Disrupted in Schizophrenia 1 (DISC1) is a well supported risk factor for schizophrenia, bipolar disorder and major recurrent depression. DISC1 is a multifunctional multicompartmentalised scaffold protein with essential roles in neuronal proliferation, differentiation, migration and integration. DISC1 also modulates pathways of vital importance for neuronal signalling and plasticity. One of the major hypotheses for the cause of psychiatric illness is N-methyl-D-aspartate (NMDA) receptor hypofunction. It was observed that NMDA receptor antagonists can induce symptoms of schizophrenia in unaffected individuals, and exacerbate symptoms in patients with schizophrenia. Recent work in our laboratory showed that DISC1 complexes with NMDA receptors within the cell body and at synapse of neurons. Here I studied whether DISC1, or DISC1 missense variants, affect the trafficking of NMDA receptors. This was done by quantifying surface NMDA receptor expression in the presence of DISC1 or variant DISC1. I found that one common variant, 607F, causes a significant reduction in surface expressed NMDA receptors. I went on to show that DISC1 reduces the number of internalised receptors associating with early RAB5-containing endosomes. This indicates that DISC1 may be involved in the trafficking and recycling of NMDA receptors, a process that may be affected by the missense DISC1 variant 607F. Further to this I studied the effects on NMDA receptor trafficking of DISC1 pathway partners Nuclear Distribution Element 1 (NDE1) and Trafficking-protein kinesin binding 1 (TRAK1), both regulators of neuronal intracellular trafficking. Phosphorylation of NDE1 at T131 has been shown to be modulated by DISC1. Using phospho-mimic and phospho-dead NDE1 expression constructs I observed a significant reduction in the surface-expressed NMDA receptors in cells expressing the phospho-mimic form of NDE1. NDE1 may therefore be involved in the trafficking of NMDA receptors, and this role may be modulated by phosphorylation of NDE1. Finally, TRAK1 was shown to associate robustly with the GluN2B subunit, and to decrease the surface expression of NMDA receptors, most likely by sequestering them. The TRAK1-induced GluN2B sequestration may be an artefact, but the association of the trafficking molecule TRAK1 with this subunit may point towards a role in NMDA receptor trafficking. These proteins have been shown to associate with each other and may form a complex in order to traffic NMDA receptors. Disruption of this complex by defective DISC1 expression may affect NMDA receptor trafficking. In the brain this could conceivably contribute to NMDA receptor hypofunction and the development of psychiatric illness.

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.

Deficits in a number of cognitive domains have been associated with core symptoms of schizophrenia, including working memory, attention, motor skills, reaction time, episodic memory and executive function. Bipolar Disorder is also associated with cognitive impairment; however the level of impairment appears to be less severe than that seen in schizophrenia. A translocation (t(1;11)) containing the Disrupted-in-Schizophrenia 1 (DISC1) gene has been found to be highly associated with schizophrenia, bipolar disorder and major depressive disorder. As such, this gene has been the focus of much research and to date DISC1 has been found to be associated with brain development, brain structure and the glutamate system - all key factors in current models of schizophrenia and affective disorders. The aim of this PhD is to identify cognitive domains that are differentially impaired or unimpaired in a large Scottish family, some of whom carry this rare DISC1 variant, a balanced translocation (t (1;11) (q 42; q14.3)), that segregates with schizophrenia and affective disorders, as well as psychiatric patients with schizophrenia and bipolar disorder and healthy control subjects. All participants have undergone standardised cognitive assessments to measure premorbid I.Q. (NART), current I.Q. (WASI) verbal memory, working memory, verbal fluency, processing speed, motor skills, executive function (BACS) and selected CANTAB tasks to assess simple and five-choice reaction time. Polygenic risk profile scores and self-report questionnaire data have also been investigated. Results indicate an impact of the DISC1 t(1;11) translocation on general intelligence and attention and processing speed. Significant differences were also identified between DISC1 t(1;11) carriers and non-carriers on self-report questionnaire data. Mean scores for polygenic risk for bipolar disorder were significantly different between DISC1 t(1;11) carriers and non-carriers and polygenic risk for schizophrenia was significantly associated with symptom severity, as measured by the Positive and Negative Symptom Scale (PANSS). Within the patient groups, a measure of processing speed (the token motor task) was found to be significantly different between those with schizophrenia and bipolar disorder and there was also a trend for attention and processing speed. As expected, I.Q. was significantly different between patients and control participants. Clinical ratings were significantly associated with neuropsychological and self-report measures. Polygenic risk for major depressive disorder was found to be significantly associated with impaired general intelligence (current IQ) and slowed reaction time in patients who were not currently depressed, suggesting there may be genetic risk markers in this population which impact on cognition. This is a novel finding and further suggests the possibility of a biological component related to the genetics of depression. In conclusion, and in line with the literature, psychosis has a negative impact on cognition with reduced performance across several neuropsychological tasks between patient groups, with schizophrenia patients performing worse than patients with bipolar disorder and both patient groups performing worse than healthy control participants. Cognition is markedly more impaired in DISC1 t(1;11) translocation carriers and especially in those with psychosis. The DISC1 t(1;11) translocation and psychosis may therefore confer a “double hit” on cognition - in addition to psychosis itself - which is known to impair cognitive function, significantly increasing the level of cognitive impairment and increasing the risk for psychosis in general.

Human genetic data suggests DISC1 (Disrupted-in-schizophrenia 1) is a susceptibility gene for schizophrenia and depression. Disc1 Q31L-/- mutants show depression-like behaviour and Disc1 L100P-/- mutants schizophrenia-like behaviour. Heterozygous mutants show an intermediate phenotype. In a gene-environment interaction study, we exposed heterozygotes to chronic social defeat (CSD) stress and phenotyped behaviour. Disc1, Bdnf(III) and Pde4b mRNA levels were also measured. Moreover, as epigenetic mechanisms may mediate some effects of CSD, we also exposed wildtype mice to CSD concurrently with the histone deacetylase inhibitor valproate. We found that CSD increased anxiety in L100P-/+ mutants, and that levels of Disc1, Bdnf(III) and Pde4b mRNA were higher in this mutant. Valproate treatment did not correct CSD-induced behavioural changes. In conclusion, we have demonstrated an interaction between a strong susceptibility gene for psychiatric disease and an environmental manipulation similar to stressors known to affect mental illness.

Human genetic data suggests DISC1 (Disrupted-in-schizophrenia 1) is a susceptibility gene for schizophrenia and depression. Disc1 Q31L-/- mutants show depression-like behaviour and Disc1 L100P-/- mutants schizophrenia-like behaviour. Heterozygous mutants show an intermediate phenotype. In a gene-environment interaction study, we exposed heterozygotes to chronic social defeat (CSD) stress and phenotyped behaviour. Disc1, Bdnf(III) and Pde4b mRNA levels were also measured. Moreover, as epigenetic mechanisms may mediate some effects of CSD, we also exposed wildtype mice to CSD concurrently with the histone deacetylase inhibitor valproate. We found that CSD increased anxiety in L100P-/+ mutants, and that levels of Disc1, Bdnf(III) and Pde4b mRNA were higher in this mutant. Valproate treatment did not correct CSD-induced behavioural changes. In conclusion, we have demonstrated an interaction between a strong susceptibility gene for psychiatric disease and an environmental manipulation similar to stressors known to affect mental illness.