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Renuka Makarand Chaphalkar, Dorota L Stankowska, Shaoqing He, Raghu R Krishnamoorthy; RNA-Seq Analysis of the Translatome in Primary Retinal Ganglion Cells (RGCs) Treated with Endothelin-1. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5294.
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Endothelin-1(ET-1) treatment has been shown to produce increased cell death in primary RGCs, however the underlying changes in gene expression are not completely understood. The purpose of the study was to assess ET-1-mediated changes in mRNA expression that occur at the translational level.
Primary RGCs were isolated from post-natal day 5 rat pups by immunopanning with an antibody to Thy1.1. RGCs obtained were allowed to attach and maintained for 7 days for neurite outgrowth to occur. The RGCs were either untreated or treated with ET-1 (100 nM) for 24 h in trophic factor-free medium. Following brief incubation with cycloheximide to inhibit protein synthesis, total polysomes were isolated by magnesium precipitation and polysomal RNA was extracted using the Trizol reagent. Libraries for RNA-Seq were prepared with Clonetech SMARTer Stranded Total RNA-Seq (Pico) Kit. The workflow consists of first-strand synthesis, template switching, adaptor ligation, cleavage of ribosomal cDNA and PCR amplification. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000 for a single read 50 run. Data quality check was done on Illumina SAV. Trimmed mean of M-values (TMM) was used to normalize the gene expression. Genes with expression changes more than 1.5 fold with p < 0.05 were considered differentially expressed.
Analysis of differential gene expression revealed a significant change in expression of several genes involved in mitochondrial function, oxidative metabolism and cell survival pathways. A decrease in expression of mitochondrial ribosomal protein 30 (mrpl30) (4-fold), COX17 cytochrome c oxidase copper chaperone (Cox17) (3-fold), and ATP synthase, H+ transporting mitochondrial F0 complex (Atp5h)(3-fold) was found. On the other hand, an increase in expression of glucose-6-phosphate dehydrogenase (5-fold), Forkhead box O1 (Foxo1) (986-fold), mitogen-activated protein kinase kinase kinase 11(Map3k11) (40-fold) and modulator of apoptosis (Moap1) (6255-fold) was observed.
Treatment of RGCs with ET-1 altered the expression of gene affecting mitochondrial metabolism, bioenergetics and cell survival which could be indicative of mechanisms underlying ET-1 mediated neurodegeneration in glaucoma.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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