Abstract
Purpose :
To generate an LHON mouse model with the same genotype and phenotype as human G3460A mutation in ND1
Methods :
The hND1G3460 gene fused with MYC epitope tag was cloned into a self-complementary AAV backbone, under the control of the mitochondrial heavy strand promoter including 3 upstream conserved sequence blocks of the D-loop responsible for replication. Mitochondrial encoded Cherry(mCherry) was put downstream of the mutant gene to visualize transgene expression in live mice. After packaging with mito-targeted AAV. rAAV was injected into the adult mouse vitreous or was microinjected into fertilized oocytes to generate transgenic mitomice. Expression and function of the mutant gene were assessed by laser capture microdissection, qPCR, immunostaining, pattern electroretinography(PERG), and spectral domain optical coherence tomography(SD-OCT)
Results :
Mutant hND1 DNA was 25 times that of endogenous mouse ND1(mND1) in the RGC layer, 3 times in the INC layer and 2 times in the ONC layer of injected mice. Longitudinal sections of the retina reacted with antibodies against MYC and mCherry showed the expression of hND1 and mCherry in RGC layer. SD-OCT showed thinning of RGC layer and inner plexiform layer in hND1G3460A injected mice. Compared to control mice injected with mCherry, the PERG amplitude dropped by 50% in mice injected with hND1G3460A(p=8.6E-5) by 12 months after injection, and the rate of ATP synthesis was reduced by 28% in the optic nerve(p=0.02). Intravitreal injection of wildtype human ND1 rescued visual function caused by hND1G3460A and significantly preserve PERG amplitude compared to unrescued mice(p=0.02). In addition, we generated 109 mitomice carrying hND1G3460A using microinjections. qPCR showed that mutant hND1 was 10 times that of mND1 in the RGC layer, 3 times in the INC layer and 0.19 times in the ONC layer of the mitomice. Immunostaining demonstrated expression hND1 and mCherry in the RGC layer of mitomouse retina. PERG showed a progressive decrease of the amplitude in mitomice as the mice aged and the reduction became significant when the mice are 12 months old(p=0.003). SD-OCT revealed optic atrophy in the mitomice at 10 months of age. Complex I activity decreased by 80% in the brain of mitomice relative to wild type controls(p=0.04).
Conclusions :
Mice carrying hND1G3460A developed visual loss, optic atrophy and decreasing respiratory chain function. This mouse model may be used to test potential treatments for human LHON
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.