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

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.

Name dc:type.qualificationname

phd

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of East Anglia

Year dc:date.issued

2013

Author dc:creator

• Ma, Ning

Language dc:language

en