Genetic modifiers of Huntington disease differentially influence motor and cognitive domains.

Genome-wide association studies (GWASs) of Huntington disease (HD) have identified six DNA maintenance gene loci (among others) as modifiers and implicated a two step-mechanism of pathogenesis: somatic instability of the causative HTT CAG repeat with subsequent triggering of neuronal damage. The largest studies have been limited to HD individuals with a rater-estimated age at motor onset. To capitalize on the wealth of phenotypic data in several large HD natural history studies, we have performed algorithmic prediction by using common motor and cognitive measures to predict age at other disease landmarks as additional phenotypes for GWASs. Combined with imputation with the Trans-Omics for Precision Medicine reference panel, predictions using integrated measures provided objective landmark phenotypes with greater power to detect most modifier loci. Importantly, substantial differences in the relative modifier signal across loci, highlighted by comparing common modifiers at MSH3 and FAN1, revealed that individual modifier effects can act preferentially in the motor or cognitive domains. Individual components of the DNA maintenance modifier mechanisms may therefore act differentially on the neuronal circuits underlying the corresponding clinical measures. In addition, we identified additional modifier effects at the PMS1 and PMS2 loci and implicated a potential second locus on chromosome 7. These findings indicate that broadened discovery and characterization of HD genetic modifiers based on additional quantitative or qualitative phenotypes offers not only the promise of in-human validated therapeutic targets but also a route to dissecting the mechanisms and cell types involved in both the somatic instability and toxicity components of HD pathogenesis.

Huntingtin HTT1a is generated in a CAG repeat-length-dependent manner in human tissues.

BACKGROUND: The disease-causing mutation in Huntington disease (HD) is a CAG trinucleotide expansion in the huntingtin (HTT) gene. The mutated CAG tract results in the production of a small RNA, HTT1a, coding for only exon 1 of HTT. HTT1a is generated by a block in the splicing reaction of HTT exon 1 to exon 2 followed by cleavage in intron 1 and polyadenylation. Translation of HTT1a leads to the expression of the highly toxic HTT exon 1 protein fragment. We have previously shown that the levels of HTT1a expression in mouse models of HD is dependent on the CAG repeat length. However, these data are lacking for human tissues. METHODS: To answer this question, we developed highly sensitive digital PCR assays to determine HTT1a levels in human samples. These assays allow the absolute quantification of transcript numbers and thus also facilitate the comparison of HTT1a levels between tissues, cell types and across different studies. Furthermore, we measured CAG repeat sizes for every sample used in the study. Finally, we analysed our data with ANOVA and linear modelling to determine the correlation of HTT1a expression levels with CAG repeat sizes. RESULTS: In summary, we show that HTT1a is indeed expressed in a CAG repeat-length-dependent manner in human post mortem brain tissues as well as in several peripheral cell types. In particular, PBMCs show a statistically significant positive correlation of HTT1a expression with CAG repeat length, and elevated HTT1a expression levels even in the adult-onset CAG repeat range. CONCLUSIONS: Our results show that HTT1a expression occurs throughout a wide range of tissues and likely with all CAG lengths. Our data from peripheral sample sources demonstrate that HTT1a is indeed generated throughout the body in a CAG repeat-length-dependent manner. Therefore, the levels of HTT1a might be a sensitive marker of disease state and/or progression and should be monitored over time, especially in clinical trials targeting HTT expression.

Speech biomarkers in Huntington's disease: A cross-sectional study in pre-symptomatic, prodromal and early manifest stages.

BACKGROUND AND PURPOSE: Motor speech alterations are a prominent feature of clinically manifest Huntington's disease (HD). Objective acoustic analysis of speech can quantify speech alterations. It is currently unknown, however, at what stage of HD speech alterations can be reliably detected. We aimed to explore the patterns and extent of speech alterations using objective acoustic analysis in HD and to assess correlations with both rater-assessed phenotypical features and biological determinants of HD. METHODS: Speech samples were acquired from 44 premanifest (29 pre-symptomatic and 15 prodromal) and 25 manifest HD gene expansion carriers, and 25 matched healthy controls. A quantitative automated acoustic analysis of 10 speech dimensions was performed. RESULTS: Automated speech analysis allowed us to differentiate between participants with HD and controls, with areas under the curve of 0.74 for pre-symptomatic, 0.92 for prodromal, and 0.97 for manifest stages. In addition to irregular alternating motion rates and prolonged pauses seen only in manifest HD, both prodromal and manifest HD displayed slowed articulation rate, slowed alternating motion rates, increased loudness variability, and unstable steady-state position of articulators. In participants with premanifest HD, speech alteration severity was associated with cognitive slowing (r = -0.52, p < 0.001) and the extent of bradykinesia (r = 0.43, p = 0.004). Speech alterations correlated with a measure of exposure to mutant gene products (CAG-age-product score; r = 0.60, p < 0.001). CONCLUSION: Speech abnormalities in HD are associated with other motor and cognitive deficits and are measurable already in premanifest stages of HD. Therefore, automated speech analysis might represent a quantitative HD biomarker with potential for assessing disease progression.

Targeting Huntingtin Expression in Patients with Huntington's Disease.

BACKGROUND: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin. METHODS: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF. RESULTS: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). CONCLUSIONS: Intrathecal administration of HTTRx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.).

[Disease-modifying treatment approaches in Huntington disease : Past and future]. / Krankheitsmodifizierende Therapieansätze bei der Huntington-Krankheit : Blicke zurück und Blicke voraus.

Huntington disease (HD) is the most frequent monogenetic neurodegenerative disease and can be unequivocally diagnosed even in the preclinical stage, at least in all individuals in whom the CAG expansion mutation in the huntingtin gene (HTT) is in the range of full penetrance. Therefore, important preconditions for an intervention early in the disease process are met, rendering modification of the course of the disease in a clinically meaningful way possible. In this respect, HD can be viewed as a model disorder for exploring neuroprotective treatment approaches. In the past emphasis was placed on the compensation of a suspected neurotransmitter deficit (GABA) analogous to Parkinson's disease and on classical neuroprotective strategies to influence hypothetical common pathways in neurodegenerative diseases (e.g., excitotoxicity, mitochondrial dysfunction, oxidative stress). With the discovery of the causative HTT mutation in 1993, therapeutic research increasingly focused on intervening as proximally as possible in the chain of pathophysiological events. Currently, an important point of intervention is the HTT mRNA with the aim of reducing the continued production of mutant huntingtin gene products and thus relieving the body of their detrimental actions. To this end, various treatment modalities (single-stranded DNA and RNA, divalent RNA and zinc finger repressor complexes, orally available splice modulators) were developed and are currently in clinical trials (phases I-III) or in late stages of preclinical development. In addition, there is the notion that it may be possible to modify the length of the somatically unstable CAG mutation, i.e. its increase in the brain during the lifetime, thereby slowing the progression of HD.

Ubiquitination and the proteasome rather than caspase-3-mediated C-terminal cleavage are involved in the EAAT2 degradation by staurosporine-induced cellular stress.

Diminished glutamate (Glu) uptake via the excitatory amino acid transporter EAAT2, which normally accounts for ~90% of total forebrain EAAT activity, may contribute to neurodegeneration via Glu-mediated excitotoxicity. C-terminal cleavage by caspase-3 (C3) was reported to mediate EAAT2 inactivation and down-regulation in the context of neurodegeneration. For a detailed analysis of C3-dependent EAAT2 degradation, we employed A172 glioblastoma as well as hippocampal HT22 cells and murine astrocytes over-expressing VSV-G-tagged EAAT2 constructs. C3 activation was induced by staurosporine (STR). In HT22 cells, STR-induced C3 activation-induced rapid EAAT2 protein degradation. The mutation of asparagine 504 to aspartate (D504N), which should inactivate the putative C3 cleavage site, increased EAAT2 activity in A172 cells. In contrast, the D504N mutation did not protect EAAT2 protein against STR-induced degradation in HT22 cells, whereas inhibition of caspases, ubiquitination and the proteasome did. Similar results were obtained in astrocytes. Phylogenetic analysis showed that C-terminal ubiquitin acceptor sites-but not the putative C3 cleavage site-exhibit a high degree of conservation. Moreover, C-terminal truncation mimicking C3 cleavage increased rather than decreased EAAT2 activity and stability as well as protected EAAT2 against STR-induced ubiquitination-dependent degradation. We conclude that cellular stress associated with endogenous C3 activation degrades EAAT2 via a pathway involving ubiquitination and the proteasome but not direct C3-mediated cleavage. In addition, C3 cleavage of EAAT2, described to occur in other models, is unlikely to inactivate EAAT2. However, mutation of the highly conserved D504 within the putative C3 cleavage site increases EAAT2 activity via an unknown mechanism.

HAP40 protein levels are huntingtin-dependent and decrease in Huntington disease.

The huntingtin-associated protein 40 (HAP40) is an abundant interactor of huntingtin (HTT). In complexes of these proteins, HAP40 tightly binds to HTT in a cleft formed by two larger domains rich in HEAT repeats, and a smaller bridge domain connecting the two. We show that HAP40 steady-state protein levels are directly dependent on HTT (both normal and mutant HTT) and that HAP40 is strongly stabilized by the interaction with HTT resulting in an at least 5-fold increase in HAP40's half-life when bound to HTT. Cellular HAP40 protein levels were reduced in primary fibroblasts and lymphoblasts of Huntington Disease (HD) patients and in brain tissue of a full-length HTT mouse model of HD, concomitant with decreased soluble HTT levels in these cell types. This data and our previous demonstration of coevolution between HTT and HAP40 and evolutionary conservation of their interaction suggest that HAP40 is an obligate interaction partner of HTT. Our observation of reduced HAP40 levels in HD invites further studies, whether HAP40 loss-of-function contributes to the pathophysiology of HD.

Different CSF protein profiles in amyotrophic lateral sclerosis and frontotemporal dementia with C9orf72 hexanucleotide repeat expansion.

OBJECTIVES: The hexanucleotide repeat expansion in the C9orf72 gene is the most common mutation associated with amyotrophic lateral sclerosis (C9-ALS) and frontotemporal dementia (C9-FTD). Until now, it is unknown which factors define whether C9orf72 mutation carriers develop ALS or FTD. Our aim was to identify protein biomarker candidates in the cerebrospinal fluid (CSF) which differentiate between C9-ALS and C9-FTD and might be indicative for the outcome of the mutation. METHODS: We compared the CSF proteome of 16 C9-ALS and 8 C9-FTD patients and 11 asymptomatic C9orf72 mutation carriers (CAR) by isobaric tags for relative and absolute quantitation. Eleven biomarker candidates were selected from the pool of differentially regulated proteins for further validation by multiple reaction monitoring and single-molecule array in a larger cohort (n=156). RESULTS: In total, 2095 CSF proteins were identified and 236 proteins were significantly different in C9-ALS versus C9-FTD including neurofilament medium polypeptide (NEFM) and chitotriosidase-1 (CHIT1). Eight candidates were successfully validated including significantly increased ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) levels in C9-ALS compared with C9-FTD and controls and decreased neuronal pentraxin receptor (NPTXR) levels in C9-FTD versus CAR. CONCLUSIONS: This study presents a deep proteomic CSF analysis of C9-ALS versus C9-FTD patients. As a proof of concept, we observed higher NEFM and CHIT1 CSF levels in C9-ALS. In addition, we also show clear upregulation of UCHL1 in C9-ALS and downregulation of NPTXR in C9-FTD. Significant differences in UCHL1 CSF levels may explain diverging ubiquitination and autophagy processes and NPTXR levels might reflect different synapses organisation processes.

Meaningful and Measurable Health Domains in Huntington's Disease: Large-Scale Validation of the Huntington's Disease Health-Related Quality of Life Questionnaire Across Severity Stages.

BACKGROUND: Although health-related quality of life is key for patients with long-term neurodegenerative conditions, measuring this is less straightforward and complex in Huntington's disease (HD). OBJECTIVES: To refine and validate a fully patient-derived instrument, the Huntington's Disease health-related Quality of Life questionnaire (HDQoL), and to elucidate health domains that are meaningful to patients' lived experience. METHODS: Five-hundred forty-one participants, from premanifest to end-stage disease, completed the HDQoL, together with generic quality-of-life measures and in-person motor, cognitive, and behavioral assessments. The psychometric properties of the HDQoL were examined using factor analysis and Rasch analysis. RESULTS: Four HDQoL domains emerged, reflecting the classical triad of HD features; they were Physical-Functional, Cognitive, and 2 different behavioral aspects, that is, the Mood-Self domain and a distinct Worries domain. These domains clarify the behavioral sequelae as experienced by patients, and all showed good to excellent internal consistency. Known-groups analyses illustrated significant and graded changes in clinical assessments and corresponding HDQoL domains across disease severity levels. Convergent and discriminant validity was demonstrated by the expected pattern of correlations between specific HDQoL domains and corresponding domain-relevant clinical assessments as well as patient-reported measures. The data demonstrate robust support for the refined HDQoL across disease stages. CONCLUSIONS: The HDQoL, with its 2 distinct behavioral domains of Mood-Self and Worries as well as the Physical-Functional and Cognitive domains, is a relevant, reliable, and valid patient-derived instrument to measure the impact of HD across all severity stages.

Body weight is a robust predictor of clinical progression in Huntington disease.

Unintended weight loss is a hallmark of Huntington disease (HD), but it is unknown to what extent weight loss impacts the rate of disease progression. Therefore, using longitudinal data from the Enroll-HD study, we assessed the association between baseline body mass index (BMI) and the rate of clinical progression in 5,821 HD mutation carriers. We found that high baseline BMI was associated with a significantly slower rate of functional, motor, and cognitive deterioration (all p < 0.001), independent of mutant HTT CAG repeat size. Our findings provide strong rationale for exploration of systemic metabolism as a therapeutic target in HD. Ann Neurol 2017;82:479-483.

Chitotriosidase (CHIT1) is increased in microglia and macrophages in spinal cord of amyotrophic lateral sclerosis and cerebrospinal fluid levels correlate with disease severity and progression.

OBJECTIVES: Neurochemical markers of amyotrophic lateral sclerosis (ALS) that reflect underlying disease mechanisms might help in diagnosis, staging and prediction of outcome. We aimed at determining the origin and differential diagnostic and prognostic potential of the putative marker of microglial activation chitotriosidase (CHIT1). METHODS: Altogether 316 patients were included, comprising patients with sporadic ALS, ALS mimics (disease controls (DCo)), frontotemporal lobar degeneration (FTLD), Creutzfeldt-Jakob disease (CJD), Alzheimer's disease (AD), Parkinson's disease (PD) and healthy controls (Con). CHIT1 and neurofilament levels were determined in cerebrospinal fluid (CSF) and blood and analysed with regard to diagnostic sensitivity and specificity and prognostic performance. Additionally, postmortem tissue was analysed for CHIT1 expression. RESULTS: In ALS, CHIT1 CSF levels were higher compared with Con (p<0.0001), DCo (p<0.05) and neurodegenerative diseases (AD p<0.05, PD p<0.01, FTLD p<0.0001) except CJD. CHIT1 concentrations were correlated with ALS disease progression and severity but not with the survival time, as did neurofilaments. Serum CHIT1 levels were not different in ALS compared with any other study group. In the spinal cord of patients with ALS, but not Con, AD or CJD cases, CHIT1 was expressed in the corticospinal tract and CHIT1 staining colocalised with markers of microglia (IBA1) and macrophages (CD68). CONCLUSIONS: CHIT1 concentrations in the CSF of patients with ALS may reflect the extent of microglia/macrophage activation in the white matter of the spinal cord. CHIT1 could be a potentially useful marker for differential diagnosis and prediction of disease progression in ALS and, therefore, seems suitable as a supplemental marker for patient stratification in therapeutic trials.

Identification of extreme motor phenotypes in Huntington's disease.

The manifestation of motor signs in Huntington's disease (HD) has a well-known inverse relationship with HTT CAG repeat length, but the prediction is far from perfect. The probability of finding disease modifiers is enhanced in individuals with extreme HD phenotypes. We aimed to identify extreme HD motor phenotypes conditional on CAG and age, such as patients with very early or very late onset of motor manifestation. Retrospective data were available from 1,218 healthy controls and 9,743 HD participants with CAG repeats ≥40, and a total of about 30,000 visits. Boundaries (2.5% and 97.5% quantiles) for extreme motor phenotypes (UHDRS total motor score (TMS) and motor age-at-onset) were estimated using quantile regression for longitudinal data. More than 15% of HD participants had an extreme TMS phenotype for at least one visit. In contrast, only about 4% of participants were consistent TMS extremes at two or more visits. Data from healthy controls revealed an upper cut-off of 13 for the TMS representing the extreme of motor ratings for a normal aging population. In HD, boundaries of motor age-at-onset based on diagnostic confidence or derived from the TMS data cut-off in controls were similar. In summary, a UHDRS TMS of more than 13 in an individual carrying the HD mutation indicates a high likelihood of motor manifestations of HD irrespective of CAG repeat length or age. The identification of motor phenotype extremes can be useful in the search for disease modifiers, for example, genetic or environmental such as medication. © 2016 Wiley Periodicals, Inc.

An exploratory double-blind, randomized clinical trial with selisistat, a SirT1 inhibitor, in patients with Huntington's disease.

AIMS: Selisistat, a selective SirT1 inhibitor is being developed as a potentially disease-modifying therapeutic for Huntington's disease (HD). This was the first study of selisistat in HD patients and was primarily aimed at development of pharmacodynamic biomarkers. METHODS: This was a randomized, double-blind, placebo-controlled, multicentre exploratory study. Fifty-five male and female patients in early stage HD were randomized to receive 10 mg or 100 mg of selisistat or placebo once daily for 14 days. Blood sampling, clinical and safety assessments were conducted throughout the study. Candidate pharmacodynamic markers included circulating soluble huntingtin and innate immune markers. RESULTS: Selisistat was found to be safe and well tolerated, and systemic exposure parameters showed that the average steady-state plasma concentration achieved at the 10 mg dose level (125 nm) was comparable with the IC50 for SirT1 inhibition. No adverse effects on motor, cognitive or functional readouts were recorded. While circulating levels of soluble huntingtin were not affected by selisistat in this study, the biological samples collected have allowed development of assay technology for use in future studies. No effects on innate immune markers were seen. CONCLUSIONS: Selisistat was found to be safe and well tolerated in early stage HD patients at plasma concentrations within the anticipated therapeutic concentration range.

A greatly extended PPARGC1A genomic locus encodes several new brain-specific isoforms and influences Huntington disease age of onset.

PGC-1α has been implicated in the pathogenesis of neurodegenerative disorders. Several single-nucleotide polymorphisms (SNPs) located in two separate haplotype blocks of PPARGC1A have shown associations with Huntington's disease (HD) and Parkinson's disease, but causative SNPs have not been identified. One SNP (rs7665116) was located in a highly conserved 233 bp region of intron 2. To determine whether rs7665116 is located in an alternative exon, we performed 5'-RLM-RACE from exon 3 and discovered multiple new transcripts that initiated from a common novel promoter located 587 kb upstream of exon 2, but did not contain the conserved region harboring rs7665116. Using real-time polymerase chain reaction, RNase protection assays and northern blotting, we show that the majority of these transcripts are brain specific and are at least equally or perhaps more abundant than the reference sequence PPARGC1A transcripts in whole brain. Two main transcripts containing independent methionine start codons encode full-length brain-specific PGC-1α proteins that differ only at their N-termini (NTs) from PGC-1α, encoded by the reference sequence. Additional truncated isoforms containing these NTs that are similar to NT-PGC-1α exist. Other transcripts may encode potential dominant negative forms, as they are predicted to lack the second LXXLL motif that serves as an interaction site for several nuclear receptors. Furthermore, we show that the new promoter is active in neuronal cell lines and describe haplotypes encompassing this region that are associated with HD age of onset. The discovery of such a large PPARGC1A genomic locus and multiple isoforms in brain warrants further functional studies and may provide new tissue-specific targets for treating neurodegenerative diseases.

Neuropsychiatric symptoms in a European Huntington's disease cohort (REGISTRY).

BACKGROUND: The majority of Huntington's disease (HD) mutation carriers experience some psychopathology during their lifetime, varying from irritability to psychosis, but prevalences of particular symptoms vary widely due to diverse study populations in different stages of HD and the use of different assessment methods. METHODS: The study population consisted of 1993 HD mutation carriers from 15 European countries, all participating in the observational REGISTRY study. The behavioural section of the Unified HD Rating Scale was used to examine the prevalence and correlates of five neuropsychiatric features: depression, irritability/aggression, obsessive/compulsive behaviours, apathy and psychosis. RESULTS: Twenty-seven per cent of the participants did not have any neuropsychiatric symptom in the last month. Moderate to severe apathy occurred in 28.1% of the participants, whereas moderate to severe depression was found in 12.7%. Irritable/aggressive symptoms were present in 13.9% of the participants, and 13.2% showed obsessive/compulsive behaviours. Moderate to severe psychotic symptoms were found in only 1.2%. Only 54.9% of all participants with moderate to severe depression used antidepressants, suggesting undertreatment of depression. Obsessive/compulsive behaviours and irritability/aggression were inversely correlated with the Total Functional Capacity score, but with apathy showing the strongest inverse association. CONCLUSIONS: A variety of neuropsychiatric symptoms are highly prevalent in different stages of HD in this European HD population, with apathy as the most frequent symptom. Depression, irritability/aggression and OCBs are prevalent in all stages of HD. Apathy was the key neuropsychiatric symptom occurring most often in advanced HD stages. Due to possible selection of relatively healthy participants, prevalences reported in this study might be an underestimation of prevalence in the entire HD population.

A single nucleotide polymorphism in the coding region of PGC-1α is a male-specific modifier of Huntington disease age-at-onset in a large European cohort.

BACKGROUND: Genetic modifiers are important clues for the identification of therapeutic targets in neurodegenerative diseases. Huntington disease (HD) is one of the most common autosomal dominant inherited neurodegenerative diseases. The clinical symptoms include motor abnormalities, cognitive decline and behavioral disturbances. Symptom onset is typically between 40 and 50 years of age, but can vary by several decades in extreme cases and this is in part determined by modifying genetic factors. The metabolic master regulator PGC-1α, coded by the PPARGC1A gene, coordinates cellular respiration and was shown to play a role in neurodegenerative diseases, including HD. METHODS: Using a candidate gene approach we analyzed a large European cohort (n = 1706) from the REGISTRY study for associations between PPARGC1A genotype and age at onset (AO) in HD. RESULTS: We report that a coding variant (rs3736265) in PPARGC1A is associated with an earlier motor AO in men but not women carrying the HD mutation. CONCLUSIONS: These results further strengthen the evidence for a role of PGC-1α in HD and unexpectedly suggest a gender effect.