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; 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.

项目摘要/摘要 这项研究的目的是确定SORL1基因中与AD风险相关的遗传变异是否与 和其他内吞基因导致人体内体网络功能障碍和细胞AD表型 神经元。内体异常记录在验尸后脑组织和多个内吞 监管基因与人群研究中的AD风险增加有关。 Sorl1是囊泡贩运 在内体，高尔基体和质膜之间运输货物的基因。 Sorl1播放 通过内吞网络和损失 SORL1的AD记录在AD脑组织中，可能导致老年斑块形成。这条路 代表了广告中治疗发展的新颖途径。我们以前的研究使用了人类诱导的 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非编码区域中AD相关变体的存在相关。在这个 工作，我们假设内体网络基因中的风险变异预测细胞内吞和AD相关 表型。为了检验这一假设，我们将利用我们在HIPSC衍生的神经元方面的长期专业知识 与我们新开发的方法的分化，以生成HIPSC-神经元并直接转化 验尸后的神经元进行测试i）AD相关的变体是否导致SORL1功能的损失 导致内体和与广告相关的表型； ii）细胞年龄是否加剧了这些表型；和 iii）在内吞途径预测内吞作用型中，AD风险变体的累积燃烧是否是否存在。 已经确定了具有SORL1编码变体的AD患者，并从这些患者那里获得了成纤维细胞 HIPSC生成。我们将使用CRISPR/CAS9基因编辑来纠正患者细胞中的变体并引入 对照单元中的变体，生成一系列等位基因细胞系，其中包括一两个副本 变体等位基因以及SORL1敲除细胞系。我们将区分神经元与这些线和测定 内体和AD病理的定义表型：内体大小扩大，内吞回收降低， 增加了e骨分泌并增加了tau磷酸化。此外，我们将产生诱导的 通过直接转换的患者和控制成纤维细胞的神经元（INS）测试内吞作用型是否为 维持细胞年龄时会增强。最后，我们将从尸检病例中得出HIPSC-NS和INS 确认的AD和高风险伯恩在内吞基因中与AD相关的SNP。我们将执行内体 测定和生成表型组。这项工作很重要，因为它将研究功能性基因型 - 内体网络中遗传变异的表型关系，已知在AD早期被破坏 发病。研究这种疾病发病机理及其与人类遗传背景的关系 对于开发AD的新和精确处理至关重要。