Functional evaluation of genes adjacent to risk-modulating SNPs in the pathogenesis of PSP

进行性核上性瘫痪(PSP)是一种以运动、认知为特征的神经退行性疾病 由神经元丢失、小胶质细胞增多症和不溶沉积物堆积引起的眼球运动缺陷和病理改变 微管相关蛋白Tau(4R-Tau)的4个重复亚型。仅限当前管理层 解决症状，迫切需要有效的治疗方法来防止疾病的发展。 遗传证据确凿地表明牛磺酸在发病机制中有牵连，尽管导致直立性变态反应和 神经元的丢失是未知的。全基因组关联研究(GWAS)已确定PSP相关的单个 与相邻基因的功能变化相关的核苷酸多态(SNP)。风险等位基因 STX6内SNP的存在与STX6表达降低有关。MOBP中SNP的风险等位基因是 与邻近基因SLC25A38的表达增加有关。EIF2AK3单核苷酸多态性的风险等位基因在 与损害蛋白质功能的非同义编码变体的连锁不平衡。自.以来 这三个基因的产物并不汇聚在单一的细胞功能上，它们很可能各自对 PSP发病机制的不同组成部分，包括多个神经元内源性和细胞非内源性 自主活动。因此，我们假设：受PSP相关SNPs影响的基因 有助于明确和具体的机制，在体内的发病机制的调整。关键特征 PSP的发病机制在体外不易复制，因此希望在体内验证这一假说。 然而，考虑到越来越多与PSP相关的SNP，这是一项艰巨的任务。为了解决这个障碍， 我们开发了一种新的斑马鱼PSP模型，该模型针对快速评估遗传修饰物进行了优化。 表达人4R-Tau的转基因斑马鱼复制了PSP的关键特征，包括神经缺陷， 神经退行性变、神经炎症和直肠畸形。在这个探索性的提案中，我们将测试 该模型用于确定PSP相关SNPs附近的基因如何影响转位症的发病机制 在活体内。我们已经鉴定了STX6、SLC25A38和EIF2AK3的斑马鱼同源物，并产生了一个 利用CRISPR-Cas9突变技术对等位基因序列进行突变。通过将这些新的突变等位基因与我们的 斑马鱼PSP模型中，我们将确定这三个基因的表达变化如何改变：生存、运动 和动眼神经异常(目标1)；神经炎、突触丢失、氧化应激和神经细胞 死亡(目标2)；以及体内Tau的表达、磷酸化、截断、聚集和错位(目标3)。 我们将使用无偏见、自动化和定量的表型分析工具，统计稳健的样本，以及 多个生物复制，以增强严密性和重复性。总而言之，这些数据将阐明不同的 STX6、EIF2AK3和SLC25A38在脊椎病中的作用重要的是，这项研究还将提供一种新的 用于评估候选基因对疾病发病机制的贡献的实验工作流程，以及 利用GWAS数据阐明潜在机制和确定药物靶点的途径。

Progressive supranuclear palsy (PSP) is a neurodegenerative disease characterized clinically by motor, cognitive an oculomotor deficits and pathologically by neuronal loss, microgliosis, and accumulation of insoluble deposits of the 4-repeat isoform of the microtubule-associated protein Tau (4R-Tau). Current management only addresses symptoms and there is an urgent need for effective treatments that prevent disease progression. Genetic evidence firmly implicates Tau in pathogenesis, although the mechanisms resulting in tauopathy and neuronal loss are unknown. Genome-wide association studies (GWAS) have identified PSP-associated single nucleotide polymorphisms (SNPs) that are associated with functional changes in adjacent genes. The risk allele of a SNP within STX6 is associated with decreased STX6 expression. The risk allele of a SNP in MOBP is associated with increased expression of a neighboring gene, SLC25A38. The risk allele of a SNP in EIF2AK3 is in linkage disequilibrium with non-synonymous coding variants that impair the function of the protein. Since the products of these three genes do not converge on a single cellular function, it is likely they each contribute to a different component of PSP pathogenesis, which encompasses multiple neuron-intrinsic and cell non- autonomous events. Consequently, we hypothesize that: genes influenced by PSP-associated SNPs each contribute to distinct and specific mechanisms mediating the pathogenesis of tauopathy in vivo. Key features of PSP pathogenesis are not easily replicated in vitro, so it would be desirable to test this hypothesis in vivo. However, this is a daunting task given the growing list of PSP-linked SNPs. In order to address this roadblock, we developed a novel zebrafish model of PSP that is optimized for rapid evaluation of genetic modifiers. Transgenic zebrafish expressing human 4R-Tau replicate key features of PSP, including neurological deficits, neurodegeneration, neuroinflammation and tauopathy. In this exploratory proposal, we will test the utility of this model for determining how genes adjacent to PSP-associated SNPs influence the pathogenesis of tauopathy in vivo. We have identified conserved zebrafish homologs of STX6, SLC25A38 and EIF2AK3, and generated an allelic series of mutations using CRISPR-Cas9 mutagenesis. By crossing these new mutant alleles with our zebrafish PSP model, we will determine how changes in the expression of these three genes alter: survival, motor and oculomotor abnormalities (aim 1); neuroinflammation, synapse loss, oxidative stress, and neuronal cell death (aim 2); and Tau expression, phosphorylation, truncation, aggregation and mislocalization (aim 3) in vivo. We will employ unbiased, automated, and quantitative phenotyping tools, statistically robust samples, and multiple biological replicates, to enhance rigor and reproducibility. Together, these data will elucidate distinct roles for STX6, EIF2AK3 and SLC25A38 in tauopathy. Importantly, this study will also provide a new experimental workflow for evaluating the contributions of candidate genes to disease pathogenesis, and a pathway for exploiting GWAS data to elucidate underlying mechanisms and identify drug targets.