Development of a highly sensitive immunoassay to measure ataxin2 as a target engagement marker in Spinocerebellar Ataxia Type 2

Abstract Background Spinocerebellar Ataxia Type 2 (SCA2) belongs to a group of neurodegenerative diseases, inherited as an autosomal dominant trait. SCA2 is a trinucleotide repeat disease with a CAG repeat expansion in exon 1 of the ATXN2gene resulting in an ataxin-2 protein with an expanded polyglutamine (polyQ)-stretch. The disease is late manifesting leading to premature death. Today, therapeutic interventions to cure the disease or even to decelerate disease progression are not available yet. Furthermore, primary readout parameter for disease progression and therapeutic intervention studies are limited. Thus, there is an urgent need for quantifiable molecular biomarkers such as ataxin-2 becoming even more important due to numerous potential protein reducing therapeutic intervention strategies. Objective Aim of this study was to establish a sensitive technique to measure the amount of polyQ-expanded ataxin-2 in human biofluids to evaluate ataxin-2 protein levels as prognostic and/ -or therapeutic biomarker in SCA2. Methods Time-Resolved Fluorescence Energy Transfer (TR-FRET) was used to establish a polyQ-expanded ataxin-2-specific immunoassay. Two different ataxin-2 antibodies and two different polyQ-binding antibodies were validated in three different concentrations and tested in cellular and animal tissue as well as in human cell lines, comparing different buffer conditions as well as total protein concentrations to evaluate the best assay conditions. Results We established a TR-FRET-based immunoassay for polyQ-expanded ataxin-2 and validated measurements in human cell lines including primary skin fibroblasts, induced pluripotent stem cells (iPSCs) and iPSC-derived cortical neurons. Additionally, our immunoassay was sensitive enough to monitor small ataxin-2 expression changes by siRNA or starvation treatment. Conclusion We successfully established the first sensitive ataxin-2 immunoassay to measure specifically polyQ-expanded ataxin-2 in human biomaterials.