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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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 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 Exo 1 KO mice 
Organisation Albert Einstein College of Medicine
Country United States 
Sector Academic/University 
PI Contribution Testing the effect of nullizygosity for Exon 1 on somatic instability
Collaborator Contribution Provision of an Exo 1 knock-out mouse and an exo 1 knock-in mouse
Impact none yet
Start Year 2018
 
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 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