Tolls and neurotrophins in central nervous system regeneration and repair in Drosophila

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

Abstract

Cells in the central nervous system (CNS) have a natural ability to respond to change. Glial and neuronal number, axons, dendrites, circuits and synapses can be formed or eliminated during development and throughout life, such as with learning, exercise and experience. Normally, the balance between generative and destructive plasticity maintains structural integrity and appropriate behaviour. This balance fails with ageing, neurodegeneration and brain tumours. The CNS does not regenerate after damage, so injury to the brain or spinal cord, stroke and neurodegeneration (e.g. Alzheimer's disease) result in devastating permanent disability. Discovering and understanding genetic mechanisms underlying cell plasticity is key to promote regeneration and repair.

Toll Like Receptors (TLRs) and neurotrophin (NT) ligands both promote generative and destructive cell change. TLRs underlie innate immunity. In the brain TLRs are in all cells, and alterations in TLRs underlie brain diseases, e.g. stroke, neurodegeneration, multiple sclerosis and anxiety. TLRs induce microglia activation and debris phagocytosis, cell survival and death, neurite growth and collapse. However their in vivo functions are poorly understood and their neuronal functions and endogenous CNS ligands, are both unknown. The NTs are the main neuroprotective factors, and NT problems underlie most brain diseases, from neurodegeneration to epilepsy and depression. NTs promote from neuronal survival and connectivity to synaptic transmission, in development, learning and with experience. NTs also have destructive functions. How NT functions are balanced in vivo, across cell types and circuits, is poorly understood.

We discovered that Tolls are receptors for Drosophila neurotrophins (DNTs) in the fruit-fly, and that human NTs and TLRs can interact too. Drosophila is a very powerful model organism to identify gene networks and test gene function in vivo, and it is often used to investigate regeneration and repair. Genes discovered in fruit-flies are tested in mammals, expediting research findings for human health and minimizing animal use.

We discovered a novel mechanism balancing cell survival and death during neural circuitry involving DNTs and Tolls. We also discovered a gene network underlying the glial regenerative response to injury that involves NFkB, the universal effector of Tolls and TLRs. Preliminary findings indicate that DNTs and Tolls could be involved in both glial and neuronal regeneration.

Harnessing our recent findings, we aim to work out how glia and neurons elicit coordinated change, to promote regeneration and repair. We will test the hypothesis that distinct DNT/Toll/adaptor modules regulate the response of glial cells, neurons and neuron-glia interactions to injury. 22 genes and over 33 proteins are potentially involved. Our strategy will be to identify the modules most relevant for neurons or glia and test 2-5 in regeneration and repair. The objectives are: (1) to visualize a map of DNTs and Tolls in the ventral nerve cord (VNC), and select neuronal and glial pairs. (2) We anticipate some overlap and some specificity in DNT-Toll interactions, and differential affinities of Tolls for the adaptor Wek can shift their function from neuroprotective to pro-apoptotic. So to narrow down, we will test and select the most specific DNT-Toll, and Toll-Wek pairs. (3) Test whether the selected 2-5 DNT/Toll/adaptor modules regulate in vivo glial (cell debris phagocytosis, glial proliferation, axonal enwrapment) or neuronal (neuroprotection, neurogenesis, axonal/dendritic patterns and circuitry) responses to injury, and whether manipulating these genes promotes regeneration and repair.

The outcome will be a gene network involving DNTs and Tolls for CNS regeneration and repair. Even if not all details were to be evolutionarily conserved, our framework will provide incisive predictions that can be tested in mammals, ultimately for the benefit of human health.

Technical Summary

The aim is to work out how Tolls with Drosophila neurotrophins (DNTs) facilitate central nervous system (CNS) regeneration and repair. The CNS does not regenerate upon damage, but cells modify number, neurite patterns and circuitry during development and learning. Discovering the underlying genes is key to mend the CNS, and Toll-Like Receptors (TLRs) and Neurotrophins (NTs) are most promising. TLRs underlie innate immunity, NTs are the main neuroprotective factors, and their alterations underlie brain disease. However, the functions of TLRs in CNS development, neurons and regeneration, and their endogenous ligands, are unknown; the in vivo NT circuits are unknown; and how TLRs and NTs balance opposing cellular outcomes is not understood.

We discovered that Tolls are DNT receptors in Drosophila, regulating cell number plasticity, connectivity and behaviour. Distinct cellular outcomes depend on the combination of DNTs, Tolls and adaptors, Wek being the most critical. We discovered a gene network underlying glial regeneration upon injury involving NFkB, the universal target of Tolls/TLRs. Preliminary data indicate TLRs and DNTs influence glial and neuronal regeneration.

We will test the hypothesis that distinct DNT/Toll/adaptor modules regulate the response of glia and neurons to injury driving regeneration and repair by: (1) mapping their cell-type distribution in the ventral nerve cord (VNC). Of the initial 22 genes, those not expressed and/or unresponsive to injury will be ruled out. (2) Identifying DNT/Toll and Toll/Wek pairs to select 2-5 most specific, using cryo-Electron Microscopy, plasmon surface resonance and super-resolution microscopy. (3) Using genetics, test 2-5 DNT/Toll/adaptor modules, to investigate glial (phagocytosis, proliferation, enwrapment) and neuronal (neuroprotection, neurogenesis, neurite patterns and circuitry) responses to VNC injury, and use opto-, thermo-genetics and calcium imaging to test whether neuronal activity affects outcomes

Planned Impact

Who might benefit from this research?
Beneficiaries will be: (1) Scientists working with Drosophila, mammalian model organisms or humans, on brain development, structural brain plasticity, brain diseases including neurodegeneration, neuroinflammation, ageing, stem cell and regenerative biology. (2) Protected animals, by implementing the "3Rs: replacing protected animals with invertebrate models", as only Drosophila will be used to address questions relevant to mammals including humans. (3) The BBSRC: this project meets the BBSRC Strategic Priorities of "Driving bioscience discovery" and "Frontier Bioscience"; the Strategic Research Priority 3 "Biosciences for health: generate new knowledge on the mechanisms of development and the maintenance of health across the life-course; generate new knowledge to advance regenerative biology, including stem cells and tissue engineering research; improve our understanding of how the ageing process results in increased frailty and loss of adaptability in areas such as brain, immune and sensory systems", the Responsive Mode Priority area of "Healthy ageing across the life-course", and the over-arching priority "3Rs: Replacement, Refinement and Reduction in research using animals". (4) The appointed post-doctoral researcher and technician will benefit from employment and training. (5) Potential BBSRC MIBTP post-graduate students will benefit from training in research.

How might they benefit from this research?
The project aims' are of global importance: to discover genetic mechanisms to promote CNS regeneration and repair after damage or disease. We will provide a molecular framework of how innate immunity Toll receptors and neuroprotective neurotrophin ligands control from neuroinflammation to cellular plasticity and neural circuit connectivity. The findings will help scientists using mammals develop drugs to influence inflammation and gene function in vivo, and control stem cells for transplantation, to restore brain health, and promote regeneration and repair. The Academic community and general society will benefit from scientific discoveries into CNS regeneration and repair. Our findings will be disseminated at conferences and Open Access peer reviewed research articles.

The BBSRC will benefit from funding internationally competitive research in world-class bioscience on regenerative neurobiology. The project uses the fruit-fly Drosophila as a model organism, but it will result in discoveries with important long-term implications for the understanding and treatment of diseases of the ageing nervous system, brain damage and regenerative biology. The BBSRC will benefit from increased international collaboration, as this project is a collaboration between PIs at the Universities of Birmingham (AH) and Cambridge (NJG); we have established further collaborations with Dr M G. Forero Vargas (University of Ibagué, Colombia), Prof. A Fiala (University of Göttingen, Germany), and Dr M Zlatic and Dr A Cardona (Janelia Research Campus, USA). AH also collaborates with Prof. A Logan (University of Birmingham) and Dr F Matsuzaki (Riken, Japan) using rodents, and NJG with Prof. C Bryant and Dr M Gangloff (University of Cambridge), investigating mammalian Toll-Like Receptors. AH's links to the consortia for Neuroscience and Ophthalmology, closely linked to the University Queen Elisabeth Hospital, and Centre for Human Brain Health at the University of Birmingham, and NJG's links to Drug Discovery at the University of Cambridge, offer unique opportunities to translate fundamental research findings into medicine.

UK and other countries will benefit from highly skilled researchers resulting from this project (including PhD students and short term students).

The general public will benefit from our outreach events, e.g. school visits, "Brain awareness week", "British Science Festival" and "Community Day", where we will explain to the public our findings from BBSRC funded research.

Publications

10 25 50
 
Title The 3D-printed fruit-fly 
Description In collaboration with an artist, we are designing a 30cm fruit-fly with anatomical precision, to be 3D printed in plastic. 
Type Of Art Artefact (including digital) 
Year Produced 2018 
Impact The 3D-printed fruit-fly will be used at outreach events for the general public. We will consider the potential to distribute it to schools. 
 
Description We have continued to progress on the molecular mechanism of CNS regeneration and repair. Up to date, we have established a new method to investigate central nervous system regeneration, after contusion injury in the adult Drosophila ventral nerve cord. This will enable us to test the ability of cells to regenerate throughout the life-course. We have generate molecular tools using CRISPR/Cas9 gene editing, carried out an in-depth analysis of the intriguing Toll signalling adaptor Wek, and purified protein to test binding affinities between Tolls and Wek. We published an article: Harrison N, Connolly E, Gascón Gubieda A, Yang Z, Altenhein B, Losada-Perez M, Moreira M, Hidalgo A (2021) Regenerative neurogenesis is induced from glia by Ia-2 driven neuron-glia communication. eLife 10: e58756.
Exploitation Route Our first publication was first in bioRxiv, accessible for others to follow up:

• Harrison, Connolly, Gascón Gubieda, Yang, Altenhein, Losada-Perez, Moreira and Hidalgo (under revision) Regenerative neurogenesis is induced from glia by Ia-2 driven neuron-glia communication. bioRkiv https://doi.org/10.1101/721498

Now it is published as an open access article in eLife:

• Harrison N, Connolly E, Gascón Gubieda A, Yang Z, Altenhein B, Losada-Perez M, Moreira M, Hidalgo A (2021) Regenerative neurogenesis is induced from glia by Ia-2 driven neuron-glia communication. eLife 10: e58756
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://elifesciences.org/articles/58756
 
Description We wrote a review on Toll receptor signalling: Anthoney, Foldi and Hidalgo (2018) Toll and Toll-like receptor signalling in development. Development, 145, dev156018. Doi:10.1242/dev.156018 and a research article: Harrison N, Connolly E, Gascón Gubieda A, Yang Z, Altenhein B, Losada-Perez M, Moreira M, Hidalgo A (2021) Regenerative neurogenesis is induced from glia by Ia-2 driven neuron-glia communication. eLife 10: e58756 Public engagement: We carried out a School visit: All Saints C of E Primary School in Staffordshire, 30th November 2018. We attended a Science festival day at All Saints primary school with the aim of showing and teaching the children what we do as a lab, how flies can be used in science and how we use fruit flies in our area of research. Children had the chance to look at stages of the life cycle from larvae through to adult and had a chance to look under the microscope to identify different markers such as curly wings or different eye colours. Children had the chance to see fly behaviour, the courtship dance under the microscope as well as watch feeding behaviours, and performed locomotion tests. We were invited to stay after school for another part of the festival where parents and guardians had the chance to see all of the visitors and researchers that had come. Overall, the whole day was delightful, the children were really engaged and fascinated with the flies. The parents were also really fascinated and most had never heard of flies being used in research. It was a really rewarding experience. The Princes Teaching Institute: December 2018. I was invited to give a talk at a school teacher training workshop, organised by the Prince's Teaching Institute. The feedback received was outstanding: "The speakers were incredibly engaging and a great reminder of why I love my subject", "The speakers were really passionate and inspiring, and I feel I learnt a lot from them", "Speakers were great and the sessions were really useful and informative". We have carried out multiple public engagement activities: Green Heart Celebrations University of Birmingham 2019; Museum Lates at the ThinkTank Museum 2019; International Brain Awareness Week 2019. At these events, we gave talks for the general public and ran hands-on activity for the public. We had microscopes, fruit-lies and explained the principles of genetics and how the fruit-fly Drosophila leads to discoveries into molecular mechanisms which is essential in medical research and for therapeutic solutions. In 2020 we were unable to carry out any outreach activities due to the Covid-19 pandemic. This year, we are organising virtual outreach events online.
First Year Of Impact 2018
Sector Education
Impact Types Cultural,Societal

 
Description Member of the University of Birmingham Senate, representing College of Life and Environmental Sciences and School of Biosciences
Geographic Reach Local/Municipal/Regional 
Policy Influence Type Membership of a guideline committee
 
Description SERU Prize
Geographic Reach Europe 
Policy Influence Type Membership of a guideline committee
Impact This was to award a prize to the best young, emerging talent amongst Spanish scientists in UK. It had a great impact in that particular person, and the overall recognition of the importance of scientific research.
 
Description "Automatic quantification of cell number in the brain of Drosophila
Amount £75,000 (GBP)
Funding ID Marco Logico 
Organisation Government of Colombia 
Sector Public
Country Colombia
Start 10/2019 
End 10/2022
 
Description The 3D-printed fruit-fly
Amount £1,950 (GBP)
Organisation University of Birmingham 
Sector Academic/University
Country United Kingdom
Start 12/2018 
End 11/2021
 
Title Novel method to investigate regeneration and repair in adult Drosophila flies 
Description We are setting up a protocol to investigate regeneration and repair in adult Drosophila fruit-flies. We carry out contusion injury in the fly ventral nerve cord, and monitor survival, recovery and cellular responses to injury. Lizzie Connolly is supervising an MIBTP rotation student who is currently carrying on work to set this method up in our lab, and with which Lizzie will carry on working. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2020 
Provided To Others? No  
Impact We are currently setting up and testing the method. It has not optimised yet, and it has not been used in any research publication yet. 
 
Description "Automatic quantification of cell number in the brain of Drosophila" 
Organisation University of Ibagué
Country Colombia 
Sector Academic/University 
PI Contribution Contribution by my team: biological research data.
Collaborator Contribution Contribution by collaborating team: software produced to analyse our data.
Impact Recent publication in eLife: Guiyi Li, Manuel G Forero, Jill S Wentzell, Ilgim Durmus, Reinhard Wolf, Niki C Anthoney, Mieczyslaw Parker, Ruiying Jiang, Jacob Hasenauer, Nicholas James Strausfeld, Martin Heisenberg, Alicia Hidalgo (2020) "A Toll-receptor map underlies structural brain plasticity" eLife DOI: 10.7554/eLife.52743
Start Year 2018
 
Description Tolls and neurotrophins in central nervous system regeneration and repair in Drosophila 
Organisation University of Cambridge
Department Department of Biochemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution This grant has not started yet, will be activated within 2018. My research team wil investigate the functions of Tolls and neurotrophins in central nervous system regeneration and repair using the fruit-fly Drosophila as a model organism.
Collaborator Contribution My partner, Prof. N.J. Gay (University of Cambridge) will contribute with biochemical approaches to determine the affinity of each of the 9 Tolls for Wek.
Impact Work in progress
Start Year 2018
 
Description "From tiny fruit-flies to great discoveries" at Green Heart Celebrations 
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 Celebrations for the Green Heart at the University of Birmingham, June 2019
Year(s) Of Engagement Activity 2019
 
Description "Plastic Brains" at Museum Lates, ThinTank Museum and University of Birmingham 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Talk for the public on "Plastic brains" at an open night at the ThinkTank Science Museum, Birmingham
Year(s) Of Engagement Activity 2019
 
Description Brain Awareness Week 2019 at the ThinkTank Museum, March 2019 
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 "From tiny fruit-flies to great discoveries" celebration of international Brain Awareness Week. Outcome to increase public awareness of brain research
Year(s) Of Engagement Activity 2019
 
Description Brain Awareness Week 2021 - Videos 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact We generated two videos for international Brain Awareness Week (March 2021): (1) One 5min video where I, Professor Alicia Hidalgo, explain the research carried out by my team; (2) One 19min video where my team describes their research. Target audience is 14-16 year olds, and the video is to be played at schools.

We are releasing it on March 11, 2021 so we do not know of its impact yet.
Year(s) Of Engagement Activity 2021
URL https://www.birmingham.ac.uk/research/centre-for-human-brain-health/events/2021/brain-awareness-week...
 
Description Debate from panel of scientists after showing a film, for Brain Awareness Week 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Panel discussion on concussion for Brain Awareness Week. Raising public awareness of brain research, and here focusing on stem cell research, brain damage, regeneration and repair.
Year(s) Of Engagement Activity 2020
 
Description Hands-on science at Museum Lates, ThinkTank Science Museum and University of Birmingham, October 2019 
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 "From tiny fruit-flies to great discoveries" Hands-on science at Museum Lates, ThinkTank Science Museum and University of Birmingham, October 2019
Year(s) Of Engagement Activity 2019
 
Description Open days and applicant visits at University of Birmingham 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Undergraduate students
Results and Impact Open days and research talks in the lab to visiting applicants.
Year(s) Of Engagement Activity 2018,2019
 
Description School Teacher Training 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact I gave a talk at a Teacher Training Workshop organised by The Princes' Teaching Institute. The workshop took place at the Pimlico Academy, London.
Year(s) Of Engagement Activity 2018
 
Description School Visit 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact My research team spent a day at a secondary school (teenagers), talking about science and why using the fruit-fly Drosophila helps research,
Year(s) Of Engagement Activity 2018