The molecular control of glial progenitor proliferation in Drosophila and mammals

Devastating nervous system injury (e.g. spinal cord injury, brain damage), neurodegenerative diseases of the ageing brain (e.g. Alzheimer's disease) and demyelinating diseases (e.g. multiple sclerosis) cannot be cured and future therapy requires understanding of the underlying biology. The key therapeutic approach to repair central nervous system (CNS) damage and disease is the transplantation of stem cells or glial progenitors to the site of injury. For instance, transplantation of stem cells or glial progenitor cells in paraplegic mice repairs the broken axons and restores normal movement. However, the current lack of knowledge of how the transplanted cells behave prevents a guarantee of repair, and prevents control over undesirable outcomes such as cancer (i.e. gliomas). Thus a molecular understanding of neural stem cell and glial progenitor proliferation is urgently required. There are glial progenitors in the adult human CNS, which upon injury or disease divide in what is known as the glial-repair response (GRR), leading to a spontaneous brief recovery. Although the GRR does not result in functional repair, it reveals an intrinsic tendency of the nervous system to repair itself. If we knew what the underlying genes are and how they work, we could manipulate them to induce repair. A golden opportunity to discovering the gene network controlling glial progenitor cell division and CNS repair is provided by the GRR. Working out gene networks important for human development and disease is frequently done using the fruit-fly Drosophila because most gene networks are evolutionarily conserved. Drosophila research enables powerful genetic approaches to investigating gene function, it has high cellular resolution, it is technically sophisticated, cheap, quick, it can be done in whole and in living animals, and it does not raise ethical concerns. We have discovered a GRR in Drosophila and we have a detailed working model of the underlying molecular genetic mechanism: a gene network involving a tight relationship between the genes Notch, Prospero/Prox1, Eiger/ TNF and Dorsal/NFkB. The aim of this proposal is to work out the molecular genetic mechanism underlying the control of glial progenitor division and the GRR in the Drosophila and mammalian CNSs. To meet this aim, the following experimental objectives will be addressed: (1) To test our working model on the involvement of the candidate genes in the control of proliferation of quiescent glial precursors in Drosophila. (2) To translate our findings to the mammalian CNS, by testing the functions of the mammalian homologues in the context of glial progenitors and the GRR of the mouse spinal cord. (3) To use Drosophila to test and identify further genes involved in the GRR and glial proliferation, which can then be extrapolated to mammalian glial proliferation and GRR. This project is a collaboration between a Drosophila and a mammalian expert to use the powerful genetics of Drosophila to advance mammalian glial progenitor research. Research into repair of the damaged or diseased CNS typically relies on mammalian animal models, requiring a severity of damage to animals ranging from sacrifice at different stages of development to inflicting physical damage (e.g. breaking the spinal cord). While Drosophila research does not raise ethical concerns, basic research using fruit-flies requires an active involvement of Drosophilists to promote the effective translation to mammalian gene discovery. Here, we will use Drosophila to propel mammalian research while in this way replacing and reducing the use of mice. Our Drosophila paradigm is simple and will become available to the wider research community for further research into the GRR and drug testing for therapeutic purposes using fruit-flies. This proposal responds to the call for the '3Rs: replacing protected animals with invertebrate models' and the strategic priority of 'Ageing and lifelong wellbeing' research.

We aim to work out the molecular mechanism underlying the control of glial cell proliferation in the central nervous system (CNS) of Drosophila and mice. The hypothesis is that the glial repair response (GRR) is evolutionarily conserved and Drosophila can be exploited to understand mammalian NG2 oligodendrocyte progenitor proliferation and the mammalian GRR. With preliminary findings from Drosophila we have established a working model: quiescent glial precursors are normally on the brink to divide due to a positive feed-back loop between Notch (activator of cell division) and Prospero/Prox1 (repressor of cell division). This balance is broken and pushed towards cell division by Eiger/TNFa released upon injury, which activates Dorsal/NFkB, the expression of which depends on Prospero/Prox1. The experimental objectives are: (1) To consolidate our Drosophila model using: genetics, immunohystochemistry and confocal microscopy; an experimental paradigm of the GRR developed in AH's lab; and automatic counting of glial cells using software developed in AH's lab. (2) To translate our Drosophila findings to the mammalian CNS, by testing the functions of the mammalian homologues in NG2 glial progenitors and the GRR of the mouse spinal cord. This will be done by: purifying NG2 cells from the spinal cord of normal mice, followed by immunohystochemistry and microscopy; carrying out transfections for gain of function and siRNA analyses in isolated cells and in situ in the spinal cord, and one transection experiment as a GRR proof of principle. (3) To use Drosophila to test and identify further genes involved in the GRR and glial proliferation, using genetics, which can then be extrapolated to mammalian NG2 cells. We already know that at least two genes involved in the control of NG2 proliferation - EGFR and FGFR - also regulate glial proliferation in Drosophila. The output will be a molecular platform for the control of glial proliferation and the GRR in Drosophila and mammals.