Investigating a neuronal subcellular transcriptome by the novel technique of RNA TU-tagging, in a normal and ALS-related mouse model.

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

Abstract

Amyotrophic Lateral Sclerosis (ALS, 'motor neuron disease') is a devastating neurodegenerative disorder which causes progressive loss of muscle function and paralysis. ALS leads to death, usually caused by the inability to breathe, on average only 3 years after diagnosis, with a lifetime risk of ~1 in 250 by 85 years old.

The principal cells affected in this disease are nerve cells called motor neurons (MNs). MNs connect the brain to the muscles therefore making movement possible. MNs progressively die during the course of ALS. MNs are amongst the largest cells of the body. Their main body lies in the spinal cord and contains numerous thin branching processes called dendrites. They also have one thin process, named the axon, which extends from the spinal cord out to each of our muscles. A single axon can measure over a meter, running from the spinal cord to ends of our fingers or toes. The connection between the MN and muscles is called the neuromuscular junction (NMJ).

All cells in an individual's body, although very diverse from each other, contain the same DNA, the genetic material that gives instructions to each cell. So the identity of each cell type (whether the cell is a nerve cell or a heart cell, for example) is the result of which regions 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 characterizes each cell type.

Knowing one cell transcriptome provides insights into its biology and helps determine the causes of disease. 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.

Importantly, we now know that RNA is transported and functions in different regions within an individual cell; in MNs it is transported in axons and to NMJs for specific roles. NMJs and axons are thought to be the first parts of the MNs to be affected in ALS.

Therefore it is important to know which RNAs are present in cell bodies and dendrites, and in axons, and at neuromuscular junctions of MNs, to understand how they function normally and what goes wrong in ALS.

It is possible to isolate and identify RNA from neurons and their axons when these are artificially grown in a culture dish or when cell bodies are dissected out under a microscope from fixed tissues. These findings have shown that thousands of different RNA species are actively transported to the axons, but it is difficult from these experiments to extract information that is relevant to mature neurons in their natural context in vivo and therefore to disease.

We will work with a new technique, 'TU-tagging', which has already been successfully used in mouse and which allows us to 'tag' RNA in specific cells in the context of a living animal. The tagged RNA can then be isolated and identified through high-throughput sequencing. We will apply this technique to MNs so for the first time we can isolate and identify RNA from MNs cell bodies and dendrites, and from their axons and NMJs in the living adult mouse.

We will work with normal mice, and with a new mouse model that we have developed that has a defect in a gene, Tardbp (also known as Tdp-43) that causes ALS. From our current work we know defects in this gene give an aberrant RNA profile in the MN cell bodies of this mouse. Currently no Tdp-43 mutant mice exactly model human ALS, but they teach us a great amount about how Tdp-43 functions in the normal and abnormal state.

These results will be extremely helpful in furthering our understanding of MN biology and of what causes these cells to be so specifically vulnerable in ALS.

This project will help research in the many other diseases in which RNA metabolism in different cell regions is important, and in response to nerve injury where again it plays a key role.

Technical Summary

Coding and non-coding RNAs are transported to different subcellular localisations for local translation and regulation. Localised 'RNA metabolism' is important in polarised cells such as neurons, but techniques to study RNA localisation are not able to isolate subcellular transcriptomes in vivo.

Motor neurons (MNs) have cell bodies in the spinal cord, numerous arborizing dendrites and they connect to muscle fibres at the neuromuscular junction (NMJ) through an axon that can exceed 1 meter in length.

We are interested in MNs because of our research into Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disorder that is characterized by the progressive loss of motor neurons that 'die back' from NMJs. ALS-causative mutations in RNA-binding and -transport genes (TARDBP 'TDP-43', FUS), and in C9ORF72 that induce RNA foci - have highlighted aberrant subcellular RNA metabolism in ALS pathogenesis.

Thus there is a clear need to identify the neuronal subcellular transcriptome in vivo to understand normal function, and dysfunction in our paradigm disease, ALS.

We will use a new technique, 'TU-tagging' (already successful in mouse) to identify the subcellular transcriptome of three compartments of adult motor neurons in normal mouse and in a mouse with a mutation in Tdp-43. Our preliminary data from a Tdp-43 mutant we are characterising shows disruption of many downstream genes including b-synuclein and tau.

TU-tagging works through the cell-specific expression of an enzyme (UPRT) and treatment with a drug (4TU), allowing incorporation of thio-uridine into RNA in specific cell types. Thio-uridine RNA is biotinylated and then pulled-down. We will use TU-tagging to identify RNA from (1) MN cell bodies and dendrites, (2) MN axons, (3) NMJs.

This will allow us insight in the biology of MNs, axons and NMJs and their susceptibility in disease.

TU-tagging is applicable to all models of normal function and disease, and of neuronal injury.

Planned Impact

The impact of this research lies in two areas (1) understanding the biology of the transcriptome in subcellular locations, (2) amyotrophic lateral sclerosis and the TDP-43 proteinopathies. These two impacts are quite different in that (1) provides a new technique (albeit previously used in mouse) that we believe will become a standard approach to isolating the subcellular transcriptome and so with very broad impact, whereas (2) tackles a biomedical issue, well-known neurodegenerative diseases for which no cure exists.


(1) TU-tagging is a technique that is relevant to every study with a need to catalogue and quantify the localised RNA transcriptome within a cell type. This has obvious application to early development of the Drosophila and mammalian embryos, but also to ALL other studies in which subcellular RNA pools are important.

Therefore we envisage this becoming a standardly used technique, only limited currently by the availability of suitable mouse models. We note that with the number of freely available mice with individual gene mutations and Cre transgenics with precisely defined patterns of expression set to rise dramatically over the next few years, TU-tagging will be very widely applicable to both academic and industrial applications. Knowing the subcellular transcriptome will have impact on academic studies of all areas of biology and also on industry studies for example in highlighting new mechanisms and targets, notably in non-coding RNAs. One immediate application for both academic and commercial interests is in the neuronal response to injury which involves local translation to form a signalling complex that is carried to the cell body.


(2) ALS is a devastating incurable mid-life disorder for which no cure or effective treatment exists. It is clear that localised RNA biology is involved within neurons and so our research will shed light on pathways and networks that are important for motor neuron health and disease. This will have impact on the ALS community.

As our paradigm, we have chosen to work with a Tdp-43 model related to ALS - this gene/protein is also dysfunctional in many common neurodegenerative disorders, including Alzheimer disease and Parkinson disease, and so this project may well also have a wider impact as more commonalities are found in the 'TDP-43 proteinopathy' neurodegenerative diseases, particularly those clearly involving disruption to RNA metabolism disruption.

Thus 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. It is intriguing also that several of the new ALS genes also play roles in specific cancers when translocated and so it is possible this research may have an impact on cancer studies also.



We note also that the sub-cellularly localised RNA transcriptome also plays a role in the response to nerve injury and thus our approach should be of interest in studying the impact of neuronal damage.

Finally, increasingly sophisticated techniques are being used for making RNA a therapeutic molecule, and it is likely that in the future knowledge of the localised transcriptome may further refine these efforts.

Publications

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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

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Fratta P (2014) Profilin1 E117G is a moderate risk factor for amyotrophic lateral sclerosis. in Journal of neurology, neurosurgery, and psychiatry

 
Description MRC ResearchGrant (Uprt) MR/K018523/1
Amount £466,205 (GBP)
Funding ID MR/K018523/1 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 04/2014 
 
Description Rosetrees award
Amount £9,601 (GBP)
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2015 
End 02/2017
 
Description Understanding disease mechanism: Sub-cellular translation in motor neurons in health and in amyotrophic lateral sclerosis
Amount £16,595 (GBP)
Funding ID M438-F1 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 06/2017 
End 07/2019
 
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 model Sod1 D83G 
Description Mouse with endogenous mutation in ALS gene (Sod1 D83G) 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2011 
Provided To Others? Yes  
Impact Paper submitted currently, after which the mouse will be made freely available. Second paper being written. We anticipate this mouse will be of interest to the ALS research community. 
 
Title UPRT development 
Description With Pietro Fratta, developing the UPRT transcriptomic analysis system in our mouse models. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact pending 
 
Title mouse embryonic stem cells for Down syndrome 
Description Manipulated mouse embryonic stem cells to model aspects of Down syndrome 
Type Of Material Cell line 
Year Produced 2006 
Provided To Others? Yes  
Impact academic papers 
 
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 Analysis of the FUS mouse translatome, Fratta, UCL 
Organisation University College London
Department Marie Curie Palliative Care Research Department
Country United Kingdom 
Sector Academic/University 
PI Contribution contribution of the unique FUS Delta14 mouse model
Collaborator Contribution RiboTagging and ChatCre breeding to pull down polysomes from the Delta14 mouse
Impact Multidisiplinary output. No outcomes yet as just started.
Start Year 2016
 
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 UPRT and localised transcript study 
Organisation University College London
Department Institute of Neurology
Country United Kingdom 
Sector Academic/University 
PI Contribution Co-grant awardee, mice and space.
Collaborator Contribution direct supervision of joint staff
Impact pending
Start Year 2014
 
Description studying ribosomal proteins 
Organisation University of Padova
Department Department of Neurosciences
Country Italy 
Sector Hospitals 
PI Contribution Access to a unique mouse model of FUS ALS (Delta14)
Collaborator Contribution Analysis of ribosomal proteins
Impact No outputs yet
Start Year 2017
 
Description Set up new ALS Seminar Series 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Set up an academic seminar series on Amyotrophic Lateral Sclerosis and similar topics, but as this is open to the lay public we have had a surprising interest and attendance from sixth form pupils and undergraduates.
Year(s) Of Engagement Activity 2014
 
Description Visit by fundraisers from MNDA 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Fundraisers from MNDA visited to learn more of current research. These people are well-informed on issues regarding MND and care for people with MND, but are not scientists and do not necessarily have easy access to researchers.

Better informed fundraisers.
Year(s) Of Engagement Activity 2014