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Transcriptional profile changes in dedifferentiating Müller glia in mammalian cell culture after treatment with an α7 nicotinic acetylcholine receptor agonist

ABSTRACT: Purpose: Eye drop application of PNU-282987, an α7 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 α7 nAChR on RPE can lead to MG dedifferentiation and that the HB-EGF/Ascl1/Lin28 pathway is upregulated in these cells.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE151477 | GEO | 2020/09/10

REPOSITORIES: GEO

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Project description: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.