Development of Techniques for Analysis of the Human Retinal Ganglion Cell Transcriptome: Application to the Role of Calcium in RGC Death in Glaucoma

Ma, Ning (2013) Development of Techniques for Analysis of the Human Retinal Ganglion Cell Transcriptome: Application to the Role of Calcium in RGC Death in Glaucoma. Doctoral thesis, University of East Anglia.

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Abstract

Purpose: Irreversible retinal ganglion cell (RGC) death is the reason for visual loss in
glaucoma. However, the mechanisms of RGC death remain unclear. The aim of this research
was to develop methods to study mRNA expression profiles in human RGCs, then to use the
data to investigate the role of calcium in RGC death.
Methods: A planar sectioning technique was developed to isolate mRNA from serial sections
of the human retina. QRT-PCR of neuronal markers validated the technique. Global gene
expression analysis, using Illumina arrays, compared expression in the retina ganglion cell
layer (RGCL) and entire macula (Mac). Immunohistochemistry and QRT-PCR validated
gene array data. RGC death was investigated using a simulated ischemia (oxygen glucose
deprivation, OGD) model in human organotypic retinal cultures (HORCs). Cell survival was
measured by LDH, and RGC loss by immunohistochemistry and QRT-PCR. Western blot
assessed proteases.
Results: The sectioning technique developed enabled isolation of relatively large quantites of
high quality mRNA from 20μm retinal sections from the macular region of the human retina.
Marker genes for retinal neurons verified accurate profiling of gene expression across the
retina. Gene arrays provided a list of genes that were most enriched in the RGCL. AHNAK2
and HSPA1B were the two most enriched genes in the RGCL. CAPN1 (calpain 1), a calciumdependent
cysteine proteases, was in the gene list. Its expression was confirmed to be mainly
in the inner retina. OGD caused calpain activation and induced RGC death. Two TRP
channels, TRPM-2 and TRPC-3, which mediate Ca2+ influx, were found that predominantly
expressed in the RGCL. Involvement in RGC death in the OGD model using the TRP
inhibitor ACA could not be confirmed.
Conclusions: The technique developed has enabled determination of the human RGCL
transcriptome and has allowed expression profiling of gene of interest across the retina. This
could prove to be a powerful tool in the investigation of pathways involved in
neurodegeneration in the retina.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Pharmacy
Depositing User: Mia Reeves
Date Deposited: 12 Mar 2014 15:17
Last Modified: 12 Mar 2014 15:17
URI: https://ueaeprints.uea.ac.uk/id/eprint/48120
DOI:

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