TFAP2A is involved in neuropathic pain by regulating Grin1 expression in glial cells of the dorsal root ganglion.

Neuropathic pain is a highly prevalent and refractory condition, yet its mechanism remains poorly understood. While NR1, the essential subunit of NMDA receptors, has long been recognized for its pivotal role in nociceptive transmission, its involvement in presynaptic stimulation is incompletely elucidated. Transcription factors can regulate the expression of both pro-nociceptive and analgesic factors. Our study shows that transcription factor TFAP2A was up-regulated in the dorsal root ganglion (DRG) neurons, satellite glial cells (SGCs), and Schwann cells following spinal nerve ligation (SNL). Intrathecal injection of siRNA targeting Tfap2a immediately or 7 days after SNL effectively alleviated SNL-induced pain hypersensitivity and reduced Tfap2a expression levels. Bioinformatics analysis revealed that TFAP2A may regulate the expression of the Grin1 gene, which encodes NR1. Dual-luciferase reporter assays confirmed TFAP2A's positive regulation of Grin1 expression. Notably, both Tfap2a and Grin1 were expressed in the primary SGCs and upregulated by lipopolysaccharides. The expression of Grin1 was also down-regulated in the DRG following Tfap2a knockdown. Furthermore, intrathecal injection of siRNA targeting Grin1 immediately or 7 days post-SNL effectively alleviated SNL-induced mechanical allodynia and thermal hyperalgesia. Finally, intrathecal Tfap2a siRNA alleviated SNL-induced neuronal hypersensitivity, and incubation of primary SGCs with Tfap2a siRNA decreased NMDA-induced upregulation of proinflammatory cytokines. Collectively, our study reveals the role of TFAP2A-Grin1 in regulating neuropathic pain in peripheral glia, offering a new strategy for the development of novel analgesics.

Conformational basis of subtype-specific allosteric control of NMDA receptor gating.

N-methyl-D-aspartate receptors are ionotropic glutamate receptors that are integral to synaptic transmission and plasticity. Variable GluN2 subunits in diheterotetrameric receptors with identical GluN1 subunits set very different functional properties, which support their individual physiological roles in the nervous system. To understand the conformational basis of this diversity, we assessed the conformation of the common GluN1 subunit in receptors with different GluN2 subunits using single-molecule fluorescence resonance energy transfer (smFRET). We established smFRET sensors in the ligand binding domain and modulatory amino-terminal domain to study an apo-like state and partially liganded activation intermediates, which have been elusive to structural analysis. Our results demonstrate a strong, subtype-specific influence of apo and glutamate-bound GluN2 subunits on GluN1 rearrangements, suggesting a conformational basis for the highly divergent levels of receptor activity, desensitization and agonist potency. Chimeric analysis reveals structural determinants that contribute to the subtype differences. Our study provides a framework for understanding GluN2-dependent functional properties and could open new avenues for subtype-specific modulation.

Accumulation of NMDA receptors in accumbal neuronal ensembles mediates increased conditioned place preference for cocaine after prolonged withdrawal.

Cue-induced cocaine craving gradually intensifies following abstinence, a phenomenon known as the incubation of drug craving. Neuronal ensembles activated by initial cocaine use, are critically involved in this process. However, the mechanisms by which neuronal changes occurring in the ensembles after withdrawal contribute to incubation remain largely unknown. Here we labeled neuronal ensembles in the shell of nucleus accumbens (NAcSh) activated by cocaine conditioned place preference (CPP) training. NAcSh ensembles showed an increasing activity induced by CPP test after 21-day withdrawal. Inhibiting synaptic transmission of NAcSh ensembles suppressed the preference for cocaine paired-side after 21-day withdrawal, demonstrating a critical role of NAcSh ensembles in increased preference for cocaine. The density of dendritic spines in dopamine D1 receptor expressing ensembles was increased after 21-day withdrawal. Moreover, the expression of Grin1, a subunit of the N-methyl-D-aspartate (NMDA) receptor, specifically increased in the NAcSh ensembles after cocaine withdrawal in both CPP and self-administration (SA) mouse models. Targeted knockdown or dysfunction of Grin1 in NAcSh ensembles significantly suppressed craving for cocaine. Our results suggest that the accumulation of NMDA receptors in NAcSh ensembles mediates increased craving for cocaine after prolonged withdrawal, thereby providing potential molecular targets for treatment of drug addiction.

Reduced Expression of the Htr2a, Grin1, and Bdnf Genes and Cognitive Inflexibility in a Model of High Compulsive Rats.

Compulsivity is a core symptom in different psychopathological disorders, characterized by excessive behaviors and behavioral inflexibility. The selection of high drinker (HD) versus low drinker (LD) rats by schedule-induced polydipsia (SIP) is a valid model for studying the compulsive phenotype. The compulsive HD rats showed cognitive inflexibility and reduced serotonin 2A (5-HT2A) receptor binding levels in the frontal cortex (FC). According to that, we hypothesize that compulsive HD rats might have an alteration in the cognitive control domain regarding inflexibility, assessed by spatial memory on the Morris Water Maze (MWM), working and reference memory by the Radial Arm Maze, and behavioral deficits in stimulus processing by the Novel Object Recognition test. The possible underlying mechanisms might be linked to the brain gene expression of 5HT2A, 5HT2C, glutamate NMDA receptors, and brain-derived neurotrophic factor (BDNF) in FC, hippocampus, and amygdala. HD rats confirmed a cognitive inflexibility profile on the reversal condition in the MWM compared to LD rats, while no differences were observed on stimulus processing, spatial, and working memory. Moreover, HD rats showed a reduced expression of the Htr2a, Grin1, and Bdnf genes in FC. Furthermore, there was a negative correlation between the relative expression of the Htr2a, Grin1, and Bdnf genes in FC and the level of compulsive water intake in HD rats on SIP. These data reveal that cognitive inflexibility may not be associated with a memory or stimulus processing deficit in compulsive individuals but may result by a region-specific alteration of the Htr2a, Grin1, and Bdnf gene expression in FC.

Quantitative proteomic and phosphoproteomic analyses of the hippocampus reveal the involvement of NMDAR1 signaling in repetitive mild traumatic brain injury.

The cumulative damage caused by repetitive mild traumatic brain injury can cause long-term neurodegeneration leading to cognitive impairment. This cognitive impairment is thought to result specifically from damage to the hippocampus. In this study, we detected cognitive impairment in mice 6 weeks after repetitive mild traumatic brain injury using the novel object recognition test and the Morris water maze test. Immunofluorescence staining showed that p-tau expression was increased in the hippocampus after repetitive mild traumatic brain injury. Golgi staining showed a significant decrease in the total density of neuronal dendritic spines in the hippocampus, as well as in the density of mature dendritic spines. To investigate the specific molecular mechanisms underlying cognitive impairment due to hippocampal damage, we performed proteomic and phosphoproteomic analyses of the hippocampus with and without repetitive mild traumatic brain injury. The differentially expressed proteins were mainly enriched in inflammation, immunity, and coagulation, suggesting that non-neuronal cells are involved in the pathological changes that occur in the hippocampus in the chronic stage after repetitive mild traumatic brain injury. In contrast, differentially expressed phosphorylated proteins were mainly enriched in pathways related to neuronal function and structure, which is more consistent with neurodegeneration. We identified N-methyl-D-aspartate receptor 1 as a hub molecule involved in the response to repetitive mild traumatic brain injury , and western blotting showed that, while N-methyl-D-aspartate receptor 1 expression was not altered in the hippocampus after repetitive mild traumatic brain injury, its phosphorylation level was significantly increased, which is consistent with the omics results. Administration of GRP78608, an N-methyl-D-aspartate receptor 1 antagonist, to the hippocampus markedly improved repetitive mild traumatic brain injury-induced cognitive impairment. In conclusion, our findings suggest that N-methyl-D-aspartate receptor 1 signaling in the hippocampus is involved in cognitive impairment in the chronic stage after repetitive mild traumatic brain injury and may be a potential target for intervention and treatment.

Exposure to anandamide on young rats causes deficits in learning, temporal perception and induces changes in NMDA receptor expression.

Human use of marijuana at an early age has been reported to lead to cognitive impairment. However, researchers have not yet clearly determined whether this impairment is due to marijuana-induced alterations in the developing nervous system and whether this deficit persists into adulthood after marijuana use has ceased. We administered anandamide to developing rats to assess the effect of cannabinoids on development. We subsequently evaluated learning and performance on a temporal bisection task in adulthood and assessed the expression of genes encoding principal subunits of NMDA receptors (Grin1, Grin2A, and Grin2B) in the hippocampus and prefrontal cortex. Rats in two age groups, namely, 21-day-old and 150-day-old rats, received intraperitoneal injections of anandamide or the vehicle for 14 days. Both groups performed a temporal bisection test, which included listening to tones of different durations and classifying them as short or long. The expression of the Grin1, Grin2A and Grin2B mRNAs was evaluated using quantitative PCR in both age groups after extracting mRNA from the hippocampus and prefrontal cortex. We observed a learning impairment in the temporal bisection task (p < 0.05) and changes in the response latency (p < 0.05) in rats that received anandamide. Furthermore, these rats exhibited decreased expression of Grin2b (p = 0.001) compared to those that received the vehicle. In human subjects, the use of cannabinoids during development induces a long-term deficit, but this deficit is not observed in subjects who use cannabinoids in adulthood. Rats treated with anandamide earlier in development took longer to learn the task, suggesting that anandamide exerts a harmful effect on cognition in developing rats. Administration of anandamide during early stages of development induced deficits in learning and other cognitive processes that depend on an adequate estimation of time. The cognitive demands of the environment must be considered when evaluating the cognitive effects of cannabinoids on developing or mature brains. High cognitive demands might induce differential expression of NMDA receptors that improves cognitive capacity, overcoming altered glutamatergic function.

Glutamate ionotropic receptor NMDA type subunit 1: A novel potential protein target of dapagliflozin against renal interstitial fibrosis.

Renal interstitial fibrosis (RIF) is the final pathway for chronic kidney diseases (CKD) to end-stage renal disease, with no ideal therapy at present. Previous studies indicated that sodium glucose co-transporter-2 inhibitor (SGLT2i) dapagliflozin had the effect of anti-RIF, but the mechanism remains elusive and the renal protective effect could not be fully explained by singly targeting SGLT2. In this study, we aimed to explore the mechanism of dapagliflozin against RIF and identify novel potential targets. Firstly, dapagliflozin treatment improved pro-fibrotic indicators in unilateral ureteral obstruction mice and transforming growth factor beta 1 induced human proximal tubular epithelial cells. Then, transcriptomics and bioinformatics analysis were performed, and results revealed that dapagliflozin against RIF by regulating inflammation and oxidative stress related signals. Subsequently, targets prediction and analysis demonstrated that glutamate ionotropic receptor NMDA type subunit 1 (GRIN1) was a novel potential target of dapagliflozin, which was related to inflammation and oxidative stress related signals. Moreover, molecular dynamics simulation revealed that dapagliflozin could stably bind to GRIN1 protein and change its spatial conformation. Furthermore, human renal samples and Nephroseq data were used for GRIN1 expression evaluation, and the results showed that GRIN1 expression were increased in renal tissues of CKD and RIF patients than controls. Additionally, further studies demonstrated that dapagliflozin could reduce intracellular calcium influx in renal tubular cells, which depended on regulating GRIN1 protein but not gene. In conclusion, GRIN1 is probably a novel target of dapagliflozin against RIF.

An experimental study of the effects of SNPs in the TATA boxes of the GRIN1, ASCL3 and NOS1 genes on interactions with the TATA-binding protein.

The GRIN1, ASCL3, and NOS1 genes are associated with various phenotypes of neuropsychiatric disorders. For instance, these genes contribute to the development of schizophrenia, Alzheimer's and Parkinson's diseases, and epilepsy. These genes are also associated with various cancers. For example, ASCL3 is overexpressed in breast cancer, and NOS1, in ovarian cancer cell lines. Based on our findings and literature data, we had previously obtained results suggesting that the single-nucleotide polymorphisms (SNPs) that disrupt erythropoiesis are highly likely to be associated with cognitive and neuropsychiatric disorders in humans. In the present work, using SNP_TATA_Z-tester, we investigated the influence of unannotated SNPs in the TATA boxes of the promoters of the GRIN1, ASCL3, and NOS1 genes (which are involved in neuropsychiatric disorders and cancers) on the interaction of the TATA boxes with the TATA-binding protein (TBP). Double-stranded oligodeoxyribonucleotides identical to the TATA-containing promoter regions of the GRIN1, ASCL3, and NOS1 genes (reference and minor alleles) and recombinant human TBP were employed to study in vitro (by an electrophoretic mobility shift assay) kinetic characteristics of the formation of TBP-TATA complexes and their affinity. It was found, for example, that allele A of rs1402667001 in the GRIN1 promoter increases TBP-TATA affinity 1.4-fold, whereas allele C in the TATA box of the ASCL3 promoter decreases the affinity 1.4-fold. The lifetime of the complexes in both cases decreased by ~20 % due to changes in the rates of association and dissociation of the complexes (ka and kd, respectively). Our experimental results are consistent with the literature showing GRIN1 underexpression in schizophrenic disorders as well as an increased risk of cervical, bladder, and kidney cancers and lymphoma during ASCL3 underexpression. The effect of allele A of the -27G>A SNP (rs1195040887) in the NOS1 promoter is suggestive of an increased risk of ischemic damage to the brain in carriers. A comparison of experimental TBP-TATA affinity values (KD) of wild-type and minor alleles with predicted ones showed that the data correlate well (linear correlation coefficient r = 0.94, p <0.01).

Abnormal sensory perception masks behavioral performance of Grin1 knockdown mice.

The development and function of sensory systems require intact glutamatergic neurotransmission. Changes in touch sensation and vision are common symptoms in autism spectrum disorders, where altered glutamatergic neurotransmission is strongly implicated. Further, cortical visual impairment is a frequent symptom of GRIN disorder, a rare genetic neurodevelopmental disorder caused by pathogenic variants of GRIN genes that encode NMDA receptors. We asked if Grin1 knockdown mice (Grin1KD), as a model of GRIN disorder, had visual impairments resulting from NMDA receptor deficiency. We discovered that Grin1KD mice had deficient visual depth perception in the visual cliff test. Since Grin1KD mice are known to display robust changes in measures of learning, memory, and emotionality, we asked whether deficits in these higher-level processes could be partly explained by their visual impairment. By changing the experimental conditions to improve visual signals, we observed significant improvements in the performance of Grin1KD mice in tests that measure spatial memory, executive function, and anxiety. We went further and found destabilization of the outer segment of retina together with the deficient number and size of Meissner corpuscles (mechanical sensor) in the hind paw of Grin1KD mice. Overall, our findings suggest that abnormal sensory perception can mask the expression of emotional, motivational and cognitive behavior of Grin1KD mice. This study demonstrates new methods to adapt routine behavioral paradigms to reveal the contribution of vision and other sensory modalities in cognitive performance.

Sex differences in the neuroadaptations associated with incubated cocaine-craving: A focus on the dorsomedial prefrontal cortex.

Introduction: Women have a shorter course from initial cocaine use to meeting the criteria for cocaine use disorder as compared to men. Preclinical findings similarly indicate that females develop key features of an addiction-like phenotype faster than males, including an enhanced motivation for cocaine and compulsive use, indicating that this phenomenon is biologically based. The goals of this study were to determine whether cocaine-craving, another key feature of addiction, also develops sooner during withdrawal in females than males and to determine whether there are sex differences in the molecular mechanisms associated with its development focusing on markers known to mediate cocaine-craving in males (i.e., dorsomedial prefrontal cortex, dmPFC, expression of brain-derived neurotrophic factor exon-IV, Bdnf-IV, and NMDA receptor subunits, Grin2a, Grin2b, and Grin1). Methods: Cocaine-craving was assessed following extended-access cocaine self-administration and 2, 7, or 14 days of withdrawal using an extinction/cue-induced reinstatement procedure. Tissue was obtained from the dmPFC immediately after reinstatement testing and gene expression changes were analyzed using real-time qPCR. Results: In males, cocaine-craving (total extinction and cue-induced reinstatement responding) progressively increased from early to later withdrawal time-points whereas in females, cocaine-craving was already elevated during early withdrawal (after 2 days) and did not further increase at later withdrawal time-points. Levels of cocaine-craving, however, were similar between the sexes. Gene expression changes differed markedly between the sexes such that males showed the expected relapse- and withdrawal-associated changes in Bdnf-IV, Grin2a, Grin2b, and Grin1 expression, but females only showed a modest increase Grin1 expression at the intermediate withdrawal timepoint. Discussion: These findings indicate that cocaine-craving is similarly expressed in males and females although the time-course for its incubation appears to be accelerated in females; the molecular mechanisms also likely differ in females versus males.

A homozygous GRIN1 null variant causes a more severe phenotype of early infantile epileptic encephalopathy.

Pathogenic variants in glutamate receptor, ionotropic, NMDA-1 (GRIN1) cause an autosomal dominant or recessive neurodevelopmental disorder with global developmental delay, with or without seizures (AD or AR GRIN1-NDD). Here, we describe a novel homozygous canonical splice site variant in GRIN1 in a 12-month-old boy with early infantile epileptic encephalopathy and severe global developmental delay. This represents only the second family with a homozygous predicted-null variant in GRIN1 reported to date. We review the published literature on AR GRIN1-NDD and find that the phenotype in our patient is much more severe than those seen with homozygous missense variants. A similarly severe phenotype of intractable epilepsy and infantile death has only been reported in one other family with a homozygous nonsense variant in GRIN1. We, therefore, propose that biallelic predicted-null variants in GRIN1 can cause a markedly more severe clinical phenotype than AR GRIN1-NDD caused by missense variants.

The effect of human GRIN1 gene 5' functional region on gene expression regulation in vitro.

INTRODUCTION: Abnormal expression of ionotropic glutamate receptor NMDA type subunit 1, the key subunit of the NMDA receptor, may be related to many neuropsychiatric disorders. In this study, we explored the functional sequence of the 5' regulatory region of the human GRIN1 gene and discussed the transcription factors that may regulate gene expression. MATERIALS AND METHODS: Twelve recombinant pGL3 vectors with gradually truncated fragment lengths were constructed, transfected into HEK-293, U87, and SK-N-SH cell lines, and analyzed through the luciferase reporter gene assay. JASPAR database is used to predict transcription factors. RESULTS: In SK-N-SH and U87 cell lines, regions from -337 to -159 bp, -704 to -556 bp inhibited gene expression, while -556 to -337 bp upregulated gene expression. In HEK-293 and U87 cell lines, the expression of fragment -1703 to + 188 bp was significantly increased compared to adjacent fragments -1539 to + 188 bp and -1843 to + 188 bp. The protein expressions of fragments -2162 to + 188 bp and -2025 to + 188 bp, -1539 to + 188 bp and -1215 to + 188 bp, -1215 to + 188 bp and -1066 to + 188 bp were significantly different in HEK-293 and SK-N-SH cells. According to the predictions of the JASPAR database, the transcription factors REST, EGR1, and CREB1/HIC2 may bind the DNA sequences of GRIN1 gene from the -337 to -159, -556 to -337, and -704 to -556, respectively. In addition, zinc finger transcription factors may regulate the expression of other differentially expressed fragments. CONCLUSIONS: Abnormal transcription regulation in the proximal promoter region of GRIN1 (-704 to + 188 bp) may be involved in the course of neuropsychiatric diseases.

Potential Ago2/miR-3068-5p Cascades in the Nucleus Accumbens Contribute to Methamphetamine-Induced Locomotor Sensitization of Mice.

Dysregulation of microRNA (miRNA) biogenesis is involved in drug addiction. Argonaute2 (Ago2), a specific splicing protein involved in the generation of miRNA, was found to be dysregulated in the nucleus accumbens (NAc) of methamphetamine (METH)-sensitized mice in our previous study. Here, we determined whether Ago2 in the NAc regulates METH sensitization in mice and identified Ago2-dependent miRNAs involved in this process. We found a gradual reduction in Ago2 expression in the NAc following repeated METH use. METH-induced hyperlocomotor activity in mice was strengthened by knocking down NAc neuronal levels of Ago2 but reduced by overexpressing Ago2 in NAc neurons. Surprisingly, miR-3068-5p was upregulated following overexpression of Ago2 and downregulated by silencing Ago2 in the NAc. Knocking down miR-3068-5p, serving as an Ago2-dependent miRNA, strengthened the METH sensitization responses in mice. These findings demonstrated that dysregulated Ago2 in neurons in the NAc is capable of regulating METH sensitization and suggested a potential role of Ago2-dependent miR-3068-5p in METH sensitization.

Memantine Prevents the WIN 55,212-2 Evoked Cross-Priming of Ethanol-Induced Conditioned Place Preference (CPP).

The activation of the endocannabinoid system controls the release of many neurotransmitters involved in the brain reward pathways, including glutamate. Both endocannabinoid and glutamate systems are crucial for alcohol relapse. In the present study, we hypothesize that N-methyl-D-aspartate (NMDA) glutamate receptors regulate the ability of a priming dose of WIN 55,212-2 to cross-reinstate ethanol-induced conditioned place preference (CPP). To test this hypothesis, ethanol-induced (1.0 g/kg, 10% w/v, i.p.) CPP (unbiased method) was established using male adult Wistar rats. After CPP extinction, one group of animals received WIN 55,212-2 (1.0 and 2.0 mg/kg, i.p.), the cannabinoid receptor 1 (CB1) agonist, or ethanol, and the other group received memantine (3.0 or 10 mg/kg, i.p.), the NMDA antagonist and WIN 55,212-2 on the reinstatement day. Our results showed that a priming injection of WIN 55,212-2 (2.0 mg/kg, i.p.) reinstated (cross-reinstated) ethanol-induced CPP with similar efficacy to ethanol. Memantine (3.0 or 10 mg/kg, i.p.) pretreatment blocked this WIN 55,212-2 effect. Furthermore, our experiments indicated that ethanol withdrawal (7 days withdrawal after 10 days ethanol administration) down-regulated the CNR1 (encoding CB1), GRIN1/2A (encoding GluN1 and GluN2A subunit of the NMDA receptor) genes expression in the prefrontal cortex and dorsal striatum, but up-regulated these in the hippocampus, confirming the involvement of these receptors in ethanol rewarding effects. Thus, our results show that the endocannabinoid system is involved in the motivational properties of ethanol, and glutamate may control cannabinoid induced relapse into ethanol seeking behavior.

The relationship of childhood trauma and DNA methylation of NMDA receptor genes in first-episode schizophrenia.

Aim: We investigated GRIN1, GRIN2A, GRIN2B and LINE-1 DNA methylation in first-episode schizophrenia patients, their nonaffected siblings and age- and sex-matched controls testing for associations between DNA methylation and exposition to childhood trauma. Materials & methods: The Childhood Trauma Questionnaire evaluated the history of childhood trauma. Genomic DNA was bisulfite converted and pyrosequencing was employed to quantify DNA methylation. Results:GRIN2A, GRIN2B and LINE-1 DNA methylation was not associated with childhood trauma in patients, siblings and controls. Siblings with childhood trauma had hypermethylation at CpG1 of GRIN1 compared with siblings without trauma. Conclusion: Childhood trauma may influence GRIN1 methylation in subjects with liability to psychosis, but not in frank schizophrenia or controls.

Lay abstract Schizophrenia results from a combination of genetic and environmental influences. We investigated how some changes in genes can be silenced by a process named DNA methylation and may be linked to schizophrenia. For this reason, we hypothesized that childhood trauma, an environmental risk factor, would be associated with DNA methylation in schizophrenia patients compared with their unaffected siblings and controls. Our research has shown that altered blood DNA methylation of one candidate gene for psychiatric disorders may be associated with childhood trauma in the unaffected siblings of schizophrenia patients, but not in frank schizophrenia or controls. We believe that this gene plays an important role in helping identify vulnerable as well as resilient individuals to schizophrenia disorder.

Chronic Alcohol Use Induces Molecular Genetic Changes in the Dorsomedial Thalamus of People with Alcohol-Related Disorders.

The Mediodorsal (MD) thalamus that represents a fundamental subcortical relay has been underrepresented in the studies focusing on the molecular changes in the brains of subjects with alcohol use disorder (AUD). In the current study, MD thalamic regions from AUD subjects and controls were analyzed with Affymetrix Clariom S human microarray. Long-term alcohol use induced a significant (FDR ≤ 0.05) upregulation of 2802 transcripts and downregulation of 1893 genes in the MD thalamus of AUD subjects. A significant upregulation of GRIN1 (glutamate receptor NMDA type 1) and FTO (alpha-ketoglutarate dependent dioxygenase) was confirmed in western blot analysis. Immunohistochemical staining revealed similar heterogenous distribution of GRIN1 in the thalamic nuclei of both AUD and control subjects. The most prevalent functional categories of upregulated genes were related to glutamatergic and GABAergic neurotransmission, cellular metabolism, and neurodevelopment. The prevalent gene cluster among down-regulated genes was immune system mediators. Forty-two differentially expressed genes, including FTO, ADH1B, DRD2, CADM2, TCF4, GCKR, DPP6, MAPT and CHRH1, have been shown to have strong associations (FDR p < 10-8) with AUD or/and alcohol use phenotypes in recent GWA studies. Despite a small number of subjects, we were able to detect robust molecular changes in the mediodorsal thalamus caused by alcohol emphasizing the importance of deeper brain structures such as diencephalon, in the development of AUD-related dysregulation of neurocircuitry.

Forebrain expression of serine racemase during postnatal development.

N-methyl-D-aspartate receptors (NMDARs) are important for synaptogenesis, synaptic maturation and refinement during the early postnatal weeks after birth. Defective synapse formation or refinement underlie cognitive and emotional abnormalities in various neurodevelopmental disorders (NDDs), including schizophrenia (Sz) and autism spectrum disorder (ASD). Serine racemase (SR) is a neuronal enzyme that produces D-serine, a co-agonist required for full NMDAR activation. NMDAR hypofunction as a result of genetic SR elimination and reduced synaptic availability of D-serine reduces neuronal dendritic arborization and spine density. In adult mouse brain, the expression of SR parallels that of NMDARs across forebrain regions including the striatum, amygdala, hippocampus, and medial prefrontal cortex (mPFC). However, there have yet to be studies providing a detailed characterization of the spatial and temporal expression of SR during early periods of synaptogenesis. Here, we examined the postnatal expression of SR in cortical and subcortical brain regions important for learning, memory and emotional regulation, during the first four weeks after birth. Using dual-antigen immunofluorescence, we demonstrate that the number of SR+ neurons steadily increases with postnatal age across the mPFC, amygdala, hippocampus and striatum. We also identified differences in the rate of SR protein induction both across and within brain regions. Analyzing existing human post-mortem brain in situ data, there was a similar developmental mRNA expression profile of SRR and GRIN1 (GluN1 subunit) from infancy through the first decade of life. Our findings further support a developmental role for D-serine mediated NMDAR activation regulating synaptogenesis and neural circuit refinement, which has important implications for the pathophysiology of Sz and other NDDs.

Acute D-Serine Co-Agonism of ß-Cell NMDA Receptors Potentiates Glucose-Stimulated Insulin Secretion and Excitatory ß-Cell Membrane Activity.

Insulin-secreting pancreatic ß-cells express proteins characteristic of D-serine regulated synapses, but the acute effect of D-serine co-agonism on its presumptive ß-cell target, N-methyl D-aspartate receptors (NMDARs), is unclear. We used multiple models to evaluate glucose homeostasis and insulin secretion in mice with a systemic increase in D-serine (intraperitoneal injection or DAAO mutants without D-serine catabolism) or tissue-specific loss of Grin1-encoded GluN1, the D-serine binding NMDAR subunit. We also investigated the effects of D-serine ± NMDA on glucose-stimulated insulin secretion (GSIS) and ß-cell depolarizing membrane oscillations, using perforated patch electrophysiology, in ß-cell-containing primary isolated mouse islets. In vivo models of elevated D-serine correlated to improved blood glucose and insulin levels. In vitro, D-serine potentiated GSIS and ß-cell membrane excitation, dependent on NMDAR activating conditions including GluN1 expression (co-agonist target), simultaneous NMDA (agonist), and elevated glucose (depolarization). Pancreatic GluN1-loss females were glucose intolerant and GSIS was depressed in islets from younger, but not older, ßGrin1 KO mice. Thus, D-serine is capable of acute antidiabetic effects in mice and potentiates insulin secretion through excitatory ß-cell NMDAR co-agonism but strain-dependent shifts in potency and age/sex-specific Grin1-loss phenotypes suggest that context is critical to the interpretation of data on the role of D-serine and NMDARs in ß-cell function.

From bedside-to-bench: What disease-associated variants are teaching us about the NMDA receptor.

NMDA receptors (NMDARs) are glutamate-gated ion channels that contribute to nearly all brain processes. Not surprisingly then, genetic variations in the genes encoding NMDAR subunits can be associated with neurodevelopmental, neurological and psychiatric disorders. These disease-associated variants (DAVs) present challenges, such as defining how DAV-induced alterations in receptor function contribute to disease progression and how to treat the affected individual clinically. As a starting point to overcome these challenges, we need to refine our understanding of the complexity of NMDAR structure function. In this regard, DAVs have expanded our knowledge of NMDARs because they do not just target well-known structure-function motifs, but rather give an unbiased view of structural elements that are important to the biology of NMDARs. Indeed, established NMDAR structure-function motifs have been validated by the appearance of disorders in patients where these motifs have been altered, and DAVs have identified novel structural features in NMDARs such as gating triads and hinges in the gating machinery. Still, the majority of DAVs remain unexplored and occur at sites in the protein with unidentified function or alter receptor properties in multiple and unanticipated ways. Detailed mechanistic and structural investigations are required of both established and novel motifs to develop a highly refined pathomechanistic model that accounts for the complex machinery that regulates NMDARs. Such a model would provide a template for rational drug design and a starting point for personalized medicine.

Functional Analysis of the 3' Untranslated Region of the Human GRIN1 Gene in Regulating Gene Expression in vitro.

PURPOSE: Abnormal expression of the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor may potentially increase the susceptibility to neuropsychiatric diseases. The purpose of this study was to investigate the functional sequence of the 3'UTR of the human GRIN1 gene, which encodes the GluN1 receptor to determine the effect on the expression of GluN1 receptor. METHODS: We transferred seven recombinant pmirGLO recombinant vectors containing the 3'UTR truncated fragment of the GRIN1 gene into HEK-293, SK-N-SH, and U87 cell lines and compared the relative fluorescence intensity of adjacent length fragments. The TargetScan database was used to predict miRNAs. Then, miRNA mimics/inhibitors were co-transfected into the three cell lines with the 3'UTR of GRIN1 (pmirGLO - GRIN1), to investigate their influence on GRIN1 gene expression. RESULTS: Compared with the pmirGLo-Basic vector, the relative fluorescence intensity of the complete GRIN1 gene 3'UTR recombinant sequence -27 bp - +1284 bp (the next base of the stop codon is +1) was significantly decreased in all three cell lines. The relative fluorescence intensities were significantly different between -27 bp - +294 bp and -27 bp - +497 bp regions, and between -27 bp - +708 bp and -27 bp - +907 bp regions. According to the prediction of the TargetScan database and analysis, miR-212-5p, miR-324-3p and miR-326 may bind to +295 bp - +497 bp, while miR-491-5p may bind to +798 bp - +907 bp. After co-transfection of miRNA mimic/inhibitor or mimic/inhibitor NC with a recombinant vector in the 3'UTR region of GRIN1 gene, we found that has-miR-491-5p inhibited GRIN1 expression significantly in all three cell lines, while has-miR-326 inhibitor upregulated GRIN1 expression in HEK-293 and U87 cells. CONCLUSION: miR-491-5p may bind to the 3'UTR of the GRIN1 gene (+799 bp - +805 bp, the next base of the stop codon is +1) and down-regulate gene expression in HEK-293, SK-N-SH, and U87 cell lines, which implicates a potential role of miR-491-5p in central nervous system diseases.