Research Round-Up

Research Round-Up

By: Lise Munsie, PhD

In neurons…

The HD Induced Pluripotent Stem Cell (iPSC) consortium differentiated HD patient iPSCs into neuronal cultures.1 RNA-seq and ChIP-seq analysis from these cultures indicated subtle alterations in expression and epigenetic signature of genes involved in neural development, function, and striatal maturation in the presence of mHTT. The small molecule isoxazole-9 is known to target some of the disrupted gene networks, and when tested in this system, was able to normalize some of the genetic phenotypes. This compound also showed beneficial effects in HD-related pathologies in the R6/2 mouse model, indicating that some of these pathways are linked to pathology.

Yu and Tanese used mouse embryonic stem cells provided by the Zeitland group to investigate HTT involvement in differentiation.2 The lines they used came from the same background, but were either huntingtin-null (Htt-null) or had tagged wild-type Htt (Q7) or mHtt (Q140). The group found that the presence of HTT, either wild-type or mutant, allowed all three germ layers to form, but Htt knockout blocked the ability of the cells to differentiate down the ectoderm pathway to form neural stem cells (NSC). Although mHTT still allowed NSC formation, it inhibited further differentiation into neurons. RNA-seq data from these lines revealed that MaoA and Oligo1/2 downregulation and upregulation of the PRC2 repressor complex may be involved in these developmental changes.

One well-described effect of mHTT is the dysregulation of Ca2+ signaling. A new drug lead that corrects deficiencies in store-operated calcium entry (SOCE) in YAC128 medium spiny neurons (MSNs) has been described in a recent publication.3 SOCE was found to be increased specifically in MSNs but not in other neuronal subtypes in this model. The group identified a tetrahydrocarbazole that attenuated this phenotype in addition to normalizing mitochondrial function. The group postulates that the dual benefit might come from preventing abnormal Ca2+ accumulation in the mitochondria and endoplasmic reticulum.


1HD iPSC Consortium. Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice. Nat Neurosci. 2017 Mar 20. doi: 10.1038/nn.4532. [Epub ahead of print].

2Yu MS, Tanese N. Huntingtin Is Required for Neural But Not Cardiac/Pancreatic Progenitor Differentiation of Mouse Embryonic Stem Cells In vitro. Front Cell Neurosci. 2017;11(33).

3Czeredys M, Maciag F, Methner A, Kuznicki J. Tetrahydrocarbazoles decrease elevated SOCE in medium spiny neurons from transgenic YAC128 mice, a model of Huntington’s disease. Biochem Biophys Res Commun. 2017 Feb 19;483(4):1194-1205. 23;116:233-246.

In gene editing…

Allele-specific silencing of mHTT with methods that degrade mHTT mRNA is a promising treatment for HD, but that silencing is neither complete nor permanent. The discovery of the CRISPR/Cas9 nuclease system has created the possibility of altering genomic DNA in affected cells, and permanently silencing mHTT. Two groups have recently published studies that attempt this in mice.1,2 Both groups utilized the existence of allele-specific single-nucleotide polymorphisms (SNPs) that create or destroy protospacer adjacent motif (PAM) elements in an allele-specific manner in the mHtt promoter. The groups then used a two-guide RNA (gRNA) system, with one gRNA targeted against the SNP in the promoter, and one in a downstream intron, leading to excision of the mHtt promoter and first few exons. The Davidson group used this technique, excising exon1 of mHtt in an allele-specific manner, in immortalized cells, in human HD fibroblasts, and in vivo in a BACHD mouse model.1 Shin and colleagues excised a 44 kb segment of mHtt, including exons 1–3, and successfully used their platform in induced pluripotent stem cells and neural precursor cells.2 Together, these data demonstrate the reproducibility and utility of using the CRISPR nuclease system to potentially permanently alter DNA to silence mHTT for therapeutic effect.

CRISPR/Cas9 also can be used to create tools for drug screening. The Pouladi group coupled CRISPR/Cas9 technology with piggyback transposon technology to create corrected isogenic HD lines.3 The corrected lines maintained their pluripotency, differentiation potential, and karyotype, with no off-target effects. The correction restored HD-induced phenotypes, including neural rosette formation deficiencies, apoptotic susceptibility, and energetic defects in the isogenic iPSC line. Performing global differential gene expression analysis on the parental line, the corrected line, and a non-related control line allowed the scientists to determine which genes were differentially expressed because of the HD mutation, and which genes were differentially expressed because of genetic background.


1Monteys AM, Ebanks SA, Keiser MS, Davidson BL. CRISPR/Cas9 Editing of the Mutant Huntingtin Allele In Vitro and In Vivo. Mol Ther.  2017 Jan 4;25(1):12-23. doi: 10.1016/j.ymthe.2016.11.010. Epub 2017 Jan 4.

2Shin JW, Kim K-H, Chao MJ, et al. Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9. Hum Mol Genet. 2016 Oct 15;25(20):4566-4576.

3Xu X, Tay Y, Sim B, et al. Reversal of Phenotypic Abnormalities by CRISPR/Cas9-Mediated Gene Correction in Huntington Disease Patient-Derived Induced Pluripotent Stem Cells. Stem Cell Reports.2017 Mar 14;8(3):619-633.

In imaging…

Dr. Sarah Tabrizi’s group continues to data-mine the extensive human imaging and disease phenotype data available from the two large multi-center cohort studies, Track-HD and Track-On HD. In the largest imaging study ever done to assess depressive symptoms in HD, the group examined variation in structural and functional brain networks in relation to symptoms of depression in premanifest HD (preHD) and healthy controls.1 Onset of depression precedes onset of motor symptoms, and neuroimaging studies may reveal the early-affected brain networks. The group found that depressive symptoms in preHD are correlated with specific connectivity changes in the default mode network and the basal ganglia.

Another group published its imaging and electrophysiological study in Nature’s Scientific Reports, looking for functional and structural changes that are present prior to clinical diagnosis.2 This study looked at preHD patients who had a very low disease burden score, far from disease onset. All methods used in this study agree that brain function and structure is normal in this cohort, meaning that symptoms are reflective of an ongoing neurodegenerative process. This may have implications for timing of treatment.

Finally, another large-scale patient-based collaborative effort aimed to identify patients with extreme HD motor phenotypes with respect to their CAG repeat length and age, in order to search for disease modifiers.3 By looking at more than 10,000 HD patients, the group found that the total motor score (TMS) could vary by up to 30 years in individuals with the same CAG repeat length. They found that a TMS score of more than 13 would predict motor onset regardless of age and CAG length. The identified patients in the upper and lower quantiles can be further examined for biological, medical, or environmental factors that may lead to their extreme motor phenotype, which will inform patient care and prognosis.


1McColgan P, Razi A, Gregory S, et al. Structural and functional brain network correlates of depressive symptoms in premanifest Huntington’s disease. Hum Brain Mapp. 2017 Mar 15. doi: 10.1002/hbm.23527. [Epub ahead of print]

2Gorges M, Müller H-P, Mayer IMS, et al. Intact sensory-motor network structure and function in far from onset premanifest Huntington’s disease. Sci Rep. 2017 Mar 7;7:43841.

3Braisch U, Hay B, Muche R, et al. Identification of extreme motor phenotypes in Huntington’s disease. Am J Med Genet B Neuropsychiatr Genet. 2017 Apr;174(3):283-294.

Research Round-Up

By: Lise Munsie, PhD

In pharma…

The well-tolerated, FDA-approved drug metformin is an antidiabetic drug that mimics caloric restriction. Jin and colleagues tested their hypothesis that it may also counteract pathways affected by the presence of mHTT using the striatal Q111 cell model.1 Under serum starvation conditions, the drug corrected phenotypes such as decreased ATP production, increased LDH release, and decreased mitochondrial membrane potential, and modulated the balance between mitochondrial fusion and fission genes by activating the AMPK pathway. The group confirmed that metformin crossed the blood-brain barrier, and that increased levels of p-AMPK could be found in the brain post-administration, suggesting that this drug could be used as a neuroprotectant in HD.

The drug topotecan, a topoisomerase 1 inhibitor, leads to the activation of Ube3a, a ubiquitin ligase implicated in mHTT clearance. In a recent article in Human Molecular Genetics, Shekhar and colleagues report on their study in which topetecan delivery was optimized and tested for symptomatic improvement in the R6/2 HD mouse model.2 The drug partially rescued striatal atrophy and decreased mHTT aggregate load. The group saw upregulation of Ube3a in the brain, and a decrease in wildtype and mutant htt levels. Although side effects on other affected genes must be carefully monitored, this drug may move forward as one that delays HD progression.

The Tingle group investigated the hypothesis that dysfunctional cholinergic neurotransmission through a loss of agonist contributes significantly to HD pathology.3 The group attempted pharmacological replacement using the nicotine analogue and partial agonist varenicline. Chronic exogenous administration to YAC128 HD model mice resulted in motor and behavioral improvements combined with improved neuorophysiological phenotypes. Varenicline is approved for other disorders, and its multi-targeted and direct effects makes it a promising candidate for further study.


1Jin J, Gu H, Anders NM, et al. Metformin protects cells from mutant huntingtin toxicity through activation of AMPK and modulation of mitochondrial dynamics. Neuromolecular Med. 2016 Dec;18(4):581-592.

2Shekhar S, Vatsa N, Kumar V, et al. Topoisomerase 1 inhibitor topotecan delays the disease progression in a mouse model of Huntington’s disease. Hum Mol Genet. 2016 Dec 22.

3McGregor AL, D’Souza G, Kim D, Tingle MD. Varenicline improves motor and cognitive deficits and decreases depressive-like behaviour in late-stage YAC128 mice. Neuropharmacology. 2016 Dec 23;116:233-246.

In patient studies…

Detailed genetic analysis of HD is important for improving targeted therapies. The Hayden lab reports on HD haplotypes in the Latin American population.1 By genotyping Peruvian HD patients, the group identifies the HD mutation on the A1 HTT haplotype, a haplotype also common in European HD patients. The group defines an Amerindian-specific A1 variant based on single nucleotide polymorphism (SNP) analysis in this population. This is the most frequent HD haplotype in the studied population. This finding shows a distinct genetic origin between HD patients on Amerindian and European origin in Latin America, but still a common genetic target.

Studying genes that affect the age of onset of disease can uncover pathways involved in HD progression. In a recent article, a Spanish group probes the association between SNPs in the melanocortin 1 receptor gene (MCR1) and age of onset in the Spanish HD population.2 MCR1 is involved in anti-oxidant and cell stress pathways. Variants in this allele occur at a normal frequency in the HD population. Using a multiple linear regression model, the group show that the p.R151C polymorphism, a mutation that affects the protein’s function, may decrease the age of onset of clinical signs in Spanish HD patients, which suggests this protein and pathway may be worthy of further study.

The Milek group examined cardiovascular pathology of HD in patients.3 Expression of mHTT has negative consequences in non-neuronal tissues, and heart disease may factor into deaths in HD patients. This led the group to compare individuals with HD at different stages of disease progression, and age-matched controls. Although not significant between all groups, there was an overall increased risk for coronary heart disease in HD patients. Specific measures showed arterial dysfunction including significantly decreased distensibility of the carotid arterial wall in presymptomatic HD patients. Further and larger studies will be required to elucidate full vascular pathology in HD patients.


1Kay C, Tirado-Hurtado I, Cornejo-Olivas M, et al. The targetable A1 Huntington disease haplotype has distinct Amerindian and European origins in Latin America. Eur J Hum Genet. 2016 Dec 21.

2Tell-Marti G, Puig-Butille JA, Gimenez-Xavier P, et al. The p. R151C polymorphism in MC1R gene modifies the age of onset in Spanish Huntington’s Disease patients. Mol Neurobiol. 2016 Dec 6:1-5.

3Kobal J, Cankar K, Pretnar J, et al. Functional impairment of precerebral arteries in Huntington disease. J Neurol Sci. 2017 Jan 15;372:363-368.

In pathways…

An exciting study from the Truant lab examines endogenous HTT function in live human cells.1 They identify HTT at sites of DNA damage, localized to DNA repair proteins during induced oxidative stress. This localization is dependent on the activity of ataxia telangiectasia mutated (ATM) protein, a kinase that has been shown to contribute to HD progression in mouse models. The group hypothesize that inhibiting ATM activity, which slows or inhibits mHTT recruitment to sites of DNA damage, may have therapeutically beneficial effects in HD.

Transcriptional dysregulation is a hallmark of HD. The Jones group focuses their pathway study on differential gene expression upon epidermal growth factor (EGF) stimulation in two cellular models of HD.2 The group found that regulatory genes within the TGFβ signaling pathway were differentially expressed in cells harboring the expanded allele, and many were associated with SMAD transcription factors (TFs). The group shows that stimulation with TGFβ1 activates SMAD TFs, leading to nuclear localization. This nuclear translocation is altered in cells that express mHTT. They additionally demonstrate direct binding of SMAD3 to HTT in the cells, showing that these proteins may directly affect HTT expression. The group concludes that TGFβ signaling is a possible target for disease modification and could prove to be a useful biomarker for disease progression.

HD patients have decreased levels of striatal phosphodiesterase 10 (PDE10), an enzyme that hydrolyzes cyclic AMP and GMP. Acute inhibition of PDE10 can partially restore the basal ganglia circuitry in HD models. A manuscript in Neuron3 extensively examines the neurophysiology associated with acute PDE10 inhibition in two mouse models that recapitulate the pre-existing PDE10 deficit. Using electrophysiological and proteomic approaches, the group defines how corticostriatal transmission is altered and subsequently improved in these models under PDE10 inhibition, specifically probing the striatal indirect pathway.


1Maiuri T, Mocle AJ, Hung CL, et al. Huntingtin is a scaffolding protein in the ATM oxidative DNA damage response complex. Hum Mol Genet. 2016 Dec 25.

2Bowles KR, Stone T, Holmans P, Allen ND, Dunnett SB, Jones L. SMAD transcription factors are altered in cell models of HD and regulate HTT expression. Cell Signal. 2016 Dec 14;31:1-14.

3Beaumont V, Zhong S, Lin H, et al. Phosphodiesterase 10A inhibition improves cortico-basal ganglia function in Huntington’s disease models. Neuron. 2016 Dec 21;92(6):1220-1237.

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.

Research Round-Up

By: Lise Munsie, PhD

In human studies…

Messages Image(250749082)Human studies provide the most useful research data and help to elucidate the natural history, biology, and potential molecular mechanisms of disease, which inform treatment development.

Dr. Jong-min Lee’s group investigated the correlation between CAG repeat length in the mutant and wildtype alleles, and age at death and disease duration.1 Similarly for age at onset of motor symptoms of HD, age at death had a strong negative correlation with mutant allele CAG repeat length. Interestingly, CAG repeat length did not correlate with disease duration, even in juvenile HD.

Nielsen and colleagues investigated the hypothesis that metabolic changes characteristic of HD may be caused in part by liver damage.2 The group used a range of common blood tests to evaluate liver function in symptomatic HD patients. Although the study has some limitations—including using data from patients on medications that may impair liver function—they found evidence of increased liver dysfunction in symptomatic HD patients. More sensitive markers of liver failure may be needed to solidly ascertain how and when the liver is affected over the course of disease.

De Souza and colleagues investigated the role of differential DNA methylation in HD, characterizing tissue specificity by examining genome-wide methylation changes in brain and liver tissue samples from HD patients.3 The study revealed DNA methylation differences in the HTT gene region between matched brain and liver samples. The study also found evidence that the transcription factor CTCF participates in DNA methylation and contributes to tissue-specific methylation patterns near the HTT proximal promoter.

1Keum JW, Shin A, Gillis T, et al. The HTT CAG-expansion mutation determines age at death but not disease duration in Huntington disease. Am J Hum Genet. 2016;98(2):287-298.

2Nielsen SM, Vinther-Jensen T, Nielsen JE, et al. Liver function in Huntington’s disease assessed by blood biochemical analyses in a clinical setting. J Neurol Sci. 2016;362:326-332.

3De Souza RA, Islam SA, McEwen LM, et al. DNA methylation profiling in human Huntington’s disease brain. Hum Mol Genet. 2016 Mar 6. pii: ddw076. [Epub ahead of print].

In animal models…Messages Image(59764496)

With the exception of using patient data, model organisms are the most biologically relevant method for studying disease. In an article published in Nature Neuroscience, Langfelder and colleagues used a variety of HD models—mainly murine—to perform tissue-, CAG length-, and age- dependent, large-scale, transcriptome and proteome comparisons.1 They identified a set of genes and corresponding proteins that are affected in a tissue- , CAG-, and age- dependent manner. The set includes previously identified genes such as CTCF, validating this method. All the data is freely available for viewing or data mining at www.hdinhd.org.

Although mammalian models have shed light on many aspects of HD, the classic models have yet to elucidate the neural mechanism by which striatal degeneration leads to a movement disorder. The Mooney group published their work exploring this mechanism in an unusual model—the songbird—in Proceedings of the National Academy of Sciences.2 They used lentivirus to express mHTT exon1 in the basal ganglia nucleus of a male zebra finch, and analyzed the bird’s vocal behaviors. They uncovered the affected neural pathways, and infer how this relates to movement disorders in human HD.

Although mice are easy to use in the lab, larger animal models are more indicative of human pathology. To this end, a transgenic HD sheep has been created. Though the sheep is not itself symptomatic, it can be used to study presymptomatic HD. In Nature Scientific Reports, Handley and colleagues describe their work examining metabolic profiles in different tissues from the HD sheep using gas chromotography mass spectrometry.3 They found specifically that amino acids in the cerebellum were altered, whereas fatty acids in the liver were altered. Their results suggest a hyper-metabolic defect in the sheep.

1Langfelder P, Cantle JP, Chatzopoulou D, et al. Integrated genomics and proteomics define huntingtin CAG length-dependent networks in mice. Nat Neurosci. 2016 Apr;19(4):623-633.

2Tanaka M, Singh Alvarado J, et al. Focal expression of mutant huntingtin in the songbird basal ganglia disrupts cortico-basal ganglia networks and vocal sequences. Proc Natl Acad Sci USA. 2016 Mar 22;113(12):E1720-7.

3Handley RR, Reid SJ, Patassini S, et al. Metabolic disruption identified in the Huntington’s disease transgenic sheep model. Sci. Rep. 2016 Feb 11;6:20681.

In cell models…

Messages Image(1774552513)Huntingtin knockdown in either a pan- or allele-specific manner is one of the hottest areas of HD research. Using fibroblasts derived from HD patients and new techniques in genome editing, the Nolta group identified single nucleotide polymorphisms (SNPs) specific to the mutant allele, and were able to deliver transcription activator-like effector nucleases (TALEN) to the cells to specifically silence the mutant allele.1 The group identified a novel method for targeting and shortening the polyglutamine tract, particularly in the mutant allele.

Although knocking down the wild-type Huntingtin gene (HTT) may be therapeutic, off-target effects remain unknown. Lopes and colleagues used neural precursor cells derived from HD human embryonic stem cells to investigate the impact of mHTT on HTT’s normal function in regulating molecular motors and spindle pole orientation.2 They demonstrated that a repeat length of 46 has aberrant effects on mitotic spindle orientation, and that HTT is required for normal distribution of mitotic components, arguing for allele-specific silencing as a therapy.  This work also demonstrates that using physiologically relevant CAG-repeat lengths (between 36-60) can be important when modeling HD.

Another way to optimize allele-specific silencing is by optimizing the chemistry of oligonucleotides. Thiophosphonacetate (thio-PACE) modifications replace a non-bridging oxygen molecule with an acetate and a phosphorothiate substitution, modifications hypothesized to increase uptake and hybridization of the oligonucleotide with its target. A recent article describes the delivery of these altered oligonucleotides to fibroblasts derived from HD patients and to an immortalized striatal cell model.3 Although oligonucleotides with different numbers of these modifications were able to silence mHTT, not all oligonucleotides had positive results compared to the unmodified control. These results allude to the importance of, and potential to modify, the chemistry of oligonucleotides for silencing mHTT.

1Fink KD, Deng P, Gutierrez J, et al. Allele-specific reduction of the mutant huntingtin allele using transcription activator-like effectors in human Huntington’s disease fibroblasts. Cell Transplant. 2016;25(4):677-686.

2Lopes C, Aubert S, Bourgois-Rocha F, et al. Dominant-negative effects of adult-onset huntingtin mutations alter the division of human embryonic stem cells-derived neural cells. PloS one. 2016;11(2):e0148680.

3Matsui M, Threlfall RN, Caruthers MH, Corey DR. Effect of 2′-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells. Artif DNA PNA XNA. 2014;5(3):e1146391.

Research Round-Up

By: Lise Munsie, PhD

In the lab…

The Ranum laboratory published their discovery of Repeat Associated Non-ATG (RAN) translation in HD in Neuron.1 This was the first demonstration of RAN translation in a disease where the causative triplet repeat expansion occurs in the coding region. They showed that poly-Ala, poly-Cys, poly-Leu and poly-Ser proteins are translated in neurons that express huntingtin (Htt) mini-genes with disease-causing CAG expansion lengths. Accumulation of these proteins increases with expansion size. They found RAN-translated proteins in brain regions affected by disease in post-mortem HD brain tissue, with abundance and protein specificity correlating with disease severity.

RoundUp3Bowles and colleagues used the immortalized striatal cell line derived from Q111 mice to examine phosphorylation of Htt by stimulating the epidermal growth factor (EGF) pathway.2 They found alterations in MEK and AKT phosphorylation of wild-type compared to mutant Htt (mHtt) after EGF stimulation, leading to altered nuclear localization and altered transcription of a subset of genes. Inhibiting these kinases in the presence of EGF affects Htt localization, especially in the mutant cell line. The authors hypothesize it may be possible to attenuate these changes using MEK and AKT inhibitors. Kinase inhibitors may correct Htt localization, attenuating pathology associated with altered transcription.

Whether over-expression or activation of 5′ adenosine monophosphate-activated protein kinase (AMPK) is neuroprotective or toxic in HD remains controversial. Using nematode models and murine striatal neuron models, a recent report showed that AMPK over-expression has positive outcomes in models.3 Using fluorescence resonance energy transfer (FRET) techniques, Vázquez-Manrique and colleagues showed a decrease in soluble mHtt in the presence of AMPK activation. In vivo, they demonstrated that active AMPK co-transfected with toxic Htt species results in smaller brain lesions, but no alteration in the number of ubiquitin-positive aggregates.

1Bañez-Coronel M, Ayhan F, Tarabochia Alex D, et al. RAN translation in Huntington disease. Neuron. 2015 Nov 18;88(4):667-77. doi: 10.1016/j.neuron.2015.10.038.

2Bowles KR, Brooks SP, Dunnett SB, Jones L. Huntingtin subcellular localisation is regulated by kinase signalling activity in the StHdhQ111 model of HD. PloS one. 2015;10(12):e0144864.

3Vázquez-Manrique RP, Farina F, Cambon K, et al. AMPK activation protects from neuronal dysfunction and vulnerability across nematode, cellular and mouse models of Huntington’s disease. Hum Mol Genet. 2015 Dec 17. pii: ddv513. [Epub ahead of print].

In the pipeline…

Using an unbiased screen for huntingtin (HTT)-interacting transcription factors, the La Spada group identified peroxisome proliferator-activated receptor delta (PPAR-δ) as a novel HTT interactor.1 An alteration in PPAR-δ transactivation in a polyglutamine-dependent manner leads to mitochondrial dysfunction, enhancing cell death. When injected into the central nervous system of mice, a dominant-negative PPAR construct produced motor dysfunction, neurodegeneration, and mitochondrial abnormalities consistent with HD. Pharmacologically inducing PPAR-δ activity ameliorated HD-induced dysfunction and cell death in mouse and stem cell striatal cell models. The authors propose that KD3010, a compound which enhances PPAR-δ activation, may be useful as a treatment for aspects of HD pathology.

RoundUp2The Messer laboratory performed studies examining the safety of active vaccination as a treatment for HD in mice.2 They assessed the safety and immunogenicity of 11 immunization protocols, including different combinations of peptide, protein and DNA vaccines. They found that a combination of three non-overlapping Htt exon-1 peptides resulted in the greatest immunogenicity, but also led to transcriptional dysregulation of immune-related genes. This treatment strategy may necessitate adjuvant therapies aimed at restoring immune controls.

Finding heterozygous polymorphisms that exist in cis with the mHTT allele is essential to development of allele-specific silencing therapies. A recent report in Molecular Therapy prioritized target selection by examining diverse HD patient populations across Canada, France, Sweden, and Italy.3 The group defined three target haplotypes, A1, A2, and A3, that could be targeted by allele-specific antisense oligonucleotides (ASOs) to treat the 80% of patients who have one of these haplotypes. The group demonstrated that ASOs against polymorphisms in the A1 haplotypes selectively inhibit mHTT in a potent and specific manner.

1Dickey AS, Pineda VV, Tsunemi T, et al. PPAR-[delta] is repressed in Huntington’s disease, is required for normal neuronal function and can be targeted therapeutically. Nat Med. 2015 Dec 7. doi: 10.1038/nm.4003. [Epub ahead of print].

2Ramsingh AI, Manley K, Rong Y, et al. Transcriptional dysregulation of inflammatory/immune pathways after active vaccination against Huntington′s disease. Hum Mol Genet. 2015 Nov 1;24(21):6186-97. doi: 10.1093/hmg/ddv335. Epub 2015 Aug 24.

3Kay 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;23(11):1759-1771.

In stem cells…

The Ellerby group created isogenic induced pluripotent stem cell (iPSC) lines from HD patients’ cells.1 In a recent Stem Cell report, the group performed transcriptome analysis on the HD and the isogenic lines. They found a 10-fold increase in differentially expressed genes in the HD line compared to the isogenic line after differentiation to neural stem cells, compared to differentially expressed genes prior to differentiation. The group showed that transforming growth factor (TGF-β) and netrin-1 signaling pathways are differentially regulated at the neural stem cell stage, and can be modulated to rescue HD-related phenotypes in both the in vitro stem cell model and an in vivo mouse model.

RoundUp1Another recent report utilized iPSCs derived from the YAC128 mouse model, an adult-onset HD patient, and a juvenile-onset HD patient, to investigate pathways involved in HD pathogenesis.2 Multiple signaling pathways were altered in these lines, including the MAPK/ERK pathways, and p53 signaling. Other pathways (such as TGF-β) were not affected in the undifferentiated cells. This paper indicates that some, but not all, pathways are affected early and, possibly, peripherally in HD.

Stimulation of the A2A adenosine receptor (A2AR) in cell culture models of HD has positive anti-apoptotic outcomes. A paper published in Human Molecular Genetics used iPSCs derived from HD and control patients differentiated into DARPP32 GABAergic neurons, and examined how stimulating A2AR differentially effects the two cell lines.3 Treating the cells with hydrogen peroxide introduced oxidative stress and caused double-stranded DNA breaks, with more double-stranded breaks in the HD-derived neurons. A2AR agonists ameliorated this phenotype in the differentiated neurons through PKA activation. The authors suggest the use of this platform for drug screening.

1Ring Karen L, An Mahru C, Zhang N, et al. Genomic analysis reveals disruption of striatal neuronal development and therapeutic targets in human Huntington’s disease neural stem cells. Stem Cell Reports. 2015 Dec 8;5(6):1023-38. doi: 10.1016/j.stemcr.2015.11.005.

2Szlachcic WJ, Switonski PM, Krzyzosiak WJ, et al. Huntington disease iPSCs show early molecular changes in intracellular signaling, the expression of oxidative stress proteins and the p53 pathway. Dis Model Mech. 2015 Sep;8(9):1047-57. doi: 10.1242/dmm.019406. Epub 2015 Jun 18.

3Chiu F-L, Lin J-T, Chuang C-Y, et al. Elucidating the role of the A2A adenosine receptor in neurodegeneration using neurons derived from Huntington’s disease iPSCs. Hum Mol Genet. 2015 Nov 1;24(21):6066-79. doi: 10.1093/hmg/ddv318. Epub 2015 Aug 11.

 

Research Round-Up: Insights of the Year 2014-15

In our final issue of 2015, HD Insights highlights influential HD research in the 2014-2015 academic year. The winners of last year’s Insights of the Year competition nominated nine papers in lab, clinical, and imaging and biomarkers research. The Editorial Board and other researchers voted for the most influential papers, selecting one each in basic and clinical science and two in imaging and biomarkers as most influential. Authors of the most influential papers will present their research in a special panel discussion at the 2015 HSG annual meeting. Congratulations to all the nominees and winners!

In the lab…
Most influential paper
Targeting ATM ameliorates mutant huntingtin toxicity in cell and animal models of HD
By: Lu XH, Mattis VB, Wang N, Al-Ramahi I, van den Berg N4, Fratantoni SA, Waldvogel H, Greiner E, Osmand A, Elzein K, Xiao J, Dijkstra S, de Pril R, Vinters HV, Faull R, Signer E, Kwak S, Marugan JJ, Botas J, Fischer DF, Svendsen CN, Munoz-Sanjuan I, Yang XW

(Summary by X. William Yang, MD, PhD)

Elevated ATM signaling in HD cells, mouse & patient brains

Figure: Diagram of TrkBR and p75NTR signaling in HD. TrkBR activation of PI3K and AKT leads to the induction of long-term potentiation (LTP) in striatal projection neurons (SPNs). This is attenuated in iSPNs in HD by downstream targets of p75NTR signaling.

Emerging evidence suggests that DNA damage and repair pathways can contribute to the pathogenesis of a number of neurodegenerative disorders. We investigated the pathogenic role in HD of ataxia-telangiectasia mutated (ATM), a protein kinase involved in DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been observed in Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS), and in polyglutamine disorders such as HD and spinocerebellar ataxia type 3 (SCA3). We showed that ATM signaling was consistently elevated in cells derived from the BACHD mouse model and in disease-vulnerable brain tissues from BACHD mice and HD patients (Figure).
ATM reduction mitigated toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we showed that genetic reduction of one copy of the Atm gene ameliorated multiple behavioral deficits, and partially improved neuropathology. Small-molecule ATM inhibitors, originally developed for radiosensitization in cancer therapy, reduced mHTT-induced cell death in a rat cortico-striatal neuronal co-culture assay and in induced pluripotent stem cells (iPSCs) derived from HD patients. Our study provides converging genetic and pharmacological evidence that partial reduction of ATM signaling could ameliorate mHTT toxicity in a number of cellular and animal models of HD, suggesting that ATM maybe a useful therapeutic target for HD.

 

 

 

Aberrant Astrocytes

Figure: Blood oxygen level-dependent magnetic resonance imaging with carbon challenge revealed impaired vascular reactivity in the cortical vessels of R6/2 mice (right) as compared to wild-type (left). The arrowheads represent the vessels with low vascular reactivity.

Aberrant astrocytes impair vascular reactivity in Huntington’s disease
By: Hsiao HY, Chen YC, Huang CH, Chen CC, Hsu YH, Chen HM, Chiu FL, Kuo HC, Chang C, Chern Y

(Summary by Chien-Hsiang Huang, PhD, Chen Chang, PhD, and Yijuang Chern, PhD)

Increased blood vessel density has been recognized in animals and patients with HD. Our study further reveals that a fraction of the cortical blood vessels in the brains of HD mice (R6/2) is nonreactive (Figure). Such impaired blood vessels in HD brains result from aberrant astrocytes that release too much vascular endothelial growth factor and inflammatory mediators. The impaired vascular reactivity causes abnormal regulation of cerebral blood flow and may be responsible for brain atrophy in R6/2 mice. Whether the formation of the nonreactive vessels is detrimental or beneficial to HD remains to be elucidated. The nonreactive vessels warrant future investigation in clinical settings, and the integrity of vascular reactivity may be used as a new biomarker to evaluate the progression of HD.

 

 

 

 

Figure: Diagram of TrkBR and p75NTR signaling in HD. TrkBR activation of PI3K and AKT leads to the induction of long-term potentiation (LTP) in striatal projection neurons (SPNs). This is attenuated in iSPNs in HD by downstream targets of p75NTR signaling.

Figure: Diagram of TrkBR and p75NTR signaling in HD. TrkBR activation of PI3K and AKT leads to the induction of long-term potentiation (LTP) in striatal projection neurons (SPNs). This is attenuated in iSPNs in HD by downstream targets of p75NTR signaling.

Impaired TrkB receptor signaling underlies corticostriatal dysfunction in Huntington’s disease
By: Plotkin JL, Day M, Peterson JD, Xie Z, Kress GJ, Rafalovich I, Kondapalli J, Gertler TS, Flajolet M, Greengard P, Stavarache M, Kaplitt MG, Rosinski, J, Chan CS, Surmeier DJ

(Summary by Joshua L. Plotkin, PhD)

Reduced trophic support of striatal projection neurons (SPNs) is thought to play a key role in the progression of HD. This has been widely attributed to diminished levels of brain-derived neurotrophic factor (BDNF). In a recent study, we showed that BDNF signaling through tyrosine-related kinase B receptors (TrkBRs) was indeed attenuated in the striatum of early symptomatic BACHD and Q175 mouse models of HD, but without changes in BDNF expression or delivery to the striatum. This attenuation led to a synapse-specific loss of corticostriatal long-term potentiation, and progressive weakening of cortical synapses to indirect pathway SPNs (iSPNs), a circuit pathology consistent with the early choreic symptoms of HD.

Although TrkBRs were activated normally, their signaling was blunted by engagement of another target of BDNF: p75 neurotrophin receptors (p75NTRs). Elevated p75NTR signaling occurred due to increased expression of its downstream target: phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is an inhibitor of TrkBR signaling, and was specifically up-regulated in iSPNs (Figure). Synaptic potentiation could be rescued in HD iSPNs by inhibiting 75NTRs, PTEN, or intermediate signaling partners. This study suggests that targeting postsynaptic p75NTR signaling, rather than presynaptic BDNF delivery, offers a promising therapeutic strategy for HD.

 

 

In the clinic…
Most influential paper

Figure: Levels of mHTT measured in cererebrospinal fluid from control, premanifest, and manifest HD patients

Figure: Levels of mHTT measured in cererebrospinal fluid from control, premanifest, and manifest HD patients

Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients
By: Wild EJ, Boggio R, Langbehn D, Robertson N, Haider S, Miller JR, Zetterberg H, Leavitt BR, Kuhn R, Tabrizi SJ, Macdonald D, Weiss A

(Summary by Edward J. Wild, PhD)

The genetic cause of HD – mHTT, encoded by the CAG-expanded mHTT gene – has been known since 1993. Clinical trials of “gene silencing” drugs that aim to reduce the production of mHTT in the brain are now underway in HD patients.

Quantifying mHTT in the central nervous system (CNS) would be helpful for understanding whether these therapies are having the desired effect, and would aid in studying the biology of mHTT in HD. However, mHTT accumulates inside cells, and its concentration in cerebrospinal fluid (CSF) is very low. Until now, mHTT could not be detected in the CNS.

We have developed a new, ultra-sensitive, “single molecule counting” immunoassay to detect and measure the concentration of mHTT in CSF. The immunoassay uses an antibody pair specific to mHTT over wild-type huntingtin. Fluorescence events corresponding to single molecules of antibody-bound protein are counted, enabling precise quantification at femtomolar concentrations.

We analyzed CSF from volunteers from two cohorts and for the first time, successfully quantified mHTT in mHTT carriers. We showed that mHTT levels in premanifest HD were intermediate between control and manifest HD levels (Figure). Moreover, the concentration of mHTT independently predicted disease burden as well as motor and cognitive scores. Associations with known neuronal proteins suggest the mHTT detectable in CSF is derived from dying neurons. We now plan to use this new assay to determine whether the level of mHTT in CSF can predict HD onset or rate of progression, and to study the first “gene silencing” drugs in clinical trials.

 

 

Figure: (Background) Pathological tau aggregates in an HD brain (Foreground left) George Huntington, MD (1850−1916), who first described the disease that now bears his name, in a report titled “On chorea” published in The Medical and Surgical Reporter of Philadelphia on April 13, 1872

Figure: (Background) Pathological tau aggregates in an HD brain (Foreground left) George Huntington, MD (1850−1916), who first described the disease that now bears his name, in a report titled “On chorea” published in The Medical and Surgical Reporter of Philadelphia on April 13, 1872

The role of tau in the pathological process and clinical expression of Huntington’s disease
By: Vuono R, Winder-Rhodes S, de Silva R, Cisbani G, Drouin-Ouellet J; REGISTRY Investigators of the European Huntington’s Disease Network,
Spillantini MG, Cicchetti F, Barker RA

(Summary by Romina Vuono, PhD)

More than twenty years after the discovery of the genetic defect responsible for HD, there are still many unanswered questions. For example, why do HD patients differ dramatically in their age at onset of manifest disease and disease progression, despite similar CAG repeat lengths? Recent studies have suggested that other genetic influences may play a role. So far, more than 24 genes have been linked to HD, but their relevance remains unclear as to whether, and how, they may drive the HD pathogenic cascade and resultant clinical features.

We have recently shown that the microtubule-associated protein tau gene (MAPT), known to be associated with many neurodegenerative diseases, can give rise to pathology in the brains of HD patients. We have reported pathological tau aggregates, as well as tau oligomers (thought to be the most toxic form of tau), in postmortem HD brain tissues (Figure). These findings were common to young-onset as well as older HD patients, confirming that tau pathology was related to the disease process, rather than related to age. We highlighted the clinical significance of this pathology by demonstrating that a genetic variation of the MAPT gene (MAPT H2 haplotype) affected the rate of cognitive decline in a large cohort of HD patients. Our findings highlight a novel and important role for tau in the pathogenic process and clinical expression of HD, which in turn may open up new therapeutic approaches.

 

 

Figure: Tau nuclear rods (TNRs), a newly identified kind of tau deposit in the brains of individuals with HD

Figure: Tau nuclear rods (TNRs), a newly identified kind of tau deposit in the brains of individuals with HD

Huntington’s disease is a four-repeat tauopathy with tau nuclear rods
By: Fernández-Nogales M, Cabrera JR, Santos-Galindo M, Hoozemans JJ, Ferrer I, Rozemuller AJ, Hernández F, Avila J, Lucas JJ

(Summary by Marta Fernández-Nogales, PhD)

Intronic mutations that produce an alteration in exon 10 alternative splicing of the MAPT (Tau) gene in frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17) result in an increase of tau isoforms with four repeats of microtubule binding domains. This is sufficient to cause neurodegeneration, though the reason is not yet known. It is possible that these tau binding domains increase the binding energy of tau to the microtubules.

We discovered an increase in tau isoforms with four microtubule binding domains in the cortex of HD patients and a concomitant decrease in isoforms with three microtubule binding domains, as well as an increase in total tau levels, similar to that observed in other dementias. This change is accompanied by the appearance of a new tau-histopathological hallmark in the brains of HD patients, consisting of deposits of rod-shaped tau protein that fill previously reported invaginations of the nuclei of some striatal and cortical neurons (Figure). We have named these inclusions “tau nuclear rods.”

Furthermore, we were able to demonstrate that tau protein plays a role in the pathogenesis of HD by verifying that the partial or total reduction of tau in an HD mouse model produces a significant improvement in motor coordination. This discovery opens the possibility of using emergent pharmacological tools under development for the treatment of other tauopathies in HD patients.

 

 

In imaging and biomarkers…
Most influential paper

Figure: The main cerebrovascular compromise in HD patients and in an HD mouse model of the disease, comprised of mhtt expression in cell types associated with blood vessels, the alteration of blood vessel morphology and an increase in blood-brain barrier permeability

Figure: The main cerebrovascular compromise in HD patients and in an HD mouse model of the disease, comprised of mhtt expression in cell types associated with blood vessels, the alteration of blood vessel morphology and an increase in blood-brain barrier permeability

Cerebrovascular and blood-brain barrier impairments in Huntington’s disease: potential implications for its pathophysiology
By: Drouin-Ouellet J, Sawiak SJ, Cisbani G, Lagacé M, Kuan WL, Saint-Pierre M, Dury RJ, Alata W, St-Amour I, Mason SL, Calon F, Lacroix S, Gowland, PA, Francis ST, Barker RA, Cicchetti F

(Summary by Janelle Drouin-Ouellet, PhD)

We report here on our work using a combination of approaches to show that the cerebrovasculature is compromised at four different levels in the context of HD. We show that:
1) mHTT protein aggregates are found in all compartments of the neurovascular unit
2) blood vessels are more dense but smaller in size
3) this change in size and density is accompanied by an increased blood-brain barrier permeability that leads to
peripheral blood cell infiltration, and
4) there are mHTT aggregates in transcytotic vesicles.

These observations appeared on MRI and in postmortem tissue in both the R6/2 mouse model and HD patients (Figure).
These findings raise critical questions regarding the impact of vascular alterations on neuronal network activity and degeneration in HD, and could indicate a more significant crosstalk between elements of the blood and the CNS than originally thought. This could in turn create an increased inflammatory response as a result of immune cell infiltration to the brain and facilitate mHTT accumulation due to the leaky blood-brain barrier. We aim to further elucidate the contribution of blood-brain barrier compromise, and the resulting presence of inflammatory elements, to the pathophysiology and progression of HD.

 

 

 

Most influential paper

Figure: Schematic of detection of mHTT in CSF by immunoprecipitation and flow cytometry

Figure: Schematic of detection of mHTT in CSF by immunoprecipitation and flow cytometry

Ultrasensitive measurement of huntingtin protein in cerebrospinal fluid demonstrates increase with Huntington disease stage and decrease following brain huntingtin suppression
By: Southwell AL, Smith SE, Davis TR, Caron NS, Villanueva EB, Xie Y, Collins JA, Li Ye M, Sturrock A, Leavitt BR, Schrum AG, Hayden MR

(Summary by Amber L. Southwell, PhD)

The lack of quantitative, robust, and reliable biomarkers for use in early subclinical diagnosis is a major obstacle for development of disease-modifying therapies for HD. Such markers are needed to assist in monitoring disease Progression, patient stratification, and evaluating efficacy of therapeutics in the clinic.

The Hayden lab at the University of British Columbia (UBC) has collaborated with colleagues at UBC and the
Mayo Clinic to develop an ultrasensitive method of measuring mHTT in the cerebrospinal fluid (CSF) of HD patients and model mice, called microbead-based immunoprecipitation and flow cytometry (IP-FCM), that could meet these needs. See Figure for a schematic that summarizes the method.

Using IP-FCM, we have shown that mHTT in the CSF originates in the brain and is released by injured or dying brain cells. Levels of mHTT in the CSF increase with worsening HD symptoms, suggesting that this approach will be useful as a measure of disease progression.

Additionally, lowering mHTT in the brain using gene-silencing treatments results in a measurable reduction of mHTT in the CSF, indicating that this method could be used to verify and quantify changes in brain mHTT levels in response to experimental therapies. This advance is clinically relevant and will enable more rigorous assessment of important endpoints in evaluating therapies for HD.

 

 

Figure: Schematic of mutant Huntingtin (mHTT) aggregation assays using whole-cell lysates (supernatants, middle of figure) and enhanced aggregation in whole cells prepared from the PC-12 cell model of HD

Figure: Schematic of mutant Huntingtin (mHTT) aggregation assays using whole-cell lysates (supernatants, middle of figure) and enhanced aggregation in whole cells prepared from the PC-12 cell model of HD

Huntington’s disease cerebrospinal fluid seeds aggregation of mutant huntingtin

By: Tan Z, Dai W, van Erp TG, Overman J, Demuro A, Digman MA, Hatami A, Albay R, Sontag EM, Potkin KT, Ling S, Macciardi F, Bunney WE, Long JD, Paulsen JS, Ringman JM, Parker I, Glabe C, Thompson LM, Chiu W, Potkin SG

(Summary by Zhiqun Tan, PhD, and Steven G. Potkin, MD)

The onset of symptoms in individuals who carry the mutant HD allele (mHTT) remains variable and unpredictable. To speed development of new therapies, biomarkers that reflect the development of the HD pathological process are quantitatively associated with the course of illness are needed. We reported that oligomeric peptides that contain the polyglutamine expansion coded by mHTT, and CSF fluid from individuals with HD, and BACHD transgenic rats, enhance seeded aggregation in a PC-12 cell model and its cell lysate. Oligomers derived from other proteins, including Aβ and α-synuclein, do not induce seeding. We demonstrate that seeding is mHTT template-specific and may reflect an underlying cell-to-cell mechanism of huntingtinopathy propagation.

Light and cryo-electron microscopy (cryo-EM) confirm that synthetic seeds nucleate and enhance mHTT aggregation in cell lysates. This seeding assay (Figure) distinguishes individuals with HD from healthy controls. Seeding measures in asymptomatic gene-positive individuals fall between HD patients and controls. This quantifiable seeding property in the CSF of individuals with HD may serve as a molecular biomarker assay to monitor HD progression, and to evaluate therapies that target mHTT. The automation of the assay is in process.

 

Research Round-Up

By: Lise Munsie, PhD

In stem cell research…

517236-vol-11-springGenerating GABAergic medium-sized spiny neurons (MSNs) from human pluripotent stem cell (hPSC) sources is a rapidly evolving technique that holds much promise for HD modeling and treatment. Arber and colleagues describe a new method of differentiating hPSCs to MSNs using Activin A (activin).1 Activin is part of the transforming growth factor beta signaling pathway, involved in neurogenesis and neuronal cell type fate. Arber’s paper describes the ability of activin to produce a much higher proportion of MSNs than current techniques, and the ability of these neurons to survive in grafts in rodent models of HD.

Dr. Ihn Sik Seong’s group makes an elegant cell model of HD using embryonic stem cells, producing six isogenic lines from one lineage. One line expresses two copies of wild-type mouse huntingtin (Htt) (Q7), an Htt null line, while four heterozygous lines express one Q7 allele and one CAG expanded allele of 20, 50, 91, or 111 CAG repeats.2 This paper explores histone modifications in the presence of mHtt and Htt, based on research indicating that Htt acts as a polycomb recessive complex-2 facilitator. The authors show subtle alterations to the chromatin landscape and histone methylation states depending on whether Htt or mHtt is present, and whether cells are in a pluripotent or differentiated state.

Mattis and his group used HD-patient-derived induced pluripotent stem cells produced from non-integrating technology to assess how HD and juvenile-onset HD CAG repeat lengths affect neuronal differentiation.3 The group found a significantly higher proportion of nestin-positive cells in cultures derived from JHD patients after 42 days of differentiation. Cells with juvenile-onset HD CAG repeat length suffer an increase in cell death with brain-derived neurotrophic factor withdrawal, due to glutamate toxicity. The authors posit that this increase may be mediated through the TrkB receptor.

1Arber C, Precious SV, Cambray S, et al. Activin A directs striatal projection neuron differentiation of human pluripotent stem cells. Development. 2015Apr 1; 142(7):1375-86.

2Biagioli M, Ferrari F, Mendenhall EM, et al. Htt CAG repeat expansion confers pleiotropic gains of mutant huntingtin function in chromatin regulation. Hum Mol Genet. 2015 May 1; 24(9):2442-57.

3Mattis VB, Tom C, Akimov S, et al. HD iPSC-derived neural progenitors accumulate in culture and are susceptible to BDNF withdrawal due to glutamate toxicity. Hum Mol Genet. 2015 Mar 3. pii: ddv080. [Epub ahead of print]


 

In the clinic…

mriA recent study assesses the clinical manifestation of HD in patients who express intermediate CAG repeat lengths.1 A group led by Panegyres examined subjects who had repeats in the 27–35 range (at risk for prodromal HD), 36–39 range (mixed penetrance), or 40+ range (HD). No patients with CAG repeat length below 35 developed manifest HD. Smoking and being older than 65 correlated with manifest HD among those with intermediate repeat lengths. Of note, a high level of education was associated with lower odds of manifest HD, and the authors propose several hypotheses about environmental, cognitive or epigenetic factors.

A paper by Hobbs and colleagues in a group led by Prof. Sarah Tabrizi assessed candidate outcomes in HD patients in the imaging, clinical and cognitive streams, at both short (six-month) and longer (15-month) time periods, in order to further the guidelines for assessing outcomes in HD clinical trials.2 This is the first study to report significant effect size in short time periods. The strongest longitudinal changes were present in caudate atrophy and ventricular expansion. Small effect sizes were noted in other imaging and clinical measures that will be useful over longer time periods.

To date, there have been few studies assessing changes in the hypothalamus, a brain structure that may be responsible for the sleep, emotional and metabolic changes associated with HD. Hypothalamic changes are noted up to 15 years before predicted onset of manifest HD. In a recent PLoS One article, 3 Gabery and colleagues explored whether alterations to hypothalamic volume may be linked to these symptoms. Using 3T-MRI image data from IMAGE-HD, the group compared hypothalamic volume between patients before and after onset of manifest HD. The group did not find any statistically significant alterations in hypothalamic volume, indicating that the hypothalamic changes noted in previous studies are not likely due to atrophy.

1Panegyres PK, Shu CC, Chen HY, Paulsen JS. Factors influencing the clinical expression of intermediate CAG repeat length mutations of the Huntington’s disease gene. J Neurol, 2015. 262(2): p. 277-84.

2Hobbs NZ, Farmer RE, Rees EM, et al. Short-interval observational data to inform clinical trial design in Huntington’s disease. J Neurol Neurosurg Psychiatry. 2015 Feb 10. pii: jnnp-2014-309768. doi: 10.1136/jnnp-2014-309768. [Epub ahead of print]

3Gabery S, Georgiou-Karistianis N, Lundh SH, et al. Volumetric analysis of the hypothalamus in Huntington Disease using 3T MRI: the IMAGE-HD Study. PLoS One, 2015. 10(2): p. e0117593.


 

In the pipeline…

Wild and colleagues report in the Journal of Clinical Investigation on an ultra-sensitive method of quantifying mHTT levels in the cerebrospinal fluid (CSF) of HD patients.1 Using a single-molecule counting immunoassay based on the MW1 antibody specific for the expanded CAG tract, the group demonstrated the ability to detect mHTT in a disease-and onset-dependent manner in the CSF of HD patients in two cohorts. This assay will be useful as a pharmacodynamic biomarker in clinical trials, specifically in trials assessing HTT-lowering strategies.

It has been shown that allele-specific silencing of mHTT can be successful using small interfering RNA (siRNA) targeting the expanded CAG tract, or single nucleotide polymorphisms associated with the mHTT allele. The Davidson group aimed to explore whether specificity for the mHTT allele was maintained when previously tested siRNA sequences were moved into the group’s artificial mRNA expression system.2 This mRNA expression system may be more amenable to sustained delivery of knockdown than previously tested methods of in vivo knockdown. The group tested their system both in vitro and in vivo, and found that only some of the previously tested sequences were efficacious long term.

A report published in Nature Chemical Biology by Jimenez-Sanchez and colleagues describes a new therapeutic HD target extracted from an siRNA screen focusing on genes that are amenable to small molecule modulation.3 The most significant suppressor of mHTT toxicity was glutaminyl-peptide cyclotransferase (QPCT), an enzyme with glutaminyl cyclase activity. The knockdown of this protein inhibits mHTT aggregation, while overexpression exacerbates aggregation. Using pharmacophore models and other known structures, the group designed QPCT inhibitors. These inhibitors have positive effects in several models of HD, and with further development may become clinically relevant.

1Wild EJ, Boggio R, Langbehn D, et al. Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients. J Clin Invest. 2015 Apr 6. pii: 80743. doi: 10.1172/JCI80743. [Epub ahead of print]

2Monteys AM, Wilson MJ, Boudreau RL, et al. Artificial miRNAs targeting mutant huntingtin show preferential silencing in vitro and in vivo. Mol Ther Nucleic Acids. 2015 Apr 7;4:e234. doi: 10.1038/ mtna.2015.7.

3Jimenez-Sanchez M, Lam W, Hannus M, et al. siRNA screen identifies QPCT as a druggable target for Huntington’s disease. Nat Chem Biol. 2015 May;11(5):347-54.

Research Round-Up

By: Lise Munsie, PhD

In the proteome…

A number of studies suggest a relationship between huntingtin (HTT) and tau protein pathology which has yet to be elucidated. Tau is found predominantly in neurons. In tauopathies such as Alzheimer disease, tau is cleaved and then aggregates. A study by Gratuze and colleagues looks at the phosphorylation state of tau in both the R6/2 and Q175 mouse models of HD.1 Hyperphosphorylation of tau at different positions can be seen in the presence of mutant huntingtin (mHtt). The authors attribute this hyperphosphorylation to a down-regulation of calcineurin phosphatase caused by mHtt. Hyperphosphorylation of tau is not associated with increased cleavage or aggregation.

Two recent publications from the Outeiro group expand these observations. The first examines how mHTT impacts tau localization, molecular interactions and phosphorylation pattern.2 Using biophotonics, they show that mHTT leads not only to altered phosphorylation of tau, but also alters tau’s cellular localization and microtubule stabilizing functions. They describe a new kind of aggregate containing tau and mHTT, and hypothesize that an aberrant interaction with mHTT may leave tau unable to interact with phosphatases.

In a second manuscript in Human Molecular Genetics, the group investigates the interaction between HTT and α- synuclein, a protein that aggregates and causes toxicity in Parkinson disease (PD), in a Drosophila model.3 The authors show that co-expression of α-synuclein and mHtt leads to an increase in insoluble aggregates containing both proteins, leading to motor deficits and decreased life span. The coexpression of these proteins synergistically enhances toxicity, accelerating the progression of the disorder. This system may be useful for screening potential drug candidates for both HD and PD.

1 Gratuze M, Noël A, Julien C, et al. Tau hyperphosphorylation and deregulation of calcineurin in mouse models of Huntington’s disease. Hum Mol Genet. 2015 Jan 1; 24(1):86-99. doi:10.1093/hmg/ddu456. Epub 2014 Sep 8.

2 Blum D, Herrera F, Francelle L, et al. Mutant huntingtin alters Tau phosphorylation and subcellular distribution. Hum Mol Genet. 2015 Jan 1; 24(1):76-85. doi: 10.1093/hmg/ddu421. Epub 2014 Aug 20.

3 Pocas GM, Branco-Santos J, Herrera F, et al. α-Synuclein modifies mutant huntingtin aggregation and neurotoxicity in Drosophila. Hum Mol Genet. 2014 Dec 1. pii: ddu606. [Epub ahead of print].


In the neurons…

neuronA report by Yao and colleagues in Molecular and Cellular Neuroscience describes an unbiased proteomics approach to screen for HTT interactors in synaptosome preparations of brain regions affected in HD.1 A large amount of HTT was found in the synaptosome fraction. HTT was also found to interact with components of the presynaptic cytomatrix: specifically, the Bassoon, Piccolo and Ahnak proteins, components of the cytomatrix at active zone complex. The authors posit that HTT acts as a scaffold, and forms part of the complex that regulates endo- and exocytosis of synaptic vesicles. Pietropaolo and colleagues report in Neuropharmacology on their investigation of the role of the endocannabanoid system (ECS) in the etiology of HD.2 The ECS modulates brain function in brain regions affected by HD, and has been implicated in HD gene expression. Pietropaolo’s group administered the cannabinoid receptor agonist WIN to R6/1 mice acutely or chronically, then monitored motor and social behavior and neurodegeneration. Chronic administration showed improvements in motor behaviors and decreased degeneration of medium spiny neurons with an increase in inclusions, suggesting a positive influence of aggregates and potential therapeutic benefit of ECS modulation.

A study by the Brouillet group in Human Molecular Genetics examines preferential degeneration of the striatum in HD.3 They look at Crym, an NADPH-dependent p38 cytosolic T3-binding protein that is preferentially expressed in the striatum. The expression of Crym is reduced in full length BACHD and knock-in models of HD, even prior to neurodegeneration. Overexpression of Crym in fragment models of HD also reduces toxicity, suggesting that Crym may be another therapeutic target for HD.

1 Yao J, Ong SE, Bajjalieh S. Huntingtin is associated with cytomatrix proteins at the presynaptic terminal. Mol Cell Neurosci. 2014 Nov 4;63C:96-100. doi: 10.1016/j.mcn.2014.10.003. [Epub ahead of print]

2 Pietropaolo S, Bellocchio L, Ruiz-Calvo A, et al. Chronic cannabinoid receptor stimulation selectively prevents motor impairments in a mouse model of Huntington’s disease. Neuropharmacology. 2015 Feb; 89:368-74. doi: 10.1016/j.neuropharm.2014.07.021. Epub 2014 Aug 11.

3 Francelle L, Galvan L, Gaiullard M, et al. Loss of the thyroid hormone binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington’s disease. Hum Mol Genet. 2014 Nov 14. pii: ddu571. [Epub ahead of print].


In the clinic…

A recent article by Sussmuth and colleagues published by the PADDINGTON consortium outlines their trial of the safety, tolerability, and deliverability of the SIRT1 inhibitor Selistat for HD patients.1 SIRT1 has been shown to acetylate mHTT, leading to altered transcription. Selistat is a selective SIRT1 inhibitor, as inhibiting SIRT1 has shown therapeutic effects in model organisms. Sussmuth’s group performed a randomized, double blind, placebo-controlled study and found that Selistat is safe and well tolerated in patients with early HD. Importantly, blood plasma contained levels of the drug that have therapeutic effects in model organisms. Tetrabenazine, currently the only FDA approved drug for treatment of HD-related chorea, has many adverse effects, including worsening psychological symptoms such as depression. The Haghighi group in Sweden performed a small study evaluating the safety and efficacy of (−)-OSU6162, a monoaminergic stabilizer that acts on dopaminergic and serotonergic receptors.2

No such psychological adverse effects were associated with the administration of the drug. The researchers noted positive trends in both psychological and motor assessments, suggesting that this class of compound may warrant larger clinical trials in HD.

The PREDICT-HD group published a new study evaluating how measures other than CAG repeat length correlate with age of onset of HD symptoms, to assist in clinical trial design and prognosis3. There were 40 different measures taken on over 1,000 patients, including imaging, motor, psychiatric, functional, and cognitive measures. The group found several measures that can improve the diagnosis of onset of HD, the strongest being total motor score, putamen volume and the Stroop word test. The results will inform the selection of outcomes for future clinical trials.

1 Sussmuth SD, Haider S, Landwehrmeyer GB, et al. An Exploratory Double blind, Randomised Clinical Trial with Selisistat, a SirT1 Inhibitor, in Patients with Huntington’s Disease. British journal of clinical pharmacology 2014.

2 Kloberg A, Constantinescu R, Nilsson MK, et al. Tolerability and efficacy of the monoaminergic stabilizer (-)-OSU6162 (PNU-96391A) in Huntington’s disease: a double-blind crossover study. Acta neuropsychiatrica 2014; 26:298-306.

3 Paulsen JS, Long JD, Ross CA, et al. Prediction of manifest Huntington’s disease with clinical and imaging measures: a prospective observational study. Lancet Neurol. 2014 Dec;

Research Round-Up: Insights of the Year 2013-2014 (In the Clinic)

In our final issue for 2014, the HD Insights team wanted to recognize the most influential papers in HD research in the 2013–2014 year. Our staff, Editorial Board, and leading HD researchers nominated the eleven papers below in three categories: lab research, clinical research, and imaging and biomarkers. The HD Insights Editorial Board 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 Huntington Study Group meeting on November 7, 2014. Congratulations to all the nominees and winners!

Mutant huntingtin is present in neuronal grafts in Huntington’s disease patients

By: Cicchetti F, Lacroix S, Cisbani G, Vallières N, Saint-Pierre M, St-Amour I, Tolouei R, Skepper JN, Hauser RA, Mantovani D, Barker RA, Freeman TB (Summary by Francesca Cicchetti, PhD)

 Ann Neurol. 2014 Jul; 76(1):31-42. doi: 10.1002/ana.24174. Epub 2014 Jun 6.

image aMutant huntingtin (mHTT), which drives HD pathology, has long been thought to exert its effects in a cell-autonomous manner, where degeneration occurs within individual cells that carry the mutant gene. We investigated the hypothesis that mHTT is capable of spreading within cerebral tissue.

The brains of four HD patients who received genetically unrelated fetal neural allografts at least a decade earlier were examined postmortem. We found a number of mHTT protein aggregates located within intracerebral allografts of striatal tissue in three of these HD patients. No grafts survived in the fourth transplant recipient. The mHTT aggregates were observed in the extracellular matrix of the genetically unrelated transplanted tissue, while in the host brain they were localized in neurons, neuropil, extracellular matrix, and blood vessels. In addition, peripheral immune cells in separate HD patients contained mHTT. There are thus a number of non-cell autonomous mechanisms that could explain these observations, including trans-synaptic propagation and hematogenous transport of mHTT, among others.

This is the first in vivo demonstration of mHTT spread in patients with a monogenic neurodegenerative disorder of the CNS. These observations raise questions about the importance of non–cell-autonomous mechanisms of pathological protein spread, and provide new targets for the development of therapeutic strategies.


Deep brain stimulation for Huntington’s disease: long-term results of a prospective open-label study

By: Gonzalez V, Cif L, Biolsi B, Garcia-Ptacek S, Seychelles A, Sanrey E, Descours I, Coubes C, de Moura AM, Corlobe A, James S, Roujeau T, Coubes P (Summary by Victoria Gonzalez, MD, PhD)

J Neurosurg. 2014 Jul; 121(1):114-22. doi: 10.3171/2014.2.JNS131722. Epub 2014 Apr 4.

image bThe role of deep brain stimulation (DBS) in the clinical management of HD has not yet been validated, although promising case reports have shown its efficacy in the treatment of severe chorea. This study aimed to analyze long-term motor outcome of a cohort of HD patients treated with globus pallidus internus (GPi) DBS. Seven patients with pharmacologically resistant chorea were included in a prospective open-label study with a median follow-up of three years. The Unified HD Rating Scale motor section was the main outcome measure. GPi DBS led to significant reduction of chorea for all patients with a mean improvement of 58.3% at one-year visit and 59.8% at three-year visit (p<0.05). Switching OFF stimulation tests confirmed sustained therapeutic effect for chorea throughout the study period. Bradykinesia and dystonia showed a non-significant trend towards progressive worsening. Increased bradykinesia was partly induced by DBS settings, and improved after adjustment of stimulation parameters. GPi DBS may provide sustained reduction of chorea in selected HD patients, with transient benefit in physical aspects of quality of life before progression of behavioral and cognitive disorders. Further studies are needed to assess the impact of GPi DBS on quality of life and cognitive measures in HD.


PRECREST: a phase II prevention and biomarker trial of creatine in at-risk Huntington disease

By: Rosas HD, Doros G, Gevorkian S, Malarick K, Reuter M, Coutu JP, Triggs TD, Wilkens PJ, Matson W, Salat DH, Hersch SM (Summary by H. Diana Rosas, MD)

Neurology 2014 Mar 11; 82(10):850-7. doi: 10.1212/WNL.0000000000000187. Epub 2014 Feb 7.

PRECREST, the first clinical trial of a drug intended to delay the onset of symptoms in individuals at risk for HD, enrolled sixty-four participants, of whom nineteen knew their gene status. Participants were randomized into two groups, regardless of gene status: one group to twice-daily oral doses of creatine, up to thirty grams per day, the other, placebo for six months.

Participants were followed for an additional twelve months on open-label creatine and assessed at regular study visits for adverse effects, and dosage levels were adjusted, if necessary, to reduce unpleasant side effects. Cognitive assessments, measurement of blood markers and MRI brain scans were conducted at the trial’s outset, at six months, and at the end of the study. Fifteen participants, including several who knew that they carried the HD mutant gene, discontinued taking creatine because of gastrointestinal discomfort, taste of the drug, inconvenience, or the stress of being constantly reminded of their HD risk. MRI scans at six months showed a slower rate of cortical and basal ganglia atrophy in gene-positive carriers who took creatine, compared to placebo. After twelve months, atrophy rates in those who crossed over to treatment were also slower than during the period of taking placebo. The results of the trial suggest that at-risk individuals can participate in clinical trials even if they do not want to learn their genetic status, and that useful biomarkers can be developed to help assess therapeutic benefits.

 

Research Round-Up: Insights of the Year 2013-2014 (In the Lab)

In our final issue for 2014, the HD Insights team wanted to recognize the most influential papers in HD research in the 2013–2014 year. Our staff, Editorial Board, and leading HD researchers nominated the eleven papers below in three categories: lab research, clinical research, and imaging and biomarkers. The HD Insights Editorial Board 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 Huntington Study Group meeting on November 7, 2014. Congratulations to all the nominees and winners!

Neuronal targets for reducing mutant huntingtin expression to ameliorate disease in a mouse model of HD

 By: Wang N, Gray M, Lu XH, Cantle JP, Holley SM, Greiner E, Gu X, Shirasaki D, Cepeda C, Li Y, Dong H, Levine MS, Yang XW (Summary by X. William Yang, MD, PhD)

Nat Med. 2014 May; 20(5):536-41. doi: 10.1038/nm.3514. Epub 2014 Apr 28. 

imageWe developed a conditional transgenic mouse model of HD (BACHD) to address the question of how ubiquitously expressed mutant huntingtin (mHTT) may selectively target striatal and cortical neurons for degeneration. The model expresses full-length human mHTT from a genomic transgene that confers endogenous-like mHTT expression patterns. The expression of mHTT in BACHD mice can be genetically shut off in cells that express Cre recombinase, allowing researchers to precisely assess the role of mHTT that is synthesized in one cell type or a combination of cell types in disease pathogenesis. Our study showed that genetically reducing mHTT in cortical neurons significantly ameliorates psychiatric-like behavioral deficits, modestly improves motor impairment, but does not improve neurodegeneration.

Importantly, we found that reducing mHTT in both cortical and striatal neurons, but not in either neuronal population alone, consistently improves all the behavioral deficits and selective brain atrophy in this HD mouse model. The study also showed that striatal synaptic dysfunction in BACHD requires both non– cell-autonomous and cell-autonomous toxicities, from cortical and striatal neurons respectively. Together, our study demonstrated distinct but interacting roles of cortical and striatal mHTT in HD pathogenesis, and suggests that optimal HD therapeutics may require targeting mHTT in both cortical and striatal neurons.


Glutathione peroxidase activity is neuroprotective in models of HD

By: Mason RP, Casu M, Butler N, Breda C, Campesan S, Clapp J, Green EW, Dhulkhed D, Kyriacou CP, Giorgini F (Summary by Robert Mason, PhD and Flaviano Giorgini, PhD)

Nat Genet. 2013 Oct;45(10):1249-54. doi: 10.1038/ng.2732. Epub 2013 Aug 25.

image 2Genetic modifiers of HD are a valuable source of potential therapeutic targets for this devastating disorder1. To uncover such modifiers we performed genetic screens in baker’s yeast, and identified 317 genes whose increased expression led to reduced mHTT toxicity2. These modifiers are involved in a wide variety of cellular processes and include members of the glutathione peroxidase (GPx) family of antioxidant enzymes, which may help protect against the increase in oxidative stress observed in HD. Glutathione peroxidases are an exciting therapeutic target due to the availability of compounds that mimic their activity. These enzymes have been tested in humans for the treatment of stroke and noise-induced hearing loss, 3, 4 possibly expediting translation into the clinic for HD patients. Notably, we found that increased levels of mouse GPX1, the most abundant mammalian glutathione peroxidase, or treatment with the GPx-mimicking compound ebselen, improved disease phenotypes in fruit fly and mammalian cell models of HD. Interestingly, increasing GPx activity was more protective than other antioxidant strategies. Unlike other antioxidant strategies, increasing GPx activity does not inhibit autophagy, an important process for the clearance of mHTT from the cell, which may contribute to the differences in protection observed. Future studies seek to evaluate the efficacy of increasing GPx activity in animal models of HD, providing critical validation before pursuing this novel candidate therapeutic approach in patients.

1 Gusella JF, MacDonald ME, Lee JM. Genetic modifiers of Huntington’s disease. Mov Disord. 2014 Sep 15; 29(11):1359-65. doi: 10.1002/mds.26001. Epub 2014 Aug 25.

2 Mason RP, Casu M, Butler N, Breda C, et al. Glutathione peroxidase activity is neuroprotective in models of Huntington’s disease. Nat Genet. 2013 Oct; 45(10):1249-54. doi: 10.1038/ng.2732. Epub 2013 Aug 25.

3 Day, B.J. Catalase and glutathione peroxidase mimics. Biochem Pharmacol. 2009 Feb 1; 77(3): 285–296.

4 Lynch ED, Kil J. Compounds for the prevention and treatment of noise-induced hearing loss. Drug Discov Today. 2005 Oct1;10(19):1291-8.


Transneuronal propagation of mutant huntingtin contributes to non-cell autonomous pathology in neurons

By: Pecho-Vrieseling E, Rieker C, Fuchs S, Bleckmann D, Esposito MS, Botta P, Goldstein C, Bernhard M, Galimberti I, Müller M, Lüthi A, Arber S, Bouwmeester T, van der Putten H, Di Giorgio FP (Summary by Lise Munsie, PhD)

Nat Neurosci. 2014 Aug;17(8):1064-72. doi: 10.1038/nn.3761. Epub 2014 Jul 13.

This thorough study demonstrates the transneuronal spreading of mHTT that may contribute to non-cell autonomous neuropathology. The authors cultured human neurons from embryonic stem cells and seeded them in organotypic brain slices from the R6/2 mouse model. They subsequently found that mHTT aggregates accumulate in human neurons, leading to pathological consequences for these neurons. These results were recapitulated in vivo in the mouse model. The cortico-striatal pathway was examined using mixed-genotype cortico-striatal brain slice cultures from R6/2 mice and their wild type counterparts. The authors found that mHTT can spread in a pre- to post-synaptic manner, as they found aggregates in wild-type medium spiny neurons after culturing with R6/2 cortex, but not the other way around. Finally, the authors used endoproteases that cleave part of the synaptic vesicle docking fusion SNARE complex, to show that this spread is mediated through synaptic vesicle recycling. The endoproteases used cleave SNAP25 and VAMP. Both treatments significantly reduced the spread of mHTT in their model.


Inhibition of mitochondrial protein import by mutant huntingtin

By: Yano H, Baranov SV, Baranova OV, Kim J, Pan Y, Yablonska S, Carlisle DL, Ferrante RJ, Kim AH, Friedlander RM.! (Summary by Lise Munsie, PhD)

Nat Neurosci. 2014 Jun; 17(6):822-31. doi: 10.1038/nn.3721. Epub 2014 May 18.

Mitochondrial dysfunction is intimately involved in the progression of HD; however, the mechanism of this dysfunction is unknown. Robert Friedlander’s group explored this in a recent Nature Neuroscience paper. Initially, the authors found that mHTT is specifically localized to mitochondria in brains from HD patients and also mouse models. Unbiased protein identification from immunoprecipitation shows that mHTT binds members of the TIM23 complex, a complex that imports matrix proteins into the inner mitochondrial membrane. Using in vitro mitochondrial protein assays, the group demonstrated that the N-terminus of mHTT is involved in this aberrant binding, leading to decreased mitochondrial protein import. This defect is enhanced in mitochondria purified from synaptosomes, compared to mitochondria purified from other parts of the cell or other cell types. The group showed that the mitochondrial protein import dysfunction is a mHTT specific function and not mediated through the polyglutamine expansion alone, and thus is a mechanism specific to HD. They additionally showed that altering protein import to the mitochondria is neurotoxic and that overexpressing major subunits of the TIM23 complex can rescue mHTT-induced neurotoxicity.

 

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