Molecular mechanisms of D-cycloserine in a fear extinction posttraumatic stress disorder (PTSD) animal model

Malan-Muller, Stefanie (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Posttraumatic stress disorder (PTSD) is a severe, chronic and debilitating psychiatric disorder that can present after the experience of a life-threatening traumatic event. D-cycloserine (DCS), a partial N-methyl-D-aspartate (NMDA) receptor agonist, has been found to augment cognitive behavioural therapy by facilitating fear extinction; however, the precise mechanisms whereby DCS ameliorates fear triggered by a traumatic context remains to be fully elucidated. This study aimed to (i) identify the molecular mechanisms of intrahippocampally administered DCS in facilitating fear extinction in a rat model of PTSD by investigating gene expression profiles in the left dorsal hippocampus (LDH) of male Sprague Dawley rats and (ii) determine whether microRNA (miRNA) expression and DNA methylation mediated these gene expression changes. An adapted version of the PTSD animal model described by Siegmund and Wotjak (2007) was utilised. The total number of 120 rats were grouped into four experimental groups (of 30 rats per group) based on fear conditioning and the intrahippocampal administration of either DCS or saline: (1) fear conditioned + intrahippocampal saline administration (FS), (2) fear conditioned + intrahippocampal DCS administration (FD), (3) control + intrahippocampal saline administration (CS) and (4) control + intrahippocampal DCS administration (CD). Behavioural tests (the light/dark [L/D] avoidance test, forced swim test and open field test) were conducted to assess anxiety and PTSD-like behaviours. The L/D avoidance test was the most sensitive behavioural test of anxiety and was subsequently used to differentiate maladapted (animals that displayed anxiety-like behaviour) and well-adapted (animals that did not display anxiety-like behaviour) subgroups. In order to identify genes that were differentially expressed between FS maladapted (FSM) (n = 6) vs. FD well-adapted (FDW) (n = 6) groups, RNA sequencing was performed on the Illumina HiSeq 2000 which generated more than 60 million reads per sample. This was followed by subsequent bioinformatics analyses (using the software programs TopHat, Bowtie, Cuffdiff and Bio-Ontological Relationship Graph (BORG) database (that identifies genes that may be biologically relevant) to identify biologically relevant differentially expressed genes between the treatment groups. Epigenetic mechanisms mediating observed differences in gene expression were investigated by conducting DNA methylation and miRNAseq analyses in the FDW and FSM experimental groups. DNA methylation was investigated using real-time quantitative PCR (qPCR) amplification followed by high resolution melt analysis on the Rotor-GeneTM 6000. Differences in miRNA expression levels between the FDW and FSM groups were investigated by sequencing the miRNA fraction on the MiSeq platform. The bioinformatics pipeline used to analyse the RNAseq data identified 93 genes that were significantly downregulated in the FDW group compared to the FSM group. Forty-two of these genes were predicted to be biologically relevant (based on BORG analysis). Integrative network analyses revealed subsets of differentially expressed genes common across biological functions, pathways and disorders. The co-administration of DCS and behavioural fear extinction downregulated immune system genes and genes that transcribe proinflammatory and oxidative stress molecules. These molecules mediate neuroinflammation and subsequently cause neuronal damage. DCS also regulated genes involved in learning and memory processes. Additionally, a subset of the genes, which have been found to be associated with disorders that commonly co-occur with PTSD (such as cardiovascular disease, metabolic disease, Alzheimer‘s and Parkinson‘s disease), was downregulated by the co-administration of DCS and behavioural fear extinction. In order to determine whether real-time qPCR analysis would be sensitive enough to detect differential expression in those genes found to be differentially expressed in RNAseq analysis, the expression of nine genes was analysed using SYBR Green qPCR technology. In the LDH, six of the nine genes were found to be differentially expressed between FDW and FSM groups and one gene, matrix metallopeptidase 9 (MMP9), was observed to be differentially expressed between these two groups in the blood. Three of the nine genes for which differential expression levels were investigated using SYBR Green real-time qPCR, contained CpG islands and were used for CpG island DNA methylation analysis. Results indicated that CpG island DNA methylation did not mediate differential gene expression of TRH, NPY or MT2A. Bioinformatics analysis of miRNAseq data identified 23 miRNAs that were differentially expressed between the FDW and FSM groups. Several of these miRNAs have previously been found to be involved in brain development and behavioural measures of anxiety. Furthermore, functional luciferase analysis indicated that the upregulation of rno-mi31a-5p could have facilitated the downregulation of interleukin 1 receptor antagonist gene (IL1RN) as detected in RNAseq. RNAseq and miRNAseq analyses in this PTSD animal model identified differentially expressed genes and miRNAs that serve to broaden our understanding of the mechanism whereby DCS facilitates fear extinction. To this end, immune system genes and genes transcribing proinflammatory and oxidative stress molecules were among the genes that were found to be differentially expressed between the FDW and FSM groups. Based on the results obtained, it can be hypothesised that DCS attenuates neuroinflammation and subsequent neuronal damage, and also regulates genes involved in learning and memory processes. Concomitantly, these gene expression alterations mediate optimal neuronal functioning, plasticity, learning and memory (such as fear extinction memory) which contribute to the fear extinction process. Furthermore, biologically relevant differentially expressed genes that were associated with DCS facilitation of fear extinction and with other chronic medical conditions, such as cardiovascular disease and metabolic diseases, might help to explain the co-occurrence of these disorders with PTSD. In conclusion, Identifying the molecular underpinnings of DCS-mediated fear extinction brings us closer to understanding the process of fear extinction and could, in future work be used to explore novel therapeutic targets to effectively treat PTSD and related disorders.

AFRIKAANSE OPSOMMING: Posttraumatiese stressindroom is 'n ernstige, kroniese aftakelende psigiatriese toestand wat kan ontwikkel na 'n lewensgevaarlike traumatiese gebeurtenis. Daar is bevind dat die gesamentlike toediening van D-sikloserien (DCS), 'n N-metiel-D-aspartaat (NMDA) reseptor agonis, en kognitiewe gedragsterapie effektief is in die bemiddeling van vrees uitwissing; maar die presiese meganisme waar deur DCS die vrees wat deur 'n traumatiese konteks ontlok word verminder, is egter onduidelik. Hierdie studie het beoog om (i) die molekulêre meganismes te identifiseer waardeur intra-hippokampaal toegediende DCS vrees uitwissing fasiliteer, in 'n rot model van posttraumatiese stressindroom, deur geen uitdrukkingsprofiele in the linker dorsale hippokampus (LDH) van manlike Sprague Dawley rotte te ondersoek en (ii) om te bepaal of mikroRNA (miRNA) uitdrukking en DNA metilering die veranderinge in geen uitdrukking bemiddel het. 'n Gewysigde weergawe van die posttraumatiese stressindroom diere model, beskryf deur Siegmund en Wotjak (2007), was gebruik tydens die studie. Rotte was in vier groepe verdeel, vrees kondisionering + soutwater (FS), vrees kondisionering + DCS (FD), kontrole + soutwater (CS) en kontrole + DCS (CD). Gedragstoetse was uitgevoer om angstige, vreesvolle en posttraumatiese stressindroom-tipe gedrag te evalueer. Gedurende die lig/donker (L/D) vermydingstoets het die FS groep aansienlik meer tyd in die donker kompartement deurgebring ('n indikasie van vreesvolle gedrag) in vergelyking met die CS en die FD groepe wat meer tyd in die verligte kompartement deurgebring het ('n indikasie van vreeslose gedrag). Die L/D toets was die mees sensitiewe gedragstoets vir angstige en vreesvolle gedrag en was gevolglik gebruik om die diere te sub-groepeer in wanaangepaste (diere wat angstige en vreesvolle gedrag vertoon het) en goedaangepaste (diere wat nie angstige en vreesvolle gedrag vertoon het nie) subgroepe. Nuwe generasie RNA volgordebepaling (RNAseq) van die LDH RNA en daaropvolgende bioinformatiese analise was uitgevoer om gene te identifiseer wat differensieel uitgedruk is tussen die twee behandelingsgroepe van belang in die betrokke studie, naamlik FS wanaangepaste (FSM) teenoor FD goedaangepaste (FDW) groepe. Epigenetiese analises was uitgevoer om te bepaal of differensieel uitgedrukte miRNAs of CpG-eiland DNA metilasie die differensiële geenuitdrukking bemiddel het. Bioinformatiese analises van die RNAseq data het 93 gene geïdentifiseer waarvan die geen uitdrukking beduidend onderdruk was in die FDW groep in vergelyking met die FSM groep; 42 van hierdie gene was voorspel om biologies relevant te wees. Geïntegreerde netwerk analise het onthul dat sekere van die differensieel uitgedrukte gene gemeenskaplik was tussen verskeie biologiese funksies, padweë en versteurings. DCS het die uitdrukking van immuun-sisteem gene en pro-inflammatoriese en oksidatiewe stres gene verlaag. Hierdie molekules medieer neuro-inflammasie wat gevolglik tot neurale skade lei. DCS het ook gene gereguleer wat betrokke is by leer en geheue prosesse. DCS het onder meer ook die geenuitdrukking verlaag van 'n sub-groep van gene wat voorheen geassosier is met komorbiede versteurings van PTSD. SYBR Green real-time qPCR (werklike tyd kwantitatiewe polimerase ketting reaksie) analise was ondersoek om te bepaal of hierdie metode sensitief genoeg sou wees om die verlaagde geen-uitdrukking van verskeie van die biologies relevante differensieel uitgedrukte gene te identifiseer, in dieselfde LDH komplementêre DNA (cDNA) monsters as wat in die RNAseq gebruik is, asook in die bloed cDNA monsters. SYBR Green real-time qPCR was in staat om ses, van die nege, differensieel uitgedrukte gene in die LDH cDNA monsters en een geen, matriks metallopeptidase 9 (MMP9), in die bloed cDNA monsters op te tel. Drie van die gene waarvoor SYBR Green real-time qPCR gebruik is om differensiële geenuitdrukking te toets, het CpG eilande bevat en was gevolglik gebruik in CpG eiland DNA metilering analises. Resultate het getoon dat CpG eiland DNA metilering nie die differensiële geenuitdrukking van TRH, NPY of MT2A gedryf het nie. Bioinformatiese analises van die miRNAseq data het 23 miRNAs geïdentifiseer wat differensieël uitgedruk was tussen die FDW en FSM groepe. Verskeie van hierdie miRNAs is reeds voorheen beskryf om betrokke te wees in brein ontwikkeling en angs gedrags metings. Funksionele luciferase analises het verder aangedui dat die verhoogde uitdrukking van rno-mi31a-5p moontlik die verlaagde geen uitdrukking van IL1RN, soos waargeneem in die RNAseq data, kon bewerkstellig het. RNAseq en miRNAseq analises in hierdie posttraumatiese stressindroom dieremodel het differensieël uitgedrukte gene en miRNAs geïdentifiseer wat dien om die verstaanswyse te verbreed van hoe DCS die vrees uitwissings proses fasiliteer. Die meganismes waardeur DCS vrees uitwissings bewerkstellig het sluit die verlaging van immuun-sisteem geen-uitdrukking in, sowel as verlaagde uitdrukking van gene wat pro-inflammatoriese en oksidatiewe stress gene transkribeer. DCS het daardeur neuro-inflammasie en gevolglike neurale skade voorkom. DCS het daarmee saam ook gene gereguleer wat betrokke is by leer en geheue prosesse. Hierdie gesamentlike veranderings in geen uitdrukking het gelei tot die uiteindelike bewerkstelling van optimale neurale funksionering, plastisiteit, leer en geheue prosesse wat uiteindelik bygedra het tot vrees uitwissing. Biologies relevante differensieël uitgedrukte gene wat ook geassosieer was met ander kondisies, soos middel verwante versteurings en metaboliese versteurings, kan help om die komorbiditeit met posttraumatiese stressindroom te verklaar. Identifisering van die molekulêre grondslae van DCS bemiddelde vrees uitwissing verbreed ons begrip en verstaan van vrees uitwissing en kan moontlik, in toekomstige navorsing gebruik word om nuwe innoverende terapeutiese teikens te verken om sodoende posttraumatiese stressindroom meer effektief te kan behandel.

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