Toll and kinase-less Trk receptors in concert drive a novel mechanism of structural synaptic plasticity.

The nervous system changes throughout life, as neurons, neurites and synapses are generated and eliminated. Structural brain plasticity enables us to learn and adapt to change, and destructive change maintains structural homeostasis and integrity, enabling further adaptation. Such neuronal remodelling could be structural correlates of brain function. Thus, unravelling the relationship between structural plasticity/homeostasis and neuronal activity is a gateway to understanding how the brain works. The balance between structural plasticity and homeostasis is also essential for brain health, and its breakdown leads to brain tumours, neurodegeneration, motor and psychiatric disorders. Conversely, increasing brain plasticity is a key strategy to tackle brain disease. Brain disease constitutes the greatest disease burden in Europe, costing over double of cancer and cardiac diseases put together. Most brain diseases - from anxiety and depression, to epilepsy, autism, neurodegeneration, e.g. Alzheimer's and Parkinson's diseases, and neuroinflammation - involve problems with the neurotrophins (NTs) and/or Toll-Like-Receptors (TLRs). NTs are key plasticity factors, and promote neuronal survival, connectivity, synaptic formation, learning and long-term memory, through Trk receptors and tyrosine kinase signaling downstream. However, paradoxically, the most abundant Trk isoforms in the human adult brain lack the tyrosine kinase, but their neuronal functions are unknown. TLRs are best known for underlying innate immunity. TLRs are also found in all neurons, however their neuronal functions are largely unknown, and their ligands in the brain are also unknown. Altogether, the molecular mechanisms regulating nervous system structural plasticity and homeostasis are little understood. Discovering novel mechanisms to enhance brain plasticity is an urgent neuroscience goal.

We recently discovered a previously unforeseen relationship between NTs, kinase-less Trk and Toll receptors in the central nervous system (CNS) of the fruit-fly, Drosophila. There are no full-length Trks in Drosophila, and instead, Trk homologues encoded by the kekkon (kek) genes lack the tyrosine kinase. Thus, Drosophila offers a golden opportunity to investigate the functions of truncated Trk receptors in vivo. The fruit-fly is the most powerful model organism for functional genetic analysis in vivo, offering from neural circuit to synaptic resolution and behaviour.

We discovered that Drosophila neurotrophins (DNTs) bind Toll receptors to regulate neuronal number, connectivity and behaviour, and bind Keks to promote structural synaptic plasticity, e.g. synaptic bouton formation and axonal arbor complexity. We suspect they also function together. We hypothesize that a feedback loop between pre- and post-synaptic cells, involving post-synaptic translation of DNT2 and a Kek-Toll receptor complex, modulates synaptic function and promotes structural changes in neurons in response to neuronal activity. We will test this hypothesis at the glutamatergic neuromuscular junction (NMJ) of the Drosophila larva by: (1) Determining the mechanism of Kek-6 action, validating candidates we have identified. (2) Working out how Keks and Tolls interact, and resolving the Kek-Toll code. As there are multiple Keks and Tolls, distinct pairs could be characteristic of neuronal type, or of distinct responses to neuronal activity by dendrites and axons. (3) We will select 2-3 Kek-Toll pairs with their downstream factors, to test whether and how they modulate synaptic function and structural changes in neurons in response to neuronal activity.

The outcome will be the identification of a novel, unanticipated molecular mechanism for nervous system structural plasticity. Even if not all details were to be evolutionarily conserved in humans, our framework will provide compelling and incisive predictions to test in rodents, for the benefit of understanding the human CNS, in health and disease.

The aim is to test the hypothesis that a recently discovered molecular mechanism promotes neuronal activity dependent structural plasticity, to deliver appropriate behaviour. In humans, neurotrophins (NTs) modulate synaptic and structural plasticity via the tyrosine kinase function of the receptor TrkB. Paradoxically, the most abundant TrkB isoforms in the adult human brain lack the tyrosine kinase, but their neuronal functions are unknown. Toll-Like-Receptors could also be involved in brain plasticity, but their neuronal functions remain unknown.

We discovered that Drosophila neurotrophins (DNTs) bind Toll and kinase-less Trk-family receptors encoded by the kekkon (kek) genes. DNT2 is a retrograde ligand for a pre-synaptic receptor complex formed of Kek-6 and Toll-6 that together regulate neuronal number, synaptic structure and arbor growth. We suspect that they modulate synaptic function and respond to neuronal activity to mould structural changes in neurons to functional requirements. We will test this idea in the Drosophila larva by: (1) Determining the molecular mechanism of Kek-6 function. We have identified 13 candidate effectors. Using optogenetics, GCaMP calcium imaging, genetics and co-immunoprecipitations, we will validate 1- 5. (2) Cracking the Kek-Toll code. Kek-6 and Toll-6 signalling can cross-talk downstream, and using genetic epistasis analysis, confocal and expansion microscopy at the neuromuscular junction (NMJ), we will test the effect on synaptic structure. With image registration to the connectome, we will see whether different Keks and Tolls are in different motorneuron types, dentrite vs. axonal arbors, or whether they overlap. (3) We will select 2-3 Kek-Toll pairs to investigate at NMJ or dendrite. Using electrophysiology, opto- and thermo-genetics, to examine, activate and silence neurons, genetics to switch genes on or off and microscopy, we will test how these mechanisms affect activity-dependent structural plasticity and behaviour.

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 central nervous system (CNS) structural plasticity to maintain or restore health in ageing, and upon disease. We will provide a molecular framework of how neuroprotective neurotrophin ligands signaling through innate immunity Toll and kinase-less truncated-Trk-like receptors regulate structural changes in the CNS, to promote plasticity and counteract neurodegeneration. The findings will help scientists using mammals develop drugs to influence these pathways in vivo, to treat brain disease, from psychiatric to neurodegenerative and restore brain health. The Academic community and general society will benefit from scientific discoveries into CNS plasticity. 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 and healthy ageing. 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 nervous system, brain damage and how ageing impacts on immune, cognitive, motor ans sensory systems. The BBSRC will benefit from increased international collaboration, as this project involves collaborators: Prof R.Baines (University of Manchester, UK), Dr M.Landgraf (University of Cambridge, UK), Dr M.Zlatic (Janelia Research Campus, USA), Prof B.Gerber (Liebniz Institut für Neurobiologie, Germany). 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 skilled researchers resulting from this project (including PhD and Master 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 BBSRC funded research.