Mechanism of the DNA damage response in Huntington’s disease pathogenesis and relevance for therapeutics

Lead Research Organisation: University College London

Abstract

The work was based on statistically robust genetic evidence focused on two very interesting GWAS hits implicated in repeat instability, discovered through significant contributions by the investigators. MSH3 protects against and FAN1 worsens CAG repeat expansion, and the programme aimed to determine whether these molecules may therefore play a role in HD progression. The applicants would also investigate the role of other DNA damage response proteins. The reviewers noted that it is well-known that dysfunction of DDR proteins can result in a variety of rare neurodegenerative disorders and therefore insights discovered into the pathophysiology of HD may be applicable to other neurodegenerative disorders.

Technical Summary

The UK Dementia Research Institute (UK DRI) is an initiative funded by the Medical Research Council, Alzheimer's Society and Alzheimer's Research UK. Funding details for UK DRI programmes will be added in 2019.

Huntington’s disease (HD) is caused by the expansion of a CAG repeat tract in exon 1 of the HTT gene, and early pathogenic events that are proximal to the mutation have recently been identified. Somatic expansion of the CAG repeat in specific brain regions and peripheral tissues occurs with disease progression in both HD patients and mouse models. It has been known for many years that ablation of specific mismatch repair proteins completely ablates somatic CAG repeat expansion, and the identification of these same mismatch repeat genes as genetic modifiers of HD, by the Tabrizi lab and others, has brought DNA repair and somatic expansion sharply into focus. At the same time, we have found that the incomplete splicing of the huntingtin gene (HTT) generates a small transcript (Httexon1) encoding an exon 1 HTT protein, and that the extent to which this occurs is CAG repeat length dependent. Therefore, we have identified a mechanism by which genetic modifiers of HD are linked directly to the production of what is known to be a highly pathogenic protein.
We have optimized our method for measuring CAG repeat expansions in mouse tissue, are generating a panel of quantitative qPCR assays to measure all HTT transcripts and have established a multiplex quantigene assay that can be used to measure relative HTT expression levels directly in tissue lysates. We have established a TR-FRET assay that measures the levels of the exon 1 HTT protein and are testing whether this is more sensitive if developed for AlphaLISA, MSD, Singulex or Simoa technologies. Once established we shall determine the relationship between somatic expansion, incomplete splicing, the exon 1 HTT protein and HTT aggregation in brain regions and peripheral tissues during the course of disease in the zQ175 knock-in HD model. To complement the work of the Tabrizi lab, we plan to use mouse models to investigate the extent to which targeting somatic CAG expansion or lowering the levels of the Httexon1 transcript might have therapeutic benefit. We are attempting to import a mismatch repair line that can be used to ablate CAG repeat expansion in a well-characterized mouse model of HD, to determine the maximum benefit of this approach, and set a baseline against which all future therapeutic approaches targeting DNA repair can be measured. We shall also be able to compare this to approaches directly targeting HTT transcripts as we have a number of reagents (ASOs, siRNAs and U1 adaptors), at various stages of development, that can be used to lower the levels of the Httexon1 and the full length HTT transcript.

Publications

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Farshim PP (2018) Mouse Models of Huntington's Disease. in Methods in molecular biology (Clifton, N.J.)

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Ghosh R (2020) Expression of mutant exon 1 huntingtin fragments in human neural stem cells and neurons causes inclusion formation and mitochondrial dysfunction. in FASEB journal : official publication of the Federation of American Societies for Experimental Biology

 
Description TREAT-HD: Developing therapies to prevent neurodegeneration in Huntington's disease
Amount £3,320,291 (GBP)
Funding ID UNS120640 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2022 
End 02/2027
 
Title Bioassays for huntingtin protein isoforms 
Description We have developed HTRF, AlphaLISA and MSD assays to measure soluble and aggregated isoforms of HTT. 
Type Of Material Technology assay or reagent 
Year Produced 2021 
Provided To Others? Yes  
Impact Landles C, Milton RE, Jean A, McLarnon S, McAteer SJ, Taxy BA, Osborne GF, Zhang C, Duan W, Howland D, Bates GP (2021) Development of novel bioassays to detect soluble and aggregated huntingtin proteins on three technology platforms. Brain Communications 3, fca231. doi.org/10.1093/braincomms/fcaa231. 
 
Title Opera Phenix and Harmony Software 
Description Application of the Opera Phenix and Harmony Analysis software for the automated, unbiased quantification of AAV transduction in complex mouse tissues 
Type Of Material Technology assay or reagent 
Year Produced 2020 
Provided To Others? No  
Impact A paper describing this work has just been submitted for publication and will be place on Bioarchive if possible. 
 
Title QuantiGene multiplex assay for huntingtin transcripts 
Description QuantiGEne assay for determining relative levels of all huntingtin transcripts. 
Type Of Material Technology assay or reagent 
Year Produced 2019 
Provided To Others? Yes  
Impact Provides a means of rapidly screening for agents to reduce huntingtin transcript levels 
 
Description ASOs to target huntingtin transcripts 
Organisation Ionis Pharmaceuticals
Country United States 
Sector Private 
PI Contribution Knowledge of splicing mechanisms in Huntington's disease and screening approaches
Collaborator Contribution design and synthesis of ASOs that cover mouse and human intron 1 sequences for the huntingtin gene and against the Msh3 gene
Impact No outputs yet
Start Year 2018
 
Description BAC-CAG mouse model of HD 
Organisation University of California, Los Angeles (UCLA)
Country United States 
Sector Academic/University 
PI Contribution We have received a new mouse model of HD which we are characterizing
Collaborator Contribution Have provided a new mouse model of HD
Impact Too soon
Start Year 2021
 
Description Correlative light and electron microscopy 
Organisation Birkbeck, University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution Funding of postdoc who conducts the work. Contribution of mouse models, antibodies and fluorescently labelled peptides. Knowledge of Huntington's disease.
Collaborator Contribution Access to equipment and reagents required to for CLEM. Training to use equipment and software. Knowledge of this specialised methodology.
Impact Not yet
Start Year 2018
 
Description Development of HTT bioassays 
Organisation PerkinElmer
Country United States 
Sector Private 
PI Contribution The development of bioassays to detect HTT isoforms
Collaborator Contribution Consultancy on how best to establish the bioassays
Impact Landles C, Milton RE, Jean A, McLarnon S, McAteer SJ, Taxy BA, Osborne GF, Zhang C, Duan W, Howland D, Bates GP (2021) Development of novel bioassays to detect soluble and aggregated huntingtin proteins on three technology platforms. Brain Communications 3, fca231. doi.org/10.1093/braincomms/fcaa231.
Start Year 2017
 
Description Drug Discovery Institute UCL 
Organisation Alzheimer's Research UK
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Intellectula input and development of a resource
Collaborator Contribution Optimisation of assay and high-throughput screen
Impact None yet
Start Year 2016
 
Description Fluorophore labelled HTT peptides 
Organisation Swiss Federal Institute of Technology in Lausanne (EPFL)
Country Switzerland 
Sector Public 
PI Contribution Using these reagents in novel exploratory experiments
Collaborator Contribution Provision of range of huntingtin peptides in monmeric and aggregated states labelled with different fluorphores
Impact Too early
Start Year 2017
 
Description HD KI models 
Organisation University of Alabama at Birmingham
Department Department of Biochemistry and Molecular Genetics
Country United States 
Sector Academic/University 
PI Contribution analysed mouse models in the publication below
Collaborator Contribution provided mouse models
Impact Sathasivam K*, Neueder A*, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RLM, Roos RAC, Howland D, Detloff PJ, Housman DE, Bates GP (2013). Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington's disease. Proc. Natl. Acad. Sci. 110, 2366-2370.
Start Year 2010
 
Description HD brain juvenile 
Organisation Brigham and Women's Hospital
Country United States 
Sector Hospitals 
PI Contribution analysed brain material in the publication below
Collaborator Contribution provided HD post mortem brain material
Impact Sathasivam K*, Neueder A*, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RLM, Roos RAC, Howland D, Detloff PJ, Housman DE, Bates GP (2013). Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington's disease. Proc. Natl. Acad. Sci. 110, 2366-2370. Neueder A, Landles C, Ghosh R, Howland D, Myers RH, Faull RLM, Tabrizi SJ, Bates GP (2017) The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington's disease patients. Scientific Reports, 7: 1307 | DOI:10.1038/s41598-017-01510-z
Start Year 2011
 
Description HD brain juvenile 
Organisation Leiden University Medical Center
Department Department of Neurology
Country Netherlands 
Sector Academic/University 
PI Contribution analysed brain material in the publication below
Collaborator Contribution provided HD post mortem brain material
Impact Sathasivam K*, Neueder A*, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RLM, Roos RAC, Howland D, Detloff PJ, Housman DE, Bates GP (2013). Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington's disease. Proc. Natl. Acad. Sci. 110, 2366-2370. Neueder A, Landles C, Ghosh R, Howland D, Myers RH, Faull RLM, Tabrizi SJ, Bates GP (2017) The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington's disease patients. Scientific Reports, 7: 1307 | DOI:10.1038/s41598-017-01510-z
Start Year 2011
 
Description HSP990 
Organisation Novartis Institutes for BioMedical Research (NIBR)
Country United States 
Sector Private 
PI Contribution used the reagent to generate the data in the publication below
Collaborator Contribution provided tool reagent
Impact Labbadia J, Cunliffe H, Weiss A, Katsyuba E, Sathasivam K, Seredenina T, Woodman B, Moussaoui S, Frentzel S, Luthi-Carter R, Paganetti P, Bates GP (2011) Altered chromatin architecture underlies progressive impairment of the heat shock response in Huntington's disease mice. J. Clin. Invest. 121, 3306-3319. Carnemolla A, Labbadia JP, Lazell H, Neueder A, Moussaoui S, Bates GP (2014) Contesting the dogma of an age-related heat shock response impairment; implications for cardiac-specific age-related disorders. Hum Mol Genet, 23, 3641-3656. Neueder A, Achilli F, Moussaoui S, Bates GP (2014) Novel isoforms of heat shock transcription factor 1, HSF1?a and HSF1?ß, regulate chaperone protein gene transcription. J Biol Chem, 289, 19894-19906. Carnemolla A, Lazell H, Moussaoui S, Bates GP (2015) In vivo profiling reveals a competent heat shock response in adult neurons: implications for neurodegenerative disorders. PLOS ONE 10, e0131985. Gomez-Paredes C, Mason MA, Taxy BA, Papadopoulou AS, Paganetti P, Bates GP (2021) The heat shock response, determined by QuantiGene multiplex, is impaired in HD mouse models and not caused by HSF1 reduction. Scientific Reports, 11, 9117. doi.org/10.1038/s41598-021-88715-5.
Start Year 2009
 
Description In vivo electrophysiology 
Organisation Psychogenics
Country United States 
Sector Private 
PI Contribution We have provided genetically modified mice
Collaborator Contribution To perform in vivo electrophysiology
Impact Too soon
Start Year 2021
 
Description LacIY mice 
Organisation Van andel Research Institute (VARI)
Country United States 
Sector Academic/University 
PI Contribution None yet
Collaborator Contribution To provide mice that are transgenic for the LacIY repressor
Impact Too soon
Start Year 2021
 
Description Msh3 KO mice 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution Testing the effect of nullizygosity for Msh3 on somatic instability
Collaborator Contribution Provision of an Msh3, Exo 1 knock-out mouse and an Exo 1 knock-in mouse
Impact none yet
Start Year 2018
 
Description Post mortem HD brains 
Organisation Columbia University
Country United States 
Sector Academic/University 
PI Contribution None yet
Collaborator Contribution Provision of well-characterised brain samples
Impact Too Soon
Start Year 2021
 
Description Preparation of CRISPR/Cas13 reagents 
Organisation Francis Crick Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution Postdoctoral salary. Cell lines. Knowledge of Huntington's disease
Collaborator Contribution Postdoctoral salary. Knowledge of CRISPR/Cas13. Development of sequencing approach to identify the use of cryptic polyA sites in the huntingtin gene.
Impact None yet
Start Year 2018
 
Description Provision of lentiviral constructs 
Organisation University of Massachusetts
Country United States 
Sector Academic/University 
PI Contribution Postdoc salary to perform work. Neural stem cell models of Huntington's disease. Knowledge of Huntington's disease.
Collaborator Contribution Preparation of lentiviral constructs and packaging to provide virus.
Impact None yet
Start Year 2020
 
Description RNAseq 
Organisation Massachusetts Institute of Technology
Department Department of Biology
Country United States 
Sector Academic/University 
PI Contribution Provided RNA from reagents generated in publication below
Collaborator Contribution Performed RNAseq on RNA supplied
Impact Sathasivam K*, Neueder A*, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RLM, Roos RAC, Howland D, Detloff PJ, Housman DE, Bates GP (2013). Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington's disease. Proc. Natl. Acad. Sci. 110, 2366-2370.
Start Year 2011
 
Description RNAseq 
Organisation University of Ulm
Country Germany 
Sector Academic/University 
PI Contribution Provided RNA from reagents generated in publication below
Collaborator Contribution Performed RNAseq on RNA supplied
Impact Sathasivam K*, Neueder A*, Gipson TA, Landles C, Benjamin AC, Bondulich MK, Smith DL, Faull RLM, Roos RAC, Howland D, Detloff PJ, Housman DE, Bates GP (2013). Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington's disease. Proc. Natl. Acad. Sci. 110, 2366-2370.
Start Year 2011
 
Description U1 adaptors to target HTT incomplete splicing 
Organisation Rutgers University
Country United States 
Sector Academic/University 
PI Contribution Know how about incomplete splicing of HTT, cell cultures for screening, in vivo expertise
Collaborator Contribution Development of U1 adaptors that target HTT
Impact None yet
Start Year 2018
 
Description siRNAs against huntingtin 
Organisation University of Massachusetts
Country United States 
Sector Academic/University 
PI Contribution testing the effect of knocking down huntingtin transcripts
Collaborator Contribution provision of chemically modified and conjugated siRNAs that are stable in biological tissue and distribute throughout the brain
Impact none yet
Start Year 2018
 
Description Work experience in lab 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Hosting A-level students in the lab for one week
Year(s) Of Engagement Activity 2019