Analysis of the cellular basis for developmental disorders arising from mutations in RAC1

Rac1 is a highly conserved small GTPase whose best known function is as a regulator of the actin cytoskeleton. Rac1 has numerous roles in embryonic development, in particular the development of the nervous system. Despite this, mutations in the RAC1 gene have not previously been associated with any human developmental disorder. Recently, five different missense mutations in RAC1 gene were identified in five individuals with varying degrees of developmental delay and intellectual disability. It is currently unclear how these mutations might cause the various phenotypes observed in the individuals. In this project we aimed to explore how these five mutations impact on the cellular functions of Rac1 and its roles in development. We initially carried out in vitro experiments to investigate how these mutations affect Rac1 function during fibroblast spreading. We found that these mutations had differing effects in these assays, with two mutations; C18Y and N39S, appeared to suppress lamellipodia formation, thus phenocopying a dominant negative mutation, while another mutation, Y64D, enhanced lamellipodia formation, thus phenocopied a constitutively active mutation. We used Drosophila to investigate the effects of the mutations on development by specifically expressing the mutations in neurons. We found that one of the mutations, Y64D, resulted in delayed pupation and a severely impaired larval motility. More detailed analysis revealed that this mutation causes changes in the morphology of neuromuscular junctions, disorganisation of the embryonic central nervous system and altered axonal morphology. Expression of Rac1-Y64D also induced ectopic filopodia during dorsal closure when expressed in the epidermis, suggesting that mislocalisation of Rac1 activity may underlie the defects induced by the mutation. Collectively, our findings provide insights into the cellular and developmental effects of a group of missense mutations in RAC1 that are associated with human developmental disorders. These findings bring us closer to understanding the mechanisms underlying these conditions towards developing therapies or diagnostics. In a separate project, we also investigated the effects of a group of naturally-occurring estrogen analogues on fibroblast motility in vitro and cutaneous wound healing in ovariectomised mice. We found that one of these compounds, chrysin, enhances fibroblast motility and accelerates mouse wound healing, suggesting it could potentially be used as a therapy for impaired wound healing in the aged.