Characterisation of Retinal Degeneration in Idh3a Mutant Mice

Abstract

Purpose : Isocitrate dehydrogenase (IDH) is an enzyme required for the production of α-ketoglutarate from isocitrate. IDH3 generates the NADH used in the mitochondria for ATP production, and is a tetramer made up of two α, a β and a γ subunit. Loss of function and missense mutations in both IDH3A and IDH3B have previously been implicated in families exhibiting retinal degeneration. A child homozygous for an IDH3A mutation has also been described with severe neurological disease, including retinal degeneration. Using mouse models we have investigated the role of IDH3 in retinal disease and mitochondrial function.

Methods : Loss of function mouse models were developed using CRISPR/cas-9 technology. Electroretinogram analysis was utilised to track the progress of the retinal degeneration in mouse models; followed by immuno fluorescence and histology to pin point the onset and quantify cell loss during. Mitochondrial functional analysis was done using Agilent Seahorse technology on mouse embryonic fibroblast cells, cultured from mutant lines.

Results : We identified mice with late-onset retinal degeneration in a screen of ageing mice carrying ENU-induced mutations. Low-resolution mapping followed by whole genome sequencing found a missense mutation in the α-subunit gene, Idh3a (E229K). Mice homozygous for this mutation, exhibit signs of retinal stress, indicated by GFAP staining, as early as 3 months and photoreceptor degeneration from 7 months, but, as with the human IDH3A mutations, no other tissues appear to be affected. We used CRISPR/Cas9 technology to produce a knockout of Idh3a and find that homozygous mice do not survive past early embryogenesis. Idh3a^E229K/- compound heterozygous mutants exhibit a more severe retinal degeneration when compared to Idh3a^E229K/E229K, showing severely deteriorated ERG responses by 2 months, accompanied by photoreceptor loss and GFAP staining. Analysis of mitochondrial function, using Agilent Seahorse technology, on MEF mutant cell lines highlighted a reduction in both mitochondrial maximal respiration and reserve capacity levels in both Idh3a^E229K/E229K and Idh3a^E229K/- cells.

Conclusions : It has been previously reported that the retina operates with a limited mitochondrial reserve capacity and we suggest that this, in combination with the reduced reserve capacity in mutants, explains the degenerative phenotype observed

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