June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.
Author Affiliations & Notes
  • Renuka M Chaphalkar
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fortworth, Texas, United States
  • Dorota L Stankowska
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fortworth, Texas, United States
  • Raghu R Krishnamoorthy
    North Texas Eye Research Institute, University of North Texas Health Science Center, Fortworth, Texas, United States
  • Footnotes
    Commercial Relationships   Renuka Chaphalkar, None; Dorota Stankowska, None; Raghu Krishnamoorthy, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 238. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Renuka M Chaphalkar, Dorota L Stankowska, Raghu R Krishnamoorthy; Endothelin Mediated Changes in Gene Expression Determined by RNA-sequencing of the Translatome in Primary Retinal Ganglion Cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):238.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
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.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×