Quantification of mutant huntingtin protein in cerebrospinal fluid from Huntington’s disease patients

Abstract

BACKGROUND:Quantification of disease-associated proteins in the cerebrospinal fluid (CSF) has been critical for the study and treatment of several neurodegenerative disorders; however, mutant huntingtin protein (mHTT), the cause of Huntington's disease (HD), is at very low levels in CSF and, to our knowledge, has never been measured previously. METHODS:We developed an ultrasensitive single-molecule counting (SMC) mHTT immunoassay that was used to quantify mHTT levels in CSF samples from individuals bearing the HD mutation and from control individuals in 2 independent cohorts. RESULTS:This SMC mHTT immunoassay demonstrated high specificity for mHTT, high sensitivity with a femtomolar detection threshold, and a broad dynamic range. Analysis of the CSF samples showed that mHTT was undetectable in CSF from all controls but quantifiable in nearly all mutation carriers. The mHTT concentration in CSF was approximately 3-fold higher in patients with manifest HD than in premanifest mutation carriers. Moreover, mHTT levels increased as the disease progressed and were associated with 5-year onset probability. The mHTT concentration independently predicted cognitive and motor dysfunction. Furthermore, the level of mHTT was associated with the concentrations of tau and neurofilament light chain in the CSF, suggesting a neuronal origin for the detected mHTT. CONCLUSIONS:We have demonstrated that mHTT can be quantified in CSF from HD patients using the described SMC mHTT immunoassay. Moreover, the level of mHTT detected is associated with proximity to disease onset and diminished cognitive and motor function. The ability to quantify CSF mHTT will facilitate the study of HD, and mHTT quantification could potentially serve as a biomarker for the development and testing of experimental mHTT-lowering therapies for HD.

Neurofilament light protein in blood as a potential biomarker of neurodegeneration in Huntington's disease: a retrospective cohort analysis

“…We were able to analyse in detail the associations with NfL from before disease onset and during manifest disease supported by rigorous clinical, cognitive, and neuroimaging data. Additionally, we were able to replicate the principal plasma findings in an independent cohort and confirm that concentrations in CSF are increased in individuals with HTT mutations, as has been previously reported 8, 9, 15, 16, 17. We further showed that NfL concentrations in plasma and CSF are closely correlated, which affirms the likely CNS origin of the NfL detected in both biofluids and suggests that NfL in blood could be a reliable estimator of concentrations in CSF.…”

“…Many potential markers in CSF have been proposed, but only a few (eg, mHTT itself, microtubule-associated protein tau, and chitinase-3-like protein 1) have shown associations with clinical phenotype beyond established predictors, such as age and HTT CAG triplet repeat count 7, 8, 9, 10. None of the potential biomarkers has been studied longitudinally 7 .…”

Polyglutamine expansion affects huntingtin conformation in multiple Huntington’s disease models

“…Various immunoassays, ranging from sandwich-type immunoassays 22–24 to time-resolved Forster resonance energy transfer (TR-FRET) based assays 25, 26 can inform on HTT levels in different sample types. However, studying HTT conformation remains a challenge and is predominantly addressed using biophysical approaches.…”

Cerebrospinal Fluid Biomarkers for Huntington’s Disease

“…Successful quantification of soluble CSF mutant huntingtin was only achieved in early 2015 [83]. We used a single molecule counting (SMC) immunoassay, and a combination of HTT N-terminal-detecting 2B7 antibody and polyglutamine-binding MW1 antibody, to quantify mHTT for the first time in CSF at femtomolar sensitivity (Fig.…”

“…mHTT correlated with (C) disease burden score and (D) Unified Huntington's Disease Rating Scale motor score. Adapted from Wild et al [83] with the author's and publisher's permission. mice expressing human mHTT, and that the signal in CSF was correlated significantly with brain mHTT level after intracerebroventricular administration of an HTT-lowering antisense oligonucleotide.…”