Röttgering Bas, Testerink Janwillem, Weij Rudie, Beekman Chantal, Datson Nicole
VICO Therapeutics B.V., J.H. Oortweg 21, 2333 CH, Leiden, The Netherlands.
Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.
Mol Neurobiol. 2025 May 31. doi: 10.1007/s12035-025-05089-9.
Polyglutamine (polyQ) diseases are monogenic fatal neurodegenerative disorders caused by a CAG repeat expansion that is translated into a toxic polyQ tract. There are nine polyQ diseases: Huntington's disease (HD), spinocerebellar ataxias 1, 2, 3, 6, 7 and 17 (SCA1, 2, 3, 6, 7, 17), dentatorubral-pallidoluysian atrophy (DRPLA) and spinal and bulbar muscular atrophy (SBMA). Although no disease-modifying therapies are available, lowering levels of the causative mutant polyQ protein is a promising potential treatment. Preclinically, the efficacy of polyQ protein-lowering compounds is often assessed using time-consuming Western blots (WB), which can produce variable results. Therefore, to improve throughput and accuracy of polyQ protein level quantification, Simple Western (SW) capillary immunoassays were developed. A panel of antibodies was screened for reactivity to the polyQ proteins on SW. The most promising antibodies were selected for further assay development. This resulted in optimised SW immunoassays for huntingtin (HTT), ataxin 1, 2 and 3 (ATXN1, 2, 3), atrophin 1 (ATN1) and androgen receptor (AR). Additionally, size-separation of the wild-type and polyQ-expanded mutant protein isoforms on SW was shown for ATXN1, ATXN3 and ATN1, allowing for their separate quantification. To facilitate size-separation of the larger HTT protein (≥ 348 kDa), a novel caspase 3-based assay was developed to generate N-terminal wild-type and mutant HTT fragments that could be separately quantified on SW in contrast to full-length HTT. In conclusion, SW capillary immunoassays were developed for polyQ proteins to improve preclinical research and aid the development of polyQ-lowering therapies for polyQ diseases.
聚谷氨酰胺（polyQ）疾病是由CAG重复序列扩增导致的单基因致死性神经退行性疾病，该扩增序列会被翻译成有毒的聚谷氨酰胺片段。聚Q疾病有九种：亨廷顿舞蹈症（HD）、脊髓小脑共济失调1型、2型、3型、6型、7型和17型（SCA1、2、3、6、7、17）、齿状核红核苍白球路易体萎缩症（DRPLA）以及脊髓延髓肌萎缩症（SBMA）。尽管目前尚无改善疾病的疗法，但降低致病突变聚Q蛋白的水平是一种有前景的潜在治疗方法。临床前研究中，聚Q蛋白降低化合物的疗效通常使用耗时的蛋白质免疫印迹法（WB）进行评估，其结果可能存在差异。因此，为提高聚Q蛋白水平定量的通量和准确性，开发了简单蛋白质免疫印迹法（SW）毛细管免疫分析技术。筛选了一组抗体，检测其对SW上聚Q蛋白的反应性。选择了最有前景的抗体用于进一步的分析方法开发。这产生了针对亨廷顿蛋白（HTT）、共济失调蛋白1、2和3（ATXN1、2、3）、萎缩素1（ATN1）和雄激素受体（AR）的优化SW免疫分析方法。此外，在SW上展示了野生型和聚Q扩展突变蛋白异构体对ATXN1、ATXN3和ATN1的大小分离，从而可以对它们进行单独定量。为便于对更大的HTT蛋白（≥348 kDa）进行大小分离，开发了一种基于新型半胱天冬酶3的分析方法，以生成N端野生型和突变型HTT片段，与全长HTT相比，这些片段可以在SW上进行单独定量。总之，开发了针对聚Q蛋白的SW毛细管免疫分析方法，以改善临床前研究，并有助于开发针对聚Q疾病的聚Q降低疗法。
