The impact of TDP-43 on translation and the response to axonal damage 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 that causes progressive loss of MNs, leading to impaired 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.

Motor neurons (MNs) are the nerve cells that send signals from the spinal cord to our muscles. They are amongst the largest cells in the body, with their cell soma located in the spinal cord and its fiber (called 'axon') projecting outside to the muscles. All cell compartments, including axons, need a constant supply of newly synthesised proteins in order to function properly. On some occasions, for example when responding to an unpredicted event as cell damage, new and different proteins are absolutely needed to re-establish cell functionality and long term survival. This is particularly challenging for axons, as the RNA for these proteins, which is essential to make them, may need to be synthesized very far away in the nucleus.

Multiple lines of evidence indicate that one of the crucial players in the disease mechanism of ALS is a protein called TDP-43, which is important for the specific transport of RNA to different locations in the axons and in the response of cells to stress and damage.

In this Fellowship, I will test how TDP-43 impacts on the response of MNs to damage in the axons, and the relevance of this response pathway in ALS. To do so, I will combine novel mouse models of disease and patient cell lines with state-of-the-art biological tools that will allow the investigation of these very specific functions.

In particular, we have developed novel strains of mice that carry specific mutations in the mouse TDP-43 gene and, as a result, develop crucial features of ALS. These mouse models of ALS will allow us, by looking over time at pre-symptomatic mice and at mice with overt ALS symptoms, to identify the molecular and cellular changes occurring during disease progression. Using an innovative and fully integrated approach, we will analyse the disease phenotype of these mice with new molecular biology and microscopic techniques to identify which types of RNAs and proteins are present in different MN regions, including the axon, and how these change in response to damage.

I will therefore be able to investigate the cellular alterations triggered by TDP-43 in axonal damage response by studying: 1. MNs grown in special culture dishes where the axons are separated from the cell bodies; 2. The axons and their damage response in wild type and mutant mice; and 3. By validating our results in MNs derived from patient-induced stem cells and using post mortem brain samples donated to research by ALS patients.

In summary, this project will contribute to understand how changes in TDP-43 impacts on MN survival. This long-awaited information is essential to develop effective therapeutics for motor neuron disorders.

Technical Summary

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder, which causes loss of motor neurons (MNs) leading to paralysis and ultimately death.
The RNA-binding protein TDP-43 is a central player in ALS pathogenesis, but how its mutations and malfunction lead to disease is unknown. Published work and our preliminary data show: 1) TDP-43 is involved in RNA splicing, transport and translation; 2) axonal RNA localisation and translation is crucial for neuronal response to injury; and 3) cellular responses to stress and axonal damage are impaired in ALS.
These observations converge to define my research question - I will investigate how mutations in TDP-43 impact on a MN process crucial for ALS: their response to damage.
In order to do so, I will use our novel mouse model which bears a single point mutation in endogenous TDP-43 gene and develops progressive MN loss, allowing us to investigate the consequences of mutations within a physiological in vivo setting.
I will combine novel experimental setups and more established approaches to address specific points:
a) What is the impact of mutant TDP-43 on axonal translation?
b) How does mutant TDP-43 alter the neuronal response to oxidative stress, and specifically to axonal stress?
c) Does mutant TDP-43 alter response to axonal damage in vivo?
d) Does the reversal of these changes impact on TDP-43 toxicity?
I will use novel mouse tools and refined culture and biochemical approaches to identify MN-specific and axonal translation changes both in vivo and in vitro. I will then use a novel set-up we have devised to study the MN response to axonal stress in vitro, and perform axotomies combined with laser capture microscopy to characterise the impact of TDP-43 on axonal injury in vivo.
Finally, I will use iPSC-derived MNs and post mortem material from patients to validate our findings, as this is 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 pathomechanisms underlying ALS, by using innovative biological tools and disease models; this data will have impact on researchers and clinician scientists operating in the ALS field and more in general in the study of neurodegenerative diseases, and will be important to enable drug discovery and therapy testing, resulting in a broad impact on patients and carers.

As outlined below, this work will have a significant impact on: 1) the neuroscience community; 2) ALS researchers; 3) neurodegeneration research; 4) biotech and pharma; and 5) the patients.

1) It has recently emerged that the local protein synthesis is crucial for axons and their response to damage. Our knowledge of axonal translation and axonal injury response is still very limited. In the first instance, this project will therefore provide novel biological information regarding axonal translation and neuronal response to axonal injury, and will therefore have a very broad impact in neuroscience research.

2) This project will provide insights in how disease-causing mutations impact on motor neuron viability and the capacity of motor neurons to respond to axonal damage. These areas of research are of high relevance to the understanding of ALS pathogenesis and therefore will have a significant impact on ALS research.

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

4) Our findings will be centred on a specific neuronal response and its alteration in pathological conditions. We will also test whether specific targets will modify the TDP-43 toxicity. Henceforth, the results generated during this fellowship will provide new insights for setting up new assays and identify new targets for the development of ALS therapeutics by pharma and biotech.

5) ALS 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 an effective therapy for ALS would also lead to huge economic benefit.

Publications

10 25 50
 
Description Good practice for C9orf72 gene testing
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
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 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
 
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 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 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 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 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