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Title: Activity dependent neuroprotective protein : initial characterisation of its role in physiology
Author: Gennet, Nicole
ISNI:       0000 0001 3493 7377
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2005
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ADNP is a novel vasoactive intestinal peptide (VIP)-regulated gene which has been shown to be essential for development. ADNP has also been hypothesised to mediate some of VIP's neuroprotective actions. The ADNP protein contains zinc fingers, a homeobox domain and a bipartite nuclear localisation signal, suggesting that it may act, in part, as a transcription factor. This thesis describes the subcellular localisation of ADNP in different cell lines and in rat hippocampal dissociated cultures. Furthermore, the response of ADNP staining and localisation in response to stimuli was investigated. According to the hypothesis of ADNP being a neuroprotective, stress regulated gene, we expected a change in ADNP expression in response to stimuli. Indeed, noxious stimuli like oxidative stress-inducing hydrogen peroxide (H2O2) are shown to lead to a reduction or loss of ADNP immunoreactivity. ADNP expression and localisation have also been investigated in an in vivo model of experimentally induced epilepsy. Preliminary data suggests that ADNP expression is lost in necrotic and compromised 'dark' neurons, while the surviving cells maintain ADNP expression. The intensity of cytoplasmic ADNP staining changes in response to the stimulus; the percentage of cells demonstrating nuclear localisation of ADNP increases significantly. The observed changes in ADNP localisation and expression in response to stimuli is consistent with our hypothesis that ADNP is a stress-responsive, protective protein. Additionally, resources were generated which will be useful for the future investigation of the function and mechanism of action of ADNP. An ADNP expression construct was generated, and expression from this construct was characterised. The obtained data suggest that ADNP overexpression may be detrimental for the individual ADNP overexpressing cell, possibly by affecting mitosis. However, for the non-transfected cells in the population, ADNP overexpression may be beneficial. Further, the possibility exists that different cell lines respond in different ways to ADNP overexpression. Effective siRNAs directed against ADNP have been identified, and siRNA transfection protocols optimised. Initial data from the use of the siRNAs suggest that lack of ADNP may have dramatic effects on cell morphology. A microarray analysis of ADNPknockdown cells is currently under way, and is hoped to give insight into potential functions and pathways in which ADNP may be involved. It is hoped this approach will lead to the identification of potential ADNP-regulated target genes. A P1-based artificial chromosome (PAC) containing the ADNP gene was modified with the aim of generating a transgenic mouse model that would coexpress GFP. The obtained intermediate construct may be used in cell lines or in vivo to analyse the expression of human ADNP in cell lines. In summary, this thesis presents data confirming that ADNP expression is regulated by noxious stimuli, which is consistent with ADNP acting as a stress-regulated protective molecule. Resources have been generated which will facilitate the investigation of the function of ADNP in the future.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral