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
Abstract
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.
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.
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.
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.
Organisations
- University College London (Lead Research Organisation)
- Leiden University Medical Center (Collaboration)
- Ionis Pharmaceuticals (Collaboration)
- Rutgers, The State University of New Jersey (Collaboration)
- University of Ulm (Collaboration)
- University of Alabama at Birmingham (Collaboration)
- Alzheimer's Research UK (Collaboration)
- Brigham and Women's Hospital (Collaboration)
- Massachusetts Institute of Technology (Collaboration)
Publications
Neueder A
(2018)
RNA Related Pathology in Huntington's Disease.
in Advances in experimental medicine and biology
Bates G
(2016)
B4 Detection of the aberrantly spliced exon 1 - intron 1 htt mRNA in HD patient post mortem brain tissue and fibroblast lines
in Journal of Neurology, Neurosurgery & Psychiatry
Ali N
(2016)
B3 Comparison of the effect of a pure CAG repeat and mixed cagcaa repeat on the extent to which the htt gene is aberrantly spliced in knock-in mice
in Journal of Neurology, Neurosurgery & Psychiatry
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
Neueder A
(2018)
Regulatory mechanisms of incomplete huntingtin mRNA splicing.
in Nature communications
Bondulich MK
(2017)
Myostatin inhibition prevents skeletal muscle pathophysiology in Huntington's disease mice.
in Scientific reports
Mason M
(2020)
Silencing Srsf6 does not modulate incomplete splicing of the huntingtin gene in Huntington's disease models
in Scientific Reports
Neueder A
(2017)
The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington's disease patients
in Scientific Reports
Papadopoulou AS
(2019)
Extensive Expression Analysis of Htt Transcripts in Brain Regions from the zQ175 HD Mouse Model Using a QuantiGene Multiplex Assay.
in Scientific reports
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 | 03/2018 |
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 | 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 | 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 | 2013 |
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 | 2013 |
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 |