A complete explanation of many genetic diseases requires that the underlying molecular changes be related to specific changes in cellular events. One example of where this relationship is still not fully understood is how changes at a molecular level ultimately lead to the cellular changes that underlie developmental brain abnormalities such as non-specific X-linked mental retardation (MRX). More than a dozen loci have been implicated in MRX and of the eight genes that have so far been identified, three encode regulators or effectors of the Rho family of small GTPases, so-called 'molecular switches' that regulate signalling pathways in diverse biological processes. These observations are consistent with a growing literature showing that the regulation of Rho GTPase signal transduction pathways are critical for normal neuronal development. For instance, work from a number of laboratories, including the Cline and Van Aelst laboratories at Cold Spring Harbor, has shown that perturbations of the Rho GTPase signal transduction pathways results in abnormal dendrite development. Taken together these lines of evidence support a more general model in which molecular changes that perturb the activity of Rho GTPases could defects in developing neuronal processes that in turn could underlie MRX. The aim of this project is to determine the function of oligophrenin-1, a putative Rho GTPase activating protein (RhoGAP) that is absent in unrelated MRX families. To achieve this aim, I will employ a number of complementary molecular and cellular experimental approaches that are established in the Van Aeslt laboratory to address the following central questions: (i) Where is oligophrenin-1 expressed during the development of mammalian central nervous system? (ii) Which Rho GTPase does oligophrenin-1 act upon in neuronal cells? (iii) What are the effects of the ectopic expression or absence of oligophrenin-1 on cellular morphology of developing neurons? (iv) What proteins does oligophrenin-1 interact with in neurons? More specifically: (i) Polyclonal antibodies will be raised against oligophrenin-1 to determine its expression pattern in the rat developing nervous system. (ii) Biochemical binding assays using the neuronal cell line PC12 will be performed to determine the Rho GTPase target(s) of oligophrenin-1 in neurons. By determining which Rho GTPase oligophrenin-1 acts upon, predictions can be made as to which downstream pathways may be perturbed by loss of this protein in MRX. (iii) Biolistic transfection of organotypic rat hippocampal slices with oligophrenin-1 sense and antisense expression constructs will address the effects of over-expressing and down-regulating oligophrenin-1 levels in hippocampal pyramidal neurons. The detailed dendritic structure of transfected neurons will be imaged using a combination of scanning laser confocal microscopy and two-photon microscopy. These studies will allow the formal testing of the hypothesis that aberrant oliophrenin-1 expression perturbs normal dendrite formation. (iv) The yeast two-hybrid system will be employed to identify oligophrenin-1 interacting proteins in the developing hippocampus. This study will provide further insights into the signalling pathways in which oligophrenin-1 participates as well as identifying additional candidate genes that may be mutated in MRX. Using the combination of molecular and cellular approaches outlined above, I hope to be able to relate specific molecular signalling events to the cellular mechanism underlying MRX.

The molecule nitric oxide (NO) is a biological messanger which has been identified as a regulator in the cardiovascular, gastrointestinal, nervous and immune systems. NO produced by one of the three nitric oxide synthase (NOS) isoforms can act directly (ie. S-nitrosation of proteins) or via binding to soluble guanylate cyclase (sGC). Upon activation by NO, sGC converts guanosine-5'-triphosphate (GTP) to the second messenger, guanosine-3',5'-monophosphate (cGMP). There are a number of disease states which may be caused by aberrant regulation of NO production and subsequent signalling, particularly in the cardiovascular system (eg. atherosclerosis, septic shock, impotence and stroke). Therefore an understanding of how this system is regulated under physiological and pathophysiological situations is important for the design of future therapies. The existence of an autoregulatory mechanism, by which the endogenous ligand, NO, can govern its own production has been established. I have shown previously that endogenous NO can feedback to both enhance and inhibit the activation profile of macrophages, and inducible NOS (iNOS) expression, via effects on the transcription factor nuclear factor-kappa B (NF-?B). It also appears that the expression and activity of components of the NO-sGC-cGMP signalling system can by up- and down-regulated allowing adaptations to changes in NO concentration. In this study I intend to characterise the molecular and biochemical changes responsible for the autoregulation of iNOS by NO. I also intend to determine the role of sGC-cGMP signalling in iNOS expression and consequently whether the expression and activity of components of this signalling system can be up- and down-regulated by NO. Initially I will investigate whether NO can feedback to induce changes in activity and expression of signalling proteins leading to iNOS expression such as p21ras, mitogen activated protein kinase and I?B Kinase. The effect of NO-donors and NOS inhibitors on activity and expression of these proteins in murine macrophages will be studied by RT-PCR and western blot. The same techniques will be used to determine whether NO can regulate the expression of GTP Cyclohydrolase I, the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, an important co-factor with respect to iNOS dimerisation and activity. The importance of the NO-sGC-cGMP signalling cascade will be studied by looking at the effect of inhibitors and activators of sGC and downstream components on iNOS expression and NF-?B activity. I will also investigate whether the expression and activity of components of the sGC signalling cascade (sGC, cGMP-dependent protein kinase, Phosphodiesterase V) can in turn be regulated by endogenous NO. Once a molecular model has been elucidated the relevance to pathophysiological and physiological situations will be confirmed by studying expression and activity of NOS and associated signalling proteins in tissue from endotoxin-treated, iNOS and eNOS knockout mice. This project will be important in revealing the endogenous regulatory mechanisms which control pathophysiological NO production. It will also, via the study of the effect of NO on sGC signalling, provide information relevant to physiological processes regulated by NO. Ultimately these results will provide an insight into the effects observed in diseases caused by under- and over-production of NO such as atherosclerosis, impotence, septic shock and stroke.

Branching morphogenesis is a key developmental process which requires exquisite regulation in order to successfully generate a variety of important tissues such as the lung, kidney, mammary gland and circulatory system. How this process is promoted and regulated to such an incredible degree is only beginning to be understood. One important player in this complex process is the fibroblast growth factor (FGF) receptor signalling pathway which has been shown conclusively to regulate branching morphogenesis in both the Drosophila tracheal system, and the mammalian lung. Evidence is emerging that this pathway is a conserved method of regulating branching, and that is may also function in other tissues. Murine mammary epithelial cells are also stimulated to undergo branching morphogenesis in vitro by FGF family members, and also by matrix metalloproteinases (MMPs). The aim of my project is to identify the role of FGFs, downstream signalling molecules and MMPs in regulating branching morphogenesis of the murine mammary gland, and to determine the relationship between FGF and MMP mediated branching. Firstly, the expression pattern of genes involved in the FGF pathway will be determined by RT-PCR and RNA in situ hybridisation in murine mammary tissue, initially concentrating of Fgf-10, Sprouty and Dof. The next step is to determine the function of these genes, and their role in branching morphogenesis using a variety of in vitro and in vivo assays which will also allow the relationship between genes and between signalling pathways to be studied. The effect of MMPs in isolation and in combination with those of FGFs can be analysed using recombinant proteins, transgenically modified cells and transgenic animals, to discover the relationship between these two inducers of epithelial branching. Finally, by grafting various combinations of wild-type, mutant and transgenic epithelium and/or stroma, I can identify the specific roles but the epithelium and the stroma have in regulating branching.

Completion of the human genome project is expected to reveal that approximately 25% of identified gene sequences will code for membrane proteins containing transbilayer helical domains. This project aims to link the subjects of membrane proteins (including structure, folding and oligomerisation events) with the vast field of lipid properties and dynamics. The work is based on the premise that helical transmembrane proteins fold in two stages, the first stage being helix formation/insertion and the second helix association within the membrane. The second of these stages will be the focus of this study. The interaction of helices within the membrane must involve changes in lipid-lipid interaction and lipid-helix interactions as well as helix-helix interactions. The proposed work will focus on the influences of the lipid environment on folding and oligomerisation events of a-helical membrane proteins. Helix-helix association constants in lipid bilayers will be measured with the aim of observing changes in the association constant resulting from changes in the lipid environment. The transmembrane domain of human glycophorin A dimerises in both detergent and lipid bilayer environments. It is this process that will be studied in lipid vesicle systems. Various lipid properties, such as bilayer thickness, lipid lateral pressure and bilayer stiffness can be controlled by changes in the composition of the host membrane system. The host membranes will be composed of biologically relevant phospholipids along with cholesterol, which is also prevalent in biological membranes. The physical properties of the host membrane will be determined by separate measurements. Helix association will be measured using Forster Resonance Energy Transfer with a donor and acceptor complex on the N-terminus of the synthetic glycophorin A peptide. Helix association will be measured as a function of specific physical membrane properties. A second technique that has recently been developed in the Engelman lab will also be used. This is a new genetic assay for helix association, which allows studies in the inner membrane of living E.coli. The function of many transmembrane proteins have been shown to be modulated by lipid composition. These studies will provide important information in this area along with other fields of study such as transmembrane signalling events (which often involve changes in protein association) and selective transport in membrane trafficking (where some models postulate bilayer thickness effects to segregate retained and transported proteins).