Abstract Title

Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.

RAD Assignment Number

1002

Presenter Name

Renuka Chaphalkar

Abstract

Purpose: Endothelin 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 endothelin-mediated changes in mRNA expression that occur at the translational level.

Methods: 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 endothelin-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 KAPA Stranded RNA-Seq Kit. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000/4000 for a pair end 150 run. The reads were first mapped to the latest UCSC transcript set using STAR version 2.4.1d and the gene expression level was quantified to annotation model (Partek E/M). Differentially expressed genes were identified using differential gene expression (GSA) algorithm in Partek. Genes showing altered expression with p < 0.05 and more than 2-fold changes were considered differentially expressed.

Results: Analysis of gene ontology of the changes in gene expression revealed a significant increase in expression of several mitochondrial genes including mitochondrial intermediate peptidase (MIPEP) (3.3-fold), cytochrome c oxidase assembly factor (SURF1) (8.7-fold) and Apolipoprotein O Like (APOOL) (7.5-fold). On the other hand, a decrease in expression of mitochondrial genes cytochrome c oxidase subunit 4I2 (8-fold), cytochrome c oxidase 6B2 (8-fold), and DNA polymerase gamma 2 (POLG2) (7-fold) was observed.

Conclusions: Analysis of the translatome offers a glimpse into de novo protein synthesis which is an important manifestation of changes in gene expression. Endothelin treatment produced changes in several key regulators of mitochondrial metabolism and bioenergetics which could be indicative of their involvement in neurodegeneration in glaucoma.

Research Area

Eye/Vision

Presentation Type

Poster

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Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.

Purpose: Endothelin 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 endothelin-mediated changes in mRNA expression that occur at the translational level.

Methods: 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 endothelin-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 KAPA Stranded RNA-Seq Kit. Different adaptors were used for multiplexing samples in one lane. Sequencing was performed on Illumina Hiseq3000/4000 for a pair end 150 run. The reads were first mapped to the latest UCSC transcript set using STAR version 2.4.1d and the gene expression level was quantified to annotation model (Partek E/M). Differentially expressed genes were identified using differential gene expression (GSA) algorithm in Partek. Genes showing altered expression with p < 0.05 and more than 2-fold changes were considered differentially expressed.

Results: Analysis of gene ontology of the changes in gene expression revealed a significant increase in expression of several mitochondrial genes including mitochondrial intermediate peptidase (MIPEP) (3.3-fold), cytochrome c oxidase assembly factor (SURF1) (8.7-fold) and Apolipoprotein O Like (APOOL) (7.5-fold). On the other hand, a decrease in expression of mitochondrial genes cytochrome c oxidase subunit 4I2 (8-fold), cytochrome c oxidase 6B2 (8-fold), and DNA polymerase gamma 2 (POLG2) (7-fold) was observed.

Conclusions: Analysis of the translatome offers a glimpse into de novo protein synthesis which is an important manifestation of changes in gene expression. Endothelin treatment produced changes in several key regulators of mitochondrial metabolism and bioenergetics which could be indicative of their involvement in neurodegeneration in glaucoma.