Abstract
Purpose :
The mitochondrial respiratory chain (RC) is the site of ATP synthesis and reactive oxygen species generation. The mitochondrial gene cytochrome c oxidase 1 (CO1) encodes a crucial subunit of the RC Complex IV. A CO1 missense mutation (V421A) generated on a C57BL/6 background demonstrates ~ 50% reduction in Complex IV activity in the heart, liver, muscle, and brain (PMID18276892). Evidence of mitochondrial myopathy includes ragged red fibers, altered mitochondrial morphology, and cardiomyopathy. We characterized the ocular phenotype in mice homoplasmic for this mutation.
Methods :
Neural retinas were isolated from CO1 mutants and age and sex-matched WTs. Retinal Complex IV activity was assessed using a colorimetric microplate assay (Abcam). QPCR was used to compare mRNA levels of the retinal ganglion cell (RGC) marker Brn3a and the antioxidant superoxide dismutase 2 (SOD2) from 18-month-old animals. Optokinetic responses (OKR) and pattern electroretinogram (pERG) were used to assess visual function in 10 months-old animals. RGCs were labeled with Brn3a on retina flat mounts and density was assessed in mutants and WTs.
Results :
Complex IV activity was reduced by 44% in the retinas of CO1 mutant mice (Fig.1A).Brn3a expression was reduced in CO1 mutants suggestive of RGC loss and SOD2 expression was increased suggestive of increased oxidative stress(Fig.1B). Consistent with this, averaged RGC density from 12 retinal fields was decreased by 17% in male but not female CO1 mutants (Fig1.C and D: 40x representative images centered 250mm from the optic disc in WT and CO1 mutants). OKRs were reduced in 10-month-old mutants compared to WTs (0.316 ± 0.026 versus 0.360 ± 0.016, respectively; n = 10; p < 0.0001). Preliminary pERG testing suggested delayed implicit time and decreased P1 amplitude in mutants compared to WTs (Fig.1E).
Conclusions :
Results support decreased Complex IV activity, decreased visual function, and increased oxidative stress in the retinas of CO1 mutants of both sexes. Results also demonstrate reduced number of RGCs and decreased RGC-specific gene expression in male mutants suggestive of RGC death associated with impaired Complex IV function. Interestingly, female mutants were not similarly affected. A sex difference favoring more severe disease in males has been described in mitochondrial diseases, including those causing optic neuropathies.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.