Lewy body neuropathologies and SNCA gene: variants expression and splicing

摘要 帕金森氏病(PD)、路易体痴呆(DLB)和阿尔茨海默病的路易体变种 (LBV/AD)是常见的共核病，是一组具有共同病理特征的神经退行性疾病 由a-突触核蛋白组成的标志，称为路易小体(Lbs)；然而，每种疾病都表现出不同的 特点。遗传关联研究表明SNCA基因与共核病的病因学有关。 有趣的是，定义PD GWA命中的SNCA变体与那些显著 与DLB相关联。然而，确切的因果变异和它们所通过的分子机制 发挥它们的致病作用还有待于探索。我们的首要目标是确定共同的和 不同的因果变异潜伏在共核病的病因和异质性之下，并揭示 为调解其影响的机制奠定基础。众所周知，SNCA表达水平在其中起作用 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，即损害了监管 SNCA基因表达异常是共核病的主要致病机制。我们假设 SNCA基因座上的非编码遗传变异在神经细胞类型中发挥共同和/或特定的效应。 SNCA表达异常的多种机制，如表观遗传、转录和后 转录，这反过来在遗传病因学和异质性的联核病中发挥作用。至 在共核病的背景下研究SNCA基因组、表观基因组和表达，我们将结合 对同一受试者脑组织中分离的单个神经元进行的发现分析，以实现所有三个目的，以及 使用HiPSC衍生的神经元模型的验证方法。目标1将定义神经调节元件在 SNCA区域是不同的突触核病、脑区域和神经元的共同和特有的区域 类型。我们将使用受影响和未受影响的人脑组织中的Neun+/-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，并使用等基因hiPSC- 衍生的神经元中假定的调控元件将被系统地删除。Aim 2将会发现 利用SMRT-seq(PacBio)对SNCA基因座的非编码调控变异体和单倍型进行分析 NCounter(纳米串)检测神经元核内SNCA-mRNA的相关性。最强的 候选变体将使用CRISPR/Cas9编辑的等基因HiPSC衍生神经元模型进行验证。在……里面 目的3我们将表征SNCA启动子/内含子1中神经元DNA甲基化的差异 区域，以及它们对SNCA-mRNA水平的影响。DNA甲基化在调控SNCA表达中的作用 逆转与疾病相关的细胞扰动的结果将通过DNA甲基化得到验证 使用基于CRISPR/dCas9的工具在等基因的HiPSC来源的神经元中编辑。我们的研究将破译 SNCA调节失调的机制，介导了不同的联核病的易感性。这个 结果对于微调SNCA的新的遗传生物标志物和治疗靶点都是可翻译的 表达到正常的生理水平。

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.