Investigating the impact of nervous system maturation on transmembrane receptor localisation and transport within CNS axons

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

Abstract

In the UK alone, 40,000 people live with paralysis, with 1,200 people becoming injured each year. Throughout the world, approximately 2.5 million people are living with paralysis. To date, there is no cure for paralysis and spinal cord injury, only rehabilitative therapy regimes. Once severed, the best instances of nerve fibre (axon) regrowth is limited to local sprouting at the site of injury, with few prospects for reconnecting damaged fibres or regaining lost function.

Further understanding about how the environment of the adult brain and spinal cord interacts with nerve cells is required in order to develop successful tools to treat neurological conditions. Evidence for failed central nervous system (CNS) regrowth implicates both the hostile injury environment and an inherent inability of nerve cells to repair themselves. This latter fact is due largely to a developmental reduction of growth-promoting molecules in the adult CNS. Studies reintroducing growth-promoting molecules in cultured neurons (nerve cells) show enhanced growth but, upon translation into an animal model, the resulting repair is very modest. A primary example is the re-expression of growth-inducing integrin receptors, which are critical for central nerve development and promote significant neurite growth in cultured neurons. In a model of CNS injury, however, the regrowth is not as robust but we went on to show that re-expressed integrins are only present in the nerve cell body and not transported within axons. Interestingly however, this is not the case in early postnatal neurons where integrins are present in perinatal brain and spinal cord axons. At this age, specialised structures such as myelin (the insulating cover of nerve fibres that increase the speed of nerve signals) as well as the extracellular matrix (structural support) are not fully developed. Likewise, in certain adult neuronal subtypes (neurons of the eye and peripheral nerve) this transport defect is also not present. These neurons do not contain specialised extracellular matrix (known as 'perineuronal nets' or PNNs) which cover many adult neurons. Without understanding how the transport of these molecules is affected by the mature nervous system, the success of integrin-based and similar treatments in future clinical translation is severely limited.

Thus, this project will determine what factors in the CNS inhibit axonal transport/expression of integrin molecules, and also use modifications previously shown to target molecules to axons to target integrins. More specifically, our initial aim will use two different experimental models: the first will utilise a transgenic mouse in which we can eliminate myelin-producing cells specifically in the area where neurons have been modified to express integrins; and the second will utilise an enzyme (chondroitinase ABC) to breakdown PNNs in the area where neurons have been modified to express integrins. The second aim will identify molecular strategies that target integrins into axons by modifying the integrin structure, thus overriding any external influence that the CNS may have on expression.

Results from all of the above experiments will involve advanced microscopic analysis of brain and spinal cord tissue in order to determine integrin expression within each experimental group. Fluorescently-labelled integrins will be used to visualise the expression within axons at an early (3 wks) and late (8 wks) time point, the former to allow expression of the integrins, and the latter to assess transport in conjunction with a spinal injury. These data will contribute greatly to neurological repair by determining factors both extracellular and intracellular which can affect expression of potential therapies involving re-expression of growth-promoting neuronal receptors. Additionally, these experiments will develop advanced techniques for bio-imaging and gene therapy that will be of benefit to the scientific community.

Technical Summary

Extracellular factors within the central nervous system (CNS) as well as intrinsic neuronal factors contribute to the inability of axons to regenerate. Certain treatments have demonstrated enhanced neurite outgrowth in cell culture models but fail to produce robust growth when translated into an in vivo paradigm. A prime example is the reintroduction of growth-promoting alpha9 integrin which results in a strong regenerative response in cultured neurons, with much less growth observed in an in vivo model injury.
Experiments have demonstrated an axonal localisation deficit of integrins and other transmembrane receptors which may contribute to reduced in vivo repair. It is unclear however what CNS factors modulate this localisation except that integrins localise within perinatal axons, prior to myelination or establishment of the mature CNS extracellular matrix (ECM), and also localise within adult axons of neurons devoid of ECM-rich perineuronal nets (dorsal root ganglia, retinal ganglion cells). This proposal will determine how the mature CNS environment affects axonal localisation of integrin receptors and will identify molecular modifications which target integrins into axons in vivo. We will use an in vivo approach to assess axonal localisation of integrins in adult rodent cortical neurons following either modification to the CNS environment or molecular modification of integrin receptors. CNS modifications will include demyelination using a targeted genetic approach to ablate oligodendrocytes with viral caspase 3 in a PLP-Cre mouse or enzymatic removal of ECM proteoglycans using chondroitinase ABC. Molecular modifications of integrin receptors will utilise successful strategies for axonal targeting in cultured neurons. Results which will be demonstrated using advanced bio-imaging techniques (confocal and light sheet microscopy) will identify in situ factors which preclude axonal localisation of integrins, directly impacting future therapeutic treatments.

Planned Impact

Who will benefit from this research?
The impact of this research will benefit both the scientific community as well as those concerned with future therapies for neurological conditions such as clinicians, patients, and the pharmaceutical industry.

Academic Research Community
The primary beneficiaries of the research outcomes in this proposal will be academic researchers interested in understanding how development and aging of the nervous system influences axonal function, expression and localisation of proteins, and potentially experimental clinical treatments. Researchers interested in targeting axons for different therapeutics will benefit greatly from the findings in this project. Furthermore, from a technique perspective, we will be developing tools throughout this project that will have far-reaching effects in the scientific community specifically with: 1) a novel focal demyelination mouse model that will be developed and used in our first Aim; 2) molecular strategies for driving axonal transport in vivo used in our second Aim; and 3) the potential advancement of bio-imaging techniques through Light Sheet microscopy for tissue imaging.
The results of this proposal will be disseminated through scientific publications in high impact peer-reviewed journals and through presentations at national and international conferences. There will be direct discussion of these results in internal seminars and group meetings in the University of St Andrews, fostering further collaborations locally and beyond.

Biotechnology and Pharmaceutical Industry
In the current climate of failed clinical trials, an increased understanding of how the body interacts with potential therapeutics is required to optimise treatments for human conditions. Those in biotechnology and pharmaceutical industries looking to develop novel and improved approaches for gene therapy in the nervous system and across disciplines will greatly benefit from the outcomes of this study.

Healthcare sector
Although not imminently translatable, patients and clinicians working in the area of neural injury and degenerative conditions may be particularly interested in these results which could directly contribute to optimised therapeutics in the future.

Education
The training of a postdoctoral researcher provided by this proposal will be a valuable contribution to the scientific community. Additionally, the research outcomes of this project will impact our current neuroscience knowledge and understanding of how neuronal function and intra-neuronal protein expression and transport can be affected by the external CNS environment. Furthermore, our results will contribute to the understanding of how the nervous system environment may control the intrinsic neuronal environment, and how these controls can be overcome through molecular modification.

General Public
Data and results generated in this project will be disseminated to the general public through outreach programs such as the University of St Andrews annual Science Discovery Day, as well as publicised through the University of St Andrews press office and appropriate social media. Both PI and the postdoctoral researcher will engage in these activities to provide and promote discussion of our work with the greater community.

How will they benefit from this research?
The education and awareness of the public on the research being performed within this project will stimulate interest into how basic research can impact on the treatment of human health conditions. This may also perk the interest of young biomedical scientists in their future careers. Additionally, the University of St Andrews has recently appointed a dedicated Public Engagement Officer for the Biomedical Sciences Research Complex who will assist in identifying activities and events for promoting our research.

Publications

10 25 50
 
Description One of the key aims of the project was to identify strategies to target growth-promoting proteins (specifically integrins) into nerve fibres by molecularly modifying the integrin. We have been successful in this aim, and to date have identifying key approaches to improving the targeting of integrins into nerve fibres. This has been performed in both neuronal cell lines and primary neurons, using published axonal-targeting motifs (ATMs), showing significant enhancement in integrin targeting. Further tests of this strategy are ongoing or planned, with preparations for a manuscript currently ongoing.
Exploitation Route The research outcomes thus far will continue to drive research questions in my own group to ensure completion of the initial objectives. We envisage a manuscript submission for several aspect of this funded work, within the next 12-18 months.
Sectors Other

 
Description Contribution to a Guidance document for Researchers ('Refining rodent models of spinal cord injury')
Geographic Reach Multiple continents/international 
Policy Influence Type Influenced training of practitioners or researchers
URL https://www.sciencedirect.com/science/article/pii/S0014488620301047?via%3Dihub
 
Description BBSRC DTP - PhD Studentship
Amount £90,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 08/2021 
End 09/2025
 
Description Enhancing Spinal Cord regeneration through combinatorial therapies: use of integrin-mediated repair together with chondroitinase treatment
Amount SFr. 150,000 (CHF)
Funding ID P182 
Organisation International Foundation for Research in Paraplegia (IRP) 
Sector Charity/Non Profit
Country Switzerland
Start 06/2020 
End 11/2023
 
Description Innovation Grant - Optimising stem cells for neuronal replacement in the CNS
Amount £20,000 (GBP)
Organisation Wessex Medical Research 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2017 
End 09/2019
 
Description Novel Targets to enhance axonal repair after spinal cord injury (Co-PI)
Amount $1,199,242 (NZD)
Funding ID 21/080 
Organisation Health Research Council of New Zealand (HRC) 
Sector Public
Country New Zealand
Start 11/2021 
End 10/2025
 
Description PhD Studentship - Enhancing Integrin-mediated Nervous System Regeneration
Amount £55,000 (GBP)
Organisation The Gerald Kerkut Charitable Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 03/2023
 
Description Attracting young students to STEM subjects: Aspire session at Barton Peveril Sixth form College 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Approximately 30 students involved in the Aspire programme, a select programme of the best and brightest students, attended STEM session at Barton Peveril Sixth Form College. The goal of this event was to stimulate and encourage interest in Biology and Biomedical Sciences whilst also providing personal perspectives from real world scientists. The age group in this event was final year students. In this event, I educated the students on the basics of the nervous system and provided information about my laboratory's research approaches that we use to find treatments for conditions such as brain and spinal cord injury. I also discussed my journey as a scientist, specifically in my field of nervous system repair. There was an extensive Q & A session, and since the event there has been good feedback from the school with a likely future event in the works.
Year(s) Of Engagement Activity 2023
 
Description Participation in a Public Outreach Event - New Forest and Hampshire County Show (Cells and the Amazing Brain) 
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 Over 100 members of the general public visited our Public Outreach booth (as part of the Wessex Medical Research showcase event). Here we aimed to engage the public (both adults and children) with the 'Amazing brain'. Our aim was to educate adults and children alike) about the structure of the brain and spinal cord, as well as what goes wrong after injury. We provided an opportunity for children to build their own neurons to take away as a reminder of their learning at our event. For the older crowd, we had nice discussions with people that were interested in hearing more about primary research in spinal cord injury and/or may have had a friend or family member affected by a nervous system injury.
Year(s) Of Engagement Activity 2022,2023
 
Description Presentation for University Visit Day - 'Meet your lecturer' 
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 Undergraduate students
Results and Impact In 2018 and 2019, our University Open days were run in person. I presented my laboratory's research as part of a new program called 'Meet your lecturer'. The intent of this was to show parents and prospective students the research that goes on in the School of Biological Sciences by the staff that also teaches the students, as a means of exciting the students about the real-life research they would be exposed to by enrolling in the University of Southampton. On the visit days, I presented a seminar entitled 'Repairing the Brain' directed at a lay audience. In the 2018 and 2019 visit days, upwards of 100+ parents and students attended at each session.
In 2020, our Open days have now been hosted online. As a result, I've recorded my 'Meet your lecturer' presentation to be provided to interested parties who join the online Open Day. As it is online, wee have a very good uptake with at least 100+ views.

We were fortunate to return to in person Visit Days recently and in our recent event, there were nearly 200 people in attendance (parents and prospective students) for my taster lecture on 'Repairing the Injured Brain'. This led to insightful discussions around next steps for research in the spinal cord injury field.
Year(s) Of Engagement Activity 2018,2019,2020,2023
 
Description Public Outreach Event - New Forest and Hampshire County Show (Neuroanatomy and Spinal cord repair) 
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 Over 100 members of the general public visited out Public Outreach booth where we aimed to engage the public with neuroanatomy and to discuss the impacts of spinal cord injury. Our aim was to educate the general public (adults and children alike) about the structure of the brain and spinal cord, as well as what goes wrong after injury. This allowed us to discuss our research regarding Enhancing the repair of the nervous system to a lay audience. We had insightful discussions with several people that either had a scientific background as well as those that had family members living with a traumatic brain or spinal cord injury, all of whom were very interested to hear about research being done on the subject.
Year(s) Of Engagement Activity 2018
 
Description Research talk at Pint of Science 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact 50-60 members of the general public attended the Pint of Science event entitled "Future of Medicine - Stem Cells." I spoke about spinal cord injury and regeneration in a talk entitled "How Do We Repair The Unrepairable?," which generated a wide range of questions from the audience and continued discussion in the subsequent intermission period. It was a great opportunity to share our research with a wider public audience from around Hampshire to both advertise our own work but to discuss the importance of this type of research and its wider implications in society.
Year(s) Of Engagement Activity 2018
 
Description School Visit (Southampton - Thornhill Primary) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Over 80 students (8-10 years of age; Year 5 and 6 in Primary school) participated in a visit held at their school during Science week in March 2018. It was a great opportunity for myself as University staff along with post-graduate students to visit them to encourage ambition, love of science and to talk about what it means to "become a scientist" with a university education as a goal. I hosted a session for 4 different class groups where I discussed "Brains" and the nervous system specifically why we need a brain and spinal cord, giving the students the opportunity to look and interact with brain models and a skeleton. During the day we had lunch with the students giving us an chance to have an informal chat on how wonderful university life is!
Following the event, the Science Team Lead Teacher reported that back that all of the sessions were really engaging and the children loved how interactive they were leading to an huge increase in science-related interests!
Year(s) Of Engagement Activity 2018
 
Description School Visit - Outreach event 'Science Day' at Thornhill Primary School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 98 School children (Primary School year 5 and 6) participated in our Outreach Event at Thornhill Primary School. The overarching theme of the event, held during national 'Science week' was to engage students with science and to spark their interest in going to University and studying science within a S.T.E.M. discipline. This particular primary school is in a deprived part of the city of Southampton with low University enrollment rates.

In our event 'Discovering the Brain and Spinal Cord', which ran in four sessions throughout the day, myself and my PhD student taught students about the structure of the brain using a Powerpoint slides together with brain and skeletal models. We also briefly introduced our research on brain and spinal cord repair, working into the discussion the importance of wearing helmets when cycling or riding scooters! Only part of the event was presentation-based. A large part of the event was hands-on whereas we invited students to explore the brain and skeletal models. We also incorporated an activity where students could build a neuron (with pipe cleaners, styrofoam balls, etc) to learn about the interesting structure of these cells.

Feedback from our previous engagement activity at Thornhill Primary School from both school teachers and students was extremely positive for the event, with students saying 'science can be fun and interesting.'
Year(s) Of Engagement Activity 2020
 
Description Schools visit for Outreach, Science Day at Kanes Hill Primary School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact 60 School children (Primary School year 6) participated in our Outreach Events at Kanes Hill Primary School. The events were held online and werre intended to engage Year 6 students with the brain as part of their Science Week events. It was also meant to spark the interest of the students in going to University and studying science within a S.T.E.M. discipline. This particular primary school is in a deprived part of the city of Southampton with low University enrollment rates. In our event 'Discovering the Brain and Spinal Cord', I spoke to the students about the structure of the brain using a Powerpoint slides together with brain and skeletal models. We also briefly introduced our research on brain and spinal cord repair, working into the discussion the importance of wearing helmets when cycling or riding scooters! We were unfortunately unable to run our hands one sessions as it was held online but the feedback was excellent. Feedback from the teachers said that "The whole school has been buzzing all day and I truly believe that all our children have gone home this afternoon talking about science with masses of enthusiasm!" An overall great event despite the Covid restrictions.
Year(s) Of Engagement Activity 2021
 
Description Skype a Scientist: Stem Cells and Gene Therapy 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Approximately 30 pupils attended the 'Skype a Scientist' event held virtually with Hazel grove High School (Hazel Grove, UK). The goal of this event was to provide connections between young students (Year 10) with a chance to hear from a Scientist away from their local community using an online platform. I was matched with this Year 10 class as they were currently studying Biology and were curious about Stem Cells and their application to Medicine and treatments for nervous system conditions. It was also an opportunity to engage with the students to encourage further participation in STEM-related areas. In this event, I educated the students about what stem cells are, what gene therapy is, and how each of these are being tested to find cures for nervous system conditions such as spinal cord and brain injury. There was an Q & A session, and since the event there has been good feedback from the school as it spurred an interest in these Year 10 students regarding Regenerative Medicine and potential careers in Medicine and STEM research.

A further iteration of this event has been held in person on the University of Southampton campus. In this event, between 35-50 pupils attended this 'Meet the Expert' event which included more detailed discussion into my career journey and research approaches. There was also time for Q & A as well.
Year(s) Of Engagement Activity 2023
 
Description Taster Lecture for visiting students from Reach Academy - 'Repairing the Injured Brain' 
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 Schools
Results and Impact Students from the Reach Academy participated in a Visit Day to the University. The goal of this event was to provide Taster Lectures to students in Year 12 across a wide variety of subject areas. Approximately 35 students interested in pursuing Science degrees attended my Taster lecture on 'Repairing the Injured Brain.' This was an opportunity to encourage Widening Participation in STEM subjects by given year 12 students the opportunity to attend a University lecture. In this event, I educated the students about the basics of the nervous system including its inability to repair itself after injury. I also discussed how researchers including myself are actively applying basic research to study potential cures for nervous system conditions such as spinal cord and brain injury. There was an interactive Q & A session, with excellent feedback from the students and school as it has spurred interest in application to Undergraduate Science programmes.
Year(s) Of Engagement Activity 2023