Lewy body neuropathologies and SNCA gene: variants expression and splicing

摘要 帕金森病（PD）、路易体痴呆（DLB）和阿尔茨海默病的路易体变体 (LBV/AD）是常见的突触核蛋白病，这是一组神经退行性疾病， 由α-突触核蛋白组成的标志，称为路易体（LB）;然而，每种疾病都表现出不同的 特色遗传关联研究表明SNCA基因与突触核蛋白病的病因有关。 有趣的是，定义PD GWA命中的SNCA变体与那些显著 与DLB有关。然而，精确的致病变异和它们通过的分子机制， 发挥其致病作用还有待探索。我们的首要目标是确定共同和 突触核蛋白病的病因学和异质性背后的不同的因果变异，并揭示 支持调节其影响的机制。众所周知，SNCA表达水平在细胞凋亡中起作用。 在这些疾病的发展中起着关键作用，支持了这项研究的前提， SNCA基因表达异常是突触核蛋白病的主要致病机制。我们假设 SNCA基因座中的非编码遗传变异对神经元的功能产生共同的和/或神经元类型特异性的影响。 SNCA表达通过多种机制失调，如表观遗传、转录和后 转录，这反过来又发挥了作用的遗传病因学和异质性的突触核离子病。到 研究SNCA基因组、表观基因组和在突触核蛋白病背景下的表达，我们将联合收割机 从同一受试者的脑组织中分离的单个神经元中的发现分析，用于所有三个目的，以及 使用hiPSC衍生的神经元模型的验证方法。目的1将定义神经元调控元件， SNCA区域是常见的和特异性的不同的突触核蛋白病，脑区，和神经元 类型我们将使用受影响和未受影响的人脑组织的NeuN+/-细胞核来确定染色质 可访问性，与公开可用的人类表观基因组数据集整合，并使用同基因hiPSC进行验证- 衍生的神经元，其中假定的调节元件将被系统地删除。目标2将发现 使用SMRT-seq（PacBio）分析SNCA基因座中的非编码调节变体和单倍型，并评估其 与通过nCounter（NanoString）测量的神经元核中SNCA-mRNA的相关性。最强 将使用通过CRISPR/Cas9编辑的等基因hiPSC衍生的神经元模型来验证候选变体。在 目的3我们将描述SNCA启动子/内含子1内神经元DNA甲基化谱的差异 区域，以及它们对SNCA-mRNA水平的影响。DNA甲基化在SNCA表达调控中的作用 并且逆转疾病相关细胞扰动的结果将通过DNA甲基化来验证 在等基因hiPSC衍生的神经元中使用基于CRISPR/dCas 9的工具进行编辑。我们的研究将破解 SNCA失调的机制，介导不同的突触核蛋白病的易感性。的 结果对于新的遗传生物标志物和微调SNCA的治疗靶点都是翻译性的 表达至正常生理水平。

ABSTRACT Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and Lewy bodies variant of Alzheimer disease (LBV/AD) are common synucleinopathies, a group of neurodegenerative disorders that share a pathological hallmark composed of a-synuclein protein, termed Lewy bodies (LBs); however, each disease presents distinct characteristics. Genetic association studies have implicated SNCA gene in the etiology of synucleinopathies. Interestingly, SNCA variants defining the PD GWA hits are distinct from those that were significantly associated with DLB. However, the precise causal variants and the molecular mechanisms through which they exert their pathogenic effects are yet to be explored. Our overarching goal is to identify the common and distinct causal variants underlying the etiology and the heterogeneity of synucleinopathies, and to uncover the underpinning mechanisms that mediate their effects. It has been well-known that SNCA expression levels play a key role in the development of these diseases, supporting the premise for this study that impaired regulation of SNCA gene expression is a major pathogenic mechanism of synucleinopathies. We hypothesize that noncoding genetic variants in SNCA locus exert common and/or neuronal type-specific effects on the dysregulation of SNCA expression via multiple mechanisms such as, epigenetic, transcriptional and post transcriptional, which in turn plays a role in the genetic etiology and heterogeneity of synucleionopathies. To investigate the SNCA genome, epigenome and expression in the context of synucleinopathies, we will combine discovery analyses in single neurons isolated from brain tissues of the same subjects for all three aims, and validation approaches using hiPSC-derived neuronal models. Aim 1 will define neuronal regulatory elements in SNCA region that are common and specific to the different synucleinopathies, brain regions, and neuronal types. We’ll use NeuN+/- nuclei from affected and unaffected human brain tissues to determine chromatin accessibility, integrate with publicly available human epigenome datasets, and validate using isogenic hiPSC- derived neurons in which the putative regulatory elements will be systematically deleted. Aim 2 will discover noncoding regulatory variants and haplotypes in SNCA locus using SMRT-seq (PacBio) and evaluate their associations with SNCA-mRNA in neuronal nuclei measured by nCounter (NanoString). The strongest candidate variants will be validated using isogenic hiPSC-derived neuronal models edited by CRISPR/Cas9. In Aim 3 we’ll characterize differences in neuronal DNA-methylation profiles within SNCA promoter/intron 1 region, and their effects on SNCA-mRNA levels. The role of DNA-methylation in regulating SNCA expression and the consequences on reversing disease related cellular perturbations will be validated by DNA-methylation editing using CRISPR/dCas9-based tool in isogenic hiPSC-derived neurons. Our study will decipher mechanisms of SNCA dysregulation that mediate the susceptibility to the different synucleinopathies. The results are translational for both new genetic biomarkers and therapeutic targets for fine-tuning SNCA expression to normal physiological levels.