What Makes Us Anxious : A Cross-Species Multi-omics Approach to the Biological Basis of Anxiety Disorders

Anxiety disorders manifest themselves as a prolonged or exaggerated response to a threatening situation, which can be either real or perceived. Their high prevalence (14%) places them as one of the most common mental disorders within the European Union. This conveys an important message about the necessity of finding new clinically relevant drug targets leading to the development of novel personalized treatment practices. To facilitate this process, efforts should be focused on gaining a deeper understanding of the complex molecular, biochemical, and system-level mechanisms behind the neurobiology of stress, and the role of stress as one of the main etiological factors in anxiety-related psychiatric disorders. The phenotypic heterogeneity of human populations and high variability of external environmental factors, along with limited access to brain tissue samples, presents some of the main challenges to studying anxiety disorders in humans. As these aspects can be controlled for in animals, animal models are often used to administer specific stressors in a uniform manner and to obtain brain tissue at a precisely chosen time point. Thereby, within the scope of this thesis, we take advantage of the fact that anxiety is an evolutionarily conserved response and address the integration of both human and mouse data obtained from a variety of approaches, including genomic, transcriptomic, and proteomic methods. First, to identify genetic loci predisposing to a specific phobia, the fear of heights, we conducted a genome-wide parametric and non-parametric linkage scan, followed by joint linkage and association analysis in a small population isolate with reduced genetic and environmental heterogeneity. Our results implicated three regions with suggestive evidence for linkage, including region 8q24.2-q24.3 (LOD = 2.09), which encompasses 49 genes, including several candidate genes for psychiatric disorders. Second, we identified molecules and biological pathways affected by chronic social defeat stress (CSDS), a mouse model of chronic psychosocial stress, in the following three brain regions: medial prefrontal cortex (PFCM), ventral hippocampus (HIPV), and bed nucleus of the stria terminalis (BNST). We used two inbred mouse strains with different basal anxiety levels, the innately non-anxious C57BL/6NCrl (B6) and innately anxious DBA/2NCrl (D2). Following analysis of RNA sequencing results, we discovered that differentially expressed (DE) oligodendrocyte-related genes were over-represented in gene set enrichment analysis (GSEA) of all studied brain regions. As oligodendrocytes are known for their function in axon myelination, we followed the results from transcriptomic analyses with transmission electron microscopy (TEM) and established that B6 stress-susceptible mice had thicker myelin in BNST axons compared to controls. Third, using the CSDS model, we further investigated the role of the BNST through additional studies of gene regulatory networks (GRN) of mRNAs and miRNAs and protein-protein interaction networks. Subsequently, we followed with an integration analysis of the results from both transcriptomic (mRNA sequencing, as well as AGO2 miRNA, and mRNA immunoprecipitation sequencing) and proteomic (liquid chromatography–tandem mass spectrometry) experiments. Furthermore, to translate our results to human anxiety disorders, we performed transcriptome profiling in blood cells of CSDS-subjected mice and compared it with gene expression patterns from blood cells of panic disorder patients who underwent exposure-induced panic attacks. We then followed with integrative gene set enrichment analysis of mouse and human data, which showed systemic genetic background-specific enrichment of mitochondria-related gene sets. Importantly, our results showed downregulation of the oxidative phosphorylation pathway in the CSDS-subjected D2 strain and panic disorder patients after a panic attack. To conclude, our results suggest (1) brain-region and mouse strain-specific differences in myelination in susceptibility and resilience to stress and (2) dysregulation of mitochondrial pathways associated with anxiety-related behavior in both mice and humans. Taken together, our results provide further insight into the complex genetic architecture of anxiety disorders and support the suitability of cross-species approaches to studying biological mechanisms underlying anxiety disorders. Keywords: anxiety disorders, chronic social defeat stress (CSDS), panic disorder, acrophobia, linkage analysis, gene expression, protein abundance, multi-omics studiesAhdistuneisuushäiriöt ovat joukko sairauksia, joihin liittyy jokapäiväistä elämää haittaava pitkittynyt tai voimakas pelko tai ahdistuneisuus. Ahdistuneisuushäiriöt ovat yleisimpiä mielenterveyden häiriöitä, ja niistä kärsii vuosittain 14% väestöstä. Olisikin tärkeää löytää uusia kliinisesti merkittäviä lääkkeitä yksilöllistettyjen hoitomenetelmien kehittämiseksi. Uusien lääkkeiden ja hoitomenetelmien kehitys vaatii kuitenkin syvällistä ymmärrystä ahdistuneisuuden biologisista mekanismeista sekä siitä, miten ympäristötekijät, kuten krooninen psykososiaalinen stressi, vaikuttavat ahdistuneisuushäiriön syntyyn. Suurimmat haasteet ahdistuneisuushäiriöiden ihmistutkimukselle ovat geneettinen heterogeenisyys, ympäristötekijöiden suuri vaihtelevuus ja aivokudosnäytteiden rajallinen saatavuus. Eläinkokeissa näitä tekijöitä voidaan kontrolloida, ja samalla keskittyä tiettyyn stressitekijään sekä kerätä aivokudosnäytteitä valittuna ajankohtana. Koska ahdistuneisuus on evolutiivisesti konservoitunut reaktio pelottavassa tilanteessa, sitä voidaan tutkia eläinmallien avulla. Tässä väitöskirjatyössä yhdistettiin sekä ihmis- että hiirigeneettisiä lähestymistapoja ahdistuneisuuden molekyylibiologisen taustan selvittämiseksi. Väitöskirjan ensimmäisessä osatyössä tutkittiin korkeanpaikankammon perinnöllistä taustaa geneettisessä isolaatissa kytkentäanalyysin avulla. Löysimme kolmelta genomin alueelta viitteellisen assosiaation ahdistuneisuushäiriöihin. Näistä 8q24.2-q24.3-alueella (LOD = 2.09) sijaitsee 49 geeniä, joista useat ovat psykiatristen sairauksien kandidaattigeenejä. Toisessa osatyössä tutkittiin ahdistuneisuushäiriöille tärkeän riskitekijän, kroonisen psykososiaalisen stressin, vaikutusta kolmen aivoalueen, etuaivokuoren, ventraalisen hippokampuksen ja stria terminaliksen bed-tumakkeen (BNST), geenien ilmentymiseen. Käytimme kahta sisäsiittoista hiirikantaa, jotka eroavat sisäsyntyisen ahdistuneisuuden suhteen: C57BL/6NCrl (B6) -kannalla on synnynnäisesti alhainen ahdistuneisuustaso, kun taas DBA/2NCrl (D2) -kanta on luontaisesti ahdistunut. RNA-sekvensointi osoitti, että oligodendrosyyttien toiminta ja erityisesti myelinisaatioon liittyvien geenien ilmentyminen muuttui kroonisen stressin seurauksena. Koska oligodendrosyytit muodostavat myeliinitupen hermosolujen aksonien ympärille, tutkimme transmissioelektronimikroskopian avulla myeliinin paksuutta stressin jälkeen. BNST:ssä, johon tämä väitöstyö keskittyi, oli stressille alttiilla B6-hiirillä paksumpi myeliini verrattuna kontrollihiiriin. Kolmannessa osatyössä keskityimme tutkimaan kroonisen psykososiaalisen stressin vaikutuksia BNST-aivoalueeseen käyttäen useita omiikka-menetelmiä. Yhdistämällä tulokset mikroRNA-sekvensoinnista, RNA-sekvensoinnista ja proteomiikka-analyysistä havaitsimme muutoksia mitokondrioiden toimintaa säätelevissä molekyyliverkostoissa. Koska ahdistuneisuushäiriöpotilailta on mahdotonta saada aivonäytteitä, tutkimme verisolujen geenien ilmentymistä paniikkihäiriöpotilailla ennen paniikkikohtausta, sen aikana, ja sen jälkeen. Tutkimme myös hiirten verisolujen geenien ilmentymistä kroonisen psykososiaalisen stressin jälkeen. Myös näissä kokeissa löysimme muutoksia mitokondrioiden toimintaan liittyvien geenien ilmentymisessä. Ahdistusherkillä D2-hiirillä ja paniikkihäiriöpotilailla geenien ilmentyminen oli hiljentynyt, kun taas vähemmän ahdistuneilla B6-hiirillä se oli lisääntynyt verrattuna kontrollihiiriin. Väitöskirjatyön tulokset osoittavat, että (1) eri sisäsiittoiset hiirikannat reagoivat krooniseen stressiin erilaisin selviytymisstrategioin, (2) geneettisellä taustalla on suuri merkitys stressialttiudelle ja -resilienttiydelle sekä käyttäytymisen että geeniekspression tasoilla, (3) myelinisaatioon vaikuttavien geenien ilmentyminen ja myeliinin paksuus muuttuvat kroonisen stressin seurauksena, ja (4) mitokondrioiden toimintaan vaikuttavien geenien ilmentyminen muuttuu paniikkihäiriössä ihmisillä ja kroonisen stressin seurauksena hiirillä. Nämä tulokset ovat merkittävästi lisänneet ymmärrystä kroonisen psykososiaalisen stressin ja ahdistuneisuuden molekyylitason vaikutuksista. Lisäksi ne tukevat eri lajeja hyödyntävien menetelmien sopivuutta ahdistuneisuushäiriöiden taustalla olevien biologisten mekanismien tutkimuksessa. Avainsanat: ahdistuneisuushäiriö, sosiaalisen lannistamisen hiirimalli, paniikkihäiriö, korkeanpaikankammo, kytkentäanalyysi, geenien ilmentyminen, proteomiikka, RNA-sekvensoint

The bradykinin system in stress and anxiety in humans and mice

Pharmacological research in mice and human genetic analyses suggest that the kallikrein-kinin system (KKS) may regulate anxiety. We examined the role of the KKS in anxiety and stress in both species. In human genetic association analysis, variants in genes for the bradykinin precursor (KNG1) and the bradykinin receptors (BDKRB1 and BDKRB2) were associated with anxiety disorders (p <0.05). In mice, however, neither acute nor chronic stress affected B1 receptor gene or protein expression, and B1 receptor antagonists had no effect on anxiety tests measuring approach-avoidance conflict. We thus focused on the B2 receptor and found that mice injected with the B2 antagonist WIN 64338 had lowered levels of a physiological anxiety measure, the stress-induced hyperthermia (SIH), vs controls. In the brown adipose tissue, a major thermoregulator, WIN 64338 increased expression of the mitochondrial regulator Pgc1 alpha and the bradykinin precursor gene Kng2 was upregulated after cold stress. Our data suggests that the bradykinin system modulates a variety of stress responses through B2 receptor-mediated effects, but systemic antagonists of the B2 receptor were not anxiolytic in mice. Genetic variants in the bradykinin receptor genes may predispose to anxiety disorders in humans by affecting their function.Peer reviewe

A genome-wide screen for acrophobia susceptibility loci in a Finnish isolate

Peer reviewe

Summaries of plenary, symposia, and oral sessions at the XXII World Congress of Psychiatric Genetics, Copenhagen, Denmark, 12-16 October 2014

The XXII World Congress of Psychiatric Genetics, sponsored by the International Society of Psychiatric Genetics, took place in Copenhagen, Denmark, on 12-16 October 2014. A total of 883 participants gathered to discuss the latest findings in the field. The following report was written by student and postdoctoral attendees. Each was assigned one or more sessions as a rapporteur. This manuscript represents topics covered in most, but not all of the oral presentations during the conference, and contains some of the major notable new findings reported

Multi-omics analysis identifies mitochondrial pathways associated with anxiety-related behavior

Author summary Genetic and environmental factors contribute to the etiology of psychiatric diseases but the underlying mechanisms are poorly understood. Chronic psychosocial stress is a well-known risk factor for anxiety disorders. To identify biological pathways involved in psychosocial stress-induced anxiety and resilience to it, we used a well-characterized mouse model of chronic social defeat stress (CSDS) in two inbred mouse strains, C57BL/6NCrl (B6) and DBA/2NCrl (D2), which differ in their susceptibility to stress. We focused on the bed nucleus of the stria terminalis, a key brain region behind stress-response and anxiety, and carried out genome-wide analysis of mRNA, and miRNA expression, and protein abundance. Bioinformatic integration of these data supported differences in mitochondrial pathways as a major stress response. To translate these findings to human anxiety, we investigated blood cell gene expression in mice and in panic disorder patients exposed to fearful situations and experiencing panic attacks. Concurring with our brain findings, expression of mitochondrial pathways was also affected in mouse and human blood cells, suggesting that the observed stress response mechanisms are evolutionarily conserved. Therefore, chronic stress may critically affect cellular energy metabolism, a finding that may offer new targets for therapeutic interventions of stress-related diseases.Peer reviewe

The circadian gene Cryptochrome 2 influences stress-induced brain activity and depressive-like behavior in mice

Cryptochrome 2 (Cry2) is a core clock gene important for circadian regulation. It has also been associated with anxiety and depressive-like behaviors in mice, but the previous findings have been conflicting in terms of the direction of the effect. To begin to elucidate the molecular mechanisms of this association, we carried out behavioral testing, PET imaging, and gene expression analysis of Cry2(-/-) and Cry2(+/+) mice. Compared to Cry2(+/+) mice, we found that Cry2(-/-) mice spent less time immobile in the forced swim test, suggesting reduced despair-like behavior. Moreover, Cry2(-/-) mice had lower saccharin preference, indicative of increased anhedonia. In contrast, we observed no group differences in anxiety-like behavior. The behavioral changes were accompanied by lower metabolic activity of the ventro-medial hypothalamus, suprachiasmatic nuclei, ventral tegmental area, anterior and medial striatum, substantia nigra, and habenula after cold stress as measured by PET imaging with a glucose analog. Although the expression of many depression-associated and metabolic genes was upregulated or downregulated by cold stress, we observed no differences between Cry2(-/-) and Cry2(+/+) mice. These findings are consistent with other studies showing that Cry2 is required for normal emotional behavior. Our findings confirm previous roles of Cry2 in behavior and extend them by showing that the effects on behavior may be mediated by changes in brain metabolism.Peer reviewe

Genetic Control of Myelin Plasticity after Chronic Psychosocial Stress

Anxiety disorders often manifest in genetically susceptible individuals after psychosocial stress, but the mechanisms underlying these gene-environment interactions are largely unknown. We used the chronic social defeat stress (CSDS) mouse model to study resilience and susceptibility to chronic psychosocial stress. We identified a strong genetic background effect in CSDS-induced social avoidance (SA) using four inbred mouse strains: 69% of C57BL/6NCrl (B6), 23% of BALB/cAnNCrl, 19% of 129S2/SvPasCrl, and 5% of DBA/2NCrl (D2) mice were stress resilient. Furthermore, different inbred mouse strains responded differently to stress, suggesting they use distinct coping strategies. To identify biological pathways affected by CSDS, we used RNA-sequencing (RNAseq) of three brain regions of two strains, B6 and D2: medial prefrontal cortex (mPFC), ventral hippocampus (vHPC), and bed nucleus of the stria terminalis (BNST). We discovered overrepresentation of oligodendrocyte (OLG)-related genes in the differentially expressed gene population. Because OLGs myelinate axons, we measured myelin thickness and found significant region and strain-specific differences. For example, in resilient D2 mice, mPFC axons had thinner myelin than controls, whereas susceptible B6 mice had thinner myelin than controls in the vHPC. Neither myelin-related gene expression in several other regions nor corpus callosum thickness differed between stressed and control animals. Our unbiased gene expression experiment suggests that myelin plasticity is a substantial response to chronic psychosocial stress, varies across brain regions, and is genetically controlled. Identification of genetic regulators of the myelin response will provide mechanistic insight into the molecular basis of stress-related diseases, such as anxiety disorders, a critical step in developing targeted therapy.Peer reviewe