HD Insights

Highlights of HSG 2016: Discovering Our Future

The Huntington Study Group held its 23rd Annual Meeting and 10th Annual Clinical Research Symposium November 2-5, in Nashville, TN.

By: Molly J. Elson, BA

HSG 2016 brought together world-renowned clinicians, scientists, coordinators, and community members united by a common passion for improving therapies for HD. Together over the course of several days in November, members gathered to celebrate achievements, explore new therapies, and welcome a new generation of scientists and families into the community. The theme of the conference chaired by Drs. Ray Dorsey and Blair Leavitt was “Progress in HD research and care.” Keynote presentations, interactive working group sessions, and educational courses explored novel therapeutic interventions and emerging models of care. Other members of the HD community joined on the meeting’s final day.

Photo: Researchers presented their findings at the research symposium.

The open sessions began on Wednesday, November 2 with a discussion of the merits of the modified UHDRS as an efficacy endpoint. Results from two major prospective cohort studies (TRACK-HD and COHORT) and clinical trials (CARE-HD) were used as examples. Panels brought together nonprofits, academia, and the pharmaceutical industry to discuss progress in capturing clinically meaningful data and optimizing trial efficiency. “HD Insights of the Year,” an open session exploring breakthrough papers, brought researchers from Harvard, King’s College London, and CHA University in South Korea to the table. Drs. Jong-Min Lee and Flavia Niccolini presented their research into genetic modifiers of time to clinical onset, and pre-symptomatic biomarkers. Similarly, Dr. Jihwan Song posited that mHTT propagates in a prion-like manner. Veteran project manager, Ms. Elise Kayson, led a forum looking back on the results of PHAROS and PREDICT and what individuals at risk for HD can learn. Later, Dr. Martha Nance led a session in which the audience was invited to bring their most challenging cases for group discussion. Ethical dilemmas discussed included how to give genetic testing results to a parent who wished their underage child to be present, and the importance of maintaining contact with research participants post-study.

Photo: Dr. David Oakes received a well-deserved award from Drs. Ray Dorsey and Ira Shoulson in appreciation of his years of service.

A warm reception concluded the second day of activities. Drs. Ira Shoulson and Ray Dorsey presented well-deserved awards to Dr. David Oakes, Dr. Jody Corey-Bloom, Ms. Paula Wasserman, and Ms. Elise Kayson.

The “Digital Biomarkers for HD” panel, chaired by Dr. Ralf Reilmann, stood out for its presentation of some of the most promising technologies scientists are bringing to HD research. Drs. Max Little, Larsson Omberg, Spyros Papetropoulos, and Guarav Sharma described their development of novel objective motor assessments using tools such as Apple’s ResearchKit and MC10’s wearable sensors. Dr. Dorsey’s “Progress and directions” lunch laid out a five-year plan with concrete, achievable objectives to make HD a treatable condition. He recognized key accomplishments in recent HSG trials, and challenged researchers to embrace novel technologies and revolutionize clinical trial conduct.

Dr. Bob Beall, former president and CEO of the Cystic Fibrosis Foundation (CFF), gave the HSG 2016 keynote address, “Lessons learned from cystic fibrosis.” Dr. Beall transformed care and research for cystic fibrosis by raising money to identify genes responsible for the condition, partnering with the pharmaceutical industry, and establishing a network of care centers to increase trial efficiency. His leadership helped to increase the average life expectancy of cystic fibrosis patients from 18 years to 41 years. Dr. Beall challenged HSG to leverage CFF’s successful strategies towards HSG’s own goals.

The 10th annual HD Research Symposium opened the final day of the conference with an education panel, “Regulatory path for development of medical devices and drugs.” Members of the FDA discussed the regulatory pathway through which new therapeutic technologies are evaluated. Dr. Bonnie Hennig led a panel presenting the latest in stem cell research for HD. Attendees left with an appreciation of the challenges faced by stem-cell researchers. When the scientific portion of the conference concluded, Family Education Day began. Families and friends now sat at the tables as Peter Rosenberger shared his story of compassion, hope, and humor as a caregiver. Other talks focused on how to talk about HD as a family, provide palliative care, and test those at risk. The community gathering at the event’s conclusion reinforced the conference’s theme of working together and looking towards the future in order to halt the progression of this disease.

Photo: Everyone gathered for a group photo on Thursday afternoon.

Thank you to Raptor

HD Insights thanks raptor for its generous support.

Editor’s Letter

Welcome to the 16th edition of HD Insights, timed for release at the beginning of the first Pan-American Parkinson’s Disease and Movement Disorders Congress in Miami, FL. Now in our sixth year, we continue our mission to promote, disseminate, and facilitate HD research with content that is valuable and informative to the global community of HD researchers. This 16th edition marks the beginning of a year-long focus on game-changing genetic therapies preparing to enter human trials, along with the continuation of our clinical trials update, Research Round-Ups, and the conversations with HD clinicians, scientists, and industry representatives that we have brought you since 2011.

We begin our first 2017 edition with a look back at the major developments in HD clinical research from 2016. We then bring you some of the most highly cited research of 2015, with articles from Dr. Zhenyu Yue and Dr. Margaret Pearce exploring the roles of autophagy and phagocytosis in HD pathology. Dr. Lise Munsie continues her excellent Research Round-Up series. We sit down with Dr. Pavlina Konstantinova of uniQure, a company working to create a one-time gene-silencing treatment for HD patients using micro RNA. Ms. Marleen van Walsem and colleagues describe their work evaluating the impact of assistive technology for cognition in improving HD patients’ quality of life, a highlight from the Journal of Huntington’s Disease. Ms. Molly Elson describes the highlights of the 2016 HSG Annual Meeting for those who could not attend. Finally, we continue to update the HD community on upcoming, ongoing, and recently completed clinical trials. We have streamlined our trial listing to add a “Pipeline” to this edition, allowing us more space to explore those compounds and treatments currently in preclinical as well as clinical development. We welcome your thoughts and comments on this new feature.

HD Insights is dedicated to bringing together ideas and people in the HD community to advance HD research. This edition, we are announcing the search for a new Deputy Editor, to lead the periodical into the future as Meredith Achey steps away to focus on her medical training. We also welcome your job postings, scholarship announcements, and other opportunities you would like to share with the HD community. If you have an opportunity you would like to share with nearly 3000 HD researchers and clinicians, please let us know at editor@hdinsights.org.

If you or your colleagues would like a free electronic subscription to HD Insights, please visit www.HDInsights.org and complete the subscription form. We truly appreciate your comments, suggestions, and contributions. If you would like to get in touch, please email editor@hdinsights.org. Thank you.

Job Opportunity: Deputy Editor, HD Insights

Job description: If you are passionate about HD research and want to reach nearly 3,000 HD researchers and clinicians around the world, consider joining our team as the Deputy Editor of HD Insights.

Oversee the creation of 3-4 editions per year of HD Insights. Responsible for soliciting, editing, and coordinating review and layout of each edition, as well as contributing original articles and ideas for the publication. Ideal candidates will have experience in academic writing, be familiar with Huntington disease research and have the flexibility to work remotely with individuals all over the world. Training and supervision will be provided remotely by the outgoing Deputy Editor and current Editor. Ideal start date is Q2 of 2017. Compensated position.

Please send CV and letter of interest to editor@hdinsights.org.

HD Insights Volume 15 (PDF)

To read a non-PDF version of HD Insights Vol. 15, click here.

Finding balance in HD: The role of PIAS1 in protein homeostasis

pias

Figure. The biochemistry in our brains is in a constant balance. In neurodegenerative disease, the scales often tilt toward a dysfunctional state. By restoring the fine balance of levels of PIAS1 in a mouse model of HD, we showed not only improvements in behavioral deficits, but a restoration of balance in several pathways known to be out of balance in HD. Decreasing the burden of PIAS1 on the cell appears to cause a shift in cellular homeostasis toward a protective state. This study suggests that PIAS1 and other E3 SUMO ligases may be key to tipping the balance between normal protein homeostasis and disease processes that cause neurodegeneration.

By: Joseph Ochaba, PhD
There are currently no medications or treatments to alter the devastating course of HD. Although some medications to ameliorate HD symptoms exist, they are often of limited effectiveness;1 therefore, there is a great need to develop treatments and discover targets to effectively alter the course of HD. As in many other neurodegenerative diseases, the underlying pathology of HD involves the decline or loss of protein quality control networks, homeostasis, and the consequent accumulation of highly insoluble protein complexes that in the case of HD contain modified and misfolded mHTT protein and other post-translationally modified proteins.

Previous studies in the lab demonstrated that the enzyme PIAS1 (an E3 SUMO-protein ligase) alters mHTT accumulation and SUMO modification of HTT in cells.2 In collaboration with Dr. Beverly Davidson’s group at the Children’s Hospital of Philadelphia, our lab recently investigated the role of PIAS1 as a potential new therapeutic target for HD in an animal model of the disease.
To examine its role in HD pathology, PIAS1 expression levels were modulated in the striatum of a rapidly progressing HD mouse model, R6/2.3 Using miRNA to knock down PIAS1 expression in the striatum, we found significant improvements in HD-associated phenotypes in a battery of motor and neuromuscular tasks; reduced pathogenic accumulation of insoluble mHTT; and improved longevity of the mice, with reduced levels of PIAS1 compared to controls.

Strikingly, reduced PIAS1 levels ameliorated neuroinflammatory disease – associated increases in apparent microglial accumulation and dysregulation of relevant proinflammatory cytokines. Conversely, overexpression of PIAS1 exacerbated disease phenotypes and the accumulation/aggregation profile. While the exact contribution of PIAS1 in HD remains unclear, we have demonstrated that this particular enzyme may provide a new target for RNAi-mediated suppression to treat several aspects of HD, including reversing protein aggregation; increasing synapses; and reducing inflammation – all of which contribute to maintaining a delicate biological balance – while also slowing or perhaps reversing behavioral decline.

While aberrant neuronal signaling, inflammation, energy production, and gene expression underlie the molecular basis of HD, a delicate balance of functional properties of the proteome ultimately dictates cell function.4,5 Under normal conditions, proteome integrity is maintained by the proteostasis network. In HD, chronic expression of expanded polyglutamine peptides results in an age-dependent collapse of this balance as evidenced by increased aggregation and mislocalization and dysfunction of stable proteins.6 Because many of these networks may be potential therapeutic targets, our current work aims to investigate more longitudinal approaches by assessing the effects of PIAS1 modulation and mHTT accumulation/aggregation dynamics in long-term mouse models of the disease, in addition to clinically relevant human patient – derived induced pluripotent stem cell neuronal subtypes, in order to fully understand the contribution of PIAS1 to HD and evaluate its potential as a clinical target. The cellular environment plays a significant role in determining whether disease proteins are converted into toxic or benign forms, and for this reason, understanding the role that PIAS1 plays pre- or post-symptomatically will be important moving forward.

Our results strengthen the link between accumulation of insoluble protein complexes and HD pathogenesis, and suggest that PIAS1 protein is a potential new target for future therapies in HD. Together, the fundamental properties identified here may also broadly impact other diseases, suggesting that regulating multifunctional proteins such as PIAS1 may be one key to tipping the balance between normal protein homeostasis and disease processes that cause neurodegeneration.

References

  1. Frank S. Treatment of Huntington’s disease. Neurotherapeutics. 2014 Jan;11(1):153-60. doi: 10.1007/s13311-013-0244-z.
  2. O’Rourke JG, Gareau JR, Ochaba J, et al. SUMO-2 and PIAS1 modulate insoluble mutant huntingtin protein accumulation. Cell Rep. 2013 Jul 25;4(2):362-75. doi: 10.1016/j.celrep.2013.06.034. Epub 2013 Jul 18
  3. Ochaba J, Monteys AM, O’Rourke JG, et al. PIAS1 regulates mutant huntingtin accumulation and Huntington’s disease-associated phenotypes in vivo. Neuron. 2016 May 4;90(3):507-20. doi: 10.1016/j.neuron.2016.03.016. Epub 2016 Apr 14
  4. Tsvetkov AS, Arrasate M, Barmada S, et al. Proteostasis of polyglutamine varies among neurons and predicts neurodegeneration. Nat Chem Biol. 2013 Sep;9(9):586-92. doi: 10.1038/nchembio.1308. Epub 2013 Jul 21
  5. Reiner A, Dragatsis I, Dietrich P. Genetics and neuropathology of Huntington’s disease. Int Rev Neurobiol. 2011; 98: 325–372. doi:  10.1016/B978-0-12-381328-2.00014-6
  6. Margulis J, Finkbeiner S. Proteostasis in striatal cells and selective neurodegeneration in Huntington’s disease. Front Cell Neurosci. 2014 Aug 7;8:218. doi: 10.3389/fncel.2014.00218. eCollection 2014.

Meet the Company: WAVE Life Sciences

Wave LifeVITAL SIGNS
Name: WAVE Life Sciences
Stock symbol: WVE
Share price as of 10/9/16: $30.27
Market capitalization: $574 million
U.S. Headquarters: Cambridge, MA

Jeglinski

Brenda Jeglinski serves as the Director of Clinical Operations at WAVE. She enjoys spending her free time with her husband and children.

Wendy Erler, MBA is the Vice President of Patient Advocacy and Market Insights at WAVE. When she is not working to bring precision medicines to patients, she enjoys paddleboarding and spending time with her husband and their three children.

Wendy Erler, MBA is the Vice President of Patient Advocacy and Market Insights at WAVE. When she is not working to bring precision medicines to patients, she enjoys paddleboarding and spending time with her husband and their three children.

panzara

Michael Panzara, MD, MPH is the Franchise Lead for Neurology at WAVE. When he is not thinking about precision medicines for HD, he enjoys spending time at home with his family.

Precision Medicine for HD
WAVE Life Sciences, a preclinical biopharmaceutical company publicly traded since 2015, utilizes their proprietary platform to synthesize stereopure nucleic acid therapies for currently untreatable genetic diseases. HD Insights spoke with Brenda Jeglinski, Director of Clinical Operations, Wendy Erler, Vice President of Patient Advocacy and Market Insights, and Michael Panzara, Franchise Leader for Neurology, about the company’s work in HD. The following is an edited transcript of the conversation.

HD INSIGHTS: Thank you all for joining us today. Please tell us about WAVE Life Sciences.

JEGLINSKI: We are a small company, just starting our first clinical trials. We are currently working with investigators to develop our clinical trials, and identify where and when we can begin the first human dosing with our compounds.

ERLER: One of the exciting things about WAVE is that we have a number of people who have come into the company with past experience working in oligonucleotides, and that has really informed our HD program in particular.

HD INSIGHTS: How did WAVE first become interested in HD?

PANZARA: One of the unique aspects of our oligonucleotide platform is that we are able to control properties of each oligonucleotide, including specificity. This enables an allele-specific approach when targeting genetic diseases, making the mutation in HD an ideal target. In addition, WAVE is building a neurology franchise upon this platform. Neurological diseases are an area of high unmet need and our primary focus at WAVE. Combining our platform with our focus on this area of high unmet need, HD seemed liked the ideal place for us to begin to develop the next generation of therapeutics.

HD INSIGHTS: You mentioned that your approach is allele-specific. Other companies are developing non – allele-specific compounds. Can you talk about the possible relative advantages of allele specificity?

PANZARA: Healthy huntingtin protein (HTT) has many essential functions in the nervous system, while the presence of mHTT leads to neurodegeneration, so we thought that if we could develop an approach that targeted the gene product, the transcript of the mHTT allele specifically, while leaving the transcript for the healthy protein intact, we could potentially increase the benefits of treatment while minimizing risks. One of the strengths of controlling the stereochemistry of nucleic acid synthesis is that it allows us to specifically enhance and adjust properties of the nucleic acid, such as its stability, its activity, its immunogenicity, and most importantly, its specificity, which enables an allele-specific approach to treatment of the disease.

HD INSIGHTS: Can you talk about the preclinical evidence, and the importance of allele specificity?

PANZARA: The literature suggests that HTT has many important functions in preservation of neuronal health in the central nervous system, which is what prompted us to explore the potential to target mHTT in a selective way.1-4 By controlling stereochemistry, we have been able to design an antisense oligonucleotide that appears to discriminate between HTT and mHTT. We have done experiments in fibroblasts from HD patients known to have specific single nucleotide polymorphisms (SNPs) associated with their HD mutations. We have designed our oligonucleotides to target those SNPs, and demonstrated in this system that mHTT can be reduced significantly, while leaving healthy wild-type HTT relatively intact (WAVE preclinical data). Again, the overarching goal was to create a very specific effect on the detrimental protein, while leaving the wild-type protein intact, attempting to maintain normal neuronal function.

ERLER: To date, nucleic acid therapies have comprised complex mixtures of hundreds of thousands of chemical entities called stereoisomers. Some of those stereoisomers have therapeutic effects, while some are less beneficial, or have an unknown impact, and could contribute to undesirable side effects. With WAVE’s novel chemistry platform, we eliminate those complex mixtures, giving us control over the pharmacology by creating stereopure compounds. This rational, very specific design is what gives all the attributes just described specific to our HD candidates, but also allows us to use our oligonucleotides in other ways, such as exon skipping.

HD INSIGHTS: Tell us about your regulatory status.

PANZARA: We intend to file two INDs before year’s end, and we have been granted orphan drug designation in the USA. This should enable us to initiate studies in the early part of 2017 that focus on two distinct patient populations.

HD INSIGHTS: Can you tell us about the patient populations that you are looking to enroll in your first clinical trials?

PANZARA: Our compounds are developed to target the two most common SNPs associated with the mHTT allele which encompasses nearly two-thirds of the HD population. In addition, we are looking to enroll patients over 25 years old with early manifest HD.

ERLER: This is a really good opportunity to explain why there are two INDs being filed, since so many in the community have had exposure to other programs, specifically Ionis’s (see HD Insights, Vol. 13). Our planned INDs are, in fact, for two separate drug candidates for HD patients specifically targeted to two distinct SNPs (SNP 1 or SNP 2), so that means patients who are screened for these studies, will have a blood test to determine if they are eligible for SNP 1 or SNP 2 (see Meet the Compounds, p. 19). We plan to run both studies in parallel at the same trial centers.

PANZARA: In addition, an individual who does not want to know their CAG repeat number or their full genotyping can still participate in the study. The screening test would focus only on study eligibility, and the presence of either SNP 1 or SNP 2. From there, they would then have the opportunity to enroll, assuming they meet other inclusion and exclusion criteria.

JEGLINSKI: Another key factor is that because there is SNP 1 versus SNP 2, there are two different programs, but they are identical studies. There is no benefit or anything more enticing in one study versus the other.

HD INSIGHTS: It sounds as if WAVE is focusing on precision medicine, or personalized medicine – not only just focusing on a particular condition, but focusing on particular subpopulations within that condition.

PANZARA: Exactly. We see this as an important step towards precision medicine, designing the most appropriate drug for a given patient. We also see this as the start of an important part in the history of WAVE Life Sciences. These will be our first two clinical studies in precision medicine, but I think it epitomizes our approach to diseases with high unmet need: focusing on the most appropriate drug for a given patient.

HD INSIGHTS: Can you say when these precision medicine treatments will be investigated in clinical trials?

PANZARA: Our intention for HD is open both trials in the first part of 2017. These will be phase 1 safety studies.

HD INSIGHTS: This is WAVE’s first involvement in HD. Can you tell us a little bit about your impressions of the HD community? Any surprises or disappointments?

ERLER: I feel completely privileged and honored to have engaged with the HD community. It has been one of the most gratifying personal experiences of my career, because the community is incredibly open and warm, and very, very interested in sharing their stories so that we as an organization can better understand the true experience of living with HD, not just as a patient, but as a caregiver, or a spouse, son or daughter of someone who is living with HD. It has completely encouraged me to fight on behalf of these families, because I have just been so incredibly impressed with their fortitude and tenacity, and universal positive attitudes in light of their burden. At WAVE, we have been fortunate to have HD community members, patients and care givers, come to our office to meet with our team and share their stories and we have also been able to have WAVE employees participate in several HD fund raisers.
So while I knew this was a devastating disease, I was definitely surprised at just the sheer will and force these families have, and how positive they are, and how everybody without question comments about wanting to get involved with our clinical trials, not to help themselves, but to help future generations.

PANZARA: The only thing I would add is that because I am relatively new to WAVE and to this particular therapeutic area from a drug development standpoint, I have been extremely impressed by the sense of collaboration and purpose of the HD community. Everyone is universally focused on doing whatever they can for patients with this disease.

HD INSIGHTS: Thank you all very much for your efforts and for WAVE’s interest in developing precision treatments for HD. We wish you all the best, and great success in the future.
References

1. Dragatsis I, Levine MS, Zeitlin S. Inactivation of Hdh in the brain and testis results in progressive neurodegeneration and sterility in mice. Nat Genet. 2000;26(3):300-306.
2. Leavitt BR, van Raamsdonk JM, Shehadeh J, et al. Wild-type huntingtin protects neurons from excitotoxicity. J Neurochem. 2006;96(4):1121-1129.
3. Rigamonti D, Sipione S, Goffredo D, Zuccato C, Fossale E, Cattaneo E. Huntingtin’s neuroprotective activity occurs via inhibition of procaspase-9 processing. J Biol Chem. 2001;276(18):14545-14548.
4. Zhang Y, Leavitt BR, van Raamsdonk JM, et al. Huntingtin inhibits caspase-3 activation. EMBO J. 2006;25(24):5896-5906.

Meet the Compounds: WVE-120101 and WVE-120102

Image source: Robinson R. RNAi Therapeutics: How Likely, How Soon? PLoS Biol. 2004 Jan;2(1):E28. Epub 2004 Jan 20. DOI: 10.1371/journal.pbio.0020028. Published under a Creative Commons 2.5 License.

Image source: Robinson R. RNAi Therapeutics: How Likely, How Soon? PLoS Biol. 2004 Jan;2(1):E28. Epub 2004 Jan 20. DOI: 10.1371/journal.pbio.0020028. Published under a Creative Commons 2.5 License.

By: Meredith A. Achey, BM
Manufacturer: WAVE Life Sciences
Molecular formula: Stereopure antisense oligonucleotide (ASO) targeting single nucleotide polymorphisms (SNPs).
Mechanism of action: WVE-120101 and WVE-120102 act by selectively binding mHTT mRNA, thereby selectively inhibiting production of mHTT. WAVE Life Sciences has developed a strategy to synthesize stereopure nucleotide-based therapies that may improve their efficacy and safety.1

Nucleotide-based gene silencing therapies such as RNA interference (RNAi) and antisense oligonucleotides (ASOs) represent some of the most promising potential therapies for genetic diseases such as HD. However, methods for their synthesis developed to date produce mixtures of stereoisomers, some of which are therapeutic, and others that may be less effective or potentially detrimental.1,2 WAVE Life Sciences has developed a propriety technology for the synthesis of stereopure nucleic acid therapeutics3-5 with the potential to improve therapeutic efficacy and reduce harmful effects.6
The company hopes to develop stereopure nucleic acid therapies for a variety of genetic diseases, including central nervous system disease such as HD, and a number of genetic disorders that affect other organ systems.7

WAVE’s most advanced HD programs currently encompass two compounds that target different single nucleotide polymorphisms (SNPs) common to the mutant allele. The company plans to file investigational new drug applications for both of these compounds this year, and has received orphan drug designation in the US for its lead candidate, WVE-120101.8 These compounds target the two most common SNPs associated with mHTT.9 Current efforts by Ionis in the first human trial of a gene-silencing nucleotide-based therapy do not target mHTT selectively (see HD Insights, Vol. 13), although preclinical data have not suggested a significant decrease in efficacy nor increase in potential harm from a non-selective approach.10 However, interest in more selective approaches has continued because the possibility for off-target effects with non-selective silencing remains problematic.11-15

Pfister and colleagues9 reported that they have been able to develop small interfering RNAs (siRNAs) that target specific SNPs to treat approximately 75% of individuals with HD. In 2015, targeting approaches were further refined with the discovery that targeting three common haplotypes could enable selective silencing of the HD gene in approximately 80% of patients.13 The use of stereopure synthesis to enhance the delivery and efficacy of these highly targeted compounds may further refine ongoing efforts to develop an effective and safe gene-silencing therapy for HD.

Table. Selected studies of allele-specific gene-silencing therapies

Study Year Type of therapy Most promising target SNP(s) Summary
Skotte NH et al.16 2014 ASO rs7685686_A Allele-specific, high affinity ASOs targeting different SNPs associated with HD could together offer allele-selective silencing to approximately 50% of patients, and non-selective silencing to the remainder.
Drouet et al.12 2014 shRNA rs363125, rs362331, rs2276881, rs362307 shRNA delivered with a lentiviral vector to cellular and animal models showed in vitro and in vivo silencing of mHTT.
Yu D et al.17 2012 ss-siRNA Targets CAG repeat ss-siRNA targeting expanded CAG repeats led to selective silencing of mHTT expression in an HD mouse model.
Abbreviations: ASO: Antisense oligonucleotide; shRNA: small hairpin RNA; ss-siRNA: single-stranded small interfering RNA; SNP: single nucleotide polymorphism

References
1. Wild EJ, Tabrizi SJ. Targets for future clinical trials in Huntington’s disease: What’s in the pipeline? Mov Disord. 2014 Sep 15;29(11):1434-45. doi: 10.1002/mds.26007. Epub 2014 Aug 25.
2. De Mesmaeker A, Altmann K-H, Waldner A, Wendeborn S. Backbone modifications in oligonucleotides and peptide nucleic acid systems. Curr. Opin. Struct. Biol. 1995;5(3):343-355.
3. Butler D, Iwamoto N, Meena M, et al. Chiral control. Google Patents; 2015.
4. Verdine GL, Meena M, Iwamoto N. Novel nucleic acid prodrugs and methods of use thereof. Google Patents; 2012.
5. Verdine GL, Meena M, Iwamoto N, Butler DCD. Methods for the synthesis of functionalized nucleic acids. Google Patents; 2014.
6. Wave Life Sciences. Wave Life Sciences – Platform. [Website]. 2016; www.wavelifesciences.com/platform. Accessed August 19, 2016.
7. Wave Life Sciences. Wave Life Science – Pipeline. 2016; www.wavelifesciences.com/pipeline. Accessed August 19, 2016.
8. WAVE Life Sciences receives orphan drug designation from FDA for its lead candidate designed to treat Huntington’s disease [press release]. June 21, 2016.
9. Pfister EL, Kennington L, Straubhaar J, et al. Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr. Biol. 2009;19(9):774-778.
10. Kordasiewicz Holly B, Stanek Lisa M, Wancewicz Edward V, et al. Sustained therapeutic reversal of Huntington’s disease by transient repression of Huntingtin synthesis. Neuron. 2012 Jun 21;74(6):1031-44. doi: 10.1016/j.neuron.2012.05.009.
11. Carroll JB, Warby SC, Southwell AL, et al. Potent and selective antisense oligonucleotides targeting single-nucleotide polymorphisms in the Huntington disease gene / Allele-specific silencing of mutant huntingtin. Mol Ther. 2011 Dec;19(12):2178-85. doi: 10.1038/mt.2011.201. Epub 2011 Oct 4.
12. Drouet V, Ruiz M, Zala D, et al. Allele-specific silencing of mutant huntingtin in rodent brain and human stem cells. PLoS One. 2014 Jun 13;9(6):e99341.
13. Kay C, Collins JA, Skotte NH, et al. Huntingtin haplotypes provide prioritized target panels for allele-specific silencing in Huntington disease patients of European ancestry. Mol Ther. 2015 Nov;23(11):1759-71.
14. Miniarikova J, Zanella I, Huseinovic A, et al. Design, characterization, and lead selection of therapeutic mirnas targeting huntingtin for development of gene therapy for Huntington’s disease. Mol Ther Nucleic Acids. 2016 Mar 22;5:e297.
15. Pfister EL, Kennington L, Straubhaar J, et al. Five siRNAs targeting three SNPs may provide therapy for three-quarters of Huntington’s disease patients. Curr Biol. 2009 May 12;19(9):774-8.
16. Skotte NH, Southwell AL, Østergaard ME, et al. Allele-specific suppression of mutant huntingtin using antisense oligonucleotides: providing a therapeutic option for all Huntington disease patients. PLoS One. 2014 Sep 10;9(9):e107434.
17. Yu D, Pendergraff H, Liu J, et al. Single-stranded rnas use rnai to potently and allele-selectively inhibit mutant huntingtin expression. Cell. 2012 Aug 31;150(5):895-908.

Highlights from the Journal of Huntington’s Disease

jhdInduced pluripotent stem cells in HD research
By: Kimberly Kegel-Gleason, PhD
Original article: Tousley A, Kegel-Gleason KB. Induced Pluripotent Stem Cells in Huntington’s Disease Research: Progress and Opportunity. J Huntingtons Dis. 2016 Jun 28;5(2):99-131. doi: 10.3233/JHD-160199.

Although many cell types are affected in HD, the impact of the disease on CNS neurons is remarkable. Neurons of the cortex and striatum degenerate and eventually die, causing the majority of HD symptoms. Until recently, it has been difficult to study human CNS neurons because of ethical considerations – it is not ethical to perform a biopsy on human patients to obtain brain cells for research. Embryonic stem cells (ESCs) are pluripotent, meaning that they have the ability to become neurons; however, the use of ESCs to obtain neurons is also fraught with ethical challenges, including the destruction of an embryo. Enter induced pluripotent stem cells (iPSCs). Skin or blood cells from controls and HD patients can be made into iPSCs by the introduction of just a few factors. iPSCs are very similar to ESCs and can be differentiated to resemble CNS neurons or other CNS cell types in order to study disease mechanisms, and to screen compounds that might be developed into new therapies.

A major advantage to iPSCs is that normal and mutant proteins are expressed at endogenous levels just as they are in the human patient; furthermore, the effects of varied genetic backgrounds on the behavior of the mutant protein can be assessed. Studies with iPSCs from patients with neurodegenerative diseases other than HD have provided new insights previously not found using animal models. For instance, using three-dimensional cultures of CNS neurons from iPSCs from human Alzheimer disease (AD) patients, intracellular tangles, which are major feature of AD, were observed.1 Intracellular tangles had never been recapitulated in mouse models of AD, and the results pointed to a particular protein only found in humans as a major target of pathology.

As with research in other neurodegenerative diseases, iPSCs from controls and HD patients have been in development to uncover previously unknown human-specific pathological mechanisms, to validate phenotypes identified in animal models, and for compound screening. In our review,2 we characterize the state of the HD iPSC field. We describe the current inventory of cells available to HD researchers, many of which cells are freely available. We also highlight changes that have been identified in HD cells compared to controls, in pathways, individual gene changes, functional phenotypes, and the role of stress and aging. Furthermore, we compare results obtained with various neuronal differentiation protocols.

Table: Summary of major progress and opportunities for expanded research using HD IPSCs

 

Progress Opportunities
Numerous HD iPSCs created Increase the number of HD and control lines used within each study
A few genetically corrected, isogenic iPSCs created Increase number of genetically corrected, isogenic iPSCs created and available (CRISPR/Cas9)
Several studies on neuronal cultures using diverse differentiation protocols Use of reproducible protocols for comparison of results across laboratories
Stress-induced phenotypes identified Expand the phenotypes identified in the absence of stress
SiRNAs and miRNAs targeting alleles with SNPs, Zinc Finger Proteins Develop additional allele-specific reagents and tools to target mHTT
Two studies with iPSC astrocytes Studies with iPSC glia (astrocytes, oligodendrocytes, microglia)
Co-culturing, 3-D cultures, and organoids
Artificial aging

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We also note the pervasive use of stress to unveil functional phenotypes in HD cells. Because stress exacerbates HD symptoms in patients, this may reflect important disease mechanisms. One argument in favor of this approach is that iPSC induced neurons are very immature compared to those in the brain of an adult with HD, so stress might mimic aging to enhance a phenotype. However, more time spent investigating what may be more subtle phenotypes in the absence of stress may lead to a better understanding of underlying pathology that leaves a cell vulnerable to stress and eventually triggers disease.

Although much progress has been made, culturing and differentiating iPSCs is still extremely expensive, time consuming and difficult, thus limiting the number of investigators who can take advantage of this valuable resource. We hope this review will enable those new to the iPSC field to consider and control the inherent problems with iPSC lines, and so enable reproducible research across the field.

One major limitation we found for interpreting data across the HD field is the relative paucity of cell lines used – many times, reports include data from just one cell line. In order to increase the reproducibility of research across the field, we suggest that results from at least three HD cell lines from three individual patients and three cell lines from three individual controls be used for robust phenotypes (six lines total). For more subtle phenotypes it may be necessary to use many more cell lines. For comparison, 10–12 control cell lines are currently being used by investigators in other fields.3 Alternatively, a combination of control cell lines (cell lines from unaffected individuals), and genetically corrected cell lines (using homologous recombination or CRISPR), and use of effective mHTT-lowering reagents such as siRNAs, miRNAs or zinc finger proteins in HD lines could be used. We note that data from genetically corrected cell lines should be interpreted with caution because the cell lines undergo several rounds of selective pressure during their generation that could alter a particular phenotype.

Our review of the field identifies areas of opportunity for which additional research would be of great value. For instance, very few studies used other brain cell types that can be differentiated from iPSCs such as astrocytes and oligodendrocytes, which may also impact HD pathology. The field should welcome more studies using iPSC-derived glial cells.

References
1. Choi SH, Kim YH, Hebisch M, et al. A three-dimensional human neural cell culture model of Alzheimer’s disease. Nature. 2014;515(7526):274-278.
2. Tousley A, Kegel-Gleason KB. Induced Pluripotent Stem Cells in Huntington’s Disease Research: Progress and Opportunity. J Huntington’s dis. 2016;5(2):99-131.
3. Boulting GL, Kiskinis E, Croft GF, et al. A functionally characterized test set of human induced pluripotent stem cells. Nat Biotechnol. 2011;29(3):279-286.

Research Round-Up: Insights of the Year 2015-2016

In this edition, we recognize the most influential papers in HD research in the 2015-2016 year with our largest Insights of the Year competition yet. The winners of last year’s competition nominated 17 articles reporting on basic science, clinical, and imaging and biomarkers research. Fourteen authors provided summaries, included in this edition, and the remaining three are cited in their respective sections. The HD Insights Editorial Board and prior winners then voted to select the three most influential papers, one in each category. The authors of the winning papers will present their research in a panel discussion at the HSG Annual Meeting on November 2, 2016. Congratulations to all the nominees and winners!

In the lab…

Figure. Mutant htt proteins (EM48) derived from human fibroblast carrying 143 CAG repeats (HD143F) attack medium spiny neurons (DARPP-32) in the host mouse brain, shown here at 40 weeks post-implantation.

Figure. Mutant htt proteins (EM48) derived from human fibroblast carrying 143 CAG repeats (HD143F) attack medium spiny neurons (DARPP-32) in the host mouse brain, shown here at 40 weeks post-implantation.

Human-to-mouse prion-like propagation of mHTT
Most influential insight
By: Iksoo Jeon, PhD, Francesca Cicchetti, PhD, and Jihwan Song, DPhil

To date, the pathophysiology of HD has been thought to be primarily driven by cell-autonomous mechanisms. However, we have demonstrated that HD patient-derived fibroblasts, or their induced pluripotent stem cells (iPSCs), can transmit mutant huntingtin (mHTT) protein aggregates to genetically unrelated and healthy host tissue following implantation into the cerebral ventricles of neonatal mice in a non – cell-autonomous fashion. We found that transmitted mHTT aggregates gave rise to loss of striatal medium spiny neurons, and increased inflammation and gliosis in associated brain regions, which led to motor and cognitive impairments, thereby recapitulating the behavioral and pathological phenotypes that characterize HD.

In addition, we showed that exosomes can carry mHTT as cargo between cells, triggering the manifestation of HD-related behavior and pathology.
This is the first evidence of human-to-mouse prion-like propagation of mHTT in the mammalian brain, a finding that will help unravel the molecular basis of HD pathology, as well as to lead to the development of a new range of therapies for CNS neurodegenerative diseases. We are currently developing ways to overcome the pathogenic host brain background to better treat HD, using stem cells and other methods.

Nominated article: Jeon I, Cicchetti F, Cisbani G, et al. Human-to-mouse prion-like propagation of mutant huntingtin protein. Acta Neuropathol. 2016 Oct;132(4):577-92. doi: 10.1007/s00401-016-1582-9. Epub 2016 May 24.

 

Figure. 3D EM maps of Q23 and Q78-huntingtin. 3D EM maps, reconstructed from negative stained Q23- (grey) and Q78- (cyan) huntingtin particles, are presented in different x-axis angles. The final resolutions are calculated at 33.5, and 32.0 Å, respectively.

Figure. 3D EM maps of Q23 and Q78-huntingtin. 3D EM maps, reconstructed from negative stained Q23- (grey) and Q78- (cyan) huntingtin particles, are presented in different x-axis angles. The final resolutions are calculated at 33.5, and 32.0 Å, respectively.

Huntingtin’s spherical solenoid structure enables polyglutamine tract − dependent modulation of its structure and function
Nominee, “In the lab…”
By: Ravi Vijayvargia, PhD and Taeyang Jung, PhD
HD is caused by an expanded polyglutamine tract in the amino terminus of huntingtin (HTT) protein. How the addition of extra glutamines in such a large protein (3,144 amino acids) results in such a drastic change in structure and function of HTT has remained an unresolved question. In order to gain insights into the role of polyglutamine tract length in the modulation of HTT structure and function, we utilized purified full-length human HTT that had polyglutamine tract lengths of 2, 23, 46, 67 and 78. Using several structural and biochemical approaches, we found that HTT is folded back, forming a spherical shape with an internal cavity that may serve as a binding pocket for other molecules.

Our study used Circular Dichromism Spectroscopy and 3D-EM analysis to show that the expanded polyglutamine tract alters the entire structure of HTT, instead of a local change near the polyglutamine tract. Furthermore, cross-linking mass spectrometry studies provide a glimpse into the domain structure of HTT and how the five sub-domains are oriented with respect to each other, implying that the polyglutamine expansion influences the arrangement of these sub-domains relative to each other. Thus, altered structure, as seen in mHTT, can produce distinct alterations of normal protein-protein interactions, as well as result in differential post-translational modifications that may serve as therapeutic targets (unpublished observations). While more work is needed to obtain a high-resolution structure of HTT, these insights may contribute to understanding the role of mHTT in HD pathogenesis.

Nominated article: Vijayvargia R, Epand R, Leitner, et al. Huntingtin’s spherical solenoid structure enables polyglutamine tract − dependent modulation of its structure and function. Elife. 2016 Mar 22;5:e11184. doi: 10.7554/eLife.11184.

Figure A. Knockdown of Htt with siRNA improves PPAR transcriptional activity both with and without PPAR agonist in primary cortical neurons from wild type and BAC-HD mice.

Figure A. Knockdown of Htt with siRNA improves PPAR transcriptional activity both with and without PPAR agonist in primary cortical neurons from wild type and BAC-HD mice.

Figure B. Results from quadriceps of preclinical trial mice (wild type and HD N171-82Q) indicate that PDK4 and SCD1 would perform better as biomarkers. (* = p< 0.05 compared to WT ctrl; # = p < 0.05 compared to HD ctrl)

Figure B. Results from quadriceps of preclinical trial mice (wild type and HD N171-82Q) indicate that PDK4 and SCD1 would perform better as biomarkers. (* = p< 0.05 compared to WT ctrl; # = p < 0.05 compared to HD ctrl)

PPAR-δ is repressed in HD, is required for normal neuronal function, and can be targeted therapeutically
Nominee, “In the lab…”
By: Audrey S. Dickey, PhD
Polyglutamine-expanded huntingtin (mHTT) physically interacts with peroxisome proliferator-activated receptor delta (PPARδ) as indicated by co-immunoprecipitation in either mouse cortex or using only in vitro-generated mHTT and PPARδ proteins. mHTT also represses transcriptional activity of PPARδ indicated by reductions in a transactivation reporter assay, and reduced expression of target genes. Pharmacologically increased PPARδ transactivation ameliorated mitochondrial dysfunction, and improved neuron survival in mouse models of HD. Genetic expression of dominant-negative PPARδ in the brains of mice was sufficient to induce motor dysfunction, neurodegeneration, mitochondrial abnormalities, and transcriptional alterations that recapitulated HD-like phenotypes.

A preclinical trial to evaluate therapeutic potential found that pharmacologic activation of PPARδ with the agonist KD3010 in a N-terminal mHTT fragment mouse model improved motor function, reduced neurodegeneration, and increased survival.
In medium-spiny-like neurons generated from induced pluripotent stem cells (iPSCs) derived from individuals with HD, PPARδ activation also reduced mHTT-induced neurotoxicity, increasing optimism that positive results in mouse models can translate to humans.

Advancing towards clinical trials of KD3010, another preclinical trial in a full-length mHTT mouse model is underway, and we have identified compelling biomarkers to assist with the clinical translation of our research. PPARδ activation may be therapeutically beneficial in HD and related neurodegenerative disorders.

Nominated article: Dickey AS, Pineda VV, Tsunemi T, et al. PPAR-δ is repressed in Huntington’s disease, is required for normal neuronal function and can be targeted therapeutically. Nat Med. 2016 Jan;22(1):37-45. doi: 10.1038/nm.4003. Epub 2015 Dec 7.

Figure. The “two-hit” model of HD. The first “hit” consists of mHTT-associated impairments during developmental neurogenesis, leading to mature neurons with enhanced vulnerability to death. The second “hit” consists of physiological stressors and mHTT-associated deleterious effects during adulthood.

Figure. The “two-hit” model of HD. The first “hit” consists of mHTT-associated impairments during developmental neurogenesis, leading to mature neurons with enhanced vulnerability to death. The second “hit” consists of physiological stressors and mHTT-associated deleterious effects during adulthood.

Selective expression of mHTT during development recapitulates characteristic features of HD
Nominee, “In the lab…”
By: Aldrin Molero, MD, PhD
Emerging evidence shows that mHTT disrupts key neural developmental processes. We investigated the role of HD-associated developmental deficits in disease pathogenesis using a mouse model, restricting the expression of full mHTT from the embryonic period until post-natal day 21. We showed that similar to mice that express mHTT throughout their life, mice that expressed mHTT only during the stages of brain development exhibited the characteristics of HD such as motor deficits, neurophysiological abnormalities, and neurodegenerative changes. Further, these mice displayed during the adult life enhanced vulnerability to NMDA-mediated excitotoxicity, and impaired corticostriatal functional connectivity and plasticity.

These findings strongly suggest that mHTT expression during development may exert long-term disease-modifying effects, including engendering selective neuronal vulnerability to degeneration. We also observed that despite similarities between mice that expressed mHTT only during the period of brain development, and those that expressed mHTT throughout life, defects observed in the former were as not as severe as those observed in the latter model.

Thus, our studies provide support for a model of HD pathogenesis that encompasses two pathogenic components or “hits”, one developmental, and the other reflecting the ongoing effects of mHTT (see Figure). Further studies are necessary to better define the developmental underpinnings of HD-associated vulnerabilities, such as the underlying developmental substrate leading to enhanced vulnerability to cell death.

Nominated article: Molero AE, Arteaga-Bracho EE, Chen CH et al. Selective expression of mutant huntingtin during development recapitulates characteristic features of Huntington’s disease. Proc Natl Acad Sci USA. 2016 May 17;113(20):5736-41. doi: 10.1073/pnas.1603871113. Epub May 2 2016.

Figure. A human glial mouse chimeric striatum. Human glia in red, much larger and more complex than mouse glia, in green.

Figure. A human glial mouse chimeric striatum. Human glia in red, much larger and more complex than mouse glia, in green.

Glial therapeutics for HD
Nominee, “In the lab…”
By: Steve Goldman MD, PhD
Our group studies the role of glial cells in neurological disease.1,2 We asked whether modification of the local glial environment might potentiate the survival of medium spiny neurons, one of the major neuronal cell types lost in HD. We were especially interested in this since in another recent study, we identified a strategy for regenerating new medium spiny neurons in brains affected by HD.3 In our new study reported in Nature Communications,4 we generated human glial progenitor cells (hGPCs), a cell type that can make both astrocytes, which are the support cells of the brain, and oligodendrocytes, which are the brain’s myelin-producing cells, from both normal and mHTT-carrying human embryonic stem cells (hESCs), using cell differentiation strategies that we developed for the purpose. We then transplanted mHTT-expressing human glia into the brains of neonatal mice, to establish mHTT human glial chimeric mice. The mHTT glial chimeras manifested significant deficiencies in coordination as well as abnormalities in neuronal physiology relative to normal hESC-derived controls. These data established a significant role for human glial dysfunction in HD.

We next asked whether normal glia might replace diseased glia, and thereby either slow or halt disease progression if introduced into the HD brain. We found that when normal hGPCs were transplanted into neonatal R6/2 mice, a transgenic mouse line that carries the mHTT gene and otherwise develops a severe form of the disease, the animals indeed manifested slower disease progression and lived significantly longer. The mHTT-expressing diseased medium spiny neurons in these glial-transplanted HD mice showed restored functional competence, with a return to normal levels of excitability. Importantly, the mice manifested significant improvements in both cognition and coordination, as reflected by their performance in a variety of behavioral and motor tests that were first conducted in the lab, and then verified by an independent contract research organization engaged by our funding sponsor, CHDI.

These findings together suggest that the restoration of a normal glial environment in the HD brain by the intracerebral transplantation of glial progenitor cells might offer significant benefit in the treatment of HD.

Nominated article: Benraiss A, Wang S, Herrlinger S, et al. Human glia can both induce and rescue aspects of disease phenotype in Huntington disease. Nat Commun. 2016 Jun 7;7:11758. doi:10.1038/ncomms11758.

References
1. Goldman SA, Nedergaard M, Windrem MS. Glial progenitor cell-based treatment and modeling of neurological disease. Science. 2012;338(6106):491-495.
2. Goldman SA. Stem and Progenitor Cell-Based Therapy of the Central Nervous System: Hopes, Hype, and Wishful Thinking. Cell Stem Cell. 2016;18(2):174-188.
3. Benraiss A, Toner MJ, Xu Q, et al. Sustained mobilization of endogenous neural progenitors delays disease progression in a transgenic model of Huntington’s disease. Cell Stem Cell. 2013;12(6):787-799.
4. Benraiss A, Wang S, Herrlinger S, et al. Human glia can both induce and rescue aspects of disease phenotype in Huntington disease. Nat Commun. 2016;7:11758.

Figure. Expression of mHTT aggregates (red) using UAS-mRFP.Htt.138Q in a subset of gustatory receptor neurons. Gustatory neurons are targeted using the gr63a-Gal4 driver, and are labeled with GFP (green). In 24-day-old Drosophila flies, mHTT aggregates are seen throughout the brain, in a unique pattern far beyond the boundaries of the neurons in which they are expressed. Neuropil (blue) is marked by anti-bruchpilot.

Figure. Expression of mHTT aggregates (red) using UAS-mRFP.Htt.138Q in a subset of gustatory receptor neurons. Gustatory neurons are targeted using the gr63a-Gal4 driver, and are labeled with GFP (green). In 24-day-old Drosophila flies, mHTT aggregates are seen throughout the brain, in a unique pattern far beyond the boundaries of the neurons in which they are expressed. Neuropil (blue) is marked by anti-bruchpilot.

Transcellular spreading of huntingtin aggregates in the Drosophila brain
Nominee, “In the lab…”
By: Daniel Babcock, PhD
Our research focuses on mechanisms by which mHTT spreads within cell populations. We previously expressed a fluorescently tagged human huntingtin (HTT) protein with a polyglutamine expansion in a small population of neurons in the Drosophila brain in order to monitor the spread of mHTT aggregates throughout the nervous system.1 We found that these aggregates spread throughout the brain and are internalized by other types of neurons. While some groups of neurons accumulated a large number of mHTT aggregates, other types of neurons died rapidly. Both of these processes were prevented by inhibiting exocytosis and endocytosis in the affected neurons by expressing a dominant-negative form of Shibire, the Drosophila homolog of Dynamin, demonstrating that exocytosis and endocytosis are major routes of transmission for mHTT.

Some of our updated results include examination of spread of mHTT aggregates using various populations of neurons. One example is shown in the accompanying figure that shows spread of mHTT aggregates from gustatory receptor neurons labeled using the gr63a-Gal4 driver. Determining how the capacity of aggregates to spread differs between neuronal populations will help us to find new ways to halt the spread of toxic mHTT aggregates in HD.

Nominated article: Babcock DT, Ganetzky B. Transcellular spreading of huntingtin aggregates in the Drosophila brain. Proc Natl Acad Sci USA. 2015 Sep 29; 112(39): E5427–E5433. Published online 2015 Sep 8. doi: 10.1073/pnas.1516217112.

 

Figure. This chromosome ideogram shows genomic regions with suggestive significance (blue bars, association analysis p-value < 0.00001) and with genome-wide significance (red bars, association analysis p-value < 0.00000005), supporting the presence of genetic modifiers of age at HD onset. The chromosome 15 region involves two independent genome-wide significant association signals. The height of each bar represents the significance in the modifier GWA analysis. The entire HD modifier GWA analysis results are available at the Genetic Modifiers of Motor Onset Age (GeM MOA) website.

Figure. This chromosome ideogram shows genomic regions with suggestive significance (blue bars, association analysis p-value < 0.00001) and with genome-wide significance (red bars, association analysis p-value < 0.00000005), supporting the presence of genetic modifiers of age at HD onset. The chromosome 15 region involves two independent genome-wide significant association signals. The height of each bar represents the significance in the modifier GWA analysis. The entire HD modifier GWA analysis results are available at the Genetic Modifiers of Motor Onset Age (GeM MOA) website.

In the clinic…
Identification of genetic factors that modify clinical onset of Huntington’s disease.
Most influential insight
By: Jong-Min Lee, PhD, on behalf of the Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium
The size of the expanded CAG repeat largely determines the rate of the pathogenic process that leads to clinical symptoms in HD; however, the CAG repeat count does not perfectly control age at onset, strongly suggesting the existence of genetic and environmental modifiers. Such modifiers are hypothesized to interact genetically with HD pathogenesis to modulate the timing of clinical manifestations. Thus, identification of genetic factors that modify HD will shed light on components involved in HD pathogenesis, and ways to delay the disease processes.

In order to identify genetic factors capable of modulating age at onset of motor symptoms, the GeM-HD Consortium performed genome-wide association (GWA) analysis of HD patients from several large natural history studies and genetic research collections. By rigorously searching for genetic variations that show correlation with the difference between observed and CAG-predicted age at onset, three genome-wide significant modification signals were discovered. These findings imply that HD can be modified prior to clinical disease onset, supporting the potential of genetic modifier pathways as therapeutic targets.

Additional genetic analysis is ongoing to reveal additional modifier loci that remain undetected in this initial GWA study due to sample size.

Nominated article: Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium. Identification of genetic factors that modify clinical onset of Huntington’s disease. Cell. 2015 Jul 30;162(3):516-26. doi: 10.1016/j.cell.2015.07.003.

Figure. Group differences in sleep quality parameters measured by polysomnography of the night restricted to the first 8 hours of sleep. Results show an increased time spent awake (left panel) and decreased sleep continuity during the night (right panel). Analyses are controlled for age. Log transformed estimates and SEM are indicated. Pre-HD A: lower disease burden score; Pre-HD B: higher disease burden score; Manifest HD: a small subgroup of patients with early manifest stage HD; Stars indicate significance in all figures: *p ≤ 0.05, T-test; N Controls = 25, N Pre-HD A = 15, N Pre-HD B = 16, N Manifest HD = 8.

Figure. Group differences in sleep quality parameters measured by polysomnography of the night restricted to the first 8 hours of sleep. Results show an increased time spent awake (left panel) and decreased sleep continuity during the night (right panel). Analyses are controlled for age. Log transformed estimates and SEM are indicated. Pre-HD A: lower disease burden score; Pre-HD B: higher disease burden score; Manifest HD: a small subgroup of patients with early manifest stage HD; Stars indicate significance in all figures: *p ≤ 0.05, T-test; N Controls = 25, N Pre-HD A = 15, N Pre-HD B = 16, N Manifest HD = 8.

Sleep disturbances are early key features of HD: why is this important?
Nominee, “In the clinic…”
By: Alpar S Lazar, PhD and Roger A Barker, MRCP, PhD, FMedSci

There is growing evidence that chronic neurodegenerative disorders of the central nervous system are associated with sleep disturbances, and HD is no exception. There are now several studies that report abnormal sleep quality in manifest HD patients.1-6 The abnormal sleep quality begins early in the disease course.7,8 This is intriguing, given that sleep has such an important function in normal brain health, and raises the question of whether sleep dysfunction could elicit or magnify early aspects of HD. We have previously shown in a comprehensive cognitive, sleep, and metabolic study that sleep deficits were among the earliest abnormalities detected in premanifest HD patients. They appeared at the same time as cognitive disturbances,9 years before any motor abnormalities (see HD Insights, Vol. 13).

To better understand the nature and significance of these sleep disturbances in HD, we have followed up this study in several ways. First, we have undertaken a detailed characterization of sleep profile and brain activity during sleep, and investigated the relative contribution of the pathological CAG repeat, age, and sex in the modulation of those HD specific sleep abnormalities. Second we extended our analyses to a new group of premanifest and manifest HD patients who had previously undergone sleep studies in Paris with Prof Isabelle Arnulf. Third, we followed up a smaller group of premanifest patients and controls to assess the reliability of sleep features as early biomarkers of HD. Our preliminary results suggest that there are specific alterations of sleep and sleep-dependent brain activity in HD driven by the CAG repeat length and independent of age and sex.

Nominated article: Lazar AS, Panin F, Goodman AO, et al. Sleep deficits but no metabolic deficits in premanifest Huntington’s disease. Ann Neurol. 2015 Oct;78(4):630-48. doi: 10.1002/ana.24495. Epub 2015 Aug 21.

References
1.Arnulf I, Nielsen J, Lohmann E, et al. Rapid eye movement sleep disturbances in Huntington disease. Arch Neurol. 2008 Apr;65(4):482-8. doi: 10.1001/archneur.65.4.482. Erratum in: Arch Neurol. 2008 Nov;65(11):1478.. Schieffer, Johannes [corrected to Schiefer,Johannes].
2. Wiegand M, Möller A, Lauer C, et al. Nocturnal sleep in Huntington’s disease. J Neurol. 1991 Jul;238(4):203-8.
3. Piano C, Losurdo A, Della Marca G, et al. Polysomnographic findings and clinical correlates in Huntington disease. A cross-sectional cohort study. Sleep. 2015 Sep 1;38(9):1489-95. doi: 10.5665/sleep.4996.
4. Hansotia P, Wall R, Berendes J. Sleep disturbances and severity of Huntington’s disease. Neurology. 1985 Nov;35(11):1672-4.
5. Neutel D, Tchikviladze M, Charles P, et al. Nocturnal agitation in Huntington disease is caused by arousal-related abnormal movements rather than by rapid eye movement sleep behavior disorder. Sleep Med. 2015 Jun;16(6):754-9. doi: 10.1016/j.sleep.2014.12.021. Epub 2015 Mar 3.
6. Morton AJ, Wood NI, Hastings MH, Hurelbrink C, Barker RA, Maywood ES. Disintegration of the sleep-wake cycle and circadian timing in Huntington’s disease. J Neurosci. 2005 Jan 5;25(1):157-63.
7. Abbott SM, Videnovic A. Chronic sleep disturbance and neural injury: links to neurodegenerative disease. Nat Sci Sleep. 2016 Jan 25;8:55-61. doi: 10.2147/NSS.S78947. eCollection 2016.
8. Goodman AO, Rogers L, Pilsworth S, et al. Asymptomatic sleep abnormalities are a common early feature in patients with Huntington’s disease. Curr Neurol Neurosci Rep. 2011 Apr;11(2):211-7. doi: 10.1007/s11910-010-0163-x.
9. Lazar AS, Panin F, Goodman AO, et al. Sleep deficits but no metabolic deficits in premanifest Huntington’s disease. Ann Neurol. 2015 Oct;78(4):630-48. doi: 10.1002/ana.24495. Epub 2015 Aug 21.

Figure. Number of individuals with a genotype of 36 CAG repeats or greater, out of a total of 7,315 individuals examined from the general population.

Figure. Number of individuals with a genotype of 36 CAG repeats or greater, out of a total of 7,315 individuals examined from the general population.

HD reduced penetrance alleles occur at high frequency in the general population
Nominee, “In the clinic…”
By: Chris Kay
All patients affected by HD have an expanded CAG repeat of 36 or greater – but do all people with 36 or more repeats go on to develop HD? Investigators at the University of British Columbia, the University of Aberdeen, and the Coriell Institute for Medical Research asked this question, and recently reported their findings in Neurology. To determine the number of people who have an expanded CAG repeat in the HD range, CAG repeat length was evaluated in 7,315 individuals from the general population of Canada, the United States, and Scotland.

In total, 18 individuals had 36 or more CAG repeats, revealing that approximately 1 in 400 people (0.246%) have an expanded CAG repeat in the HD range. This is much higher than previous estimates based on the prevalence of HD patients seen in the clinic, who represent approximately 1 in 7,300 of the general population. Strikingly, most individuals with an expanded repeat had 36 and 39 CAG, called reduced penetrance alleles, which usually result in HD onset over the age of 60.

These results suggest that many people with the mutation in the reduced penetrance range may never develop the disease, but also that more people in old age may have signs of HD than previously believed. Increased testing for HD in elderly individuals with suggestive signs or symptoms may be warranted.

Nominated article: Kay C, Collins JA, Miedzybrodzka Z, et al. Huntington disease reduced penetrance alleles occur at high frequency in the general population. Neurology. 2016 Jul 19;87(3):282-8. doi: 10.1212/WNL.0000000000002858. Epub 2016 Jun 22.

Figure. (A) Diagram of virtual reality arena showing platform location (blue square) within the circular pool and corresponding landmarks. (B) Screenshot of MWM task where subjects have to use a joystick to search for the submerged platform. (C) HD patients show impaired learning of the hidden platform compared controls and preHD. (D) Representative illustrations of the paths taken towards the hidden platform (blue square) demonstrating learning of the hidden platform location by controls and impairment of learning the platform location by HD patients.

Figure. (A) Diagram of virtual reality arena showing platform location (blue square) within the circular pool and corresponding landmarks. (B) Screenshot of MWM task where subjects have to use a joystick to search for the submerged platform. (C) HD patients show impaired learning of the hidden platform compared controls and preHD. (D) Representative illustrations of the paths taken towards the hidden platform (blue square) demonstrating learning of the hidden platform location by controls and impairment of learning the platform location by HD patients.

Hippocampal dysfunction defines disease onset in HD
Nominee, “In the clinic…”
By: Faye Begeti, PhD
Much of our understanding of the relationship between pathology and function in HD is a result of transgenic mouse studies. However, in many cases, the findings from such studies have not been verified in patients. For example, there is extensive literature that shows impairments of hippocampal-dependent cognitive tests in a number of HD mouse models, something that had not been investigated in patients. Therefore, we studied hippocampal functioning in patients with manifest and premanifest HD, using both a virtual reality version of the Morris water maze (MWM) task where participants have to swim through a virtual pool to find a submerged platform using a joystick (Figures A and B), and a computerized spatial memory task called the paired associates learning (PAL) task, both of which replicate tests that have been used in rodent studies.

We found that similar hippocampal deficits exist in patients with early manifest HD to those that have been described in transgenic mouse models. Specifically, performance in both the MWM and the PAL was impaired compared to controls. Whereas controls demonstrated that they learned the location of the platform by exhibiting a decreased latency, manifest HD participants were not able to learn its location (Figures C and D). Furthermore, during a probe test where the platform was removed, participants spent a large proportion of their search in the platform location, whereas HD exhibited a random search pattern. Similar findings were reflected in the CANTAB PAL where HD participants made significantly more errors than controls. Importantly, there was a significant correlation between decreasing performance in each of these tasks, and estimated time to disease onset in premanifest HD. These results highlight the potential use of either test in future therapeutic trials of treatments that target cognitive impairment in HD.

Nominated article: Begeti F, Schwab LC, Mason SL, Barker RA. Hippocampal dysfunction defines disease onset in Huntington’s disease. J Neurol Neurosurg Psychiatr. 2016 Sep;87(9):975-81. doi: 10.1136/jnnp-2015-312413. Epub 2016 Feb 1.

Effect of deutetrabenazine on chorea in patients with HD
Nominee, “In the clinic…”
By: Samuel Frank, MD
Deutetrabenazine is a novel formulation of the tetrabenazine molecule. It contains deuterium, which reduces activity in the enzyme CYP2D6, a key step in drug metabolism, and increases active metabolite half-lives, leading to stable systemic exposure while preserving key pharmacological activity. Deutetrabenazine is the first deuterated compound to be evaluated in late-stage development.

The First-HD trial, conducted at 34 Huntington Study Group sites, enrolled 90 ambulatory adults diagnosed with manifest HD and who had a baseline Total Maximal Chorea (TMC) score ≥ 8, and randomized them to receive deutetrabenazine (n = 45) or placebo (n = 45) in a double-blind fashion. Study drug was titrated to optimal dose level over 8 weeks and maintained for 4 weeks, followed by 1-week washout. The primary endpoint was TMC change from baseline to maintenance therapy, and was reduced in the deutetrabenazine group by 4.4 points vs 1.9 points in the placebo group (P < 0.001). Secondary endpoints were: the proportion of patients who achieved treatment success on Patient Global Impression of Change (P=0.002); the proportion of patients who achieved treatment success on Clinical Global Impression of Change (P=0.002); change in SF-36 physical functioning subscale score (P=0.03); and change in Berg Balance Test (NS). Adverse event rates were similar for deutetrabenazine and placebo, including depression, anxiety, and akathisia. Among HD patients, the use of deutetrabenazine compared with placebo resulted in improved chorea at 12 weeks. Patients and clinicians both indicated the overall clinical importance of improved motor measures. A study of longer-term exposure safety and efficacy is ongoing.

Nominated article: Huntington Study Group. Effect of deutetrabenazine on chorea among patients with Huntington disease: a randomized clinical trial. JAMA. 2016 Jul 5;316(1):40-50. doi: 10.1001/jama.2016.8655.
Nominated article: Huntington Study Group. Effect of deutetrabenazine on chorea among patients with Huntington disease: a randomized clinical trial. JAMA. 2016 Jul 5;316(1):40-50. doi: 10.1001/jama.2016.8655.
Also nominated: Bettencourt C, Hensman-Moss D, Flower M et al. DNA repair pathways underlie a common genetic mechanism modulating onset in polyglutamine diseases. Ann Neurol. 2016;79: 983–990. doi: 10.1002/ana.24656

Figure. Axial summed [11C]IMA107 PET images co-registered and fused with 3-T MRI images for the striatum of a 33-year-old healthy male showing normal striatum [11C]IMA107 binding (BPND = 2.24) (left); a 35-year-old male premanifest HD gene carrier (CAGr: 40; DBS: 153; 90% probability to onset: 43 years) showing mild to moderate decreases in striatal [11C]IMA107 binding (BPND = 1.45) (middle-left); a 33-year-old female premanifest HD gene carrier (CAGr: 43; DBS: 247.5; 90% probability to onset: 25 years) showing moderate decreases in striatal [11C]IMA107 binding (BPND = 1.32) (middle-right); and a 52-year-old male early premanifest HD gene carrier (CAGr: 41; DBS: 282.2; 90% probability to onset: 21 years) showing severe decreases in striatal [11C]IMA107 binding (BPND = 0.57) (right). NB: Predicted onset was estimated using the validated variant of the survival analysis formula described by Langbehn et al.6 This formula can be transformed into a probability distribution for age of diagnosis and subsequently years from symptomatic onset that depends on both the subject’s CAG expansion length and current age.7

Figure. Axial summed [11C]IMA107 PET images co-registered and fused with 3-T MRI images for the striatum of a 33-year-old healthy male showing normal striatum [11C]IMA107 binding (BPND = 2.24) (left); a 35-year-old male premanifest HD gene carrier (CAGr: 40; DBS: 153; 90% probability to onset: 43 years) showing mild to moderate decreases in striatal [11C]IMA107 binding (BPND = 1.45) (middle-left); a 33-year-old female premanifest HD gene carrier (CAGr: 43; DBS: 247.5; 90% probability to onset: 25 years) showing moderate decreases in striatal [11C]IMA107 binding (BPND = 1.32) (middle-right); and a 52-year-old male early premanifest HD gene carrier (CAGr: 41; DBS: 282.2; 90% probability to onset: 21 years) showing severe decreases in striatal [11C]IMA107 binding (BPND = 0.57) (right).
NB: Predicted onset was estimated using the validated variant of the survival analysis formula described by Langbehn et al.6 This formula can be transformed into a probability distribution for age of diagnosis and subsequently years from symptomatic onset that depends on both the subject’s CAG expansion length and current age.7

In imaging and biomarkers…
Altered PDE10A expression detectable early before symptomatic onset in HD
Most influential insight
By: Marios Politis, MD, MSc, DIC, PhD, FEAN and Flavia Niccolini, PhD

Phosphodiesterase 10A (PDE10A) is an intracellular enzyme highly expressed in striatal medium spiny neurons. It hydrolyses cAMP and cGMP signaling cascades, thus playing a key role in the regulation of the direct and indirect striatal output pathways, and in promoting neuronal survival. By using combined molecular and structural imaging in vivo, we showed changes in PDE10A expression in premanifest HD gene carriers, which are associated with the risk of symptomatic conversion, and are detectable up to 43 years (range: 17–43 years) before the predicted onset of clinical symptoms. PDE10A expression in early premanifest HD gene carriers was decreased in the striatum and globus pallidus, similar to initial observations in animal HD models and postmortem HD brain tissue,1-3 and increased in motor thalamic nuclei compared to a group of matched healthy controls.

Connectivity-based functional analysis revealed prominent PDE10A decreases confined in the sensorimotor striatum and in both direct and indirect projecting segments of striatum. The altered balance of PDE10A signaling between motor thalamic nuclei and striatopallidal projecting segments of the striatum was the strongest reported association, with predicted risk of symptomatic conversion at an alpha level of 0.001.

A pilot PDE10A PET study reported 60–70% decreases in striatal PDE10A expression in five patients with manifest HD with significant striatal atrophy.4 Using PET with [18F]MNI- 659, Russell et al. have found 47.6% decreases in striatal and pallidal PDE10A expression in eight patients with early manifest HD and lower striatal PDE10A expression was associated with disease severity and disease burden of pathology.5 Our findings demonstrate in vivo a novel and early pathophysiological mechanism underlying HD, with direct implications for the development of new pharmacological treatments that can promote neuronal survival, and therefore improve outcomes in HD gene carriers.

For a discussion of other research exploring PDE10A in HD, see HD Insights, Vol. 5.

Nominated article: Niccolini F, Haider S, Reis Marques T, et al. Altered PDE10A expression detectable early before symptomatic onset in Huntington’s disease. Brain. 2015 Oct;138(Pt 10):3016-29. doi: 10.1093/brain/awv214. Epub 2015 Jul 21.

References
1. Hebb AL, Robertson HA, Denovan-Wright EM. Striatal phosphodiesterase mRNA and protein levels are reduced in Huntington’s disease transgenic mice prior to the onset of motor symptoms. Neuroscience. 2004;123(4):967-981.
2. Hu H, McCaw EA, Hebb AL, Gomez GT, Denovan-Wright EM. Mutant huntingtin affects the rate of transcription of striatum-specific isoforms of phosphodiesterase 10A. Eur J Neurosci. 2004;20(12):3351-3363.
3. Leuti A, Laurenti D, Giampa C, et al. Phosphodiesterase 10A (PDE10A) localization in the R6/2 mouse model of Huntington’s disease. Neurobiol Dis. 2013;52:104-116.
4. Ahmad R, Bourgeois S, Postnov A, et al. PET imaging shows loss of striatal PDE10A in patients with Huntington disease. Neurology. 2014;82(3):279-281.
5. Russell DS, Barret O, Jennings DL, et al. The phosphodiesterase 10 positron emission tomography tracer, [18F]MNI-659, as a novel biomarker for early Huntington disease. JAMA Neurology. 2014;71(12):1520-1528.
6. Langbehn DR, Brinkman RR, Falush D, Paulsen JS, Hayden MR. A new model for prediction of the age of onset and penetrance for Huntington’s disease based on CAG length. Clin Genet. 2004;65(4):267-277.
7. Paulsen JS, Langbehn DR, Stout JC, et al. Detection of Huntington’s disease decades before diagnosis: the Predict-HD study. J Neurol Neurosurg Psychiatry. 2008;79(8):874-880.

 

Figure. A systems biology approach revealed a novel HD-relevant gene network in a human HD cohort (GSE3790) that is astrocyte-specific, conserved across HD mouse models, and associated with stress susceptibility and sleep in a (B6xA/J)F2 mouse population. This non-neuronal gene network is downstream of the TGFβ-FOXO3 pathway and is regulated by several potentially therapeutic small compounds.

Figure. A systems biology approach revealed a novel HD-relevant gene network in a human HD cohort (GSE3790) that is astrocyte-specific, conserved across HD mouse models, and associated with stress susceptibility and sleep in a (B6xA/J)F2 mouse population. This non-neuronal gene network is downstream of the TGFβ-FOXO3 pathway and is regulated by several potentially therapeutic small compounds.

Systems genetic analyses highlight a TGFβ-FOXO3 — dependent striatal astrocyte network conserved across species and associated with stress, sleep, and HD
Nominee, “In imaging and biomarkers…”
By: Joseph Scarpa, PhD

HD patients notably develop motor abnormalities. They also develop significant non-motor symptoms, including depression, anxiety, and sleep disturbance, that often precede the motor phenotype by many years. Understanding the biological basis of these early non-motor symptoms may reveal therapeutic targets that prevent disease onset or slow disease progression, but the molecular mechanisms underlying this complex clinical presentation remain largely unknown and are difficult to examine directly in a single human HD cohort.

This work leverages multiple large transcriptomic datasets across mouse and human cohorts to describe how genetic and transcriptional networks contribute to complex psychiatric traits in HD. These analyses led to several novel findings. We show that HD significantly changes molecular networks in the human cerebellum and frontal cortex, as well as the caudate.

Further, we demonstrate that an astrocyte gene network in the caudate is most significantly altered in HD and is strongly correlated in a mouse population with many common non-motor HD phenotypes involved in the early phases of the disease. Finally, we identify genes and drugs that can regulate this network, and also show that deep brain stimulation of the subthalamic nucleus affects this pathway. This study provides evidence that multi-focal and non-neuronal molecular networks contribute to HD and argues that an understanding of the molecular mechanisms of non-motor HD phenotypes can reveal novel therapeutic pathways.
Follow up work will seek to understand the functional role of these regulator genes and the effectiveness of drugs in modulating motor traits, non-motor phenotypes, and disease progression.

Nominated article: Scarpa JR, Jiang P, Losic B, et al. Systems genetic analyses highlight a TGFβ-FOXO3 dependent striatal astrocyte network conserved across species and associated with stress, sleep, and Huntington’s disease. PLoS Genet. 2016 Jul 8;12(7):e1006137. doi: 10.1371/journal.pgen.1006137. eCollection 2016.

Figure. Functional gene sets associated with innate immunity and inflammation are enriched in resting HD monocytes. A network diagram of significant biological themes is shown, indicating number of genes (node size), statistical significance (darkest shading = lowest p-value) and gene content similarity (edge thickness). A false discovery rate cut-off of < 0.05 was used to determine inclusion in the diagram, before filtering for sets with similar gene content.

Figure. Functional gene sets associated with innate immunity and inflammation are enriched in resting HD monocytes. A network diagram of significant biological themes is shown, indicating number of genes (node size), statistical significance (darkest shading = lowest p-value) and gene content similarity (edge thickness). A false discovery rate cut-off of < 0.05 was used to determine inclusion in the diagram, before filtering for sets with similar gene content.

RNA-Seq of HD patient myeloid cells reveals innate transcriptional dysregulation associated with proinflammatory pathway activation
Nominee, “In imaging and biomarkers…”
By: James Miller, PhD

HD patients are known to have peripheral immune abnormalities, including increased plasma levels of proinflammatory cytokines and chemokines. These abnormalities occur many years before the onset of motor symptoms. HD myeloid cells are also hyper-reactive to immune stimuli, but the molecular mechanisms behind this are incompletely understood.

We used RNA-sequencing to analyze the transcriptome of peripheral blood monocytes from 30 manifest HD patients and 33 control subjects. We found that monocytes in HD patients have a significantly abnormal transcriptional profile even in the absence of stimulation, including previously undetected increases in the basal expression of proinflammatory cytokines such as IL-6. Further bioinformatic analysis revealed significant resting enrichment of functional gene sets relating to innate immunity and inflammation. A summary of relevant enriched gene sets is shown in the Figure. These data suggest that mHTT has a ‘priming’ effect on HD myeloid cells, whereby resting dysfunction of intracellular signaling pathways leads to an exaggerated inflammatory response to stimulation. Functional studies indicate that this is due to abnormal basal activity in the NFĸB pathway. This study enhances our understanding of peripheral HD pathogenesis, and supports targeting the peripheral innate immune system as a potential disease-modifying treatment for HD in future research.

Nominated article: Miller JR, Lo KK, Andre R et al. RNA-Seq of Huntington’s disease patient myeloid cells reveals innate transcriptional dysregulation associated with proinflammatory pathway activation. Hum Mol Genet. 2016 May 11. pii: ddw142. [Epub ahead of print].

Also nominated: Rosas HD, Doros G, Bhasin S, et al. A systems-level “misunderstanding”: the plasma metabolome in Huntington’s disease. Ann Clin Transl Neurol. 2015 Jul;2(7):756-68. doi: 10.1002/acn3.214. Epub 2015 May 28.
Also nominated: Wagner L, Björkqvist M, Lundh SH, et al. Neuropeptide Y (NPY) in cerebrospinal fluid from patients with Huntington’s Disease: increased NPY levels and differential degradation of the NPY1–30 fragment. J Neurochem. 2016 137: 820–837. doi: 10.1111/jnc.13624.

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