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- 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). Unpaired t-test, ***P<0.001. (B) Fundus imaging comparing Idh3aE229K/E229K retina with that of a wild-type littermate; the mutant retina exhibits signs of retinal degeneration, as evidenced by a dark patchy appearance. (C) Structure of the IDH3A:IDH3G heterodimer (PDB ID: 5GRI) with the positions of the E229K mutation identified in this study (red) and previously identified missense mutations (blue) highlighted. (D) Predicted effects of mutations on the stability of the IDH3A monomer and on the strength of the interaction with IDH3G. Higher values indicate greater disruption of protein stability or interaction strength.
- Fig. 2.
Idh3a−/E229K and Idh3aE229K/E229K mice show early and rapid photoreceptor loss. (A) In Idh3a−/E229K mice, reactive gliosis, as indicated by green GFAP staining, is present from P30 and continues through P60-90 (n=3). This staining is not present in P30-60 Idh3aE229K/E229K, or in P90 wild-type littermates (rightmost), but is seen in P90 Idh3aE229K/E229K retinal sections (n=3). Blue staining is DAPI. (B) Histological analysis of H&E-stained eyes. The numbers of nuclei in the ONL are clearly decreased in Idh3a−/E229K at P60 and almost absent by P90. (C,D) Numbers of photoreceptor nuclei in the ONL (y-axis) at fixed distances either side of the optic nerve head (x-axis). Counts at P30 (C) show that all genotypes have normal nuclei numbers, except for the periphery of Idh3a−/E229K retinas (n=3-6, data in Table S2). By P60 and continuing to P90 (D), the numbers of nuclei are significantly decreased in Idh3aE229K/E229K retinas (n=7, F7, M2) compared with those of wild-type littermate controls (n=7, F4, M3). Unpaired t-test, *P<0.05, **P<0.01. Scale bars: 50 µm. INL, inner nuclei layer; ONL, outer nuclei layer; RGC, retinal ganglion cell layer; RPE, retinal pigmented epithelium.
- Fig. 3.
Idh3a−/E229K and Idh3aE229K/E229K mice show normal then decreased photoreceptor function. (A-C) A-wave amplitudes of dark-adapted Idh3a−/E229K mice and littermate controls, exposed to a 3 cd/m3 flash of light at P30 (A) and P60 (B), with accompanying traces showing mutant (red) and wild-type (blue) scotopic responses at P30 (C, top) and P60 (C, bottom). At P30, Idh3a−/E229K mice exhibit a-wave amplitudes comparable to those of control littermates, as shown by one-way ANOVA [F(3,10)=0.7970, P=0.5232] [n=4 (F2, M2), 3 (F2, M1), 3 (F1, M2), 4 (F2, M2)], but by P60 this has significantly decreased [F(3,9)=32.66, ***P<0.001] [n=3 (M3), 3 (F1, M2), 3 (F1, M2), 5, (F1, M4)]. (D,E) Comparison of a-wave amplitudes between Idh3aE229K/E229K and wild-type controls at P60 (D) and P120 (E), respectively. At P60, mutant mice exhibit no loss of retinal function [265.5±18.18 (mean±s.e.m., n=5, F2, M3)] compared with wild-type littermates [267.0±21.3 (mean±s.e.m., n=4, F4)]. By P120, photoreceptor response to a light stimulus is significantly reduced in mutant [153.2±35.11 (mean±s.e.m., n=4, F2, M2)] compared with wild-type [247.7±15.51 (mean±s.e.m., n=4, F3, M1)] mice. *P<0.05. (F) Accompanying traces show Idh3aE229K/E229K (green) and wild-type control traces (blue) at P30 (top) and P120 (bottom).
- Fig. 4.
Mutant MEF cells exhibit reduced reserve and maximal respiration. Idh3a mutants exhibit reduced mitochondrial maximum respiration and reserve capacity. (A) Oxygen consumption (OCR) of MEF cells was measured using an Agilent Seahorse XFe24 Extracellular Flux Analyser in basal and stimulated conditions. (B-F) Histograms show maximal respiration [FCCP measurement 2 – antimycin+rotenone (Ant/Ret) measurement 2] (B), reserve capacity (FCCP measurement 2 – basal measurement 2) (C), basal respiration (basal measurement 2 – Ant/Ret measurement 2) (D), ATP respiration (basal measurement 2 – oligo measurement 2) (E), leaked respiration (oligo measurement 2 – Ant/Ret measurement 2) (F) pmol min−1 OD540−1 for each part of the experiment (A). Idh3a−/E229K and Idh3aE229K/E229K mutant cells show a lower, but not significant, trend in basal, ATP-dependent or leaked respiration compared with wild type (D-F). Both Idh3a−/E229K and Idh3aE229K/E229K showed significantly reduced maximum respiration [F(4,10)=5.701, P=0.0118] (B) and spare respiratory capacity [F(4,10)=6.536, P=0.0075] (C), as shown by one-way ANOVA. *P<0.05, **P<0.01.
- Fig. 5.
Idh3b−/− mice show no signs of retinal degeneration up to 6 months. Idh3b−/− mice show no retinal degeneration up to P180. (A) GFAP green immunofluorescence on retinal sections shows no upregulation in mutant mice, indicating the absence of retinal stress (n=3). (B) ERG analysis of P180 Idh3b−/− in response to 3 cd/m3 flash under scotopic conditions shows a similar a-wave amplitude [233.4±19.63 (mean±s.e.m., n=6, F2, M4)] to that of wild-type littermates [207.7±14.42 (mean±s.e.m., n=6, F3, M3)] (left). ERG traces of mutant mice (red) also show no defects when compared with wild type (black) (right). (C-E) OCR was measured in Idh3b−/− MEF cells and compared with that of control littermates. In contrast to Idh3a mutant cells, Idh3b−/− cells did not show significantly reduced maximum respiration or spare respiratory capacity. (F) Western blot analysis for IDH3B and α-tubulin in retinal tissue shows the absence of IDH3B protein in Idh3b−/− retinas. Scale bar: 50 µm.
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