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The molecular and genetic mechanisms involved in the pathogenesis of primary dystonia were investigated. This was carried out with two core studies. The pathogenesis of DYT1 dystonia was examined by the creation of a cell model. A single amino acid deletion in the DYT1 gene, which codes for a protein called torsinA, is responsible for most cases of primary, early-onset, generalized torsion dystonia. The gene in its wildtype and mutant forms were transfected into HEK 293 cells and the proteins locations and associations were studied. Wildtype torsinA was found to reside in the endoplasmic reticulum (ER). Mutant torsinA, however, was found in the nuclear envelope (NE) and in perinuclear inclusions that were not associated with the endoplasmic reticulum. In SH-SY5Y cells these inclusions were found to be associated with vesicular monoamine transporter type 2 (VMAT2), a transmembrane protein responsible for packaging catecholamine into vesicles. This association was studied in greater detail by immunoprecipitation and the functional implications were investigated by dopamine release experiments. The ultrastructure of the inclusions was also investigated by electron microscopy. Cervical dystonia and blepharospasm are the most common forms of focal primary dystonia. Most cases are apparently sporadic, although familial cases have been described. An allelic association study of genes involved in dopamine neurotransmission was carried out in order to determine whether genetic factors might contribute to an individual’s susceptibility to developing dystonia. An allele near the dopamine receptor 5 (DRD5) gene was found to be associated with cervical dystonia and blepharospasm. The presence of a causative mutation in the DRD5 gene was investigated by restriction fragment length polymorphism (RFLP) analysis and by sequencing.

Stressful experiences in animals trigger responses that lead to adaptive changes. These changes to physiology, metabolism and behaviour are mediated by the stress axis, which acts to maintain or restore homeostasis. Stress in early life is linked to the development of adult-onset psychiatric disorders in humans. Evidence from animal models has demonstrated that stress can affect behaviour, endocrine function and gene expression. Studies in mice also suggest that the combination of stress and a genetic predisposition can result in abnormal behaviours that resemble psychiatric disease. One such genetic factor is Disrupted-In-Schizophrenia-1 (DISC1). A translocation in DISC1 segregates with a high prevalence of mental illness in a large human family. The DISC1 protein is implicated in a wide variety of roles in the nervous system. Mouse models of DISC1 exhibit various behavioural abnormalities that have been likened to anxiety in humans. Recently, it has been demonstrated that mutant DISC1 mice display abnormal phenotypes in response to stress, thereby opening an avenue to investigate the role of DISC1 in the stress response. The zebrafish is powerful study system to address such a topic. Zebrafish are genetically tractable, exhibit quantifiable behaviours and have relatively simple brains that offer a good level of functional homology with humans [1]. The work described in this thesis utilises novel zebrafish models of disc1, the orthologue of the genetic risk factor in humans, which have not been previously examined with respect to stress responses. I describe studies in which I have analysed disc1 mutant zebrafish in terms of behavioural and endocrine responses to stress and investigated the expression of genes linked to hypothalamic development and the stress response. When exposed to an acute chemical stressor, wild type zebrafish modulated their behaviour and upregulate cortisol synthesis. Conversely, disc1 mutants typically do not modulate their behaviour or cortisol synthesis when exposed to the stressor. Mutants also displayed abnormal expression of a number of genes in the hypothalamus, which are critical to normal hypothalamic development. These findings suggest that disc1 alters stress axis function via abnormal hypothalamic development.

Arterial occlusion accounts for high rates of mortality in the western world. Strategies to bypass an occlusion by activating collateral vessels could reduce the consequences of arterial diseases. This thesis investigates the genes involved in collateral vessel remodelling in the chick embryo to gain insight into the process. Ligation of the right proximal vitelline artery of HH st 17 chick embryos occluded blood flow to the right hand side of the extra-embryonic tissue and vitelline vessel network. Collateral vessels were seen to develop from the pre-existing, left (unligated) vitelline artery and extended across the midline to carry arterial blood to the un-perfused side of the extra-embryonic tissue. The remodelling process was active over 48 hours and developed many small collateral vessels into a few, main conducting arteries. The number of collateral vessels peaked at 12 hours post tied-ligation and then decreased, whilst collateral vessel diameter continued to increase over the time period. Analysis of the global transcriptional profile of collateral vessels in the chick embryo was assessed following ligation, during early stages of collateral vessel development (4 hours), at the point of pruning and remodelling of the collateral network (12 hours). Collateral vessel formation in the chick embryo was found to be associated with a unique and specific gene expression profile. Phosphodiesterase 10A (PDE10A), an cAMP hydrolysing enzyme, was upregulated in tied-ligated embryos at 4 hours post tied-ligation and hypothesised to play a role in the remodelling process. To study PDE10A papaverine hydrochloride was used to inhibit the enzyme. Papaverine had no effect on normal vessel development but significantly impaired collateral vessel diameter from 6 hours post-ligation. This effect was rescued by co-treatment with Protein Kinase A inhibitor, Rp-8-Br-cAMPS. To begin an assessment of the role of PDE10A in collateral vessel remodelling, proliferation was investigated, in vivo. However, a mechanism of action for PDE10A has yet to be elucidated.