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

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.

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

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.