Effects of hypothyroidism on cell signalling in the developing rat brain.

Rat pups bom of dams maintained on a propylthiouracil/low iodine diet regime were used to assess the effects of hypothyroidism on signalling components in the developing brain. The success o f the animal model of perinatal hypothyroidism was confirmed following measurements of free T 3 and T 4 plasma levels. Alterations in development and behaviour noted in hypothyroid pups were similar to those observed previously by other groups. Plasma metabolite concentrations indicated that the impairments in body and brain development noted in the hypothyroid state, were not the result of serious under-nutrition. Crude and synaptosomal membranes were isolated from forebrain and hindbrain regions o f euthyroid and hypothyroid animals at various ages. Changes in a-subunit abundance of Gq, Gq, Gjl and Gj2 were measured via quantitative immunoblotting. No differences in (%-subunit levels of euthyroid and hypothyroid cmde membranes were seen in either brain region. Compared to euthyroid animals, between 10 and 20 days postpartum a number of significant alterations were detected in synaptosomal membranes isolated from forebrains of hypothyroid pups; with the exception of G{la, all o f these changes were that of subunit up-regulation. By contrast, the hindbrain showed very few hypothyroid-induced changes; the only significant effects were seen in G jla at day 1 0 (total hindbrain) and day 25 (medulla oblongata). Hypothyroidism also altered the normal developmental profiles of all four o:-subunits in both forebrain and hindbrain regions. Synaptosomal membranes of 15 day old pups were also used to assess hypothyroidinduced effects on adenylyl cyclase activity. Changes in the pattern of activation/inhibition of adenylyl cyclase by GTP were observed in hypothyroid forebrain and hindbrain membranes in the presence of forskolin. Furthermore, a significant decrease in specific cyclase activity was observed in hypothyroid forebrain membranes. By contrast, in the hindbrain, a significant increase in cyclase activity was evident. Together, the changes observed may contribute to some of the neurological defects seen during perinatal hypothyroidism. ACKNOWLEDGEMENTS I would like to thank my two supervisors Professor E. Dave Saggerson and Professor John B. Clark for giving me the opportunity of doing this Ph.D. and for their advice and support throughout the duration of the project. My thanks also go to the people, past and present, in Lab 302 (UCL), the Dept of Biochemistry (UCL) and in the Dept, o f Neurochemistry (ION), whom I have had the good fortune to know and befriend. I am grateful for all their inputs both stimulatory and inhibitory, i.e. advice, humour, alcohol etc. I apologise for not naming you all in person, but you know who you are. I am particularly grateful to June Smalley for her help with the more elaborate diagrams in this thesis and to the members o f the Denny Brown Laboratory for their assistance. I am indebted to the Wellcome Trust Foundation for their generous funding o f this project and for the workshops and meetings which were extremely helpful over the three years. Finally I would like to extend my gratitude to my family and friends for their continual moral support and numerous attempts to understand what it was exactly I was doing for my Ph.D. This thesis is dedicated to my Mum, Dad and brother Gabriel. To the one person without whom all this would not be possible, who persevered (eventually) through good times and bad, toiled, sweated, cursed and agonized over numerous hours. It was worth it. Scientists.peeping-toms at the keyhole o f Mother Nature. CONTENTS Page Title P a g e . 1 Abstract . 2 Acknowledgem ents.4 List o f Figures and T a b le s . 10 Abbreviations. 13 CHAPTER