Mechanisms of long-range retrograde signal propagation and morphological plasticity in hippocampal neurons

Lead Research Organisation: University of Southampton
Department Name: School of Biological Sciences

Abstract

The aim of this work is to understand how long-range growth factor signalling shapes neurons of the brain. Throughout life we encounter new experiences, meet new people, learn and forget. All this information is encoded within neuronal networks of the brain, and the ability of the brain to remodel and adapt these networks to store new information is termed plasticity. To restructure neuronal networks, components of the network, i.e. individual neurons change their shape. They extend processes where new synaptic connections are built, and retract other processes where old ones are abolished. This is a strictly controlled and tightly balanced procedure important in normal brain development, and its deregulation contributes to cognitive dysfunction.
Neurons are specialised cells that extend axons and dendrites over very large distances. Particularly the axon, the "delivering end" of the neuron, can reach up to several centimeters in the human brain, and up to over a meter in motor and sensory neurons, from the spinal cord to the toes. This poses multiple logistical challenges, for example with regards to information processing within the neuron. Neurons communicate with their environment via electrical signal propagation and neurotransmitter release, or via a slower mode of communication, involving growth factors. These molecules are detected by receptors that sit on the neuronal surface and can prompt signalling cascades inside the cell. As their names suggest, growth factors are key to the structural organisation of the cell. In contrast to electrical signals, growth factor signalling is not by default unidirectional, and therefore allows the cell body to gather information about events happening at the axon terminal. This direction of information flow is termed "retrograde". Each growth factor detected near the axon terminal ("distally") may initiate signals that are processed locally, to be translated into e.g. "start building a new synapse at this place". Alternatively, it can be sent back to the cell body, to instruct the entire neuron to start growing. This retrograde long-distance communication from the terminal back to the cell body is crucial for neuronal health, and defects in this process have been found associated with a large number of diseases, including motor neuron degeneration and dementia. However, surprisingly little is know about retrograde signalling within neurons of the brain.
Brain-derived neurotrophic factor (BDNF) is a major growth factor of the brain that is first detected early postnatally and is expressed throughout life, and is known to promote network plasticity. The work proposed here will address how signals activated by BDNF at the distal axon are processed within the neuron, and how this relates to changes in cell shape and thus neuronal plasticity. We are particularly keen to understand what information is sent back to the cell body, where it can alter gene expression and affect the makeup of the entire neuron, and how this process of sending back information works on a mechanistic level. We will address this question by growing hippocampal neurons in specialised devices that allow isolation and controlled perfusion of the axon with BDNF. We chose the hippocampus as it is a highly plastic area of the brain, crucial for learning and remembering. We will identify the intracellular mechanisms that are activated locally and those relayed back to the soma using this approach, and monitor how interfering with select information impacts on changes in the shape of individual neurons. This will help understand how all signals, supportive or adverse, are communicated over distances in neurons of the brain, and why this process is so crucial for the proper function of individual nerve cells. A broader knowledge of mechanisms required for normal maintenance and plasticity of individual neurons will also provide information about processes that are failing in cognitive dysfunction.

Technical Summary

Neuronal networks are maintained and remodelled in a controlled manner throughout life, a process called plasticity that is tightly coupled to neuronal activity. BDNF is an example of a growth factor that impacts morphology and is released at synaptic areas in response to activity. This stimulus may be processed locally, or alternatively the soma detects and reacts to such signals initiated at distal axons; our previous studies suggest that a combination of both is the case. The neuron then translates this information into defined changes in morphology to maintain appropriate connectivity, a process that remains poorly understood.
Retrograde signal propagation is critical to neuronal function in both central and peripheral neurons. My preliminary work suggests that there are key differences in retrograde receptor trafficking between central and peripheral neurons, which implies also differences in long-range signalling. We have recently identified a broad range of signals generated by BDNF in central neurons using quantitative mass spectrometry, and characterised a mechanism of BDNF-mediated axon branching. Combining my experience in neuronal signalling and membrane trafficking and the results of our proteomic screen with the local expertise in bioengineering, we will now ask how retrograde signal propagation impacts the morphology of hippocampal neurons. We will develop microfluidic devices that allow us to investigate retrograde axonal signalling in response to tightly controlled stimulation (spatial and temporal) using Western blotting to identify signals and live cell imaging approaches to monitor morphological effects over time.
This work will begin to illuminate retrograde signal propagation along axons of brain-derived neurons, and define underlying mechanisms how retrograde signalling impacts on the morphology of these neurons, both outstanding questions in neuronal cell biology.

Planned Impact

Pharmaceutical Industries and Charities: The project addresses fundamental cell biological/ neurobiological questions of signalling and morphological plasticity. However, there is a tight correlation of reduced BDNF levels with a number of neurodegenerative diseases and mental health dysfunctions, and observations exists that for example (i) genetically raising BDNF levels ameliorates symptoms in mouse models of Huntington's disease and (ii) antidepressants generally raise BDNF levels. Combined with the fact that BDNF itself does not cross the blood-brain barrier and no functional biomimetics exist, advancing the understanding basic BDNF cell biology may help identify alternative strategies and highlight new potentially druggable targets and processes that may ultimately lead to therapies ameliorating disease progression. I will engage with the translational community at Southampton General Hospital through the Southampton Neuroscience Group and joint seminars to explore this further.
Economic Impact: As addressed above, the proposed project is basic neurobiology research, and as such the future economic impact is difficult to assess. However, publications and communications arising from this work are expected to be of interest to the international research community and contribute to the competitiveness of the UK Higher Education Institutions. Further, in line with the potential impact on general health, mental health disorders affect a significant, often relatively young, part of the population and their immediate relatives, and as such negatively impact on the skilled workforce, while neurodegenerative disorders place a huge burden on the health system. Thus any potential new, druggable avenues uncovered by advancing the basic understanding of morphological plasticity may also ultimately, indirectly, have a significant economic impact.
General Public/ Quality of Life: A better general understanding of molecular mechanisms underlying brain plasticity may lead to a broader acceptance of mental health disorders as a true illness, and thus help de-stigmatise those suffering from these disorders. In addition, given the high number of people affected by neurodegenerative diseases or mental health disorders, any advances in the basic understanding of neuronal plasticity that may ultimately lead to new therapeutic approaches have the potential to significantly contribute to the overall general health and well-being of the population. I will engage with the general public through local outreach activities such as "Bringing Research to Life. University of Southampton Roadshow" and the Science and Engineering day open house.

Publications

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Bowling HL (2019) Proteomic Approaches to Dissect Neuronal Signalling Pathways. in Advances in experimental medicine and biology

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Hallinan GI (2019) Tau Misfolding Efficiently Propagates between Individual Intact Hippocampal Neurons. in The Journal of neuroscience : the official journal of the Society for Neuroscience

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Hallinan GI (2019) Minimalistic in vitro systems for investigating tau pathology. in Journal of neuroscience methods

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Martinez-Nunez RT (2017) Modulation of nonsense mediated decay by rapamycin. in Nucleic acids research

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Peter Baksh, Michal Odstrcil, Aaron Parsons, Jo Bailey, Katrin Deinhardt, John E. Chad, William S. Brocklesby And Jeremy G. Frey (2016) Quantitative Evaluation of Hard X-ray Damage to Biological Samples using EUV Ptychography

 
Description We have developed the use of microfluidic devices for analysis of long range growth factor signalling in collaboration with Professor Hywel Morgan, Centre for Hybrid Biodevices at the University of Southampton. We have performed several iterations of device design and validated new generation devices. This has led to a collaboration with the Hislop lab in Aberdeen.
We have identified BDNF long-range signals that feed into morphological plasticity. This has opened up questions regarding the integrity of these signalling pathways in neurodegeneration and their potential as a therapeutic target, which has led to a Gerald Kerkut PhD studentship.
The award has further led to local (Brocklesby, Frey labs) and international (Difato lab, IIT Genova) collaborations that develop imaging methods and protocols for remote mechanical stimulation of neurons, that have both led to joint publications and funding applications.
Exploitation Route New device designs could be exploited by others to look at long range signalling or signal transfer between different neuronal subtypes.
Sectors Other

 
Description Scientific Cafes on the ARUK South Coast Dementia Day, discussed research with interested general public (March 2017, January 2018, January 2019) "Changing minds" project on Mental Health Awareness with the Peter Symonds college (2016) external invited speaker at PhD symposium "Cell to cell communication"; in December 2015, San Raffaele, Milan, Italy Invited speaker at Pint of Science, Southampton, audience of 50+ general public (May 2015) running the "dementia" table on the Wessex Medical stand at the New Forest show (August 2015)
Sector Education
Impact Types Societal

 
Description Course - Milan
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Production of Fellowship Booklet for British Neuroscience Association
Geographic Reach Europe 
Policy Influence Type Influenced training of practitioners or researchers
 
Description Alzheimer's Research UK PhD Scholarship
Amount £60,000 (GBP)
Funding ID ARUK-PhD2014-10 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description Alzheimer's Society Biomedical PhD studentship
Amount £84,953 (GBP)
Funding ID AS-PhD-2015-029 
Organisation Alzheimer's Society 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 09/2019
 
Description Gerald Kerkut PhD studentship
Amount £27,000 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2014 
End 09/2017
 
Description Wessex Medical Research Innovation Grant
Amount £19,000 (GBP)
Organisation Wessex Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2015 
End 08/2017
 
Description Ultrasound stimulation 
Organisation Italian Institute of Technology (Istituto Italiano di Tecnologia IIT)
Country Italy 
Sector Academic/University 
PI Contribution In collaboration with the Difato lab at the IIT and Dario Carugo at ISVR, UoS we express mechanosensitive channels in neurons to assess selective stimulation by Ultrasound. We provide the expertise in neuronal cell biology and physiology, as well as the primary cultures themselves.
Collaborator Contribution Our partners provide the expertise in mechanosensitive channels, and in engineering ultrasound devices and defining and characterising stimulation paradigms.
Impact This is a multidisciplinary collaboration between neuronal cell biologists and biomedical and electromechanical engineers. We established this collaboration in 2016 and are part of a multi-partner funding bid to the EU. The collaboration has resulted in a publication.
Start Year 2016
 
Description Xray 
Organisation Diamond Light Source
Country United Kingdom 
Sector Private 
PI Contribution Provision of biological samples and expertise on their subcellular makeup
Collaborator Contribution development of imaging techniques to visualise different features of these samples, imaging and data analysis expertise
Impact multidisciplinary collaboration with physicists developing label-free imaging techniques
Start Year 2015
 
Description Xray 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of biological samples and expertise on their subcellular makeup
Collaborator Contribution development of imaging techniques to visualise different features of these samples, imaging and data analysis expertise
Impact multidisciplinary collaboration with physicists developing label-free imaging techniques
Start Year 2015
 
Description ARUK public engagement event, AMEX stadium Brighton 
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 100 participants attended the engagement afternoon where we presented our research on neuronal network function and dysfunction, which led to lively discussions
Year(s) Of Engagement Activity 2019
URL http://www.sussex.ac.uk/broadcast/read/47644
 
Description Joanne Bailey elected as Early Careers representative on to British Neuroscience Association committee. 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Joanne Bailey elected as the Early Careers representative on the British Neuroscience Association Committee.
Year(s) Of Engagement Activity 2016,2017
 
Description Mental Health Public Engagement Project with Peter Symonds College 
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 The University of Southampton researchers and A-Level students from Peter Symonds College (Winchester) have teamed up to create a neuroscience-inspired artistic display.
The aim of the exhibition is to reduce stigma and increase awareness and understanding of mental health and brain disorders. Researchers from the University of Southampton visited Peter Symonds College to provide insight into how their research is revealing the molecular underpinnings of neurodegenerative and psychiatric disorders. The student's learnt about the different cells in the brain and how their function and morphology can change in disease. The aim was to inspire the students to depict/interpret a certain aspect of brain disorder/diseases, through the medium of textiles and art. With the use of bright colours and words, the student's designs have portrayed their impressions of neurological condition, ranging from Alzheimer's, Huntington's disease and depression.

This display, where 'art meets the mind', is a powerful and disarming medium to engage the public in discussions about mental health. This is a novel way to open science up to new audiences - those who thought that science was inaccessible to them as well as those who do not feel comfortable about discussing mental health issues.

The display at the Birmingham ICC will include garments, depicting aspects of brain disorders and their effects, as well as posters and sketch books explaining the pieces of art.
Year(s) Of Engagement Activity 2016,2017
 
Description Museum visit (Basingstoke Willis Museum) 
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 Dr Jo Bailey, with the help of a PhD and an undergraduate student, hosted a brain imaging stand at the museum, open to the general public, as part of the Wellcome Image Awards Exhibition. The stand was aimed at Primary and Secondary school children and included demonstrating the complex shapes and forms neuronal brain cells can take, how such cells are probed and researched by growing them in a dish and how neurons can be made to 'glow-in-the-dark'. Using the power of play doh and pipe cleaner straws approximately 80 adults and children had the opportunity to design and make their own 'glowing' neuronal cell. Some of the best were exhibited on a board-of-fame. Hopefully all who visited the brain imaging stand gained an insight into the beautiful world of brain imaging, and an appreciation for the complex architecture that makes up our brains.

none returned yet - event happened less than a week ago
Year(s) Of Engagement Activity 2014
 
Description Neuroscience Sixth Form Outreach Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Out of the 100 students who attended the outreach day 18 wrote in their feedback forms that the event had convinced them to study Bio-medical Sciences/ Neuroscience at University. Even better were the 8 who were inspired after seeing the facilities and laboratory research at Southampton to apply to the University of Southampton for their degrees.

In total over 100 students and teachers attended the event from 12 schools, some as far as Bristol and London, and with over 30 students still remaining on the waiting list. 12 interactive workshops were brought together by PhD students, Research Fellows and Undergraduates to promote science and inspire the young minds of our future. The feedback from the event was fantastic; some were even convinced to study Science at Degree level and were instilled with a passion for Neuroscience.
Year(s) Of Engagement Activity 2015
URL http://www.southampton.ac.uk/biosci/news/2016/01/song-sixth-form.page?
 
Description New Forest Show Dementia Stand 
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 The New Forest Show is spread over three days and is frequented by 1000s of local people and holiday makers. Members of the University of Southampton Neuroscience Group (SoNG) brought three themes to the event. The first day saw early career researchers Shmma Quraishe and Joanne Bailey, along with PhD students Prutha Patel and Grace Hallinan teaching children and adults of all ages what the brain is made of, and how the brains cells, neurons, are able to form memories. A particular focus of the event was to try and pick apart the social stigma associated with mental health disorders. Dresses designed by students from the Winchester School of Art, as part of a collaborative project entitled 'Changing Minds through Neuroscience Inspired Fashion' were displayed which depict the symptoms of the disease, social isolation and the cognitive and behavior changes the affected person feels. The dresses stimulated conversation and questions from the general public who hopefully left with a better understanding of brain disorders.
For the children, our scientists of the future, playdoh was on hand to demonstrate the different compartments of a neuronal cell and their function. Overall, the day was a great success, and hopefully this will be reflected in future donations to Wessex Medical Research.

The event summary was published in the BNA bulletin.

The main impact was made by talking to the general public about neurodegenerative and psychological diseases. The Changing Minds Project, which acts to stimulate conversion through Neuroscience inspired fashion, engaged the public to talk about these disorders and the stigma and misconceptions surrounding them.
Year(s) Of Engagement Activity 2015
URL http://www.deinhardtlab.moonfruit.com
 
Description Outreach activity- detangling the knots 
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 Providing scientific input, hosting artists in the lab and providing scientific images for a dementia related outreach project, funded by the Arts Council England, that was on display as part of the Human Worlds Festival (first prize, creativity award) and will be taken into Care Homes next.
Year(s) Of Engagement Activity 2020
URL https://letscrochetuk.wordpress.com/starting-detangling-the-knots/
 
Description Pint of Science Festival 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The audience were very engaged on all three nights of the festival, asking interesting questions and evoking discussions and debates

Feedback forms indicated that the audience were very impressed by the festival and we have been both requested to manage the team for the 2016 festival and to keep in touch with the pub landlord and audience about hosting in the same location
Year(s) Of Engagement Activity 2015
URL http://www.southampton.ac.uk/biosci/news/2015/05/22-pint-of-science-jo-bailey.page
 
Description exhibiting at ARUKs "Reframe Dementia" 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact our microscopy images contributed the the ARUK "Reframe Dementia" exhibition at the gallery@oxo, South Bank, London
Year(s) Of Engagement Activity 2018