Breaking Cellular Symmetry: Intracellular Pathways Underlying Neuritogenesis

The proper functioning of the nervous system depends on nerve cells forming appropriate connections with each other and with other cell types, such as muscle, during embryonic development. Nerve cells form connections in the embryo by extending fine processes called neurites that grow through the embryo seeking an appropriate cell to connect with. At the tip of a growing neurite is a motile structure called a growth cone that masterminds the search in the embryo for a connecting cell by leading the neurite along an appropriate route. The growth cone is guided by signals coming from other cells in the embryo and responds to these signals by changes in growth, turning left or right, up or down in the embryo. Growth cones are first formed on the surface of a new-born nerve cell in a process called neuritogenesis which involves localised changes in the nerve cell's cytoskeleton. The timing and location of growth cone formation on the surface of the new-born nerve cell is crucial in order that neurites grow out in the correct direction and at the appropriate time. To form a growth cone on their surface, new-born nerve cells have to break their radial symmetry and locally re-organise their cytoskeleton where the growth cone will form. The research proposed will improve our understanding of how the cytoskeleton changes during neuritogenesis to form a growth cone. In particular, we will learn how a protein called drebrin enables an interaction between two important classes of cytoskeletal filaments, called microtubules and microfilaments, that are essential for neuritogenesis. This research will not only help us to understand a fundamental process in nervous system development but it will also contribute to the effort to repair damaged axons in the adult nervous system that have lost the ability to form a growth cone.

Growth cone formation during neuritogenesis depends on the co-operation of microtubules and F-actin. The microtubule +TIP protein EB3, located on the growing ends of dynamic microtubules, binds to the actin-binding protein drebrin in growth cone filopodia. Knockdown of drebrin and dominant negative constructs of EB3 inhibit neuritogenesis. Conversely, over-expression of drebrin leads to supernumerary neurites. Drebrin has two domains that independently bind to F-actin and act co-operatively to bundle F-actin. This activity is repressed by an intramolecular interaction that is relieved by Cdk5 phosphorylation of drebrin that might target it to F-actin in filopodia. Consistent with this idea, pS142-drebrin accumulates, along with dynamic microtubules, at the base of filopodia where growth cones are forming and phospho-mimetic and phospho-dead mutants of S142 enhance and inhibit neuritogenesis respectively. An unanswered question is how these cytoskeletal elements, including the drebrin/EB3/Cdk5 pathway, are coordinated to drive neuritogenesis.
We will identify activating signalling pathways up-stream of the drebrin/EB3/Cdk5 pathway underlying neuritogenesis. We will test the involvement of two potential up-stream pathways, both driven by BDNF-elicited neuritogenesis: a Ca2+/CaMKIIbeta/cofilin pathway that competes with drebrin for F-actin binding and a PKCdelta/p35 pathway that activates Cdk5. We will correlate Ca2+ transients and active cofilin with neuritogenesis and test whether blocking components of these pathways inhibits neuritogenesis. We will characterise the involvement in neuritogenesis of drebrin interactors identified using proteomic screens that differentiate between interactors binding to pS142 drebrin and unphosphorylated drebrin. We will establish the spatiotemporal sequence of cytoskeleton reorganisation in relation to the drebrin/EB3/Cdk5 pathway underlying neuritogenesis using live cell imaging of fluorescent proteins.

Neuritogenesis is a critical stage in neuronal development because unless growth cones emerge at the appropriate time and place they will not be in a position to respond to guidance cues that orchestrate a correctly connected nervous system. Furthermore, understanding neuritogenesis will shed light on how to enhance growth cone formation by injured axons in the adult central nervous system and thereby improve regeneration. The molecular mechanisms and signalling pathways underlying neuritogenesis are not well understood.
The postdoc and technician working on the project will both develop and extend their research skills and learn various cellular and molecular in vitro and in vivo assays. They will develop their communication and networking skills by interacting with our collaborator Dr Maddy Parsons and will be expected to present their work at the MRC Centre's internal seminars once a year. There are opportunities to supervise Ph.D students in the group, including the four rotating, 4-year Ph.D students funded by the Centre and undergraduate and M.Sc students doing laboratory based projects. Prof. Gordon-Weeks has supervised 12 Ph.D students many of whom have gone on to successful careers in academic and industrial science and was awarded the "Ph.D Supervisory Excellence Award" of the School of Biomedical and Health Sciences, King's College London in 2009. There are ample opportunities to teach undergraduates and postgraduates in tutorials and practical classes including contributing to the EMBO practical course: Developmental Neurobiology: From Worms to Mammals which has now been organised by the MRC Centre on three occasions. Each year there are formal appraisals at which the personal and professional development of the person is discussed and means of support from the MRC Centre, the School (IoPPN) and the College are identified. A range of powerful tools and reagents of benefit to the scientific community will be derived and developed by the work including dominant negative constructs, KillerRed and LOV domain constructs. Some of these might have commercial value. We have a long-standing relationship with SeroTec, Novus Biologicals and Millipore who currently market our antibodies. We have recently published work that includes characterization and use of our monoclonal and polyclonal pS142 drebrin antibodies (Worth et al., 2013) and Millipore has already approached us with a view to marketing these antibodies. We have deposited expression plasmids, including those for drebrin, in the AddGene repository and will continue to do so. Reagents such as these are added to our curated DNA and antibody banks in -80oC freezers whose temperature is centrally monitored.