Investigations of Olfactory Mucosa to Test the Neurodevelopmental Nature of Psychoses

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Mackay-Sim, Alan
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McGrath, John
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Evidence from various sources suggests that schizophrenia may result from altered brain development. The adult olfactory epithelium provides an available 'window' on neuronal development because new neurons are formed there throughout life. This thesis set out to test the neurodevelopmental hypothesis of psychotic disease. Two cell-based models, skin fibroblast and olfactory mucosa culture, were employed to investigate this hypothesis. In order to first demonstrate the utility of olfactory mucosa culture as a model of neurodevelopment, and to allow the candidate to gain proficiency in the culture of this tissue, an investigation of the mitogenic and differentiating properties of insulin-like growth factor-I within this system was undertaken. Insulin-like growth factor-I has multiple effects within the developing nervous system but its role in neurogenesis in the adult nervous system is less clear. The adult olfactory mucosa is a site of continuing neurogenesis that expresses insulin-like growth factor-I, its receptor, and its binding proteins. The action of insulin-like growth factor-I was assayed in several serum-free culture systems combined with bromodeoxyuridine labelling of proliferating cells and immunochemistry for specific cell types. Once proficiency in olfactory mucosa culture was gained, this model was applied to biopsied olfactory mucosa from schizophrenia and bipolar disorder patients in order to test the developmental parameters of adhesion, cell proliferation, and cell death in a neural tissue. It was previously shown that olfactory cultures from individuals with schizophrenia had increased cell proliferation and attached less frequently than cultures from healthy controls suggesting disrupted neurogenesis. An aim of this study was to replicate those observations in individuals with schizophrenia and and extend them to individuals with bipolar disorder. After completion of the cell and tissue culture assays, microarray analysis of these cell-based models was used to reveal gene expression differences present between patients and healthy controls. Microarray analysis is a complicated technique and the limited amounts of RNA that can be extracted from a single nasal biopsy further compounds this issue. In order to obtain enough material for microarray hybridization RNA samples underwent antisense amplification. Therefore, with the aim of allowing the candidate to gain proficiency in both these techniques prior to microarray analysis of olfactory biopsies from patients with schizophrenia and bipolar disorder, a pilot microarray study of cultured skin fibroblasts from schizophrenia patients and healthy controls was performed. The present findings show that insulin-like growth factor-I and its receptor were expressed by globose basal cells (the neuronal precursor), by neurons and by olfactory ensheathing cells, the special glia of the olfactory nerve. Insulin-like growth factor-I reduced the numbers of proliferating neuronal precursors, induced their differentiation into neurons, and promoted morphological differentiation of neurons. In contrast, this growth factor was mitogenic for olfactory ensheathing cells. The evidence suggests that insulin-like growth factor-I is an autocrine/paracrine signal that induces neuronal precursors to differentiate into olfactory sensory neurons and induces olfactory ensheathing cells to proliferate and that olfactory mucosa culture is valuable in modelling neurodevelopmental processes. When the olfactory musoca culture model was applied to patients with psychosis, a two-fold increase in proliferation of neural cells was found in schizophrenia compared to controls and bipolars. In bipolar cultures there was a 3-fold increase in cell death compared to controls and schizophrenia. Microarray analysis of cultured skin fibroblasts revealed differential expression of over 1000 genes between patients and controls. Inspection of the significant data showed alterations to gene expression between groups in the cell cycle, oxidative phosphorylation, TCA cycle and oxidative stress pathways. Gene expression in each of these pathways was predominately decreased in schizophrenia. Quantitative PCR analysis of selected differentially expressed genes involved with cell cycle regulation validated the increased expression of vitamin D receptor, and decreased expression of proliferating cell nuclear antigen and DEAD (Asp-GIu-Ala-Asp) box polypeptide 5 in skin fibroblasts from patients with schizophrenia. Microarray analysis of biopsied olfactory mucosa showed 146 and 139 differentially expressed genes in schizophrenia and bipolar disorder respectively, compared to controls. Consistent with increased mitosis in schizophrenia biopsy cultures three genes that function to positively influence cell cycle had increased expression. In the bipolar disorder group a dysregulation of the phosphatidylinositolsignalling pathway was seen; five genes that either directly function within or interact with this pathway had decreased expression. There is speculation that the therapeutic effect of psychotropic drugs acting upon this pathway in bipolar disorder involves reduction of neuronal cell death. Increased mitosis of neural cells has now been observed in two separate groups of schizophrenic patients indicating a robust finding. The use of fibroblast and olfactory mucosal tissue can be used to study biological and genetic aspects of neurodevelopment in living humans both with and without psychotic disease. Biopsied olfactory mucosa provides benefits over the use of autopsied material for study of psychotic disease because post-mortem duration and agonal factors that lead to tissue, protein and nucleic acid degradation are not an issue. This study provides evidence for a neurodevelopmental aetiology of schizophrenia and bipolar disorder acting at the level of cell cycle control. Subtle changes in the timing of cell cycle regulation could account for the brain pathologies observed in these diseases. Olfactory mucosa culture is a valuable model of neurodevelopmental processes.

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Thesis (PhD Doctorate)
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Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
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adult olfactory epithelium
insulan-like growth factor-1
gene expression
psychotic disease
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