Abstract
BackgroundHuntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. Since non-invasive methods to quantify HTT in the CNS do not exist, measuring amount of soluble HTT in peripheral cells represents an important step in development of disease-modifying interventions in HD.ResultsAn ELISA assay using commercially available antibodies was developed to quantify HTT levels in complex matrices like mammalian cell cultures lysates and human samples. The immunoassay was optimized using a recombinant full-length HTT protein, and validated both on wild-type and mutant HTT species. The ability of the assay to detect significant variations of soluble HTT levels was evaluated using an HSP90 inhibitor that is known to enhance HTT degradation. Once optimized, the bioassay was applied to peripheral blood mononuclear cells (PBMCs) from HD patients, demonstrating good potential in tracking the disease course.ConclusionsThe method described here represents a validated, simple and rapid bio-molecular assay to evaluate soluble HTT levels in blood cells as useful tool in disease and pharmacodynamic marker identification for observational and clinical trials.
Highlights
Huntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT)
HD is caused by an unstable CAG repeat expansion in the gene encoding huntingtin (HTT) on chromosome 4, leading to an extended polyglutamine stretch in the amino terminus of the HTT protein [3]; the disease is associated with a mutant form of the HTT protein that contains 36 or more glutamine residues
The analysis of human samples indicates that levels of soluble HTT in peripheral blood mononuclear cells (PBMCs) cells was quantifiable using our assay without any need of enrichment and that it was possible to detect different levels of the protein in healthy controls compared to HD patients
Summary
Huntington’s disease (HD) is a monogenic disorder caused by an aberrant expansion of CAG repeats in the huntingtin gene (HTT). Pathogenesis is associated with expression of the mutant (mHTT) protein in the CNS, with its levels most likely related to disease progression and symptom severity. HD is caused by an unstable CAG repeat expansion in the gene encoding huntingtin (HTT) on chromosome 4, leading to an extended polyglutamine (polyQ) stretch in the amino terminus of the HTT protein [3]; the disease is associated with a mutant form of the HTT protein that contains 36 or more glutamine residues. Whilst mHTT expression in the CNS [25] is the primary pathological hallmark in HD development [26], the presence of abnormalities in several other compartments [27] provide a source of accessible tissue for HTT quantification potentially to monitor disease progression and treatment efficacy. We report the development of a robust and simple ELISA assay that is sensitive enough to detect differences in endogenous HTT levels in blood from HD patients at different stages of disease, highlighting its potential suitability for monitoring both disease progression and therapeutic intervention in clinical trials
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