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

用于鉴定 APP/BACE-1 相互作用的遗传修饰剂的 CRISPR-Cas9 筛选

• 批准号: 9074668
• 负责人: Subhojit Roy
• 金额: $ 22.31万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-15 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9074668
• 关键词: Abeta synthesis; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Attenuated; Biological Assay; Brain; CRISPR screen; CRISPR/Cas technology; Cells; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; DNA Double Strand Break; Endocytosis; Endosomes; Ensure; Enzymes; Event; Figs - dietary; Fluorescence; Frequencies; Gene Proteins; Gene Silencing; Genes; Genetic; Goals; Guide RNA; Hippocampus (Brain); Human; Human Genome; Image; Israel; Knock-out; Lead; Letters; Libraries; Life; Methods; Molecular; Nature; Neurons; Optics; Paper; Pathogenesis; Pathologic; Pathway interactions; Physiological; Play; Protein Fragment; Proteins; Proteolysis; Proxy; RNA Interference; Recycling; Reporting; Research; Role; Running; Science; Seminal; Site; Sorting - Cell Movement; Specificity; Students; System; Technology; Validation; Vesicle; base; beta-site APP cleaving enzyme 1; endonuclease; enzyme substrate; gene discovery; genome-wide; induced pluripotent stem cell; insight; loss of function; meetings; new therapeutic target; novel; protein expression; protein protein interaction; public health relevance; research study; screening; secretase; stem; tool; trafficking

 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.

 描述（由申请人提供）：本提案的总体目标是发现沿着导致阿尔茨海默病（AD）发病机制中两种关键蛋白质-淀粉样前体蛋白（APP）和β位点APP裂解酶-1（BACE-1）会聚的运输途径的分子。这种聚合以及随后的APP酶促β裂解是淀粉样蛋白β（Aβ）产生的限速步骤-这是AD大脑的病理标志，也是AD研究的主要焦点。可视化APP/BACE-1在海马神经元中的运输，我们最近发现，在合成后，APP和BACE-1被分选到不同的囊泡中，BACE-1选择性地进入再循环内体。在稳态下，APP和BACE-1会聚是在基础水平下产生Aβ的低频事件（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会聚事件的分子途径将允许：1）鉴定APP和BACE-1相遇以启动淀粉样蛋白生成级联的运输途径的所有组成部分;和2）发现可被操纵以减少APP/BACE-1会聚和Aβ产生的新型“可药物靶点”。为此，我们开发了一种细胞内光学测定来可视化APP和BACE-1的会聚（OptiCAB）。基于荧光互补，该检测报告APP/BACE-1相互作用作为简单的开/关读数，与APP β-裂解相关，适用于大规模分析。将该检测与新开发的使用CRISPR-Cas9敲除（GeCKO）文库的强大基因组规模筛选（与麻省理工学院张峰合作）相结合，我们的目标是发现参与“贩运相关”上游途径的基因，最终导致APP/BACE-1融合和Aβ产生。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淆脱靶效应的限制，这些效应在历史上限制了RNAi的效用。“命中”（即减弱APP/BACE-1相互作用的基因）的二级验证 将在人诱导多能干细胞（iPSC）中进行;其中将在相关CRISPR敲除后分析APP切割产物。我们的目标是：目标一：使用OptiCAB和GeCKO发现导致APP和BACE-1融合的途径;和目标#2：在人类神经元分化的iPSC中从目标1中获得“命中”。我们的实验不仅将提供对人类生理“淀粉样蛋白通路”的见解，而且还可能为AD提供新的靶点。最后，请注意，我们对导致APP/BACE-1近似的运输途径的关注源于我们自己的实时成像研究;并且与目前对分泌酶的酶活性的狭隘关注不同。