The jasmonates are a class of oxylipin phytohormones known to exhibit anti-inflammatory, antioxidant, and anti-cancer effects in mammalian cells. We investigated the ability of three jasmonate compounds (jasmonic acid, methyl jasmonate, and 12-OPDA) and two structurally distinct jasmonate precursors (alpha-linolenic acid and palmitic acid) to attenuate inflammation in an in vitro model of neurodegenerative disease, for which the mechanisms of action have not been well identified. The study modeled chronic neuroinflammation in SH-SY5Y neuroblastoma cells using exogenous prostaglandin E 2 (PGE 2 ) treatment. Prostaglandin E 2 caused concentration-dependent levels of inflammation and SH-SY5Y cell death, which were attenuated by the jasmonates and their precursors. To this end, structural similarities between the jasmonates and PGE 2 were correlated with increased potency of their anti-inflammatory effects. Downstream biomarkers of signaling through the pro-inflammatory E prostanoid receptor subtype 2 (EP2) were then quantified using enzyme-linked immunosorbent assay methods. Of the compounds tested, only jasmonic acid and methyl jasmonate attenuated inflammation in the SH-SY5Y cells via crosstalk with the PGE 2 /EP2 signaling axis. Additionally, structural models and molecular binding simulations serve as evidence for our hypothesis that JA and MeJA achieve this crosstalk through competitive inhibition of the receptor EP2. This novel finding has implications in the study of neurodegenerative diseases for which the disease pathology is related to chronic neuroinflammation, including Alzheimer’s Disease (AD), Parkinson’s Disease (PD), amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS). In addition, these findings add to the understanding of the relationship between pro-inflammatory prostaglandin E 2 signaling and disease severity.

Background: The GluA2 subunit of AMPA receptors (AMPARs) undergoes RNA editing at a specific base mediated by the enzyme ADAR2, changing the coded amino acid from a glutamine to arginine at the so-called Q/R site, which is critical for regulating calcium permeability. ADAR2 exists as multiple alternatively-spliced variants within mammalian cells with differing editing efficiency. New method: In this study, phosphorodiamidate morpholino oligomers (PMOs) were used to increase Q/R site editing, by affecting the alternative splicing of ADAR2. Results: PMOs targeting the ADAR2 pre-mRNA transcript successfully induced alternative splicing around the AluJ cassette leading to expression of a more active isoform with increased editing of the GluA2 subunit compared to control. Comparison with existing method(s): Previously PMOs have been used to disrupt RNA editing via steric hindrance of the GluA2 RNA duplex. In contrast we report PMOs that can increase the expression of more catalytically active variants of ADAR2, leading to enhanced GluA2 Q/R RNA editing. Conclusions: Using PMOs to increase Q/R site editing is presented here as a validated method that would allow investigation of downstream cellular processes implicated in altered ADAR2 activity.

Alix/AIP1 is an adaptor protein involved in apoptosis, endocytic membrane trafficking and brain development. Alix has been found within the human postsynaptic density (PSD) and, since NMDA receptors (NMDARs) are central components of the PSD, we hypothesized that the close proximity of both proteins may allow Alix to influence the downstream pathways following NMDAR activation. NMDARs play important roles in excitotoxicity and we evaluated the effects of recombinant Alix in an NMDAR cell death assay. Overexpression of Alix with NMDARs increases the potency of NMDAR‐ induced cell death compared to cells expressing only NMDARs, and this requires expression of the Alix C‐terminal region. Therefore, we demonstrate a previously unreported role for Alix as a potential modulator of NMDAR function.

Perampanel is an adjunctive treatment for epilepsy that works through the direct inhibition of AMPA receptors. The same molecular mechanism has recently been shown for a fatty acid, decanoic acid, prescribed in the medium chain triglyceride ketogenic diet for the treatment of patients with drug-resistant epilepsy. Because each compound has been proposed to act through a distinct AMPA receptor binding site, we predicted that perampanel and decanoic acid would act synergistically against AMPA receptors and, consequently, seizures. Here, we show a synergistic interaction between perampanel and decanoic acid in direct AMPA receptor inhibition, in an ex vivo model of seizure activity, and against seizure-induced activity in human brain slices. These data support a potential role for combination treatment using perampanel and dietary decanoic acid to provide enhanced seizure control.

Previous research has showed that AMPA receptor can be used as a molecular target for anti-epileptic drugs, and there is evidence of an inhibitory effect of several fatty acids on AMPA receptor, for example, Decanoic Acid (DA), 4-Ethyl Octanoic Acid (4EOA) and 4-Butylcy Cloheaxne Carboxylic Acid (4BCCA). However, as NMDA receptor and AMPA receptor are quite similar in structures and are widely distributed in CNS, there is a concern on whether the three fatty acids also have an effect on NMDA receptor, and NMDA receptor inhibition may result in some unwanted side effects, such as hallucinations, nightmares, and memory loss. Two-electrode voltage clamp was performed on the oocytes of Xenopus Laevis expressing the NMDAR subunit GluN1/N2A, to record agonist evoked inward currents generated from oocytes exposed to different concentrations of fatty acid solutions. On further examination it was found that all of the three acids have some inhibitory effect on NMDA recceptor, and seem to act as non-competitive antagonists. The 4EOA has the strongest inhibition effect on NMDA receptor, followed by 4BCCA and DA. However, the three fatty acids only have a clear inhibitory effect when the concentration of the corresponding fatty acid is higher than 500 µM, which is considered high for clinical use. Moreover, the fatty acids tested had a stronger inhibitory effect on NMDA receptor containing the GluN1/N2B subunits.

The medium chain triglyceride ketogenic diet is an established treatment for drug-resistant epilepsy that increases plasma levels of decanoic acid and ketones. Recently, decanoic acid has been shown to provide seizure control in vivo, yet its mechanism of action remains unclear. Here we show that decanoic acid, but not the ketones beta-hydroxybutryate or acetone, shows antiseizure activity in two acute ex vivo rat hippocampal slice models of epileptiform activity. To search for a mechanism of decanoic acid, we show it has a strong inhibitory effect on excitatory, but not inhibitory, neurotransmission in hippocampal slices. Using heterologous expression of excitatory ionotropic glutamate receptor AMPA subunits in Xenopus oocytes, we show that this effect is through direct AMPA receptor inhibition, a target shared by a recently introduced epilepsy treatment perampanel. Decanoic acid acts as a non-competitive antagonist at therapeutically relevant concentrations, in a voltage-and subunit-dependent manner, and this is sufficient to explain its antiseizure effects. This inhibitory effect is likely to be caused by binding to sites on the M3 helix of the AMPA-GluA2 transmembrane domain; independent from the binding site of perampanel. Together our results indicate that the direct inhibition of excitatory neurotransmission by decanoic acid in the brain contributes to the anti-convulsant effect of the medium chain triglyceride ketogenic diet.

Regulation of the activity of N-methyl-D-aspartate receptors (NMDARs) at glutamatergic synapses is essential for certain forms of synaptic plasticity underlying learning and memory and is also associated with neurotoxicity and neurodegenerative diseases. In this report, we investigate the role of Src-like adaptor protein (Slap) in NMDA receptor signaling. We present data showing that in dissociated neuronal cultures, activation of ephrin (Eph) receptors by chimeric preclustered eph-Fc ligands leads to recruitment of Slap and NMDA receptors at the sites of Eph receptor activation. Interestingly, our data suggest that prolonged activation of EphA receptors is as efficient in recruiting Slap and NMDA receptors as prolonged activation of EphB receptors. Using established heterologous systems, we examined whether Slap is an integral part of NMDA receptor signaling. Our results showed that Slap does not alter baseline activity of NMDA receptors and does not affect Src-dependent potentiation of NMDA receptor currents in Xenopus oocytes. We also demonstrate that Slap reduces excitotoxic cell death triggered by activation of NMDARs in HEK293 cells. Finally, we present evidence showing reduced levels of NMDA receptors in the presence of Slap occurring in an activity-dependent manner, suggesting that Slap is part of a mechanism that homeostatically modulates the levels of NMDA receptors.

The medium chain triglyceride (MCT) ketogenic diet is used extensively for treating refractory childhood epilepsy. This diet increases the plasma levels of medium straight chain fatty acids. A role for these and related fatty acids in seizure control has not been established. We compared the potency of an established epilepsy treatment, Valproate (VPA), with a range of MCT diet-associated fatty acids (and related branched compounds), using in vitro seizure and in vivo epilepsy models, and assessed side effect potential in vitro for one aspect of teratogenicity, for liver toxicology and in vivo for sedation, and for a neuroprotective effect. We identify specific medium chain fatty acids (both prescribed in the MCT diet, and related compounds branched on the fourth carbon) that provide significantly enhanced in vitro seizure control compared to VPA. The activity of these compounds on seizure control is independent of histone deacetylase inhibitory activity (associated with the teratogenicity of VPA), and does not correlate with liver cell toxicity. In vivo, these compounds were more potent in epilepsy control (perforant pathway stimulation induced status epilepticus), showed less sedation and enhanced neuroprotection compared to VPA. Our data therefore implicates medium chain fatty acids in the mechanism of the MCT ketogenic diet, and highlights a related new family of compounds that are more potent than VPA in seizure control with a reduced potential for side effects. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

Mandarin translation of abstract Background and purpose: The carboxy terminal domain (CTD) of NR2 N ‐methyl‐ d ‐aspartate receptor (NMDAR) subunits interacts with numerous scaffolding and signal transduction proteins. Mutations of this region affect trafficking and downstream signalling of NMDARs. This study determines to what extent characteristic pharmacological properties of NR2A‐containing NMDARs are influenced by this key functional domain. Experimental approach: Using recombinant receptor expression in Xenopus laevis oocytes and two electrode voltage clamp recordings we characterized pharmacological properties of rat NR1/NR2A NMDARs with altered CTDs. We assessed the effects of truncating [at residue Iso1098; NR2A(trunC)] and deleting [from residue Phe822; NR2A(delC)] the CTD of NR2A NMDAR subunits on agonist potencies, channel block by Mg 2+ and memantine and potentiation of NMDAR‐mediated responses by chelating contaminating divalent cations. Key results: Truncation or deletion of the CTD of NR2A NMDAR subunits did not affect glutamate potency [EC 50 = 2.2 µmol·L −1 , NR2A(trunC); 2.7 µmol·L −1 , NR2A(delC) compared with 3.3 µmol·L −1 , NR2A(WT)] but did significantly increase glycine potency [EC 50 = 500 nmol·L −1 , NR2A(trunC); 900 nmol·L −1 , NR2A(delC) compared with 1.3 µmol·L −1 , NR2A(WT)]. Voltage‐dependent Mg 2+ block of NR2A(WT)‐ and NR2A(trunC)‐containing NMDARs was similar but low concentrations of Mg 2+ (1 µmol·L −1 ) potentiated NR1/NR2A(delC) NMDARs. Memantine block was not affected by changes to the structure of the NR2A CTD. EDTA‐induced potentiation was similar at each of the three NMDAR constructs. Conclusions and implications: Of the parameters studied only minor influences of the CTD were observed; these are unlikely to compromise interpretation of studies that make use of CTD‐mutated recombinant receptors or transgenic mice in investigations of the role of the CTD in NMDAR signalling. Mandarin translation of abstract

The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1(N598R) allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.

Many studies examine the actions of ethanol on N-methyl-D-aspartate (NMDA) receptors using concentrations that are highly toxic (>= 100 mM). This study re-assesses the actions of ethanol at concentrations based around the US/UK 'drink-drive' limit (17 mM). Using two-electrode voltage-clamp recordings we examined the actions of ethanol on recombinant GluN1/GluN2A and GluN1/GluN2B NMDA receptors expressed in Xenopus laevis oocytes. We also investigated its actions on NMDA receptors containing GluN2A subunits with truncated or deleted carboxy terminal domains. Ethanol inhibition was voltage-independent and for GluN1/GluN2A NMDA receptors mean inhibition (20 mM at - 60 mV) was 9.5 +/- 0.8% (n = 33) while corresponding values for GluN1/GluN2B NMDA receptors were 6.5 +/- 0.8% (n = 21). EC50 values for glutamate at GluN1/GluN2A and glutamate and glycine at GluN1/GluN2B NMDA receptors were unaffected by the presence of ethanol. We did however observe a small increase in glycine potency, in the presence of ethanol, at GluN1/GluN2A NMDA receptors. Neither voltage-dependent Mg2+ block nor memantine block was affected by ethanol. Reduced ethanol inhibition was observed however at NMDA receptors containing GluN2A subunits with mutated carboxy terminal domains. We conclude that the levels of inhibition seen with ethanol concentrations near to the US/UK drink-driving limit are very modest and even at higher (intoxicating) concentrations do not alter characteristic NMDA receptor properties. (C) 2009 Elsevier B.V. All rights reserved.

N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg(2+) and memantine that are dependent on the identity of the NR2 subunit present in the receptor-channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg(2+) and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg(2+) block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a approximately 10-fold reduction in Mg(2+) potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a approximately 2-fold increase in Mg(2+) potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg(2+) also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg(2+) is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.

Background: Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wld(s)) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wld(s)-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, crossbreeding of Wld(s) mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wld(s) mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wld(s) alleles is using copy number. Current approaches to genotype Wld(s) mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).Results: We have developed a rapid, robust and efficient genotyping method for Wld(s) using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous Wld(s) mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous Wld(s) mutation as well as animals expressing the Wld(s) transgene.Conclusion: We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wld(s) copy number. This technique will be of particular benefit in studies where Wld(s) mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the Wld(s) mutation.

Selective receptor antagonists are one of the most powerful resources in a pharmacologist's toolkit and are essential for the identification and classification of receptor subtypes and dissecting their roles in normal and abnormal body function. However, when the actions of antagonists are measured inappropriately and misleading results are reported, confusion and wrong interpretations ensue. This article gives a general overview of Schild analysis and the method of determining antagonist equilibrium constants. We demonstrate why this technique is preferable in the study of competitive receptor antagonism than the calculation of antagonist concentration that inhibit agonist-evoked responses by 50%. In addition we show how the use of Schild analysis can provide information on the outcome of single amino acid mutations in structure-function studies of receptors. Finally, we illustrate the need for caution when studying the effects of potent antagonists on synaptic transmission where the timescale of events under investigation is such that ligands and receptors never reach steady-state occupancy.

Heteromeric NMDARs are composed of coagonist glycine‐binding NR1 subunits and glutamate‐binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A–D). We show that the potency of a variety of endogenous and synthetic glycine‐site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D‐containing and the lowest at NR2A‐containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine‐binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand‐binding domains and coexpressed these with NR1 subunits inXenopus laevisoocytes. Glycine concentration–response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC50values similar to NR2A(WT)‐containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)‐containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand‐binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating.

The four N-methyl-d-aspartate (NMDA) receptor NR2 subunits (NR2A-D) have different developmental, anatomical, and functional profiles that allow them to serve different roles in normal and neuropathological situations. Identification of subunit-selective NMDA receptor agonists, antagonists, or modulators could prove to be both valuable pharmacological tools as well as potential new therapeutic agents. We evaluated the potency and efficacy of a wide range of glutamate-like compounds at NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors. Twenty-five of 53 compounds examined exhibited agonist activity at the glutamate binding site of NMDA receptors. Concentration-response relationships were determined for these agonists at each NR2 subunit. We find consistently higher potency at the NR2D subunit for a wide range of dissimilar structures, with (2S,4R)-4-methylglutamate (SYM2081) showing the greatest differential potency between NR2A- and NR2D-containing receptors (46-fold). Analysis of chimeric NR2A/D receptors suggests that enhanced agonist potency for NR2D is controlled by residues in both of the domains (Domain1 and Domain2) that compose the bilobed agonist binding domain. Molecular dynamics (MD) simulations comparing a crystallography-based hydrated NR1/NR2A model with a homology-based NR1/NR2D hydrated model of the agonist binding domains suggest that glutamate exhibits a different binding mode in NR2D compared with NR2A that accommodates a 4-methyl substitution in SYM2081. Mutagenesis of functionally divergent residues supports the conclusions drawn based on the modeling studies. Despite high homology and conserved atomic contact residues within the agonist binding pocket of NR2A and NR2D, glutamate adopts a different binding orientation that could be exploited for the development of subunit selective agonists and competitive antagonists.

We have examined the function of a conserved serine residue (Ser670) in the S2 ligand‐binding region of the NR2A N‐methyl‐d‐aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124‐fold. Single‐channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild‐type NMDA receptors. NR1/NR2A(S670G) shut‐time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single‐channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 ± 0.5 and 29.6 ± 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 ± 0.06). Consistent with a shortening of NMDA receptor‐channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside‐out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single‐channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.

We have quantified the effects of the N-methyl-d-aspartate (NMDA) receptor antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) at rat recombinant N-methyl-D-aspartate receptor (NR)1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either preapplied or coapplied with glutamate. The IC50 values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses, being 31 +/- 2 nM (with glutamate at its EC50 concentration) and 214 +/- 10 nM (at 10 times the EC50 concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K(B)) of 15 +/- 2 nM. Furthermore, Schild analysis of an NMDA receptor carrying a threonine-to-alanine point mutation in the NR2A ligand binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K(B) increased by around 15-fold. At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC50 value of 215 +/- 13 nM was obtained in the presence of the EC50 concentration of glutamate (1.5 microM), whereas a value of 2.2 +/- 0.14 microM was obtained with higher (15 microM) glutamate concentrations. Schild analysis gave a K(B) for NVP-AAM077 at NR2B-containing receptors of 78 +/- 3 nM. Finally, using a kinetic scheme to model "synaptic-like" activation of NMDA receptors, we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors.