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.