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

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

Abstract

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.

Technical Summary

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.

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 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.

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 discovered that a novel mechanism driven by the Toll receptor family underlies structural brain plasticity. Structural brain plasticity reflects the ability of the brain to change throughout the life course. Normally, plasticity is in balance with structural homeostasis, which constrains plasticity, and together they maintain brain integrity whilst enabling it to respond to the environment. When homeostasis takes over, it can drive neurodegeneration and brain disease. The underlying mechanisms controlling the swing between plasticity and degeneration were unknown.
We discovered that Toll receptors - best known for their involvement in innate immunity - control brain plasticity and neurodegeneration in the fruit-fly brain. The same mechanism could operate in the human brain.

These findings were recently published 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

We were invited to write a review on this discovery, published as: Li, G and Hidalgo, A (2020) Adult neurogenesis in the Drosophila brain: the evidence and the void. IJMC 21(18), 6653.

We are also currently writing a second review: Li, G and Hidalgo, A (2021) A Toll route to structural brain plasticity. Frontiers in Physiology, to be submitted soon.

We have work in progress: (1) investigation of Toll receptors in the larval neurodegenerative-muscular junction and their function in structural synaptic plasticity; (2) investigation of key receptors at the larval neurodegenerative muscular junction and their function in structural synaptic plasticity. This work is in progress and we have not published findings yet.
Exploitation Route Others can get inspiration from our recent publication Li et al (2020) eLife DOI: 10.7554/eLife.52743.

We have recently been invited to write two reviews: (1) Li G and Hidalgo A (2020) Adult neurogenesis in the Drosophila brain: the evidence and the void. International Journal of Molecular Sciences 21(18), 6653. (2) Li, G and Hidalgo, A (2021) A Toll route to structural brain plasticity. Frontiers in Physiology. These will make our findings known more widely.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description So far, this grant has resulted in two publications: 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 Li G and Hidalgo A (2020) Adult neurogenesis in the Drosophila brain: the evidence and the void. International Journal of Molecular Sciences 21(18), 6653 We have also been invited to write a review for Frontiers in Physiology, which we are currently working on: Guiyi Li and Alicia Hidalgo (2021) A Toll route to the plastic Drosophila brain. Frontiers in Physiology. We have been engaged in outreach and public engagement activities. 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 participated in Museum Lates: night at the ThinkTank Science Museum in Birmingham, with a talk for the public "Plastic Brains" by Alicia Hidalgo, and a hands-on event for the public "From tiny fruit-flies to great discoveries", 24 October 2019. We regularly participate in Brain Awareness Week, in Birmingham: 11-17 March 2019 at the ThinkTank Science Museum, and 22 March at the MAC. This is an international event to celebrate the importance of brain research. This year we have entitled it "Mysteries of the brain". We made two videos for Brain Awareness Week 2021 that will be made publicly available through the internet. Visit: https://www.birmingham.ac.uk/research/centre-for-human-brain-health/events/2021/brain-awareness-week-2021.aspx
First Year Of Impact 2020
Sector Education,Healthcare
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 "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 Toll and kinase-less Trk receptors in concert drive a novel mechanism of structural synaptic plasticity.
Amount £475,100 (GBP)
Funding ID BB/R017034/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2018 
End 08/2021
 
Title New DeadEasy plugins to investigate brain plasticity 
Description We develop several new DeadEasy plugins, to count automatically cell number in the adult brain of the fruit-fly Drosophila: (1) DeadEasy Optic Lobe; (2) DeadEasy Central Brain; (3) DeadEasy Kenyon Cells. These were used in the publication Li et al 2020 eLife. 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? Yes  
Impact This has only just been published, so impacts have not materialised yet. 
URL https://elifesciences.org/articles/52743
 
Title Novel DeadEasy plugins to investigate brain plasticity in Drosophila 
Description We have generated novel DeadEasy plugin to investigate brain plasticity in Drosophila. They enable us to automatically count cells throughout the brain: (1) DeadEasy Optic lobes for smaller cells; (2) DeadEasy Central Brain; (3) DeadEasy Kenyon Cells, for tightly packed cells. These plugins have been used in our publication Li et al 2020 eLife 
Type Of Material Computer model/algorithm 
Year Produced 2020 
Provided To Others? Yes  
Impact This has only just been published so impact still unknown 
URL https://elifesciences.org/articles/52743
 
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
 
Title DeadEasy Central Brain 
Description DeadEasy Central Brain counts automatically in vivo all the nuclei in the Drosophila adult central brain, labelled with histone-YFP. Will count other nuclei of similar size, stained with nuclear markers. 
Type Of Technology Software 
Year Produced 2020 
Impact recently published in Li et al 2020 eLife 
URL https://elifesciences.org/articles/52743
 
Title DeadEasy Kenyon Cells 
Description DeadEasy Kenyon Cells counts automatically in vivo the tightly packed cells of the mushroom bodies of Drosophila, labelled with histone-YFP. Will count other similar nuclei. 
Type Of Technology Software 
Year Produced 2020 
Impact recently published, impacts not materialised yet 
URL https://elifesciences.org/articles/52743
 
Title DeadEasy Optic Lobe 
Description DeadEasy Optic Lobes counts automatically the many and tiny cell nuclei labelled with Histone-YFP of the Drosophila adult optic lobes, in 3D, in vivo. It will count other cells of similar size, visible with fluorescent nuclear markers. 
Type Of Technology Software 
Year Produced 2020 
Impact Published in Li et al 2020 eLife 
URL https://elifesciences.org/articles/52743
 
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