Transcriptome and proteome profiling of neural stem cells from the human subventricular zone in Parkinson's disease

It is currently accepted that the human brain has a limited neurogenic capacity and an impaired regenerative potential. We have previously shown the existence of CD271-expressing neural stem cells (NSCs) in the subventricular zone (SVZ) of Parkinson’s disease (PD) patients, which proliferate and differentiate towards neurons and glial cells in vitro. To study the molecular profile of these NSCs in detail, we performed RNA sequencing and mass spectrometry on CD271+ NSCs isolated from human post-mortem SVZ and on homogenates of the SVZ. CD271+ cells were isolated through magnetic cell separation (MACS). We first compared the molecular profile of CD271+ NSCs to the SVZ homogenate from control donors to assess the CD271+ NSCs gene signature and finally made a comparison between controls and PD patients to establish a specific molecular profile of NSCs and the SVZ in PD. While our transcriptome analysis did not identify any differentially expressed genes in the SVZ between control and PD patients, our proteome analysis revealed several proteins that were differentially expressed in PD. Some of these proteins are involved in cytoskeletal organization and mitochondrial function. Transcriptome and proteome analyses of NSCs from PD revealed changes in the expression of genes and proteins involved in metabolism, transcriptional activity and cytoskeletal organization. Our results not only confirm pathological hallmarks of PD (e.g. impaired mitochondrial function), but also suggest that NSCs may transit into a primed-quiescent state, that is in an “alert” non-proliferative phase in PD. From post-mortem human SVZ of control and Parkinson disease donors we isolated CD271+ NSCs and Cd11b+ microglia by MACS and the whole SVZ to generate RNA sequencing libraries using Celseq2 method. We aimed for low coverage sequencing (~2 million mapped to the coding regions) per sample to investigate the gross changes in the transcriptome. Libraries (rpi small primer) were sequenced in 3 runs, 2 on an Illumina NextSeq500 using 75-bp paired-end sequencing at the Utrecht Seuqencing center (USEQ) and the third on a HiSeq4000 using 150-bp paired-end sequencing at Genomescan. All the samples were mapped in a single run to an average depth of ~10 million reads per sample. Reads were mapped to the latest human coding transcriptome using bwa, normalized and analyzed using the standard DESEQ2 package.

目前学界公认，人类大脑具备有限的神经发生能力与受损的再生潜能。我们此前已证实，帕金森病（Parkinson’s Disease, PD）患者脑室下区（subventricular zone, SVZ）中存在表达CD271的神经干细胞（neural stem cells, NSCs），这类细胞在体外可增殖并分化为神经元与神经胶质细胞。为深入解析这类神经干细胞的分子特征，我们对从人类死后脑室下区分离得到的CD271阳性神经干细胞，以及脑室下区组织匀浆进行了RNA测序与质谱分析。CD271阳性细胞通过磁性细胞分选（magnetic cell separation, MACS）完成分离。我们首先将CD271阳性神经干细胞的分子特征与对照供体的脑室下区组织匀浆进行比对，以明确CD271阳性神经干细胞的基因表达特征；最终通过对比对照组与帕金森病患者样本，确立帕金森病患者中神经干细胞与脑室下区的特异性分子图谱。尽管转录组分析未在对照组与帕金森病患者的脑室下区中鉴定出任何差异表达基因，但蛋白质组分析揭示了多种在帕金森病患者中存在差异表达的蛋白质，其中部分参与细胞骨架组织与线粒体功能调控。对帕金森病患者神经干细胞的转录组与蛋白质组分析显示，参与代谢、转录活性及细胞骨架组织的基因与蛋白质表达发生了改变。我们的研究结果不仅验证了帕金森病的病理标志（如线粒体功能受损），还提示帕金森病患者体内的神经干细胞可能进入了一种预激活静息状态，即处于“警觉性”的非增殖阶段。我们从对照及帕金森病供体的死后人类脑室下区中，通过磁性细胞分选分离得到CD271阳性神经干细胞与CD11b阳性小胶质细胞，并获取完整脑室下区组织，采用Celseq2方法构建RNA测序文库。我们旨在对每个样本进行低覆盖度测序（约200万条reads比对至编码区），以探究转录组的整体变化。文库（使用rpi小引物）分三批完成测序：两批在乌得勒支测序中心（Utrecht Sequencing Center, USEQ）的Illumina NextSeq500平台上采用75 bp双端测序，第三批在Genomescan实验室的HiSeq4000平台上采用150 bp双端测序。所有样本统一在一次分析中完成比对，平均比对深度约为每个样本1000万条reads。我们使用bwa工具将reads比对至最新版人类编码转录组，随后采用标准DESEQ2软件包完成数据标准化与分析。