A CRISPR-Cas9 screen to identify genetic modifiers of APP/BACE-1 interactions

 描述(申请人提供)：这项提议的总体目标是沿着导致阿尔茨海默病(AD)发病机制中两个关键蛋白-淀粉样前体蛋白(APP)和β位点APP裂解酶-1(BACE-1)汇聚的运输途径发现分子。这种收敛，以及随之而来的APP的酶促β裂解，是淀粉样β蛋白(Aβ)产生的限速步骤--淀粉样β蛋白是AD大脑的病理标志，也是AD研究的一个主要焦点。可视化APP/BACE-1在海马神经元的运输，我们最近发现，合成后，APP和BACE-1被分成不同的囊泡，BACE-1选择性地进入循环内小体。在稳定状态下，APP和BACE-1的融合是一种低频事件-在基础水平上产生Aβ(Das等人，Neuron 2013；PMID：23931995)。在这些研究的基础上，我们推断，确定导致这一开创性融合事件的分子途径将允许：1)鉴定APP和BACE-1相遇启动淀粉样蛋白致级联反应的运输途径；2)发现可以被操纵以减少APP/BACE-1融合和Aβ产生的新的“可用药靶点”。为此，我们开发了一种细胞内光学分析方法来可视化APP和BACE-1(OptiCAB)的融合。基于荧光互补原理，本实验将APP/BACE-1相互作用报告为简单的开/关读数，与APPβ-裂解相关，适用于大规模分析。我们的目标是将这一检测与使用CRISPR-Cas9基因敲除(壁虎)文库(与麻省理工学院张峰合作)新开发的强大的基因组规模筛查相结合，以发现与人口贩运相关的上游途径中涉及的基因，这些上游途径最终导致APP/BACE-1融合和Aβ生产。值得注意的是，基于CRISPR-Cas9的筛选不受蛋白质不完全耗尽和混杂的靶外效应的限制，这些影响历来限制了RNAi的用途。“HITS”(即减弱APP/BACE-1相互作用的基因)的二次验证 将在人类诱导多能干细胞(IPSC)中完成；APP裂解产物将在相关CRISPR基因敲除后进行分析。我们的目标是：目标1：使用OptiCAB和壁虎发现导致APP和BACE-1融合的途径；目标2：在人类神经元分化的IPSCs中验证来自目标1的“HITS”。我们的实验不仅将为人类的生理“淀粉样蛋白通路”提供洞察力，而且还可能为AD提供新的靶点。最后，请注意，我们关注的是导致-t app/BACE-1近似的运输途径的曲目，源于我们自己的现场成像研究；而不同于目前对分泌酶的酶活性的狭隘关注。

 DESCRIPTION (provided by applicant): The overall goal of this proposal is to discover molecules along trafficking pathways leading to the convergence of two key proteins in Alzheimer's disease (AD) pathogenesis - Amyloid Precursor Protein (APP) and β-site APP-cleaving enzyme-1 (BACE-1). This convergence, and consequent enzymatic β-cleavage of APP, is the rate-limiting step of amyloid beta (Aβ) production - a pathological hallmark of AD brains and a prevailing focus in AD research. Visualizing APP/BACE-1 trafficking in hippocampal neurons, we recently found that after synthesis, APP and BACE-1 are sorted into distinct vesicles, with BACE-1 selectively routed into recycling endosomes. At steady state, APP and BACE-1 convergence is a low-frequency event - producing Aβ at basal levels (Das et al., Neuron 2013; PMID: 23931995). Following up on these studies, we reasoned that ascertaining molecular pathways leading up-to this seminal convergence event would allow: 1) identification of the repertoire of trafficking pathways by which APP and BACE-1 meet to initiate the amyloidogenic cascade; and 2) discovery of novel "druggable targets" that can be manipulated to diminish APP/BACE-1 convergence and Aβ production. Towards this we developed an in-cellulo Optical assay to visualize Convergence of APP and BACE-1 (OptiCAB). Based on fluorescence complementation, this assay reports APP/BACE-1 interactions as a simple on/off readout, correlates with APP β-cleavage, and is suitable for large-scale analyses. Combining this assay with a newly-developed powerful genome-scale screen using CRISPR-Cas9 knockout (GeCKO) library (collaboration with Feng Zhang, MIT), our goal is to discover genes involved in `trafficking-related' upstream pathways that eventually lead to APP/BACE-1 convergence and Aβ production. Notably, CRISPR-Cas9- based screens are not limited by incomplete protein depletion and confounding off-target effects that have historically limited the utility of RNAi. Secondary validation of `hits' (i.e. genes that attenuate APP/BACE-1 interactions) will be done in human induced pluripotent stem cells (iPSC's); where APP-cleavage products will be analyzed after relevant CRISPR-knockout. Our aims are: Aim #1: Discover pathways leading to APP and BACE-1 convergence using OptiCAB and GeCKO; and Aim #2: Validate `hits' from Aim 1 in human neuronally-differentiated iPSCs. Our experiments will not only provide insights into the physiologic "amyloid-pathway" in humans, but may also offer new targets for AD. Finally, note that our focus on the repertoire of trafficking pathways leading up-t APP/BACE-1 approximation stems from our own live-imaging studies; and is different from the current narrow focus on enzymatic activity of the secretases.