Investigating neuronal RNA localisation and translational deficits as gain of function mechanisms in ALS.

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

Abstract

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 fiber (called 'axon') projects outside to the muscles. Amyotrophic Lateral Sclerosis (ALS) is a relentless and incurable disease that can kill the motor neurons, resulting in progressive paralysis and death within 5 years of diagnosis. ALS has a lifetime risk in UK of 1 in 250 in the UK population.

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 cell's functions. 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.

Relatively recently it has become clear that axons, the long projections from individual neurons, need proteins to be made throughout their length, including at the very end most distant from the cell body. We do not yet know why this is, but certainly the need to respond quickly to local injury of an axon at a distant site, is one good reason why proteins need to be made in a localized region of a neuron, along the axon.

To have the capacity to be able to make proteins where they are needed, neurons have to transport RNA around the cell, especially along the axon. Motor neurons are some of the largest cells in the body and have tremendously long axons, in humans one single axon can extend from the spinal cord out to our feet, often for over 1 meter in length. So in these cells particularly, the movement of RNA, and the local production of proteins is extremely important.

Unfortunately, currently, we do not know which RNAs are moved around motor neurons and nor do we know which proteins can be made as a result. Our lack of knowledge has been largely because of the shear technical difficulty of analyzing RNA and protein in the separate cell bodies and axons. But this knowledge is critical to understanding ALS, because it turns out that proteins that bind and transport RNA in motor neurons, including a protein called FUS, can be mutated in ALS. So to understand ALS, we need to know what happens to FUS and the RNA it binds and the proteins that it makes, in health and in ALS, in different regions of motor neurons.

Here, we tackle the technical problems of looking at RNA and protein in different regions of motor neurons, including the very important axon, by combining new molecular biology and microscope techniques, with novel mouse model we have made, in an innovative approach to find out what RNA and proteins are present in different regions within motor neurons. We will look over time, at presymptomatic animals, and at animals with ALS, so that we can see the healthy state as mice age, and the progressive disease state. We can then validate our results in samples taken from human ALS patients. We badly need this information to understand the mechanisms and pathology of ALS and to develop effective therapeutics for motor neuron disorders.

Technical Summary

RNA binding proteins (RBPs) are crucial in ALS and mutations in RBP FUS cause ~5% familial cases. New evidence supports cytoplasmic gain of function as the pathomechanism in RBP-ALS, but its nature is unknown.

Here, we use our new FUS-ALS mouse to dissect changes in FUS cytoplasmic functions: in RNA localisation and in regulating local translation. These functions are crucial for maintenance and response to injury of motor neuron (MN) axons -- the majority of the neuronal cell mass. So far, technical hurdles limited this research so we know little of the composition of RNAs translated in axons, in health or disease. Here, we take combine new molecular tools to overcome these hurdles.

We use the RiboTag system to precipitate RNAs bound to ribosomes in MN cell bodies and axons from the mature mouse nervous system. Thus, we study the axonal translatome in vivo at pre-symptomatic and early disease timepoints of our FUS-ALS model. We follow our preliminary data showing reduction in ribosomal genes in this model by studying ribosome quantity and localisation in motor axons, and polysome profiling in spinal cord and nerve.

Then, in vitro, we physically separate axons from cell bodies to gain insight into processes linking FUS mutation to altered translation. We use both FUS ALS and Fus knockout MNs for polysome sequencing: a technique enabling us to calculate translation efficiency of all transcripts - to gain understanding of how FUS mutations cause alterations in local translation. We complement these molecular data by visualising protein translation by fluorescent labelling of nascent protein chains detected by confocal microscopy.

Finally, in Year 3, we validate our findings using a unique clinically and molecularly well-characterised bank of relevant human post-mortem material.

Thus, we address the molecular dysfunction that arises in FUS-ALS, using novel techniques and mouse resources, ultimately moving towards realistic translational approaches.

Planned Impact

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 RNA, and non-neurological RNA based diseases. Thus the beneficiaries are commercial Pharma and Biotech, patient and carer groups, academic scientists and the scientists and clinicians whom we train. 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 ALS mechanisms in order to get to treatments and ultimately cures for these relentless diseases. Thus we have created a new mouse model which will enable us to understand what goes wrong in this form of neurodegeneration. Our mouse system is an essential tool for understanding not only how mutant FUS causes motor neuron death, but how mutation in other genes (for example, TDP43, C9orf72), in ALS and other areas of biomedicine, give rise to pathology. We are combining this mouse model with a novel way of assaying the RNA content and protein translation of different locations within neurons, and this combination of resources and techniques will give us new information about disruption to RNA metabolism and how this results in neurodegeneration.


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 metabolism. 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 UCLBusiness. The beneficiaries are also patient organisations ultimately as treatments and cures are eventually found for ALS (and other forms of neurodegeneration).


We also note we are excellently placed to follow up findings with translational neuroscience, as the adjacent National Hospital for Neurology and Neurosurgery hosts one of the largest ALS clinics in Europe and co-PI PF is co-director of this clinic. In addition UCL hosts an Alzheimer's Research UK Drug Development Institute and the new Dementia Research Institute - we have excellent ties to both and our findings in neurodegenerative disease will be of great interest to both.


With respect to the basic biology of RNA and of non-neurological RNA based disease, the technology described here, to define the subcellular transcriptome/translatome, is applicable to all cells for which we can physically dissect the different compartments - and this technology is applicable to all organisms for which ES and iPS cells can be differentiated.


We also want to point out the impact from training as we have a range of students at all levels working in our labs including undergraduate neuroscientists and geneticists from UCL, UK and abroad, Master's students from UCL and elsewhere in UK, PhD students, MD students, and a range of clinician scientists. Thus we provide an unusual and important training in the basic science/translational interface and this directly benefits students in the short term through their degree programmes, and benefits UK science in the longer term by contributing to a trained pool of academics and clinician scientists.
 
Title A new antibody - mouse system for analysing FUS ALS, the Delta14 FUS mouse 
Description Novel antibody that detects mutant FUS only, including in our genome engineered mouse. 
Type Of Material Antibody 
Year Produced 2016 
Provided To Others? Yes  
Impact Mice/antibody used currently by collaborators e.g. Fratta/Ule/Schiavo. Will make freely available after our first publication. 
 
Title A new mouse model of motor neuron degeneration (FUS ALS) 
Description A new genetically engineered mouse model of FUS ALS 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2017 
Provided To Others? Yes  
Impact Several labs now working with this model 
 
Title Mouse humanised wildtype FUS model 
Description Humanised genomic wildtype FUS gene in mouse 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2015 
Provided To Others? Yes  
Impact Pending 
 
Title FUS homozygotes MEFs 
Description Working with a mouse model, an in vivo model, to produce IMMORTILISED cell lines so that we can drop our animal useage. 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact Reduced mouse numbers 
 
Description MMON 
Organisation MRC Harwell
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration with the Mouse Models of Neurodegeneration lab at MRC Harwell, analysis of homozygous and heterozygous mice
Collaborator Contribution Breeding, inbreeding onto another background, and phenotypic analysis of homozygous and heterozygous mice.
Impact Inbred mice on different backgrounds. Cohorts of mice of different ages, sex-matched with littermate controls, wildtype, heterozygous, homozygous, for phenotypic analysis. Analysis of different phenotypes ranging from behavioural through to physiological.
Start Year 2017
 
Description  
IP Reference  
Protection Protection not required
Year Protection Granted
Licensed Yes
Impact Antibody for FUS Delta 14 sent out for academic use.
 
Description UCL Postgraduate symposium poster 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Postgraduate students
Results and Impact De Giorgio A, Devoy A, Milioto C, Zhu F, MacKenzie K, Acevedo Arozena A, Fisher EMC (2018) Humanising the mouse Tardbp gene. UCL Sixth Annual Postgraduate Research Symposium, London, UK

Poster presented to postgraduates outside field.
Year(s) Of Engagement Activity 2018