Systematic elucidation of endosomal trafficking as a therapeutic opportunity in AD using CRISPR-based functional genomics

项目概要/摘要 阿尔茨海默病（AD）的有效的疾病改善治疗是一个迫切的，未满足的需求。出面代言 治疗是有效的，它可能必须针对AD发病机制中的早期事件。两条证据 指出了内溶酶体运输变化在AD发病机制中的中心和早期作用：首先， 与晚发型AD（LOAD）相关的基因在内吞作用和内溶酶体运输中起作用。 有趣的是，Karch实验室最近共同发现了一种运输因子基因RAB 10的变体， 降低RAB 10表达，赋予对AD的恢复力。第二，内溶酶体的病理变化 系统，如扩大的早期内体和溶酶体酶的上调，是一些最早的 人类AD大脑的病理特征因此，我们的中心假设是， 是AD早期干预的治疗靶点。这项研究的目的是阐明具体的 治疗靶点，以纠正神经元中与LOAD风险基因相关的内溶酶体缺陷， 在治疗上概括了由RAB 10的变体赋予的对LOAD的保护。坎普曼实验室合作- 开发了一个基因筛选平台，使诱导和可逆的抑制（CRISPRi）和激活 （CRISPRa）在人类细胞中的基因进行全基因组功能缺失和获得筛选，并在 人iPSC衍生的神经元。Karch实验室已经建立了大量的患者来源的成纤维细胞 和iPSC，并产生了CRISPR校正的同基因对照系，使我们能够揭示 iPSC衍生的神经元中的表型与疾病变体有关，包括内溶酶体缺陷。我们提出 联合收割机将我们的创新方法结合起来，实现两个特定目标。目标1的目标是确定治疗靶点 用于AD，其概括了保护性RAB 10变体的机制。我们假设， RAB 10中的变体抵消与AD相关的内溶酶体缺陷。我们将测试这个假设， iPSC衍生的神经元和人脑。我们发现，RAB 10中的保护性变体减少了RAB 10 表达，并且相反地，在LOAD脑中RAB 10表达升高。我们将进行无偏见的基因组- 在WT iPSC衍生的神经元中进行广泛的CRISPRi/a筛选，以鉴定控制RAB 10水平的基因， 因此成为治疗靶点。与此同时，我们将进行全基因组筛选，以确定其他治疗方法， 靶向复制RAB 10中的保护性变体。我们将重点关注与RAB 10表现出上位性的热门歌曲 保护性变体，其最有可能表型复制人中保护性RAB 10等位基因的作用 有AD风险的人目的2的目标是使用我们的遗传互作作图方法来阐明 LOAD风险基因、内溶酶体途径和相关治疗靶点之间的联系。我们 将在一组AD患者来源的iPSC来源的细胞中验证所鉴定的治疗靶点的潜力。 神经元和同基因对照的广泛阵列的内溶酶体和APP加工表型。

PROJECT SUMMARY / ABSTRACT An effective, disease-modifying treatment for Alzheimer's Disease (AD) is an urgent, unmet need. For an AD treatment to be effective, it will likely have to target early events in AD pathogenesis. Two lines of evidence point to a central and early role for changes in endolysosomal trafficking in AD pathogenesis: First, several risk genes associated with late-onset AD (LOAD) function in endocytosis and endolysosomal trafficking. Intriguingly, a variant in the trafficking factor gene RAB10 recently co-discovered by the Karch lab, which lowers RAB10 expression, confers resilience to AD. Second, pathological changes in the endolysosomal system, such as enlarged early endosomes and upregulation of lysosomal enzymes, are some of the earliest pathological hallmarks of human AD brains. Therefore, our central hypothesis is that endolysosomal trafficking is a therapeutic target for early intervention in AD. The goal of the proposed research is to elucidate specific therapeutic targets to correct endolysosomal defects associated with LOAD risk genes in neurons, and to therapeutically recapitulate protection from LOAD conferred by variants of the RAB10. The Kampmann lab co- developed a genetic screening platform enabling inducible and reversible repression (CRISPRi) and activation (CRISPRa) of genes in human cells for genome-wide loss- and gain-of-function screens, and implemented it in human iPSC-derived neurons. The Karch lab has established a large collection of patient-derived fibroblasts and iPSCs, and generated CRISPR-corrected isogenic control lines that have enabled us to uncover phenotypes in iPSC-derived neurons linked to disease variants, including endolysosomal defects. We propose to combine our innovative approaches for two Specific Aims. The goal of Aim 1 is to identify therapeutic targets for AD that recapitulate the mechanism of protective RAB10 variants. We hypothesize that the protective variants in RAB10 counteract the endolysosomal defects associated with AD. We will test this hypothesis in iPSC-derived neurons and human brains. We found that protective variants in RAB10 reduce RAB10 expression, and conversely RAB10 expression is elevated in LOAD brains. We will conduct unbiased genome- wide CRISPRi/a screens in WT iPSC-derived neurons to identify genes that control RAB10 levels and may therefore be therapeutic targets. In parallel, we will conduct genome-wide screens to identify other therapeutic targets that phenocopy protective variants in RAB10. We will focus on hits that show epistasis with the RAB10 protective variant, which are most likely to phenocopy the effect of the protective RAB10 allele in human individuals at risk for AD. The goal of Aim 2 is to use our genetic interaction mapping approach to elucidate connections between LOAD risk genes, the endolysosomal pathway, and associated therapeutic targets. We will validate the potential of the identified therapeutic targets in a panel of AD patient-derived iPSC-derived neurons and isogenic controls for an extensive array of endolysosomal and APP processing phenotypes.