Mechanisms Underlying Retinal Ganglion Cell-Specific Superoxide Production: Electron Transport Chain Components

Abstract

Purpose:: We previously showed that superoxide production differed between the retinal ganglion cell (RGC)-like RGC-5 cell line and brain mitochondria. To elucidate mechanisms for this difference, we quantified mitochondrial electron transport chain (METC) components, the primary source of cellular superoxide. To determine whether RGC-specific superoxide production was due to differences between cell lines and fresh tissue, we also compared superoxide production in the RGC-5 line and the SK-N-AS neuroblastoma line in the presence of METC inhibitors.

Methods:: Mitochondrial and cytosol-enriched samples were prepared using standard protocols. Complexes I-V of the METC in mitochondrial preparations were quantified by western blot. Band densities were compared between cell types using NIH ImageJ software. To indirectly assess superoxide production in mitochondrial preparations we used the H₂O₂ fluorescent probe Amplex Red. Fluorescence was recorded after the addition of METC complex-specific substrates, glutamate and malate (GLU/MAL) for complex I and succinate (SUCC) for complex III, and subsequent treatment with the appropriate complex inhibitor, rotenone (ROTE) and antimycin A (ANTI A) for complexes I and III respectively.

Results:: RGC-5 and brain mitochondria showed similar amounts of complexes II-V. However, levels of complex I in brain mitochondria were 58 fold higher than in RGC-5 mitochondria. Band density comparison revealed an 11-fold and 5-fold higher level of complexes III and IV respectively in RGC-5 mitochondria in comparison to SK-N-AS. SK-N-AS derived mitochondria increased H₂O₂ production after the addition of the complex I substrates GLU/MAL (0.552±0.044 to 0.967±0.029 in nM H₂O₂/mL/mg; p < 0.001) and decreased H₂O₂ production after subsequent treatment with ROT (0.703±0.020;p < 0.001). RGC-5-derived mitochondria increased H₂O₂ in the presence of GLU/MAL only (0.064±0.008 to 0.108±0.008; p = 0.003). SK-N-AS-derived mitochondria decreased H₂O₂ production in the presence of SUCC (0.625±0.032 to 0.512±0.020).

Conclusions:: RGC-specific differences in superoxide-producing sites in mitochondria may provide an explanation for the RGC-specific cell death observed in certain mutations of mitochondrial DNA, e.g. Leber’s hereditary optic neuropathy.

Keywords: oxidation/oxidative or free radical damage • mitochondria • ganglion cells