OPA1 Expression and Mitochondrial Distribution in the Rat Retina

Abstract

Abstract: : Purpose: OPA1 is a mitochondrial dynamin-related GTPase implicated in the formation and maintenance of the mitochondrial network. Mutations of OPA1 caused Autosomal Dominant Optic Atrophy (Kjer type; ADOA). In the present study, we asked why only retinal ganglions cells (RGCs) are affected by OPA1 mutations in spite of ubiquitous OPA1 distribution. One hypothesis could be that OPA1 is predominantly expressed in RGCs. To address this hypothesis, OPA1 expression in the rat retina was examined and compared to the mitochondrial distribution. Methods: Anti-OPA1 antibody (Ab) was affinity-purified from the serum of rabbits immunized with OPA1 residues 261-904. To evaluate the specificity of this anti-OPA1 Ab, extracts from Hela and human and rat tissues were analyzed by Western blot. Immunohistochemistry study in Hela cells was also performed with this Ab as well as with Ab to mitochondrial protein cytochrome b and Hsp60. Cryostat sections of rat retina (3-4 weeks) were incubated with the anti-OPA1 Ab and the Ab to mitochondrial protein cytochrome c. Fluorescence micrographs were obtained using confocal microscope. Results: In Western blots, the anti-OPA1 Ab specifically recognized OPA1 (82KD) in Hela cell mitochondrial fraction and in all examined human and rat tissues. Immunofluorescence study in Hela cells demonstrated the colocalization of OPA1 and Hsp60 or cytochrome b. In rat retinal sections, OPA1 staining was seen in ganglion cell layer (GCL), inner plexiform layer (IPL), outer plexiform layer (OPL), photoreceptor (PR) inner segments and pigment epithelium (PE), in which cytochrome c staining was also found. However, cytchrome c expression was stronger than that of OPA1 in PRs and PE, while in other layers, including ganglion cells, it was similar. Conclusions: In the rat retina, expression of OPA1 did not predominate in the RGCs as compared to other retinal cells. However, PR inner segments and PE showed milder expression of OPA1 than others layers when compared to the pattern of cytochrome c expression.

Keywords: ganglion cells • genetics • mitochondria