© 2018. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2018) 11, dmm036426. doi:10.1242/dmm.036426 RESEARCH ARTICLE Mouse Idh3a mutations cause retinal degeneration and reduced mitochondrial function Amy S. Findlay1, Roderick N. Carter2, Becky Starbuck3, Lisa McKie1, Klára Nováková1, Peter S. Budd1, Margaret A. Keighren1, Joseph A. Marsh1, Sally H. Cross1, Michelle M. Simon3, Paul K. Potter3, Nicholas M. Morton2 and Ian J. Jackson1,4,* ABSTRACT these diseases have a wide range of pathologies (Koopman et al., Isocitrate dehydrogenase (IDH) is an enzyme required for the 2012). Common to many of the diseases, however, are neurological production of α-ketoglutarate from isocitrate. IDH3 generates the or neuromuscular manifestations, including retinal disease. We have NADH used in the mitochondria for ATP production, and is a tetramer identified a missense mutation in the gene encoding a subunit of the made up of two α, one β and one γ subunit. Loss-of-function and tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase 3 missense mutations in both IDH3A and IDH3B have previously been (IDH3) as causing a retinal degeneration phenotype in mice. There implicated in families exhibiting retinal degeneration. Using mouse are three isocitrate dehydrogenase isozymes in mammals. IDH1 and models, we investigated the role of IDH3 in retinal disease and IDH2 are both homodimers that catalyse the decarboxylation of α mitochondrial function. We identified mice with late-onset retinal isocitrate to -ketoglutarate with the concomitant reduction of NADP degeneration in a screen of ageing mice carrying an ENU-induced to NADPH. IDH1 is cytoplasmic, whereas IDH2 is localised to the mutation, E229K, in Idh3a. Mice homozygous for this mutation exhibit mitochondria. No inherited disease has been associated with IDH1 signs of retinal stress, indicated by GFAP staining, as early as mutations, but mutations in IDH2 cause a metabolic disease, D-2- 3 months, but no other tissues appear to be affected. We produced a hydroxyglutaric aciduria, with a range of features including epilepsy, knockout of Idh3a and found that homozygous mice do not survive hypotonia and other neurological manifestations (Kranendijk et al., past early embryogenesis. Idh3a−/E229K compound heterozygous 2010). IDH1 or IDH2 has been found to be mutated in a high mutants exhibit a more severe retinal degeneration compared with proportion of glioblastomas and other tumours of the nervous system. α β Idh3aE229K/E229K homozygous mutants. Analysis of mitochondrial By contrast, IDH3 is a heterotetramer, composed of two ,one and γ function in mutant cell lines highlighted a reduction in mitochondrial one subunit, encoded by three genes. IDH3 is located in the α maximal respiration and reserve capacity levels in both mitochondria, where the conversion of isocitrate to -ketoglutarate is Idh3aE229K/E229K and Idh3a−/E229K cells. Loss-of-function Idh3b necessary for the TCA cycle to progress and generate NADH, which mutants do not exhibit the same retinal degeneration phenotype, feeds into oxidative phosphorylation to generate ATP. No recurrent with no signs of retinal stress or reduction in mitochondrial respiration. mutations in any of the IDH3 genes have been described in tumours. It has previously been reported that the retina operates with a limited A number of patients have been described with a variety of mitochondrial reserve capacity and we suggest that this, in mutations in IDH3A (Fattal-Valevski et al., 2017; Pierrache et al., combination with the reduced reserve capacity in mutants, explains 2017). A patient with the most severe of these, a homozygous missense the degenerative phenotype observed in Idh3a mutant mice. mutation (p.Pro304His), exhibited neurological defects from birth, possibly due to a failure during development, as well as retinal This article has an associated First Person interview with the first degeneration. However, the patient showed no symptoms of muscle author of the paper. weakness, typically associated with mitochondrial deficiencies. The remaining patients, with bi-allelic variants, all exhibited childhood KEY WORDS: Krebs cycle, Mouse model, Retinitis pigmentosa onset of retinal degeneration and some had pseudocoloboma of the macula. Pseudocoloboma is not a developmental defect but is caused INTRODUCTION by degeneration of the retina. Seven different alleles were found in four Mitochondrial diseases are highly diverse: over 220 different genes different families all predicted to be pathogenic, including two that have been identified that can cause diseases of the mitochondria, and could potentially cause nonsense-mediated decay. The other five, all missense mutations, are anticipated to be damaging. Two families 1MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, with homozygous mutations in IDH3B, and which exhibited University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK. 2Molecular non-syndromic retinal degeneration, have also been described Metabolism Group, Centre for Cardiovascular Sciences, Queens Medical Research Institute, University of Edinburgh, Edinburgh EH16 4TJ, UK. 3MRC Mammalian (Hartong et al., 2008). None of these patients showed symptoms of Genetics Unit, MRC Harwell Institute, Harwell Campus, Oxfordshire OX11 0RD, UK. mitochondrial dysfunction other than retinitis pigmentosa (RP). 4Roslin Institute, University of Edinburgh, Edinburgh EH25 9RG, UK. We have identified a missense mutation, E229K, in mouse *Author for correspondence ([email protected]) Idh3a, which is associated with late-onset retinal degeneration. In addition, we generated a complete loss-of-function mutation in A.S.F., 0000-0002-9382-5911; P.S.B., 0000-0001-8951-2413; I.J.J., 0000-0001- this gene and show that it is early embryonic lethal. This mutation, 6526-0688 when combined with the missense mutation Idh3aE229K,results This is an Open Access article distributed under the terms of the Creative Commons Attribution in a more rapid retinal degeneration than that in the missense License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed. homozygotes. Photoreceptors of the retina have a particularly high energy requirement, and the mitochondria of these cells Received 9 July 2018; Accepted 12 November 2018 function at 70-80% of their maximal rate, leaving limited reserve Disease Models & Mechanisms 1 RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm036426. doi:10.1242/dmm.036426 capacity (Kooragayala et al., 2015). This most likely accounts phenotypes were detected in the mice up to 18 months of age for the sensitivity of the retina to mitochondrial mutations. On the (Table S1). other hand, a loss-of-function mutation in Idh3b is viable and Genotyping of the affected littermates in this cross revealed a has no detectable abnormal phenotype. The retina of these mice single common region of homozygosity on chromosome 9, between are normal up to 6 months of age. We have analysed the 50.1 Mb and 74.8 Mb. Analysis of whole-genome sequence of the mitochondrial function in cells with Idh3 mutations, and find the founder male for this line found two candidate protein coding maximum and reserve capacity of Idh3a mutant cells to be mutations in the interval, heterozygous in the founder: sorting nexin reduced. However, cells lacking IDH3B show no detectable 33 (Snx33) and the α subunit of isocitrate dehydrogenase 3 (Idh3a). mitochondrial defect. The Snx33 mutation was not present in the mutant line, presumably because it is on the unaffected chromosome. We considered the RESULTS missense mutation, E229K, in Idh3a to be the likely causative Following a screen for N-ethyl-N-nitrosourea (ENU)-induced mutation, as human patients with mutations in either IDH3A or recessive mutations with an age-dependent phenotype, we IDH3B exhibit retinal degeneration (Fattal-Valevski et al., 2017; identified a mouse line that exhibited loss of vision and retinal Hartong et al., 2008; Pierrache et al., 2017). To confirm Idh3a as the degeneration associated with increasing age (Potter et al., 2016). causative gene, we used CRISPR/Cas9 to introduce a loss-of- When subjected to a moving visual stimulus within an optokinetic function mutation. We generated an insertion of 5 bp into exon 7 of drum (OKD) at 12 months of age, a subset of the littermates in this the gene, c.702_703InsGTACT, termed Idh3aem1Jkn, abbreviated cross responded with the stereotypical head movement only at ∼0.2 herein as Idh3a−. cycles per degree (c/d), compared with >0.3 c/d for phenotypically We bred compound heterozygous mice for Idh3a− and Idh3aE229K wild-type littermates. These mice maintained a diminished response and examined their eyes and visual function. As documented below, at 18 months (Fig. 1A). The same mice had abnormal retina, these mice have rapid retinal degeneration, demonstrating lack of identified by indirect ophthalmoscopy and documented by fundal complementation between the two alleles, and indicating that the imaging at 7 months (Fig. 1B). The dark patches on the retinal E229K mutation is the cause of retinal degeneration in the mice found images are indicative of retinal degeneration. No other overt in the screen. Fig. 1. Idh3aE229K/E229K mice exhibit signs of retinal degeneration. (A) Idh3aE229K/E229K mice (red) showed a decline in visual acuity, as determined by optokinetic drum (OKD) score, by 12 months {0.2071±0.01872 c/d [mean±s.e.m., n=9, female (F) 4, male (M) 5]} compared with wild-type (blue) [0.3090±0.009441 c/d (mean±s.e.m., n=23, F14, M9)] (P<0.0001) and heterozygous (yellow) [0.3173±0.007472 c/d (mean±s.e.m., n=35, F24, M11)] (P<0.0001) mice. This significant decline continued and, by 18 months, homozygous mice (red) had degenerated further [0.1592±0.02396 c/d (mean ±s.e.m., n=9 F4, M5)] compared with wild-type (blue) [0.3004±0.01223 c/d (mean±s.e.m., n=23, F14, M9)] (P<0.0001) and heterozygous (yellow) [0.2836 ±0.009699 c/d (mean±s.e.m., n=35, F24, M11)] mice (P<0.0001).