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Investigation of the expression pattern and functional importance of the Gnasxl-encoded XLαs protein of the imprinted Gnas Locus

Burton, Katie (2012) Investigation of the expression pattern and functional importance of the Gnasxl-encoded XLαs protein of the imprinted Gnas Locus. Doctoral thesis, University of Liverpool.

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Abstract

XLαs is a NH2-terminal splice variant of the stimulatory G-protein α-subunit Gsα. Both are encoded by the imprinted Gnas locus. Similar to Gsα, XLαs can couple 7-TM receptors to adenylate cyclase in cultured cells. Previously, a knock-out mouse specific for the Gnasxl transcript was generated (Gnasxlm+/p-) (PLAGGE et al. 2004). This mouse model exhibits a lean, hypermetabolic phenotype with elevated sympathetic nervous system (SNS) activity (XIE et al. 2006). Changes in phenotype between neonatal and adult stages (e.g. mortality; failure to thrive as neonates vs. healthy but hypermetabolic adults) are not yet fully understood. More recently a conditional gene trap knock-out mouse line (XLlacZGT) was established. Immunohistochemistry, XGal and immunofluorescence data were collected to document the changes in expression pattern of XLαs and determine possible signalling pathways affected by lack of XLαs in the brain; histological analysis of the lacZ-containing genetrap line revealed new sites of XLαs expression. A comparison of neonatal and adult brain expression patterns revealed that the lateral hypothalamus (LH), dorsomedial hypothalamus (DMH), arcuate nucleus (Arc), locus coeruleus and ventrolateral medulla express XLαs in both stages; the laterodorsal tegmental nucleus, hypoglossal and facial nucleus (motor nuclei important for feeding) express XLαs only in neonates; and expression in the amygdala and preoptic area are only found in adult brain. Colocalisation studies in the brain for XLαs, neuropeptides and other markers related to regulation of food intake and energy expenditure. Orexin partly colocalised with XLαs in the LH and DMH (22% orexin neurons XLαs positive); tyrosine hydroxylase/dopaminergic neurons colocalised with XLαs in the Arc (60% TH neurons XLαs positive); and phosphorylated S6, a component of the leptin signalling pathway, colocalised with XLαs in the Arc (30% XLαs neurons pS6 positive). MCH and CRF did not colocalise with XLαs. Changes in pS6/S6K1 and the indicators of ghrelin signalling in knock-out Arc neurons did not reach statistical significance. Analysis of the lacZ-genetrap line at neonatal stages revealed that XLαs is also expressed in spinal cord and peripheral tissues e.g. skeletal muscle, tongue muscle and blood vessel smooth muscle cells. Expression in muscle tissues, including blood vessel smooth muscle cells is silenced in adults, but the spinal cord remains positive for XLαs. XLαs expression pattern changes in the brain and peripheral tissues concur with changes in phenotype seen between neonatal and adult mice. pS6 is a good indicator of S6K1 activity, which influences leptin and insulin signalling. A decrease of S6K1 activity in Gnasxlm+/p- mice might explain their leptin sensitivity (Frontera et al. in prep). XLαs colocalises with orexigenic peptides – including orexin and NPY/AgRP neurons (Frontera et al. in prep) – in the hypothalamus; however, the function of XLαs in energy expenditure and SNS activity remains elusive.

Item Type:Thesis (Doctoral)
Departments, Research Centres and Related Units:Academic Faculties, Institutes and Research Centres > Faculty of Medicine > School of Biomedical Sciences
Status:Unpublished
ID Code:6213
Deposited On:03 Aug 2012 10:41
Last Modified:03 Aug 2012 10:41

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