Using hiPSCs to investigate the protective mechanisms of the ApoEch mutation

使用 hiPSC 研究 ApoEch 突变的保护机制

• 批准号: 10303436
• 负责人: DAVID A BRAFMAN
• 金额: $ 43.18万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10303436
• 关键词: 3-Dimensional; Address; Alleles; Alzheimer' s Disease; Alzheimer' s disease patient; Alzheimer' s disease risk; Amino Acids; Amyloid beta-Protein; Animal Model; Animals; Apolipoprotein E; Astrocytes; Biochemical; Biomedical Engineering; Carrier Proteins; Cell Line; Cells; Cholesterol; Cholesterol Homeostasis; Coculture Techniques; DNA; DNA Double Strand Break; Data; Development; Disease; Disease Progression; Disease model; Double Strand Break Repair; Engineering; Ensure; Epigenetic Process; Future; Generations; Genes; Genetic; Genetic Polymorphism; Genetic Transcription; Genotype; Gold; Heterozygote; Human; Human Engineering; Individual; Investigation; Karyotype; Lipoprotein (a); Mediating; Methods; Modeling; Modification; Molecular; Mutation; Nerve Degeneration; Neurodegenerative Disorders; Onset of illness; Phenotype; Primary Cell Cultures; Probability; Regulator Genes; Reporter; Reporting; Research; Risk; Risk Factors; Series; Signal Pathway; Signaling Protein; System; TNFRSF10A gene; Technology; Testing; Therapeutic Intervention; Variant; apolipoprotein E-3; apolipoprotein E-4; base; design; experience; genome editing; genome wide association study; induced pluripotent stem cell; molecular targeted therapies; mutation carrier; neuroprotection; non-demented; novel; pluripotency; presenilin-1; protective effect; rare variant; relating to nervous system; repaired; stem cell model; stem cells; targeted treatment; tau Proteins; tau phosphorylation; transcriptome; transcriptome sequencing; translational impact; uptake

Although the majority of AD patient are sporadic, genome-wide association studies and related approaches have identified numerous genetic factors that modulate the risk of AD onset and progression. While the majority of these risk factors are associated with increased AD risk, several variants have been identified that reduce the risk of AD. As such, variation in Apolipoprotein E (ApoE), a cholesterol transport protein secreted primarily by astrocytes in the CNS, has been identified as one the more powerful of these risk factors. ApoE has three common alleles which differ with respect to two amino acid residues. Compared to the risk neutral ApoE3/3 genotype, each additional copy of the ApoE4 increases risk of AD, while the presence the APO2 allele is protective. More recently, an additional rare variant of APOE—the ApoE Christchurch (ApoCh)—was identified as potentially mitigating neurodegeneration in a PSEN1 mutation carrier. As such, understanding the protective mechanisms of the ApoCh variant will have a significant effect on therapeutic interventions. We and others have used hiPSC-based models, which are highly complementary to existing animal and primary cell culture models, to study AD is a simplified and accessible system. As such, we will use our collective experience in stem cell bioengineering, neurodegenerative disease modeling, and genome editing to elucidate the potential mechanisms by which ApoEch mitigates AD risk. In the first aim, we will use our recently developed highly efficient gene editing approach to introduce the ApoEch mutation into isogenic hiPSCs. In the second aim, we will perform biochemical, molecular, and transcriptome analysis of neural cultures derived from these isogenic lines to test the hypotheses that ApoEch exerts its protection effects through (i) modulation of Aβ processing, secretion, and uptake and (ii) alteration in tau hyperphosphorylation and internalization. In addition, we will use RNA-seq analysis to identify signaling pathways, gene regulatory networks, and transcriptional target that are independently influenced by the presence of the ApoEch mutation and disease status. Overall, a better understanding of the mechanism by which ApoEch enhances neuroprotection against AD will have a significant translational impact on the design of therapeutic interventions.

尽管大多数AD患者是散发性的，但全基因组关联研究和相关方法 确定了许多调节AD发病和进展风险的遗传因素。虽然大多数人 这些风险因素与AD风险增加有关，已发现几种变种可降低 AD的风险。因此，载脂蛋白E(ApoE)的变异，一种主要由 中枢神经系统中的星形胶质细胞，已被确定为这些危险因素中更强大的因素之一。APOE有三个 两个氨基酸残基不同的常见等位基因。与风险中性的ApoE3/3相比 ApoE4的每一个额外拷贝都会增加AD的风险，而APO2等位基因的存在则是 防护性的。最近，APOE的另一个罕见变体-ApoE基督城(ApoCH)-被发现 有可能减轻PSEN1突变携带者的神经退化。因此，理解保护性 载脂蛋白CH变异的机制将对治疗干预产生重大影响。我们和其他人有 使用基于HiPSC的模型，这是对现有动物和原代细胞培养模型的高度补充， 研究AD是一个简单易行的系统。因此，我们将利用我们在干细胞方面的集体经验 生物工程、神经退行性疾病建模和基因组编辑，以阐明潜在的 ApoEch降低AD风险的机制。在第一个目标中，我们将使用最近开发的高度 将ApoEch突变引入等基因hPSCs的有效基因编辑方法。在第二个目标中，我们 将对来自这些同源基因的神经培养物进行生化、分子和转录组分析 用于测试ApoEch通过(I)调制β处理来施加其保护效果的假设， 分泌、摄取和(Ii)tau过度磷酸化和内化的变化。此外，我们还将使用 Rna-seq分析以确定信号通路、基因调控网络和转录靶点 独立受ApoEch突变的存在和疾病状态的影响。总体而言，更好的 了解ApoEch增强对AD的神经保护的机制将具有重要意义 翻译对治疗干预措施设计的影响。