Image_1_In Vivo Two Photon Imaging of Astrocytic Structure and Function in Alzheimer’s Disease.tif

<p>The physiological function of the neurovascular unit is critically dependent upon the complex structure and functions of astrocytes for optimal preservation of cerebral homeostasis. While it has been shown that astrocytes exhibit aberrant changes in both structure and function in transgenic murine models of Alzheimer’s disease (AD), it is not fully understood how this altered phenotype contributes to the pathogenesis of AD or whether this alteration predicts a therapeutic target in AD. The mechanisms underlying the spatiotemporal relationship between astrocytes, neurons and the vasculature in their orchestrated regulation of local cerebral flow in active brain regions has not been fully elucidated in brain physiology and in AD. As there is an incredible urgency to identify therapeutic targets that are well-tolerated and efficacious in protecting the brain against the pathological impact of AD, here we use the current body of literature to evaluate the hypothesis that pathological changes in astrocytes are central to the pathogenesis of AD. We also examine the current tools available to assess astrocytic calcium signaling in the living murine brain as it has an important role in the complex interaction between astrocytes, neurons and the vasculature. Furthermore, we discuss the altered function of astrocytes in their interaction with neurons in the preservation of glutamate homeostasis and additionally address the role of astrocytes at the vascular interface and their contribution to functional hyperemia within the living murine brain in health and in AD.</p

Decrease in GABA_A immunoreactivity in APP mice.

<p>GABA_A immunoreactivity in the somatosensory cortex of a 4 month old wildtype littermate control mouse (<b>A</b>), and a 4 month old APP mouse (<b>B</b>). (<b>C</b>) Bar graph comparing intensity of GABA_A immunoreactivity between conditions as a percentage of wildtype level at 4 months (n = 3–4 mice/group). (<b>D</b>) Voltage-sensitive dye traces showing a decrease in power of slow oscillations 60 minutes after topical application of 50 μM picrotoxin to a 4 month old wildtype mouse brain. (<b>E</b>) Slow oscillation power (normalized to wildtype) and (<b>F</b>) mean slow oscillation frequency before (WT baseline) and after picrotoxin (PTX) application to brains of 2–4 month old wildtype mice (n = 4 mice). (<b>G</b>) Slow oscillation power (normalized to APP) and (<b>H</b>) mean slow oscillation frequency before (APP baseline) and after picrotoxin (PTX) application to brains of 2–4 month old APP mice (n = 4 mice). Scale bar, 50 μm. * p<0.05, *** p≤0.001.</p

Decrease in GABA_B expression in APP mice.

<p>GABA_B immunoreactivity in the somatosensory cortex of a 4 month old wildtype littermate control mouse (<b>A</b>), and a 4 month old APP mouse (<b>B</b>). B represents an extreme case. (<b>C</b>) Bar graph comparing GABA_B immunoreactivity between conditions as a percentage of wildtype level at 4 months (n = 3–4 mice/group, p≤0.001). (<b>D</b>) Slow oscillation power (normalized to wildtype) and (<b>E</b>) mean slow oscillation frequency before (baseline) and after 50 μM saclofen application to the brain of wildtype mice (n = 4 mice). (<b>F</b>) Slow oscillation power (normalized to wildtype) and (<b>G</b>) mean slow oscillation frequency before (baseline) and after 50 μM saclofen application to the brain of APP mice (n = 6 mice). Scale bar, 30 μm. * p<0.05, *** p≤0.001.</p

Decrease in GABA in APP mice.

<p>GABA immunoreactivity in the cortex of a 4 month old wildtype littermate control (<b>A</b>), and a 4 month old APP mouse (<b>B</b>). An example in B shows an extreme case. (<b>C</b>) Bar graph comparing cortical GABA levels measured with HPLC in mice older than 4 months (n = 5–6 mice/group). (<b>D</b>) Slow oscillation traces before and after topical application of 0.5 mM GABA to somatosensory cortices of WT and APP mouse brains. (<b>E</b>) Cortical GABA levels measured with HPLC in 2 month old WT and APP mice. (<b>F</b>) Bar graph showing a dose response to a topical application of 0 (PBS+/+), 0.05, 0.5 and 5mM GABA to the brains of APP mice; 0 and 5mM GABA to brains of WT mice (n = 3–4 mice/group). (<b>G</b>) Bar graph showing a dose response to a topical application of 0 (PBS+/+), 0.05, 0.5 and 5mM GABA to the brains of APP mice, normalized to the power after PBS+/+ application (n = 3–4 mice/group). (<b>H</b>) Slow oscillation power (normalized to 0 mM GABA or PBS+/+) before (0 mM GABA or PBS+/+) and after 5mM GABA application to wildtype mice (n = 3 mice/group). (<b>I</b>) Slow oscillation power (normalized to baseline) before (baseline) and after PBS application to APP mice (n = 7 mice/group). (<b>J</b>) Mean slow oscillation frequency in response to various GABA applications to APP mice (n = 3–4 mice/group). (<b>K</b>). Mean slow oscillation frequency before (0mM GABA or PBS+/+) and after 5mM GABA application to wildtype mice (n = 3 mice). (<b>L</b>) Slow oscillation power (normalized to baseline) before (baseline) and after PBS application to wildtype mice (n = 3 mice/group). Scale bar, 50 μm. * p<0.05.</p

Optogenetic manipulation of slow oscillations.

<p>(<b>A</b>) Voltage-sensitive dye signal showing spatiotemporal resolution of neural circuit oscillations driven with light activation of ChR2 at 1.2 Hz in the somatosensory cortex of a Thy1-ChR2-YFP mouse. The images correspond to the first three oscillations in <b>B</b>. Scale bar, 20 μm. (<b>B</b>) WT: Trace of voltage-sensitive dye signal showing that slow oscillations can be driven at twice the normal rate with light activation of ChR2 at 1.2 Hz when light pulses are present (light pulses are depicted in blue) in the somatosensory cortex. The frequency of waves slows after cessation of light stimulation. APP: VSD trace depicting restoration of slow oscillations in APP mice expressing ChR2 virus under CamKIIα promoter when stimulated with light at 0.6 Hz. A decrease in power is evident after light stimulation is stopped. (<b>C</b>) Site of ChR2 viral injection under the CamKIIα promoter tagged with mCherry, or empty vector. (<b>D</b>) mCherry expression in the cortex in a coronal section of postmortem brain tissue. Scale bar, 100 μm. (<b>E</b>) Site of viral injection of YC3.6 in the contralateral hemisphere posterior to the site of ChR2 expression. (<b>F</b>) YFP expression in the cortex in a coronal section of postmortem brain tissue. Scale bar, 100 μm. (<b>G-H</b>) Neuronal activity driven with light activation of ChR2 in wildtype mice (n = 3–5 mice). Control corresponds to mCherry vector lacking ChR2. Light activation of ChR2 at 1.2 Hz increases the power and frequency of slow oscillations. Powers (<b>G</b>) and frequencies (<b>H</b>) of neuronal activity generated in the presence or absence of light with or without ChR2. (<b>I</b>) Slow oscillation power (normalized to APP without light activation) and (<b>J</b>) mean slow oscillation frequency before or after light activation of ChR2 in 4 month old APP mice expressing the virus (n = 6 mice). * p<0.05, ** p≤0.01.</p

Calcium overload is prevented in APP mice whose slow oscillations were driven with light activation of ChR2.

<p>(<b>A,B</b>) In vivo multiphoton images of cortical neurites, pseudocolored according to [Ca2+]i, show the presence of elevated levels of calcium (yellow-red neurites) in a 6 months old APP mouse (<b>A,</b> arrows) in addition to neurites displaying normal calcium levels (for instance, blue neurites). Restoring slow oscillations with light activation of ChR2 prevented elevations of calcium (calcium overload) in cortical neurites (<b>B</b>). (<b>C</b>) Histograms showing distribution of YFP/CFP ratios in neurites with YC3.6 in APP mice at 5 and 6 months of age (n = 746 neurites in 7 mice). Calcium overload was defined as ratios greater than 2 standard deviations above the mean in wildtype mice (1.79) (n = 321 neurites in 5 mice). (<b>D</b>) Distribution of neurite YFP/CFP ratios in 5, 6, and 7 month old APP mice whose slow oscillations were restored with light activation of ChR2 (n = 369 neurites in 6 mice). (<b>E</b>) A bar graph showing the percentage of neurites exhibiting calcium overload across conditions at 6 months. (<b>F,G</b>) In vivo multiphoton images of cortical neurites, pseudocolored according to [Ca2+]i, show limited calcium overload within neurites in a 6 months old WT mouse (<b>F</b>). Driving slow oscillations with light activation of ChR2 failed to significantly alter calcium overload in cortical neurites in wildtype mice (<b>G</b>). (<b>H</b>) Histograms showing distribution of YFP/CFP ratios in neurites with YC3.6 in WT mice at 5 and 6 months of age (n = 321 neurites in 5 mice). (<b>I</b>) Distribution of neurite YFP/CFP ratios in 5 and 6 month old WT mice whose slow oscillations were driven with light activation of ChR2 at normal frequency (n = 326 neurites in 5 mice). (<b>J</b>) A bar graph showing the percentage of neurites exhibiting calcium overload across conditions in wildtype mice at 6 months. (<b>K</b>) Percentage of total time spent mobile during the day and night by APP and wildtype littermates whether treated with light or not. (Scale bar, 100 μm) ** p≤0.01.</p

Driving slow oscillations with light restores GABA receptor levels.

<p>(<b>A-C</b>). GABA immunoreactivity in the cortex of a 7 month old wildtype littermate control mouse (<b>A</b>), an APP transgenic (<b>B</b>) and an APP mouse whose slow oscillations were recovered with light (<b>C</b>). (<b>D-F</b>) GABA_A immunoreactivity in the cortices of a 7 month old wildtype littermate control mouse (<b>D</b>), an APP mouse (<b>E</b>), and an APP mouse whose slow oscillations were restored with light (<b>F</b>). (<b>G-I</b>) GABA_B immunoreactivity in the cortex of a 7 month old wildtype littermate control mouse (<b>G</b>), an APP mouse (<b>H</b>), and an APP mouse whose slow oscillations were restored with light activation of ChR2 (<b>I</b>). (<b>J</b>) Bar graph comparing cortical GABA levels measured with HPLC (n = 4 mice/group). (<b>K</b>). A bar graph comparing GABA_A immunoreactivity between conditions as a percentage of wildtype level at 7 months (n = 3–4 mice/group). (<b>L</b>) A bar graph comparing GABA_B immunoreactivity between conditions as a percentage of wildtype level at 7 months (n = 3–4 mice/group). ** p≤0.01, *** p≤0.001.</p

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

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 < 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