Elucidation of the structures and biological activities of huntingtin oligomers

描述(申请人提供)：亨廷顿病(HD)是一种致命的遗传性神经退行性疾病，由亨廷顿蛋白中的聚谷氨酰胺(PolyQ)链扩张引起，导致其聚集成淀粉样纤维。历史上，纤维被认为直接介导了HD和其他神经退行性疾病，如阿尔茨海默病(AD)和帕金森病(PD)的神经退行性变。然而，最近对AD和PD的研究提出了另一种假说，即潜在的可扩散纳米级组件，如球形低聚物、原纤维和孔状环状结构，是介导神经退行性变的结构实体。突变的Huntingtin蛋白是否形成了这样的结构，并与AD和PD具有类似的致病机制，目前尚不清楚。在我们的初步数据中，我们用非原位原子力显微镜(AFM)证明了带有扩展的多Q重复序列的突变Huntingtin片段形成了球形和环状低聚结构，这使人想起与AD和PD相关的蛋白质形成的低聚结构。我们还表明，分子伴侣Hsp70和Hsp40在HD和其他神经退行性疾病的动物模型中具有保护作用，可以减少球形和环状低聚物的形成。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不是在溶液中进行的。现在，我们希望通过在近生理条件下利用原位原子力显微镜来阐明不同类型的多聚Q聚集体的结构和生物活性，以加深我们对HD发生的神经退变的理解。我们将首先表征突变的亨廷顿蛋白片段在不同的表面化学和不同的溶液条件下的聚集。接下来，我们将结合原位原子力显微镜和膜片钳分析，通过表征不同聚集体对核膜中核孔复合体功能的影响来确定不同聚集体的生物学特性。最后，我们还将表征分子伴侣和抗亨廷顿蛋白抗体对突变的亨廷顿蛋白片段的结构和生物学活性的影响。我们的长期目标是，对突变的亨廷顿蛋白形成的聚集体的结构以及它们如何介导神经退化有一个基本的了解，这将导致设计出潜在的治疗方法来抑制它们在体内的毒性。

DESCRIPTION (provided by applicant): 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.