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Huijun Yuan, Hong Yu, John Guy; A Mutant Human T8993G ATP6 Transgenic Mouse Model With Visual Loss, Paralysis and Death. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):247.
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© ARVO (1962-2015); The Authors (2016-present)
To develop and characterize a transgenic mouse model of maternally inherited Leigh syndrome (MILS) and neurogenic ataxia & retinitis pigmentosa (NARP).
To generate MILS & NARP mice, the mutant human T8993G ATP6 gene was fused to the FLAG epitope (mutATP6FLAG) followed by mitochondrially encoded mCherry under the mitochondrial heavy strand promoter (HSP) into self-complementary AAV. ScAAV2-cox8mut ATP6FLAG+mcherry was microinjected into zygotes to generate 53 founders. Female A6M mice were backcrossed with normal males. Expression and integration of mutATP6 was assessed using PCR, sequencing, immunohistochemistry, blue native polyacrylamide gel electrophoresis, confocal laser scanning ophthalmoscopy (CLSO), respiration analysis and histopathology. Visual function and retinal structure was monitored using serial flash and pattern electroretinography (PERG) and OCT. Motor function was evaluated by Rota-Rod testing.
As in patients the mortality rate of A6M mice was high, 78%. Sequencing confirmed human T8993G ATP6 DNA. Immunostaining showed mCherry and Flag tagged human ATP6 with a perinuclear distribution and colocalization to the mitochondrial marker porin in retina, brain, spinal cord, lung, heart and muscles. Western blotting confirmed mutant human ATP6 in mitochondrial isolates. Quantification of band intensities of ATP6E in A6M mice was reduced two-fold compared to wild-type mice. In gel activity showed dissociation of complex V with a lower sized F1 band in A6M mice compared to the fully assembled active complex in wild-type mice (F0+F1). 2D-BN-PAGE using FLAG antibodies showed human mutant ATP6 in complex V of A6M mice. These mice developed ataxia and paralysis. Rota Rod testing showed latency-to-fall time was significantly decreased from 8 min to 0.1 min with increasing speed (4 to 14 rpm) in A6M mice compared to wild-type mice from 10 to 5 min at the age of 5 month (P<0.05). Video showed quadriparetic mice prior to their spontaneous deaths. Histopathology of the brain revealed a spongiform degeneration with components of demyelination and loss of neurons. PERG amplitudes decreased to 10 uV compared to 31 uV in wild-type mice (P=0.0001). FERG was 20% lower in A6M mice than controls, p=0.65.
A mutant human ATP6 mouse model was generated with a genotype and phenotype of T8993G NARP and MILS patients.
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