Elucidation of the structures and biological activities of huntingtin oligomers

亨廷顿氏病（HD）是一种致命的遗传性神经退行性疾病， 亨廷顿蛋白中的多聚谷氨酰胺（polyQ）区，导致其聚集成淀粉样纤维。 历史上，原纤维被认为直接介导HD和其他神经退行性疾病中的神经退行性变。 阿尔茨海默病（AD）和帕金森病（PD）等疾病。然而，最近的研究表明， 和PD提出了另一种假设，即潜在的扩散纳米级组件，如 球形低聚物、原纤维和孔样环状结构是介导 神经变性突变的亨廷顿蛋白是否形成这样的结构， AD和PD的致病机制仍知之甚少。在我们的初步数据中，我们证明， 用离位原子力显微镜（AFM）观察到一个带有扩展的polyQ重复序列的突变亨廷顿蛋白片段， 形成球形和环形寡聚结构，使人联想到与AD连接的蛋白质形成的结构， 警局我们还表明，在动物模型中具有保护作用的分子伴侣Hsp70和Hsp40， HD和其他神经退行性疾病，可以减弱球形和环形的形成， 低聚物虽然我们的初步结果是挑衅性的，但对这些实验的一个重要警告是， AFM不在溶液中进行。我们现在希望进一步了解 通过阐明不同类型的结构和生物活性， 在接近生理条件下使用原位AFM对polyQ聚集体进行了分析。我们将首先描述 突变亨廷顿蛋白片段在各种表面化学和不同溶液下的聚集 条件接下来，我们将通过表征不同聚集体的生物学特性来确定它们的生物学特性 原子力显微镜结合贴片技术对核膜核孔复合体功能的影响 钳位分析最后，我们还将描述分子伴侣和抗亨廷顿蛋白的作用 抗体对突变亨廷顿蛋白片段的结构和生物活性的影响。我们的长期目标是， 对突变亨廷顿蛋白形成的聚集体结构的基本理解，以及它们如何 介导的神经变性，将导致设计潜在的治疗剂来抑制其体内毒性。

Huntington's disease (HD)is a fatal, inherited neurodegenerative disorder that is caused by an expansion of a polyglutamine (polyQ) tract in the protein huntingtin, which leads to its aggregation into amyloid fibrils. Historically, fibrils were thought to directly mediate neurodegeneration in HD and other neurodegenerative diseases such as Alzheimer's disease (AD)and Parkinson's disease (PD). However, recent studies in AD and PD suggest an alternative hypothesis whereby potentially diffusible nanoscale assemblies, such as spherical oligomers, protofibrils and pore-like annular structures, are the structural entities that mediate neurodegeneration. Whether the mutant huntingtin protein forms such structures and shares a similar pathogenic mechanism to AD and PD remains poorly understood. In our preliminary data we demonstrate by ex situ atomic force microscopy (AFM) that a mutant huntingtin fragment with an expanded polyQ repeat forms spherical and annular oligomeric structures reminiscent of those formed by proteins linked to AD and PD. We also show that the molecular chaperones Hsp70 and Hsp40, which are protective in animal models of HD and other neurodegenerative disorders, can attenuate the formation of spherical and annular oligomers. Although our preliminary results are provocative, an important caveat to these experiments is that the AFM was not performed in solution. We now wish to advance our understanding of the neurodegeneration that occurs in HD by elucidating the structures and biological activities of different types of polyQ aggregates using in situ AFM under near physiological conditions. We will first characterize the aggregation of mutant huntingtin fragments on various surface chemistries and under different solution conditions. We will next determine the biological properties of different aggregates by characterizing their effects on the function of nuclear pore complexes in nuclear envelopes by combining in situ AFM with patch clamp analyses. Finally, we will also characterize the effects of molecular chaperones and anti-huntingtin antibodies on the structure and biological activities of mutant huntingtin fragments. Our long-term goal is that a fundamental understanding of the structures of aggregates formed by mutant huntingtin, and how they mediate neurodegeneration, will result in the design of potential therapeutics to suppress their toxicity in vivo.