Department of Medical and Molecular Genetics, King's College London School of Medicine, King's College London, 8th Floor Tower Wing, Guy's Tower, Great Maze Pond, London, UK.
Hum Mol Genet. 2010 Jan 1;19(1):65-78. doi: 10.1093/hmg/ddp467.
Huntington's disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS-PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit.
亨廷顿病（HD）是一种迟发性神经退行性疾病，其神经病理学特征是存在神经丝聚集物和核内包涵体。然而，HD 患者或 HD 小鼠模型大脑中存在的聚集物结构的特征以及特定聚集物结构对疾病发病机制的相对贡献尚不清楚。我们使用 Seprion 配体开发了一种基于酶联免疫吸附测定（ELISA）的高度敏感方法，用于定量测定 HD 小鼠模型组织中的聚集多聚谷氨酰胺。我们结合使用电子显微镜、原子力显微镜（AFM）和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）来研究配体分离出的聚集物结构。我们发现，从 R6/2 和 HdhQ150 基因敲入小鼠大脑中提取的寡聚体、原纤维和纤维状聚集物非常相似。使用 AFM，我们确定从两种小鼠模型大脑中分离出的纳米级球状寡聚物的尺寸与从重组亨廷顿外显子 1 蛋白生成的尺寸相同。最后，检测外显子 1 Htt 表位的抗体在 SDS-PAGE 凝胶上对配体捕获的物质有不同的识别。Seprion 配体 ELISA 提供了一种具有良好统计功效的测定方法，可用于临床前药效学治疗试验，或评估对潜在治疗靶点的遗传操作对聚集物负荷的影响。这一点，加上在 HD 小鼠组织中识别一系列聚集物物种的能力，将有助于我们理解这些结构与 HD 发病机制的关系，以及它们的形成是否可以为治疗带来益处。
