Nanoscopic Membrane Modulations Induced by Nanoscale Oligomers

亨廷顿病(HD)是一种毁灭性的神经元疾病，由突变的亨廷顿蛋白(Htt)引起 多聚谷氨酰胺(PolyQ)束的异常扩张。突变的htt有一种内在的形成倾向 前纤维和纤维聚集体。尽管聚集体指示的毒性已被广泛建议用于 过多的神经元紊乱，淀粉样蛋白导致HD中棘神经元死亡的机制 目前仍不清楚。虽然纤维状聚集体通常见于菌斑和包涵体中，但较小的 低聚物现在被认为是许多神经退行性疾病的主要分子毒素。 根据寡聚体假说和膜组件在淀粉样蛋白毒性中的明显作用，我们 假设突变的Htt的纳米级低聚物负责调节膜的性质， 导致细胞功能的进行性丧失，如钙离子流量不受调节和形状异常 突触膜和线粒体膜的转化。我们进一步假设低聚物膜 相互作用是脂类特有的。由于脂类分布依赖于细胞和年龄，因此脂类特异性可能 可能是同一种淀粉样蛋白的可变细胞易损性的原因。尽管多Q- 膜相互作用已被广泛研究，前纤维和纤维聚集体的混合物通常是 现在时。因此，要区分哪些聚集物种是造成 观察对膜性能的影响。我们将首先从纤维和纤维中分离出可溶的多聚Q低聚物 单体。然后，我们将使用实验方法来研究齐聚物与膜的相互作用。在AIM 1，我们将寻求确定纳米级低聚物对微观和纳米性质的影响 脂膜。根据特定脂质在HD发病机制中的意义，我们将制备脂质 具有定义的脂类成分的膜。我们的脂类依赖研究将阐明特定脂类的作用 在管理齐聚物毒性方面。在目标2中，我们将进行拉曼光谱实验来研究 脂膜的原子水平变化。拉曼光谱数据将揭示低聚物如何扰乱脂质I) 链内构象顺序和II)链间堆积顺序。生物物理工具的组合将是 在本项目中使用的包括高分辨率原子力显微镜(AFM)、基于AFM的力 光谱学、囊泡渗漏分析、微吸管抽吸和拉曼光谱。生物物理学 这个项目中阐述的方法可以转移到对一系列由 不同的淀粉样蛋白。因此，我们的研究将为以后的研究做好铺垫 阐明寡聚体支配的毒性的分子基础。 1/1

Huntington’s disease (HD) is a devastating neuronal disorder caused by the mutant huntingtin (Htt) protein with an abnormal expansion of the polyglutamine (polyQ) tract. Mutant Htt has an intrinsic propensity of forming prefibrillar and fibrillar aggregates. Although aggregates-dictated toxicity has been widely suggested for a plethora of neuronal disorders, the mechanism of how amyloids cause medium spiny neuronal death in HD remains unclear. Although fibrillar aggregates are commonly found in plagues and inclusions bodies, small oligomers are now considered as the dominant molecular toxins underlying many neurodegenerative diseases. Based on the oligomer hypothesis and the apparent role of membrane assemblies in amyloid toxicity, we hypothesize that nanoscale oligomers of mutant Htt are responsible for modulating membrane properties, leading to progressive loss of cellular functions such as unregulated flux of calcium ions and abnormal shape transformation of synaptic and mitochondrial membranes. We further hypothesize that oligomer-membrane interactions are lipid specific. Because lipid profile is cell dependent and age dependent, lipid specificity could serve as a plausible cause for variable cell vulnerability to the same amyloid species. Although polyQ- membrane interactions have been widely studied, a mixture of prefibrillar and fibrillar aggregates are often present. Consequently, it is challenging to distinguish which aggregated species is responsible for the observed effects on membrane properties. We will first separate soluble polyQ oligomers from fibrils and monomers. We will then use experimental approaches to investigate oligomer-membrane interactions. In Aim 1, we will seek to determine the effects of nanoscale oligomers on microscopic and nanoscopic properties of lipid membranes. Based on the significance of specific lipids in HD pathogenesis, we will prepare lipid membranes with defined lipid compositions. Our lipid-dependent studies will elucidate the role of specific lipids in governing oligomer toxicity. In Aim 2, we will conduct Raman spectroscopy experiments to investigate atomic-level changes of lipid membranes. Raman spectral data will unveil how oligomers perturb the lipid i) intra-chain conformational order and ii) inter-chain packing order. A combination of biophysical tools will be employed in this project, including high-resolution atomic force microscopy (AFM), AFM-based force spectroscopy, vesicle leakage assay, micropipette aspiration, and Raman spectroscopy. The biophysical approaches elaborated in this project can to transferred to investigations of an array of oligomers formed by different amyloidogenic proteins. Therefore, our research will pave the way for future studies aimed at elucidating molecular bases of oligomer-governed toxicity. 1/1