Functional analysis of Arkadia2 a novel ubiquitin ligase expressed specifically in neurons

Neuronal connectivity and synaptic plasticity are believed to underlie higher cognitive functions, learning, dexterity, etc, and to be defective in diseases like autism schizophrenia, motor neuron and neuromuscular diseases. Genetic factors that control normal neuronal wiring of the brain and the muscles remain largely unknown. Genetically engineered mice are ideal for the identification and study of gene function in the context of the whole organism, and the understanding of the normal development, the architecture and function of complex organs and systems such as the brain and neurons. We previously discovered Arkadia, a novel factor essential for embryonic development in mice. We have now identified a homologue of Arkadia, here termed Arkadia2, which is present specifically in neurons of the brain and spinal cord. We generated mice deficient for Arkadia2 and found that they exhibit abnormal limb movement and posture. During the first 2 weeks after birth they become thinner and die with symptoms of progressive neuromuscular weakness with inability to feed and breath. These symptoms are similar to humans with motor neuron disease a debilitating and lethal disorder. Arkadia and most likely Arkadia2 are part of large networks of genes such as that of the TGFbeta/BMP signalling cascade. Interestingly, in the fruit fly, BMP signalling has been shown to regulate the growth of the neuromuscular synapse (which are the structures that a neuron connects with the muscle cells to communicate and control movement). It is therefore very likely that the phenotype of the Arkadia2 mutant mice is similar and due to defective muscle innervation and communication. However, the role of this gene network in mammalian neuromuscular synapse remains unknown. Here we propose to analyse the neuronal defects in these mice and study how Arkadia2 protein interacts with other proteins of the TGFbeta/BMP gene network. Our research will provide valuable insights to normal neuronal development and a system to understand the pathogenesis of diseases associated with muscle innervation and brain wiring. In addition, these studies will provide an animal model for the development of drugs and therapeutic approaches.

Molecular mechanisms that govern later stages of neuronal differentiation, such as connectivity and plasticity, remain largely unknown. Mouse models as they combine genetics and genetic manipulations, are ideal for understanding the complex wiring of the mammalian brain. Transforming Growth Factor beta (TGFbeta) signalling including the Bone Morphogenetic Protein (BMP) branch are associated with a variety cellular and developmental events in different tissues including patterning of neural precursors. Several studies in mammalian neuronal cultures suggest that the BMPs enhance axon and dendrite growth. Furthermore, MBP signalling has been shown to be upstream synaptic growth in Drosophila neuromuscular junction and CNS synapse. However, this role of the pathway has not been addressed in vivo in mammals. We have previously shown that Arkadia/Rnf111, a RING domain E3 ubiquitin ligase, enhances specifically the TGFbeta/Nodal-Activin branch during early vertebrate development. We now have identified additional proteins with homology to Arkadia's functional domains. The human Akd2 has been reported to map close to an inversion linked to psychiatric illness suggesting a role of Akd2 in later neuronal differentiation and function. In mice, we found that Akd2 is expressed specifically in the nervous system, and that its disruption causes a neurological phenotype after birth which is associated with motor and vital functions. The objectives of this proposal are (a) to examine the molecular mechanism of Akd2 function and its involvement in TGFbeta signalling regulation; and (b) to analyse its role in postmitotic neuronal differentiation and function.