Spatial centrosome proteome of human neural cells uncovers novel interactors and disease hubs

Despite the crucial importance of the centrosome in brain development and disease, its comprehensive proteome remains uncharacterized in neural cells. Here, we used spatial proteomics to elucidate protein interaction networks at the centrosome of iPSC-derived human neural stem cells (NSCs) and neurons. Centrosome-associated proteins were largely cell-type specific, with protein hubs involved in RNA-dynamics. Analysis of neurodevelopmental disease cohorts identified a significant overrepresentation of NSC centrosome proteins with variants in patients with periventricular heterotopia (PH). Expressing the PH-associated mutant splicing factor PRPF6 reproduced the periventricular misplacement in the developing mouse brain, highlighting mis-splicing of transcripts of the MAP-kinase SAD-A at centrosomal location as essential for the phenotype. Collectively, cell-type specific centrosome interactomes explain how genetic variants in ubiquitous proteins may convey brain-specific phenotypes. RNA from FACS sorted electroporated (GFP+) cells was isolated in Extraction Buffer (Arcturus), heated to 42 and stored at -80 cDNA was synthesized from 300 pg of total RNA using SMART‐Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech), according to the manufacturer's instructions. Prior to generating the final library for Illumina sequencing, the Covaris AFA system was used to perform the cDNA shearing, resulting in 200‐ to 500‐bp‐long cDNA fragments. The quality and concentration of the sheared cDNA were assessed on Agilent 2100 Bioanalyzer before proceeding to library preparation using MicroPlex Library Preparation kit v2 (Diagenode). Final libraries were evaluated and quantified using an Agilent 2100 Bioanalyzer, and the concentration was measured additionally with Quant‐iT PicoGreen dsDNA Assay Kit (InvitrogenTM) before sequencing. The uniquely barcoded libraries were multiplexed onto one lane and 150‐bp paired‐end deep sequencing was carried out on HiSeq 4000 (Illumina) that generated ~ 30 million reads per sample.

尽管中心体（centrosome）在大脑发育与疾病发生中发挥关键作用，但目前神经细胞中其完整蛋白质组仍未得到系统解析。本研究采用空间蛋白质组学（spatial proteomics）技术，解析了诱导多能干细胞（induced pluripotent stem cell, iPSC）来源的人类神经干细胞（neural stem cells, NSCs）及神经元的中心体蛋白质相互作用网络。中心体相关蛋白大多呈现细胞类型特异性，其中蛋白质枢纽（protein hubs）参与RNA动态调控过程。对神经发育疾病队列的分析显示，携带致病变异的NSCs中心体蛋白在脑室周围异位症（periventricular heterotopia, PH）患者中显著富集。过表达与PH相关的突变剪接因子PRPF6可重现发育中小鼠大脑的脑室异位表型，揭示了中心体区域内MAP激酶SAD-A转录本的异常剪接是该表型的核心致病机制。综上，细胞类型特异性的中心体相互作用组解释了泛在蛋白的遗传变异如何引发脑特异性表型。从经荧光激活细胞分选（fluorescence-activated cell sorting, FACS）分选的电转染绿色荧光蛋白（green fluorescent protein, GFP）阳性细胞中提取RNA，使用Arcturus公司的提取缓冲液进行裂解，将样品加热至42℃后保存于-80℃冰箱。按照试剂盒说明书，使用Clontech公司的SMART-Seq v4超低起始量RNA测序试剂盒，从300 pg总RNA中反转录合成互补DNA（complementary DNA, cDNA）。在构建Illumina测序最终文库前，使用Covaris AFA系统对cDNA进行片段化处理，得到长度为200~500 bp的cDNA片段。片段化后的cDNA的质量与浓度通过安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）进行评估，随后使用Diagenode公司的MicroPlex文库制备试剂盒v2完成文库构建。最终文库通过安捷伦2100生物分析仪进行质量评估与定量，并在测序前额外使用Invitrogen™品牌的Quant-iT PicoGreen dsDNA检测试剂盒测定其浓度。将带有唯一条形码的文库进行多重测序混合，上样至同一测序通道，随后在Illumina公司的HiSeq 4000测序平台上进行150 bp双端深度测序，每个样本可产出约3000万条测序读段（reads）。