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.
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.
Organisations
- University College London, United Kingdom (Lead Research Organisation)
- PerkinElmer (Collaboration)
- Alzheimer's Research Uk (Collaboration)
- Swiss Federal Institute of Technology in Lausanne (EPFL) (Collaboration)
- University of Ulm, Germany (Collaboration)
- Van Andel Institute (Collaboration)
- Ionis Pharmaceuticals (Collaboration)
- Yeshiva University (Collaboration)
- Brigham and Women's Hospital (Collaboration)
- Columbia University, United States (Collaboration)
- University of Massachusetts Lowell, United States (Collaboration)
- University of California Los Angeles, United States (Collaboration)
- Novartis Institutes for Biomedical Research (NIBR) (Collaboration)
- Psychogenics (Collaboration)
- Massachusetts Institute of Technology (Collaboration)
- Rutgers University (Collaboration)
- Francis Crick Institute (Collaboration)
- Birkbeck, University of London (Collaboration)
- University of Alabama at Birmingham, United States (Collaboration)
- Leiden University Medical Center (Collaboration)
Publications

Ast A
(2018)
mHTT Seeding Activity: A Marker of Disease Progression and Neurotoxicity in Models of Huntington's Disease.
in Molecular cell

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

Franich NR
(2019)
Phenotype onset in Huntington's disease knock-in mice is correlated with the incomplete splicing of the mutant huntingtin gene.
in Journal of neuroscience research

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

Gomez-Paredes C
(2021)
The heat shock response, determined by QuantiGene multiplex, is impaired in HD mouse models and not caused by HSF1 reduction.
in Scientific reports

Goold R
(2021)
FAN1 controls mismatch repair complex assembly via MLH1 retention to stabilize CAG repeat expansion in Huntington's disease
in Cell Reports

Goold R
(2019)
FAN1 modifies Huntington's disease progression by stabilizing the expanded HTT CAG repeat.
in Human molecular genetics

Hegde RN
(2020)
TBK1 phosphorylates mutant Huntingtin and suppresses its aggregation and toxicity in Huntington's disease models.
in The EMBO journal

Irvine EE
(2019)
Genetic deletion of S6k1 does not rescue the phenotypic deficits observed in the R6/2 mouse model of Huntington's disease.
in Scientific reports

Landles C
(2021)
Development of novel bioassays to detect soluble and aggregated Huntingtin proteins on three technology platforms.
in Brain communications
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 |