IL-17 Signaling in Midbrain Neurons in Parkinson's Disease

To determine IL-17-induced global transcriptome changes in midbrain neurons derived from induced pluripotent stem cells (iPSC) from three sporadic Parkinson's disease (PD) patients and three age- and sex-machted controls, deep RNA sequencing (RNA-Seq) of IL-17-treated and untreated PD and control iPSC-dderived midbrain neurons was performed. Total RNA was isolated from untreated and IL-17-treated cells with the TruSeq RNA Sample Preparation Kit v2 (Illumina). RNA libraries were quantified using the KAPA SYBR FAST ABI Prism Library Quantification Kit (Kapa Biosystems) and cluster generation was performed on the cBot with the TruSeq SR Cluster Kit v3 (Illumina). The sequencing run was performed on a HiSeq 1000 instrument (Illumina) using the indexed, 50 cycles single read (SR) protocol and the TruSeq SBS v3 Kit (Illumina). Image analysis and base calling resulted in .bcl files that were then converted into .fastQ files by the CASAVA1.8.2 software. FastQ files were aligned to the human genome (hg19) using STAR.and annotated with gencode.v19. DESeq2 was used to determine differential expression. Criteria to determine significantly dysregulated genes were as follows: adjusted p-value below 0.05 and log2FC (fold change) of greater than one. Only genes with a mean expression value of greater than one RPKM (reads per kilobase per million mapped reads) throughout the dataset were considered. Control and PD samples were analyzed as two independent datasets. Upon IL-17 treatment only 17 genes were found to be dysregulated in controls but 125 genes were dysregulated in iPSC-derived midbrain neurons from PD patients. The 125 IL-17-dependent genes in PD iPSC-derived neurons separated the treated from untreated PD samples using an unsupervised, hierarchical clustering applying an Euclidean distance metric. More detailed study information can be found in Sommer A, Maxreiter F, Krach F, Fadler T, Grosch J, Maroni M, Graef D, Eberhardt E, Riemenschneider MJ, Yeo GW, Kohl Z, Xiang W, Gage FH, Winkler J, Prots I, Winner B. Th17 Lymphocytes Induce Neuronal Cell Death in a Human iPSC-Based Model of Parkinson's Disease. Cell Stem Cell. 2018 Jul 5;23(1):123-131.e6. doi: 10.1016/j.stem.2018.06.015. PMID: 29979986]]> Inclusion Criteria: Human midbrain neurons were generated from induced pluripotent stem cells (iPSC) from three sporadic Parkinson's disease (PD) patients and three sex- and age-matched controls. Patients were included into PD group based on the PD diagnosis. PD was diagnosed by board examined movement disorder specialists according to consensus criteria of the German Society of Neurology, which are similar to the UK PD Society Brain Bank criteria for diagnosis of PD. PD patients were defined as having a sporadic disease by the absence of known PD-causing genetic mutations (PARK 1-18). All PD patients were at early stages of PD (Hoehn & Yahr: 1-2) and were under dopaminergic substitution therapy. Control individuals were defined as those if they did not have any neurological disease. Exclusion Criteria: Controls and patients with a history of cancer, infectious or autoimmune diseases, or anti-inflammatory treatment within the past three months prior to the study inclusion were excluded.]]>为探究白细胞介素17（Interleukin-17, IL-17）诱导源自3名散发性帕金森病（Parkinson's disease, PD）患者及3名年龄、性别匹配对照的诱导多能干细胞（induced pluripotent stem cells, iPSC）来源中脑神经元的全转录组变化，研究人员对经IL-17处理与未处理的PD及对照iPSC来源中脑神经元实施了深度RNA测序（RNA-Seq）。 总RNA采用TruSeq RNA样本制备试剂盒v2（Illumina）从经IL-17处理及未处理的细胞中提取。RNA文库使用KAPA SYBR FAST ABI Prism文库定量试剂盒（Kapa Biosystems）完成定量，并在cBot平台上使用TruSeq SR Cluster Kit v3（Illumina）完成簇生成。测序在HiSeq 1000仪器（Illumina）上进行，采用带索引的50个循环单端读长（single read, SR）方案，并搭配TruSeq SBS v3试剂盒（Illumina）。图像分析与碱基识别生成了.bcl文件，随后通过CASAVA1.8.2软件将其转换为.fastQ文件。使用STAR软件将FastQ文件比对至人类基因组（hg19），并以gencode.v19进行注释。采用DESeq2进行差异表达分析。筛选显著差异表达基因的标准如下：校正后p值小于0.05，且log2FC（折叠变化）大于1。仅纳入在整个数据集中平均表达值大于1 RPKM（每百万比对读长的每千碱基读段数，reads per kilobase per million mapped reads）的基因。将对照样本与PD样本作为两个独立数据集进行分析。经IL-17处理后，对照样本中仅发现17个差异表达基因，而PD患者的iPSC来源中脑神经元中则存在125个差异表达基因。这125个受IL-17调控的PD患者iPSC来源中脑神经元基因，通过采用欧氏距离度量的无监督层次聚类算法，可将处理组与未处理组的PD样本清晰区分。更详细的研究信息可参阅：Sommer A, Maxreiter F, Krach F, Fadler T, Grosch J, Maroni M, Graef D, Eberhardt E, Riemenschneider MJ, Yeo GW, Kohl Z, Xiang W, Gage FH, Winkler J, Prots I, Winner B. Th17淋巴细胞在基于人类iPSC的帕金森病模型中诱导神经元细胞死亡. Cell Stem Cell. 2018年7月5日;23(1):123-131.e6. doi: 10.1016/j.stem.2018.06.015. PMID: 29979986. 纳入标准：本研究的人类中脑神经元源自3名散发性PD患者及3名年龄、性别匹配对照的iPSC。PD组患者的纳入基于确诊的PD：由经认证的运动障碍专科医师依据德国神经病学学会的共识标准（与英国PD学会脑库的PD诊断标准一致）进行诊断。PD患者被定义为无已知PD致病基因突变（PARK 1-18）的散发性病例。所有PD患者均处于PD早期阶段（Hoehn & Yahr分级：1-2级），且正在接受多巴胺能替代治疗。对照个体定义为无任何神经系统疾病者。 排除标准：有癌症、感染性或自身免疫性疾病病史，或在研究纳入前3个月内接受过抗炎治疗的对照个体及患者均被排除。