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

项目概要/摘要 本研究的目的是确定SORL 1基因中与AD风险相关的遗传变异是否 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 神经元内体异常记录在死后AD脑组织和多个内吞 在人群研究中，调节基因与AD风险增加相关。SORL 1是一种囊泡运输 在核内体、高尔基体和质膜之间转运货物的基因。SORL 1游戏 在通过内吞网络运输淀粉样蛋白β和淀粉样蛋白前体蛋白中不可或缺， 在AD脑组织中记录了SORL 1的表达，可能有助于老年斑的形成。该途径 代表了AD治疗开发的新途径。我们以前的研究使用人类诱导的 多能干细胞（hiPSC）衍生的神经元（hiPSC-Ns），以显示SORL 1表达的缺陷 诱导与SORL 1非编码区中AD相关变体的存在相关。在这 工作，我们假设内体网络基因的风险变异预测细胞内吞和AD相关 表型为了验证这一假设，我们将利用我们在hiPSC衍生的神经元细胞方面的长期专业知识， 用我们新开发的方法产生hiPSC神经元并直接转分化 i）AD相关变体是否导致SORL 1功能丧失 导致内体和AD相关表型; ii）细胞年龄是否加剧这些表型;和 iii）内吞途径中AD风险变体的累积负荷是否预测内吞表型。我们 已经鉴定出具有SORL 1编码变体的AD患者，并从这些患者中获得成纤维细胞， hiPSC生成。我们将使用CRISPR/Cas9基因编辑来纠正患者细胞中的变异， 对照细胞中的变体，产生等位基因系列的细胞系，其将包括一个或两个拷贝的 变体等位基因以及SORL 1敲除细胞系。我们将从这些细胞系中分化出神经元， 确定的内体和AD病理学表型：内体尺寸增大，内吞再循环减少， 增加的A β肽分泌和增加的Tau磷酸化。此外，我们还将生成诱导 通过直接转化从患者和对照成纤维细胞中分离神经元（iN），以测试内吞表型是否 当细胞年龄保持不变时会增强。最后，我们将从尸检病例中获得hiPSC-Ns和iNs 已证实的AD和内吞基因中AD相关SNP的高风险负担。我们将进行我们的内体 测定并产生表型组。这项工作的意义在于它将研究一种功能基因型- 内体网络中遗传变异的表型关系，已知其在AD早期被破坏 发病机制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 在开发新的精确的AD治疗方法中至关重要。

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.