Fig. 2
Mitochondrial oxidative stress in AD transgenic mouse neurons after Aβ plaque deposition. a Experimental procedure to determine oxidative stress in neuronal mitochondria in mice. APP/PS1 Tg and non-Tg mice were injected with AAV.hSyn.mt-roGFP and a cranial window was implanted. Three weeks later, oxidative stress was assessed by two photon microscopy. Texas Red (TR) Dextran 70 kDa was used to create a fluorescent angiogram. HS169 was used to label Aβ plaques. b In vivo images of neurites and cell bodies expressing AAV.hSyn.mt-roGFP in mitochondria in non-Tg (top) and APP/PS1 Tg mice (bottom). Field of view shows two-photon images of the expression of the AAV (green), blood vessels (Dextran, red) and Aβ plaques (HS169, red) in the cortex. Ratiometric roGFP imaging shows pseudocolor images according to the pseudocolor scale on the bottom. Scale bar represents 20 μm. c, d Mitochondrial oxidative stress (Ratio 800/900) comparison between non-Tg and APP/PS1 Tg mice at 10 months of age (i.e., after plaque deposition) in neuronal mitochondria (c. average per field of view. non-Tg: 1.00 ± 0.024, n = 69 z-stacks; APP/PS1: 1.17 ± 0.034, n = 60 z-stacks from 11 and 12 mice respectively, ***p = 0.0001. d. average per mouse. non-Tg: 0.99 ± 0.046, n = 11 mice (5 males, 6 females); APP/PS1: 1.19 ± 0.056, n = 12 mice (9 males, 3 females), p = 0.0224. Blue dots denote male and pink dots denote female). Error bars represent mean ± SEM. e Histogram of mitochondrial oxidative stress frequency distribution (indicated by Ratio 800/900) in neurons in non-Tg (blue) and APP/PS1 Tg mice (red). f Within 70 µm distance from the edge of a plaque, the probability of finding mitochondrial oxidative stress in neurons was higher the closer to the plaque (mean ± SEM; r (Pearson’s correlation coefficient) -0.90, R2 0.81, ****p < 0.0001, n = 33 plaques analysed from 8 Tg mice). g Representative high magnification pseudocolor images of somas (top) and neurites (bottom) expressing AAV.hSyn.mt-roGFP in mitochondria in vivo in non-Tg (left) and APP/PS1 Tg mice (right). Scale bars represent 15 or 10 μm. h, i Comparison of mitochondrial oxidative stress (Ratio 800/900) in somas and neurites in 10-mo-old non-Tg and APP/PS1 Tg mice. APP/PS1 Tg mice showed significantly higher oxidative stress levels in mitochondria in both compartments. Error bars represent mean ± SEM (h. Neuronal somas: 0.87 ± 0.024, n = 13 z-stacks from 4 non-Tg mice (3 males, 1 females), and 1.08 ± 0.067, n = 12 z-stacks from 6 APP/PS1 Tg mice (4 males, 2 females). **p = 0.006. i. Neurites: 0.99 ± 0.039, n = 13 z-stacks from 4 non-Tg mice (3 males, 1 females), and 1.35 ± 0.084, n = 12 z-stacks from 6 APP/PS1 Tg mice (4 males, 2 females). **p = 0.002). j, k Comparison of mitochondrial oxidative stress (Ratio 800/900) in the different cell compartments (somas and neurites) in 10-month-old (old) non-Tg (j) and APP/PS1 Tg mice (k). Neurites showed significantly higher oxidative stress levels in mitochondria in both conditions. Error bars represent mean ± SEM. (j. Old non-Tg: 0.87 ± 0.024 for somas and 0.99 ± 0.039 for neurites, n = 13 z-stacks from 4 mice (3 males, 1 females), ***p = 0.0003. k Old APP/PS1: 1.08 ± 0.065 for somas and 1.35 ± 0.084 for neurites, n = 12 z-stacks from 6 mice (4 males, 2 females), ****p < 0.0001). Blue dots denote male and pink dots denote female