Proposed mechanism for the action of Wnt3a in regulating mitochondrial mPTP opening.

<p>Wnt signaling is activated by binding of the Wnt3a ligand to its receptor, Fz, and to its co-receptor, LRP6. This interaction activates Dvl, which causes the dissociation of the destruction complex, which includes Axin, APC, and GSK-3β. This dissociation prevents the proteasomal degradation of β-catenin, thereby inducing its cytoplasmic accumulation and eventual translocation to the nucleus, where it regulates the expression of Wnt target genes. This pathway is known as Wnt/β-catenin signaling. However, Wnt can also act through another signaling mediator, GSK-3β, which has functions that are independent of β-catenin and thus of Wnt target gene transcription [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168840#pone.0168840.ref094" target="_blank">94</a>]. The activation of Wnt signaling triggers the inhibitory phosphorylation of GSK-3β at serine 9 in the cytosol. In our proposed model, the inhibited GSK-3β accumulates in mitochondria in response to the activation of Wnt signaling by Wnt3a. Mitochondrial p-GSK3β-S9 then interacts with ANT to inhibit the opening of the mPTP. This interaction has also been correlated with inhibition of the binding between ANT and CypD that is necessary for the conformational shift and opening of the mPTP. GSK-3β activity can also control the detachment or translocation of HKII to the mitochondria. Activation of Wnt signaling increases mitochondrial HKII, probably through the inhibition of GSK-3β activity, thus also favoring the closed conformation of the pore. (Fz: Frizzled; Dvl: Dishevelled; APC: adenomatous polyposis coli; GSK-3β: glycogen synthase kinase-3β; p-GSK3β-S9: GSK-3β phosphorylated at Ser9; ANT: adenine nucleotide translocase; CypD: cyclophilin D; VDAC: voltage-dependent anion channel; HKII: hexokinase II).</p

The inhibition of Wnt target genes transcription does not abolish the protective effect of Wnt3a on mPTP opening.

<p>(A) Wnt signaling cascade representing the mechanism of action of different Wnt antagonists and inhibitors: sFRP2 binds to Wnt ligand preventing the binding with the receptor; DKK1 binds to the co-receptor LRP6; ICG001 down-regulates β-catenin/T cell factor signaling; and 6-BIO is a specific inhibitor of GSK-3β, thereby it activates the Wnt signaling. (B) mPTP opening assay performed in the presence of Wnt inhibitors: sFRP2 (250 nM), DKK1 (100 ng/mL), ICG001 (20 μM) and 6-BIO (10 nM). The graph represents the measurements of each condition at the end point (500 s) of the experiment, normalized to the basal average registered previous to Aβos addition. mPTP opening is visualized as a decay in the fluorescence. (C) _mΔΨ was evaluated in the same conditions described in (B). Statistical analysis was performed using one-way ANOVA <i>post hoc</i> Bonferroni correction: *p<0.05; **p<0.005; ***p<0.0005. n = 3–7 independent experiments.</p

Wnt3a-mediated mPTP inhibition also prevents mitochondrial membrane potential (_mΔΨ) collapse, cytochrome-c release, and cell death.

<p>(A) _mΔΨ, DIV10 hippocampal neurons were treated with control media or with Wnt3a protein for 24 h and were loaded with MitoTracker. Representative red pseudocolored neurons from images obtained before (10 s) and after (500 s) Aβo exposure. The graph represents the time-lapse quantification in neurites for each condition and shows the mean ± SEM of 5 independent experiments. Scale bar, 10 μm. (B) cytochrome-c release, immunodetection of cytochrome-c in neurons treated with 5 μM Aβos in the presence or absence of Wnt3a for 24 h and loaded with MitoTracker-Orange (50 nM). Representative neurites show the colocalization between cytochrome <i>c</i> (green) and the mitochondrial marker (red). Manders’ Coefficient M2 was calculated to determine the colocalization of cytochrome <i>c</i> with mitochondria. Quantification represents the results of three independent experiments, with 10–15 neurons analyzed per experiment. Scale bar, 5 μm. (C) Live/Dead assay of neurons treated with Wnt3a+Aβos for 24 h or with CsA (20 μM) for 30 min before Aβo treatment. Neurons loaded with calcein/EthD1 were analyzed by epifluorescence microscopy to detect neuronal viability. Neurons stained in green (positive for calcein) represent live cells, whereas the red nuclei correspond to dead cells. The graph shows the percentages of live neurons (calcein/EthD1 ratio) under different treatment conditions. Scale bar, 100 μM. The measurements represent the results of 6 independent experiments. Statistical analysis was performed using one-way ANOVA and <i>post hoc</i> Bonferroni correction: *p<0.05; **p<0.005; ***p<0.0005.</p

Percentile values and statistical analysis of mitochondrial area based on electron microscopy.

<p>Percentile values and statistical analysis of mitochondrial area based on electron microscopy.</p

Wnt Signaling Prevents the Aβ Oligomer-Induced Mitochondrial Permeability Transition Pore Opening Preserving Mitochondrial Structure in Hippocampal Neurons

<div><p>Alzheimer’s disease (AD) is a neurodegenerative disorder mainly known for synaptic impairment and neuronal cell loss, affecting memory processes. Beside these damages, mitochondria have been implicated in the pathogenesis of AD through the induction of the mitochondrial permeability transition pore (mPTP). The mPTP is a non-selective pore that is formed under apoptotic conditions, disturbing mitochondrial structure and thus, neuronal viability. In AD, Aβ oligomers (Aβos) favor the opening of the pore, activating mitochondria-dependent neuronal cell death cascades. The Wnt signaling activated through the ligand Wnt3a has been described as a neuroprotective signaling pathway against amyloid-β (Aβ) peptide toxicity in AD. However, the mechanisms by which Wnt signaling prevents Aβos-induced neuronal cell death are unclear. We proposed here to study whether Wnt signaling protects neurons earlier than the late damages in the progression of the disease, through the preservation of the mitochondrial structure by the mPTP inhibition. To study specific events related to mitochondrial permeabilization we performed live-cell imaging from primary rat hippocampal neurons, and electron microscopy to analyze the mitochondrial morphology and structure. We report here that Wnt3a prevents an Aβos-induced cascade of mitochondrial events that leads to neuronal cell death. This cascade involves (a) mPTP opening, (b) mitochondrial swelling, (c) mitochondrial membrane potential loss and (d) cytochrome <i>c</i> release, thus leading to neuronal cell death. Furthermore, our results suggest that the activation of the Wnt signaling prevents mPTP opening by two possible mechanisms, which involve the inhibition of mitochondrial GSK-3β and/or the modulation of mitochondrial hexokinase II levels and activity. This study suggests a possible new approach for the treatment of AD from a mitochondrial perspective, and will also open new lines of study in the field of Wnt signaling in neuroprotection.</p></div

Mitochondrial volume variation in response to Aβos and Wnt3a.

<p>Neurons transfected with mito-Cherry at DIV10 were treated for 24 h with 5 μM Aβos and Wnt3a or were pre-treated for 30 min with 20 μM CsA and then exposed to Aβos. Images show individual 3D-reconstructed mitochondria. Scale bar, 0.3 μm. The graph represents the measurement of mitochondrial volume performed with the Imaris software and used for the mitochondrial network reconstruction. The results are shown as the mean of n = 4 independent experiments and the statistical analysis was performed using one-way ANOVA and <i>post hoc</i> Bonferroni correction: *p<0.05, ***p<0.0005.</p

Wnt3a protects mitochondria from morphological and structural alterations induced by Aβos.

<p>Hippocampal slices (400 μm) were pre-incubated for 4 h with recombinant Wnt3a and then treated with 5 μM Aβos for 1 h. The slices pre-treated with CsA (20 μM, 30 min) were maintained in ACSF for 4 h (to keep the same experimental conditions as Wnt3a-treated slices) until Aβos treatment. (A) Electron micrographs show three representative mitochondria for each treatment. The red asterisk indicates specific regions with disrupted mitochondrial membrane in the Aβos group. Crista disorganization is also clearly observed in this group. Scale bar, 500 nm. (B) Morphological analysis of mitochondria shows the average area of mitochondria in each condition. (C) Ultrastructural analysis of mitochondrial membrane integrity indicates the percentage of mitochondria that exhibit intact membranes. (D) The organization of mitochondrial cristae is represented in a graph showing the percentage of mitochondria with intact cristae. Statistical analysis was performed on data from three independent slices by using one-way ANOVA and <i>post hoc</i> Bonferroni correction: *p< 0.05, **p<0.005, ***p<0.0005.</p

Resistance to calcium-induced mPTP opening in response to Wnt3a.

<p>(A) Representative images showing DIV10 hippocampal neurons treated with control media, recombinant Wnt3a protein (300 ng/ml) or pretreated 30 min with DKK1 (100 ng/mL) and coincubated with Wnt3a for 24 h and loaded with calcein-Co²⁺ to stain mitochondria. The fluorescence intensity decay of mitochondrial calcein corresponds to mPTP opening in response to 20 μM CaCl₂ exposure. Yellow rectangles indicate magnified regions. Scale bar, 8 μm. Close-up images are shown in black and white (x4 magnification) as well as pseudocoloured images. Intensity plots represent the fluorescence intensity profile of each magnified region. (B) Time-lapse quantification of the fluorescence intensity variations. The arrows indicate the addition of 20 and 100 μM CaCl₂. The results represent the analysis of 5–10 neurites from 2 neurons per experiment. (C) The graph represents the measurements of each condition after the addition of 20 μM CaCl₂ (140 s) of the experiment, normalized to the basal average registered previous to the stimulus. mPTP opening is visualized as a decay in the fluorescence. The graph shows the mean ± SEM of n = 3–5 independent experiments. Statistical analysis was performed using one-way ANOVA *p<0.05.</p

Activation of Wnt signaling modulates mitochondrial GSK-3β phosphorylation and p-GSK-3β/ANT interaction.

<p>Hippocampal slices were treated with DKK1 (100 ng/mL) for 30 min before and during Wnt3a treatment. (A) Western blot analysis of mitochondrial fractions obtained from hippocampal slices shows p-GSK3β-S9 levels in mitochondria in response to 300 ng/mL of Wnt3a. The graph shows a significant increase in mitochondrial p-GSK3β-S9 after 4 h of Wnt3a treatment. (B) Inhibition of Wnt3a-mediated induction of mitochondrial p-GSK3β-S9 by DKK1 (100 ng/mL) at 4 h of co-treatmet (n = 3). The graphs represent the densitometric analysis of p-GSK3β-S9 in mitochondrial fractions. Protein levels were normalized to COXIV. (C) Immunoprecipitation assay with phosphorylated-GSK3β-S9 from hippocampal slices treated with Wnt3a for 4 h. <i>Input</i> corresponds to whole-slice lysate, and <i>IPP</i>: <i>p-GSK3β-S9</i> corresponds to the fraction immunoprecipitated with a phosphorylated GSK3β-S9 antibody. Immunoblotting (IB) was performed to detect p-GSK3β-S9, ANT and CypD. Total GSK-3β and GAPDH were used as positive and negative controls, respectively, for the immunoprecipitation assay. Lane C: control; 3a: recombinant Wnt3a. The densitometric analysis was performed from 4 independent experiments. Statistical analyses were conducted using one-way ANOVA and <i>post hoc</i> Bonferroni correction: *p<0.05; **p<0.005.</p

Wnt signaling prevents Aβo-induced mitochondrial permeability transition pore opening in living neurons.

<p>(A) Representative images showing DIV10 hippocampal neurons treated with control media or with recombinant Wnt3a protein (300 ng/ml) for 24 h and loaded with calcein-Co²⁺ to stain mitochondria. 20 μM CsA and 0.5 μM ionomycin were used as negative and positive controls for mPTP induction, respectively. The fluorescence intensity decay of mitochondrial calcein corresponds to mPTP opening in response to Aβos exposure. Yellow rectangles indicate magnified regions. Scale bar, 10 μm. Close-up photos are shown as 8-bit black and white images (x4 magnification) as well as pseudocoloured images. Intensity plots represent the fluorescence intensity profile of each magnified region. (B) Time-lapse quantification of the fluorescence intensity variations. The white horizontal bar on the graph indicates the addition of Aβos. The results represent the analysis of 5–7 neurites from 3–4 neurons per experiment. The graph shows the mean ± SEM of n = 7 independent experiments. Statistical analysis was performed with two-way ANOVA with <i>post hoc</i> Bonferroni correction: ***p<0.0005.</p