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

神经元内αS聚集体（路易体和路易神经突）是家族性和家族性的病理标志。 散发性（“特发性”）PD以及其他“突触核蛋白病”，包括路易体痴呆、多发性 系统萎缩甚至阿尔茨海默病。用于治疗人突触核蛋白病的疾病改善药物 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，以概括 从生理状态到非生理状态的动态转变。在这里，我们提出了两种新的小鼠模型 基于我们对αS结构的新见解，进行高效的细胞药物筛选。 PI最近进行了一项诱变筛选，以确定60/80/100 kDa推定αS多聚体的丢失， 现在被几个实验室观察到是明显的本地物种。他发现了关键的氨基酸， 降低α S 60/80/100水平，并显著扰乱细胞αS稳态，导致毒性后果。重要的是， 我们在本基金中提出研究的αS变异体基本上是引起fPD/DLB的突变的“扩增” KTKEGV中的E46 K重复#4。它们是通过将1或2个额外的E46 K样突变插入到侧翼序列中来制备的。 重复#3和5。与单一αS fPD点突变不同， 表型的细胞培养，提出的“扩增”策略很容易产生病理性αS的关键特征， “在培养皿中”：αS不溶性增加、进行性神经毒性和形成圆形包涵体。结构性 与E46 K的类比将使我们的发现与突触核蛋白病的建模和治疗相关。 我们广泛的初步数据将被利用在3个主要目标：1.新型αS小鼠模型， 易包含的α SE 35 K + E46 K（“α S2 K”）和α SE 35 K + E46 K + E61 K（“α S3 K”）变体，加上“α SKLK”，另一个 神经毒性αS基序突变体。2.毒性αS变体的神经元模型。特别关注的是 在神经元胞体和神经突中形成的引人注目的αS包涵体，确定它们的关系，如果有的话， 富含β-片层的αS Lewy聚集体，并将内含物定义为对神经元有毒或保护。3.执行 筛选可以纠正蛋白质稳态异常的因子（基因，但主要是小的药物样分子） 这是包涵体形成和神经毒性的基础，目的是找到突触核蛋白病修饰药物。 所有三个目标都是基于详细的和技术上可行的初步研究。我们相信新的研究 本文提出的方法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 突触核蛋白病发病机制中的神经元内疾病起始。因此，我们的模式将是互补的 关注细胞外非细胞自主扩散模型的方法。 PI在细胞生物学，生物化学和神经退行性疾病研究方面具有很强的背景， 在他的新的独立实验室中，与αS小鼠模型专家合作开展工作（Silke Nuber）、αS药物筛选（Susan Lindquist实验室）和αS生物物理学（Tim Bartels）。 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