Investigating putative pathogenic mechanisms within a family in which a chromosomal translocation confers risk of major mental illness
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Date
02/07/2016Author
Briggs, Gareth James
Metadata
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.