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

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.

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

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.