Mutant huntingtin fragmentation in immune cells tracks Huntington’s disease progression

Weiss, Andreas; Träger, Ulrike; Wild, Edward J.; Grueninger, Stephan; Farmer, Ruth; Landles, Christian; Scahill, Rachael I.; Lahiri, Nayanjot; Haider, Salman; Macdonald, Douglas; Frost, Chris; Bates, Gillian P.; Bilbe, Graeme; Kühn, Rainer; André, Ralph; Tabrizi, Sarah J.

doi:10.1172/jci64565

Cited by 125 publications

(128 citation statements)

References 24 publications

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“…The expanded subjects (table 2) had a median CAGn of 42 (range 37-48), which is typical of the premanifest and manifest adult subjects who participate in clinical research. There were no subjects with extreme CAG lengths (CAG .48) that might have skewed the results 25 and indeed there was no correlation between CAGn and the assay results in expanded subjects in the PHAROS sample set (data not shown). Previous results in mice and humans, which included subjects with much higher repeat lengths than 48, have demonstrated increased mtHtt signals using HTRF assays with increased CAG length.…”

Section: Results Demographic and Experimental Characteristicsmentioning

confidence: 98%

“…28,29 When normalized to protein concentrations, mtHtt levels tended to remain stable through the HD prodrome and increase after phenoconversion suggesting impaired proteolysis of Htt. We speculate that mtHtt turnover may be slowed in leukocytes or more specifically in monocytes 25 perhaps because of impaired proteolysis or autophagy, leading to accumulation of soluble holoprotein and fragments. 25,29 Although this is a cross-sectional study, these data suggest that the Htt HTRF assay could possibly mark progression in prodromal HD or phenoconversion, depending on the normalization used.…”

Section: Results Demographic and Experimental Characteristicsmentioning

confidence: 99%

“…We speculate that mtHtt turnover may be slowed in leukocytes or more specifically in monocytes 25 perhaps because of impaired proteolysis or autophagy, leading to accumulation of soluble holoprotein and fragments. 25,29 Although this is a cross-sectional study, these data suggest that the Htt HTRF assay could possibly mark progression in prodromal HD or phenoconversion, depending on the normalization used. Prospective longitudinal studies in premanifest and manifest subjects will be necessary to examine these possibilities.…”

Section: Results Demographic and Experimental Characteristicsmentioning

confidence: 99%

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HTRF analysis of soluble huntingtin in PHAROS PBMCs

et al. 2013

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Objective: We measured the levels of mutant huntingtin (mtHtt) and total huntingtin (tHtt) in blood leukocytes from Prospective Huntington At-Risk Observational Study (PHAROS) subjects at 50% risk of carrying the Huntington disease mutation using a homogeneous time-resolved fluorescence (HTRF) assay to assess its potential as a biomarker.Methods: Peripheral blood mononuclear cells from consenting PHAROS subjects were analyzed by HTRF using antibodies that simultaneously measured mtHtt and tHtt. mtHtt levels were normalized to tHtt, double-stranded DNA, or protein and analyzed according to cytosine-adenineguanine repeat length (CAGn), demographics, predicted time to clinical onset or known time since clinical onset, and available clinical measures.Results: From 363 assayed samples, 342 met quality control standards. Levels of mtHtt and mt/tHtt were higher in 114 subjects with expanded CAG repeats (CAG $37) compared with 228 subjects with nonexpanded CAG repeats (CAG ,37) (p , 0.0001). Analysis of relationships to predicted time to onset or to phenoconversion suggested that the HTRF signal could mark changes during the Huntington disease prodrome or after clinical onset. Conclusions:The HTRF assay can effectively measure mtHtt in multicenter sample sets and may be useful in trials of therapies targeting huntingtin. Huntington disease (HD) is caused by the expression of the toxic mutant huntingtin (mtHtt) protein, which contains an expanded polyglutamine repeat sequence near its N-terminus.1 mtHtt misfolds, undergoes posttranslational modifications, fragments, and forms soluble oligomers and insoluble intracellular aggregates, 2-4 which are differentially toxic. 5,6 Huntingtin (Htt) is the most salient target for neuroprotective therapies 7-9 and it is both essential and challenging to reliably measure it 1,2,10 to enable the development of therapies. We adapted a semiquantitative cell-based immunoassay that measures soluble mtHtt and total Htt (tHtt) using homogeneous time-resolved fluorescence (HTRF) Förster resonance energy transfer. 11,12 This HTRF assay is sensitive, reliable, and specific for soluble mtHtt in tissues and blood from HD mouse models, 11 in postmortem tissue, and in single-site studies using human peripheral blood mononuclear cells (PBMCs) from subjects with premanifest and manifest HD. [11][12][13] We optimized and technically validated the HTRF assay according to Good Laboratory Practice (GLP) standards for analyzing mtHtt and tHtt in clinical PBMC samples.12 To validate the HTRF assay in the context of a blinded multicenter study encompassing subjects with and without the HD mutation, to assess normalization methods for Htt values, and to examine whether the HD prodrome or the development of clinical

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Section: Results Demographic and Experimental Characteristicsmentioning

confidence: 98%

Section: Results Demographic and Experimental Characteristicsmentioning

confidence: 99%

Section: Results Demographic and Experimental Characteristicsmentioning

confidence: 99%

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HTRF analysis of soluble huntingtin in PHAROS PBMCs

et al. 2013

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“…mHTT is a large, aggregation-prone protein expressed mostly intracellularly, and since HD generally progresses slowly, mHTT would be expected to be released very gradually into the CSF from dying neurons. Consequently its CSF concentration is certainly extremely low and several generations of improvement in antibodies and mHTT-quantifying assays failed to yield a method sensitive enough to quantify it [19].…”

Section: Mutant Huntingtinmentioning

confidence: 99%

“…TR-FRET detection of mHTT in blood-derived leukocytes might, for instance, be used to assess target engagement by a peripherally-administered huntingtin-lowering compound [19] that was known to be brain-penetrant.…”

Section: Introductionmentioning

confidence: 99%

Cerebrospinal Fluid Biomarkers for Huntington’s Disease

Byrne¹,

Wild

2016

JHD

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Abstract. Cerebrospinal fluid (CSF) is enriched in brain-derived components and represents an accessible and appealing means of interrogating the CNS milieu to study neurodegenerative diseases and identify biomarkers to facilitate the development of novel therapeutics. Many such CSF biomarkers have been proposed for Huntington's disease (HD) but none has been validated for clinical trial use. Across many studies proposing dozens of biomarker candidates, there is a notable lack of statistical power, consistency, rigor and validation. Here we review proposed CSF biomarkers including neurotransmitters, transglutaminase activity, kynurenine pathway metabolites, oxidative stress markers, inflammatory markers, neuroendocrine markers, protein markers of neuronal death, proteomic approaches and mutant huntingtin protein itself. We reflect on the need for large-scale, standardized CSF collections with detailed phenotypic data to validate and qualify much-needed CSF biomarkers for clinical trial use in HD.

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