Amyloid-β-Acetylcholinesterase complexes potentiate neurodegenerative changes induced by the Aβ peptide. Implications for the pathogenesis of Alzheimer's disease

The presence of amyloid-β (Aβ) deposits in selected brain regions is a hallmark of Alzheimer's disease (AD). The amyloid deposits have "chaperone molecules" which play critical roles in amyloid formation and toxicity. We report here that treatment of rat hippocampal neurons with Aβ-acetylcholinesterase (Aβ-AChE) complexes induced neurite network dystrophia and apoptosis. Moreover, the Aβ-AChE complexes induced a sustained increase in intracellular Ca2+ as well as a loss of mitochondrial membrane potential. The Aβ-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Aβ-AChE fibrillar complexes. These alterations in calcium homeostasis were reversed when the neurons were treated previously with lithium, a GSK-3β inhibitor; Wnt-7a ligand, an activator for Wnt Pathway; and an N-methyl-D-aspartate (NMDA) receptor antagonist (MK-801), demonstrating protective roles for activation of the Wnt signaling pathway as well as for NMDA-receptor inhibition. Our results indicate that the Aβ-AChE complexes enhance Aβ-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Aβ alone. From a therapeutic point of view, activation of the Wnt signaling pathway, as well as NMDAR inhibition may be important factors to protect neurons under Aβ-AChE attack

Genetic background modulates phenotypic expressivity in OPA1 mutated mice, relevance to DOA pathogenesis

Dominant optic atrophy (DOA) is mainly caused by OPA1 mutations and is characterized by the degeneration of retinal ganglion cells (RGCs), whose axons form the optic nerve. The penetrance of DOA is incomplete and the disease is marked by highly variable expressivity, ranging from asymptomatic patients to some who are totally blind or who suffer from multisystemic effects. No clear genotype–phenotype correlation has been established to date. Taken together, these observations point toward the existence of modifying genetic and/or environmental factors that modulate disease severity. Here, we investigated the influence of genetic background on DOA expressivity by switching the previously described DOA mouse model bearing the c.1065 + 5G → A Opa1 mutation from mixed C3H; C57BL/6 J to a pure C57BL/6 J background. We no longer observed retinal and optic nerve abnormalities; the findings indicated no degeneration, but rather a sex-dependent negative effect on RGC connectivity. This highlights the fact that RGC synaptic alteration might precede neuronal death, as has been proposed in other neurodegenerative diseases, providing new clinical considerations for early diagnosis as well as a new therapeutic window for DOA. Furthermore, our results demonstrate the importance of secondary genetic factors in the variability of DOA expressivity and offer a model for screening for aggravating environmental and genetic factors

How the Wnt signaling pathway protects from neurodegeneration: The Mitochondrial Scenario

Alzheimer´s disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as mitochondrial dynamics is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration

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

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

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

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