Investigation of the mechanism by which huntingtin fragments are produced and their pathogenic relevance to Huntington's disease

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


Huntington's disease (HD) is a neurodegenerative disorder with an average age of onset of 40 years. Affected individuals lose their ability to control movement, develop an impaired mental capacity and psychiatric problems and lose weight. The disease progresses for 15-20 years until death. There are effective treatments for some psychiatric problems (e.g. depression) but there is no way to halt or slow the disease progression. HD is inherited and offspring of affected individuals have a 50% chance of developing the disease. The only difference between the huntingtin gene (HTT) in people who become affected and those who remain disease free is the increased length of the DNA sequence CAG close to its beginning. This results in an extra long tract of glutamines (a building block for proteins) in the huntingtin protein (HTT). The extra glutamines cause the HTT protein to interact with itself and other proteins in an aberrant fashion but the mechanism by which this causes HD remains poorly understood.

Fragments of HTT have been detected in the brains of HD patients and HD mouse models and a considerable amount of research indicates that HTT fragments are more important for the disease process than the full-length protein. Most research into the origin of these fragments has focussed on identifying the relevant proteases (enzymes that cleave proteins), but despite this, the origin of most fragments remains unknown.

We have recently found that the smallest HTT fragment is generated by altered processing of the HTT gene and not by protease digestion. Most genes are composed of exons (DNA sequences that code for the protein) that are separated by introns. As the gene is transcribed into messenger RNA (mRNA), the introns are removed by splicing prior to protein production. The HTT gene is comprised of 67 exons and we have discovered that when the HD mutation is present, exon 1 of HTT does not always splice to exon 2, producing a small novel mRNA that contains exon 1 and intron 1 sequences and terminates in a polyA tail, that marks the end mRNA. This is translated to produce a small exon 1 HTT protein that has been shown to be highly pathogenic in multiple model systems. These results are important because understanding how this HTT fragment is generated will allow us to devise strategies to prevent its formation and thereby determine the extent to which it contributes to the disease process.

An understanding of the processing of the HTT gene and the production of HTT fragments is a basic requisite to unraveling the pathogenic process that causes HD. In this proposal we shall:
1. Further investigate the mechanism by which the HD mutation (extra long CAG sequence) results in the incomplete splicing of exon 1 HTT to exon 2. We have identified a splicing factor (SRSF6) that recognizes CAG repeat sequences, is known to modulate the splicing of genes and we have shown that it binds to the beginning of the HTT gene. We shall investigate the mechanism by which SRSF6 influences HTT splicing and identify other splicing factors that may be involved.
2. Over the past ten years, we have performed an extensive characterization of various mouse models of HD. We shall use a new technology (zinc finger nucleases) to manipulate the Htt gene in HD mice in order to prevent the exon 1 HTT protein from being generated via this mis-splicing mechanism. This will ultimately allow us to determine the extent to which the exon 1 HTT protein contributes to the HD disease process.
3. We have identified a number of additional HTT fragments in the brains of the HD mice. With the exception of exon 1 HTT, the identity and origin of these fragments remains unknown. We shall use a combination of chemical modification and protein sequencing to identify the ends of as many of these other fragments as possible. This may reveal strategies by which the formation of specific fragments can be prevented and their contribution to pathogenesis determined.

Technical Summary

Huntington's disease (HD) is an inherited late onset neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene that leads to an extra long polyglutamine tract in the huntingtin (HTT) protein. Considerable evidence has accumulated to indicate that N-terminal fragments of mutant HTT are pathogenic and may trigger the disease process. We have recently identified a novel mechanism by which a small HTT fragment is generated. We found that the HD mutation leads to incomplete splicing of HTT exon 1 to exon 2, resulting in a small polyadenylated exon 1-intron 1 mRNA that is translated to produce an exon 1 HTT protein. This may have considerable implications for HD pathogenesis as exon 1 HTT fragments have been found to be highly pathogenic in multiple model systems.

SRSF6 is a splicing factor that recognises a CAG repeat sequence and modulates splicing and premature polyadenylation. We shall use molecular biology approaches to further investigate the role of SRSF6 in the aberrant splicing of HTT and identify other splicing factors that may contribute to this process. We shall use zinc finger nuclease technology to produce a model in which the exon 1 HTT protein cannot be generated by mis-splicing and use this to determine the extent to which this small HTT fragment contributes to HD pathogenesis. Finally, we shall use chemical modification and mass spectrometry to determine the identity of additional larger HTT fragments.

Huntingtin is a validated therapeutic target for HD. Understanding the contribution that exon 1 HTT makes to disease pathogenesis is essential. A considerable effort is currently being directed at using gene therapy approaches to lower the levels of HTT, not all of which prevent the production of the exon 1-intron 1 mRNA. A complete understanding of HTT gene processing and the production of HTT fragments is a basic requisite to unraveling HD pathogenesis and may lead to novel therapeutic strategies.

Planned Impact

The potential beneficiaries of our research include Huntington's disease patients and families, scientists and clinicians working on HD, scientists working in the splicing field, as well as biotech and pharma companies with interests in these areas.

A deep understanding of the processing of the HTT gene and the life cycle of the HTT protein are essential to uncovering the pathogenic basis of HD and for developing therapeutic strategies. From this perspective, the entire community of researchers who work on HD in either academia or industry will benefit.

All researchers in both academia and industry who work on therapeutic target validation or the preclinical assessment of therapeutics for HD will benefit as this research will be highly informative as to the relevance of HD models.

Researchers currently devising strategies by which to decrease the levels of HTT will benefit, as this work may reveal which strategies are likely to be optimal.

Disease-modifying treatments for HD currently do not exist. It is a dreadful disease that places an intolerable burden on not only those suffering from the disorder but also on their entire families. Significant breakthroughs in our understanding of the basic disease mechanism convey hope and will encourage participation in clinical trials. An in depth understanding of the basic biology of HD will assist in the development of treatments as discussed above.

This grant will provide Andreas Neueder with the resources to capitalise on his preliminary work in identifying splicing as a novel pathogenic mechanism for HD. He has had a large input into preparing this grant application and this funding will allow him to build a highly competitive CV from which he can launch his independent career. It will benefit the field by ensuring that a highly capable young investigator is committed to work on HD for the near future.


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Description Specific project funding
Amount $60,000 (USD)
Organisation CHDI Foundation 
Sector Charity/Non Profit
Country United States
Start 02/2017 
End 08/2017
Description Wellcome Trust Collaborative Award
Amount £3,365,490 (GBP)
Funding ID 200181/Z/15/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2018 
End 03/2021
Title Delta intron 1 HTT mice 
Description Mouse lines in which all of the cryptic polyA sites have been removed from intron 1 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact The models still need to be characterised 
Title Splicing assay 
Description Minigene system for assessing splicing of HTT gene 
Type Of Material Cell line 
Year Produced 2016 
Provided To Others? Yes  
Impact None yet 
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 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 Lecture at the Young European Scientists meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Undergraduate students
Results and Impact Approximately 400 young scientists from across Europe at a conference to discuss a wide range of topics in the biological and medical sciences
Year(s) Of Engagement Activity 2017
Description School visit (Guildford) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Very engaged sixth formers invited speakers to discuss topics in which they were interested. the lecture continued as a careers discussion.
Year(s) Of Engagement Activity 2015
Description work experience for sixth formers 
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 Arrangement with Tiffin Girls School for sixth form students to visit for work experience during the summer. Students from other schools take part on an ad hoc basis
Year(s) Of Engagement Activity 2006,2007,2008,2009,2010,2011,2012,2013,2014,2015,2016,2017,2018,2019