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Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients.

ABSTRACT: In Huntington's disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHTT seeding species in HD patient biosamples. We showed that the seeding-competent mHTT species in HD CSF are significantly elevated upon disease onset and with the progression of neuropathological grades. Mechanistically, we showed that mHTT seeding activities in patient CSF could be ameliorated by the overexpression of chaperone DNAJB6 and by antibodies against the polyproline domain of mHTT. Together, our study developed a selective and scalable cell-based tool to investigate mHTT seeding activities in HD CSF, and demonstrated that the CSF mHTT seeding species are significantly associated with certain disease states. This seeding activity can be ameliorated by targeting specific domain or proteostatic pathway of mHTT, providing novel insights into such pathological activities.

SUBMITTER: Lee CYD

PROVIDER: S-EPMC7679413 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature

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Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients.

Lee C Y Daniel CYD Wang Nan N Shen Koning K Stricos Matthew M Langfelder Peter P Cheon Kristina H KH Cortés Etty P EP Vinters Harry V HV Vonsattel Jean Paul JP Wexler Nancy S NS Damoiseaux Robert R Frydman Judith J Yang X William XW

Scientific reports 20201120 1

In Huntington's disease (HD), the mutant Huntingtin (mHTT) is postulated to mediate template-based aggregation that can propagate across cells. It has been difficult to quantitatively detect such pathological seeding activities in patient biosamples, e.g. cerebrospinal fluids (CSF), and study their correlation with the disease manifestation. Here we developed a cell line expressing a domain-engineered mHTT-exon 1 reporter, which showed remarkably high sensitivity and specificity in detecting mHT ...[more]

PMID: 33219289

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Project description:BackgroundQuantification 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.MethodsWe 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.ResultsThis 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.ConclusionsWe 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.Trial registrationNot applicable.FundingCHDI Foundation Inc.; Medical Research Council (MRC) UK; National Institutes for Health Research (NIHR); Rosetrees Trust; Swedish Research Council; and Knut and Alice Wallenberg Foundation.

Project description:Neoplastic invasion into leptomeninges and subarachnoid space, resulting in neoplastic meningitis (NM) is a fatal complication of advanced solid and hematological neoplasms. Identification of malignant involvement of the cerebrospinal fluid (CSF) early in the disease course has crucial prognostic and therapeutic implications, but remains challenging. As indicators of extracellular matrix (ECM) degradation and breakdown of the blood-brain-barrier, Matrix Metalloproteases (MMPs) and A Disintegrin and Metalloproteases (ADAMs) are potential analytes for cerebral pathophysiology and metastatic dissemination of tumor cells into the CSF.We compared protease activities in CSF samples from patients with NM and control individuals using FRET-based metalloprotease substrates with distinct enzyme selectivity profiles in a real-time, multiplex approach termed "proteolytic activity matrix assay" (PrAMA). Protease activity dynamics can be tracked by fluorescence changes over time. By simultaneously monitoring a panel of 5 FRET-substrate cleavages, a proteolytic signature can be identified and analyzed to infer the activities of multiple specific proteases. Distinct patterns of substrate cleavage comparing disease vs. control samples allow rapid, reproducible and sensitive discrimination even in small volumes of CSF.Individual substrate cleavage rates were linked to distinct proteases, and PrAMA computational inference implied increased activities of MMP-9, ADAM8 and ADAM17 (4-5-fold on average) in CSF samples from NM patients that were inhibitable by the metalloprotease inhibitor batimastat (BB-94). The activities of these proteases correlated with blood-brain barrier impairment. Notably, CSF cell counts were not found to directly reflect the protease activities observed in CSF samples from NM patients; this may explain the frequent clinical observation of negative cytology in NM patients.PrAMA analysis of CSF samples is a potential diagnostic method for sensitive detection of NM and may be suitable for the clinical routine.

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Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients.

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Disease-related Huntingtin seeding activities in cerebrospinal fluids of Huntington's disease patients.

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