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Schizophrenia is a common and debilitating mental illness, affecting 1% of the population and affecting all ethnic groups (Wilkinson et al 1996). It is a highly complex disorder containing genetic and environmental components (Birkett et al. 2000), making it difficult to identify those genes responsible for the development of schizophrenia using linkage alone. This thesis presents work on three candidate genes Disrupted in Schizophrenia 1 (DISC1), Neuregulin 1 (NRG1) and 5-HT5A, using a case control approach to test for association between schizophrenia and the candidate genes, in the Scottish population. All findings for 5-HT5A were negative. The exact function of DISC1 is unknown but it appears to have a role in neurodevelopment. Acting in complex with NUDEL to affect neurite growth (Ozeki et al. 2003) and modifying Translin binding when intragenically spliced with TRAX (Chennathukuzhi et al. 2001). Genotypic and haplotypic analysis of DISC1 using a combination of pooling and individual genotyping. Led to the discovery of a novel nonsynonymous SNP A40961G located in exon 2 of DISC1 which shows significant association with schizophrenia (p = 0.000232, O.R. = 0.71861, n = 600). Haplotypic analysis identified a 4 marker at-risk haplotype (p = 0.0010, O.R = 2.56, n = 200) containing the A40961G marker. NRG1 was investigated at the request of DeCode Genetics in order to replicate there positive at the 5' end of NRG1 (p = 0.000087) (Stefansson et al. 2002). The genotyping was carried out blind of diagnosis by myself at DeCode Genetics, analysis was carried out independently. The results confirmed the Icelandic findings for a 7 marker at-risk haplotype located at the 5' end NRG1 (p = 0.00031).

Recent advances in gene expression analysis using microarray technology should assist in our understanding of complex psychiatric diseases such as schizophrenia. The aim of this thesis was to identify differentially expressed genes in schizophrenic patients using post-mortem prefrontal cortex (DLPFC) (BA 10) with a genome approach and compare the expression pattern obtained with a microarray study using prefrontal cortex tissue from a chronic phencyclidine (PCP) model of schizophrenia (Cochran et aI., 2003). It is hypothesised that the identification of common altered genes in these two independent strategies will reveal genes that relate to the cognitive deficits observed in schizophrenia. A microarray study using total RNAs extracted from control and schizophrenic prefrontal cortices and Affymetrix Human Genome U-133A GeneChips was performed. Significance Analysis of Microarray (SAM) and two-tailed t test for data mining identified 236 probe sets (SAM; false positive rate (FDR) , 39%) and 918 probe sets (p<0.05, t test) as being differentially expressed. Further analyses revealed that Pyk/Nck pathway and Ca2+ sensitive gene signalling pathways may be dysfunctional in subjects with schizophrenia. Moreover, 123/671 genes showing more than 10% change were located on chromosomal loci linked to schizophrenia. These were relatively concentrated on 9q34.3, 11q22-23, 22q12-13 or proximal regions to these. Importantly 11 q22- 23 and 22q 12-13 have been described as strongly linked genetic loci (Prasad et al. 2002). In RT-qPCR confirmation stuclies, almost 2/3 of selected genes were validated. This suggested that the microarray study was successfully detecting gene expression changes despite the fold changes being small (≤l.5-fold), In vitro functional studies were performed on selected candidate genes using molecular based experiments. These demonstrated that the activity of CaMKIV was regulated by Serine/Threonine phosphatase, POPX 1, in CRE-dependent gene expression via direct interaction with POPXl, and that SLCIAl (neuronal excitatory amino acid transporter EAAT3)"could be involved in GABAergic machinery by participation in the supply of the substrate of GABA, glutamate, into cells via its transport activity. In conclusion, this project enabled construction of novel hypotheses about candidate genes relate to schizophrenia. The results suggest that there is prefrontal cortex dysregulation in glutamatergic and GABAergic neurotransmission associated with the candidate genes participating in Pyk/Nck pathway and Ca2+ sensitive gene signalling pathways. Moreover these could be potential targets for drug development in schizophrenia.

Aberrant neurotransmitter function promotes cognitive deficits in schizophrenia. These abnormalities in functioning are seen as disruptions in attentional and information processing, as well as disruptions in the consolidation and retrieval of information. Tasks of attentional salience and memory that are used to model these disruptions include the latent inhibition (LI) task of attentional salience, prepulse inhibition (PPI) task of sensorimotor gating and an Episodic memory (EM) task, which is an index of memory for episodes at a particular point in time. Aberrant functioning of candidate genes that are associated with risk for schizophrenia may be seen as behavioural alterations in these tasks of schizophrenia relevant phenotypes. dopaminergic hyperactivity and hypofunction have been implicated in mediating disruptions on these cognitive tasks. Increased transmission in the dopamine system in the striatal region promotes schizophrenia symptoms, and indirect dopamine (DA) agonist Amphetamine worsens these symptoms in patients, and disrupts schizophrenia relevant behaviours on these cognitive tasks. We investigated the effects of deletion of two genes relevant to schizophrenia on cognitive tasks known to be disrupted in the disorder. The effect of deletion of the dopamine D2 receptor (D2R) and trans membrane (TM) domain Neuregulin-1 (Nrg-1) receptor were investigated in mediating disruptions in cognitive processes in an animal model of schizophrenia. The role of the D2R in an attentional model of sensorimotor gating was assessed. PPI was attenuated in D2R knock out (KO), in a one day sensorimotor gating task. In a one day PPI test protocol, amphetamine disruptions on PPI were spared in D2R WT and KO mice. Following on from previous reports of disrupted LI by a single low dose amphetamine injection, separated by 24h interval, we established a single vs. two low dose PPI protocol in order to facilitate a direct comparison of amphetamine induced disruption in LI with PPI. A one injection (prior to test only) vs. two injection (prior to habituation and prior to test) task was established. In the two day protocol, a single low dose of amphetamine disrupted PPI in D2R KO mice and reduced startle reactivity to the 120 dB pulse alone trials. Two low dose injections of amphetamine however, do not disrupt PPI in D2R KO or their WT littermates, and do not mimic low dose amphetamine disruptions in the LI task. These findings demonstrate that prior conclusions about the requirement of the D2R for amphetamine effects in PPI does not generalise to all dose regimens. Episodic memory was also investigated as a measure of cognitive impairment in schizophrenia. D2R KO mice show sex specific dissociations on an EM task. Male D2R WT and KO animals show equal exploration of old vs. recent objects on the what-when component of the EM task, and female KO animals show enhanced memory for old vs. recent objects. Both D2R WT and KO mice show intact memory for displaced objects. These deficits were also investigated in the TM domain Nrg-1 model. Nrg-1 has been implicated as a candidate gene for schizophrenia, and behavioural phenotypes assessing its role in cognitive impairment in schizophrenia were established. Intact LI is seen in both Nrg-1 WT and Het animals. Nrg-1 TM domain Het mutants also show deficits on the schizophrenia relevant PPI task. Nrg-1 Het mutants show attenuated % PPI compared to their WT littermates, which reflects interrupted sensorimotor gating in schizophrenia. Lastly, we found some evidence that reduced function of TM-domain of the Nrg-1 gene disrupted episodic-like memory (what- where-when recognition) in males and improved it in females.