Transcriptional profile changes in dedifferentiating Müller glia in mammalian cell culture after treatment with an a7 nicotinic acetylcholine receptor agonist
Purpose: Eye drop application of PNU-282987, an a7 nAChR agonist, causes regeneration through activation of Müller glia (MG) in the adult rodent retina. The goals of this study are to examine the transcrptional changes through RNA-seq after treatment with PNU-282987 in mammalian MG cell culture that result in dedifferentiated MG that have a retinal progentior cell(RPC)-like fate and to vallidate RNA-seq differentially expressed genes with qRT-PCR and immunocytochemistry. Methods: Retinal pigment epithelium (RPE-J) cells were treated with PNU-282987 for 24 hours and then washed extensivley. These treated RPE cells were co-cultured via a transwell system with MG (rMC-1) cell culture for 0, 12, 24 and 48 hours. RNA was extracted from MG 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 and found 1050 significantly differentially expressed genes and 12 of these were validated with qRT–PCR to compare trends. Notably, the HB-EGF/Ascl1/Lin28 pathway was signficantly upregulated. ICC showed expression of RPC genes that were also seen in the RNA-seq profile. Conclusions: PNU-282987 activation of RPE causes signficant transcript profile changes in MG cell culture that leads to upregulation of the HB-EGF/Ascl1/Lin28 pathway that was seen with RNA-seq profiles and validated with qRT-PCR. Further, these gene expression changes lead to dedifferentation of MG to an RPC-like fate and expression of several retinal RPC markers were found in RNA-seq data set and was validated with immunocytochemistry. Our study is the first to show that activaiton of the a7 nAChR on RPE can lead to MG dedifferentiation and that the HB-EGF/Ascl1/Lin28 pathway is upregulated in these cells. Overall design: RPE-J cell lines were treated with DMSO (vehicle control) or PNU-282987 for 24 hours before being extensivley washed. RPE-J cells were allowed to grow in reponse to treatment for 24 hours before being put into a transwell co-culture system with rMC-1 cells. rMC-1 cells were allowed to be in contact with PNU treated RPE-J cells for 8, 12, 24, 48h or with DMSO treated RPE-J cells for 48h. rMC-1 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.