Regulator Of G-protein Signaling 4 Research Articles

MGST3 regulates BACE1 protein translation and amyloidogenesis by controlling the RGS4-mediated AKT signaling pathway

Microsomal glutathione transferase 3 (MGST3) regulates eicosanoid and glutathione metabolism. These processes are associated with oxidative stress and apoptosis, suggesting that MGST3 might play a role in the pathophysiology of Alzheimer's disease (AD). Here, we report that knockdown (KD) of MGST3 in cell lines reduced the protein level of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) and the resulting amyloidogenesis. Interestingly, MGST3 KD did not alter intracellular ROS level but selectively reduced the expression of apoptosis indicators which could be associated with the receptor of cysteinyl leukotrienes (cysLTs), the downstream metabolites of MGST3 in arachidonic acid pathway. We then showed that the effect of MGST3 on BACE1 was independent of cysLTs but involved a translational mechanism. Further RNA-seq analysis identified that regulator of G-protein signaling 4 (RGS4) was a target gene of MGST3. Silencing of RGS4 inhibited BACE1 translation and prevented MGST3 KD-mediated reduction of BACE1. The potential mechanism was related to AKT activity, as the protein level of phosphorylated AKT (p-AKT) was significantly reduced by silencing of MGST3 and RGS4, and the AKT inhibitor abolished the effect of MGST3/RGS4 on p-AKT and BACE1. Together, MGST3 regulated amyloidogenesis by controlling BACE1 protein expression, which was mediated by RGS4 and downstream AKT signaling pathway.

RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms

Regulators of G protein signaling (RGS proteins) constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase activating (GAP) activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle (ASM) contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and ASM contraction whereas RGS4 knockout (KO) mice unexpectedly have decreased AHR due to increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GAP activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared to either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3 kinase (PI3K) and inhibited PI3K-dependent PGE2 secretion elicited by TGFβ in airway epithelial cells. Together these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

Depression-related RGS2 Gene Serves as a Novel Molecular Biomarker of Stomach Adenocarcinoma Prognosis and Immune Infiltration

Depression-related RGS2 Gene Serves as a Novel Molecular Biomarker of Stomach Adenocarcinoma Prognosis and Immune Infiltration

Abstract 5249: Polyphenon E effects MXD1 and RGS4 gene expression in PC-3 prostate cancer cells

Abstract Polyphenon E (Poly E) is a standardized, caffeine-free green tea extract with defined polyphenol content. Oral Poly E administration confers chemoprotective activity against prostate cancer (PCa) progression in the TRAMP model of human PCa. Poly E has limited activity against human PCa, but only in those previously diagnosed with atypical small acinar proliferation or high-grade prostate intraepithelial neoplasia. The molecular mechanisms underpinning Poly E chemopreventive activities against PCa are not fully understood. We hypothesized Poly E treatment of PCa cells induces gene expression changes, which could involve molecular mechanisms underpinning the limited Poly E chemoprevention activity against PCa. PC-3 cells were cultured in complete growth media supplemented with varied Poly E concentrations (0, 100, 200, or 300 mg/L) for 24 hr. RNA was isolated for comparative DNA microarray (0 vs. 200 mg/L Poly E) and subsequent qRT-PCR analyses for validation. Microarray data for 54,613 genes were filtered for > 2-fold expression level changes, with 8,319 increased and 6,176 genes decreased. Seven genes (CASP8, CBLB, CCNB1, HDAC4, MXD1, RGCC, RGS4) involved in key cellular signaling pathways and with > 2-fold expression level changes, as well as a control gene (RB1), were selected for qRT-PCR analyses. These eight genes are listed with corresponding fold-change gene expression values for each microarray probe set (Table 1). The qRT-PCR analyses identified two genes with significantly increased expression, MXD1 (13.98-fold; p=0.0003) and RGS4 (21.98-fold; p=0.0011), between poly E treatment levels. The significantly increased MXD1 and RGS4 gene expression in Poly E-treated PC-3 cells, and the Poly E-dose dependence of MXD1 gene expression increases, could implicate MXD1 and RGS4 in the PCa chemopreventive activity of Poly E. Future experimental directions include cell cycle profile and Annexin V analyses, immunoblotting analyses, and including other established PCa cell lines in these studies. Microarray Data from Poly E-treated PC-3 Cells Affymetrix Probe ID Gene symbol Log2-Fold Change 213373_s_at CASP8 -2.23 1553306_at CASP8 -3.20 208348_s_at CBLB 19.01 209682_at CBLB 3.83 214710_s_at CCNB1 -2.77 228729_at CCNB1 -3.47 1554322_a_at HDAC4 -2.47 204225_at HDAC4 -3.23 228813_at HDAC4 -4.45 206877_at MXD1 13.56 228846_at MXD1 7.35 226275_at MXD1 1.91 211540_s_at RB1 1.85 203132_at RB1 -1.43 218723_s_at RGCC 23.12 239827_at RGCC 1.22 204338_s_at RGS4 20.54 204337_at RGS4 6.12 204339_s_at RGS4 3.69 Citation Format: L. Michael Carastro, Ethan J. Vallebuona, Ricardo Cordova, Ashley N. Gannon, Corrine M. Costello, Ricardo A. Declet-Bauzo, Seung Joon Kim, Nagi Kumar, Jong Y. Park. Polyphenon E effects MXD1 and RGS4 gene expression in PC-3 prostate cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5249.

Concurrent inactivating mutations and expression losses of RGS2, HNF1A, and CAPN12 candidate tumor suppressor genes in colon cancers.

Microsatellite instability-high (MSI-H) colorectal cancer (CRC) is different from microsatellite stable (MSS) CRC concerning biological, and clinical features. In MSI-H CRCs, defects of mismatch repair genes produce increased mutation accumulation in repetitive DNA sequences. To see whether candidate tumor suppressor genes (TSGs) are altered in MSI-H CRC, we studied frameshift mutation and protein expression of candidate TSGs of RGS2, HNF1A, HNF1B, CAPN12, RCBTB2, ATE1, PKNOX1, and USP19. We found frameshift mutations of RGS2 in 5 (5%), HNF1A in 6 (6%), HNF1B in 2 (2%), CAPN12 in 3 (3%), RCBTB2 in 4 (4%), ATE1 in 2 (2%), PKNOX1 in 2 (2%), and USP19 in 2 (2%) MSI-H CRCs. However, we found no such mutations in MSS CRCs. RCBTB2, CAPN12, HNF1A, and HNF1B frameshift mutations revealed the regional difference in the same tumors. In addition, we identified loss of RGS2, HNF1A, and CAPN12 protein expression irrespective of MSI phenotype in 13-29% of CRCs. The results indicate that many TSGs harbor concurrent inactivating mutations and protein loss in MSI-H CRCs with intratumoral mutational heterogeneity, and that MSS CRCs are altered by protein losses. These alterations could contribute to CRC development and underlying mechanisms and consequences of the TSG alterations remain to be clarified.

IDENTIFICATION OF NOVEL DRUG/LEAD MOLECULE FOR SCHIZOPHRENIA TARGETING RGS4 GENE THROUGH IN-SILICO ANALYSIS

Schizophrenia is a neuropsychiatric disorder that is caused by many factors including changes in the chemistry of the brain, structure of the brain, and genetic factors. The neuropathology of the disorder has been identified morphologically and physiologically through a number of parameters from a reduction in brain size and abnormalities in the neurotransmitter network affecting the brain to changes in the molecular composition of specific cell populations in the brain. Over a long period of time, clinicians and researchers have identified schizophrenia as a complex neurological genetic disorder as a result of the functionality of several susceptible genes. Various scientists are of the opinion that there are about 20 candidate genes that cause schizophrenia. Several genes like Dysbindin, Neuregulin, DAAO, G72, PRODH, COMT, and RGS4 have been proven to be the risk factors for schizophrenia disease. RGS4 was identified as a target gene for schizophrenia. Hence the present study was taken up to identify a Novel Drug/Lead Molecule from 12 selected drugs based on a bioinformatics approach for schizophrenia targeting the RGS4 gene. The genomics and proteomics approach of the RGS4 gene was carried out for nucleotide and protein sequences and the protein was modeled using SPDBV with 80% accuracy. The potential active site amino acids were predicted using molecular cavities based on energy and surface area and the protein was targeted using the best active site amino acids. The protein-ligand interactions were also performed using the Argus Lab engine with flexible docking. The data of the present study reveals that ziprasidone is showing maximum inhibition of the RGS4 gene among the 12 drugs selected for screening. It is thus concluded that ziprasidone with the lowest free energy and high affinity is the best inhibitor for the schizophrenia protein and hence it can be suggested for the treatment of schizophrenia.

Elevated Expression of RGS2 May Underlie Reduced Olfaction in COVID-19 Patients.

Anosmia is common in COVID-19 patients, lasting for weeks or months following recovery. The biological mechanism underlying olfactory deficiency in COVID-19 does not involve direct damage to nasal olfactory neurons, which do not express the proteins required for SARS-CoV-2 infection. A recent study suggested that anosmia results from downregulation of olfactory receptors. We hypothesized that anosmia in COVID-19 may also reflect SARS-CoV-2 infection-driven elevated expression of regulator of G protein signaling 2 (RGS2), a key regulator of odorant receptors, thereby silencing their signaling. To test our hypothesis, we analyzed gene expression of nasopharyngeal swabs from SARS-CoV-2 positive patients and non-infected controls (two published RNA-sequencing datasets, 580 individuals). Our analysis found upregulated RGS2 expression in SARS-CoV-2 positive patients (FC = 14.5, Padj = 1.69 × 10−5 and FC = 2.4; Padj = 0.001, per dataset). Additionally, RGS2 expression was strongly correlated with PTGS2, IL1B, CXCL8, NAMPT and other inflammation markers with substantial upregulation in early infection. These observations suggest that upregulated expression of RGS2 may underlie anosmia in COVID-19 patients. As a regulator of numerous G-protein coupled receptors, RGS2 may drive further neurological symptoms of COVID-19. Studies are required for clarifying the cellular mechanisms by which SARS-CoV-2 infection drives the upregulation of RGS2 and other genes implicated in inflammation. Insights on these pathway(s) may assist in understanding anosmia and additional neurological symptoms reported in COVID-19 patients.

Associations of GNAS and RGS Gene Polymorphisms with the Risk of Ritodrine-Induced Adverse Events in Korean Women with Preterm Labor: A Cohort Study.

Ritodrine, a β2-adrenergic receptor agonist, is among most commonly prescribed tocolytic agents. This study aimed to evaluate the associations of single nucleotide polymorphisms in GNAS, RGS2, and RGS5 with the risk of ritodrine-induced adverse events (AEs) and develop a risk scoring system to identify high-risk patients. This is the prospective cohort study conducted at the Ewha Woman’s University Mokdong Hospital between January 2010 and October 2016. Pregnant women were included if they were treated with ritodrine for preterm labor with regular uterine contractions (at least 3 every 10 min) and cervical dilation. A total of 6, 3, and 5 single nucleotide polymorphisms (SNPs) of GNAS, RGS2, and RGS5 genes were genotyped and compared in patients with and without ritodrine-induced AEs. A total of 163 patients were included in this study. After adjusting confounders, GNAS rs3730168 (per-allele odds ratio (OR): 2.1; 95% confidence interval (95% CI): 1.0–4.3) and RGS2 rs1152746 (per-allele OR: 2.6, 95% CI: 1.1–6.5) were significantly associated with ritodrine-induced AEs. According to the constructed risk scoring models, patients with 0, 1, 2, 3, 4, and 5 points showed 0%, 13%, 19%, 31%, 46%, and 100% risks of AEs. This study suggested that GNAS and RGS2 polymorphisms could affect the risk of AEs in patients treated with ritodrine.

Phenotypic plasticity of Monilinia spp. in response to light wavelengths: From in vitro development to virulence on nectarines

The development of brown rot in stone fruit caused by the necrotrophic fungus Monilinia spp. is influenced by many abiotic factors, such as temperature, humidity, and light. Specifically, filamentous fungi perceive light as a signal for ecophysiological and adaptive responses. We have explored how specific light wavelengths affect the in vitro development, the regulation of putative development genes and the virulence of the main species of Monilinia (M. laxa, M. fructicola and M. fructigena). After subjecting Monilinia spp. to different light wavelengths (white, black, blue, red, far-red) for 7 days, several differences in their phenotype were observed among light conditions, but also among species. These species of Monilinia exhibited a different phenotypic plasticity in response to light regarding pigmentation, growth, and specially conidiation of colonies. In this sense, we observed that the conidial production was higher in M. laxa than M. fructicola, while M. fructigena showed an inability to produce conidia under the tested conditions. Growth rate among species was significantly lower in M. fructicola under red light wavelength while among light conditions it was increased under far-red light wavelength for M. laxa and under black light for M. fructicola; in contrast, no statistical differences were observed for M. fructigena. Gene expression analysis of 13 genes involved in fungal development of Monilinia spp. revealed a significant difference among the three species of Monilinia, and especially depended on light wavelengths. Among them, a high expression of OPT1, RGS2, RGS3 and SPP1 genes was observed in M. laxa, and LTF1 and STE12 in M. fructicola under black light. In contrast, a high expression of REG1 and C6TF1 genes occurred in both M. fructicola and M. laxa subject to red and far-red light wavelength, respectively. When nectarines were artificially infected with M. laxa and M. fructicola subjected to black light, the virulence was clearly reduced, but not in M. fructigena. Overall, results presented herein demonstrate that light wavelengths are a key abiotic factor for the biology of Monilinia spp., specially modulating its capacity to form conidia, and thus, influencing its spreading and the onset of the disease on nectarines during postharvest.

Exploring the expression and preliminary function of chicken regulator of G protein signalling 3 (RGS3) gene in follicular development

ABSTRACT 1. The following study explored the expression and preliminary function of RGS3. The spatial and temporal expression patterns of the RGS3 gene were analysed in the ovarian stroma of Shendan No. 6 Green shell hens and Hy-line Brown hens at four time points (6, 28, 40 and 52 weeks old), as well as in various organs and follicles of Hy-line Brown hens. 2. Based on the genomic and protein sequences of RGS3 in NCBI database, phylogenetic trees were constructed using MEGA-X. The protein interaction network was analysed using STRING. According to the results of protein-protein interaction network and pathways, the mRNA expression levels of RGS3 and three interaction proteins were explored by qRT-PCR in vitro. 3. Spatio-temporal expression data revealed that RGS3 mRNA was expressed in all the organs tested, being highest in the hypothalamus. In different follicles, RGS3 mRNA was highly expressed in post-ovulatory follicles, followed by ovarian stroma and large white follicles. The expression levels of RGS3 mRNA in the ovarian stroma were significantly higher in Shendan No. 6 Green shell hens than that in the Hy-line Brown hens at all egg-laying stages. 4. The phylogenetic tree results showed that ducks, geese and chickens had higher homology based on the genomic and protein sequence of RGS3. Moreover, chicken RGS3 interacted with GSK3B, RAF1 and BRAF based on STRING prediction. In vitro follicle stimulating hormone (FSH) treatment showed that mRNA expression levels of RGS3 and those of its predicted interacting proteins BRAF and GSK3B decreased with increasing FSH concentration. The results suggested that RGS3 responds to FSH and may play an important role in the regulation follicular development in chicken.

126 Regulator of G Protein Signaling 4 (RGS4) Gene Expression is Upregulated in the Superficial Spinal Dorsal Horn and in Dorsal Root Ganglion Neurons in a Murine Model of Neuropathic Pain

INTRODUCTION: Regulator of G protein signaling 4 (RGS4), a potent negative regulator of G-protein coupled receptor signaling, is a key gene in the maintenance of mechanical and cold allodynia associated with chronic pain states in mice. RGS4 is abundantly expressed across the pain matrix, and is upregulated in response to peripheral inflammation and nerve injury. METHODS: To understand the dynamics of RGS4 gene expression after nerve injury, we perform the spared-nerve injury (SNI) model of neuropathic pain in C57BL/6. At 21 days (DRG) or 2.5 months (spinal cord) post-surgery (SNI vs sham), animals were sacrificed and specimens were harvested for tissue processing. RNAScope, an ultrasensitive RNA ISH method, was used to visualize and quantify single-molecule expression of RGS4, prodynorphin/PDYN (exclusively expressed by inhibitory dorsal horn interneurons), and somatostatin/SST (expressed by excitatory interneurons) within the cellular topography of the lumbar superficial dorsal horn. RESULTS: RGS4 expression increased significantly in the dorsal horn following spared-nerve injury (p = 0.0384). In sham-treated mice, there were no differences in cell-type-specific (PDYN vs. SST) RGS4 expression patterns in the dorsal horn (p = 0.4170). Cell-type-specific RGS4 expression in the dorsal horn was significantly different (F (2,27) = 8.353; p = 0.0015; one-way ANOVA) following SNI. SNI-induced RGS4 expression was significantly enriched in PDYN vs SST-expressing interneurons (p = 0.0300), and overall RGS4 expression was significantly downregulated in SST-expressing cells compared to all cells (p = 0.0012). In addition, neuronal-specific RGS4 expression was significantly increased in the DRG in SNI vs Sham-injured mice (p = 0.0050). CONCLUSION: Our data suggest that upregulation of RGS4 expression in the superficial dorsal horn and DRG may play a crucial role in maintenance of chronic pain following nerve injury.

Regulator of G-Protein Signalling 4 (RGS4) negatively modulates nociceptin/orphanin FQ opioid receptor signalling: Implication for l-Dopa-induced dyskinesia.

Regulator of G-protein signalling 4 (RGS4) is a signal transduction protein that accelerates intrinsic GTPase activity of Gαi/o and Gαq subunits, suppressing GPCR signalling. Here, we investigate whether RGS4 modulates nociceptin/orphanin FQ (N/OFQ) opioid (NOP) receptor signalling and if this modulation has relevance for l-Dopa-induced dyskinesia. HEK293T cells transfected with NOP, NOP/RGS4 or NOP/RGS19 were challenged with N/OFQ and the small-molecule NOP agonist AT-403, using D1-stimulated cAMP levels as a readout. Primary rat striatal neurons and adult mouse striatal slices were challenged with either N/OFQ or AT-403 in the presence of the experimental RGS4 chemical probe, CCG-203920, and D1-stimulated cAMP or phosphorylated extracellular signal regulated kinase 1/2 (pERK) responses were monitored. In vivo, CCG-203920 was co-administered with AT-403 and l-Dopa to 6-hydroxydopamine hemilesioned rats, and dyskinetic movements, striatal biochemical correlates of dyskinesia (pERK and pGluR1 levels) and striatal RGS4 levels were measured. RGS4 expression reduced NOFQ and AT-403 potency and efficacy in HEK293T cells. CCG-203920 increased N/OFQ potency in primary rat striatal neurons and potentiated AT-403 response in mouse striatal slices. CCG-203920 enhanced AT-403-mediated inhibition of dyskinesia and its biochemical correlates, without compromising its motor-improving effects. Unilateral dopamine depletion caused bilateral reduction of RGS4 levels, which was reversed by l-Dopa. l-Dopa acutely up-regulated RGS4 in the lesioned striatum. RGS4 physiologically inhibits NOP receptor signalling. CCG-203920 enhanced NOP responses and improved the antidyskinetic potential of NOP receptor agonists, mitigating the effects of striatal RGS4 up-regulation occurring during dyskinesia expression. This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Aging whole blood transcriptome reveals candidate genes for SARS-CoV-2-related vascular and immune alterations.

The risk of severe COVID-19 increases with age as older patients are at highest risk. Thus, there is an urgent need to identify how severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) interacts with blood components during aging. We investigated the whole blood transcriptome from the Genotype-Tissue Expression (GTEx) database to explore differentially expressed genes (DEGs) translated into proteins interacting with viral proteins during aging. From 22 DEGs in aged blood, FASLG, CTSW, CTSE, VCAM1, and BAG3 were associated with immune response, inflammation, cell component and adhesion, and platelet activation/aggregation. Males and females older than 50 years old overexpress FASLG, possibly inducing a hyperinflammatory cascade. The expression of cathepsins (CTSW and CTSE) and the anti-apoptotic co-chaperone molecule BAG3 also increased throughout aging in both genders. By exploring single-cell RNA-sequencing data from peripheral blood of SARS-CoV-2-infected patients, we found FASLG and CTSW expressed in natural killer cells and CD8 + T lymphocytes, whereas BAG3 was expressed mainly in CD4 + T cells, naive T cells, and CD14 + monocytes. In addition, T cell exhaustion was associated with increased expression of CCL4L2 and DUSP4 over blood aging. LAG3, PDCD1, TIGIT, VCAM1, HLA-DRA, and TOX also increased in individuals aged 60–69 years old; conversely, the RGS2 gene decreased with aging. We further identified a distinct gene expression profile associated with type I interferon signaling following blood aging. These results revealed changes in blood molecules potentially related to SARS-CoV-2 infection throughout aging, emphasizing them as therapeutic candidates for aggressive clinical manifestation of COVID-19.Key messages• Prediction of host-viral interactions in the whole blood transcriptome during aging.• Expression levels of FASLG, CTSW, CTSE, VCAM1, and BAG3 increase in aged blood.• Blood interactome reveals targets involved with immune response, inflammation, and blood clots.• SARS-CoV-2-infected patients with high viral load showed FASLG overexpression.• Gene expression profile associated with T cell exhaustion and type I interferon signaling were affected with blood aging.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00109-021-02161-4.

Association between RGS4 gene polymorphisms and schizophrenia: A protocol for systematic review and meta-analysis.

Background:Schizophrenia is a complex brain disorder, the pathogenesis of which remains unclear. Regulator of G-protein signaling 4 is regarded as a candidate gene for schizophrenia risk. The association between the regulator of G-protein signaling 4 gene and the risk of schizophrenia is complicated and controversial, thus, an updated meta-analysis is needed.Methods:A search strategy using Medical Subject Headings was developed in English (PubMed, SZGene) and Chinese (CNKI, Wanfang, and Weipu) databases. Inclusion and exclusion criteria were used to screen for eligible studies. Parameters, such as P value of Hardy–Weinberg equilibrium, odds ratios, 95% confidence intervals, P values of association, heterogeneity (Ph), and publication bias, were analyzed by the Stata software using a random effects model. Subgroup analyses were performed to detect heterogeneity.Results:There were 15 articles regarding rs10917670 (8046 cases and 8837 controls), 16 regarding rs951436 (8990 cases and 10,568 controls), 15 regarding rs951439 (7995 cases and 8646 controls), 15 regarding rs2661319 (8320 cases and 9440 controls), and 4 regarding rs10759 (2752 cases and 2866 controls). The frequencies of rs10917670 and rs951439 were not significantly different between the case and control groups (P > .05). As shown by the East Asian and hospital-based subgroup analyses, the genotype TT of rs951436 might be related to the risk of schizophrenia. The genotypes CC + CT of rs2661319 and CC + CA of rs10759 were statistically different between the 2 groups, and the East Asian population contributed to these differences.Conclusion:The genotypes CC + CT of rs2661319 and CC + CA of rs10759 might be associated with the risk of schizophrenia.

Prenatal interleukin 6 elevation increases glutamatergic synapse density and disrupts hippocampal connectivity in offspring

SummaryEarly prenatal inflammatory conditions are thought to be a risk factor for different neurodevelopmental disorders. Maternal interleukin-6 (IL-6) elevation during pregnancy causes abnormal behavior in offspring, but whether these defects result from altered synaptic developmental trajectories remains unclear. Here we showed that transient IL-6 elevation via injection into pregnant mice or developing embryos enhanced glutamatergic synapses and led to overall brain hyperconnectivity in offspring into adulthood. IL-6 activated synaptogenesis gene programs in glutamatergic neurons and required the transcription factor STAT3 and expression of the RGS4 gene. The STAT3-RGS4 pathway was also activated in neonatal brains during poly(I:C)-induced maternal immune activation, which mimics viral infection during pregnancy. These findings indicate that IL-6 elevation at early developmental stages is sufficient to exert a long-lasting effect on glutamatergic synaptogenesis and brain connectivity, providing a mechanistic framework for the association between prenatal inflammatory events and brain neurodevelopmental disorders.

Circulating microRNAs predispose to takotsubo syndrome following high-dose adrenaline exposure.

Aims Takotsubo syndrome (TTS) is an acute heart failure, typically triggered by high adrenaline during physical or emotional stress. It is distinguished from myocardial infarction (MI) by a characteristic pattern of ventricular basal hypercontractility with hypokinesis of apical segments, and in the absence of culprit coronary occlusion. We aimed to understand whether recently discovered circulating biomarkers miR-16 and miR-26a, which differentiate TTS from MI at presentation, were mechanistically involved in the pathophysiology of TTS.Methods and results miR-16 and miR-26a were co-overexpressed in rats with AAV and TTS induced with an adrenaline bolus. Untreated isolated rat cardiomyocytes were transfected with pre-/anti-miRs and functionally assessed. Ventricular basal hypercontraction and apical depression were accentuated in miR-transfected animals after induction of TTS. In vitro miR-16 and/or miR-26a overexpression in isolated apical (but not basal), cardiomyocytes produced strong depression of contraction, with loss of adrenaline sensitivity. They also enhanced the initial positive inotropic effect of adrenaline in basal cells. Decreased contractility after TTS-miRs was reproduced in non-failing human apical cardiomyocytes. Bioinformatic profiling of miR targets, followed by expression assays and functional experiments, identified reductions of CACNB1 (L-type calcium channel Cavβ subunit), RGS4 (regulator of G-protein signalling 4), and G-protein subunit Gβ (GNB1) as underlying these effects.ConclusionmiR-16 and miR-26a sensitize the heart to TTS-like changes produced by adrenaline. Since these miRs have been associated with anxiety and depression, they could provide a mechanism whereby priming of the heart by previous stress causes an increased likelihood of TTS in the future.

Impaired regulation of heart rate and sinoatrial node function by the parasympathetic nervous system in type 2 diabetic mice

Heart rate (HR) and sinoatrial node (SAN) function are modulated by the autonomic nervous system. HR regulation by the parasympathetic nervous system (PNS) is impaired in diabetes mellitus (DM), which is denoted cardiovascular autonomic neuropathy. Whether blunted PNS effects on HR in type 2 DM are related to impaired responsiveness of the SAN to PNS agonists is unknown. This was investigated in type 2 diabetic db/db mice in vivo and in isolated SAN myocytes. The PNS agonist carbachol (CCh) had a smaller inhibitory effect on HR, while HR recovery time after CCh removal was accelerated in db/db mice. In isolated SAN myocytes CCh reduced spontaneous action potential firing frequency but this effect was reduced in db/db mice due to blunted effects on diastolic depolarization slope and maximum diastolic potential. Impaired effects of CCh occurred due to enhanced desensitization of the acetylcholine-activated K+ current (IKACh) and faster IKACh deactivation. IKACh alterations were reversed by inhibition of regulator of G-protein signaling 4 (RGS4) and by the phospholipid PIP3. SAN expression of RGS4 was increased in db/db mice. Impaired PNS regulation of HR in db/db mice occurs due to reduced responsiveness of SAN myocytes to PNS agonists in association with enhanced RGS4 activity.

Morphological and biological properties of silica nanoparticles for CRTC3-siRNA delivery and downregulation of the RGS2 expression in preadipocytes.

The aim of this study was to characterize the morphological properties of amorphous silica nanoparticles (SiO2 NPs), their cytotoxicity and intracellular location within Human Osteoblasts (HOB). Additionally, SiO2 NPs were explored for their effectivity as carriers of CRTC3-siRNA on Human Preadipocytes (HPAd), and thus downregulate RGS2 gene expression. SiO2 NPs were synthesized using the method of Stöber at 45 °C, 56 °C, and 62 °C. These were characterized via TEM with EDS, Zeta Potential and FT-IR. Cytotoxicity was evaluated by XTT at three concentrations 50, 100 and 500 µg/mL; SiO2 NPs intracellular localization was observed through Confocal Laser Scanning Microscope. Delivering siRNA effectivity was measured by RT-qPCR. Morphology of SiO2 NPs was spherical with a range size from 64 to 119 nm; their surface charge was negative. Confocal images demonstrated that SiO2 NPs were located within cellular cytoplasm. At a SiO2 NPs concentration of 500 µg/mL HOB viability decreased, while at 50 µg/mL and 100 µg/mL cell viability was not affected regardless SiO2 NPs size. SiO2 NPs-CRTC3-siRNA are effective to down-regulate RGS2 gene expression in HPAd without cytotoxic effects. The developed SiO2 NPs-CRTC3-siRNA are a promising tool as a delivery vehicle to control obesity.

The Antipsychotic Drug Clozapine Suppresses the RGS4 Polyubiquitylation and Proteasomal Degradation Mediated by the Arg/N-Degron Pathway.

Although diverse antipsychotic drugs have been developed for the treatment of schizophrenia, most of their mechanisms of action remain elusive. Regulator of G-protein signaling 4 (RGS4) has been reported to be linked, both genetically and functionally, with schizophrenia and is a physiological substrate of the arginylation branch of the N-degron pathway (Arg/N-degron pathway). Here, we show that the atypical antipsychotic drug clozapine significantly inhibits proteasomal degradation of RGS4 proteins without affecting their transcriptional expression. In addition, the levels of Arg- and Phe-GFP (artificial substrates of the Arg/N-degron pathway) were significantly elevated by clozapine treatment. In silico computational model suggested that clozapine may interact with active sites of N-recognin E3 ubiquitin ligases. Accordingly, treatment with clozapine resulted in reduced polyubiquitylation of RGS4 and Arg-GFP in the test tube and in cultured cells. Clozapine attenuated the activation of downstream effectors of G protein-coupled receptor signaling, such as MEK1 and ERK1, in HEK293 and SH-SY5Y cells. Furthermore, intraperitoneal injection of clozapine into rats significantly stabilized the endogenous RGS4 protein in the prefrontal cortex. Overall, these results reveal an additional therapeutic mechanism of action of clozapine: this drug posttranslationally inhibits the degradation of Arg/N-degron substrates, including RGS4. These findings imply that modulation of protein post-translational modifications, in particular the Arg/N-degron pathway, may be a novel molecular therapeutic strategy against schizophrenia.

GENETICS OF GENERALIZED ANXIETY DISORDER

The essential feature of generalized anxiety disorder (GAD) is excessive anxiety and worry about various spheres of own life. Its prevalence ranges from 0,4% to 3,6%. The genetic share of GAD is about 32%. The rest is an influence of environmental factors, of which the most significant are childhood adversities and parental overprotection. In consequence of such features the genetic study of GAD is very difficult.To reveal candidate genes for GAD the genetic study of brain neurotransmitters is dominated. Now there are proposed over 20 genes, which has been associated with GAD.First candidate gene was proposed by D.C. Rowe with colleagues in 1998. It was the dopamine transporter gene (DAT1; modern gene symbol is SLC6A3; 5p15.33). The disorder symptoms are increased with a greater number of 10 tandem repeats of a 40 bp fragment in the 3’UT region of DAT1 gene. In following year K. Ohara with colleagues obtained a significant similar result for the serotonin transporter gene (5-HTT; modern gene symbol is SLC6A4; 17q11.2). In this study tandem repeats of 17 bp have been identified in the second intron of the gene. The 5-HTT gene variants are associated also with over 80 different diseases. Later A. Tadic with colleagues found a significant association between GAD and single nucleotide polymorphism (SNP) T941G (rs6323) in exon 8 of the monoamine oxidase A gene (MAOA; Xp11.3). In the MAOA gene uVNTR promoter J. Samochowiec with colleagues discovered an association between GAD and an increase of tandem repeats of 30 bp number over 3. In the study of J.S. You with colleagues there was showed the significant involvement of short alleles of the 5-HTT gene (allase SLC6A4) in GAD, but there is not confirmed the involvement of variable number tandem repeats in this gene, as shown by Japanese authors. Short allele of 5-HTT contains 14 tandem repeats of a 22 bp vs normal 16 in the 5-HTTLPR intron region near the promoter.In addition, the J.M. Hettema’s group of American psychiatrists proposed three GAD candidate genes – the GAD1 (Glutamate Decarboxylase 1; 2q31.1), the HTR1A (Serotonin Receptor 1A; 5q12.3) and the COMT (Catechol-O-methyltransferase; 22q11.21). Several SNPs were related to GAD: three SNPs (rs2241165, rs2058725 and rs3791850) were localized in the GAD1 gene introns, one (rs6295) – in the HTR1A gene promoter and two in the COMT gene (rs4680 – in exon and rs165599 – in the 3’UTR).A study dedicated to assessing the effects of exposure to the 2004 Florida hurricane reported increased risk of GAD in relation to SNP in the 3’UTR (rs4606) of the RGS2 gene (Regulator of G-Protein Signaling 2; 1q31.2) and SNP in the promoter region (rs16147) of the NPY gene (Neuropeptide Y; 7p15.3).A. J. Cooper with colleagues suggested some more two candidate genes for GAD: the hypophysis activating hormone (PACAP; allase ADCYAP1; 18p11.32) and the opioid receptor 1(OPRM1; allase MOR1; 6q25.2). In the PACAP gene SNP Asp54Gly (rs2856966) was involved in GAD. It was located in exon 2 of the gene. There was also showed a small correlation between SNP A118G (rs1799971; Asn40Asp) in exon 1 of the OPRM1 gene and GAD.All these results should be taken quite critically. First, the study was carried out on a statistically small samples of a few hundred people. Second, the results of these studies was not confirmed by other authors for samples of a few thousand people. GAD problems remains relevant and requires further intense research efforts.