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

Lead Research Organisation: Imperial College London
Department Name: Dept of Medicine


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.

Technical Summary

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.
Description Motor neurons control movement via long axons that extend from the spinal cord to muscles as far as in distant limbs. Little is known about factors that regulate this extensive axonal growth in the periphery. Here we report that the ubiquitin ligase Ark2C (Arkadia2) is expressed in neurons and can serve to amplify neuronal responses to specific signals. We find that these signals belong to the Bone Morphogenetic Protein (BMP) family of secreted factors, which are highly expressed in the periphery and known to regulate the development of the limbs. Loss of Ark2C gene function in mice results in inefficient growth of motor axons to distant muscles, and we show that this process is regulated by BMP signaling. Ark2C targets BMP inhibitors for destruction, and therefore the presence of Ark2C helps to enhance BMP signaling, which in turn is necessary for the innervation of distal muscles. Our experiments reveal a previously unknown function of BMP in motor axon growth and describe a molecular mechanism for how axons and limbs coordinate their growth.
Exploitation Route these findings would be used for further research into understanding further the molecular mechanisms that govern motor neuron connectivity.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

Description findings have been used 1) for publications which advanced further the research into understanding molecular factors that govern motor neuron connectivity 2) for obtaining an new grant MR/J013331/1 to continue this research 3) for initiating a collaboration with the prominent Prof. Thomas Jessell at Columbia University, New York, USA, who is an expert in motor neuron connectivity.
Sector Education,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

Title TGF-ß/Smad2/3 Signaling Directly Regulates Several miRNAs in Mouse ES Cells and Early Embryos 
Description The Transforming Growth Factor-ß (TGF-ß) signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs) have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR) from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-ß signaling in mouse embryonic stem (ES) cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-ß activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341~3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-ß inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341~3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-ß/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-ß/Smad2/3 pathway underlie tumor suppression and metastasis, this research also provides a resource for miRNAs involved in cancer. 
Type Of Material Database/Collection of data 
Year Produced 2013 
Provided To Others? Yes  
Impact Sequencing data has been submitted to the Gene Expression Omnibus (GEO) (; accession ID: GSE39994). publication can be found in the following URL: 4,566 views so far 4 citations so far 6 citations including self 
Description TGFbeta induced miRNA 
Organisation University of Oxford
Department Wellcome Trust Centre for Human Genetics
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We contribute the experimental materials and insights
Collaborator Contribution The collaborators perform deep sequencing service and do the bioinformatic analysis.
Impact publication Redshaw N, Camps C, Sharma V, Motallebipour M, Guzman-Ayala M, Oikonomopoulos S, Thymiakou E, Ragoussis J, Episkopou Vet al., 2013, TGF-beta/Smad2/3 Signaling Directly Regulates Several miRNAs in Mouse ES Cells and Early Embryos, PLOS ONE, Vol: 8, ISSN: 1932-6203
Start Year 2007
Description spinal cord development 
Organisation Medical Research Council (MRC)
Department MRC National Institute for Medical Research (NIMR)
Country United Kingdom 
Sector Academic/University 
PI Contribution Designing of the experiments generation of genetically modified mouse strain Sox1-Cre ERT-2 and writing and authoring a publication
Collaborator Contribution Designing experiments performing experiments and generating data analysis of data and writing and authoring a publication
Impact a high profile and impact publication Coordination of progenitor specification and growth in mouse and chick spinal cord. Kicheva A, Bollenbach T, Ribeiro A, Valle HP, Lovell-Badge R, Episkopou V, Briscoe J. Science. 2014 Sep 26;345(6204):1254927. doi: 10.1126/science.1254927. PMID: 25258086 [PubMed - indexed for MEDLINE]
Start Year 2006