Extended Data Fig. 6: Chronic Idh3 overexpression modestly ameliorates axonal ATP deficits in EAE lesions.
(a) Experimental design for [ATP/ADP]axon measurements in chronic EAE in Thy1- PercevalHR mice virally overexpressing Idh3a or a control protein (Cre recombinase) and tdTomato in a subset of axons. (b) In vivo multi-photon image projections of chronically Idh3a-overexpressing Thy1-PercevalHR spinal axons. Left: Grayscale LUT of tdTomato. Right: Ratiometric [ATP/ADP]axon LUT (λex ratio 950 nm/840 nm). Details: Image pairs of tdTomato-negative (tdTom−; left) and -positive (tdTom+; right) normal-appearing, swollen, and fragmented axons. (c) Comparison of [ATP/ADP]axon in tdTom− and tdTom+ axons (λex ratio 950 nm/840 nm, normalized to control axon mean indicated by black line; values above 1.5 lined up on the “≥1.5” dashed line). Left: [ATP/ADP]axon of single tdTom− (gray) and tdTom+ (orange) axons. Right: Lesion-specific paired analysis of mean [ATP/ADP]axon in tdTom− (gray) and tdTom+ (orange) axon populations of the three morphological stages. Mean ± s.e.m. Comparison of 130 tdTom−versus 115 tdTom+ axons in 12 lesions from four mice in c using two-tailed, unpaired Student’s t-test or Mann-Whitney test where normal distribution could not be confirmed (c, left graphs; p = 7 × 10-6, 9.9 × 10-5 and 3.8 × 10-5 for stages 0, 1 and 2) and a paired t-test (right graph; 0.001, 0.0273 and 0.0547 for stages 0, 1 and 2.) (d) Paired analysis of the frequency of stage 0, 1 and 2 in tdTom− (gray) and tdTom+ (orange) axon populations. Mean ± s.e.m. Comparison of 551 tdTom− versus 539 tdTom+ axons in 38 lesions from six mice in d using two-tailed, paired Student’s t-test or Wilcoxon test where normal distribution could not be confirmed (p = 0.2108, 0.743 and 0.0575 for stage 0, 1 and 2). Scale bars: 25 μm in b. *, p < 0.05; ***, p < 0.005; ****, p < 0.001. See source data for individual data points and further statistical parameters. Illustration created with BioRender.
