Benzo[a]pyrene (BaP) is a genotoxic carcinogen and a neurotoxicant. The neurotoxicity of BaP is proposed to arise from either genotoxicity leading to neuronal cell death, or perturbed expression of N-methyl-d-aspartate receptor (NMDAR) subunits. To explore these hypotheses, we profiled hippocampal gene expression of adult male Muta(™) Mouse administered 0, 1, 35, or 70 mg BaP/kg bw per day by oral gavage for 3 days. Transcriptional profiles were examined by RNA-sequencing (RNA-seq), DNA microarrays, and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). BaP-DNA adducts in the cerebellum were quantified by (32) P-post-labeling to measure genotoxicity. RNA-seq revealed altered expression of 0, 260, and 219 genes (P-value < 0.05, fold-change ≥ ± 1.5) following exposure to the low, medium, and high doses, respectively; 54 genes were confirmed by microarrays. Microarray and RT-PCR analysis showed increased expression of NMDAR subunits Grina and Grin2a. In contrast, no effects on DNA-damage response genes were observed despite comparable BaP-DNA adduct levels in the cerebellum and in the lungs and livers of mice at similar BaP doses in previous studies. The results suggest that DNA-damage response does not play a major role in BaP-induced adult neurotoxicity. Meta-analysis revealed that BaP-induced transcriptional profiles are highly correlated with those from the hippocampus of transgenic mice exhibiting similar neurotoxicity outcomes to BaP-exposed mice and rats (i.e., defects in learning and memory). Overall, we suggest that BaP-induced neurotoxicity is more likely to be a consequence of NMDAR perturbation than genotoxicity, and identify other important genes potentially mediating this adverse outcome. Environ. Mol. Mutagen. 57:350-363, 2016. © 2016 Her Majesty the Queen in Right of Canada. Environmental and Molecular Mutagenesis © 2016 Environmental Mutagen Society.

Evidences have indicated a high degree of comorbidity of alcoholism and depression. N-acetylcysteine (NAC) has shown its clinical efficiency in the treatment of several psychiatric disorders and is identified as a multi-target acting drug. The ability of NAC to prevent alcohol abstinence-induced depression-like effects and underlying mechanism(s) have not been adequately addressed. This study was aimed to investigate the beneficial effects of NAC in the alcohol abstinence-induced depression developed following long-term voluntary alcohol intake. For evaluation of the effects of NAC, Sprague-Dawley rats were enabled to voluntary drinking of 4.5%, 7.5% and 9% v/v alcohol for fifteen days. NAC (25, 50, and 100 mg/kg) and fluoxetine (5 mg/kg) were injected intraperitoneally for three consecutive days during the alcohol abstinence period on the days 16, 17, 18. The behavioral studies were conducted employing forced swim test (FST), and tail suspension test (TST) on day 18 to determine the effects of N-acetylcysteine and fluoxetine in the ethanol withdrawal induced-depression. Blood alcohol concentration, alcohol biomarkers like SGPT, SGOT, ALP, GGT, and MCV were estimated by using commercially available kits. Serotonin concentrations were measured in the plasma, hippocampus and pre-frontal cortex using the rat ELISA kit. The expression of GRIN1, GRIN2A, GRIN2B genes for the N-methyl d-aspartate receptors (NMDAR) subunits in the hippocampus and the prefrontal cortex were also examined by reverse-transcription quantitative polymerase chain reaction. The results revealed that alcohol abstinence group depicted increased immobility time in FST and TST. Further, NAC exerted significant protective effect at the doses 50 mg/kg and 100 mg/kg, but 25 mg/kg showed insignificant protection against alcohol abstinence-induced depression. The increased level of biochemical parameters following ethanol abstinence were also reversed by NAC at the dose of 100 mg/kg. The significant reversal effect of NAC on the serotonin level following alcohol abstinence was greater in the hippocampus as compared to the third-day alcohol withdrawal group. The increased expression levels of GRIN2A and GRIN2B following ethanol abstinence were reversed with a higher dose of NAC (100 mg/kg) treatment. In conclusion, the results of the study reveal that NAC has remarkable protective effects in the alcohol abstinence-induced depression by modulating alcohol markers, serotonin levels and GRIN2A, GRIN2B gene expression of NMDAR signaling pathway in rats.

NRAS mutation in melanoma has been associated with aggressive tumor biology and poor prognosis. Although targeted therapy has been tested for NRAS mutated melanoma, response rates still appear much weaker, than in BRAF mutated melanoma. While plenty of cell lines exist, however, only few melanogenic cell lines retain their in vivo characteristics. In this work we present an intensively pigmented and well-characterized cell line derived from a highly aggressive NRAS mutated cutaneous melanoma, named MUG-Mel2. We present the clinical course, unique morphology, angiogenic properties, growth characteristics using in vivo experiments and 3D cell culture, and results of the exome gene sequencing of an intensively pigmented melanogenic cell line MUG-Mel2, derived from a cutaneous metastasis of an aggressive NRAS p. Q61R mutated melanoma. Amongst several genetic alterations, mutations in GRIN2A, CREBP, PIK3C2G, ATM, and ATR were present. These mutations, known to reinforce DNA repair problems in melanoma, might serve as potential treatment targets. The aggressive and fast growing behavior in animal models and the obtained phenotype in 3D culture reveal a perfect model for research in the field of NRAS mutated melanoma.

N-methyl-D-aspartate (NMDA) glutamate receptors play crucial roles in neuronal synaptic plasticity, learning and memory. However, as to whether different NMDA subunits are implicated in specific forms of memory is unclear. Moreover, nothing is known about the interspecific genetic variability of the GRIN2A subunit and how this variation can potentially explain evolutionary changes in behavioral phenotypes. Here, we used 28 primate GRIN2A sequences and various proxies of memory across primates to investigate the role of GRIN2A. Codon-specific sequence analysis on these sequences showed that GRIN2A in primates coevolved with a likely ecological proxy of spatial memory (relative home-range size) but not with other indices of non-spatial learning and memory such as social memory and social learning. Models based on gene averages failed to detect positive selection in primate branches with major changes in relative home-range size. This implies that accelerated evolution is concentrated in specific parts of the protein expressed by GRIN2A. Overall, our molecular evolution study, the first on GRIN2A, supports the notion that different NMDA subunits may play a role in specific forms of memory and that phenotypic diversity along with genetic evolution can be used to investigate the link between genes and behavior across evolutionary time.

Lead (Pb) is neurotoxic and children are highly susceptible to this effect, particularly within the context of continuous low-level Pb exposure. A current major challenge is identification of children who may be uniquely susceptible to Pb toxicity because of genetic predisposition. Learning and memory are among the neurobehavioral processes that are most notably affected by Pb exposure, and modification of N-methyl-D-aspartate receptors (NMDAR) that regulate these processes during development are postulated to underlie these adverse effects of Pb. We examined the hypothesis that polymorphic variants of genes encoding glutamate receptor, ionotropic, NMDAR subunits 2A and 2B, GRIN2A and GRIN2B, exacerbate the adverse effects of Pb exposure on these processes in children. Participants were subjects who participated as children in the Casa Pia Dental Amalgam Clinical Trial and for whom baseline blood Pb concentrations and annual neurobehavioral test results over the 7 year course of the clinical trial were available. Genotyping assays were performed for variants of GRIN2A (rs727605 and rs1070503) and GRIN2B (rs7301328 and rs1806201) on biological samples acquired from 330 of the original 507 trial participants. Regression modeling strategies were employed to evaluate the association between genotype status, Pb exposure, and neurobehavioral test outcomes. Numerous significant adverse interaction effects between variants of both GRIN2A and GRIN2B, individually and in combination, and Pb exposure were observed particularly among boys, preferentially within the domains of Learning & Memory and Executive Function. In contrast, very few interaction effects were observed among similarly genotyped girls with comparable Pb exposure. These findings support observations of an essential role of GRIN2A and GRIN2B on developmental processes underlying learning and memory as well as other neurological functions in children and demonstrate, further, modification of Pb effects on these processes by specific variants of both GRIN2A and GRIN2B genes. These observations highlight the importance of genetic factors in defining susceptibility to Pb neurotoxicity and may have important public health implications for future strategies aimed at protecting children and adolescents from potential health risks associated with low-level Pb exposure.

OBJECTIVE

The present study aimed to test whether exposure to benzo(a)pyrene [B(a)P] affects spatial learning and short-term memory by modulating the expression of the Gria1 and Grin2a glutamate receptor subunit genes in the hippocampus.

METHODS

Thirty-six 21-24-day-old, male rats were randomly assigned into high-, medium-, and low-dose toxin exposure groups (6.25, 2.5, and 1 mg/kg, respectively) and a control group, each containing nine rats. The behavioral performance of adult rats exposed to sub-chronic administration of B(a)P was monitored by learning and memory tests (Morris water maze). Real-time PCR assays were used to quantify Gria1 and Grin2a gene expression in the hippocampus.

RESULTS

At medium and high doses, B(a)P impaired spatial learning performance. The crossing-platform-location frequency and the time spent swimming in the platform area, which both relate to short-term memory, were significantly decreased in B(a)P-treated rats compared with controls. The level of Gria1 mRNA increased 2.6-5.9-fold, and the level of Grin2a mRNA increased 10-14.5-fold, with a greater fold increase associated with higher doses of B(a)P.

CONCLUSIONS

We demonstrated that sub-chronic administration of B(a)P inhibits spatial learning and short-term memory, and increases Gria1 and Grin2a expression in the hippocampus. This suggests a relationship of B(a)P exposure levels with Gria1 and Grin2a expression and impairment of short-term and spatial memory.

We studied the expression of mRNA for genes, whose protein products regulate the glutamate/NO/cGMP signal cascade in the frontal cortex, striatum, midbrain, and hippocampus of rats with various degrees of spontaneous morphine withdrawal syndrome. The concentration of Grin2a mRNA (subunit of the NMDA glutamate receptor) in the frontal cortex increased mainly in animals with severe abstinence. By contrast, the expression of mRNA for the Grin2b subunit in the striatum decreased in animals with mild abstinence. Variations in the content of mRNA for other products of the cascade (isoforms of NO-dependent guanylate cyclase and cGMP-dependent protein kinase) after morphine withdrawal were not related to the degree of abstinence. Our results suggest that the region-specific expression of mRNA for certain subunits of the NMDA glutamate receptor after morphine withdrawal can determine the degree of abstinence.

Studies to date have reported hundreds of genes connected to bipolar disorder (BP). However, many studies identifying candidate genes have lacked replication, and their results have, at times, been inconsistent with one another. This paper, therefore, offers a computational workflow that can curate and evaluate BP-related genetic data. Our method integrated large-scale literature data and gene expression data that were acquired from both postmortem human brain regions (BP case/control: 45/50) and peripheral blood mononuclear cells (BP case/control: 193/593). To assess the pathogenic profiles of candidate genes, we conducted Pathway Enrichment, Sub-Network Enrichment, and Gene-Gene Interaction analyses, with 4 metrics proposed and validated for each gene. Our approach developed a scalable BP genetic database (BP_GD), including BP related genes, drugs, pathways, diseases and supporting references. The 4 metrics successfully identified frequently-studied BP genes (e.g. GRIN2A, DRD1, DRD2, HTR2A, CACNA1C, TH, BDNF, SLC6A3, P2RX7, DRD3, and DRD4) and also highlighted several recently reported BP genes (e.g. GRIK5, GRM1 and CACNA1A). The computational biology approach and the BP database developed in this study could contribute to a better understanding of the current stage of BP genetic research and assist further studies in the field.

Inhalation of cadmium (Cd) is associated with lung diseases, but less is known concerning pulmonary effects of Cd found in the diet. Cd has a decades-long half-life in humans and significant bioaccumulation occurs with chronic dietary intake. We exposed mice to low-dose CdCl2 (10 mg/L in drinking water) for 20 weeks, which increased lung Cd to a level similar to that of nonoccupationally exposed adult humans. Cd-treated mice had increased airway hyperresponsiveness to methacholine challenge, and gene expression array showed that Cd altered the abundance of 443 mRNA transcripts in mouse lung. In contrast to higher doses, low-dose Cd did not elicit increased metallothionein transcripts in lung. To identify pathways most affected by Cd, gene set enrichment of transcripts was analyzed. Results showed that major inducible targets of low-dose Cd were neuronal receptors represented by enriched olfactory, glutamatergic, cholinergic, and serotonergic gene sets. Olfactory receptors regulate chemosensory function and airway hypersensitivity, and these gene sets were the most enriched. Targeted metabolomics analysis showed that Cd treatment also increased metabolites in pathways of glutamatergic (glutamate), serotonergic (tryptophan), cholinergic (choline), and catecholaminergic (tyrosine) receptors in the lung tissue. Protein abundance measurements showed that the glutamate receptor GRIN2A was increased in mouse lung tissue. Together, these results show that in mice, oral low-dose Cd increased lung Cd to levels comparable to humans, increased airway hyperresponsiveness and disrupted neuronal pathways regulating bronchial tone. Therefore, dietary Cd may promote or worsen airway hyperresponsiveness in multiple lung diseases including asthma.

BACKGROUND

Intelligence is a core construct of individual differences in cognitive abilities and a strong predictor of important life outcomes. Within recent years, rates of cesarean section have substantially increased globally, though little is known about its effect on neurodevelopmental trajectories. Thus, we aimed to investigate the influence of delivery by cesarean section on the genetics of intelligence in children.

METHODS

Participants were recruited through the Avon Longitudinal Study of Parents and Children (ALSPAC). Intelligence was measured by the Wechsler Intelligence Scale for Children (WISC). Genotyping was performed using the Illumina Human Hap 550 quad genome-wide SNP genotyping platform and was followed by imputation using MACH software. Genome-wide interaction analyses were conducted using linear regression.

RESULTS

A total of 2,421 children and 2,141,747 SNPs were subjected to the genome-wide interaction analyses. No variant reached genome-wide significance. The strongest interaction was observed at rs17800861 in the GRIN2A gene (β = -3.43, 95% CI = -4.74 to -2.12, p = 2.98E-07). This variant is predicted to be located within active chromatin compartments in the hippocampus and may influence binding of the NF-kappaB transcription factor.

CONCLUSIONS

Our results may indicate that mode of delivery might have a moderating effect on genetic disposition of intelligence in children. Studies of considerable sizes (>10,000) are likely required to more robustly detect variants governing such interaction. In summary, the presented findings prompt the need for further studies aimed at increasing our understanding of effects various modes of delivery may have on health outcomes in children.

Silver nanoparticles (Ag-NPs) can enter the brain and subsequently induce neurotoxicity. However, the toxicity of Ag-NPs on the blood-brain barrier (BBB) and the underlying mechanism(s) of action on the hippocampus in vivo are not well understood. To investigate Ag-NP suspension (Ag-NPS)-induced toxicity on the BBB and neurons, Sprague-Dawley rats were randomly divided into 3 groups, and Ag-NPS, Ag-ion, and 5% sucrose solution (vehicle control) were administrated intravenously, respectively. After 24 h exposure to Ag-NPS, the BBB permeability was not significantly changed. However, the Ag concentrations in the brain tissues were only detected in the Ag-NPS group. Gene expression results indicated that the expression of Claudin 4 (tight junction protein) was significantly decreased. Furthermore, astrocyte foot swelling, neuron shrinkage and Ag-NP like particles were observed under transmission electron microscopy. Global gene expression analysis showed that 502 genes were up-regulated and 703 genes were down-regulated in the hippocampi treated with Ag-NPS. In the Ag-NPS-treated group, 78 biological functions were changed based on gene ontology (GO) and 34 signaling pathways were significantly changed using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, which were associated with the neuroactive ligand-receptor interaction, cytokine-cytokine receptor interaction, calcium signaling pathway and MAPK signaling pathway. In comparison, 27 GO and 9 KEGG pathways were changed in the released Ag-ion-treated group. Ag-NPS decreased C1qtnf3 expression and increased Adra1d expression to affect MAPK signaling pathway, which promoted inflammation and apoptosis in the hippocampus. Moreover, Ag-NPS significantly increased Spp1, Cacna1s and Tacr3 mRNAs expression, which may result in intracellular calcium increasing and initiate cell death. Furthermore, Ag-NPS affected calcium signaling pathway and neuroactive ligand-receptor (Grin2a, Drd2, and Adra1d), which are crucial in diverse cellular functions in the brain including cognition and neurodevelopment. These results draw our attention to the importance of Ag-NP-induced toxicity in the rat hippocampus and provide a better understanding of its toxicological mechanisms in vivo.

Previous studies revealed a peripheral nerve regeneration (PNR)(1) promoting activity of Clostridium botulinum C3(2) exoenzyme or a 26(mer) C-terminal peptide fragment covering amino acids 156-181 (C3(156-181)),(3) when delivered as one-time injection at the lesion site. The current study was performed to 1) investigate if prolonged availability of C3 and C3(156-181) at the lesion site can further enhance PNR in vivo and to 2) elucidate effects of C3 and C3(156-181) on Schwann cells (SCs)(4)in vitro. For in vivo studies, 10 mm adult rat sciatic nerve gaps were reconstructed with the epineurial pouch technique or autologous nerve grafts. Epineurial pouches were filled with a hydrogel containing i) vehicle, ii) 40 μM C3 or iii) 40 μM C3(156-181). Sensory and motor functional recovery was monitored over 12 weeks and the outcome of PNR further analyzed by nerve morphometry. In vitro, we compared gene expression profiles (microarray analysis) and neurotrophic factor expression (western blot analysis) of untreated rat neonatal SCs with those treated with C3 or C3(156-181) for 72 h. Effects on neurotrophic factor expression levels were proven in adult human SCs. Unexpectedly, prolonged delivery of C3 and C3(156-181) at the lesion site did not increase the outcome of PNR. Regarding the potential mechanism underlying their previously detected PNR promoting action, however, 6 genes were found to be commonly altered in SCs upon treatment with C3 or C3(156-181). We demonstrate significant down-regulation of genes involved in glutamate uptake (Eaac1,(5)Grin2a(6)) and changes in neurotrophic factor expression (increase of FGF-2(7) and decrease of NGF(8)). Our microarray-based expression profiling revealed novel C3-regulated genes in SCs possibly involved in the axonotrophic (regeneration promoting) effects of C3 and C3(156-181). Detection of altered neurotrophic factor expression by C3 or C3(156-181) treated primary neonatal rat SCs and primary adult human SCs supports this hypothesis.

Cortical dopaminergic systems are critically involved in prefrontal cortex (PFC) functions, especially in working memory and neurodevelopmental disorders such as schizophrenia. GSK-3β (glycogen synthase kinase-3β) is highly associated with cAMP (cyclic adenosine monophosphate)-independent dopamine D₂ receptor (D₂R)-mediated signaling to affect dopamine-dependent behaviors. However, the mechanisms underlying the GSK-3β modulation of cognitive function via D₂Rs remains unclear.

This study explored how conditional cell-type-specific ablation of GSK-3β in D₂R+ neurons (D₂R-GSK-3β^-/-) in the brain affects synaptic function in the medial PFC (mPFC). Both male and female (postnatal days 60-90) mice, including 140 D₂R, 24 D₁R, and 38 DISC1 mice, were used.

This study found that NMDA receptor (NMDAR) function was significantly increased in layer V pyramidal neurons in mPFC of D₂R-GSK-3β^-/- mice, along with increased dopamine modulation of NMDAR-mediated current. Consistently, NR2A and NR2B protein levels were elevated in mPFC of D₂R-GSK-3β^-/- mice. This change was accompanied by a significant increase in enrichment of activator histone mark H3K27ac at the promoters of both Grin2a and Grin2b genes. In addition, altered short- and long-term synaptic plasticity, along with an increased spine density in layer V pyramidal neurons, were detected in D₂R-GSK-3β^-/- mice. Indeed, D₂R-GSK-3β^-/- mice also exhibited a resistance of working memory impairment induced by injection of NMDAR antagonist MK-801. Notably, either inhibiting GSK-3β or disrupting the D₂R-DISC1 complex was able to reverse the mutant DISC1-induced decrease of NMDAR-mediated currents in the mPFC.

This study demonstrates that GSK-3β modulates cognition via D₂R-DISC1 interaction and epigenetic regulation of NMDAR expression and function.

Testis-specific protein, Y-encoded-like 2 (TSPYL2) is an X-linked gene in the locus for several neurodevelopmental disorders. We have previously shown that Tspyl2 knockout mice had impaired learning and sensorimotor gating, and TSPYL2 facilitates the expression of Grin2a and Grin2b through interaction with CREB-binding protein. To identify other genes regulated by TSPYL2, here, we showed that Tspyl2 knockout mice had an increased level of H3K27 trimethylation (H3K27me3) in the hippocampus, and TSPYL2 interacted with the H3K27 methyltransferase enhancer of zeste 2 (EZH2). We performed chromatin immunoprecipitation (ChIP)-sequencing in primary hippocampal neurons and divided all Refseq genes by k-mean clustering into four clusters from highest level of H3K27me3 to unmarked. We confirmed that mutant neurons had an increased level of H3K27me3 in cluster 1 genes, which consist of known EZH2 target genes important in development. We detected significantly reduced expression of genes including Gbx2 and Prss16 from cluster 1 and Acvrl1, Bdnf, Egr3, Grin2c, and Igf1 from cluster 2 in the mutant. In support of a dynamic role of EZH2 in repressing marked synaptic genes, the specific EZH2 inhibitor GSK126 significantly upregulated, while the demethylase inhibitor GSKJ4 downregulated the expression of Egr3 and Grin2c. GSK126 also upregulated the expression of Bdnf in mutant primary neurons. Finally, ChIP showed that hemagglutinin-tagged TSPYL2 co-existed with EZH2 in target promoters in neuroblastoma cells. Taken together, our data suggest that TSPYL2 is recruited to promoters of specific EZH2 target genes in neurons, and enhances their expression for proper neuronal maturation and function.

Maternal alcohol consumption throughout pregnancy can result in long term behavioural deficits in offspring. However, less is known about the impact of alcohol during the periconceptional period (PC). The aim of this study was to examine the effect of PC ethanol (PC:EtOH) exposure on long term cognitive function; including memory and anxiety. Rats were exposed to a liquid diet containing ethanol (EtOH) (12.5% vol;vol) or a control diet from 4 days prior to mating until day 4 of pregnancy. Separate cohorts of animals were tested at 6 months (adult) or 15-18 months of age (aged). Offspring underwent a series of behavioural tests to assess anxiety, spatial and recognition memory. The hippocampus was collected, and mRNA expression of epigenetic modifiers and genes implicated in learning and memory were examined. PC:EtOH exposure resulted in a subtle anxiety like behaviour in adult female offspring with a significant reduction in directed exploring/head dipping behaviour during holeboard testing. In aged male offspring, PC:EtOH exposure resulted in a tendency for increased directed exploring/head dipping behaviour during holeboard testing. No differences between treatments were observed in the elevated plus maze. Aged female offspring exposed to PC:EtOH demonstrated short term spatial memory impairment (P < 0.05). PC:EtOH resulted in an upregulation of hippocampal mRNA expression of bdnf, grin2a and grin2b at 18 months of age along with increased expression of epigenetic modifiers (dnmt1, dnmt3a and hdac2). In conclusion, PC:EtOH can lead to sex specific anxiety-like behaviour and impairments in spatial memory and altered hippocampal gene expression.

Problem solving and innovation are key components of intelligence. We compare wild-caught individuals from two species that are close relatives of Darwin's finches, the innovative Loxigilla barbadensis, and its most closely related species in Barbados, the conservative Tiaris bicolor. We found an all-or-none difference in the problem-solving capacity of the two species. Brain RNA sequencing analyses revealed interspecific differences in genes related to neuronal and synaptic plasticity in the intrapallial neural populations (mesopallium and nidopallium), especially in the nidopallium caudolaterale, a structure functionally analogous to the mammalian prefrontal cortex. At a finer scale, we discovered robust differences in glutamate receptor expression between the species. In particular, the GRIN2B/GRIN2A ratio, known to correlate with synaptic plasticity, was higher in the innovative L. barbadensis. These findings suggest that divergence in avian intelligence is associated with similar neuronal mechanisms to that of mammals, including humans.

Serotonin (5-HT) and the habenula (Hb) contribute to motivational and emotional states such as depression and drug abuse. The dorsal raphe nucleus, where 5-HT neurons originate, and the Hb are anatomically and reciprocally interconnected. Evidence exists that 5-HT influences Hb glutamatergic transmission. Using serotonin transporter knockout (SERT-/- ) rats, which show depression-like behavior and increased cocaine intake, we investigated the effect of SERT reduction on expression of genes involved in glutamate neurotransmission under both baseline conditions as well as after short-access or long-access cocaine (ShA and LgA, respectively) intake. In cocaine-naïve animals, SERT removal led to reduced baseline Hb mRNA levels of critical determinants of glutamate transmission, such as SLC1A2, the main glutamate transporter and N-methyl-D-aspartate (Grin1, Grin2A and Grin2B) as well as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (Gria1 and Gria2) receptor subunits, with no changes in the scaffolding protein Dlg4. In response to ShA and LgA cocaine intake, SLC1A2 and Grin1 mRNA levels decreased in SERT+/+ rats to levels equal of those of SERT-/- rats. Our data reveal that increased extracellular levels of 5-HT modulate glutamate neurotransmission in the Hb, serving as critical neurobiological substrate for vulnerability to cocaine addiction.

Background GRIN2A encodes the GluN2A subunit of the NMDA receptor (NMDAR), an ionotropic glutamate receptor that has important roles in synaptogenesis and synaptic plasticity. Some individuals with early onset epilepsies and intellectual disability carry heterozygous missense mutations in this gene, including a de-novo mutation in the receptor pore region (GluN2A(N615K)). We hypothesised that this mutation underlies the carrier's brain disorder and sought to explore its functional consequences.

METHODS

We made two-electrode voltage clamp recordings from Xenopus laevis oocytes expressing GluN1/GluN2A(N615K) (N615K) NMDARs and compared them with wild-type (WT) NMDARs to assess the mutation's effect on potency of inhibition by Mg(2+) and other channel blockers. We then used whole-cell patch-clamping to evaluate NMDAR-mediated currents in mouse primary cortical pyramidal neurons transfected with either GluN2A(WT) or GluN2A(N651K) subunits. Means were compared by use of independent two-tailed t tests.

RESULTS

In oocytes, Mg(2+) (1 mM) block at -60 mV was significantly decreased (N615K [n=13], mean 5% [SE 8] vs WT [n=15], 89 [4]; p<0·0001). Furthermore, in N615K (n=17) and WT (n=17) oocytes, block by 10 μM memantine was also reduced (mean 26% [6] vs 75 [7], p<0·0001) as was block by 100 μM amantadine (18% [4] vs 44 [12], p<0·0001). Block by ketamine (N615K, n=14; WT, n=14) was not significantly affected, whereas block by dextromethorphan was increased (N615K [n=9], 56% [8] vs WT [n=8], 44 [6]; p=0·003). In N615K (n=10) and WT (n=10) neurons we observed a significant decrease in Mg(2+) sensitivity (49% [18] vs 95 [5], p<0·0001) and a significant decrease in current density (42 pA/pF [19] vs 61 [20], p=0·044).

CONCLUSIONS

This study suggests that the disease-associated mutation GluN2A(N615K) has substantial effects on NMDAR inhibition by both endogenous and exogenous channel blockers, and on NMDA current density. It is plausible that these changes underlie the carrier's phenotype.

BACKGROUND

Wellcome Trust via an Edinburgh Clinical Academic Training PhD Fellowship.

GRIN2A mutations are frequent in melanoma tumours but their role in disease is not well understood. GRIN2A encodes a modulatory subunit of the N-methyl-d-aspartate receptor (NMDAR). We hypothesized that certain GRIN2A mutations increase NMDAR function and support melanoma growth through oncogenic effects. This hypothesis was tested using 19 low-passage melanoma cell lines, four of which carried novel missense mutations in GRIN2A that we previously reported. We examined NMDAR expression, function of a calcium ion (Ca2+) channel and its contribution to cell growth using pharmacological modulators; findings were correlated with the presence or absence of GRIN2A mutations. We found that NMDAR expression was low in all melanoma cell lines, independent of GRIN2A mutations. In keeping with this, NMDAR-mediated Ca2+ influx and its contribution to cell proliferation were weak in most cell lines. However, certain GRIN2A mutations and culture media with lower glutamate levels enhanced NMDAR effects on cell growth and invasion. The main finding was that G762E was associated with higher glutamate-mediated Ca2+ influx and stronger NMDAR contribution to cell proliferation, compared with wild-type GRIN2A and other GRIN2A mutations. The pro-invasive phenotype of mutated cell lines was increased in culture medium containing less glutamate, implying environmental modulation of mutation effects. In conclusion, NMDAR ion channel function is low in cultured melanoma cells but supports cell proliferation and invasion. Selected GRIN2A mutations, such as G762E, are associated with oncogenic consequences that can be modulated by extracellular glutamate. Primary cultures may be better suited to determine the role of the NMDAR in melanoma in vivo.

N-methyl-d-aspartate receptors (NMDARs) mediate a slow component of excitatory synaptic transmission that plays important roles in normal brain function and development. A large number of disease-associated variants in the GRIN gene family encoding NMDAR GluN subunits have been identified in patients with various neurological and neuropsychiatric disorders. Many of these variants reduce the function of NMDARs by a range of different mechanisms, including reduced glutamate potency, reduced glycine potency, accelerated deactivation time course, decreased surface expression, and/or reduced open probability. We have evaluated whether three positive allosteric modulators of NMDAR receptor function (24(S)-hydroxycholesterol, pregnenolone sulfate, tobramycin) and three co-agonists (d-serine, l-serine, and d-cycloserine) can mitigate the diminished function of NMDARs harboring GRIN variants. We examined the effects of these modulators on NMDARs that contained 21 different loss-of-function variants in GRIN1, GRIN2A, or GRIN2B, identified in patients with epilepsy, intellectual disability, autism, and/or movement disorders. For all variants, some aspect of the reduced function was partially restored. Moreover, some variants showed enhanced sensitivity to positive allosteric modulators compared to wild type receptors. These results raise the possibility that enhancement of NMDAR function by positive allosteric modulators may be a useful therapeutic strategy.

Keywords: Channelopathy; Endogenous neurosteroid; Glutamate receptors; Rescue pharmacology; Translational study.

The dopamine and glutamate hypotheses reflect only some of the pathophysiological changes associated with schizophrenia. We have proposed a new "comprehensive progressive pathophysiology model" based on the "dopamine to glutamate hypothesis." Repeated administration of methamphetamine (METH) at a dose of 2.5 mg/kg in rats has been used to assess dynamic changes in the pathophysiology of schizophrenia. Previous use of this model suggested N-methyl-d-aspartate receptor (NMDA-R) dysfunction, but the mechanism could only be inferred from limited, indirect observations. In the present study, we used this model to investigate changes in the expression of NMDA-R subunits. Repeated administration of METH significantly decreased the gene expression levels of glutamate ionotropic receptor NMDA type subunit (Grin) subtypes Grin1 and Grin2c in the prefrontal cortex (PFC), Grin1 and Grin2a in the hippocampus (HPC), and Grin1, Grin2b, and Grin2d in the striatum (ST).We observed a significant difference in Grin1 expression between the PFC and ST. Furthermore, repeated administration of METH significantly decreased the protein expression of GluN1 in both cytosolic and synaptosomal fractions isolated from the PFC, and significantly decreased the protein expression of GluN1 in the cytosolic fraction, but not the synaptosomal fraction from the ST. These regional differences may be due to variations in the synthesis of GluN1 or intracellular trafficking events in each area of the brain. Considering that knockdown of Grin1 in mice affects vulnerability to develop schizophrenia, these results suggest that this model reflects some of the pathophysiological changes of schizophrenia, combining both the dopamine and glutamate hypotheses.

Keywords: Animal model; GluN1; Methamphetamine; NMDA receptor; Schizophrenia.

NMDA receptors play crucial roles in excitatory synaptic transmission. Rare variants in GRIN2A encoding the GluN2A subunit are associated with a spectrum of disorders, ranging from mild speech and language delay to intractable neurodevelopmental disorders, including but not limited to developmental and epileptic encephalopathy. A de novo missense variant, p.Ser644Gly, was identified in a child with this disorder, and Grin2a knock-in mice were generated to model and extend understanding of this intractable childhood disease. Homozygous and heterozygous mutant mice exhibited altered hippocampal morphology at 2 weeks of age, and all homozygotes exhibited lethal tonic-clonic seizures by mid-third week. Heterozygous adults displayed susceptibility to induced generalized seizures, hyperactivity, repetitive and reduced anxiety behaviours, plus several unexpected features, including significant resistance to electrically-induced limbic seizures and to pentylenetetrazole induced tonic-clonic seizures. Multielectrode recordings of neuronal networks revealed hyperexcitability and altered bursting and synchronicity. In heterologous cells, mutant receptors had enhanced NMDA receptor agonist potency and slow deactivation following rapid removal of glutamate, as occurs at synapses. NMDA receptor-mediated synaptic currents in heterozygous hippocampal slices also showed a prolonged deactivation time course. Standard anti-epileptic drug monotherapy was ineffective in the patient. Introduction of NMDA receptor antagonists was correlated with a decrease in seizure burden. Chronic treatment of homozygous mouse pups with NMDA receptor antagonists significantly delayed the onset of lethal seizures but did not prevent them. These studies illustrate the power of using multiple experimental modalities to model and test therapies for severe neurodevelopmental disorders, while revealing significant biological complexities associated with GRIN2A developmental and epileptic encephalopathy.

Keywords: NR2A; autistic spectrum disorder; childhood epilepsy; experimental models; synaptic transmission.

Rare variants in GRIN genes, which encode NMDAR subunits, are strongly associated with neurodevelopmental disorders. Among these, GRIN2A, which encodes the GluN2A subunit of NMDARs, is widely accepted as an epilepsy-causative gene. Here, we functionally characterize the de novo GluN2A-S1459G mutation identified in an epilepsy patient. We show that S1459 is a CaMKIIα phosphorylation site, and that endogenous phosphorylation is regulated during development and in response to synaptic activity in a dark rearing model. GluN2A-S1459 phosphorylation results in preferential binding of NMDARs to SNX27 and a corresponding decrease in PSD-95 binding, which consequently regulates NMDAR trafficking. Furthermore, the epilepsy-associated GluN2A-S1459G variant displays defects in interactions with both SNX27 and PSD-95, resulting in trafficking deficits, reduced spine density, and decreased excitatory synaptic transmission. These data demonstrate a role for CaMKIIα phosphorylation of GluN2A in receptor targeting and implicate NMDAR trafficking defects as a link to epilepsy.

Keywords: CaMKIIα; GRIN2A rare variants; NMDA receptors; endocytic sorting; epilepsy; protein trafficking.