Additional file 1 of Real-time imaging of mitochondrial redox reveals increased mitochondrial oxidative stress associated with amyloid β aggregates in vivo in a mouse model of Alzheimer’s disease
Creators: name: Calvo-Rodriguez, Maria
name: Kharitonova, Elizabeth K.
name: Snyder, Austin C.
name: Hou, Steven S.
name: Sanchez-Mico, Maria Virtudes
name: Das, Sudeshna
name: Fan, Zhanyun
name: Shirani, Hamid
name: Nilsson, K. Peter R.
name: Serrano-Pozo, Alberto
name: Bacskai, Brian J.
Publisher: name: figshare
Year: 2024
Subjects: Biochemistry
Medicine
Cell Biology
Neuroscience
Chemical Sciences not elsewhere classified

description: Additional file 1: Fig. S1. Validation of AAV.hSyn.mt-roGFP in vitro. a. Mitochondrial co-transfection verified proper targeting of mt-roGFP to mitochondria. N2a cells (top) and primary cortical neurons (bottom) were co-transfected with mt-roGFP (green) and mRuby-Mito-7 (red) and subjected to confocal microscopy imaging. Scale bar represents 10 μm. b. Double immunolabelling of mt-roGFP (green) and mRuby-ER5 (red, targeting endoplasmic reticulum, ER) in N2a cells shows lack of colocalization and supports the mitochondrial localization of mt-roGFP. Scale bar represents 10 μm. c. In vitro imaging of cellular oxidative stress with mt-roGFP. Primary cortical neurons were exposed to either the oxidant DTDP or the reducing agent DTT. Images at 800 nm (red), 900 nm (green) and merged are shown. d. The relative changes in ratio 800/900 were represented by histograms of ratio 800/900 frequency distribution in control conditions (grey) and 20 min after exposure to DTT 1 mM (blue) and DTDP 100 μM (red) (Control, n = 143 cells; DTT 1 mM, n = 125; DTDP 100 μM, n = 109 cells). Fig. S2. Validation of pAAV.hSyn.mt-roGFP ex vivo. AAV.hSyn.mt-roGFP targets neuronal mitochondria in vivo. a. Colocalization of AAV.hSyn.mt-roGFP (green), NeuN (red) and GS (glutamine synthetase, magenta) in the mouse cortex shown by immunohistochemistry. Note that AAV.hSyn.mt-roGFP only colocalizes with the neuronal marker NeuN. Scale bar represents 10 μm. b. Colocalization of AAV.hSyn.mt-roGFP (green), HSP60 (mitochondrial marker, red) and NeuN (magenta) in the cortex shown by immunohistochemistry. Scale bar represents 10 μm. c. Inset. Colocalization of AAV.hSyn.mt-roGFP (green) and HSP60 (red) in cortex shown by immunohistochemistry (top). Scale bar 5 μm. Graph shows intensity profile of the ROI across the cell. Green line represents the fluorescence intensity of AAV.hSyn.mt-roGFP and red line represents the fluorescence intensity of HSP60. Fig. S3. Original images excited at 800nm and 900nm of Fig. 1b. Fig. S4. Original images excited at 800nm and 900nm of Fig. 2b. Fig. S5. Mitochondrial oxidative stress in male and female mice. Mitochondrial oxidative stress (Ratio 800/900) in neurons was compared between non-Tg and APP/PS1 Tg mice at 10 months of age within males (a) or females (b). Note that only for males the difference is significantly different (a. Males: average per field of view: non-Tg: 0.95 ± 0.026, n = 31 z-stacks; APP/PS1: 1.17 ± 0.046, n = 41 z-stacks from 5 and 9 mice respectively, ***p = 0.0001; Average per mouse: non-Tg: 0.95 ± 0.037, n = 5 mice; APP/PS1: 1.19 ± 0.073, n = 9 mice, *p=0.0190. b. Females: average per field of view: non-Tg: 1.038 ± 0.038, n = 38 z-stacks; APP/PS1: 1.17 ± 0.043, n = 19 z-stacks from 6 and 3 mice respectively; Average per mouse: non-Tg: 1.02 ± 0.08, n = 6 mice; APP/PS1: 1.19 ± 0.067, n = 3 mice). Error bars represent mean ± SEM. Fig. S6. The overall mitochondrial redox levels are not elevated in AD transgenic mouse neurons before Aβ plaque deposition. a. In vivo images of neurites and cell bodies expressing pAAV.hSyn.mt-roGFP in mitochondria in non-Tg (top) and APP/PS1 Tg mice (bottom) in young mice. Scale bar represents 10 μm. b, c. Scatter dot plot represents overall mitochondrial oxidative stress (Ratio 800/900) in non-Tg and APP/PS1 Tg mice at 3 months of age, before plaque deposition, in mitochondria in neurons (b, average per field of view, non-Tg: 0.83 ± 0.024, n = 18 z-stacks from 3 mice (3 male); APP/PS1: 0.87 ± 0.024, n = 42 z-stacks from 6 mice (3 male, 3 female); c. average per mouse, non-Tg: 0.82 ± 0.039, n = 3 mice (3 male); APP/PS1: 0.87 ± 0.034, n = 6 mice (3 male, 3 female)). Error bars represent mean ± SEM. Blue dots denote male and pink dots denote female. d. Histogram of mitochondrial oxidative stress frequency distribution (indicated by Ratio 800/900) in the young non-Tg and APP/PS1 Tg mice. e. Representative high resolution pseudocolor images of somas (top) and neurites (bottom) expressing AAV.hSyn.mt-roGFP in mitochondria in vivo in young non-Tg (left) and APP/PS1 Tg mice (right). Scale bar represents 15 or 10 μm. f. Comparison of mitochondrial oxidative stress (Ratio 800/900) within somas or neurites in 3-month-old non-Tg and APP/PS1 Tg mice. APP/PS1 Tg mice showed higher oxidative stress levels in mitochondria in neurites. Error bars represent mean ± SEM. (somas: 0.79 ± 0.023, n = 9 z-stacks from 3 non-Tg mice (3 male), and 0.75 ± 0.026, n = 10 z-stacks from 3 APP/PS1 Tg mice (1 male, 2 females); neurites: 0.82 ± 0.040, n = 9 z-stacks from 3 non-Tg mice (3 male), and 0.92 ± 0.030, n = 10 z-stacks from 3 APP/PS1 Tg mice (1 males, 4 females); *p = 0.0467). g. Comparison of mitochondrial oxidative stress (Ratio 800/900) in the different cell compartments (somas and neurites) in 3-month-old (old) non-Tg and APP/PS1 Tg mice. Neurites showed significantly higher oxidative stress levels in mitochondria in the APP/PS1 Tg mouse when compared to the somas. Error bars represent mean ± SEM. (Young non-Tg: 0.79 ± 0.023 for somas and 0.82 ± 0.040 for neurites, n = 9 z-stacks from 3 mice (3 male); Young APP/PS1: 0.75 ± 0.026 for somas and 0.92 ± 0.030 for neurites, n = 10 z-stacks from 3 mice (1 male, 2 female), ***p = 0.0003). Blue dots denote male and pink dots denote female. Fig. S7. Original images excited at 800nm and 900nm of Fig. 3b. Fig. S8. Original images excited at 800nm and 900nm of Fig. 4a. Fig. S9. Original images excited at 800nm and 900nm of Fig. 5a. Fig. S10. SS31 reduces Aβ-associated dystrophic neurite number but not amyloid burden in the AD transgenic mouse. a.  Representative images of the global amount of amyloid in the cortex of SS31 and SS20 treated APP/PS1 mice at 10 mo of age after Aβ immunostaining. Scale bar represents 100 μm.  b. Scatter dot plots represent the quantification of amyloid load in the cortex after anti-Aβ immunostaining or ThioS labeling. The number of dense-core plaques detected by ThioS (top) and the overall load of Aβ (bottom) was comparable among SS31 and SS20 APP/PS1 treated mice. n = 7 mice per condition. Histograms represent the dense core plaque (top) and diffuse amyloid deposit (bottom) size in both conditions. c. Representative images of neuritic dystrophies (arrow heads, neurofilaments in green) around amyloid plaques (blue) in APP/PS1 mouse after either SS31 or SS20 treatment. Scale bar 20 μm. d. Scatter dot plot represents the quantification of the number of dystrophic neurites observed per plaque, n = 362 plaques from 4 SS31 APP/PS1 treated mice and n = 295 plaques from 4 SS31 APP/PS1 treated mice, **p &lt; 0.05. e. Scatter dot plot represents the percentage of plaques showing dystrophic neurites, n = 4 – 5 areas per 4 mouse per condition, *p = 0.022.