Abstract
Purpose :
To evaluate the vision function of a transgenic mouse harboring human mutant ATP6 (A6) which was created by microinjection of mito-targeted AAV2 carrying sc-HSP-mutATP6FLAG+mcherry into mouse zygotes. The transgenic A6 mice exhibit the most common features of leigh syndrome and neuropathy, ataxia, and retinitis pigmentosa (NARP) including premature death, mortality high to 78%, paralysis, seizures, tumor and involvement of multiple organs.
Methods :
PCR and DNA sequencing were used to confirm human mutant ATP6 gene in A6 mice. Laser capture microdissection was applied to collect positive mCherry cells from retina. Taqman quantitative PCR was performed to detect mutant ATP6 copy number and the ratio of human mutant ATP6 to mouse endogenous ATP6. Immunofluorescences and two dimensional native/SDS-PAGE western blot were performed to detect human mutant ATP6 expression with Flag, mCherry and Porin or ATP5A antibodies. Mouse vision function was assessed by Pattern and Flash ERG. H&E staining was performed to analyze A6 mouse retina pathology.
Results :
Human mutant ATP6 gene was amplified by PCR from A6 mouse brain, spinal cord and liver. The mutation of human ATP6 was further confirmed by DNA sequencing. Taqman quantitative PCR following laser capture microdissection showed 26% of human mutant ATP6 compared to mouse endogenous wild type ATP6 in retina ganglion cells. Immunofluorescences showed human mutant ATP6 was expressed and co-localized with mitochondrial marker porin in A6 mouse tissues including RGC, brain and spinal cord. Two dimensional native/SDS-PAGE western blot suggested that the mutant hATP6 assembled into murine mitochondrial complex V in A6 mitochondria. Complex V activity in gel assay showed complex V activity significantly decreased in A6 mouse. Patten and flash ERG showed that the vision function was significantly decreased in A6 mice. Histology analysis showed significantly photoreceptor cell death or complete loss.
Conclusions :
Retina degeneration and photoreceptor cell death in a transgenic mouse model of Leigh syndrome and NARP caused by human mutant T8993G in subunit 6 in mitochondrial ATP synthase.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.