Elucidating early stage ALS pathomecanisms that drive mitochondrial dysfunction

Motor neurons are the nerves that send signals from the spinal cord to our muscles. They are amongst the largest nerve cells in the body and are highly specialized, in that their cell body is located in the spinal cord and its fibre (called the 'axon') projects outside to the muscles; these cells have huge energy demands because they are so big and active.

Amyotrophic Lateral Sclerosis (ALS) is a relentless and incurable disease that kills the motor neurons, resulting in progressive paralysis and death typically within 5 years of diagnosis. ALS has a lifetime risk of 1 in 250 in the UK population, and although it is mainly considered a disorder of mid-life, children as young as 11 years of age have been diagnosed with ALS. We have a desperate need to find some kind of treatment for this disease - however, we do not yet know the fundamental reasons why motor neurons die in ALS, or even when the disease processes start, so we cannot tackle these processes with targeted therapies.

Every cell in our body contains DNA, RNA and proteins. DNA is in the nucleus of cells and carries the information on how to build the proteins. Proteins are the building block of our cells and they can also perform important tasks for the normal functioning of cells. RNA is the intermediate and carries the instructions for making individual proteins from the DNA, to the various locations inside a cell where these proteins are made and needed for specific jobs.

90% of ALS is 'sporadic' -- it occurs in people who have no family history of the disease. However, ~10% occurs in families and usually if a parent is affected there is a 50% chance a child will have ALS. There are many mutant genes that can cause ALS in these familial cases. Some of these genes give the instructions to make proteins that bind to RNA and enable RNA to work properly in the cell. While we know that several of these 'RNA binding proteins', can be mutated in ALS, and cause motor neurons to die, we do not know how this happens, or when it begins.

Here, we have created a unique system, for starting to understand why motor neurons die in one form of familial ALS, caused by mutations in an RNA binding protein called 'FUS'. This system is based on a new antibody that just binds to the mutant FUS protein. We have a FUS mouse model - which has a human mutation recognised by the antibody - that causes progressive motor neuron loss in the animal. We will now make a complementary human cell line with the same mutation. Having this antibody means we can now look at how mutant FUS behaves differently from the normal FUS protein. We can start to see what aberrant processes the mutant protein is involved in, and where these happen in the motor neuron and other cells.

This antibody system has already told us that changes in proteins and energy balance, and in fats, occur early, before we see motor neuron death in our mouse model. Thus, working with our system, and comparing our findings with our human FUS-ALS cells, will make an important contribution to our understanding of ALS, particularly in the early stages before motor neurons have died. This this research will help us to target therapies in this form of ALS, and, we believe, shed light on other forms of ALS that arise from aberrant RNA binding proteins.

In regards to molecular disease mechanisms, ALS is a poorly understood disease. However, we recently identified that (1) mutant FUS Delta14 protein accumulates in the mitochondrial fraction to a greater extent than wildtype FUS, (2) the level of wildtype FUS in mitochondria is significantly altered in the FUS Delta14 mice and (3) lipidomic analysis of CNS tissues suggest significant mitochondrial dysfunction through key alterations in mitochondria-associated lipids in FUS Delta14 mice before MN loss. All together these data suggest that mutant FUS is likely to play key roles at mitochondria and that mitochondrial dysfunction has an important role in early stage disease processes.
To dissect the role of mitochondria in FUS-ALS we have designed five independent, yet inter-related aims.
Aim 1.Identify FUS wildtype, and mutant, protein-protein interactions through tissue fractionation and immunoprecipitation
Aim 2.Visualise key interactions of mutant and wildtype FUS associated with mitochondria using super resolution microscopy (SIM and STORM)
Aim 3.ssess structural changes in ER-mitochondrial tethering and mitochondrial morphology by electron microscopy (EM)
Aim 4.Generation of human FUS Delta14 frameshift iPSC line
Aim 5.In vitro functional studies using human iPSC derived MN
We combine comparable in vivo and in vitro systems, with the same genetic modifications, in order to identify key disease pathomechanisms. Our mouse model allows working in vivo with multiple cell types, throughout aging and disease development, while our cellular systems will allow detailed mechanistic investigation of disease-associated pathways.
Overall these experiments take advantage of our novel FUS frameshift model systems, provide fundamental new insight into earliest pathological events in FUS-ALS. As mitochondria dysfunction has been reported in many neurodegenerative diseases, it may also provide important insights for the whole ALS and neurodegeneration research field.

1. Who will benefit from this research?
The impact of this application lies in the fields of motor neuron disease/amyotrophic lateral sclerosis and neurodegenerative/neurological disease, the basic biology of wildtype and mutant FUS protein, with implications for other RNA binding proteins and diseases arising from mutations in these proteins. Thus, the beneficiaries are the scientific community, the scientists whom we train, commercial Pharma and Biotech, and patient and carer groups. The work described here will contribute to health and wealth outcomes and have an impact on biomedicine contributing to quality of life outcomes.
2. How will they benefit from this research?
With respect to motor neuron disease/ALS and neurodegenerative disease/neurological disease, we are specifically interested in dissecting early stage ALS mechanisms in order to get to treatments and ultimately cures for these relentless diseases, with a focus on knowing when and how disease begins, before motor neurons are dead. This project will advance our understanding of the role of FUS and RBPs in general in the development of ALS and associated dementias such as FTD. It will also provide critical information about energy homeostasis and mitochondrial dysfunction in neurodegeneration. The project will generate new resources for the research community, such as novel -omics datasets both from the FUS Delta14 mouse and iPSC neuronal lines as well as new frameshift FUS isogenic iPSC lines to complement the frameshift FUS antibody and the FUS Delta14 mouse model. We also note that information about FUS function, in particular the -omics datasets, may also be of interest to the field of cancer research, as chromosomal rearrangement and the resultant gene fusion of FUS to transcription factors such as CHOP (DDIT3) lead to aggressive forms of liposarcoma.
Thus, this project will give new insight and new data for those in Pharma, Biotech and academia who are interested in neurodegenerative mechanisms involving disruption to RNA binding proteins. The direct translation of our findings to trials in patient groups will likely take many years but cannot happen unless we understand the basic biology of disease. We note also the increasing propensity of Pharma to outsource basic research to small Biotech and academia, from which they can effectively develop therapeutics. We have excellent ties to Pharma and Biotech, through current collaborations and through the extensive opportunities for networking and development provided by UCL and KCL. The beneficiaries are also patient organisations ultimately as treatments and cures are eventually found for ALS (and other forms of neurodegeneration).
We have excellent connections to bio-pharma through a number of routes including existing collaborations through the UCL IoN/National Hospital for Neurology and Neurosurgery and KCL IoPPN/King's College Hospital, including drug trials in both animal models and patients. The Ruepp group is based within the new UK Dementia Research Institute (DRI) based at KCL and Plun-Favreau has drug discovery funding from Takeda and is also a member of the Therapeutic Innovation Group (TIG), a UCL Eisai partnership. In addition, Plun-Favreau has joint funding with the Alzheimer's Research UK Drug Development Institute (DDI) based at UCL. Thus, we are excellently placed to follow up findings with translational neuroscience.
Finally, an important impact will be the knowledge transfer and academic career development of the postdoctoral academic staff. This is enabled through the exchange of the two postdoctoral associates between the two sites, ensuring a true and sustainable technology and knowledge transfer. This application is an important step towards independence for Dr Anny Devoy (Co-I), ensuring she receives the recognition for her work and scientifc input that led to this proposal and further develop her project management skills by co-ordinating the research across the two sites.