Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany.
Centre for Rare Diseases, Medical Faculty, University of Tübingen, Tübingen, Germany.
Mol Neurobiol. 2023 Jun;60(6):3553-3567. doi: 10.1007/s12035-023-03294-y. Epub 2023 Mar 9.
Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominantly inherited neurodegenerative disease, which belongs to the trinucleotide repeat disease group with a CAG repeat expansion in exon 1 of the ATXN2 gene resulting in an ataxin-2 protein with an expanded polyglutamine (polyQ)-stretch. The disease is late manifesting leading to early 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-lowering therapeutic intervention strategies. The aim of this study was to establish a sensitive technique to measure the amount of soluble polyQ-expanded ataxin-2 in human biofluids to evaluate ataxin-2 protein levels as prognostic and/or therapeutic biomarker in SCA2. 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 to evaluate the best assay conditions. We established a TR-FRET-based immunoassay for soluble polyQ-expanded ataxin-2 and validated measurements in human cell lines including iPSC-derived cortical neurons. Additionally, our immunoassay was sensitive enough to monitor small ataxin-2 expression changes by siRNA or starvation treatment. We successfully established the first sensitive ataxin-2 immunoassay to measure specifically soluble polyQ-expanded ataxin-2 in human biomaterials.
脊髓小脑性共济失调 2 型（SCA2）是一种常染色体显性遗传的神经退行性疾病，属于三核苷酸重复疾病组，其 ATXN2 基因外显子 1 中的 CAG 重复扩展导致具有扩展多聚谷氨酰胺（polyQ）-延伸的 ataxin-2 蛋白。该疾病表现较晚，导致早逝。目前，尚无治愈该疾病甚至减缓疾病进展的治疗干预措施。此外，疾病进展和治疗干预研究的主要读出参数有限。因此，迫切需要可量化的分子生物标志物，如由于许多潜在的蛋白质降低治疗干预策略，ataxin-2 变得更加重要。本研究的目的是建立一种灵敏的技术来测量人生物体液中可溶性 polyQ 扩展 ataxin-2 的量，以评估 ataxin-2 蛋白水平作为 SCA2 的预后和/或治疗生物标志物。时间分辨荧光能量转移（TR-FRET）用于建立 polyQ 扩展 ataxin-2 特异性免疫测定法。两种不同的 ataxin-2 抗体和两种不同的 polyQ 结合抗体在三种不同浓度下进行验证，并在细胞和动物组织以及人细胞系中进行测试，比较不同的缓冲条件以评估最佳测定条件。我们建立了一种基于 TR-FRET 的可溶性 polyQ 扩展 ataxin-2 免疫测定法，并在包括 iPSC 衍生的皮质神经元在内的人细胞系中验证了测量结果。此外，我们的免疫测定法足够灵敏，可以通过 siRNA 或饥饿处理监测小的 ataxin-2 表达变化。我们成功建立了第一个灵敏的 ataxin-2 免疫测定法，可特异性测量人生物材料中的可溶性 polyQ 扩展 ataxin-2。
