability behavior in neurodevelopmental diseases. These findings suggest the relevance of GRIN2B genetic variations in neurodevelopmental problems.

CaMKII T286 phosphorylation has distinct essential functions in three forms of long-term plasticity

The Ca2+/calmodulin-dependent protein kinase II (CaMKII) mediates long-term potentiation or depression (LTP or LTD) after distinct stimuli of hippocampal NMDA-type glutamate receptors (NMDARs). NMDAR-dependent LTD prevails in juvenile mice, but a mechanistically different form of LTD can be readily induced in adults by instead stimulating metabotropic glutamate receptors (mGluRs). However, the role that CaMKII plays in the mGluR-dependent form of LTD is not clear. Here we show that mGluR-dependent LTD also requires CaMKII and its T286 autophosphorylation (pT286), which induces Ca2+-independent autonomous kinase activity. In addition, we compared the role of pT286 among three forms of long-term plasticity (NMDAR-dependent LTP and LTD, and mGluR-dependent LTD) using simultaneous live imaging of endogenous CaMKII together with synaptic marker proteins. We determined that after LTP stimuli, pT286 autophosphorylation accelerated CaMKII movement to excitatory synapses. After NMDAR-LTD stimuli, pT286 was strictly required for any movement to inhibitory synapses. Similar to NMDAR-LTD, we found the mGluR-LTD stimuli did not induce CaMKII movement to excitatory synapses. However, in contrast to NMDAR-LTD, we demonstrate that the mGluR-LTD did not involve CaMKII movement to inhibitory synapses and did not require additional T305/306 autophosphorylation. Thus, despite its prominent role in LTP, we conclude that CaMKII T286 autophosphorylation is also required for both major forms of hippocampal LTD, albeit with differential requirements for the heterosynaptic communication of excitatory signals to inhibitory synapses.

Chronic treatment with enhancer drugs modifies the gene expression of selected parameters related to brain plasticity in rats under stress conditions

Selegiline, also known as L-deprenyl, and (2R)-1-(1-benzofuran-2-yl)-N-propylpentane-2-amine (BPAP) were found to induce enhancement of monoamine neurotransmission in low and very low doses. In addition, these enhancers may modify glutamatergic neurotransmission. The aim of the present study was to test the hypothesis that under stress conditions, chronic treatment with enhancer drugs has a positive impact on the glutamatergic system and other parameters related to brain plasticity, stress-related systems, and anxiety behavior. We exposed male Wistar rats to a chronic mild stress procedure combined with chronic treatment with two synthetic enhancer drugs. The gene expression of GluR1, an AMPA receptor subunit was reduced by repeated treatment with deprenyl in the hippocampus and with both BPAP and deprenyl in the prefrontal cortex. A significant reduction of NMDA receptor subunit GluN2B expression was observed in the hippocampus but not in the prefrontal cortex. Deprenyl treatment led to an enhancement of hippocampal BDNFmRNA concentrations in stress-exposed rats. Treatment with enhancer drugs failed to induce significant changes in stress hormone concentrations or anxiety behavior. In conclusion, the present study in chronically stressed rats showed that concomitant treatment with enhancer drugs did not provoke substantial neuroendocrine changes, but modified gene expression of selected parameters associated with brain plasticity. Observed changes may indicate a positive influence of enhancer drugs on brain plasticity, which is important for preventing negative consequences of chronic stress and enhancement of stress resilience. It may be suggested that the changes in glutamate receptor subunits induced by enhancer drugs are brain region-specific and not dose-related.

Evaluation of (rac)-, (R)-, and (S)-18F-OF-NB1 for Imaging GluN2B Subunit-Containing N-Methyl-d-Aspartate Receptors in Nonhuman Primates.

Despite 2 decades of research, no N-methyl-d-aspartate (NMDA) glutamate receptor (GluN) subtype 2B (GluN1/2B) radioligand is yet clinically validated. Previously, we reported on (rac)-18F-OF-NB1 as a promising GluN1/2B PET probe in rodents and its successful application for the visualization of GluN2B-containing NMDA receptors in postmortem brain tissues of patients with amyotrophic lateral sclerosis. In the current work, we report on the invivo characterization of (rac)-, (R)-, and (S)-18F-OF-NB1 in nonhuman primates. Methods: PET scans were performed on rhesus monkeys. Plasma profiling was used to obtain the arterial input function. Regional brain time-activity curves were generated and fitted with the 1- and 2-tissue-compartment models and the multilinear analysis 1 method, and the corresponding regional volumes of distribution were calculated. Blocking studies with the GluN1/2B ligand Co 101244 (0.25 mg/kg) were performed for the enantiopure radiotracers. Receptor occupancy, nonspecific volume of distribution, and regional binding potential (BP ND) were obtained. Potential off-target binding toward σ1 receptors was assessed for (S)-18F-OF-NB1 using the σ1 receptor ligand FTC-146. Results: Free plasma fraction was moderate, ranging from 12% to 16%. All radiotracers showed high and heterogeneous brain uptake, with the highest levels in the cortex. (R)-18F-OF-NB1 showed the highest uptake and slowest washout kinetics of all tracers. The 1-tissue-compartment model and multilinear analysis 1 method fitted the regional time-activity curves well for all tracers and produced reliable regional volumes of distribution, which were higher for (R)- than (S)-18F-OF-NB1. Receptor occupancy by Co 101244 was 85% and 96% for (S)-18F-OF-NB1 and (R)-18F-OF-NB1, respectively. Pretreatment with FTC-146 at both a low (0.027 mg/kg) and high (0.125 mg/kg) dose led to a similar reduction (48% and 49%, respectively) in specific binding of (S)-18F-OF-NB1. Further, pretreatment with both Co 101244 and FTC-146 did not result in a further reduction in specific binding compared with Co 101244 alone in the same monkey (82% vs. 81%, respectively). Regional BP ND values ranged from 1.3 in the semiovale to 3.4 in the cingulate cortex for (S)-18F-OF-NB1. Conclusion: Both (R)- and (S)-18F-OF-NB1 exhibited high binding specificity to GluN2B subunit-containing NMDA receptors. The fast washout kinetics, good regional BP ND values, and high plasma free fraction render (S)-18F-OF-NB1 an attractive radiotracer for clinical translation.

Synaptic Dysfunction by Mutations in GRIN2B: Influence of Triheteromeric NMDA Receptors on Gain-of-Function and Loss-of-Function Mutant Classification.

GRIN2B mutations are rare but often associated with patients having severe neurodevelopmental disorders with varying range of symptoms such as intellectual disability, developmental delay and epilepsy. Patient symptoms likely arise from mutations disturbing the role that the encoded NMDA receptor subunit, GluN2B, plays at neuronal connections in the developing nervous system. In this study, we investigated the cell-autonomous effects of putative gain- (GoF) and loss-of-function (LoF) missense GRIN2B mutations on excitatory synapses onto CA1 pyramidal neurons in organotypic hippocampal slices. In the absence of both native GluN2A and GluN2B subunits, functional incorporation into synaptic NMDA receptors was attenuated for GoF mutants, or almost eliminated for LoF GluN2B mutants. NMDA-receptor-mediated excitatory postsynaptic currents (NMDA-EPSCs) from synaptic GoF GluN1/2B receptors had prolonged decays consistent with their functional classification. Nonetheless, in the presence of native GluN2A, molecular replacement of native GluN2B with GoF and LoF GluN2B mutants all led to similar functional incorporation into synaptic receptors, more rapidly decaying NMDA-EPSCs and greater inhibition by TCN-201, a selective antagonist for GluN2A-containing NMDA receptors. Mechanistic insight was gained from experiments in HEK293T cells, which revealed that GluN2B GoF mutants slowed deactivation in diheteromeric GluN1/2B, but not triheteromeric GluN1/2A/2B receptors. We also show that a disease-associated missense mutation, which severely affects surface expression, causes opposing effects on NMDA-EPSC decay and charge transfer when introduced into GluN2A or GluN2B. Finally, we show that having a single null Grin2b allele has only a modest effect on NMDA-EPSC decay kinetics. Our results demonstrate that functional incorporation of GoF and LoF GluN2B mutants into synaptic receptors and the effects on EPSC decay times are highly dependent on the presence of triheteromeric GluN1/2A/2B NMDA receptors, thereby influencing the functional classification of NMDA receptor variants as GoF or LoF mutations. These findings highlight the complexity of interpreting effects of disease-causing NMDA receptor missense mutations in the context of neuronal function.

Fentanyl induces autism-like behaviours in mice by hypermethylation of the glutamate receptor gene Grin2b

BackgroundEnvironmental factors contribute to autism spectrum disorder. Fentanyl, one of the most widely used opioid analgesics in anaesthesia, can induce neurotoxicity, but its role in autism remains unknown. We determined whether fentanyl induced autism-like behaviours in young mice and the underlying mechanisms. MethodsYoung male and female mice received fentanyl at postnatal days 6, 8, and 10, and performed behavioural tests, including three-chamber social preference, elevated plus maze, grooming behaviour, and open-field test, from postnatal days 30–32. Expression of Grin2b, the gene encoding the GluN2B subunit of the N-methyl-d-aspartate receptor, was assessed in the anterior cingulate cortex of male mice using fluorescence in situ hybridisation histochemistry. We used bisulfite target sequencing to determine Grin2b hypermethylation sites after fentanyl treatment. In the specific activation and rescue experiments, we injected the mu opioid receptor agonist [D-Ala,2 N-MePhe,4 Gly-ol]-enkephalin (DAMGO) or Grin2b overexpression lentivirus into the anterior cingulate cortex of male mice. ResultsFentanyl induced autism-like behaviours in both young male and female mice, and downregulated Grin2b expression (0.49-fold [0.08] vs 1.00-fold [0.09]; P<0.01) and GluN2B protein amounts (0.38-fold [0.07] vs 1.00-fold [0.12]; P<0.01) in the anterior cingulate cortex through hypermethylation of Grin2b. The mu-opioid receptor antagonist naloxone and overexpression of Grin2b in anterior cingulate cortex attenuated the fentanyl-induced effects, whereas DAMGO injection into the anterior cingulate cortex induced autism-like behaviours. ConclusionsThese data suggest that fentanyl induces autism-like behaviours in young mice via an epigenetic mechanism. Further research is required to determine possible clinical relevance to autism risk.

Negative allosteric modulators of NMDA receptors with GluN2B subunit: Alanine-derived benzoxazolone bioisosteres of 2-methyl-3-benzazepine-1,7-diols.

Inspired by besonprodil, the phenol of potent negative allosteric modulators of GluN2B-N-methyl-d-aspartate (NMDA) receptors was replaced by a benzoxazolone system. To increase the similarity to the lead compounds, an additional methyl moiety was installed in the 8-position of tricyclic oxazolobenzazepines, resulting in compounds 6. The additional methyl moiety originates from alanine, which was introduced by a Mitsunobu reaction of benzoxazolylethanol 7 with N-triflyl-protected alanine methyl ester. A crucial feature of the synthesis was the protection of the oxazolone ring by an allyl moiety, which was cleaved off at the end of the synthesis by RhCl3 -catalyzed isomerization. Due to the additional methyl moiety, the intramolecular Friedel-Crafts acylation of acid 10 to afford ketone 11 required careful optimization to minimize the formation of the side product tetrahydroisoquinoline 16. Alkylation or reductive alkylation of secondary amine 13 led to diastereomeric oxazolobenzazepines cis-14 and trans-14, which were separated by flash chromatography. Phenylbutyl derivatives cis-6a and trans-6a revealed twofold higher GluN2B affinity than analog 5a without 8-CH3 group. The methylated oxazolobenzazepines 6 and 14 did not interact with the phencyclidine binding site of NMDA receptors and σ2 receptors. However, the σ1 receptor preferred cis-configured oxazolobenzazepines. The highest σ1 receptor affinities were obtained for cis-14a (Ki = 26 nM) and cis-6b (Ki = 30 nM).

HMGB1‐mediated microglial activation as a mechanism for cognitive dysfunction in neuropsychiatric lupus

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by the presence of anti‐nuclear antibodies as well as a broad array of clinical manifestations spanning nearly every organ system. Neuropsychiatric syndromes affect over one half of SLE patients, presenting most commonly as cognitive dysfunction or headache. The pathophysiology of neuropsychiatric lupus is not fully understood; however, a critical mechanism has been identified, which involves passage of inflammatory cytokines and anti‐brain antibodies through the blood‐brain barrier.We previously developed a mouse model to study the effects of anti‐dsDNA antibodies that cross‐react with the NMDA receptor (NMDAR), termed DNRAbs, which are present in approximately 30% of SLE patients and whose presence in the CSF is associated with non‐focal CNS manifestations of neuropsychiatric lupus. In this mouse model, non‐spontaneously autoimmune mice are immunized with DWEYS peptide, a DNA mimotope that elicits production of DNRAb antibodies, which act as positive allosteric modulators of the NMDAR. DNRAbs only enter the brain when the blood‐brain barrier is porous; so, we administer LPS, which breaches the blood‐brain barrier in the hippocampus. In this model, we discovered two stages of brain injury. In the first stage, lasting up to a week, we observed excitotoxic neuronal death, secondary to DNRAb‐mediated NMDAR activation. In the second stage, lasting months, we identified an inflammatory homeostasis, consisting of microglial activation, loss of dendritic arborization in surviving hippocampal neurons, and neuronal secretion of HMGB1, a chromatin protein that can be secreted to act as a damage‐associated molecular pattern (DAMP). Once secreted by neurons, HMGB1 interacts with two important receptors —RAGE and TLR4 — inducing a cascade of downstream inflammatory events. In addition, HMGB1 binds to the GluN2B subunit of NMDARs and the complement component C1q, forming a molecular bridge that facilitates synaptic pruning.Here we show that HMGB1 acts directly on microglia, where it activates microglia through RAGE and TLR4 signaling. We found that HMGB1 stimulates microglia to secrete IFNa, transcriptionally upregulating interferon regulatory factors, including IRF7. We also show that IFNa secreted by microglia acts in an autocrine fashion, regulating IFN‐response genes such as MX1; IFNa also induces C3 and C1q transcription in microglia. HMGB1 also stimulates microglia to secrete TNFa and IL‐1b, which enhances the inflammatory milieu in surrounding neurons.In addition, we found that the ACE‐inhibitor captopril reverses dendritic pruning in DWEYS‐immunized mice­­ — an essential component of the second stage of DNRAb‐related damage — by regulating expression of the inhibitory receptor LAIR1 in microglia. Whereas captopril reverses dendritic pruning in DWEYS‐immunized wildtype mice, captopril has no such effect on LAIR1 knockout mice. We are currently analyzing RNA sequencing data from microglia isolated from captopril‐treated, DWEYS immunized mice, to characterize the transcriptional program responsible for this process.

Elucidating the Mechanisms of CaMKII‐CaMKAP Interactions

Dynamic changes of intracellular calcium ion (Ca2+) concentrations are critical signaling mechanisms in all cells and organisms, and Ca2+ oscillations must be precisely decoded by intracellular signaling molecules, such as the ubiquitous Ca2+/calmodulin‐dependent protein kinase II (CaMKII). Four mammalian CaMKII genes encode diverse alternative mRNA splice variants, which are thought to be targeted to different subcellular compartments to regulate numerous cell responses (exocytosis, gene expression, cell cycle). Our lab hypothesized that the localization, activity, and biological functions of CaMKII are precisely modulated by CaMKII‐associated proteins (CaMKAPs), and we have detected over 100 CaMKAPs associated with brain CaMKII holoenzymes. Some CaMKII‐CaMKAP interactions are critical for normal physiological function. CaMKIIδ binding to the L‐type Ca2+ channel (LTCC) b2a subunit is essential for cardiac function1, and binding of neuronal CaMKII to the GluN2B subunit of the NMDA‐type glutamate receptor is necessary for synaptic plasticity2. While b2a, GluN2B and most other CaMKAPs preferentially interact with activated CaMKII, the exact molecular mechanisms for these interactions, and their physiological roles, have yet to be determined. Several residues in the catalytic domain of CaMKII have been implicated in CaMKAP binding. Previous mutagenesis studies from our lab indicated that mutation of valine 102 (V102) to glutamate disrupts CaMKII binding to LTCC α1 subunits, but not to b2a, GluN2B, or densin, and also disrupts neuronal excitation‐transcription coupling3. Additionally, a naturally‐occurring CaMKIIα Glu183Val mutation linked to autism spectrum disorder and intellectual disability disrupted interactions with all CaMKAPs tested4. To further probe the molecular basis of CaMKII‐CaMKAP interactions, we are exploiting available structural information to design additional CaMKII catalytic domain mutations, and then test their effect on CaMKII‐CaMKAP interactions, as well as CaMKII activity toward multiple substrates. We are also examining the impact of additional human CaMKII mutations. Initial studies indicate that some CaMKII mutations differentially affect binding to various CaMKAPs, CaMKII autophosphorylation, and phosphorylation of a representative substrate, the GluA1 subunit of AMPA‐type glutamate receptors. Our ongoing studies are further exploring the impact of these CaMKII mutations. Expanding our knowledge of mechanisms underlying CaMKII‐CaMKAP interactions is required to better understand CaMKII biology, the role of CaMKAPs, and the molecular basis of Ca2+signaling. Koval et al., 2010. PNAS 107:4996. Halt et al., 2012. EMBO J. 31:1203. Wang et al., 2017. J Biol Chem 292:17324. Stephenson et al., 2017. J Neurosci 37:2216.

Positive modulation of N-methyl-D-aspartate receptors in the mPFC reduces the spontaneous recovery of fear

N-methyl-D-aspartate receptor (NMDAR) modulators have recently received increased attention as potential therapeutics for posttraumatic stress disorder (PTSD). Here, we tested a novel NMDAR-positive modulator, NYX-783, in the following two rodent models of PTSD: an auditory fear-conditioning model and a single-prolonged stress (SPS) model. We examined the ability of NYX-783 to reduce subsequent fear-based behaviors by measuring enhanced fear extinction and reduced spontaneous recovery (spontaneous return of fear) in male mice. NYX-783 administration significantly reduced spontaneous recovery in both PTSD models and enhanced fear extinction in the SPS model. Furthermore, NYX-783 increased the NMDA-induced inward currents of excitatory and inhibitory neurons in the infralimbic medial prefrontal cortex (IL mPFC) and that the GluN2B subunit of NMDARs on pyramidal neurons in the IL mPFC is required for its effect on spontaneous recovery. The downstream expression of brain-derived neurotrophic factor was required for NYX-783 to achieve its behavioral effect. These results elucidate the cellular targets of NYX-783 and the molecular mechanisms underlying the inhibition of spontaneous recovery. These preclinical findings support the hypothesis that NYX-783 may have therapeutic potential for PTSD treatment and may be particularly useful for inhibiting spontaneous recovery.

Synthesis and biological evaluation of conformationally restricted GluN2B ligands derived from eliprodil

N-Methyl-d-aspartate (NMDA) receptors containing one or two GluN2B subunits play a crucial role in a variety of neurodegenerative diseases, such as Alzheimer's and Huntington's disease. In order to increase selectivity for GluN2B NMDA receptors, the piperidine ring of eliprodil (2) was conformationally restricted by introduction of an ethano bridge across C-2 and C-6 resulting in a tropane scaffold. Benzylidenetropanes 15 and 16 and benzyltropanes 17 and 18 were prepared by nucleophilic opening of enantiomerically pure phenyloxiranes 13 and 14 with racemic secondary amines (Z/E)-11 and diastereomeric mixtures (r/s)-12. The diastereomers were separated by preparative HPLC to obtain enantiomerically pure test compounds 15–18. The absolute and relative configuration of the products were determined by X-ray crystal structure analysis. Benzylidenetropanes 15 and 16 as well as benzyltropanes 17 and 18 display very high GluN2B affinity in receptor binding studies. Benzylidinetropanes with the phenyl moiety oriented towards C-5 of the tropane system showed higher GluN2B affinity than their analogs with the phenyl moiety oriented towards C-1. In benzyltropanes endo-configured stereoisomers exhibit higher GluN2B affinity than exo-configured diastereomers. Unfortunately, tropanes 15–18 show also high σ1 and σ2 affinity with the same trends for the stereoisomers as for GluN2B affinity. The high-affinity GluN2B ligand (R,r)-17b was able to inhibit the ion flux in two-electrode voltage clamp experiments using GluN1a/GluN2B expressing oocytes.

Anti-inflammatory drugs prevent memory and hippocampal plasticity deficits following initial binge-like alcohol exposure in adolescent male rats.

Binge drinking during adolescence impairs learning and memory on the long term, and many studies suggest a role of neuroinflammation. However, whether neuroinflammation occurs after the very first exposures to alcohol remains unclear, while initial alcohol exposure impairs learning for several days in male rats. To investigate the role of neuroinflammation in the effects of only two binge-like episodes on learning and on neuronal plasticity in adolescent male rat hippocampus. Animals received two ethanol i.p. injections (3g/kg) 9h apart. Forty-eight hours later, we recorded long-term depression (LTD) and potentiation (LTP) in CA1 area of hippocampus slices. In isolated CA1, we measured immunolabelings for microglial activation and Toll-like receptor 4 (TLR4) and mRNA levels for several cytokines. Forty-eight hours after the two binges, rats performed worse than control rats in novel object recognition, LTD was reduced, LTP was increased, and excitatory neurotransmission was more sensitive to an antagonist of the GluN2B subunit of the NMDA receptor. Exposure to ethanol with minocycline or indomethacin, two anti-inflammatory drugs, or with a TLR4 antagonist, prevented all effects of ethanol. Immunolabelings at 48h showed a reduction of neuronal TLR4 that was prevented by minocycline pretreatment, while microglial reactivity was undetected and inflammatory cytokines mRNA levels were unchanged. Two binge-like ethanol exposures during adolescence in rat involved neuroinflammation leading to changes in TLR4 expression and in GluN2B functioning inducing disturbances in synaptic plasticity and cognitive deficits. Anti-inflammatory drugs are good candidates to prevent brain function and memory deficits induced by few binge-drinking episodes.

Amyloid-β oligomers interact with NMDA receptors containing GluN2B subunits and metabotropic glutamate receptor 1 in primary cortical neurons: Relevance to the synapse pathology of Alzheimer’s disease

Recent evidence suggests that soluble amyloid-β oligomers (AβOs) act as a key factor in the pathogenetic mechanism of Alzheimer's disease (AD). AβOs induce neurotoxic and synaptotoxic effects probably through binding to certain receptors, however it remains unclarified which receptors are most critically involved. In addition, dysregulation in glutamatergic signaling is implicated in AD. In this study, we used a rat primary cortical neuron model to investigate AβO-induced aberrations of synaptic proteins and binding of extracellular AβOs to candidate receptors in the glutamatergic system. Immunocytochemical analyses showed that both presynaptic (SNAP-25, synapsin I) and postsynaptic (spinophilin, homer 1b/c) proteins appeared to aberrantly dislocate from synapses upon AβO treatment. Double immunofluorescence staining of AβO-treated neurons without permeabilization pretreatment revealed that extracellular AβOs exist over neuronal soma and neurites and clearly colocalized with GluN1 and GluN2B subunits of NMDA receptors and metabotropic glutamate receptor 1 (mGluR1), but not with NMDA GluN2A subunits and mGluR5. AβO treatment altered neither total protein levels nor intracellular localizations of these receptors. These results suggest that extracellular AβOs specifically bind to both NMDA receptors containing GluN2B subunits and mGluR1. It is likely that binding of AβOs to these receptors induces various pathological responses, consequently leading to synaptic disruptions. Our study thus highlights the important roles of GluN2B-containing NMDA receptors and mGluR1 receptors in the synapse pathology in AD.

Ifenprodil tartrate treatment of adolescents with post-traumatic stress disorder: A double-blind, placebo-controlled trial

BackgroundSeveral lines of evidence suggest that glutamatergic neurotransmission via the N-methyl-D-aspartate (NMDA) receptor plays a role in certain behavioral manifestations common to Post-Traumatic Stress Disorder (PTSD). Ifenprodil tartrate is a neuroprotective agent that binds to the GluN2B subunit of the NMDA receptor. The aim of this study is to confirm whether ifenprodil tartrate is effective in the adolescent PTSD patients. MethodsThis is a randomized, double-blind, placebo‐controlled trial. Ten adolescent (13 to 18 years old) PTSD patients were randomized into two arms: placebo (n = 4), 40 mg/day ifenprodil tartrate (n = 6) for 4 weeks. All of the patients were assessed by IES-R-J (Primary outcome measure), TSCC-J, CDRS-R, DSRS-C-J and CGI-I. ResultsA comparison of baseline IES-R-J total scores and 4-week end-point scores showed a mild trend of improvement (p = 0.0895) and the difference score was -9.314. A comparison of baseline scores and 2-week intermediate-point scores showed that IES-R-J hyperarousal subscores and TSCC-J subscores (dissociation subscores, sexual concerns subscores) improved significantly. A comparison of baseline TSCC-J sexual concerns subscores and 4-week end-point scores improved significantly. ConclusionsOur study may prove to be an short-term effective alternative safe treatment for adolescent patients with PTSD.