Mitochondrial transfer of the human ND6T14484C gene causes visual loss and optic neuropathy

Abstract

Purpose : To generate an LHON mouse model with the same genotype and phenotype as theT14484C mutation in hND6

Methods : The hND6T14484C gene fused with HA 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(HSPCSB) 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. Expression and function of the muthND6 were assessed by qPCR on laser microdissected cells, PCR, immunostaining, pattern electroretinography(PERG), spectral domain optical coherence tomography(SD-OCT) and respiratory function assay

Results : PCR with primers targeting the HSPCSB and hND6 amplified a fragment with expected size. Sanger sequencing showed the amplified fragment was hND6T14484C. qPCR showed that muthND6 DNA was 31 times that of endogenous mouse ND6(mND6) in the RGC layer, 1 time in the INC layer and 6 times in the ONC layer of injected mice. Retinal longitudinal sections reacted with antibodies against HA and mCherry showed the expression of hND6 and mCherry in the RGC layer. Serial SD-OCT showed optic nerve head swelling 1 to 4 months after injection of muthND6, followed by progressive thinning of the RGC layer and inner plexiform layer 8 to 14 months after injection. Compared to mCherry injected control mice, the PERG amplitude dropped at 1m and became significant at 3m(p=0.0023), 6m(p=0.0058) and 15m(p=0.031) postinjection. In addition, muthND6 injected mice showed a 24% decrease in complex I activity and an 18% decrease in complex I-dependent ATP synthesis in the optic nerve relative to mCherry injected control mice. These results were consistent with what reported that ND6T14484C is associated with a 20-30% reduction in complex I activity. Postmortem analysis at 15 months post injection showed marked atrophy of the entire optic nerve from globe to the optic chiasm in eyes injected with mutanthND6, but not in eyes injected with mCherry. Cell counting in the RGC layer revealed a significant loss of cell number in muthND6 injected mice(19336±1819cells/mm² versus 26851±4332cells/mm² of control mice; mean±SD)(p=0.0209)

Conclusions : human ND6T14484C results in visual loss and optic neuropathy characteristic of human LHON

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.