Investigating the mechanisms of axonal degeneration in amyotrophic lateral sclerosis using Drosophila and mouse transgenics

Lead Research Organisation: Babraham Institute
Department Name: Molecular Signalling

Abstract

ALS is a devastating, progressive, paralysing disease, which kills over 1200 people in the UK each year, usually within 3-5 years of onset. It usually affects people in their 50s and older, but it can affect adults of any age. Despite decades of research, ALS remains incurable and medical care is essentially palliative (ventilator machines and feeding tubes). The causes of ALS are not fully understood, but in about 10% of cases there may be a family history of ALS suggesting a genetic cause. Excitingly, the past five years has seen an explosion in our understanding of ALS with the discovery of many genes linked to ALS. One of the most important genes is TDP-43. Recent breakthrough research has found that 95% of patients with ALS have clumps of TDP-43 in their brains and spinal cords. In some patients the gene that produces TDP-43 is also mutated and we now believe that TDP-43 plays an important role in causing almost all cases of ALS. Thus, researchers believe that TDP-43 models of ALS will be critical to working out the fundamental causes of ALS, and, ultimately, to develop desperately needed treatments for ALS.

Another important consideration for scientists is understanding how ALS begins, because tackling ALS in the early stages is likely to be the most effective way of treating it. ALS is characterised by the loss of nerve cells called motoneurones. These cells have long, delicate processes, called axons, which connect them to other nerve cells and also muscles, allowing us to move, swallow and breathe. In ALS the very earliest signs of disease appear in these axons. We therefore believe that by protecting these axons we can prevent the very earliest stages of ALS. Excitingly, two protective factors have been discovered, which dramatically protect axons when they are cut. However, these factors have never been tested in TDP-43 models of ALS. I plan to test these factors in genetically modified TDP-43 fruitflies and mice, which mimic ALS. I also plan to use these models to find new ways of protecting axons in ALS, which could open up whole new avenues of research. I shall be conducting this research in the labs of Dr Coleman and Dr Freeman, the two world-leading axon experts who discovered these protective factors.

Flies are an excellent model for scientists as they have very similar genes to humans, including TDP-43, and have similar types of motoneurons, muscles and brains. We will use novel cutting edge techniques to rapidly test the potential of axon protective factors. We still have to confirm our fly discoveries in mice (as mammals, mice are more similar to humans than flies and of course we ultimately want to develop treatments for patients). However, by starting our experiments in flies, we will limit the numbers of mice we use to an absolute minimum. We will also use nerve cells from patients with ALS, which have been created from skin biopsies using revolutionary stem cell technology.

One of the most exciting aspects of our planned research is that it aims to not only push forward our understanding of the mechanisms underlying ALS, it also aims to simultaneously identify ways of protecting axons in animal models. This research therefore holds great promise for finding therapeutic targets that can ultimately benefit patients with ALS. Furthermore, our work may well benefit patients with a whole range of other brain diseases, which also display axon degeneration with or without TDP-43 clumps, including Alzheimer's disease and Parkinson's disease. The research we propose therefore has the potential to benefit many thousands of people with incurable and devastating degenerative diseases.

Technical Summary

Introduction
ALS is an incurable motoneuron disease characterised by pathological TDP-43 inclusions. TDP-43 models are emerging as important tools to understand the fundamental causes of ALS and will be important to develop treatments. Significantly, axonal degeneration is one of the earliest processes in ALS and is therefore important to target therapeutically, as early treatment is likely to be the most effective strategy. The Coleman and Freeman labs are pioneers in axonal biology and have recently characterised two ways of potently protecting axons: Ax-NMNAT1 and dSarm/Sarm1 knockout. Significantly, both are conserved in flies and mice and have never been tested in TDP-43 ALS models.

Aims and objectives
We aim to identify molecular pathways that can protect axons in animal models of amyotrophic lateral sclerosis (ALS).

Methods
We will use cutting-edge technologies invented by the Freeman lab to develop novel TDP-43 transgenic fly models of ALS, which permit single motoneuron/axon resolution analysis using mosaic animals. These flies will be used to test the axon-protective potential of Ax-NMNAT1 and dSarm-knockout and to perform a sophisticated Drosophila forward-screen (using wing-blade cut) for novel candidates ameliorating TDP-43-mediated axonal degeneration. The top genetic candidates from Drosophila studies will be validated at Babraham using: 1) TDP-43 transgenic mice with fluorescently-labelled upper and lower motoneurons/axons; 2) human post-mortem tissue; 3) induced pluripotent stem cell-derived human ALS motoneurons.

Scientific and medical opportunities
Our forward screen will be comprehensive, utilising new technologies that will change the way other Drosophila biologists perform screens. My results could benefit researchers working on Parkinson's and Alzheimer's disease, which involve axonal degeneration (in many cases with TDP-43 pathology). Finally, this research holds great promise for identifying new therapeutic avenues for ALS

Planned Impact

Aside from academic beneficiaries a number of other parties are likely to benefit from this research.

In the short term medical charities will benefit from this research. The Babraham Institute and the Coleman group have a long history of links with a range of medical charities including the Alzheimer's trust and I expect to create similar links (in particular with the MNDA) during the course of my fellowship. I expect to work with the MNDA to publicise our work and in parallel increase the public profile of the work that the MNDA is doing. This will help patients and carers in the short term, by raising awareness of the disease, and encourage funding from donors, which will promote further research in the medium to long term.

Pharmaceutical companies and industry will benefit from important molecular discoveries made in this research. They will be able to exploit these findings, using their resources to push forward into drug discovery. The Coleman group already have strong links to a number of pharmaceutical organisations and other commercial collaborators, and in the medium term I will be able to tap into these contacts. Similarly, collaboration with industry will give the commercial sector access to cutting-edge thinking or tools and training of researchers, with relevant skills, who they have employed.

The MRC will benefit from the research through delivery of its missions, in particular, to encourage neurodegeneration research.

The wider public will benefit from our work in a number of ways including the following; our public engagement activities; jobs and economic growth in the UK through the successful development of our discoveries by the pharmaceutical sector; and the improvement of clinical practice through our collaborations with clinicians.

Ultimately, the aim of our research is to benefit patients through the development of effective therapies for what remain incurable diseases. This will include patients with ALS and other TDP-43 proteinopathies, in particular FTLD. Furthermore, given that TDP-43 and axonal degeneration are relevant to Alzheimer's disease and Parkinson's disease, patients with tau-opathies and synucleinopathies may also benefit in the longer term. We would hope that with continued advances in molecular biology and drug delivery techniques that effective therapies for neurodegeneration can be developed within the next 10-15 years.

Given the immense burden of neurodegenerative diseases on patients, carers and professional health providers, in the longer term our research will benefit the NHS and the wider UK economy.
 
Description MND Association small grant
Amount £19,890 (GBP)
Funding ID Sreedharan/Jun15/912-793 
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2015 
End 11/2016
 
Description MNDA project grant
Amount £54,901 (GBP)
Funding ID Sreedharan/Apr16/849-791 
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2016 
End 11/2017
 
Title A novel Drosophila TDP-43 model of ALS using the fly leg 
Description As outlined in the original proposal we have succeeded in generating a novel model of TDP-43 neurodegeneration in the fly that permits rapid screening for genetic suppressors. By overexpressing ALS-linked TDP-43 in select neurons in the fly leg we are able to recapitulate dying back degeneration of motor nerves as seen in human disease. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
Impact The screen was conducted at pace and three novel genetic suppressors of TDP-43 toxicity were identified. We are in the process of writing a manuscript, which we aim to submit before the end of November. 
 
Title A novel mouse TDP-43 model of ALS 
Description We have created a novel knockin mouse, which harbours a point mutation in the mouse gene that is homologous to a point mutation found in a patient with ALS. We used CRISPR/CAS9 technology to create this mouse at UMass medical school and have shipped mutant animals to the Babraham Institute where we will begin characterising it in collaboration with Dr Robert Brown's group at UMass. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact no impact as yet - we are only in the early stages of characterising this animal 
 
Title TDP-43 knock-in mouse, publication and datasets 
Description We generated a vast RNA sequencing transcriptomic dataset that has been made publically available. It has elucidated several novel mechanisms of disease in our mouse model, which have relevance to human neurodegeneration as well. We hope these datasets will accelerate the development of therapeutic agents for neurodegenerative diseases. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
Impact Our data has already been used by other groups to validate their own findings and has led to new collaborations with other researchers 
 
Description Neuroprotective effects of GSK3 inhibition 
Organisation University of Michigan
Country United States 
Sector Academic/University 
PI Contribution We have provided important data to our collaborator, who has used this to identify a way to protect neurons from degeneration in his high throughput in vitro system.
Collaborator Contribution Our collaborator has helped confirm our own data by quantifying suppression of toxicity in his system. He has also demonstrated effects of our candidate suppressor that we were not aware of and led to further corroborative experiments in our lab.
Impact No publications as yet, but we anticipate submitting a manuscript before the end of 2016
Start Year 2015
 
Description The role of GANP in TDP-43 processing and expression 
Organisation Hutchison/MRC Research Centre
Country United Kingdom 
Sector Multiple 
PI Contribution We have shared our data with our collaborator and then made use of his reagents to conduct new experiments that indicate an interaction between his protein of interest and TDP-43
Collaborator Contribution Our partners have provided important reagents for our experiments as well as advice and expertise. They have taught us new techniques as well.
Impact Our collaborator is interested in cancer, while we are interested in neurodegeneration. Thus, our collaboration is multi-disciplinary. No publications have arisen as yet, but we anticipate at least one manuscript being submitted before the end of 2016.
Start Year 2014
 
Description The role of mutant TDP-43 in metabolism 
Organisation University of Cambridge
Department Metabolic Research Laboratories
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified hyperphagia and weight gain in our mutant mice. TDP-43 has been linked with obesity. We have been in discussion with the Institute of Metabolic Studies (IMS) in Cambridge, specifically with Prof Vidal-Puig and Prof Farooqi. We have been breeding mice to send to the IMS to have comprehensive monitoring and undergo assays to look for metabolic derangements.
Collaborator Contribution They have contributed knowledge and expertise and the data from the studies will be analysed with their assistance
Impact no outcomes as yet.
Start Year 2016
 
Description motor function in TDP-43 mutant mice 
Organisation University of Sheffield
Department Sheffield Institute for Translational Neuroscience (SITraN)
Country United Kingdom 
Sector Academic/University 
PI Contribution we have sent a number of mice from our breeding program to Sheffield (Dr Richard Mead's laboratory) for detailed motor studies. We have been analysing the data he gets from his studies and his contributions will form part of our first publication on this model
Collaborator Contribution Richard Mead has CatWalk gait assessment tools and running wheel cages.
Impact Data are to be submitted for publication this year
Start Year 2016
 
Description the role of GSK3 in TDP-43 mediated neurodegeneration 
Organisation Michigan State University
Country United States 
Sector Academic/University 
PI Contribution We have confirmed, in vitro, that GSK3 loss of function suppresses TDP-43 toxicity. We have sought the help of Dr Sami Barmada, Michigan, to test compounds that we have been using to inhibit GSK3 in his high-throughput in vitro live imaging system. This collaborative efforts aims to identify therapeutic targets for ALS-FTD. We will be submitting a paper for publication by June 2018
Collaborator Contribution Dr Barmada has used the compounds we have suggested/provided and shown that they also work to inhibit TDP-43 toxicity.
Impact There has been free exchange of results between our labs, and we have engaged actively using email and Skype. We have advised Dr Barmada, and he us to make experimental progress. We will soon be beginning to write a joint manuscript.
Start Year 2017
 
Description the role of tau in TDP-43 mediated neurodegeneration 
Organisation University of Cambridge
Country United Kingdom 
Sector Academic/University 
PI Contribution we have tested the toxicity of TDP-43 in the presence or absence of tau in in vitro primary neuronal cultures.
Collaborator Contribution Prof Michael Coleman of the Brain Repair Centre has provided us with tau null mice to allow us to perform our studies.
Impact There has been free exchange of data between our labs. We aim to submit a joint paper for publication by June 2018
Start Year 2017
 
Description using MRI/MRS to identify in vivo biomarkers of disease in the TDP-43Q331K knock-in mouse 
Organisation King's College London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have generated TDP-43 knockin mice at Babraham without microchips of various genotypes and sent them to BRAIN at KCL for imaging.
Collaborator Contribution The BRAIN team have performed imaging studies of the brains of our mice
Impact we have generated substantial pilot data that has been used to write a project grant to ARUK for further MRI studies. This pilot data itself will be submitted for publication later this year.
Start Year 2017
 
Description Cambridge Science Festival 2015 
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 Using a specially constructed display we explained our research to lay people (adults and children). This involved discussions with the use of toys and games, and questions to be answered in a book that was freely given to willing participants. This was an all day event.
Year(s) Of Engagement Activity 2015
URL http://www.sciencefestival.cam.ac.uk/system/files/csf_2015_web_programme.pdf
 
Description Cambridge TV interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact My Current Biology paper was discussed in the TV programme and I was interviewed to give further insight into the research. I was interviewed by the 'Cambridge TV' channel in the lab and was videod conducting a simple experiment. I was asked about how my research contributes to medical research to identify much needed treatments for neurodegenerative conditions such as MND.
Year(s) Of Engagement Activity 2015
URL http://www.cambridge-tv.co.uk
 
Description Naked Scientists podcasts 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact I was invited to contribute to a special edition of The Naked Scientists BBC radio programme, celebrating the life and work of Prof Stephen Hawking, who died of ALS this year. We also created a podcast discussing the mouse and the exciting potential this model holds. I am incredibly keen to increase public awareness of the importance of dementia research, which, considering the implications of an ageing population, remains under funded. The programme was aired live on BBC radio, and the podcast was recorded and remains available to download and listen to throughout the world.
Year(s) Of Engagement Activity 2018
URL https://www.thenakedscientists.com/podcasts/short/mice-motorneurone-disease