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

Lead Research Organisation: University of Birmingham
Department Name: Sch of Biosciences

Abstract

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.

Technical Summary

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.

Planned Impact

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.

Publications

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Hidalgo A (2017) Go and stop signals for glial regeneration. in Current opinion in neurobiology

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Kato K (2018) Gene network underlying the glial regenerative response to central nervous system injury. in Developmental dynamics : an official publication of the American Association of Anatomists

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Losada-Perez M (2017) Glial gene network for central nervous system repair. in Neural regeneration research

 
Description From this grant, we discovered: (1) completed a project that we started with a previous BBSRC grant on the genetic mechanisms regulating glial differentiation and proliferation in mice (Kato et al 2015). (2) Established a novel CNS repair/regeneration paradigm following crush injury in the Drosophila larva. (3) Discovered a molecular mechanism for CNS repair and glial regeneration after crush injury in Drosophila. This mechanism involves evolutionarily conserved genes, including the NG2 homologue kon-tiki, prospero/prox-1 and Notch, with an impact in understanding CNS regeneration in mammals. This resulted in a publication Losada-Perez, Harrison and Hidalgo 2016 "Molecular mechanism of CNS repair by the Drosophila NG2 homologue kon-tiki" Journal of Cell Biology 214, 587. (4) As a result of this publication, we were invited to write 4 reviews, and wrote 3 of them. (5) We then identified genes expressed in neurons that interact with Kon to induce neurogenesis after injury. Neurogenesis is a key process in regeneration. We are currently preparing this work for publication (Harrison, Connolly, Losada-Perez, Jiang and Hidalgo, in preparation).
Exploitation Route The findings from this grant were published in Losada-Perez M, Harrison N, Hidalgo A. (2016) Molecular mechanism of central nervous system repair by the Drosophila NG2 homologue kon-tiki. Journal of Cell Biology 214, 587. This publication received considerable attention, and as a result we wrote three review articles: Losada-Perez et al 2017 Neural Regeneration Research; Kato et al 2018 Developmental Dynamics; Hidalgo and Logan, 2018, Current Opinion in Neurobiology.

Some authors in a further publication were also funded by this grant. This was Foldi I, Anthoney N, Harrison N, Gangloff M, Verstak B, Ponnadai Nallasivan M, AlAhmed S, Phizacklea M, Losada-Perez M, Moreira M, Gay NJ and Hidalgo A (2017) Three-tier regulation of cell number plasticity by neurotrophins and Tolls in Drosophila. Journal of Cell Biology 216(5):1421. This article had considerable impact:
o Highlight review on our findings by Keeler and Deppmann DOI: 10.1083/jcb.201702115
o Selected by JCB Editors as "One of the Top 10 Articles of 2017"
o Selected for Special Collection "The year in cell biology: 2017" http://jcb.rupress.org/cc/the-year-in-cell-biology-2017
o Selected for Special Collection on "Cellular Neurobiology"
"In this collection, the editors have selected some of the most exciting cellular neurobiology research published in the Journal of Cell Biology over the past year." http://jcb.rupress.org/cc/cellular-neurobiology

We are preparing a further article for publication Harrison, Connolly, Losada-Perez, Jiang and Hidalgo (in preparation).

Others benefit from our published findings to progress their research.
Sectors Healthcare

URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Regeneration_and_Glia.html
 
Description We discovered a molecular mechanism of glial regeneration and central nervous system repair, which we published in Losada-Perez, Harrison and Hidalgo 2016 Journal of Cell biology 214, 587; and in three invited reviews: Losada-Perez, Harrison and Hidalgo 2017 Neural Regeneration Research, Kato, Losada-Perez and Hidalgo 2017 Developmental Dynamics and Hidalgo and Logan 2017 Current Opinion in Neurobiology). Ours is fundamental research and any non-academic impact will be longer term. In the longer term, our findings will have implications for understanding how to find therapeutic solutions to brain damage and spinal cord injury. In the course of this grant we have participated in outreach events including school visits to talk about our research, university Community Day to present research to the public and families, British Science Festival and Brain Awareness Week.
First Year Of Impact 2016
Sector Healthcare
Impact Types Societal

 
Description BBSRC Committee A Core Member
Geographic Reach National 
Policy Influence Type Membership of a guidance committee
 
Description BBSRC Pool of Experts
Geographic Reach National 
Policy Influence Type Participation in advisory committee
 
Description Wellcome Trust Committee Pool of Experts
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description BBSRC ISIS
Amount £3,071 (GBP)
Funding ID BB/P004997/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2016 
End 08/2016
 
Description EU Marie Curie IntraEuropean Post-doctoral Fellowship
Amount € 221,606 (EUR)
Funding ID 622082 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 03/2014 
End 02/2016
 
Description MIBTP PhD Studentship
Amount £13,500 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2017 
End 09/2021
 
Title BALM confocal microscopy 
Description During the course of my MRC-CEG, I coordinated the successful application of an Equipment Grant from the Wellcome Trust, that enabled us to purchase a new confocal microscope, employ an imaging specialist and create the Biosciences Advanced Light Microscopy Facility. 
Type Of Material Improvements to research infrastructure 
Year Produced 2006 
Provided To Others? Yes  
Impact The Leica SP2 confocal microscope has been essential for all the projects carried out by my research team. Thjs confocal and the general management and access to BALM has been open to everyone in our University, and has helped many other research groups in the University of Birmingham (a total of 67 users). 
 
Title DeadEasy Glia 
Description software programme to count the number of glial cells in Drosophila in vivo 
Type Of Material Technology assay or reagent 
Year Produced 2012 
Provided To Others? Yes  
Impact sharing tools 
URL http://www.biosciences-labs.bham.ac.uk/hidalgo/Software.html
 
Title Nerve cord injury paradigm in Drosophila 
Description During the course of my MRC-CEG, and funded by an EU Marie Curie Post-doctoral fellowship and subsequently (ie 2010) by a BBSRC Project Grant, we established an injury paradigm in the Drosophila larva that enables us to investigate the genetic and molecular basis underlying central nervous system regeneraiton. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact This work is not published yet, the manuscript is currently under review, but the method and reagents will become available after publication. 
 
Title Regeneration and repair in the living Drosophila larval CNS 
Description CNS injury in Drosophila living larvae to investigate regeneration and repair 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact this model was presented at a conference for the first time in 2014 
 
Description Behavioural assays for structural plasticity and repair in the central nervous system of Drosophila 
Organisation University of Wurzburg
Country Germany 
Sector Academic/University 
PI Contribution The aim of this collaboration was for my research team to learn from our collaborators about behaviour assays in fruit-flies. Once we have data ready for publication, we will offer them authorship in a joint paper.
Collaborator Contribution We collaborated with Prof Martin Heisenberg and Dr Reinhard Wolf. One member of my team visited their lab to learn behaviour assays for fruit-flies. This helps our research by enriching the experimental approaches we can use to address our questions.
Impact Behavioural analysis in flies. Outcomes not ready yet, work in progress.
Start Year 2016
 
Description Brain Awareness Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Brain Awareness Week is celebrated world-wide between 13-19 March 2017 to raise awareness on the importance of brain research. We organised: (1) a cafe scientific; (2) evening talks every day of that week; (3) hands on science activities for everyone at The Think Tank museum.
Year(s) Of Engagement Activity 2017
URL http://www.birmingham.ac.uk/university/colleges/les/news/2017/16Feb-brain-awareness-week.aspx
 
Description British Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Engagement with the general public

Public understanding of science and research
Year(s) Of Engagement Activity 2014
 
Description Community Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Communicating science to the general public

outreach
Year(s) Of Engagement Activity 2014
 
Description Community Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Communicating scientific discovery to the public using fruit-flies

outrach
Year(s) Of Engagement Activity 2013
 
Description Lab web-site 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Soon after starting this project, I created a lab web-site where information on this project, and other lab projects, can be found. www.biosciences.bham.ac.uk/labs/hidalgo

making our work known to the public
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010
 
Description Organise international symposium for EuroGlia 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact CO-ORGANISER for symposium for the international conference EuroGlia 2017: S18
Year(s) Of Engagement Activity 2017
URL http://www.glia2017.eu/?s=scientific-program