Investigating deficits of axonal RNA metabolism and axonal signalling in amyotrophic lateral sclerosis

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

Abstract

Amyotrophic Lateral Sclerosis (ALS), also known as motor neuron disease (MND), is a devastating neurodegenerative disorder which causes progressive loss of muscle function and paralysis. ALS is incurable and leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of about 1 in 400.

The main cells affected in this disease are nerve cells called motor neurons (MNs), which progressively die during the course of ALS. MNs are amongst the largest cells of the body and connect the brain and the spinal cord to the muscles therefore making movement possible. In order to do this, MNs rely on one thin process, named the axon, which extends from the spinal cord out to each and every muscle of our body. In adults, a single axon can measure over a meter, running from the spinal cord to our fingers or toes, and needs sophisticated transport and communication systems to survive and function.

Importantly, research has shown that abnormalities in axons are found in the very early stages of ALS and other incurable human diseases. All cells in an individual's body, although very diverse from each other, contain the same genetic material called DNA, that gives instructions to each individual cell. Therefore the identity of each cell type (whether a MN or a heart cell, for example) is the result of which portions of DNA are active and produce another type of chemical called RNA. RNA carries all the necessary information for the cell to function. The sum of all the RNA in a cell, named the transcriptome, is the signature that characterises each cell type.

Knowing the transcriptome of a certain cell type provides insights into its biology and helps determine the causes of diseases. This is particularly relevant with MNs in ALS since there is good evidence showing that the biological processes linked to RNA 'metabolism' are primarily affected in ALS.

Further, RNA is transported in axons and this is essential for axon maintenance and its response to injuries.

The findings summarized above, highlighting that: 1) axons are involved in early stages of disease; and 2) ALS is caused by alterations of the RNA repertoire in MNs, alongside with novel preliminary data from my Sponsor's laboratory which shows that 3) key ALS molecules localise to cellular organelles which are involved in the communication system of axons, all converge to form my research questions.

I will use a novel animal model of ALS to investigate:

a) Which changes occur in the RNA of axons;

b) How these changes can play a role in ALS;

c) How the communication system between axons and the cell body is affected in ALS.

The feasibility of this project is ensured by the recent technological advances provided by my Sponsor's laboratory and collaborators. These cutting edge approaches will allow me to isolate and study RNA specifically found in MNs and their axons. Further, I will be able to isolate the small particles that contribute to transmitting survival signals in MN axons.

In summary this project will contribute to understand how axons function normally and what goes wrong in ALS. This will greatly help us to understand disease mechanisms and discover novel targets for effective therapies for ALS.

Technical Summary

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease (MND), is a relentlessly progressive neurodegenerative disorder, which causes loss of motor neurons (MN) leading to paralysis and ultimately death. It is currently untreatable, hence there is a desperate need for understanding its underlying mechanisms to develop novel and effective therapeutic strategies.

Published research and preliminary data show that: 1) RNA metabolism alterations play a role in ALS; 2) MN axons are affected in the early stages of disease; 3) axonal RNA transport is altered in ALS and deficits in axonal RNA localisation make axons more susceptible to noxious stimuli; 4) key ALS proteins associate with signalling endosomes (SEs), endosomal organelles which are responsible for axonal signalling and transport of survival messages.

These observations converge to define my research questions. I will investigate:

a) the axonal RNA changes occurring in ALS;

b) the mechanisms contributing to these changes, with a focus on RNA stress granules (cytoplasmic bodies, altered in ALS, where RNAs are protected during cell stress);

c) the novel link between key ALS proteins and axonal signalling.

In order to do so, I will combine the cutting edge tools and unique resources available in my Sponsor's laboratory and through collaborators. I will isolate MNs from a unique novel ALS mouse model which expresses an aggressive ALS-causative FUS mutation at physiological levels. I will then use microfluidic chambers and the UPRT RNA tagging technology in order to specifically isolate axonal RNA from MNs; I will then analyse SEs using magnetic isolation techniques and quantitative proteomics.

Finally I will be able to validate my results using differentiated MNs derived from human iPSC isolated from ALS patients. These findings will be further tested in patient tissue and will be paramount for the identification of novel pathways and potential therapeutic targets in ALS.

Planned Impact

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with a 1:400 lifetime risk, for which no effective treatment is available. The aim of this proposal is to generate new knowledge about the mechanisms underlying ALS, by using innovative biological tools and disease models; this data will have impact on researchers and clinician scientists and will be important to enable drug discovery and therapy testing in the future, with therefore a broad impact on patients and carers.

1) Although axons are a crucial compartment of neuronal cells, our knowledge of axonal RNA and signalling molecules, is still very limited. In the first instance, this project will provide novel biological information and data regarding the axonal transcriptome and will therefore have a very broad impact in neuroscience research.

2) This project will provide insights in axonal biology in ALS, and specifically in RNA metabolism and signalling alterations occurring in the disease and therefore have impact on ALS research.

3) Axonal dysfunction has been postulated to be involved in numerous neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease, therefore extending the impact of these findings more widely to the neurodegeneration field.

4) Our findings may benefit biotech/pharma by providing new insights and new targets for the development of therapeutics for ALS, and possibly other RNA disorders.

5) Amyotrophic lateral sclerosis patients will ultimately benefit from the development of drugs that can slow or halt their disease; this would directly contribute to improving the health and well-being of society. The development and commercialisation of a therapy for ALS would also lead to huge economic benefit.

Publications

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Birsa N (2020) Cytoplasmic functions of TDP-43 and FUS and their role in ALS. in Seminars in cell & developmental biology

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Bunton-Stasyshyn RK (2015) SOD1 Function and Its Implications for Amyotrophic Lateral Sclerosis Pathology: New and Renascent Themes. in The Neuroscientist : a review journal bringing neurobiology, neurology and psychiatry

 
Description Good practice for C9orf72 gene testing
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description Do the heterogeneous ribonuclear proteins play a role in the pathogenesis of Alzheimer's disease?
Amount £250,000 (GBP)
Organisation Alzheimer's Society 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2018 
End 11/2021
 
Description Identification of biomarkers for spinal bulbar muscular atrophy
Amount £53,000 (GBP)
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 06/2018 
End 05/2020
 
Description Investigating neuronal RNA localisation and translational deficits as gain of function mechanisms in ALS
Amount £513,524 (GBP)
Funding ID MR/R005184/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description Investigating skeletal muscle imaging and blood biomarkers as a multimodal approach to sensitively evaluate ALS progression in clinical trials
Amount £253,000 (GBP)
Organisation National Institute for Health Research 
Department NIHR Biomedical Research Centre
Sector Public
Country United Kingdom
Start 04/2014 
End 03/2016
 
Description Investigation of early-stage protein translation deficits in ALS using a combined mouse/iPSC approach
Amount £209,295 (GBP)
Funding ID Fratta/Apr19/868-791 
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2019 
End 09/2022
 
Description Motor Neurone Disease Association - Research Grant
Amount £191,000 (GBP)
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 02/2017 
End 01/2020
 
Description PhD supplement funding
Amount £19,203 (GBP)
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2016 
End 10/2019
 
Description Project grant
Amount £378,214 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2014 
End 09/2016
 
Description Project grant
Amount € 160,000 (EUR)
Organisation Thierry Latran Foundation 
Sector Charity/Non Profit
Country France
Start 05/2014 
End 04/2016
 
Description RNA dysfunction in MND: understanding the changes through novel RNA-seq technologiesThe Masonic Charitable Foundation PhD Studentship
Amount £100,000 (GBP)
Funding ID Fratta/Apr19/893-792 
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2019 
End 11/2022
 
Description RNA dysfunction in motor neuron disease: identification of novel changes in transcript processing and localisation through long-read RNA-seq
Amount £15,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2019 
End 06/2019
 
Description The impact of TDP-43 on translation and the response to axonal damage in amyotrophic lateral sclerosis
Amount £1,926,269 (GBP)
Funding ID MR/S006508/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2019 
End 09/2024
 
Description The role of miRNAs in ALS and their use as a biomarker of disease progression
Amount £229,328 (GBP)
Funding ID Greensmith/Apr15/839-791 
Organisation Motor Neurone Disease Association (MND) 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2016 
End 12/2018
 
Title MN specific transcription and translation detection 
Description We have crossed the CHAT-Cre mouse line with UPRT and RiboTag transgenic mice in order to generate mice that allow the isolation of MN-specific and axon-specific RNA and RNA coupled to Ribosomes. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The combination of these two methods will allow to obtain novel insight in MN specific RNA 
 
Title Motor axon specific transcriptome analysis 
Description We have combined microfluidic chambers, Motor neuron culturing and low-input RNA-seq in order to reliably sequence RNA deriving from pure motor axon populations. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact This method allows to study how a very well defined subcompartment of the motor neuron responds to genetic or external stimuli. 
 
Title Novel ALS mouse models - TDP-43 
Description New allelic series of TDP-43 endogenous mutations. 3 lines: a) Q331K line - endogenous ALS-causative mutation knock-in model b) M323K line - endogenous C-terminal mouse mutation: this mouse develops a progressive neuromuscular phenotype, including MN loss. c) F210I line - this is not a disease mode, but a mammalian in vivo model of TDP-43 loss of RNA binding function 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? Yes  
Impact Mouse lines deposited and publicly available 
 
Title Novel MRI assessment for ALS and KD patients 
Description Diagnostic value of muscle MRI for KD and ALS patients 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Protocol adopted by biotech setting up clinical trial 
 
Title Sod1 D83G mice 
Description A new mouse line carrying a pathogenic ALS mutation and developing signs of motorneuron degeneration. Different from all previous published lines in that the mutation is in the endogenous mouse gene. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact This has highlighted the importance of dosage in pathogenesis of animal models, and also underlined the integarted loss and gain of function occurring in SOD1 ALS. 
 
Title few cell transcriptomics 
Description isolation of few cells from mouse tissue followed by RNA extraction, amplification and sequencing 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact possibility of obtaining a transcriptome from few mouse tissue cells 
 
Title ALS and FTD Brain Transcriptomics 
Description Major contributors to a large initiative for ALS brain transcriptomics initiative (ALS-NYGC). 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? Yes  
Impact Access is granted to all members and through an application process. a anumber of papers have originated from this initiative. 
 
Description Bioinformatics analysis 
Organisation University College London
Department Genetics Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We provided data generation, data analysis and wet-lab validation of data.
Collaborator Contribution Bioinformatic analysis and processing of sequencing data
Impact This collaboration has resulted in significant progress of our understanding and capacity to interpret complex sequencing data. This has resulted in 2 publications (acceptes, but not yet in press)
Start Year 2012
 
Description Generation of novel mouse models 
Organisation MRC Harwell
Country United Kingdom 
Sector Academic/University 
PI Contribution Studied molecular features and RNA profile of novel lines
Collaborator Contribution Generated novel mouse mutants for the study of ALS related genes Fus, Tardbp and Sod1
Impact This collaboration has led to numerous findings - manuscripts are in preparation.
Start Year 2011
 
Description PiggyBac iPSC motor neurons 
Organisation Italian Institute of Technology (Istituto Italiano di Tecnologia IIT)
Department Neuroscience and Brain Technologies IIT
Country Italy 
Sector Academic/University 
PI Contribution NA
Collaborator Contribution Contributed expertise and isogenic cell lines to study FUS mutations in high-quality and high-purity motor neurons
Impact NA
Start Year 2019
 
Description Polysome profiling 
Organisation University of Trento
Country Italy 
Sector Academic/University 
PI Contribution In order to investigate the the role of FUS in influencing translation in ALS, we have started a collaboration with Gabriella Viero, a t the University of Trento, who is a leading ribosomal biology expert. We have provided our capacity to isolate specific tissue and subcellular compartments from our unique ALS model and are working together on uderstanding the role of mutant protein FUS on protein translation.
Collaborator Contribution See above.
Impact We have submitted an MRC research grant application with Gabriella VIero as collaborator using initial data from our work.
Start Year 2017
 
Description RNA sequencing 
Organisation Massachusetts Institute of Technology
Country United States 
Sector Academic/University 
PI Contribution Collection, isolation and processing of different Central Nervous System samples originating from our novel mutant mouse lines.
Collaborator Contribution Sequencing and initial analysis of the RNA sequencing data.
Impact Outputs have been full datasets obtained on our mutant mouse lines.
Start Year 2011
 
Description iCLIP 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution We have been able to apply this technique to our unique mouse mutants
Collaborator Contribution Technical and scientific input and support
Impact Ongoing publication
Start Year 2016
 
Description miRNA collaboration 
Organisation Weizmann Institute of Science
Country Israel 
Sector Academic/University 
PI Contribution We are working with Eran Hornstein, from the Weizmann Institute in to investigate the changes in miRNAs occurring in ALS and their potential use as biomarkers. We are using our mouse model resources and our patient sample collections and Eran Hornstein is assisting in the high throughput sequencing of miRNAs.
Collaborator Contribution As above
Impact NA
Start Year 2016
 
Description 1st UK Kennedy's Disease Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact A day to update patients, carers and healthcare professionals to all clinical and research aspects of this rare disease.
Year(s) Of Engagement Activity 2017
 
Description 2nd UK Kennedy's disease day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact Day of information and scientific communication for patients, carers, gene carriers and healthcare professionals.
International speakers and participation.

Forum for UK and US patient groups and charities to meet.

I organised and obtained funding for the event, due to take place on April 24 2020
Year(s) Of Engagement Activity 2020
 
Description Curation of quarterly research and clinical update letter for KD patients, families and carers 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Update on latest research and clinical developments
Year(s) Of Engagement Activity 2018,2019
 
Description Italian 2019 KD Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Participated to a National event for KD patients and carers
Year(s) Of Engagement Activity 2019
 
Description MND open day - National Hospital of Neurology and Neurosurgery 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Type Of Presentation Poster Presentation
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact 50 patients and carers took part in the Open Day.

Understanding of our reasearch activities and requests to actively participate in research
Year(s) Of Engagement Activity 2012
 
Description MNDA spring conference research update 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Participants in your research and patient groups
Results and Impact Very long and constructive question session

Number of patients were interested in participating in research
Year(s) Of Engagement Activity 2014
 
Description Regular commenting on MNDA and KD Disease association blogs and newsletters 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Explained findings and research plans to patient audiences.
Year(s) Of Engagement Activity 2013,2014,2015,2016