Interpretation of guidance cues by the actin-binding protein Drebrin to direct collective neuronal migration

Forming a human brain with its billions of nerve cells and trillions of connections is a fantastically complex process with little margin for error. During brain growth in the embryo and early childhood, many nerve cells have to migrate from their site of birth to their correct final location within the circuitry. To do this they use proteins on their surface to respond to signals in the tissue around them. These cues are then translated by other proteins inside the cells to move and steer them to the appropriate place. If this goes wrong, then nerve cells do not make the right connections and the brain cannot function: it cannot form memories, process sensory inputs or produce motor outputs. Every aspect of brain function from sophisticated cognitive behaviour to basic control of the body can be compromised. The connections between nerve cells are continually renewed and replaced throughout life. The ability to form new links or reinforce existing ones is widely believed to underlie object recognition and memory formation. However, as we grow older the connections become less dynamic, they tend to break down more often and be repaired less efficiently, resulting in the memory loss and dementia associated with ageing. One of the reasons for this is thought to be that the proteins driving brain formation early in life become less abundant in the adult hence its ability to maintain and repair itself becomes increasingly compromised. Therefore, understanding the proteins that are responsible for wiring up the brain is likely to shed light on the other end of the life cycle and reveal ways to reactivate the repair processes. Our research focusses on such a protein that could play an important role shaping brains and then keeping them functioning throughout life. This protein, called Drebrin, is essential for the migratory ability of certain nerve cells during development and then maintaining the contacts between them but how it does so remains unknown. There is a clear need to understand more about this protein and the role it plays in co-ordinating brain formation and how and why it deteriorates with age. We will use genetic tools to change the amount of Drebrin produced by nerve cells and analyse the effect this has on their shape and movement during brain development. Drebrin and the proteins with which it interacts will be labelled with different coloured fluorescent tags and introduced into growing nerve cells in tissue culture dishes. The tags will act like molecular light bulbs that can be digitally filmed using a special microscope illuminated by lasers. The resultant movies will enable us to visualise the relative motions of the tagged proteins inside living cells and watch their behaviour in normal cells. These will then be compared to movies of cells in which Drebrin and/or its partner proteins have been removed or mutated so that the effects on nerve cell shape and movement can be quantified. We will then genetically remove these proteins from nerve cells in embryonic chick brains to see how they influence actual brain development. This research is of vital importance because it will provide novel and exciting information about how nerve cells find their way around the developing nervous system. We will analyse whether they perform this feat individually or if they navigate as a collective mass following a set of pioneer cells. We will investigate how the cells manage to translate a myriad of external cues into the correct changes in their shape and movement. Better knowledge of the key proteins involved in development will greatly enhance our ability to apply this to understanding the ageing brain and how and why it loses its function. People are living longer; the population as a whole is ageing so this research is both necessary and timely in order to improve quality of life and tackle the social and economic impacts of this demographic shift.

Neuronal migration is a fundamental constituent of normal brain development, yet remains poorly understood. Its failure underlies many congenital disorders so there is a clear need for investigation into how it is controlled. It is a complex process requiring cells to respond to environmental guidance cues and translate them into motile changes. Our focus will be tangential neuronal migration, in which neurons extend a leading process to their target which the cell body follows. This is a key feature of the developing oculomotor system, the model used here. Our extensive preliminary data point to a central role for the actin-binding protein, Drebrin, in forming leading processes, coordinating the motile response to extracellular signals and ensuring the collective migration of neuronal pools. Drebrin is also important beyond development; its loss is linked to degenerative conditions of old age, particularly dementia. A better understanding of its role is thus critical for improving quality of life from birth right through to old age. We will examine the function of Drebrin and its interaction with both axon guidance receptors and intracellular effector proteins. In ovo electroporation will be used to examine the role of Drebrin - and the effect of specific mutations - in neuronal migration. This technique will be enhanced by using genetic tools to control the timing and level of Drebrin expression and enable us to dissect its involvement at discrete stages e.g. initiation of migration compared to choice of trajectory. These studies on the intact brain will be complemented by fluorescent live cell imaging to analyse Drebrin kinetics within neurites in vitro. Such data will reveal how molecular interactions produce macroscopic behavioural changes in cells at individual and population levels. The interactions of Drebrin with guidance receptors and adapter proteins will be confirmed biochemically using co-immunoprecipitation and Western blotting.

The immediate impact of this application is in the opportunities it presents for communicating science to the public. As described in the Impact Plan, I am committed to public engagement and actively expanding this from schools work to wider sections of the community, frequently its more senior members. Indeed this sector has been a common thread in new activities: the BBSRC-funded roadshow attracted many retired people passing through the shopping centre; the SciBAR to discuss dementia and ageing, understandably had a largely elderly audience; most of the Women's Insititute members who visit the labs or to whom I talk are retired. Two key features have emerged from all these cases: firstly, people are fascinated by how their brain ages and the changes taking place, the more so as they feel it happening. Secondly, they are generally well-informed and eager to engage in dialogue about the subject and how current research aims to approach this. This project presents an excellent chance to further these links by using ongoing, cutting edge research to build upon initial interest. By explaining the aims, strengths and limitations of the work we can engage and challenge people. Different facets of the project provide opportunities to introduce a variety of topics: the vast complexity of the brain and how it forms; how connections are maintained, regulated and deteriorate with age; what strategies might one day be employed to combat this. This is an area that can be tailored to suit all audiences from schoolchildren to adults of any age, each with their own interests and concerns. Importantly, it will reveal to the public the processes underlying the normal development and ageing of the brain and relevance of understanding these. The project will feature in the schools activities organised for National Science Week and throughout the year. The excitement of the project in terms of what it seeks to achieve scientifically, as well as the tools used to reach these goals, will be conveyed to the schoolchildren. It will demonstrate how a reductionist approach and testing suitable hypotheses can be used to try and understand complex and fundamental scientific problems. The equipment used, particularly the fluorescent live cell imaging, is way beyond the scope of an average school lab and so it provides a vivid insight into modern research methodologies. A clear outcome of using this project to underpin such activities will be to inspire pupils to consider science - in its widest sense - for further study and as a possible career. In terms of contributing to the overall body of scientific knowledge the gains of this project are long term. The brain is a fantastically complex organ and understanding how such intricacy reliably arises during normal development is an incremental process. Nevertheless, studies such as this one are vital because they will increase understanding of how molecular processes are translated into the macroscopic behaviour of cells as individuals and collectives. This project focusses on a specific mode of behaviour, namely tangential migration, a developmental characteristic of many neurons. By studying it within the well-defined context of the oculomotor system and analysing a selection of proteins it will be possible to establish underlying prinicples that can be extrapolated to other areas of the brain less experimentally tractable. Furthermore, it will provide a framework into which proteins identifed in genomic and proteomic screens with sequence or structural similarity to those studied here can be readily slotted. One of the strengths of this project is that from the outset we will have already clearly defined the model system, the proteins of interest and crucially established the links between all these components. We are thus in a very strong position to deliver upon the aims of this project and provide novel insights that are widely applicable across developmental biology and neuroscience.