Regulation of axonal transport by neurotrophic factors in health and disease

Lead Research Organisation: University College London
Department Name: Institute of Neurology

Abstract

Charcot-Marie-Tooth disease (CMT) is a debilitating, inherited neuromuscular condition that is characterised by muscle weakness and sensation deficiencies mainly in the hands and feet. The mechanisms linking gene alterations (mutations) to this neurodegeneration remain unresolved, and there are currently no effective treatments for the disease. To better understand CMT and develop therapies, I study the 2D subtype (CMT2D) caused by mutations in the gene GARS, which produces glycyl-tRNA synthetase (GlyRS) protein. GlyRS is found in almost all cells of the body, yet it is the nerves responsible for movement (motor neurons) and sensation (sensory neurons) that are affected by the disease. By understanding exactly why these peripheral nerves deteriorate, we will be better able to engineer targeted, molecular therapies.

It has previously been shown that CMT2D-linked mutations in GARS affect the structure of the resulting GlyRS protein. I recently discovered that this conformational change causes mutant GlyRS to aberrantly interact with specific proteins called tropomyosin receptor kinase (Trk) receptors found on the surface of nerve cells. Trk receptors are crucial to life because they bind to survival molecules called neurotrophins, hence they are also known as neurotrophin receptors. I have shown that mutant GlyRS binding to Trk proteins disturbs the usual nerve response to neurotrophins, impairing sensory nervous system development in CMT2D mice. In addition, this non-physiological interaction leads to defects in a process called axonal transport, which is also essential for nerve cell function and survival. I have successfully treated these defects in CMT2D mice by injecting purified neurotrophic factors to overcome the detrimental impact of mutant GlyRS binding. This work has not only identified a possible way to treat CMT2D, but it has uncovered a previously unappreciated role for neurotrophic factors in regulating the dynamics of axonal transport.

I now propose to unravel the molecular mechanism responsible for the modulation performed by neurotrophic factors on axonal transport in healthy nerves and, in the process, determine exactly how mutant GlyRS binding to Trk receptors reduces nerve survival in CMT2D.

I will begin by testing the effects of a variety of neurotrophic factors on axonal transport in healthy and CMT2D mice in order to identify the signalling nodes most relevant to this essential cellular process. Complementing this, I will assess where the neurotrophic factor receptors are found in the muscle, and determine whether receptor localisation and levels are linked with the susceptibility of different muscles to weakness and degeneration in CMT2D mice. I will then grow nerve cells in tissue culture and use them to determine exactly how impairments in neurotrophic factor-regulated pathways disrupt axonal transport. Finally, I will design gene therapies, which will be delivered by harmless viruses, to boost levels of specific neurotrophic factors in CMT2D mice muscles and test this strategy as a potential therapy for the disease.

This proposal will not only enhance our understanding of how neurotrophic factors regulate axonal transport in healthy nerves, but it has the very real potential of generating innovative gene therapies for CMT.

Technical Summary

Charcot-Marie-Tooth disease type 2D (CMT2D) is a debilitating and presently incurable peripheral nerve disorder caused by dominantly-inherited, gain-of-function mutations in the housekeeping gene GARS, which encodes glycyl-tRNA synthetase (GlyRS). The mechanisms linking motor and sensory degeneration to GlyRS dysfunction remain unresolved; however, I identified that pathological mutations in GARS permit the mutant protein to aberrantly bind to the extracellular domains of mainly neuron-specific neurotrophin receptors. The activity of these important receptors is integral to the survival, development, and differentiation of motor and sensory neurons in all mammalian organisms. Perturbations in neurotrophin signalling account for pervasive in vivo defects in CMT2D axonal transport of signalling endosomes, which are reversed by administration of specific neurotrophic factors (NTFs). I have thus uncovered an unappreciated role for NTFs in regulating the dynamics of this essential neuronal process, and discovered a neuropathic pathway amenable to therapeutic intervention. I now propose to interrogate NTFs integral to transport in vivo in healthy and diseased peripheral axons. Detailed expression experiments of NTFs and their receptors will identify molecular determinants of differential CMT2D muscle vulnerability. These experiments will be coupled with mechanistic studies in primary peripheral neurons in vitro to elucidate how NTFs control transport and how mutant GlyRS disturbs this process. Virus-mediated gene therapies will be designed and tested in CMT2D mice to determine whether augmenting neurotrophic input in a muscle-specific fashion alleviates, in part or completely, neuropathic phenotypes. This combined approach provides a powerful, integrated platform to unravel the non-cell autonomous mechanisms governing axonal transport, with real possibility of producing a viable gene therapy for a currently untreatable neurological condition.

Planned Impact

The major impact goals of this research are to:

1. Provide evidence for the therapeutic efficacy of boosting neurotrophic support in CMT using virus-mediated gene therapy, with the long-term objective of improving the health and well-being of CMT patients.
2. Empower CMT patient charities, and thus patients and their families, with clear and concise research summaries.
3. Increase awareness of in vivo imaging of axonal transport as a research tool in neuroscience, and provide opportunities for enhancing capacity, knowledge, and skills of interested academic and private sector researchers.
4. Encourage public awareness and understanding of neuroscience, neurodegenerative disease, and gene therapy, and related issues.

With these impact goals in mind, the following stakeholders have been identified as likely beneficiaries of my research program:

1. CMT patients
2. CMT charities
3. Private sector organisations
4. Interested public

CMT2D is a debilitating neurological condition that causes muscle weakness and sensory deficits leading to difficulty walking, foot deformities, and reduced dexterity. Patient quality of life is therefore severely reduced, and there is currently no available cure or treatment to limit this. The experiments outlined in this proposal are highly likely to identify an effective gene therapy for CMT2D, and other CMTs given that underlying mechanisms may be shared between subtypes. Patients will thus benefit clinically in the medium to long term from the development of a gene therapy that targets the molecular mechanisms linking disease mutations to neuropathology.

I will interact with CMT patient charities about my research so that they will be better able to inform CMT patients and their families about the latest CMT discoveries. I know from my long-standing interaction with the charity Spinal Muscular Atrophy Support UK that well-informed patients and families feel that they are better supported, and are generally happier and less worried by their condition. Additionally, publicity surrounding CMT will likely increase awareness and support for research, especially with a plausible disease therapy on the horizon.

The ability to image and assess the dynamics of axonal transport in an in vivo model is highly coveted by academic and private company researchers. This is evidenced by my frequent discussions at conferences, and me having been contacted by pharmaceutical company Merck (USA) and numerous international academic teams to consult on the technique and help to validate their conclusions in an in vivo setting. By facilitating the adoption of this technique in industry science, I can have a significant impact on the quality and reliability of experiments assessing axonal transport in a range of neurological disease and aging models.

Finally, and more generally, by actively engaging with the public using a variety of platforms (outlined in Pathways to Impact annex), I will continue to have a meaningful impact on society's understanding and enjoyment of science. Discussing science and my work with non-academics not only improves my communication skills and overall understanding of neuroscience, but it can help to create a more conscientious and knowledgeable population that will facilitate informed political, environmental, and health-related decision making, as well as inspiring more young people to pursue careers in STEM.

Publications

10 25 50
 
Description Robert Burgess (Jackson Laboratory) 
Organisation The Jackson Laboratory
Country United States 
Sector Charity/Non Profit 
PI Contribution Dr. Burgess and I are in a long-standing collaboration, stemming from my visiting his laboratory in 2012 as a PhD student. We have published several papers together in the past, and we are currently collaborating on a couple of different ventures. I set up the current collaboration and am conducting the research.
Collaborator Contribution Dr. Burgess is an official collaborator on my Career Development Award and is providing tissues and mouse models for my research project.
Impact Pending
Start Year 2018
 
Description Xiang-Lei Yang (Scripps Research Institute) 
Organisation Scripps Research Institute
Country United States 
Sector Charity/Non Profit 
PI Contribution Prof. Yang and I are in a long-standing collaboration, stemming from my first contacting her in 2016 to collaborate on a manuscript (published in 2017 in the Proceedings of the National Academy of Science). We have published two papers together, and we are currently collaborating on several different ventures. I set up the current collaboration and am conducting the research.
Collaborator Contribution Prof. Yang is an official collaborator on my Career Development Award and is providing recombinant proteins and performing protein-binding experiments for my research project.
Impact Pending
Start Year 2018
 
Description Frontiers for Young Minds article on axonal transport 
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 I wrote an article on axonal transport targeted at children interested in neuroscience. This is published in the specialist journal for kids aged 8-14 called Frontiers for Young Minds. Sleigh (2020) Axonal transport- The delivery system keeping nerve cells alive. Front Young Minds 8, 12.
Year(s) Of Engagement Activity 2020
URL https://kids.frontiersin.org/article/10.3389/frym.2020.00012
 
Description Rare Disease Day 2019 for charity SMA UK 
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 High schools from the Reading/Slough region attended Rare Disease Day events at Royal Holloway, where I ran a stall to provide information on the disease spinal muscular atrophy (SMA) as a representative of the UK charity SMA UK. I also gave advice on applying to universities and careers in research.
Year(s) Of Engagement Activity 2019
URL https://www.royalholloway.ac.uk/research-and-teaching/departments-and-schools/biological-sciences/ev...
 
Description Rare Disease Day 2020 for charity SMA UK 
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 High schools from the Reading/Slough region attended Rare Disease Day events at Royal Holloway, where I ran a stall to provide information on the disease spinal muscular atrophy (SMA) as a representative of the UK charity SMA UK. I also gave advice on applying to universities and careers in research.
Year(s) Of Engagement Activity 2020
URL https://www.royalholloway.ac.uk/research-and-teaching/departments-and-schools/biological-sciences/ev...