Phosphotyrosine Phosphatases of the AtT-20 Murine Corticotroph Cell Line

Somatostatin (somatotropin release inhibitory factor; SRIF) is a peptide-signaling molecule that activates a family of heterotrimeric guanine nucleotide binding protein (G­ protein) coupled receptors (sst.-sst-). SRIF receptors control essential intracellular signaling events and, by reducing cyclic nucleotide levels, ion concentrations and protein phosphorylation, ultimately controlling cell proliferation and secretion. In the current study, we investigated the intracellular phosphatase activity present in the AtT-20 cell, as well as whether these enzymes were under direct SRIF receptor control. AtT-20 cells retain many of the properties of anterior pituitary corticotrophs, yet are an established cell line that expresses at least two SRIF receptor subtypes (sst, and sst.). Both SRIF receptor subtypes potently suppress hormonally induced adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells. Since intracellular protein kinase activation plays a crucial role in ACTH release from AtT-20 cells, identifying the corresponding protein phosphatases will provide valuable information on corticotroph function. The novel fluorescent phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiF­ MUP) was employed to identify soluble or membrane-associated phosphatase activities. Treatment with a selective tyrosine phosphatase inhibitor (sodium vanadate; Na3 V04) attenuated DiF-MUP phosphatase activity by 75%, suggesting that the dominant activity detected in AtT-20 cells is a tyrosine phosphatase. Na3V04 inhibition was potent as concentration response studies demonstrated an inhibitory concentration for 50% activity nM. (ICso) of 90 A serine/threonine phosphatase inhibitor failed todecrease phosphatase activity, indicating that the dominant phosphatase activity present is due to protein tyrosine phosphatases. Immunoprecipitation studies, in conjunction with sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-P AGE) and immunoblotting identified two intracellular phosphotyrosine phosphatases: Src homology phosphatase- I (SHP-1) and Src homology phosphotyrosine phosphatase-2 (SHP-2). Immunoblotting with phospho­ specific antisera confirmed the presence of SHP-1 and SHP-2, while also demonstrating that neither enzyme appears to be under SRIF control in AtT-20 cells. Together, these results show that AtT-20 murine corticotrophs contain sodium vanadate sensitive, soluble tyrosine phosphatase activity that is independent of SRIF control

Phosphotyrosine Phosphatases of the AtT-20 Murine Corticotroph Cell Line

Somatostatin (somatotropin release inhibitory factor; SRIF) is a peptide-signaling molecule that activates a family of heterotrimeric guanine nucleotide binding protein (G­ protein) coupled receptors (sst.-sst-). SRIF receptors control essential intracellular signaling events and, by reducing cyclic nucleotide levels, ion concentrations and protein phosphorylation, ultimately controlling cell proliferation and secretion. In the current study, we investigated the intracellular phosphatase activity present in the AtT-20 cell, as well as whether these enzymes were under direct SRIF receptor control. AtT-20 cells retain many of the properties of anterior pituitary corticotrophs, yet are an established cell line that expresses at least two SRIF receptor subtypes (sst, and sst.). Both SRIF receptor subtypes potently suppress hormonally induced adrenocorticotropic hormone (ACTH) secretion from AtT-20 cells. Since intracellular protein kinase activation plays a crucial role in ACTH release from AtT-20 cells, identifying the corresponding protein phosphatases will provide valuable information on corticotroph function. The novel fluorescent phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiF­ MUP) was employed to identify soluble or membrane-associated phosphatase activities. Treatment with a selective tyrosine phosphatase inhibitor (sodium vanadate; Na3 V04) attenuated DiF-MUP phosphatase activity by 75%, suggesting that the dominant activity detected in AtT-20 cells is a tyrosine phosphatase. Na3V04 inhibition was potent as concentration response studies demonstrated an inhibitory concentration for 50% activity nM. (ICso) of 90 A serine/threonine phosphatase inhibitor failed todecrease phosphatase activity, indicating that the dominant phosphatase activity present is due to protein tyrosine phosphatases. Immunoprecipitation studies, in conjunction with sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-P AGE) and immunoblotting identified two intracellular phosphotyrosine phosphatases: Src homology phosphatase- I (SHP-1) and Src homology phosphotyrosine phosphatase-2 (SHP-2). Immunoblotting with phospho­ specific antisera confirmed the presence of SHP-1 and SHP-2, while also demonstrating that neither enzyme appears to be under SRIF control in AtT-20 cells. Together, these results show that AtT-20 murine corticotrophs contain sodium vanadate sensitive, soluble tyrosine phosphatase activity that is independent of SRIF control

Cluster Headache Genomewide Association Study and Meta-Analysis Identifies Eight Loci and Implicates Smoking as Causal Risk Factor

Objective: The objective of this study was to aggregate data for the first genomewide association study meta-analysis of cluster headache, to identify genetic risk variants, and gain biological insights. Methods: A total of 4,777 cases (3,348 men and 1,429 women) with clinically diagnosed cluster headache were recruited from 10 European and 1 East Asian cohorts. We first performed an inverse-variance genomewide association meta-analysis of 4,043 cases and 21,729 controls of European ancestry. In a secondary trans-ancestry meta-analysis, we included 734 cases and 9,846 controls of East Asian ancestry. Candidate causal genes were prioritized by 5 complementary methods: expression quantitative trait loci, transcriptome-wide association, fine-mapping of causal gene sets, genetically driven DNA methylation, and effects on protein structure. Gene set and tissue enrichment analyses, genetic correlation, genetic risk score analysis, and Mendelian randomization were part of the downstream analyses. Results: The estimated single nucleotide polymorphism (SNP)-based heritability of cluster headache was 14.5%. We identified 9 independent signals in 7 genomewide significant loci in the primary meta-analysis, and one additional locus in the trans-ethnic meta-analysis. Five of the loci were previously known. The 20 genes prioritized as potentially causal for cluster headache showed enrichment to artery and brain tissue. Cluster headache was genetically correlated with cigarette smoking, risk-taking behavior, attention deficit hyperactivity disorder (ADHD), depression, and musculoskeletal pain. Mendelian randomization analysis indicated a causal effect of cigarette smoking intensity on cluster headache. Three of the identified loci were shared with migraine. Interpretation: This first genomewide association study meta-analysis gives clues to the biological basis of cluster headache and indicates that smoking is a causal risk factor

Cerebral small vessel disease genomics and its implications across the lifespan

White matter hyperintensities (WMH) are the most common brain-imaging feature of cerebral small vessel disease (SVD), hypertension being the main known risk factor. Here, we identify 27 genome-wide loci for WMH-volume in a cohort of 50,970 older individuals, accounting for modification/confounding by hypertension. Aggregated WMH risk variants were associated with altered white matter integrity (p = 2.5×10-7) in brain images from 1,738 young healthy adults, providing insight into the lifetime impact of SVD genetic risk. Mendelian randomization suggested causal association of increasing WMH-volume with stroke, Alzheimer-type dementia, and of increasing blood pressure (BP) with larger WMH-volume, notably also in persons without clinical hypertension. Transcriptome-wide colocalization analyses showed association of WMH-volume with expression of 39 genes, of which four encode known drug targets. Finally, we provide insight into BP-independent biological pathways underlying SVD and suggest potential for genetic stratification of high-risk individuals and for genetically-informed prioritization of drug targets for prevention trials.Peer reviewe

Genome-wide analysis of 102,084 migraine cases identifies 123 risk loci and subtype-specific risk alleles.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked DownloadMigraine affects over a billion individuals worldwide but its genetic underpinning remains largely unknown. Here, we performed a genome-wide association study of 102,084 migraine cases and 771,257 controls and identified 123 loci, of which 86 are previously unknown. These loci provide an opportunity to evaluate shared and distinct genetic components in the two main migraine subtypes: migraine with aura and migraine without aura. Stratification of the risk loci using 29,679 cases with subtype information indicated three risk variants that seem specific for migraine with aura (in HMOX2, CACNA1A and MPPED2), two that seem specific for migraine without aura (near SPINK2 and near FECH) and nine that increase susceptibility for migraine regardless of subtype. The new risk loci include genes encoding recent migraine-specific drug targets, namely calcitonin gene-related peptide (CALCA/CALCB) and serotonin 1F receptor (HTR1F). Overall, genomic annotations among migraine-associated variants were enriched in both vascular and central nervous system tissue/cell types, supporting unequivocally that neurovascular mechanisms underlie migraine pathophysiology.US National Institute of Neurological Disorders and Stroke (NINDS) of the US National Institutes of Health (NIH) Finnish innovation fund Sitra Finska Lakaresallskapet Academy of Finland Sigrid Juselius Foundation Academy of Finland Appeared in source as:Academy of Finland Center of Excellence in Complex Disease Genetics Finnish Foundation for Cardiovascular Research Novo Nordisk Foundation Novocure Limited CANDY foundation (CEHEAD) South-Eastern Norway Regional Health Authorit

Meta-analysis of 375,000 individuals identifies 38 susceptibility loci for migraine

Migraine is a debilitating neurological disorder affecting around one in seven people worldwide, but its molecular mechanisms remain poorly understood. There is some debate about whether migraine is a disease of vascular dysfunction or a result of neuronal dysfunction with secondary vascular changes. Genome-wide association (GWA) studies have thus far identified 13 independent loci associated with migraine. To identify new susceptibility loci, we carried out a genetic study of migraine on 59,674 affected subjects and 316,078 controls from 22 GWA studies. We identified 44 independent single-nucleotide polymorphisms (SNPs) significantly associated with migraine risk (P < 5 × 10−8) that mapped to 38 distinct genomic loci, including 28 loci not previously reported and a locus that to our knowledge is the first to be identified on chromosome X. In subsequent computational analyses, the identified loci showed enrichment for genes expressed in vascular and smooth muscle tissues, consistent with a predominant theory of migraine that highlights vascular etiologies

Genome-wide analysis of 102,084 migraine cases identifies 123 risk loci and subtype-specific risk alleles

Abstract Migraine affects over a billion individuals worldwide but its genetic underpinning remains largely unknown. Here, we performed a genome-wide association study of 102,084 migraine cases and 771,257 controls and identified 123 loci, of which 86 are previously unknown. These loci provide an opportunity to evaluate shared and distinct genetic components in the two main migraine subtypes: migraine with aura and migraine without aura. Stratification of the risk loci using 29,679 cases with subtype information indicated three risk variants that seem specific for migraine with aura (in HMOX2, CACNA1A and MPPED2), two that seem specific for migraine without aura (near SPINK2 and near FECH) and nine that increase susceptibility for migraine regardless of subtype. The new risk loci include genes encoding recent migraine-specific drug targets, namely calcitonin gene-related peptide (CALCA/CALCB) and serotonin 1F receptor (HTR1F). Overall, genomic annotations among migraine-associated variants were enriched in both vascular and central nervous system tissue/cell types, supporting unequivocally that neurovascular mechanisms underlie migraine pathophysiology