Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.716638
Title: Investigating putative pathogenic mechanisms within a family in which a chromosomal translocation confers risk of major mental illness
Author: Briggs, Gareth James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2016
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Abstract:
In a large Scottish family a high incidence of schizophrenia, bipolar disorder and major depressive disorder co-segregates with a balanced autosomal translocation (t(1;11)(q42.1;q14.3). The translocation disrupts Disrupted-in-Schizophrenia-1 (DISC1) and DISC2 on chromosome 1, and DISC1FP1 (Disrupted-in-Schizophrenia-Fusion-Partner-1), also known as Boymaw, on chromosome 11. DISC1 is a leading candidate gene for major mental illness and is involved in neurodevelopment and cellular signalling, whilst DISC2 and DISC1FP1 are apparently non-coding RNA genes that undergo alternative splicing and that are expressed in the brain. This thesis aimed to investigate putative mechanisms of pathogenesis that may result from the t(1;11), with the hope that pathogenic mechanisms identified in the t(1;11) pedigree might shed light upon mechanisms conferring risk for psychiatric illness in the wider population. Previous work had identified DISC1/DISC1FP1 chimeric transcripts in t(1;11)-family derived lymphoblastoid cell lines. The detected transcripts include CP60 and CP69 which encode DISC1 aa1-597 plus an additional 60 or 69 amino acids from DISC1FP1, respectively. In this thesis a novel DISC1/DISC1FP1 transcript, CP1, was identified in t(1;11) lymphoblastoid cell lines. The CP1 transcript encodes DISC1 aa1-597 plus one glycine. A truncated form of DISC1 comprising aa1-597 was previously suggested to be a putative product of the translocation and, as such, has been the focus of multiple studies. The identification of the CP1 species is of interest as it differs from DISC1 aa1-597, by only a glycine. As glycines are simple uncharged aa’s, it is likely that these two DISC species share similar properties. In vitro exogenous expression of the three DISC1/DISC1FP1 protein species in both COS-7 and primary neuron cultures revealed contrasting cellular phenotypes. CP1 showed a diffuse cellular localisation pattern with cells containing readily visible tubular mitochondria. This is indistinguishable from the staining pattern of DISC1 aa1-597, highlighting the high degree of similarity between these species. CP60 and CP69, however, appeared to be clustered in the perinuclear region of the cell. Initial staining attempts with MitoTracker Red to visualise mitochondria in CP60 and CP69 expressing cells resulted in fewer than 30% of cells being stained. In those that did stain, the mitochondria appeared clustered. The absence of MitoTracker Red staining in mitochondria may be due to the loss of the mitochondrial membrane potential, Δψm. The adoption of a co-staining protocol with antibodies for mitochondrial proteins enabled the visualisation of mitochondrial structure in all of the cells exogenously expressing CP60 and CP69. All of these mitochondria possessed a clustered morphology, with which CP60 and CP69 expression was substantially co-localised. To see if MitoTracker staining was perturbed, in t(1;11) lymphoblastoid cell lines, as may occur if the DISC1/DISC1FP1 chimeras are expressed endogenously, the fluorescence of MitoTracker Red staining was investigated by FACS. Pooled analysis of experimental replicates revealed a negative result, with MitoTracker Red staining in t(1;11) lymphoblastoid cell lines not differing from controls. These findings indicate a need for further research using the mitochondrial membrane potential, Δψm as a metric as this would enable variations in mitochondrial mass to be accounted for. Prior to my arrival, an expression microarray had been carried out on lymphoblastoid cell line cDNA to assess gene expression differences resulting from the t(1;11). In order to identify putative pathogenic mechanisms, I carried out functional enrichment analysis of the expression array data using multiple analysis programs. Several programs detected dysregulation of the cell cycle and enrichment of altered expression of genes involved in the immune response and inflammation in t(1;11) carriers. The use of a rare variant investigative paradigm in this thesis furthers understanding of the putative pathogenic mechanisms that might act to increase risk for psychiatric illness in t(1;11) carriers. Moreover, it may aid the biological understanding of the aetiology of psychiatric illness in the general population. As such, improved understanding of the mechanisms of risk in the t(1;11) pedigree may eventually lead to the development of better treatments. In the intervening time since some of the research for thesis was published, two studies have emerged that may serve to highlight potential mechanisms of pathogenic action mediated by CP60 and CP69 expression. It has recently been observed that WT-DISC1 couples to the adaptor protein TRAK1 and the mitochondrial membrane anchor Miro1, which are part of the mitochondrial transport complex (Ogawa et al, 2014; Norkett et al, 2016). Furthermore, the exogenous expression of CP60 impairs bidirectional mitochondrial trafficking (Norkett et al, 2016). This suggests that CP60 expression may impair interactions with TRAK1 and Miro1. Given the sequence homology between CP60 and CP69, mitochondrial transport deficits also likely arise with CP69 expression. It is therefore possible that the exogenously expressed CP60 and CP69 proteins could be docked on stationary mitochondria, which may contribute to the clustered expression patterns observed.
Supervisor: Millar, Kirsty ; Porteous, David Sponsor: Medical Research Council (MRC)
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.716638  DOI: Not available
Keywords: Disrupted-in-Schizophrenia-1 ; DISC1 ; mitochondria ; microarray ; DISC1FP1
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