Purpose: Retinas of albino rats born and raised in bright cyclic light (300-800 lux) are less susceptible to light-induced apoptosis than retinas of animals born and raised in dim cyclic light (5 lux). In this study, the objective was to study the mechanisms of neuroprotection in the bright cyclic light-reared retina by identification of differentially expressed genes with differential display (DD)-PCR.
Methods: Albino rats were born and raised in 5- or 400-lux cyclic light (12 hours on/off). At 6 to 8 weeks of age, animals were either killed to harvest retinas or exposed to 1700 lux illumination for 12 or 24 hours. The neuroprotection of 400-lux cyclic light rearing was evaluated by DNA fragmentation and quantitative histology. The differentially expressed candidate genes were identified by DD-PCR. Northern blot analysis was used to quantitate differential expression of selected genes. Differential expression of protein was determined by Western blot and enzyme activity analysis. Cellular localization of transcripts was determined by in situ hybridization.
Results: DNA fragmentation and quantitative histology results indicated that 400-lux cyclic light rearing protected the retina from light-induced apoptosis compared with 5-lux cyclic light rearing. DD-PCR analysis showed that a 283-bp expressed sequence tag (EST) was downregulated in retinas of rats raised from birth in 400-lux cyclic light. A BLAST search identified the EST as the 3'-terminal sequence of mitochondria-encoded NADH dehydrogenase subunit 3 (ND-3). Northern blot analysis showed that the EST hybridized to two mRNA transcripts, the larger of which was confirmed to encompass the adenosine triphosphate (ATP) synthase subunit 6 (ATPase-6), cytochrome c oxidase subunit III (CO-III), ND-3, and tRNA-Gly. Northern blot analysis demonstrated that CO-III and ATPase-6 were downregulated 1.8- and 2.3-fold by 400-lux cyclic light compared with 5-lux cyclic light, respectively; however, there was no change in cytochrome c oxidase subunit I and II (CO-I and -II) or in 12S ribosomal RNA (12S rRNA), a mitochondrial housekeeping gene. Western blot analysis using anti-CO-III antibody showed more CO-III protein in retinal mitochondria from dim-light-raised rats. The enzyme activity of CO was two times higher in retinal homogenates from dim-light-raised rats than those from bright-light-raised rats. In situ hybridization using a (35)S-labeled CO-III probe showed that the CO-III transcript was present and downregulated in most of the retinal layers of bright-light-reared animals.
Conclusions: Rearing in cyclic light at 400-lux downregulates the expression of ATPase-6, CO-III, and ND-3 compared with rearing in 5-lux cyclic light. The authors hypothesize that these changes are adaptive responses to light stress that provide neuroprotection to retinal cells by elevating the level of stress-related factors and reducing the level of oxidized cytochrome c, the form that activates the apoptotic cascade of cell death.