Stabilizing native α-synuclein homeostasis to prevent insoluble α-synuclein aggregates

神经元内的αS集合体(路易小体和刘易神经突起)是家族性和 散发性(‘特发性’)帕金森病以及其他‘联体核病’，包括路易体痴呆、多发性 系统萎缩，甚至阿尔茨海默病。治疗人类突触核病的疾病修饰药物 都还不存在。这在一定程度上是由于缺乏令人信服的动物和细胞模型来概括 αS从生理状态到非生理状态的动态转换。在这里，我们提出了两种新的小鼠模型 和高效的细胞药物筛选，基于我们对αS结构的新见解。 PI最近进行了突变筛查，寻找60/80/100 kDa的可能的αS多聚体的缺失 现在被几个实验室观察到是明显的本土物种。他发现了关键的氨基酸，它的突变 降低αS60/80/100水平，显著扰乱细胞α的稳态，导致S中毒。重要的是 我们在这项资助中建议研究的αS变体基本上是导致fpd/dlb突变的“放大” KTKEGV Repeat#4中的E46K。它们是通过在侧翼中插入1或2个额外的类似E46K的突变而形成的 重复#3和#5。与单个α不同，S平板电脑的点突变不会产生全面和健壮的结果 在细胞培养中的表型，所提出的‘扩增’策略很容易产生病理性αS的关键特征 在菜中：αS不溶性增加，进行性神经毒性和圆形包裹体的形成。结构性的 与E46K的类比将使我们的发现与建模和治疗共核病相关。 我们将在三个主要目标上利用我们广泛的初步数据：1.新型αS小鼠模型 易于包含的αSE35K E46K(‘αS2K’)和αSE35K E46K E61K(‘αS3K’)变体，外加‘αSKLK’，另一个 神经毒性αS基序突变体。2.毒性αS变异体的神经元模型。特别关注的将是人物塑造 在神经元体和神经突起中形成的引人注目的αS包涵体中，确定了它们与 富含β-Sheet的αS·路易聚集体，并将包裹体定义为对神经元有毒或保护。3.表演 筛选可以纠正蛋白质动态平衡失调的因子(基因，但主要是类似药物的小分子) 这是包裹体形成和神经毒性的基础，目的是寻找治疗联核症的药物。 所有三个目标都基于详细的和技术上可行的初步研究。我们相信这项新研究 在这里提出的将有助于克服缺乏令人信服的啮齿动物和细胞模型来研究早期的方面 突触核病发病机制中的神经细胞内疾病始发。因此，我们的模式将是互补的 到专注于细胞外、非细胞自主传播模型的方法。 PI在细胞生物学、生物化学和神经退行性疾病研究方面有很强的背景，并将 在他新的独立实验室与αS小鼠模型(Silke)专家合作进行这项工作 αS(苏珊·林德奎斯特实验室)和αS(蒂姆·巴特尔斯)。 1

Intraneuronal αS aggregates (Lewy bodies and Lewy neurites) are pathological hallmarks of both familial and sporadic (‘idiopathic’) PD as well as other ‘synucleinopathies’ including dementia with Lewy bodies, multiple system atrophy and even Alzheimer’s disease. Disease-modifying drugs for treating human synucleinopathies do not yet exist. This is due in part to a lack of compelling animal and cellular models that recapitulate the dynamic transition from physiological to non-physiological αS states. Here we propose both new mouse models and efficient cellular drug screens based on our novel insights about αS structure. The PI recently performed a mutagenesis screen for loss of 60/80/100 kDa putative αS multimers that have now been observed by several labs as apparent native species. He identified key amino acids whose mutation lowers αS60/80/100 levels and markedly perturbs cellular αS homeostasis with toxic consequences. Importantly, the αS variants we propose to study in this grant are basically ‘amplifications’ of the fPD/DLB-causing mutation E46K in KTKEGV repeat #4. They are made by inserting either 1 or 2 additional E46K-like mutations into flanking repeats #3 and 5. Unlike single αS fPD point mutations that do not produce comprehensive and robust phenotypes in cell culture, the proposed ‘amplification’ strategy readily produces key features of pathological αS ‘in the dish’: increased αS insolubility, progressive neurotoxicity and formation of round inclusions. The structural analogy to E46K will make our findings relevant for modeling & treating synucleinopathies. Our extensive preliminary data will be exploited in 3 major aims: 1. Novel αS mouse models that express inclusion-prone αSE35K+E46K (‘αS2K’) and αSE35K+E46K+E61K (‘αS3K’) variants, plus ‘αSKLK’, another neurotoxic αS motif-mutant. 2. Neuronal models of the toxic αS variants. Special focus will be the characterization of the striking αS inclusions that form in neuronal somata and neurites, determining their relationship, if any, to β-sheet-rich αS Lewy aggregates, and defining the inclusions as toxic or protective for the neuron. 3. Performing screens for factors (genes but principally small drug-like molecules) that can correct the protein dyshomeostasis that underlies this inclusion formation and neurotoxicity, with the goal of finding synucleinopathy-modifying drugs. All 3 Aims are based on detailed and technically enabling preliminary studies. We believe that the new research proposed herein will help overcome the lack of compelling rodent and cellular models to study early aspects of intraneuronal disease initiation in the pathogenesis of synucleinopathies. Our models will thus be complementary to approaches that focus on extracellular, non-cell-autonomous spreading models. The PI has a strong background in cell biology, biochemistry and neurodegenerative disease research and will conduct the work in his new, independent laboratory, in collaboration with experts on αS mouse models (Silke Nuber), αS drug screens (Susan Lindquist lab) and αS biophysics (Tim Bartels). 1