Final Report Summary - NAS (Neuronal Alternative Splicing) The nervous system is one of the most complex tissues in animals harbouring myriad different cell types with specialized functions. However, the genetic programs that regulate the differentiation of these different cell types is mostly unknown. NAS (Neuronal Alternative Splicing) project’s major goal is to elucidate mechanisms by which an undifferentiated cell develops into a neuron. The project has investigated this problem at the level of gene expression, i.e. the mechanism by which information written in genes is used by a panel of cellular machineries to synthesize proteins. Gene expression can be regulated both at transcriptional and at post-transcriptional levels and within the NAS project we have investigated the process of neuronal gene expression at both of these levels.1. Neuronal Regulation of Gene Expression by Alternative Splicing: Alternative Splicing Process refers to the ability of the cell to synthesize different proteins from a single gene by the alternative usage of exons within the final mature mRNA. It is considered to be one of the major mechanisms by which proteome and functional diversity is achieved in evolutionarily higher organisms such as mammals. Many aspects of alternative splicing regulation are dependent on RNA binding proteins (RNABPs). We have investigated the role of a family of RNABPs, called nELAV-like (Neuronal Embryonic Lethal Abnormal Vision-Like) in the development of the mouse nervous system. A study by Gulayse Ince Dunn, Koc University and Robert Darnell, Rockefeller University, have identified genome-wide RNA targets of nELAV-like within the developing cerebral cortex and have demonstrated their roles in proper development of neuronal communication (Ince-Dunn G et al, 2012). Within the NAS project we focused on a major RNA target of nELAV-like RNABPs, called Kif2a mRNA and how it is required for neuronal differentiation and function. Kif2a gene encodes a motor protein that belongs to the Kinesin family. Kif2a is localized to the axons and has essential roles in axonal pruning (Homma N et al, 2003). We have focused on investigating the mechanism by which two different mutations in Kif2a gene which cause severe cortical development problems, mental retardation and epilepsy in humans (Poirier K et al, 2013). We have recapitulated these two different mutations, both of which localize to the motor domain, in mouse cortical neurons and investigated their effects on the neuronal cytoskeleton, axonal development and organelle trafficking. Our results suggest that motor domain mutations in Kif2a gene, result in the stabilization of the microtubule cytoskeletal network of the neuron. Further, mutant Kif2a also causes defects in axonal morphogenesis and trafficking of the mitochondrial organelle. Our results elucidate how a major target of the nELAV-like RNABPs, Kif2a, regulates neuronal differentiation and when mutated causes major defects in neuronal development and function. Our publication that will arise from this part of the project is currently in preparation.2. Neuronal Regulation of Gene Expression by Transcription: One of the most important families of gene regulatory proteins with essential roles both during neuronal differentiation and the adult functioning of the nervous system are the neurogenic basic helix loop helix transcription factors (Polleux F. et al, 2007; Ince-Dunn G. et al, 2006). We have focused on understanding the role of NeuroD2, which is a member of the neurogenic bHLH family of transcription factors, in the development of the cerebral cortex. Towards this aim, we took an unbiased genomics approach in which we identified all direct targets of NeuroD2 in the genome during mid-embryonic development of the cerebral cortex. We used a technology called chromatin immunoprecipitation and sequencing, which is based on the biochemical purification of transcription factor-DNA complexes followed by high-throughput sequencing of associated DNA fragments. Our results demonstrated that NeuroD2 transcription factor is associated with genes that regulate two essential processes during cortical development: 1. The guidance of axons of cortical projection neurons to the locations where their final synaptic partners reside and, 2. The migration of neuronal precursors from the germinal zones that they are born in, to their final layer of residence in the mature cortex. Moreover, our data suggest that NeuroD2 is controlling these developmental processes by activating members of critical cellular signalling pathways, such as the Reelin pathway, Semaphorin/Plexin and Eph/ephrin receptor axonal guidance pathways. Defects in the functioning of cortical projection neurons have been associated with numerous neurological diseases, such as autism and schizophrenia. Our study contributes significantly to our understanding of the developmental gene expression programs used by this class of neurons. We have collected our results in a manuscript which is in the stage of submission currently. ReferencesG. Ince-Dunn*, B.J. Hall*, S.C Hu, B. Ripley, R.L. Huganir, J.M. Olson, S.J. Tapscott, and A. Ghosh. (2006) Regulation of thalamocortical patterning and synaptic maturation by NeuroD2. Neuron. 49(5): 683-95. (*equal contribution). G. Ince-Dunn, H. J. Okano, K. Jensen, W.Y. Park, Z. Ru, J. Ule, A. Mele, J. Fak, C.W. Yang, C. Zhang, H. Okano, J. Yoo, J. Noebels, R.B. Darnell. (2012) Neuronal Elav-like (Hu) proteins regulate RNA splicing and abundance to control glutamate levels and neuronal excitability. Neuron. 75(6): 1067-1081.N. Homma, Y. Takei, Y. Tanaka, T. Nakata, S. Terada, M. Kikkawa, Y. Noda, N. Hirokawa. (2003) Kinesin Superfamily Protein 2A (KIF2A) Functionsin Suppression of Collateral Branch Extension. Cell. 114:229-239.P. Polleux, G. Ince-Dunn, A. Ghosh. (2007) Transcriptional regulation of axon guidance and synapse formation. Nature Neuroscience Reviews. 8(5): 331-40.Poirier K et al. (2013) Mutations in TUBG1, DYNC1H1, KIF5C and KIF2A cause malformations of cortical development and microcephaly. Nature Genetics. 45(6).