Canonical Wnt signaling protects hippocampal neurons from Aβ oligomers: role of non-canonical Wnt-5a/Ca(2+) in mitochondrial dynamics

Abstract

Alzheimer's disease (AD) is the most common type of age-related dementia. The disease is characterized by a progressive loss of cognitive abilities, severe neurodegeneration, synaptic loss and mitochondrial dysfunction. The Wnt signaling pathway participates in the development of the central nervous system and growing evidence indicates that Wnts also regulate the function of the adult nervous system. We report here, that indirect activation of canonical Wnt/β-catenin signaling using Bromoindirubin-30-Oxime (6-BIO), an inhibitor of glycogen synthase kinase-3β, protects hippocampal neurons from amyloid-β (Aβ) oligomers with the concomitant blockade of neuronal apoptosis. More importantly, activation with Wnt-5a, a non-canonical Wnt ligand, results in the modulation of mitochondrial dynamics, preventing the changes induced by Aβ oligomers (Aβo) in mitochondrial fission-fusion dynamics and modulates Bcl-2 increases induced by oligomers. The canonical Wnt-3a ligand neither the secreted Frizzled-Related Protein (sFRP), a Wnt scavenger, did not prevent these effects. In contrast, some of the Aβ oligomer effects were blocked by Ryanodine. We conclude that canonical Wnt/β-catenin signaling controls neuronal survival, and that non-canonical Wnt/Ca(2+)signaling modulates mitochondrial dysfunction. Since mitochondrial dysfunction is present in neurodegenerative diseases, the therapeutic possibilities of the activation of Wnt signaling are evident.

Keywords: Aβ oligomers; Wnt-5a signaling; Wnt/ Ca2+; hippocampal neurons; mitochondria.

Figure 1

Aβ oligomers are attached to the somatodendritic region of hippocampal neurons. (A) Characterization of Aβ oligomer species. a, Different forms of Aβ o were analyzed by Tris-Tricine SDS gels using anti-4G8 antibody. b, Densitometric measurement represent relative percentage of oligomers to total Aβ. c, Electron microscopy shows the Aβ oligomer preparation obtained under negative staining, scale bar: 100 nm. (B) Hippocampal neurons were treated with Aβ oligomers and then stained with Mitotracker (red) and 4G8 antibody against Aβ_17–24 (green). a, Control neurons; b, Neurons exposed to 500nM of Aβ oligomers for 24 h. (C) The viability of hippocampal neurons was measured in non-fixed cells using LIVE/DEAD kit assay. Neurons were treated for 24 h with various concentrations of Aβ oligomers (1–20 μM) a, Treated neurons were stained with Calcein-AM/EthD1. Calcein detects live cells (green) and ethidium stain dead cells (red). 0.5 mM H₂O₂ was used as a positive control for cell death b, Statistical analysis represents the neuronal viability using LIVE/DEAD assay. Results are the mean ± SEM. n = 3 experiments, Student's t-test, ^*p < 0.05, ^**p < 0.005.

Figure 2

Activation of canonical Wnt/β-catenin by a GSK-3b inhibitor (6-BIO) induces β-catenin stabilization and protects hippocampal neurons from Aβ oligomers damage. (A) The micrographs show representative neurons stained with β III-tubulin (green) and β-catenin (red), a, control hippocampal neurons, and treatment with Aβ oligomers, 6-BIO or 6-BIO plus Aβ oligomers; b, the graph shows the somatic fluorescence of β-catenin in the neuronal soma. (B) The micrograph shows neurons stained with Hoechst to visualize apoptotic nuclei; a, Hippocampal neurons were treated under the same early conditions; b, the graph shows the number of apoptotic nuclei under conditions specified. Results are the mean ± SEM. n = 4–6 experiments, Student's t-test, ^*p < 0.05. Bar represent 10 μm.

Figure 3

Wnt-5a modulates mitochondrial dynamics in hippocampal neurons. Hippocampal neuron cultures of 15 days in vitro were labeled with Mitotracker and treated with different Wnt ligands. (A) Photographs show mitochondria staining with Mitotracker in control and treated neurons, a, Control neurons; a', magnification of mitochondria, b, treated with Wnt-5a, b', magnification of mitochondria treated with Wnt-5a for 30 min. (B) Various populations of mitochondria were selected and measured in control and treated neurons. The graph shows the average mitochondrial length distribution observed in a time course experiment with Wnt-5a. The quantitative analysis considered a mitochondrial length ranging from <1 μm (white bars), 1–2 μm (gray bars) and greater than 3 μm (black bars). (C) Quantitative analysis of mitochondrial dynamics in neurons treated with different ligands. The graph shows the average mitochondrial length distribution observed in a time course treatment with control (white bars), Wnt-5a plus sFRP (light gray bars), Wnt-3a (dark gray bars), Wnt-5a (black bars), bar (a,b), 10 μm; bar (a',b'), 1 μm. Results are the mean ± SEM. n = 4–6 experiments, Student's t-test, ^*p < 0.05.

Figure 4

Aβ oligomers modify mitochondrial dynamics in hippocampal neurons, Wnt-5a ligand protects and stabilizes mitochondrial membrane potential from such effects. 15 day in vitro hippocampal neuron cultures were labeled with Mitotracker orange and treated with various concentrations of Aβ oligomers. (A) Photographs show mitochondria stained with Mitotracker in control and treated neurons, a, Control neurons; a‘, magnification of mitochondria; b, treated with Aβ oligomers; b', magnification of mitochondria treated with Aβ oligomers, c, treated with Wnt-5a, c', magnification of mitochondria treated with Wnt-5; d, treated with 5 μM Thapsigargin. (B) Quantitation of mitochondrial dynamics under different conditions; a, Graph shows mitochondria/neuron ratio in a time course experiment under various concentrations of Aβ oligomers; b, Neurons pretreated with Wnt-5a or Wnt-3a and then challenged with Aβ oligomers; c, Length of mitochondria under inhibitors of RyR and SERCA. Primary cultures of rat embryo hippocampal neurons (15 DIV) were treated at 37°C. bar (a,b,c,d), 10 μm; bar (a',b',c'), 2 μm. Results are the mean ± SEM. n = 4–6 experiments, Student's t-test, ^*p < 0.05.

Figure 5

Wnt-5a prevents Bcl-2 increase triggered by Aβo in mitochondrial compartments. The micrographs show representative mitochondria stained with Mitotracker orange and Bcl-2 (green). (A) control neurons, neurons treated with Wnt-5a for 2 h, neurons treated with Aβ oligomers (500 nM) and neurons treated with Aβ oligomers plus Wnt-5a. (B) graph shows the co-localization of Bcl-2 staining over mitochondria (dynamic events) in the presence of Aβ oligomers with and without Wnt-5a. Primary cultures of rat embryo hippocampal neurons (15 DIV) were treated at 37°C. Results are the mean ± SEM. n = 4–5 experiments, the number of dynamics events were measured in 100 μm of dendrites. Student's t-test, ^*p < 0.05.

Figure 6

Possible role of non-canonical Wnt/Ca²⁺ pathway in mitochondrial dynamics. In the non-canonical Wnt/Ca²⁺pathway, the binding of the ligand to its receptor Fz, activates Dishevelled (Dsh), which allows the activation of a trimeric G protein. The G protein activates phospholipase C (PLC), increasing the levels of inositol triphosphate (IP₃), which increases the intracellular Ca²⁺ concentrations, coming from the ER. This Ca²⁺ induces further Ca²⁺ releases through RyRs. The high levels of Ca²⁺ activate Ca²⁺-dependent proteins such as protein kinase C (PKC) and the phosphatase Calcineurin. These enzymes regulate the activation of Dynamin-related protein 1 (Drp1), phosphorylation by PKC and CaMK and dephosphorylation by calcineurin, as a result Drp1 is translocated from the cytoplasm to the outer mitochondrial membrane (OMM), which is a signal for mitochondrial fission.