Understanding essential roles of microtubule regulators during synapse formation and maintenance

Lead Research Organisation: University of Liverpool
Department Name: Institute of Translational Medicine

Abstract

A key prerequisite for nervous system function is the capability of neurons to communicate with other cells via specialised cell junctions called synapses. Synapses contain complex machinery for rapid transmission of signals to partner cells. Once formed, synapses have to be maintained in a plastic state, and precocious loss of synapses is considered a potential cause of neuronal decay in ageing and in neurodegenerative diseases. However, in spite of this importance, the mechanisms underlying synaptic maintenance are very little understood. The overarching aim of this project is to deliver such understanding, thus bridging an important gap in our knowledge about processes of ageing and degeneration in the brain.
During a neuron's life, its synaptic machinery is constantly recycled. To this end, its building blocks have to be efficiently transported between the neuronal cell body and the very distant synapses (up to a meter away in humans), connected only by a cable-like neuronal protrusion called the axon. Such precise movement of synaptic proteins along the axon is achieved by motor proteins which bind to transport vesicles containing synaptic proteins and trail along highways made out of parallel bundles of microtubules (MTs). MTs are dynamic filamentous polymers which are continuously built and degraded throughout a neuron's life, and these processes have to be regulated to sustain proper axonal transport. The number of MTs needs to be well controlled, they have to bear the right posttranslational modifications (PTMs) to promote the right motor protein interactions, and they have to maintain their bundled organisation - all so that blockage or slowdown of transport is prevented. For this, MTs are regulated through MT-binding proteins (MTBPs) which can control MT de/polymerisation, stabilisation, cross-linkage and PTMs. It seems therefore obvious that MTBPs, through controlling MT networks, can regulate axonal transport and consequently also synaptic maintenance and neuronal survival, and this causative chain could provide important explanations for why a number of MTBPs are associated with neurodegenerative disease. However, MTBP-based mechanisms of synaptic maintenance remain poorly understood.
For example, the MTBP Tau was discovered several decades ago. It has been associated with Alzheimer's Disease and Frontotemporal Dementia and has therefore been intensely researched. However, its function in health and disease remains surprisingly poorly understood. This is due to the complexity and robustness of the regulatory networks underpinning MT regulation which are experimentally difficult to decipher. To tackle this problem I am using a genetic model organism, the fruit fly Drosophila, I have extensive experience with this system and the role and regulation of the neuronal cytoskeleton therein. I have provided substantial proof of principle that regulatory mechanisms can be deciphered and applied to higher animals. Apart from the enormous amenability and speed of experimentation, the fundamental advantage for cytoskeletal research in Drosophila is the efficiency with which genes can be manipulated and investigated in combination. Thus, on this project, I capitalise on my finding that functions of tau become apparent when combined with loss of a second MTBP, called Shot. Only upon combined deletion does a new phenotype occur consisting in dramatic loss of synapses caused by collapse of axonal transport of synaptic proteins. This phenotype provides robust readouts to decipher the underlying mechanisms, which will be one key objective of this project. In addition, I will study the relevance of these mechanisms for neuronal survival and assess their potential conservation in mouse neurons. This work will unlock important new mechanistic understanding that will advance research on brain development, ageing and degeneration.

Technical Summary

This project aims to understand the role of two microtubule-binding proteins (MTBPs), Shot and Tau, in synapse formation and maintenance. Synapses require sustained axonal transport of synaptic proteins from the distant soma, which is a prerequisite for brain development, function and longevity, and synapse loss is considered an early step in neurodegeneration. Axonal transport is driven by motor proteins trailing along bundles of microtubules (MT), and changing the properties of these MTs can significantly impact on synaptic transport. MTs are regulated by MTBPs which are therefore in a key position to regulate synapse formation and homeostasis, although the underlying mechanisms remain speculative. Understanding these mechanism would help to explain the cell biology of synapse formation and maintenance and deliver mechanisms which sustain neurons in healthy ageing or are targeted in neurodegeneration.
Mutations in the MTBP Tau result in Alzheimer's and other neurodegenerative diseases, but its functions in neurons remain little understood. We previously suggested that Tau has functional links to the MTBP Shot (homologue of dystonin, linked to neurodegeneration in mouse and humans). Meanwhile, I have tested this in shot tau double-mutants, using efficient Drosophila genetics. I find striking suppression of synaptic transport leading to entire loss of synapses, a phenotype that is absent in shot or tau single mutants. This suggests functional synergy and opens up new research avenues into tau and the role of MTBPs in synapse regulation. Here I will use a range of synaptic proteins, mutations for different motor proteins, and refined imaging to pinpoint the precise properties of transport affected; I will investigate underlying mechanisms focussing on MT properties and the JNK pathway as candidates; I will demonstrate their in vivo relevance and test whether combined loss of the Shot and Tau homologues in mouse neurons causes comparable synaptic phenotypes.

Planned Impact

The proposed project will unravel fundamental mechanisms of the formation and maintenance of the nervous system, relevant also for work on ageing and neurodegeneration. To make sure impact is reached we will disseminate our work by a) publishing in high impact journals and contributing book chapters; b) presenting our work on national and international research conferences; c) developing on-line resources such as a lab webpage illustrating the main objectives, strategy and outcomes of the project; d) initiating and contributing to scientific multi-author blogs, discussing and informing about main breakthroughs in the area of our research (including our own work).

Since public opinion has an increasing impact on political funding decisions, we will foster public awareness and understanding of the importance of science and research. For this, our research is appealing to the general public which is fascinated by topics of brain research. To impact on the public we will: a) present our work on open days and in public lectures: the University of Liverpool has over 15 years experience in delivering high quality activities on a local, national and global level, and we will make frequent contributions within this framework; b) we will develop a special section on our webpage to inform and enthuse the public about our research; c) we will use the knowledge and experience we have on brain and cytoskeleton research to provide material and concepts for museum exhibitions whenever opportunities arise (see my contributions to the permanent exhibition "All about us" at the "At Bristol" venue).

This project addresses synapse loss and "dying-back" mechanisms likely to bring better understanding of neurodegenerative diseases, pave the way for future research and potential long-term benefits for social well-being. This will be of interest to the commercial and private sector. We know from the treatment of many cancers that the microtubule cytoskeleton is a promising drug target, and in the context of brain disorders this potential has not been well explored. Therefore, we will establish suitable contacts capitalising on the Research and Business Gateway provided by the University of Liverpool which aims to catalyse communication between researchers and business.
 
Description Synapses are key elements of functional nervous systems, and their maintenance during a neuron's life requires constant recycling of synaptic machineries. Defects in synapse maintenance are likely to lead to loss of functional synapses, and precocious loss of synapses is a key event during ageing and in many dementias. Very little is known about the mechanisms that regulate the maintenance of synapses. We have discovered a novel regulatory cascade downstream of Tau and Spectraplakins necessary for the delivering of synaptic proteins to axonal terminals in the developing and ageing brain.
We found that the loss of Tau or Spectraplakins causes the microtubules to fall apart and triggers an internal stress signalling pathway known as the JNK pathway. Activating JNK signalling blocks the transport of synaptic materials along axons, which prevents the formation of new synapses and starves existing synapses leading to their decay.
We further investigated the mechanisms by which Tau regulates microtubules. During this work we discovered a novel function of Tau in controlling the peculiar organisation of microtubules in the axon. This function involves competition of other microtubule regulators (eg. EB1) for the polymerising end of microtubules.
In view of the important roles of Tau in maintaining synapses, we investigated whether Tau and other microtubule are key players in the process of brain ageing. We found significant changes in the localisation of Tau and in other microtubule regulators during physiological ageing. Importantly, these changes could be the cause of neuronal atrophy which typically occurs during ageing and we currently investigate this possibility.
These findings stimulated us to develop a very powerful new model to study brain ageing in Drosophila. Our new in vivo model shows gradual subcellular changes (cytoskeletal decay, deterioration of axons and loss of synapses) and recapitulates many changes seen in the human brain. Therefore, its use has great potential to advance our knowledge of mechanisms of brain ageing. Unlike vertebrate models, these hallmarks manifest fast, within 4 weeks only, and their underlying molecular and cellular mechanisms can be efficiently dissected with impressive subcellular resolution and with powerful Drosophila genetics.
In addition, we also study several models of tauopathies based on the expression of human tau variants that carry disease related mutations. We are characterising the changes they induce at the subcellular level. So far we find that increasing the level of Tau causes important morphological changes and this is cause by altering the properties of microtubule networks. These findings could open up new opportunities to develop therapies for Alzheimer's and other neurodegenerative diseases.
Exploitation Route These findings providing potential explanations for precocious synapse loss in ageing and dementias. They also link Tau (key in Alzheimer's disease) to synapse maintained, providing an alternative explanation for the disease. The next step is to find out whether Tau and spectraplakins play similar roles in the nerve cells of mammals, which may open up new opportunities to develop therapies for Alzheimer's and other neurodegenerative diseases. Furthermore since physiological ageing of the human brain is characterised by changes in the localisation of Tau, our findings could help to understand the causes of cognitive decline during physiological ageing. In this context our new model to study brain ageing in Drosophila, is efficient, cheap and representative of ageing in vertebrates and could be used for research into mechanisms of ageing.
Sectors Pharmaceuticals and Medical Biotechnology,Other

URL https://elifesciences.org/content/5/e14694/article-metrics
 
Description Dual PhD studentships University of Liverpool, University of Szeged
Amount £48,000 (GBP)
Organisation University of Liverpool 
Sector Academic/University
Country United Kingdom
Start 10/2015 
End 09/2017
 
Description University of Liverpool: - Dual PhD studentships University of Liverpool, Riken, Japan
Amount £48,000 (GBP)
Organisation RIKEN 
Department RIKEN Brain Science Institute
Sector Public
Country Japan
Start 10/2017 
End 09/2021
 
Description Wellcome trust student ship
Amount £40,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Wellcome trust student ship
Amount £40,000 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2018 
End 09/2021
 
Title A model in Drosophila to study axonal ageing in the brain with high subcellular resolution 
Description We have developed in Drosophila, a model to study neuronal ageing in the brain with high sub cellular resolution. This model reproduces several of the changes observed in studies with vertebrate models, but it is cheaper faster and does not have ethical concerns associated to it. We believe this model could realistically reduce the number of animals used in research into the mechanisms leading to ageing of the brain. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2017 
Provided To Others? No  
Impact We are using this model to study mechanisms of neuronal ageing. This work is on progress. 
 
Description Collaboration with Dr Moore to investigate the links betwen APP and Tau 
Organisation RIKEN
Department RIKEN Brain Science Institute
Country Japan 
Sector Public 
PI Contribution We discovered that APP (abeta precursor involved in Alzheimer disease ) is able to regulate neuronal microtubules in axons ans dendrites probably by regulating tau function. We are characterising the details of this interaction in axons.
Collaborator Contribution Dr Moore is contributing towards the characterisation of this interaction in dendrites of sensory neurons in Drosophila
Impact NA
Start Year 2018
 
Description Collaboration with Dr. Chris Elliott to measure electrical properties of lamina neurons using Steady state visually evoked potential in tauophaty models 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution We have developed a tauophaty model using the lamina of the Drosophila brain in which morphological changes in synaptic terminals are detected. Next step in collaboration with Dr. Chris Elliott will be to characterise synaptic changes at the functional level.
Collaborator Contribution Dr. Chris Elliott is providing the infrastructure and the know how in order to quantify Steady state visually evoked potential
Impact NA
Start Year 2018
 
Description Collaboration with Prof Alice Ting to perform proximity binding assays with Tau 
Organisation Stanford University
Country United States 
Sector Academic/University 
PI Contribution We are performing proximity binding assays with Tau to determine changes in tau interactome in tauopathy models
Collaborator Contribution Prof Alice Ting has provided the miniTurbo BioID tools for human cell lines.
Impact NA
Start Year 2017
 
Description Collaboration with Prof Perrimon Norbert to perform proximity binding assays with Tau 
Organisation Harvard University
Department Harvard Medical School
Country United States 
Sector Academic/University 
PI Contribution We are performing proximity binding assays with Tau to determine changes in Tau interactome during ageing in vivo
Collaborator Contribution Prof Perrimon Norbert has provided miniTurbo BioID tools for Drosophila research
Impact NA
Start Year 2017
 
Description Collaboration with Prof Prokop and Dr Han to understand the link between aberrant microtubule regulation and the onset of epilepsy in Drosophila 
Organisation University of Manchester
Department School of Health Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We discovered that defects in microtubule regulators such as Tau induces epilepsy in Drosophila. We currently testing inhitory pathways as possible mechanisms using in vivo and cell culture approaches and localisation studies of components of the GABAergic pathway.
Collaborator Contribution Prof Prokop and Dr Han are contributing with neurocircuit assays for seizures in mutants of Drosophila
Impact NA
Start Year 2018
 
Description Collaboration with Prof Prokop and Dr Han to understand the mechanisms by which Tau regulates the organisation of axonal microtubules into parallel bundles 
Organisation University of Manchester
Department Faculty of Science and Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution We are investigating how Tau and other regulators control the organisation of axonal microtubules into parallel bundles by the use of fly genetics and in vivo models to study axonal microtubules.
Collaborator Contribution Prof Prokop and Dr Han are providing complementary experiments using neuronal cultures from Drosophila embryos
Impact NA
Start Year 2017
 
Description Collaboration with Profesor Bassem Hassan to investigate mechanisms of Drosophila brain wiring downstream of the Slit-Robo-RPTP signaling complex. 
Organisation University of Leuven
Country Belgium 
Sector Academic/University 
PI Contribution We investigated the Slit-Robo-RPTP signaling complex at the sub-cellular level using primary neuronal cultures from Drosophila embryos
Collaborator Contribution They investigated the Slit-Robo-RPTP signaling complex in vivo and using biochemistry
Impact 1 publication: Oliva, C., Soldano, A., Mora, N., De Geest, N., Claeys, A., Erfurth, M., Sierralta, J., Ramaekers, A., Dascenco, D., Ejsmont, R., Schmucker, D., Sanchez-Soriano, N., Hassan, B. (2016). Organization of Drosophila brain wiring by the mushroom body via a Slit-Robo-RPTP signaling complex. Developmental Cell, 39(2), 267-278.
Start Year 2015
 
Description Collaboration with Profesor Mihály to investigate the formin DAAM in the regulation of the microtubule cytoskeleton in axonal growth cones. 
Organisation Hungarian Academy of Sciences (MTA)
Department Department Institute of Genetics Research
PI Contribution We demonstrated that DAAM stabilises microtubules in axonal growth cones.
Collaborator Contribution They demonstrated that DAAM binds to micritubules and actin and that regulates polymerisation of microtubules in vivo ans in vitro
Impact One publication: Szikora, S., Földi, I., Tóth, K., Migh, E., Vig, A., Bugyi, B., Maléth, J , Hegyi, P , Kaltenecker, P , Sanchez-Soriano, N., Mihály, J. (2017). The formin DAAM is required for coordination of the actin and microtubule cytoskeleton in axonal growth cones. Journal of cell science, 130(15), 2506-2519.
Start Year 2017
 
Description Participation in 2 open days at my research institution ( Application Discovery Days ) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity was aimed to showcase the research carried in the lab and how with our work we can advance knowledge about the cause of neurodegenerative diseases and ageing
Year(s) Of Engagement Activity 2017
 
Description Applicant Discovery Days University of Liverpool (2 times participation in ) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact This activity was aimed to showcase the research carried in the lab and how with our work we can advance knowledge about the cause of neurodegenerative diseases and ageing
Year(s) Of Engagement Activity 2018
 
Description Art exhibition 
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 Spot On Micro - Image contest and exhibition aims to bring sciences closer to the general public by presenting sciences as objects of artistic beauty. Several images produced in my laboratory were short listed in order to be displayed at the Royal Botanic Garden of Edinburgh in Oct. 2015.
Year(s) Of Engagement Activity 2015
URL http://www.spot-on-micro.eps.hw.ac.uk
 
Description Brain Awareness Week (Biological Research Centre, Szeged, Hungary) 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact To increase awareness about research carrion to understand mechanisms of formation of neuronal networks and their maintenance ain
Year(s) Of Engagement Activity 2018
 
Description Co-organiser of the Data Reproducibility and Communication workshops 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I co-organised a workshop on Data Reproducibility and Science Communication
Year(s) Of Engagement Activity 2018
 
Description Contributor to Grant Skill Development workshop (University of Liverpool) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact I contributed with a talk and debate to a workshop on development of grant writing skills for University of Liverpool staff and students.
Year(s) Of Engagement Activity 2018
 
Description NC3Rs workshop: non-mammalian model organisms in bioscience research 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact I contributed to a NC3Rs workshop on non-mammalian model organisms in bioscience research with a talk and a hands on stand. The aim was to bring awareness of possibilities to use Drosophila to understand neurodegeneration and loss of cognition during ageing.
Year(s) Of Engagement Activity 2018
 
Description Participation in 3 independent Application Discovery Days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Applicant Discovery Days are designed for students and their parents/guardians who have chosen Liverpool as one of their 5 options to study a degree. They are invited to tour around the labs and are introduced to research topics follow by small demonstrations / talks.
Year(s) Of Engagement Activity 2016
 
Description Small Brains, Big Ideas, University of Valparaiso, Chile 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I provided materials to instruct researches from Latin America (mostly at the PhD and postdoc level) to the use of Drosophila for neuroscience research. The use and maintenance of invertebrate models is inexpensive and straightforward and this makes them ideal subjects for research in areas of the world where resources available for scientific research are limited, such as Latin America
Year(s) Of Engagement Activity 2016
 
Description The Brain Box 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact The Brain Box was an exciting outreach activity taking place in the spectacular Town Hall right in the middle of Manchester on Manchester Day - The activity was designed in order to allow the public to discover the wonders of the brain: how it works, how it is studied, what can go wrong in our brains, and how this might be fixed. The event gave a unique opportunity for the public to interact with scientists, clinicians, creative practitioners and artists with exciting and thought-provoking exhibits, displays and hands-on activities for all ages. Feedback on the event can be found at thttps://mcrbrainbox.wordpress.com/legacy/
Year(s) Of Engagement Activity 2016
URL https://mcrbrainbox.wordpress.com/
 
Description The CAJAL Advanced Neuroscience Training Program (FENS and IBRO initiative, France) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact In this activity I instructed for 10 days a group of international researchers wishing to learn techniques and approaches in order to research questions related to the cell biology of the neuron using the model organisms Drosophila. The workshop involved a series of lectures and practical work.
Year(s) Of Engagement Activity 2016
 
Description Tour of facilities and induction to research during Freshers week at University of Liverpool 
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 For new undergraduate students, as a part of the induction week we offer the students a tour of facilities and introduce them them to the varied research which they could be aiming towards in their degrees.
Year(s) Of Engagement Activity 2016