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

PSP 发病机制中风险调节 SNP 附近基因的功能评估

• 批准号: 10285679
• 负责人: Edward Alan Burton
• 金额: $ 43.41万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10285679
• 关键词: 3-Dimensional; Address; Alleles; Amino Acid Sequence; Biochemical; Biological; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Cell physiology; Cells; Cessation of life; Characteristics; Clinical; Code; Cognition; Cognitive; Complex; Data; Deglutition; Dementia; Deposition; Deterioration; Development; Disease; Disease Progression; Drug Targeting; Equilibrium; Evaluation; Event; Experimental Models; Eye Movements; Functional disorder; Future; Gene Combinations; Genes; Genetic; Genotype; Haplotypes; Homologous Gene; Human; Hypokinesia; Impairment; In Vitro; Intervention; Link; Linkage Disequilibrium; MAPT gene; Mediating; Methods; Modeling; Molecular; Motor; Movement; Mutagenesis; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurologic; Neurologic Deficit; Neurons; Ophthalmoplegia; Oxidative Stress; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Phenocopy; Phenotype; Phosphorylation; Process; Progressive Supranuclear Palsy; Protein Isoforms; Reproducibility; Risk; Role; Sampling; Series; Single Nucleotide Polymorphism; Speech; Symptoms; Synapses; Tauopathies; Testing; Tissues; Transgenic Organisms; Untranslated RNA; Variant; Work; Zebrafish; biophysical properties; effective therapy; genetic risk factor; genetic variant; genome wide association study; in vivo; individual patient; intravital microscopy; mutant; nervous system disorder; neuroinflammation; neuron loss; novel; oculomotor; personalized medicine; prevent; protein function; risk variant; symptom treatment; tau Proteins; tau expression; tau phosphorylation; therapeutic target; tool

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.

进行性核上性麻痹（PSP）是一种神经退行性疾病，临床上以运动、认知和运动障碍为特征， 眼部缺陷和病理学上的神经元丢失、小胶质细胞增生和不溶性沉积物的积累 微管相关蛋白Tau（4 R-Tau）的4-重复同种型。仅限当前管理层 解决了症状，并且迫切需要有效的治疗方法来防止疾病进展。 遗传学证据坚定地表明Tau参与了发病机制，尽管导致Tau病和Tau突变的机制还不清楚。 神经元损失是未知的。全基因组关联研究（GWAS）已经确定了PSP相关的单一基因。 核苷酸多态性（SNP）与相邻基因的功能变化相关。该风险等位基因 STX 6内的SNP与STX 6表达降低相关。MOBP中SNP的风险等位基因是 与邻近基因SLC 25 A38的表达增加相关。EIF 2AK 3中的SNP的风险等位基因是 与损害蛋白质功能的非同义编码变体的连锁不平衡。以来 这三个基因的产物并不集中在一个单一的细胞功能上，它们很可能各自贡献于一个细胞功能。 PSP发病机制的不同组成部分，其中包括多个神经元内源性和细胞非 自主事件。因此，我们假设：受PSP相关SNP影响的基因， 有助于介导体内tau蛋白病发病机制的独特和特异性机制。关键特征 PSP发病机制的假设在体外不容易复制，因此需要在体内测试这一假设。 然而，这是一项艰巨的任务，因为PSP连锁SNP的列表越来越多。为了解决这一障碍， 我们开发了一种新的PSP斑马鱼模型，该模型被优化用于快速评估遗传修饰剂。 表达人4 R-Tau的转基因斑马鱼复制PSP的关键特征，包括神经缺陷， 神经变性、神经炎症和tau蛋白病。在这个探索性的建议中，我们将测试 该模型用于确定与PSP相关SNP相邻的基因如何影响tau蛋白病的发病机制， in vivo.我们已经鉴定了STX 6、SLC 25 A38和EIF 2AK 3的保守的斑马鱼同源物，并产生了一个新的基因。 使用CRISPR-Cas9诱变的等位基因系列突变。通过将这些新的突变等位基因与我们的 在斑马鱼PSP模型中，我们将确定这三个基因表达的变化如何改变：生存，运动 和眼部异常（aim 1）;神经炎症、突触丢失、氧化应激和神经元细胞 死亡（aim 2）;和体内Tau表达、磷酸化、截短、聚集和错误定位（aim 3）。 我们将采用无偏的、自动化的和定量的表型分析工具，统计学上可靠的样本， 多个生物复制品，以提高严谨性和可重复性。总之，这些数据将阐明 STX 6、EIF 2AK 3和SLC 25 A38在tau蛋白病中的作用。重要的是，这项研究还将提供一个新的 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识别药物靶点的途径。