Probing the role of SORL1 and endosomal network genetic variation on Alzheimer's disease phenotypes in human neurons.

探讨 SORL1 和内体网络遗传变异对人类神经元阿尔茨海默病表型的作用。

• 批准号: 10221575
• 负责人: Jessica Elaine Young
• 金额: $ 64.7万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10221575
• 关键词: Adopted; Affect; Age; Aging; Alleles; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease pathology; Alzheimer' s disease patient; Alzheimer' s disease risk; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Autopsy; Biological Assay; CRISPR/Cas technology; Candidate Disease Gene; Cell Line; Cell membrane; Cells; Code; Data; Deposition; Development; Disease; Elements; Embryo; Endosomes; Epigenetic Process; Family; Fibroblasts; Functional disorder; Generations; Genes; Genetic; Genetic Variation; Genetic study; Genotype; Goals; Golgi Apparatus; Human; Human Genetics; Impairment; In Vitro; Induced pluripotent stem cell derived neurons; Investigation; Knock-out; Late Onset Alzheimer Disease; Lead; Lysosomes; Methods; Modeling; Molecular; Nerve Degeneration; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Peptides; Phenotype; Play; Population Study; Precision therapeutics; Process; Recycling; Regulator Genes; Risk; Role; Senile Plaques; Series; Sorting - Cell Movement; System; Testing; Therapeutic Intervention; Tissues; Untranslated RNA; Variant; Work; age related; age related neurodegeneration; base; brain tissue; cohort; disease phenotype; exome sequencing; experimental study; gene function; genetic manipulation; genetic variant; genome wide association study; high risk; human stem cells; induced pluripotent stem cell; knock-down; loss of function; member; network dysfunction; neuropathology; novel; novel therapeutics; preservation; rare variant; receptor; risk variant; sortilin; stem cell differentiation; tau Proteins; tau phosphorylation; tau-1; therapeutic candidate; therapeutic development; therapeutic target; trafficking

Project Summary/Abstract The purpose of this study is to determine whether genetic variants associated with AD risk in the SORL1 gene and other endocytic genes lead to endosomal network dysfunction and cellular AD phenotypes in human neurons. Endosomal abnormalities are documented in post-mortem AD brain tissue and multiple endocytic regulatory genes are associated with increased AD risk in population studies. SORL1 is a vesicular trafficking gene that functions in transporting cargo between endosomes, Golgi and the plasma membrane. SORL1 plays an integral in trafficking amyloid beta and the amyloid precursor protein through the endocytic network and loss of SORL1 is documented in AD brain tissue, possibly contributing to senile plaque formation. This pathway represents a novel avenue for therapeutic development in AD. Our previous studies have used human induced pluripotent stem cell (hiPSC)-derived neurons (hiPSC-Ns) to show that deficiencies in SORL1 expression induction are correlated with the presence of AD-associated variants in non-coding regions of SORL1. In this work, we hypothesize that risk variants in endosomal network genes predicts cellular endocytic and AD relevant phenotypes. To test this hypothesis, we will leverage our long-standing expertise in hiPSC-derived neuronal differentiations with our newly developed methods to generate hiPSC-neurons and directly transdifferentiated neurons from post-mortem AD tissue to test i) whether AD associated variants lead to loss of function of SORL1 resulting in endosomal and AD-relevant phenotypes; ii) whether cellular age exacerbates these phenotypes; and iii) whether a cumulative burden of AD risk variants in the endocytic pathway predicts endocytic phenotypes. We have identified AD patients with SORL1 coding variants and have obtained fibroblasts from these patients for hiPSC-generation. We will use CRISPR/Cas9 gene editing to correct the variants in patient cells and introduce the variants in control cells, generating an allelic series of cell lines that will include one or two copies of the variant allele as well as SORL1 knock-out cell lines. We will differentiate neurons from these lines and assay defined phenotypes of endosomal and AD pathology: Enlarged endosome size, decreased endocytic recycling, increased A peptide secretion and increased Tau phosphorylation. Furthermore, we will generate induced neurons (iNs) from patient and control fibroblasts by direct conversion to test whether endocytic phenotypes are enhanced when cellular age is maintained. Finally, we will derive hiPSC-Ns and iNs from cases with autopsy confirmed AD and high risk burdens of AD-associated SNPs in endocytic genes. We will perform our endosomal assays and generate phenotypic groups. This work is significant in that it will investigate a functional genotype- phenotype relationship of genetic variants in the endosomal network, which is known to be disrupted early in AD pathogenesis. Investigating this driver of disease pathogenesis and how it relates to human genetic background is critical in the development of new and precision treatments for AD.

项目概要/摘要 本研究的目的是确定 SORL1 基因的遗传变异是否与 AD 风险相关 和其他内吞基因导致人类内体网络功能障碍和细胞 AD 表型 死后 AD 脑组织和多个内吞细胞中记录了内体异常。 在人群研究中，调控基因与 AD 风险增加相关。 SORL1 在内体、高尔基体和质膜之间发挥转运作用。 通过内吞网络运输淀粉样蛋白和淀粉样蛋白前体蛋白的过程中不可或缺的一部分 SORL1 存在于 AD 脑组织中，可能有助于老年斑的形成。 代表了 AD 治疗开发的新途径，我们之前的研究使用了人类诱导的方法。 多能干细胞 (hiPSC) 衍生的神经元 (hiPSC-Ns) 表明 SORL1 表达缺陷 诱导与 SORL1 非编码区中 AD 相关变异的存在相关。 在工作中，我们研究内体网络基因中的风险变异可预测细胞内吞和 AD 相关性 为了检验这一假设，我们将利用我们在 hiPSC 衍生神经方面的长期专业知识。 使用我们新开发的方法产生 hiPSC 神经元并直接转分化 来自死后 AD 组织的神经元来测试 i) AD 相关变异是否导致 SORL1 功能丧失 导致内体和 AD 相关表型；ii) 细胞年龄是否会加剧这些表型；以及 iii) 内吞途径中 AD 风险变异的累积负担是否可以预测内吞表型。 已经鉴定出具有 SORL1 编码变异的 AD 患者，并从这些患者中获得了成纤维细胞用于治疗 hiPSC 一代。我们将使用 CRISPR/Cas9 基因编辑来纠正患者细胞中的变异并引入 对照细胞中的变体，产生一系列等位基因细胞系，其中包括一个或两个拷贝 我们将从这些细胞系中区分出神经元并进行分析。 内体和 AD 病理学的明确表型：内体尺寸增大、内吞循环减少、 A 肽分泌增加和 Tau 磷酸化增加此外，我们将产生诱导的。 通过直接转换来自患者和对照成纤维细胞的神经元（iN）来测试内吞表型是否 最后，我们将从尸检病例中得出 hiPSC-N 和 iN。 确认了 AD 和内吞基因中 AD 相关 SNP 的高风险负担，我们将进行内体分析。 这项工作意义重大，因为它将研究功能基因型。 内体网络中遗传变异的表型关系，已知该网络在 AD 早期就被破坏 研究疾病发病机制的驱动因素及其与人类遗传背景的关系。 对于开发 AD 的新型精准治疗方法至关重要。