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

项目摘要/摘要 这项研究的目的是确定SORL1基因中的遗传变异是否与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 神经元。死后阿尔茨海默病患者的脑组织和多个内细胞体均出现内体异常 在人群研究中，调节基因与AD风险的增加有关。SORL1是一种泡状物运输 在内体、高尔基体和质膜之间运输货物的基因。SORL1播放 淀粉样β蛋白和淀粉样前体蛋白通过胞内网络运输和丢失的积分 在AD的脑组织中发现了SORL1的表达，可能与老年斑的形成有关。这条路 为AD的治疗发展开辟了一条新的途径。我们之前的研究使用了人类诱导的 多能干细胞来源的神经元(hiPSC-NS)显示SORL1表达缺陷 诱导与SORL1非编码区AD相关变体的存在相关。在这 工作中，我们假设内体网络基因的风险变异预测细胞内吞作用和AD相关 表型。为了验证这一假设，我们将利用我们在HiPSC来源的神经元方面的长期专业知识 用我们新开发的方法分化产生HiPSC神经元和直接转分化 从死后AD组织中提取神经元进行测试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 3)内吞途径中AD危险变量的累积负荷是否可以预测内吞表型。我们 已经鉴定出带有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 控制细胞中的变异，产生一系列等位基因的细胞系，其中将包括一个或两个拷贝的 变异等位基因以及SORL1基因敲除细胞系。我们将从这些细胞系中分化出神经元并进行分析 内小体和AD病理的明确表型：内小体增大，内皮细胞循环减少， A多肽分泌增加，Tau磷酸化增加。此外，我们将生成诱导式 通过直接转化患者和对照成纤维细胞的神经元(INS)来测试内吞细胞表型是否 当细胞年龄保持不变时增强。最后，我们将从尸检病例中提取HiPSC-NS和INS 确认AD和内吞基因中AD相关SNPs的高风险负荷。我们会做内窥镜检查 分析并产生表型群。这项工作意义重大，因为它将研究一种功能基因-- 已知在AD早期被破坏的内体网络中遗传变异的表型关系 发病机制。研究这种疾病的发病机制及其与人类遗传背景的关系 是开发AD新的精确治疗方法的关键。

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.