Gain-of-function and loss-of-function variants in GRIA3 lead to distinct neurodevelopmental phenotypes.

AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors (AMPARs) mediate fast excitatory neurotransmission in the brain. AMPARs form by homo- or heteromeric assembly of subunits encoded by the GRIA1-GRIA4 genes, of which only GRIA3 is X-chromosomal. Increasing numbers of GRIA3 missense variants are reported in patients with neurodevelopmental disorders (NDD), but only a few have been examined functionally. Here, we evaluated the impact on AMPAR function of one frameshift and 43 rare missense GRIA3 variants identified in patients with NDD by electrophysiological assays. Thirty-one variants alter receptor function and show loss-of-function (LoF) or gain-of-function (GoF) properties, whereas 13 appeared neutral. We collected detailed clinical data from 25 patients (from 23 families) harbouring 17 of these variants. All patients had global developmental impairment, mostly moderate (9/25) or severe (12/25). Twelve patients had seizures, including focal motor (6/12), unknown onset motor (4/12), focal impaired awareness (1/12), (atypical) absence (2/12), myoclonic (5/12), and generalized tonic-clonic (1/12) or atonic (1/12) seizures. The epilepsy syndrome was classified as developmental and epileptic encephalopathy in eight patients, developmental encephalopathy without seizures in 13 patients, and intellectual disability with epilepsy in four patients. Limb muscular hypotonia was reported in 13/25, and hypertonia in 10/25. Movement disorders were reported in 14/25, with hyperekplexia or non-epileptic erratic myoclonus being the most prevalent feature (8/25). Correlating receptor functional phenotype with clinical features revealed clinical features for GRIA3-associated NDDs and distinct NDD phenotypes for LoF and GoF variants. GoF variants were associated with more severe outcomes: patients were younger at the time of seizure onset (median age one month), hypertonic, and more often had movement disorders, including hyperekplexia. Patients with LoF variants were older at the time of seizure onset (median age 16 months), hypotonic, and had sleeping disturbances. LoF and GoF variants were disease-causing in both sexes but affected males often carried de novo or hemizygous LoF variants inherited from healthy mothers, whereas all but one affected females had de novo heterozygous GoF variants.

Myoclonic status epilepticus and cerebellar hypoplasia associated with a novel variant in the GRIA3 gene.

AMPA-type glutamate receptors (AMPARs) are postsynaptic ionotropic receptors which mediate fast excitatory currents. AMPARs have a heterotetrameric structure, variably composed by the four subunits GluA1-4 which are encoded by genes GRIA1-4. Increasing evidence support the role of pathogenic variants in GRIA1-4 genes as causative for syndromic intellectual disability (ID). We report an Italian pedigree where some male individuals share ID, seizures and facial dysmorphisms. The index subject was referred for severe ID, myoclonic seizures, cerebellar signs and short stature. Whole exome sequencing identified a novel variant in GRIA3, c.2360A > G, p.(Glu787Gly). The GRIA3 gene maps to chromosome Xq25 and the c.2360A > G variant was transmitted by his healthy mother. Subsequent analysis in the family showed a segregation pattern compatible with the causative role of this variant, further supported by preliminary functional insights. We provide a detailed description of the clinical evolution of the index subjects and stress the relevance of myoclonic seizures and cerebellar syndrome as cardinal features of his presentation.

Acetylation of H3K27 activated lncRNA NEAT1 and promoted hepatic lipid accumulation in non-alcoholic fatty liver disease via regulating miR-212-5p/GRIA3.

Non-alcoholic fatty liver disease (NAFLD) was a world-wide health burden. H3K27 acetylation, long non-coding RNA (lncRNA), and miRNA were all implicated in NAFLD regulation, yet the detailed regulatory mechanism was not well understood. LncRNA NEAT1, miR-212-5p, and GRIA3 expression were detected both in high fatty acid-treated hepatocytes cells and NAFLD patients. Lipid droplets were stained and analyzed by oil red O staining. Expression of fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC), and GRIA3 was detected by qRT-PCR and western blot. RNA level of lncRNA NEAT1 and miR-212-5p was analyzed by qRT-PCR. The binding sequences of lncRNA NEAT1/miR-212-5p and miR-212-5p/GRIA3 were predicted bioinformatically and validated through luciferase assay. ChIP was performed to analyze H3K27 acetylation on the promoter of lncRNA NEAT1. LncRNA NEAT1 and GRIA3 was upregulated, while miR-212-5p was downregulated in NAFLD patients. FFA promoted lncRNA NEAT1 and GRIA3 expression while suppressing miR-212-5p and promoted lipid accumulation as indicated by increased oil red O staining and FAS and ACC expression. ChIP indicated enrichment of H3K27 on NEAT1 promoter. Inhibition of H3K27 acetylation suppressed lncRNA NEAT1 level. Luciferase results indicated direct interaction of NEAT1/miR-212-5p (which was confirmed by RIP) and miR-212-5p/GRIA3. LncRNA NEAT1 knockdown upregulated miR-212-5p level and inhibited FFA-induced lipid accumulation while suppressing GRIA3 expression. Such function was antagonized by miR-212-5p inhibition and GRIA3 knockdown counteracted with miR-212-5p inhibition. H3K27 acetylation was enriched within the promoter of lncRNA NEAT1 and promoted lncRNA NEAT1 transcription. LncRNA NEAT1 could then interact with miR-212-5p and suppress its cellular concentration.

The GRIA3 c.2477G > A Variant Causes an Exaggerated Startle Reflex, Chorea, and Multifocal Myoclonus.

BACKGROUND: Hemizygous mutations in GRIA3 encoding the GluA3 subunit of the amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor are known to be associated with neurodevelopmental disorders, including intellectual disability, hypotonia, an autism spectrum disorder, sleep disturbances, and epilepsy in males. OBJECTIVE: To describe a new and consistent phenotype in 4 affected male patients associated with an undescribed deleterious variant in GRIA3. METHODS: We evaluated a large French family in which segregate a singular phenotype according to an apparent X-linked mode of inheritance. Molecular analyses using next generation sequencing and in vitro functional studies using 2-electrode voltage clamp recordings on Xenopus laevis oocytes and a ß-lactamase reporter assay in transfected human embryonic kidney (HEK293) cells were performed. RESULTS: In addition to mild intellectual disability and dysarthria, affected patients presented a tightly consistent early-onset movement disorder combining an exaggerated startle reflex with generalized chorea and multifocal myoclonus. The unreported GRIA3 missense variant c.2477G > A; p.(Gly826Asp) affecting the fourth transmembrane domain of the protein was identified in index patients and their unaffected mothers. Functional studies revealed that variant receptors show decreased current response evoked by agonist (ie, kainic acid and glutamate) and reduced expression on the cell surface in favor of pathogenicity by a loss-of-function mechanism. CONCLUSIONS: Taken together, our results suggest that apart from known GRIA3-related disorders, an undescribed mutation-specific singular movement disorder does exist. We thus advocate considering GRIA3 mutations in the differential diagnosis of hyperekplexia and generalized chorea with myoclonus. © 2020 International Parkinson and Movement Disorder Society.

Case-control study of GRIA1 and GRIA3 gene variants in migraine.

BACKGROUND: As the most abundant excitatory neurotransmitter in the central nervous system, glutamate has been accepted to play a major role in the pathophysiology of migraine. The previous studies have reported the glutamate receptor ionotropic GRIA1 and GRIA3 genes variants associated with migraine. The project aims to investigate the polymorphisms in both genes for their association with migraine in the Chinese Han population. METHODS: A Han-Chinese case-control population, including 331 unrelated female migraine patients and 330 matched controls, was studied. Variants in genes (GRIA1 and GRIA3) were genotyped by Multiplex SNaPshot assay. RESULTS: In the group of patients, the frequency of allele C was 84.1 % (557 C alleles) and allele T was 15.9 % (105 T alleles) for the GRIA1 (rs2195450) in migraineurs, this was significantly as compared with the controls (P = .001, OR = 1.786, 95 % CI: 1.28-2.49). And an association was also seen in the migraine with aura (MA) subtype (P = .012, OR = 2.092, 95 % CI: 1.17-3.76) and migraine without aura (MO) subtype (P = .002, OR = 1.737, 95 % CI: 1.23-2.45). However, no evidence was found that GRIA1 (rs548294) or GRIA3 (rs3761555) is associated with migraine. CONCLUSION: Our data of this study confirmed the association of GRIA1 (rs2195450) to female migraine (MA, MO) susceptibility in the Chinese Han population. The result provides evidence that the glutamatergic system is implicated in the pathophysiology of migraine.

Female GluA3-KO mice show early onset hearing loss and afferent swellings in ambient sound levels.

AMPA-type glutamate receptors (AMPAR) mediate excitatory cochlear transmission. However, the unique roles of AMPAR subunits are unresolved. Lack of subunit GluA3 (Gria3KO) in male mice reduced cochlear output by 8-weeks of age. Since Gria3 is X-linked and considering sex differences in hearing vulnerability, we hypothesized accelerated presbycusis in Gria3KO females. Here, auditory brainstem responses (ABR) were similar in 3-week-old female Gria3WT and Gria3KO mice. However, when raised in ambient sound, ABR thresholds were elevated and wave-1 amplitudes were diminished at 5-weeks and older in Gria3KO. In contrast, these metrics were similar between genotypes when raised in quiet. Paired synapses were similar in number, but lone ribbons and ribbonless synapses were increased in female Gria3KO mice in ambient sound compared to Gria3WT or to either genotype raised in quiet. Synaptic GluA4:GluA2 ratios increased relative to Gria3WT, particularly in ambient sound, suggesting an activity-dependent increase in calcium-permeable AMPARs in Gria3KO. Swollen afferent terminals were observed by 5-weeks only in Gria3KO females reared in ambient sound. We propose that lack of GluA3 induces sex-dependent vulnerability to AMPAR-mediated excitotoxicity.

Association of a GRIA3 gene polymorphism with migraine in an Australian case-control cohort.

BACKGROUND: The excitatory neurotransmitter glutamate has been implicated in both the hyperexcitability required for cortical spreading depression as well as activation of the trigeminovascular system required for the allodynia associated with migraine. Polymorphisms in the glutamate receptor ionotropic amino-3-hydroxy-5-methyl-4-isoxazole-propionin acid 1 (GRIA1) and GRIA3 genes that code for 2 of 4 subunits of the glutamate receptor have been previously associated with migraine in an Italian population. In addition, the GRIA3 gene is coded within a previously identified migraine susceptibility locus at Xq24. This study investigated the previously associated polymorphisms in both genes in an Australian case-control population. METHODS: Variants in GRIA1 and GRIA3 were genotyped in 472 unrelated migraine cases and matched controls, and data were analyzed for association. RESULTS: Analysis showed no association between migraine and the GRIA1 gene. However, association was observed with the GRIA3 single nucleotide polymorphism (SNP) rs3761555 (P=.008). CONCLUSION: The results of this study confirmed the previous report of association at the rs3761555 SNP within the migraine with aura subgroup of migraineurs. However, the study identified association with the inverse allele suggesting that rs3761555 may not be the causative SNP but is more likely in linkage disequilibrium with another causal variant in both populations. This study supports the plethora of evidence suggesting that glutamate dysfunction may contribute to migraine susceptibility, warranting further investigation of the glutamatergic system and particularly of the GRIA3 gene.

The p.Glu787Lys variant in the GRIA3 gene causes developmental and epileptic encephalopathy mimicking structural epilepsy in a female patient.

The GRIA3 gene is located in the X chromosome and encodes for one of the subunits (iGluR3) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), an excitatory synaptic transmission receptor present in most parts of the brain. iGluR3 dysfunction has been associated with both abnormal memory formation and learning. It has been observed in patients with different neurological and cognitive disorders, including epilepsy. Three different de novo missense variants of GRIA3 have recently been reported in patients with Developmental and Epileptic Encephalopathy (DEE). We report on a female pediatric patient with DEE whose clinical picture mimicked structural epilepsy. We give a detailed description of our patient's most important electro-clinical features. Genetic analysis revealed that the patient carried a de novo missense variant in GRIA3 (c.2359G>A; p.Glu787Lys). The p.Glu787Lys variant had previously been reported in a male pediatric patient. Additionally, we studied iGluR3 expression in the patient and control fibroblasts. We found significantly lower iGluR3 expression in the patient's fibroblasts than in controls and different responses to glutamate treatment. In summary, our report expands knowledge of GRIA3 variants affecting boys and girls, describes functional studies of these variants, and provides an extensive review of the literature concerning GRIA3 genetic variants.

Analysis of the circRNA and T-UCR populations identifies convergent pathways in mouse and human models of Rett syndrome.

Noncoding RNAs play regulatory roles in physiopathology, but their involvement in neurodevelopmental diseases is poorly understood. Rett syndrome is a severe, progressive neurodevelopmental disorder linked to loss-of-function mutations of the MeCP2 gene for which no cure is yet available. Analysis of the noncoding RNA profile corresponding to the brain-abundant circular RNA (circRNA) and transcribed-ultraconserved region (T-UCR) populations in a mouse model of the disease reveals widespread dysregulation and enrichment in glutamatergic excitatory signaling and microtubule cytoskeleton pathways of the corresponding host genes. Proteomic analysis of hippocampal samples from affected individuals confirms abnormal levels of several cytoskeleton-related proteins together with key alterations in neurotransmission. Importantly, the glutamate receptor GRIA3 gene displays altered biogenesis in affected individuals and in vitro human cells and is influenced by expression of two ultraconserved RNAs. We also describe post-transcriptional regulation of SIRT2 by circRNAs, which modulates acetylation and total protein levels of GluR-1. As a consequence, both regulatory mechanisms converge on the biogenesis of AMPA receptors, with an effect on neuronal differentiation. In both cases, the noncoding RNAs antagonize MeCP2-directed regulation. Our findings indicate that noncoding transcripts may contribute to key alterations in Rett syndrome and are not only useful tools for revealing dysregulated processes but also molecules of biomarker value.

Protein profiling identified mitochondrial dysfunction and synaptic abnormalities after dexamethasone intervention in rats with traumatic brain injury.

Dexamethasone has been widely used after various neurosurgical procedures due to its anti-inflammatory property and the abilities to restore vascular permeability, inhibit free radicals, and reduce cerebrospinal fluid production. According to the latest guidelines for the treatment of traumatic brain injury in the United States, high-dose glucocorticoids cause neurological damage. To investigate the reason why high-dose glucocorticoids after traumatic brain injury exhibit harmful effect, rat controlled cortical impact models of traumatic brain injury were established. At 1 hour and 2 days after surgery, rat models were intraperitoneally administered dexamethasone 10 mg/kg. The results revealed that 31 proteins were significantly upregulated and 12 proteins were significantly downregulated in rat models of traumatic brain injury after dexamethasone treatment. The Ingenuity Pathway Analysis results showed that differentially expressed proteins were enriched in the mitochondrial dysfunction pathway and synaptogenesis signaling pathway. Western blot analysis and immunohistochemistry results showed that Ndufv2, Maob and Gria3 expression and positive cell count in the dexamethasone-treated group were significantly greater than those in the model group. These findings suggest that dexamethasone may promote a compensatory increase in complex I subunits (Ndufs2 and Ndufv2), increase the expression of mitochondrial enzyme Maob, and upregulate synaptic-transmission-related protein Gria3. These changes may be caused by nerve injury after traumatic brain injury treatment by dexamethasone. The study was approved by Institutional Ethics Committee of Beijing Neurosurgical Institute (approval No. 201802001) on June 6, 2018.

Expression of Glutamate Receptor Subtype 3 Is Epigenetically Regulated in Podocytes under Diabetic Conditions.

BACKGROUND: Recent studies suggest a role of epigenetics in the pathogenesis of diabetic kidney disease. However, epigenetic changes occurring specifically in kidney cells is poorly understood. METHODS: To examine the epigenetic regulation of genes in podocytes under diabetic conditions, we performed DNA methylation and transcriptomic profiling in podocytes exposed to high glucose conditions. RESULTS: Comparative analysis of genes with DNA methylation changes and correspondingly altered mRNA expression identified 337 hypomethylated genes with increased mRNA expression and only 2 hypermethyated genes (ESX1 and GRIA3) with decreased mRNA expression. Glutamate ionotropic receptor AMPA type subunit 3 (GRIA3) belongs to the ionotropic class of glutamate receptors that mediate fast excitatory synaptic transmission in the central nervous system. As podocytes have glutamate-containing vesicles and various glutamate receptors mediate important biological effects in podocytes, we further examined GRIA3 expression and its function in podocytes. Real-time PCR and western blots confirmed the suppression of GRIA3 expression in podocytes under high glucose conditions, which were abolished in the presence of a DNA methyltransferase inhibitor. Sites of DNA hypermethylation were also confirmed by bisulfite sequencing of the GRIA3 promoter region. GRIA3 mRNA and protein expression was suppressed in diabetic kidneys of human and mouse models, and knockdown of GRIA3 exacerbated high glucose-induced apoptosis in cultured podocytes. CONCLUSION: These results indicate that decreased GRIA3 expression in podocytes in diabetic condition heightens podocyte apoptosis and loss.

MicroRNA-330-3p promotes brain metastasis and epithelial-mesenchymal transition via GRIA3 in non-small cell lung cancer.

Brain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). We sought to identify microRNAs (miRNAs) that could serve as biomarkers to differentiate NSCLC patients with and without BM. Logistic regression was conducted with 122 NSCLC patients (60 without BM, 62 with BM) to assess the association between miRNAs and BM. We confirmed several risk factors for BM and revealed that serum miR-330-3p levels are higher in NSCLC patients with BM than that without BM. Overexpression of miR-330-3p promoted proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) of NSCLC cells in vitro and NSCLC tumorigenesis in vivo. Knocking down miR-330-3p suppressed this metastatic phenotype. We identified putative miR-330-3p target genes by comparing mRNA microarray analysis data from A549 cells after miR-330-3p knockdown with candidate miR-330-3p target genes predicted by public bioinformatic tools and luciferase reporter assays. We found that GRIA3 is a target of miR-330-3p and that miR-330-3p stimulates EMT progress by mediating GRIA3-TGF-ß1 interaction. Our results provide novel insight into the role of miR-330-3p in NSCLC metastasis, and suggest miR-330-3p may be a useful biomarker for identifying NSCLC with metastatic potential.

Chaihu-Shugan-San exerts an antidepressive effect by downregulating miR-124 and releasing inhibition of the MAPK14 and Gria3 signaling pathways.

Dysregulation of miR-124 has been reported to be involved in the pathophysiology of depression. Chaihu-Shugan-San, a traditional Chinese medicine, has antidepressive activity; however, the underlying mechanisms remain unclear. In this study, to generate a rodent model of depression, rats were subjected to a combination of solitary confinement and chronic unpredictable mild stress for 28 days. Rats were intragastrically administered Chaihu-Shugan-San (2.835 mL/kg/d) for 4 weeks, once a day. Real-time reverse-transcription quantitative polymerase chain reaction, miRNA microarray, western blot assay and transmission electron microscopy demonstrated that Chaihu-Shugan-San downregulated miR-124 expression and upregulated the mRNA and protein levels of mitogen-activated protein kinase 14 (MAPK14) and glutamate receptor subunit 3 (Gria3). Chaihu-Shugan-San also promoted synapse formation in the hippocampus. The open field test, sucrose consumption test and forced swimming test were used to assess depression-like behavior. After intragastric administration of Chaihu-Shugan-San, sucrose consumption increased, while the depressive behaviors were substantially reduced. Together, these findings suggest that Chaihu-Shugan-San exerts an antidepressant-like effect by downregulating miR-124 expression and by releasing the inhibition of the MAPK14 and Gria3 signaling pathways.

MicroRNA-330-3p promotes cell invasion and metastasis in non-small cell lung cancer through GRIA3 by activating MAPK/ERK signaling pathway.

BACKGROUND: Brain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Recent studies demonstrated that microRNA-330-3p (miR-330-3p) was involved in NSCLC brain metastasis (BM). However, the exact parts played by miR-330-3p in BM of NSCLC remain unknown. Discovery and development of biomarkers and elucidation of the mechanism underlying BM in NSCLC is critical for effective prophylactic interventions. Here, we evaluated the expression and biological effects of miR-330-3p in NSCLC cells and explored the underlying mechanism of miR-330-3p in promoting cell migration and invasion in NSCLC. METHODS: Stable over-expression and knockdown of miR-330-3p in NSCLC cells was constructed with lentivirus. Expression levels of miR-330-3p in NSCLC cells were quantified by quantitive real-time PCR (qRT-PCR). The effects of miR-330-3p on NSCLC cells were investigated using assays of cell viability, migration, invasion, cell cycle, apoptosis, western blotting, immunohistochemical, and immunofluorescence staining. A xenograft nude mouse model and in situ brain metastasis model were used to observe tumor growth and brain metastasis. The potential target of miR-330-3p in NSCLC cells was explored using the luciferase reporter assay, qRT-PCR, and western blotting. The miR-330-3p targets were identified using bioinformatics analysis and verified by luciferase reporter assay. The correlation between GRIA3 and DNA methyltransferase (DNMT) 1 and DNMT3A was tested by RT-PCR, western blotting, and co-immunoprecipitation (IP). RESULTS: miR-330-3p was significantly up-regulated in NSCLC cell lines. MTT assay, transwell migration, and invasion assays showed that miR-330-3p promoted the growth, migration, and invasion of NSCLC cells in vitro and induced tumor growth and metastasis in vivo. Luciferase reporter assays showed that GRIA3 was a target of miR-330-3p. qRT-PCR and western blotting exhibited that miR-330-3p promoted the growth, invasion, and migration of NSCLC cells by activating mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinases (ERK) signaling pathway. Furthermore, miR-330-3p up-regulated the total DNA methylation in NSCLC cells, and co-IP-demonstrated GRIA3 was directly related with DNMT1 and DNMT3A. CONCLUSIONS: miR-330-3p promoted the progression of NSCLC and might be a potential target for the further research of NSCLC brain metastasis.

Shared genetic background for regulation of mood and sleep: association of GRIA3 with sleep duration in healthy Finnish women.

STUDY OBJECTIVES: Sleeping 7 to 8 hours per night appears to be optimal, since both shorter and longer sleep times are related to increased morbidity and mortality. Depressive disorder is almost invariably accompanied by disturbed sleep, leading to decreased sleep duration, and disturbed sleep may be a precipitating factor in the initiation of depressive illness. Here, we examined whether, in healthy individuals, sleep duration is associated with genes that we earlier found to be associated with depressive disorder. DESIGN: Population-based molecular genetic study. SETTING: Regression analysis of 23 risk variants for depressive disorder from 12 genes to sleep duration in healthy individuals. PARTICIPANTS: Three thousand, one hundred, forty-seven individuals (25-75 y) from population-based Health 2000 and FINRISK 2007 samples. MEASUREMENTS AND RESULTS: We found a significant association of rs687577 from GRIA3 on the X-chromosome with sleep duration in women (permutation-based corrected empirical P=0.00001, ß=0.27; Bonferroni corrected P=0.0052; f=0.11). The frequency of C/C genotype previously found to increase risk for depression in women was highest among those who slept for 8 hours or less in all age groups younger than 70 years. Its frequency decreased with the lengthening of sleep duration, and those who slept for 9 to 10 hours showed a higher frequency of C/A or A/A genotypes, when compared with the midrange sleepers (7-8 hours) (permutation-based corrected empirical P=0.0003, OR=1.81). CONCLUSIONS: The GRIA3 polymorphism that was previously found to be associated with depressive disorder in women showed an association with sleep duration in healthy women. Mood disorders and short sleep may share a common genetic background and biologic mechanisms that involve glutamatergic neurotransmission.