The genetic mechanisms underlying the regenerative potential of ensheathing glial cells in Drosophila

The central nervous system (CNS) does not regenerate after damage. Thus, spinal cord and brain damage (e.g. injury, stroke, multiple sclerosis) and neurodegeneration (e.g. Alzheimer's and Parkinson's diseases) result in devastating permanent disability. However, cells can accommodate changes in development and throughout normal life (e.g. during learning) to maintain normal function and behaviour, and regeneration after injury in animals also reveals that cells have a natural ability to sense and restore normal organism integrity. Understanding how cells 'know' how to achieve this and why they cannot in the CNS, is a great goal of biology and neuroscience. The glial cells that ensheath CNS axons respond to damage by proliferating, leading to axonal re-enwrapment and partial functional recovery of behavior. This glial regenerative response (GRR) is limited, but it is found across species, from flies to humans, suggesting that there is a natural, genetic mechanism of CNS repair. If we could understand this mechanism, we would be able to manipulate glial cells to promote repair. In mammals, oligodendrocyte progenitor cells (OPCs) have the greatest potential to induce regeneration in the damaged CNS and transplantation of stem or OPCs to the lesion site is the most promising therapeutic approach to CNS damage. However, the current scarce knowledge of how transplanted cells behave prevents a guarantee of repair or of avoidance of cancer. Most critically, what controls the differentiation of glial cells enabling axonal re-enwrapment and how might they influence neuronal regeneration or repair, are unknown.

The fruit-fly Drosophila is a very powerful model organism to identify gene networks and test gene function in vivo, and it is successfully used to investigate responses to CNS injury, regeneration and repair. This led to the discovery of genetic mechanisms that induce glial proliferation, cell debris clearance, and axonal and dendritic regeneration. Genes discovered in fruit-flies are then tested in mammals, expediting research, and minimizing the use of protected animals.

With BBSRC funding, we recently discovered a gene network controlling the Glial Regenerative Response (GRR) in Drosophila. This involves the genes encoding Prospero (Pros, a transcription factor that inhibits proliferation and promotes differentiation), Notch and NFkB (two cell cycle activators). Together, they form a homeostatic mechanism that balances glial cell number and differentiation control, enabling enwrapment whilst preventing tumours. Upon CNS injury, this gene network activates the glia to clear up cell debris, triggers their proliferation restoring cell number and enables axonal re-enwrapment. Manipulating Notch and Pros levels is sufficient to induce glial regeneration and promote axonal neuropile repair. We have tested this gene network in the mouse, and found that the pros homologue Prox1 is present in mammalian NG2-positive OPCs. This indicates that the GRR gene network is evolutionarily conserved.

It is key to find out what genes are regulated by Pros to promote glial differentiation and enable glial and neuronal regeneration or repair. We aim to discover and investigate the functions of these genes. (1) We will select 5 out of 39 candidate genes regulated by Pros with potential functions in the GRR, including kon-tiki (kon), the Drosophila homologue of NG2. (2) We will analyse the functions of kon and four other genes in the glial responses to injury, and test their link to the GRR gene network. (3) We will investigate whether these genes can influence neuronal regeneration and/or repair.

The outcome will be the discovery of molecular genetic mechanisms underlying glial differentiation and/or neuronal regeneration. In future projects, we will test our discoveries in mice, thus implementing the 3Rs policy (Refinement, Reduction, Replacement) using flies to speed up research for the improvement of human wellbeing and health.

The aim is to discover the molecular mechanisms by which the transcription factor Prospero (Pros) confers regenerative potential upon ensheathing glial cells. Central nervous system (CNS) injury induces a Glial Regenerative Response (GRR) across species that triggers ensheathing glial proliferation and leads to spontaneous remyelination and functional recovery of locomotion. In mammals, the GRR is implemented by NG2+ oligodendrocyte progenitor cells (OPCs). However, it is not known how to induce NG2+ cell differentiation to enable remyelination. NG2+ cells can be both permissive and inhibitory to axonal growth, and how this occurs is not understood either. With previous BBSRC funding, we established a novel injury paradigm to investigate the GRR in the fruit-fly Drosophila, we discovered a gene network underlying the GRR, we found that Pros controls ensheathing glial differentiation, and that manipulating Notch and Pros levels in glia induced glial regeneration and axonal neuropile repair. We also found that the mammalian homologue of pros, Prox1, is expressed in NG2+ OPCs. Pros is the key factor that controls glial differentiation and influences neuropile repair. Here, the aim is to find out what genes are regulated by Pros in glia conferring this regenerative potential. We have identified 39 candidate Pros target genes. The experimental objectives are: (1) Using a combination of RT-PCRs, in situ hybridizations and genetics, we will validate these genes in relation to Pros and the GRR gene network and select five. (2) We will analyse the functions of five genes in glial differentiation and repair using genetics, immunostainings, confocal and TEM microscopy, and Optical Projection Tomography. (3) We will test whether the top candidate genes can influence neuronal regeneration, with a combination of DeadEasy software, time-lapse confocal microscopy and Flybow, glial manipulation with the LexA system, and locomotion recordings with FlyTracker and Trikinetics monitors.

Who might benefit from this research?

Beneficiaries will be: (1) Scientists working with Drosophila, mammalian model organisms or humans, on ageing and neurodegeneration, stem cell research, regeneration and repair. Our project will provide a molecular mechanism for CNS repair which will help mammalian scientists control neural stem cells and glial progenitors and their integration into functional neural circuits, whilst avoiding brain tumours. It will also provide an improved paradigm to for drug testing in Drosophila in the context of CNS regeneration and repair. (2) Protected animals: This project directly implements the "3Rs: replacing protected animals with invertebrate models", as only Drosophila is used to address questions directly relevant to mammals including humans. (3) The BBSRC will benefit since this project meets the Research priority Area of "Ageing across the life-course" with particular reference to the use of invertebrate model systems, the Strategic Priorities: "Basic bioscience underpinning health: Ageing research: lifelong health and wellbeing" and the "Over-arching Strategic Priority: The replacement, refinement and reduction (3Rs) in research using animals". (4) The post-doctoral researcher and technician appointed in this grant will benefit form training in research.

How might they benefit from this research?

The Academic community and general society will benefit from the scientific discoveries, creation of new knowledge and scientific advancement resulting from this project. The BBSRC will benefit from the creation of internationally competitive research in basic biomedical science. This project addresses questions that are important globally, such as how to control proliferation and differentiation of stem cells and how to promote regeneration and repair in the damaged central nervous system. The project is in basic biology, but it will result in discoveries with important longer-term implications for the understanding and treatment of diseases of the ageing nervous system and for regenerative medicine. I collaborate with the teams of Prof Ann Logan (Institute of Biomedical Research, Birmingham) and Dr Fumio Matsuzaki (Riken Center for Developmental Biology, Kobe, Japan) using rodents to investigate glial and stem biology, and CNS regeneration. I have close ties with the consortium for "Neurotrauma and neurodegeneration" at the Medical School, University of Birmingham, close to the University Queen Elisabeth Hospital offering unique opportunities to translate findings of basic research into medicine.

The findings resulting from this project will be disseminated in presentations at conferences and peer reviewed research articles in Open Access journals or as open access articles. Publication in a professional magazine will bring our BBSRC funded research to the attention of the government, the health sector, the pharmaceutical industry and other stakeholders. I will also organize a research workshop on Glia and CNS regeneration and repair, applying for additional funding.

UK and Europe will benefit form the creation of highly skilled researchers as a result of training in this project. As well as post-docs, I have hosted short-term international visitors (e.g. Diploma and PhD students from Europe), and work experience students (e.g. College and Undergraduate summer students), on top of my normal roles to the University of Birmingham to train undergraduate, Masters and PhD students in research.

The general public will benefit by attending our annual public events in which we promote the public understanding of science including: school visits, whole-day events such as "Meet the scientist for Brain awareness week" at the Think Tank Museum in Birmingham, "The twelve experiments of Christmas" and the "Community Day", both at the University of Birmingham. We will run these activities at least once a year, to explain to the public what our BBSRC funded research aims to find out.