Transcriptional profile changes in retinal pigment epithelial cell culture after treatment with an α7 nicotinic acetylcholine receptor agonist. Transcriptional profile changes in retinal pigment epithelial cell culture after treatment with an α7 nicotinic acetylcholine receptor agonist

Purpose: Eye drop application of PNU-282987, an α7 nAChR agonist, causes regeneration through development of Müller derived progeintor cells (MDPCs) that are generated as a result of signaling from activated retinal pigment epitheilal (RPE) cells . The goals of this study are to examine the transcrptional changes through RNA-seq after treatment with PNU-282987 in mammalian RPE cell culture that lead to the dedifferentation and generation of MDPCs in the adult mammalian retina. To validate the RNAseq findings, RT-qPCR was used. Finally, several genes that were identified were knocked out in RPE culture through a novel CRISPR/Cas12 system and the resulting activated supernatant was assayed for it's ability to cause neurogensis in the adult rodent retina. Methods: Retinal pigment epithelium (RPE-J) cells were treated with DMSO (vehicle control), nicotine (an α7 nAChR agonist that does not cause regeneration as a control), or PNU-282987, or MLA (an α7 nAChR antagonist as a control). The cells were treated for either 30 minutes, 1 hour, 3 hours, 8 hours, or 12 hours. RNA was extracted from RPE-J cells using Zymo Direct-zol RNA miniprep kit and mRNA proflies were generated by GeneWiz with the Illumina NextSeq 550 high-output platform. Results: Using an optimized data analysis workflow, GeneWiz mapped about 30 million sequence reads per sample to the rat genome (Rnor6.0) and identified over 15,000 transcripts. Deseq2 analysis was performed to determine log 2 fold changes in gene expression compared to DMSO control, nicotine control, and MLA control, and found over 450 significantly differentially expressed genes. Twelve genes were validated with qRT–PCR to compare trends and were found to be consistent. Eight genes identified through RNA-seq involved in inflammation response, receptor signaling, WNT signaling and early retinal development were knocked out using CRISPR/Cas12 in RPE-J culture. Intravitreal injections of KO lines supernatant after treatment with PNU-282987 was assayed for invovlment of these genes in the adult neuroregeneration response. Conclusions: PNU-282987 activation of RPE causes signficant transcript profile changes in RPE-J cell culture that leads to significant differentially expressed genes as seen in RNA-seq profiles and validated with qRT-PCR. Further, several genes identified in the RNA-seq were found to be necessary for the neurogenic response as seen with generation of KO lines of RPE. Our study is the first to show that activaiton of the α7 nAChR on RPE leads to genetic changes sufficient to induce adult mammalian neurogenesis in the rodent retina and that several novel genes are invovled in this pathway in mammals as compared to other vertebrate models of retinal regeneration. Overall design: RPE-J cell lines were treated with DMSO (vehicle control), nicotine (positive receptor activation control), MLA (negative control) or PNU-282987 for 30 minutes, 1 hour, 3 hours, 8 hours, or 12 hours before collection. RPE-J cells were trypsinized and collected for RNA extraction. RNA was shipped overnight on dry ice to GeneWiz for RNA-seq analysis resulting in Log2 fold changes for differentially expressed genes.

研究目的：α7烟碱型乙酰胆碱受体（α7 nAChR）激动剂PNU-282987可通过激活视网膜色素上皮（retinal pigment epithelium, RPE）细胞的信号通路，诱导米勒细胞源性前体细胞（Müller derived progenitor cells, MDPCs）发育，进而实现视网膜再生。本研究旨在通过RNA测序（RNA-seq）分析哺乳动物RPE细胞经PNU-282987处理后的转录组变化，以探究其如何诱导成年哺乳动物视网膜中米勒细胞源性前体细胞的去分化与生成。为验证RNA-seq分析结果，本研究采用实时定量聚合酶链反应（RT-qPCR）进行验证。最后，本研究通过新型CRISPR/Cas12系统对RPE细胞培养体系中筛选得到的若干基因进行敲除，并检测其激活后的培养上清液诱导成年啮齿类视网膜神经发生的能力。

实验方法：将视网膜色素上皮细胞（RPE-J）分别用二甲基亚砜（DMSO，溶剂对照）、尼古丁（一种无法诱导再生的α7烟碱型乙酰胆碱受体激动剂，作为受体激活对照）、PNU-282987或MLA（α7烟碱型乙酰胆碱受体拮抗剂，作为阴性对照）处理，处理时长分别设置为30分钟、1小时、3小时、8小时及12小时。采用Zymo Direct-zol RNA小量提取试剂盒从RPE-J细胞中提取总RNA，由GeneWiz公司利用Illumina NextSeq 550高通量输出平台完成mRNA表达谱测序。

实验结果：通过优化后的数据分析流程，GeneWiz将每个样本约3000万条序列读数比对至大鼠基因组（Rnor6.0），共鉴定到超过15000个转录本。采用DESeq2软件进行差异表达分析，以DMSO对照组、尼古丁对照组及MLA对照组为参照，计算基因表达的log₂倍数变化，最终鉴定到超过450个显著差异表达基因。选取12个基因进行qRT-PCR验证，结果显示其表达趋势与RNA-seq分析结果一致。通过RNA-seq筛选得到的8个参与炎症应答、受体信号传导、WNT信号通路及早期视网膜发育的基因，在RPE-J细胞培养体系中利用CRISPR/Cas12系统进行敲除。将经PNU-282987处理后的敲除细胞系培养上清液进行玻璃体内注射，以检测上述基因在成年神经再生应答中的作用。

研究结论：PNU-282987对RPE细胞的激活可诱导RPE-J细胞培养体系产生显著的转录组谱变化，表现为RNA-seq分析鉴定到的大量差异表达基因，该结果经qRT-PCR验证。进一步研究发现，RNA-seq筛选得到的若干基因对于神经源性应答是必需的，该结论通过RPE细胞基因敲除实验得到证实。本研究首次证实，激活RPE细胞表面的α7烟碱型乙酰胆碱受体可引发足够的遗传变化，从而诱导啮齿类成年哺乳动物视网膜发生神经发生；且与其他脊椎动物视网膜再生模型相比，哺乳动物该通路中存在多个全新的功能基因。

实验整体设计：RPE-J细胞系分别经DMSO（溶剂对照）、尼古丁（受体激活阳性对照）、MLA（阴性对照）或PNU-282987处理30分钟、1小时、3小时、8小时及12小时后收集细胞。采用胰酶消化收集RPE-J细胞，用于RNA提取。提取的RNA经干冰冷链运输至GeneWiz公司进行RNA-seq分析，最终得到差异表达基因的log₂倍数变化数据。