Comparative gene array analysis of TNF-alpha-induced MAPK and NF-kappaB signaling pathways between retinal ganglion cells and glial cells

Abstract

TNF-alpha has recently been identified to be a mediator of retinal ganglion cell (RGC) death, while glial cells are relatively protected against this death stimulus. To identify molecular mechanisms that control diverse responses of RGCs and glial cells to TNF-alpha, we studied differential gene expression between primary cultures of RGCs and glial cells exposed to TNF-alpha using cDNA array analysis of MAPK and NF-kappaB signaling pathways. Findings of this comparative analysis demonstrated differential regulation of various genes between RGCs and glial cells exposed to TNF-alpha. RT-PCR confirmed the differential expression of selected genes, and immunocytochemistry demonstrated gene products in cultured cells. Immunolabeling with phosphorylation site-specific antibodies also revealed differential post-translational modifications of selected proteins between cell types. Identification of signaling molecules differentially regulated in RGCs and glial cells can improve our understanding of the diverse cellular responses and provide targets for neuroprotective interventions in several neurodegenerative conditions.

Fig. 1.

Cultured retinal cells. Notice cellular processes of glial cells, and round or oval cell bodies of RGCs with a phase-bright appearance and branched neurites of uniform caliber and varying length. Scale bar; RGCs, 50 μm; glial cells, 40 μM.

Fig. 2.

An image profile of gene expression obtained using the non-radioactive GEArraye^™ cDNA chip. Genes expressed were easily identified as a specific hybridisation signal as appeared as an image of tetra-spots. In addition to 96 test genes, each chip contained two house-keeping genes (GAPDH and beta-actin as indicated) for normalization, two genes as positive controls, one PUC18 plasmid DNA as a negative control, and some blank spots as additional non-specific binding control.

Fig. 3.

Semi-quantitative RT-PCR confirmation of the differential expression of selected genes. Amplification of the selected genes was performed with their specific primers and predetermined number of cycles for linear amplification. Band intensities were then captured and analysed. The expression level of all selected genes was changed in the same direction as the changes measured by the microarrays. (A) A representative image of the expression profiles of selected genes in the control and treated samples of RGCs and glial cells. (B) Fold change in the expression of selected genes with TNF-α treatment. White bars, RGCs; black bars, glial cells. Data are presented as mean±sd. *Statistical significance (P<0.05, Student’s t test).

Fig. 4.

Immunolabeling of cultured cells with specific antibodies to demonstrate gene products. Presented are the phase-contrast and corresponding fluorescent images of RGCs and glial cells exposed to TNF-α for 24 hr. RGCs and glial cells exhibited immunolabelling for ERK, JNK, p38, and NF-κB. However, glial cells were immunolabelled with antibodies against phospho-ERK, phospho-p38, and phospho-NF-κB (p65), while RGCs exhibited immunolabelling for phospho-JNK and phospho-p38. Scale bar, 50 μm.