Characterisation of a novel model of optic atrophy

Autosomal dominant optic atrophy (ADOA) is a slowly progressive ocular disorder associated with retinal ganglion cell loss and optic atrophy, manifesting with a variable reduction in visual acuity, colour vision defects and visual field loss. Genetic studies lead to the identification of mutations in the OPA1 gene on chromosome 3q28-qter, which is the main ADOA-causing gene. OPA1 is an ubiquitously expressed, nuclear dynamin-related GTPase, targeted to the inner mitochondrial membrane, which plays a role in mitochondrial fusion and ultimately has a protective role against apoptosis. In order to study the pathophysiology of ADOA, a new ENU-induced mutant mouse carrying a protein truncating nonsense mutation in Opal has been generated in our laboratory (B6 C3-OpalQ285STOP). The heterozygous mutation leads to approximately 50% reduction in Opal protein in mitochondria from retinal samples studied by Western blot. The homozygous mutation is embryonic lethal. The phenotype of heterozygous mutant mice is not associated with a severe neuro-degenerative process, in keeping with the variable and relatively mild phenotype observed in many patients with ADOA. Thus, visual phenotyping shows reduced visual function and electron microscopy reveals significant abnormalities in myelin bundles and increased autophagy with increasing age up to two years. Despite this, heterozygous adult mutants (up to 2 years of age) do not show age-dependent loss of retinal ganglion cell (RGC) bodies when studied on retinal sections stained with H&E and there is no visibly increased apoptosis in the retina (at 2 years of age) on TUNEL staining. In retinas from mice of 4-5 months of age proteolysis of Opal is normal and no statistically significant reduction in the average mitochondrial membrane potential is observed in the retina and brain. Quantitative RT-PCR on retinal samples does not indicate any differences in the expression of the six genes involved in the mitochondrial structure in mutant versus wild type mice. However, the observation of mitochondrial network in Mitotracker stained mouse embryonic fibroblasts (MEFs) at El3.5 demonstrates a higher incidence of fragmented mitochondria in MEFs taken from heterozygous mice, with a 15% increase in the number of opal +/- MEFs showing mitochondrial fragmentation (the average length of mitochondrial particles below 1 um), when compared to Opal +/- MEFs. However, their growth and survival after oxidative stress (with H2O2 and blue light) was not significantly affected when compared to the wild type control in a MTT assay. In conclusion, in Opal +/- mice, a reduction in the mitochondrial level of Opal caused a mild defect in mitochondrial morphology without any significant decline in those cellular functions investigated