Promoter-Specific Expression and Genomic Structure of IgLON Family Genes in Mouse

IgLON family is composed of five genes: Lsamp, Ntm, Opcml, Negr1, and Iglon5; encoding for five highly homologous neural adhesion proteins that regulate neurite outgrowth and synapse formation. In the current study we performed in silico analysis revealing that Ntm and Opcml display similar genomic structure as previously reported for Lsamp, characterized by two alternative promotors 1a and 1b. Negr1 and Iglon5 transcripts have uniform 5' region, suggesting single promoter. Iglon5, the recently characterized family member, shares high level of conservation and structural qualities characteristic to IgLON family such as N-terminal signal peptide, three Ig domains, and GPI anchor binding site. By using custom 5'-isoform-specific TaqMan gene-expression assay, we demonstrated heterogeneous expression of IgLON transcripts in different areas of mouse brain and several-fold lower expression in selected tissues outside central nervous system. As an example, the expression of IgLON transcripts in urogenital and reproductive system is in line with repeated reports of urogenital tumors accompanied by mutations in IgLON genes. Considering the high levels of intra-family homology shared by IgLONs, we investigated potential compensatory effects at the level of IgLON isoforms in the brains of mice deficient of one or two family members. We found that the lack of IgLONs is not compensated by a systematic quantitative increase of the other family members. On the contrary, the expression of Ntm 1a transcript and NEGR1 protein was significantly reduced in the frontal cortex of Lsamp-deficient mice suggesting that the expression patterns within IgLON family are balanced coherently. The actions of individual IgLONs, however, can be antagonistic as demonstrated by differential expression of Syp in deletion mutants of IgLONs. In conclusion, we show that the genomic twin-promoter structure has impact on both anatomical distribution and intra-family interactions of IgLON family members. Remarkable variety in the activity levels of 1a and 1b promoters both in the brain and in other tissues, suggests complex functional regulation of IgLONs by alternative signal peptides driven by 1a and 1b promoters.Peer reviewe

Promoter polymorphism -119C/G in MYG1 (C12orf10) gene is related to vitiligo susceptibility and Arg4Gln affects mitochondrial entrance of Myg1

<p>Abstract</p> <p>Background</p> <p><it>MYG1 </it>(<it>Melanocyte proliferating gene 1</it>, also C12orf10 in human) is a ubiquitous nucleo-mitochondrial protein, involved in early developmental processes and in adult stress/illness conditions. We recently showed that <it>MYG1 </it>mRNA expression is elevated in the skin of vitiligo patients. Our aim was to examine nine known polymorphisms in the <it>MYG1 </it>gene, to investigate their functionality, and to study their association with vitiligo susceptibility.</p> <p>Methods</p> <p>Nine single nucleotide polymorphisms (SNPs) in the <it>MYG1 </it>locus were investigated by SNPlex assay and/or sequencing in vitiligo patients (n = 124) and controls (n = 325). <it>MYG1 </it>expression in skin biopsies was detected by quantitative-real time PCR (Q-RT-PCR) and polymorphisms were further analysed using luciferase and YFP reporters in the cell culture.</p> <p>Results</p> <p>Control subjects with -119G promoter allele (rs1465073) exhibited significantly higher <it>MYG1 </it>mRNA levels than controls with -119C allele (<it>P </it>= 0.01). Higher activity of -119G promoter was confirmed by luciferase assay. Single marker association analysis showed that the -119G allele was more frequent in vitiligo patients (47.1%) compared to controls (39.3%, <it>P </it>< 0.05, OR 1.37, 95%CI 1.02-1.85). Analysis based on the stage of progression of the vitiligo revealed that the increased frequency of -119G allele occurred prevalently in the group of patients with active vitiligo (n = 86) compared to the control group (48.2% <it>versus </it>39.3%, <it>P </it>< 0.05; OR 1.44, 95%CI 1.02-2.03). Additionally, we showed that glutamine in the fourth position (in Arg4Gln polymorphism) completely eliminated mitochondrial entrance of YFP-tagged Myg1 protein in cell culture. The analysis of available EST, cDNA and genomic DNA sequences revealed that Myg1 4Gln allele is remarkably present in human populations but is never detected in homozygous state according to the HapMap database.</p> <p>Conclusions</p> <p>Our study demonstrated that both <it>MYG1 </it>promoter polymorphism -119C/G and Arg4Gln polymorphism in the mitochondrial signal of Myg1 have a functional impact on the regulation of the <it>MYG1 </it>gene and promoter polymorphism (-119C/G) is related with suspectibility for actively progressing vitiligo.</p

Myg1 geeni ja valgu iseloomustamine: ekspressioonimuster, rakusisene lokalisatsioon, geeni puudulikkusega hiir ja funktsionaalsed polümorfismid inimesel

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Myg1 is one of the most conserved genes that has been evolutionarily crucial to maintain from protozoans to humans. Despite that Myg1 has not been associated with any remarkable disease conditions or fundamental cellular processes. The main purpose of the current study was to characterize Myg1 (Melanocyte proliferating gene 1) gene and to generate relevant data about this novel gene and protein. In our study group, Myg1 was first identified as a most extensively up-regulated gene in the amygdaloid area of rats after cat odour-induced anxiety (Kõks et al, 2004). Studies linked to the current dissertation were designed to shed light on the function of the Myg1 gene that had previously been cited in only one scientific abstract (Smicun, 2000) describing Myg1 as a highly expressed gene in freely proliferating melanocytes and downregulated in malignant melanoma cells. In the current study we found that in adult tissues Myg1 mRNA expression is ubiquitous and homogeneous; in different embryonic phases Myg1 expression is characterized by a specific pattern and dynamic intensity, indicating developmental impact of Myg1. Myg1 localizes in the nucleus and mitochondria and we also demonstrated the existence of nuclear and mitochondrial targeting signals in the N-terminal region of human and mouse Myg1 proteins. We showed that Myg1 4Gln allele that has remarkable prevalence in the Nigerian population, disturbs mitochondrial entrance of Myg1. Because our subjects from the Estonian population were consistently homozygous for Myg1 4Arg allele, we can not confirm the relevance of Myg1 Arg4Gln, but our current results suggest that Myg1 has indispensable functions in the mitochondria. According to our studies it is most likely that Myg1 is involved in cellular pathways implicated in cellular stress, immune response, development and metabolism. Phenotyping of Myg1-deficient mice revealed that Myg1 (-/-) mice are vital, fertile and display no gross abnormalities. Myg1-deficiency caused moderate alterations in anxiety-related behaviour and stress-reactions in mice, but also remarkable reduction of sex-dependent behavioural differences. In our primary screen Myg1-deficient mice displayed no significant alterations in immune response or metabolic activity but several mild tendencies encourage us to study these functions further in Myg1 (-/-) mice. Both MYG1 promoter polymorphism -119C/G and Arg4Gln polymorphism in the mitochondrial signal of Myg1 have a functional impact on the regulation of the MYG1 gene. Our in vivo and in vitro promoter activity analysis together with association analysis confirms that -119C/G polymorphism influences MYG1 mRNA levels. Our results suggest that more active -119G is the risk-allele for the development of vitiligo and more specifically risk-allele for the maintenance of the active progression stage of the disease.Myg1 geen tuvastati algselt hiirte melanotsüütidest (Smicun, 2000), ning sellest tuleneb ka geeni nimi: “Melanotsüüte prolifereeriv geen 1” (Melanocyte proliferating gene 1, Myg1). Meie laboris tuvastati Myg1 esmalt geenina, mille ekspressioon rottide amügdalas märkimisväärselt tõusis (1,8-kordne mRNA taseme tõus) pärast seda, kui rotid puutusid 30 minuti jooksul kokku stressitekitava kassilõhnaga (Kõks et al, 2004). Järjestuse homoloogia alusel peetakse Myg1 valku oletatavalt fosfoesteraasiks ning lisaks võib andmebaasidest leida informatsiooni, et tegemist on väga konserveerunud geeniga: Myg1 on evolutsiooni käigus säilinud kõikides eukarüootides alates pärmist inimeseni, ent esineb ka mõnedes ainuraksetes algloomades. Kuna spetsiifilisemad uurimused Myg1 valgu kohta puudusid, siis viidi käesolevas töös läbi katsed eesmärgiga iseloomustada Myg1 geeni ja valku igakülgselt, alates ekspressioonimustrist, rakulisest paiknemisest ja geeni puudlikkusega hiire fenotüübi iseärasustest ning lõpetades selle algselt melanotsüütidest tuvastatud geeni võimaliku osalusega pigmentatsioonihäire vitiliigo kujunemisel. Myg1/MYG1 geen kodeerib laialdase ekspressioonimustriga transkripti, millel on dünaamiline ekspressioonitase nii embrüonaalse kui postnataalse arengu käigus. Täiskasvanueas muutub Myg1/MYG1 geeni ekspressioon reaktsioonina erinevatele haigus- või keskkonna seisunditele. Myg1 valk paikneb raku mitokondrites ning tuumas, kuhu valk suunatakse N-terminuses asuvate signaaljärjestuste põhjal. Arg4Gln mutatsioon mõjutab Myg1 valgu transporti mitokondritesse ning Myg1 4Gln mitokondritesse ei viida. Kokkuvõtvalt võib meie tulemuste põhjal öelda, et Myg1 valk osaleb kõige tõenäolisemalt raku stressi, immuunvastuse, arengu ja metabolismiga seotud radades. Ärevuse ja stressiga seotud testides ilmnenud mõõdukad kõrvalekalded Myg1-puudulikkusega hiirtel kinnitavad, et Myg1 on vajalik stressiolukordades. Vähenenud soospetsiifilised erinevused Myg1-puudulikkusega emaste ja isaste hiirte vahel aga viitavad, et tegemist võib olla faktoriga, mis reguleerib soospetsiifilist käitumist ja füsioloogilisi reaktsioone. -119C/G promootori polümorfismi aktiivsem alleel (G) on vitiliigo puhul riskialleeliks eelkõige aktiivselt progresseeruva vitiliigoga haigete grupis. Tõenäoliselt on aktiivsema alleeli sagedasem esinemine seotud kõrgenenud MYG1 geeni ekspressioonitasemega vitiliigohaigete patsientide kahjustatud nahas ning aktiivse vitiliigo puhul ka vitiliigopatsientide terves nahas

Limbic system associated membrane protein as a potential target for neuropsychiatric disorders

The studies performed in laboratory animals and psychiatric patients suggest a possible role of limbic system-associated membrane protein (LAMP) in the mechanisms of psychiatric disorders. Stressful manipulations and genetic invalidation have revealed a role of the Lsamp gene in the regulation of anxiety in rodents. Besides that, Lsamp-deficient mice display reduced aggressiveness and impaired adaptation in novel and stressful environments. The behavioral effects of amphetamine were blunted in genetically modified mice. Recent pharmacological and biochemical studies point toward altered function of GABA-, 5-hydroxytryptamine-, and dopaminergic systems in Lsamp-deficient mice. Moreover, we found an association between the gene polymorphisms of LSAMP and major depressive disorder (MDD). Patients suffering from MDD had significantly increased ratio between risk and protective haplotypes of the LSAMP gene compared to healthy volunteers. However, the impact of these haplotypes for the function of LAMP is not clear and remains to be elucidated in future studies

Repeated Administration of D-Amphetamine Induces Distinct Alterations in Behavior and Metabolite Levels in 129Sv and Bl6 Mouse Strains

The main goal of the study was to characterize the behavioral and metabolomic profiles of repeated administration (for 11 days) of d-amphetamine (AMPH, 3 mg/kg i. p.), indirect agonist of dopamine (DA), in widely used 129S6/SvEvTac (129Sv) and C57BL/6NTac (Bl6) mouse strains. Acute administration of AMPH (acute AMPH) induced significantly stronger motor stimulation in Bl6. However, repeated administration of AMPH (repeated AMPH) caused stronger motor sensitization in 129Sv compared acute AMPH. Body weight of 129Sv was reduced after repeated saline and AMPH, whereas no change occurred in Bl6. In the metabolomic study, acute AMPH induced an elevation of isoleucine and leucine, branched chain amino acids (BCAA), whereas the level of hexoses was reduced in Bl6. Both BCAAs and hexoses remained on level of acute AMPH after repeated AMPH in Bl6. Three biogenic amines [asymmetric dimethylarginine (ADMA), alpha-aminoadipic acid (alpha-AAA), kynurenine] were significantly reduced after repeated AMPH. Acute AMPH caused in 129Sv a significant reduction of valine, lysophosphatidylcholines (lysoPC a C16:0, lysoPC a C18:2, lysoPC a C20:4), phosphatidylcholine (PC) diacyls (PC aa C34:2, PC aa C36:2, PC aa C36:3, PC aa C36:4) and alkyl-acyls (PC ae C38:4, PC ae C40:4). However, repeated AMPH increased the levels of valine and isoleucine, long-chain acylcarnitines (C14, C14:1-OH, C16, C18:1), PC diacyls (PC aa C38:4, PC aa C38:6, PC aa C42:6), PC acyl-alkyls (PC ae C38:4, PC ae C40:4, PC ae C40:5, PC ae C40:6, PC ae C42:1, PC ae C42:3) and sphingolipids [SM(OH)C22:1, SM C24:0] compared to acute AMPH in 129Sv. Hexoses and kynurenine were reduced after repeated AMPH compared to saline in 129Sv. The established changes probably reflect a shift in energy metabolism toward lipid molecules in 129Sv because of reduced level of hexoses. Pooled data from both strains showed that the elevation of isoleucine and leucine was a prominent biomarker of AMPH-induced behavioral sensitization. Simultaneously a significant decline of hexoses, citrulline, ADMA, and kynurenine occurred. The reduced levels of kynurenine, ADMA, and citrulline likely reflect altered function of N-methyl-D-aspartate (NMDA) and NO systems caused by repeated AMPH. Altogether, 129Sv strain displays stronger sensitization toward AMPH and larger variance in metabolite levels than Bl6

Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation

C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv) mice display different coping strategies in stressful conditions. Our aim was to evaluate biomarkers related to different adaptation strategies in the brain of male 129Sv and Bl6 mice. We focused on signaling pathways related to the dopamine (DA) system, N-methyl-D-aspartate (NMDA) receptor and epidermal growth factor (EGF) family, shown as the key players in behavioral adaptation. Mice from Bl6 and 129Sv lines were divided into either home cage controls (HCC group) or exposed to repeated motility testing and treated with saline for 11 days (RMT group). Distinct stress responses were reflected in severe body weight loss in 129Sv and the increased exploratory behavior in Bl6 mice. Besides that, amphetamine caused significantly stronger motor stimulation in Bl6. Together with the results from gene expression (particularly Maob), this study supports higher baseline activity of DA system in Bl6. Interestingly, the adaptation is reflected with opposite changes of DA markers in dorsal and ventral striatum. In forebrain, stress increased the gene expressions of Egf-Erbb1 and Nrg1/Nrg2-Erbb4 pathways more clearly in 129Sv, whereas the corresponding proteins were significantly elevated in Bl6. We suggest that not only inhibited activity of the DA system, but also reduced activity of EGF family and NMDA receptor signaling underlies higher susceptibility to stress in 129Sv. Altogether, this study underlines the better suitability of 129Sv for modelling neuropsychiatric disorders than Bl6

Alternative Promoter Use Governs the Expression of IgLON Cell Adhesion Molecules in Histogenetic Fields of the Embryonic Mouse Brain

The members of the IgLON superfamily of cell adhesion molecules facilitate fundamental cellular communication during brain development, maintain functional brain circuitry, and are associated with several neuropsychiatric disorders such as depression, autism, schizophrenia, and intellectual disabilities. Usage of alternative promoter-specific 1a and 1b mRNA isoforms in Lsamp, Opcml, Ntm, and the single promoter of Negr1 in the mouse and human brain has been previously described. To determine the precise spatiotemporal expression dynamics of Lsamp, Opcml, Ntm isoforms, and Negr1, in the developing brain, we generated isoform-specific RNA probes and carried out in situ hybridization in the developing (embryonic, E10.5, E11.5, 13.5, 17; postnatal, P0) and adult mouse brains. We show that promoter-specific expression of IgLONs is established early during pallial development (at E10.5), where it remains throughout its differentiation through adulthood. In the diencephalon, midbrain, and hindbrain, strong expression patterns are initiated a few days later and begin fading after birth, being only faintly expressed during adulthood. Thus, the expression of specific IgLONs in the developing brain may provide the means for regionally specific functionality as well as for specific regional vulnerabilities. The current study will therefore improve the understanding of how IgLON genes are implicated in the development of neuropsychiatric disorders