Regulation of axonal branching in vivo

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

Our nervous system is made of up to 1000 billion neurons governing thinking, feeling, moving, learning and memory amongst other activities. This is possible because neurons are highly interconnected (creating neuronal circuits) and connected to all the organs of the body in a regulated manner. Understanding how neuronal circuits are established and repaired is a major challenge of developmental and regenerative neurobiology. Furthermore, upon injury, neuronal circuits often fail to reform, leading to severe disabilities. This is particularly important after spinal cord injuries which can lead to partial or total paralysis.
In my laboratory, we are studying the molecular mechanisms regulating neuronal growth and shape (or morphology) in the complex environment of the whole organism. In particular, we are investigating how motor neurons develop and function. Motor neurons are highly specialised cells with long extensions, called axons, which can travel up to one metre away from their cell body to reach and innervate their target organs, the muscles. Therefore, the growth, guidance and morphology of these axons must be tightly controlled. We use the frog Xenopus as an experimental model because the embryos develop externally allowing us to visualise directly the events leading to motor neurons development. Furthermore, frog tadpoles start to swim 2-3 days after fertilisation. We can therefore correlate defects in motor neurons development with functional tests on their function.
We have recently identified a molecule expressed specifically in motor neurons which has an essential role in axonal growth and branching. We want to build on this recent discovery to gain insight in the regulation of axonal branching in this population of neurons. To this end, we will combine state-of-the-art experiments in living embryos and studies in neuronal cells in culture to uncover the molecular and cellular events that occur during axonal branching. Whilst we know that the molecules are conserved across species, our ultimate goal is to investigate whether the mechanism regulating axonal branching is also applicable in human motor neurons. This will allow us to explore new therapeutic avenues for conditions such as spinal cord injuries.

Technical Summary

Defects in axonal growth, guidance and branching of neurons have very severe effects on their ability to transmit information appropriately. Whilst axonal growth and pathfinding have been intensively studied, much less is known about axonal branching, despite the fact that axonal branching plays an essential role in the formation of neuronal circuits. Furthermore, it has been suggested that branch sprouting might play a role in nerve regeneration following injury.
The neurotrophin Brain Derived Neurotrophic Factor (BDNF) is a known inducer of axonal branching. However, the cellular and molecular mechanisms by which BDNF induces branching are still elusive. We have recently identified a negative regulator of BDNF signalling, Spry3, which prevents axonal branching in motor neurons (MNs). We want to build on this discovery to uncover the molecular mechanisms governing axonal branching in MNs. In particular, we will elucidate the intracellular signalling pathways involved in axonal branching from the BDNF receptor to the cytoskeleton. To this end, we will explore the role of the different intracellular signalling pathways activated by BDNF stimulation during axonal branching. We will also establish the molecular mechanisms by which Spry3 prevents axonal branching in MNs.
To achieve these objectives, we will combine genetic tools (transgenesis), high quality live imaging (in vivo and in MNs in culture) and biochemical analyses. Together, the results obtained during this grant will not only inform us on the molecular mechanisms of axonal branching but will have wider implication in the regulation of Receptor Tyrosine Kinase signalling. Finally, this work will provide a framework to explore the idea that axonal branching is a viable option to promote neural regeneration.

Planned Impact

1. Beneficiaries of the research. This project will uncover the molecular mechanisms underlying the control of neuronal morphology by receptor tyrosine kinase signalling. This is a basic research project which will benefit the academic community with an interest in cell biology, signalling and neurobiology. However, the process we are interested in (axonal branching) and the molecules involved in this process (BDNF, PI3K, MAPK, Sprouty) have all been involved in human pathologies. Therefore, the proposed work will also benefit the biomedical sector investigating neuronal regeneration, the pharmaceutical companies developing new drugs for people suffering from diseases such as Amyotrophic Lateral Sclerosis (and motor neuron diseases in general). We will work in close collaboration with UMIP (University of Manchester Intellectual Property) to identify any potential commercial application arising from our research.
2. The PDRA and TA employed on this grant will benefit from excellent training in embryology, molecular biology, microscopy and neurobiology. The University of Manchester has also a comprehensive range of training courses and career development programme which will be very beneficial to the PDRA.
3. Public engagement. Developmental biology and Neurobiology are areas of science of great interest to the general public. Furthermore, the nature of our research, involving live imaging of embryos and cells, generate movies which are fascinating for a lay audience. This year, I was one of the organisers of a Faculty-wide event for the National Science and Engineering Week (NSEW), named the Body Experience. This event involved coordinating more than 40 Faculty staff and students to present the role of different organs of the body (brain, gut, skin) to the public at the Manchester Museum. This event was a success and will be organised on a yearly basis. The PDRA will be encouraged to take part in the Researchers in Residence Scheme and other Faculty initiatives such as displays at the Manchester Science Festival. The Faculty provides training and support in such activities. We are also in contact with the press office to ensure that research appropriate for media release will be advertised to the local and national media as well as relevant patient groups. The Faculty has a dedicated media advisor who has been very successful in disseminating our research to the national and international media. The University also houses a popular museum (The Manchester Museum), which provides a direct and permanent link between our research activities and the local public.
4. Dissemination of our research. Throughout this project, we will communicate our results by several means. We will publish our finding is a timely manner in peer-reviewed, open access journals. The PI and PDRA will also communicate our results by presenting our data in conferences and by giving seminars at universities in the UK and abroad.
 
Description The purpose of the grant is to understand how a simple ion,calcium, can regulate cell shape and therefore cell function. We have made significant advances on the molecular mechanisms regulating the changes of calcium concentration in the cell. We are now concentrating our efforts in understanding the consequences in term of cell behaviour.
During the course of the grant we have developed extensive expertise in calcium imaging in different systems (cells in culture, embryos etc). We have also developed techniques to manipulate the genome of our model animal, the frog Xenopus. In the long run, this will allow us to perform genetic experiments in a model where it was not possible only a few years ago.

This grant has allowed us to obtain fund from the BBSRC to develop a collaboration with the lab of Prof Tim Gomez in Madison, Wisconsin, USA. He is a world expert in neuronal cell biology and this collaboration will greatly enhance the impact of our research.
Exploitation Route During the course of this research, we have uncover new cross talks between different signalling pathways. This will impact our understanding of how cells integrate multiple extracellular signals.
We have also developed tools to study neuronal development and regeneration (such as a transgenic frog expressing GFP specifically in motor neurons) which will be useful for the community.
Furthermore, we have secured further funding from the Wellcome Trust to continue and expand this work
Sectors Other

 
Description BBSRC-DTP PhD studentship
Amount £80,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2013 
End 09/2017
 
Description Seed Award in Science
Amount £99,844 (GBP)
Funding ID 205894/Z/17/Z 
Organisation Wellcome Trust 
Department Wellcome Trust Bloomsbury Centre
Sector Academic/University
Country United Kingdom
Start 03/2017 
End 09/2018
 
Description UK-US partnering award
Amount £29,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2014 
End 07/2018
 
Description Dorey-Gomez partnerships 
Organisation University of Wisconsin-Madison
Country United States 
Sector Academic/University 
PI Contribution this is a UK-US partnering award from the BBSRC which has just started in August 2014. The objective is to obtain joint funding from the NIH or the NSF
Collaborator Contribution we are collaborating to develop an in vivo system to study axonal growth, guidance and degeneration. The partner is bringing its expertise in celll biology
Impact n/a
Start Year 2014
 
Description Faculty Open Day 
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 Public/other audiences
Results and Impact The activity was targeted at families stimulating thinking for both parents (via presentation) and children (via hands-on activities). Both groups asked a lot of question and increase their understanding of the research in the lab.


The activity was organised in the lab, so they could experience the setting of lab research
Year(s) Of Engagement Activity 2014
 
Description National Science and Engineering Week 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact We presented our work on wound healing and regeneration, emphasising the important roles that model organisms play in this research

The audience was very varied in age and background. We did a mix of scientific presentation and hands-on experience both of which were very well received
Year(s) Of Engagement Activity 2011,2012,2013