CNTF Induced Gene Expression in Rat Retina

Abstract

Abstract: : Purpose: Neurotrophic factors including CNTF promote survival of photoreceptor cells in a variety of degenerative insults. However photoreceptor cells do not have receptors for CNTF and recent studies have revealed a detrimental effect for CNTF on ERG B-wave amplitudes. The pathways of rescue by CNTF are unknown although Jak/Stat transduction pathways have been implicated. In this study we applied gene array technology to begin to unravel the genes induced in rat retina by increased CNTF secretion. Methods: Rat eyes received one of three treatments (1) CNTF: subretinally injected with lentivirus.EF1α.sDH-CNTF (5*10⁴ iu); (2) Inactive: subretinally injected with heat-inactivated lentivirus.EF1α.sDH-CNTF (5*10⁴ iu); (3) Control: no injection. Animals were sacrificed 7 days post injection and retinal RNA was extracted and prepared for hybridization according Affymetrix protocols. Rat genomic arrays (Affymetrix U34A) were hybridized and scanned. The intensity values were processed with the ProbeProfiler Software. Two pair-wise comparisons were performed: CNTF vs Control and CNTF vs Inactive. Lists of genes for which there was a significant treatment effect (p≤0.05) were compiled. To eliminate the effect of virus and injection procedure, genes not common to both lists and genes with a significant difference between Inactive and Control were eliminated. Only genes with a twofold or greater intensity increase were considered for further analysis. Results: K-Means clustering was performed on the filtered list following normalization of signal values. The final list consisted of 131 genes and expressed sequence tags (ESTs), of which 114 were upregulated and 16 downregulated. Genes were assigned to the following functional categories based on the gene orthology information when such information was available: apoptosis (4), transcription (26), signal transduction (23), protein degradation/synthesis (16), stress response (3), metabolism (13) and cellular organization (11). Seven days post injection, Stat3 in CNTF treatment increased 2.65 fold over Inactive and GFAP mRNA levels increased 2.0 fold (p-value = 0.002). MAPK, Fos and Erk levels 7 days post CNTF treatment were similar to Control. Conclusions: The use of microarrays facilitates the identification of co-regulated genes, which may function coordinately in metabolic or signal transduction pathways. Since our paradigm applies continuous expression of CNTF, it will begin to unravel the mechanism of CNTF rescue in retinal degenerations and conceivably lead to the development of the next generation of survival factor therapy.

Keywords: gene microarray • gene transfer/gene therapy • neuroprotection