HTRF analysis of soluble huntingtin in PHAROS PBMCs

Abstract

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

Suppression of MAPK11 or HIPK3 reduces mutant Huntingtin levels in Huntington's disease models

“…As a result, the HTRF signals are proportional to the target protein concentration and can be used to quantify its level 20 . This technology has been successfully applied to the measurement of HTT levels in previous studies 21 , 22 .…”

State biomarkers for Machado Joseph disease: Validation, feasibility and responsiveness to change

“…, 2013), but properties of soluble ataxin-3 as a disease biomarker were not addressed up to date. Soluble mutant protein levels have been measured in other neurodegenerative disorders, such as in Huntington disease (HD), and were associated to clinical features (Moscovitch-Lopatin et al , 2013). Soluble huntingtin is currently being evaluated as an outcome in recent HD clinical trials (Huntington Study Group Reach2HD Investigators, 2015; Süssmuth et al , 2015).…”

What, When and How to Measure—Peripheral Biomarkers in Therapy of Huntington’s Disease

“…Single molecule counting (SMC) immunoassay [ 61 , 62 ] as well as microbead-based immunoprecipitation and flow cytometry (IP-FCM) demonstrated high specificity for mHTT in CSF [ 63 ]. The mHTT level was also determined in the blood [ 64 , 65 , 66 , 67 ] and saliva [ 68 , 69 ]. These analyses unambiguously distinguish between HD patients and healthy individuals.…”

“…Determining the level of the blood-soluble form of mHTT derived from the CNS is relatively difficult diagnostically due to the fact that its concentration is very low and constantly produced peripherally [ 70 ]. For this purpose, a highly sensitive time-resolved Förster resonance energy transfer (TR-FRET) [ 66 ] and homogeneous time-resolved Förster resonance energy transfer (HTRF) are used [ 64 , 65 ]. These assays are performed in peripheral whole blood, isolated erythrocytes or buffy coats containing majority of leukocytes and platelets after centrifugation.…”