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.