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

Dinamarca MC, Sagal JP, Quintanilla RA, Godoy JA, Arrázola MS, Inestrosa NC - Mol Neurodegener (2010)

Bottom Line: The Abeta-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Abeta-AChE fibrillar complexes.Our results indicate that the Abeta-AChE complexes enhance Abeta-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Abeta 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 Abeta-AChE attack.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Regulación Celular y Patología "Joaquín V, Luco" (CRCP), Instituto Milenio MIFAB, Centro de Envejecimiento y Regeneración (CARE), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, 8331010 Santiago, Chile. ninestrosa@bio.puc.cl.

ABSTRACT
The presence of amyloid-beta (Abeta) 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 Abeta-acetylcholinesterase (Abeta-AChE) complexes induced neurite network dystrophia and apoptosis. Moreover, the Abeta-AChE complexes induced a sustained increase in intracellular Ca2+ as well as a loss of mitochondrial membrane potential. The Abeta-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Abeta-AChE fibrillar complexes. These alterations in calcium homeostasis were reversed when the neurons were treated previously with lithium, a GSK-3beta 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 Abeta-AChE complexes enhance Abeta-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Abeta 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 Abeta-AChE attack.

No MeSH data available.


Related in: MedlinePlus

Role of the intracellularcalcium increase in the toxicity of Aβ-AChE complexes. (A) Hippocampal neurons were loaded with Calcein-AM to analyze the neuronal integrity and with FuraRed as an indicator of intracellular free calcium. The confocal photographs correspond to neurons before treatment (a, c and e) and after 1 h treatment with 5 μM Aβ-AChEf (b, d and f). (B) The figure shows a representative SDS-PAGE of AChE immunoblot to detect Aβ-AChEo complexes on neuronal surface by biotinylation. The graph shows a quantification of two independent experiments, results are the mean ± S.E.M. (C) Hippocampal neurons were loaded with Fluo-3 AM and treated with increasing Aβ-AChEf concentrations (μM), indicated by the black bars on top of the graph. The complexes were not washed out in between increasing concentrations. Fluorescence changes were recorded at 1 min intervals and are shown as ratio ΔF/Fo. (D) Final normalized fluorescence intensities reached at the end of experiments are expressed over the control situation for the following treatments: 5 μM Aβ-AChEf; 2 mM EGTA; 30 μM BAPTA-AM; * p ≤ 0.001. (E) Cell viability was evaluated by MTT assay. Hippocampal neurons were treated for 24 h as follow: 5 μM Aβ-AChEf; 2 mM EGTA. Mean MTT reduction value was expressed as % of control situation; *p ≤ 0.001 compared with the control condition; #p ≤ 0.005 compared to the Aβ-AChEf treatment.
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Figure 2: Role of the intracellularcalcium increase in the toxicity of Aβ-AChE complexes. (A) Hippocampal neurons were loaded with Calcein-AM to analyze the neuronal integrity and with FuraRed as an indicator of intracellular free calcium. The confocal photographs correspond to neurons before treatment (a, c and e) and after 1 h treatment with 5 μM Aβ-AChEf (b, d and f). (B) The figure shows a representative SDS-PAGE of AChE immunoblot to detect Aβ-AChEo complexes on neuronal surface by biotinylation. The graph shows a quantification of two independent experiments, results are the mean ± S.E.M. (C) Hippocampal neurons were loaded with Fluo-3 AM and treated with increasing Aβ-AChEf concentrations (μM), indicated by the black bars on top of the graph. The complexes were not washed out in between increasing concentrations. Fluorescence changes were recorded at 1 min intervals and are shown as ratio ΔF/Fo. (D) Final normalized fluorescence intensities reached at the end of experiments are expressed over the control situation for the following treatments: 5 μM Aβ-AChEf; 2 mM EGTA; 30 μM BAPTA-AM; * p ≤ 0.001. (E) Cell viability was evaluated by MTT assay. Hippocampal neurons were treated for 24 h as follow: 5 μM Aβ-AChEf; 2 mM EGTA. Mean MTT reduction value was expressed as % of control situation; *p ≤ 0.001 compared with the control condition; #p ≤ 0.005 compared to the Aβ-AChEf treatment.

Mentions: Given the effect of the Aβ-AChE on intracellular calcium homeostasis, we examined whether the Aβ-AChEf alter the integrity of the plasma membrane. We treated hippocampal neurons with 5 μM Aβ-AChEf and then loaded with calcein-AM and Fura-red. After 1 h treatment the fluorescence of Fura-red in neurons treated with Aβ-AChEf (Fig. 2Ad) decreased respect to the control (Fig. 2Ac), whereas the calcein fluorescence was not affected respect to the control (Fig. 2Aa and 2b), suggesting that the Aβ-AChEf treatment did not alters the integrity of the plasma membrane. We also evaluated whether Aβ-AChEo preparation interacts with the neuronal membrane through surface biotinyl assay. Fig. 2B shows that Aβ-AChEo interacts with the cell membrane, suggesting that the effects observed are because the complex is acting at the neuronal membrane level. Then, we evaluated the effect of different concentrations of Aβ-AChEf and the source of calcium responsible for the intracellular calcium increase. We determined the behaviour of calcium levels in neurons loaded with Fluo-3 AM and then treated with increasing Aβ-AChEf concentrations. We observed that the increase in the cytoplasmic calcium levels correlated with increasing Aβ-AChEf concentrations (Fig. 2C). In order to determine the source of the intracellular calcium increase induced by the complex, we treated neurons with the complex (5 μM Aβ-AChEf) in the presence of the extracellular calcium chelator, EGTA (2 mM), or the intracellular calcium chelator, BAPTA-AM (30 μM). We observed that treatment with EGTA prevented the increase of intracellular calcium induced by the complex (Fig. 2D). Additionally in neurons pre-incubated with BAPTA-AM and treated with the complex, we observed a ~ 2.8-fold increase in the intracellular calcium (Fig. 2D). These results suggest that intracellular calcium deregulation induced by the complex is dependent on extracellular calcium and that massive entrance of the ion could release intracellular calcium reservoirs. In order to study the role of the intracellular calcium deregulation in the neurotoxic properties of Aβ-AChE, we performed MTT cell viability assays in the presence of 2 mM EGTA, as indicated in Fig. 2E. Five μM Aβ-AChEf induced ~ 55% cell death, while EGTA partially reduced neuronal death triggered by the Aβ-AChEf complex (~ 25% reduction of viability), suggesting that extracellular calcium has an important role, but is not the only player in the mechanism of toxicity induced by the complex.


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

Dinamarca MC, Sagal JP, Quintanilla RA, Godoy JA, Arrázola MS, Inestrosa NC - Mol Neurodegener (2010)

Role of the intracellularcalcium increase in the toxicity of Aβ-AChE complexes. (A) Hippocampal neurons were loaded with Calcein-AM to analyze the neuronal integrity and with FuraRed as an indicator of intracellular free calcium. The confocal photographs correspond to neurons before treatment (a, c and e) and after 1 h treatment with 5 μM Aβ-AChEf (b, d and f). (B) The figure shows a representative SDS-PAGE of AChE immunoblot to detect Aβ-AChEo complexes on neuronal surface by biotinylation. The graph shows a quantification of two independent experiments, results are the mean ± S.E.M. (C) Hippocampal neurons were loaded with Fluo-3 AM and treated with increasing Aβ-AChEf concentrations (μM), indicated by the black bars on top of the graph. The complexes were not washed out in between increasing concentrations. Fluorescence changes were recorded at 1 min intervals and are shown as ratio ΔF/Fo. (D) Final normalized fluorescence intensities reached at the end of experiments are expressed over the control situation for the following treatments: 5 μM Aβ-AChEf; 2 mM EGTA; 30 μM BAPTA-AM; * p ≤ 0.001. (E) Cell viability was evaluated by MTT assay. Hippocampal neurons were treated for 24 h as follow: 5 μM Aβ-AChEf; 2 mM EGTA. Mean MTT reduction value was expressed as % of control situation; *p ≤ 0.001 compared with the control condition; #p ≤ 0.005 compared to the Aβ-AChEf treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2823746&req=5

Figure 2: Role of the intracellularcalcium increase in the toxicity of Aβ-AChE complexes. (A) Hippocampal neurons were loaded with Calcein-AM to analyze the neuronal integrity and with FuraRed as an indicator of intracellular free calcium. The confocal photographs correspond to neurons before treatment (a, c and e) and after 1 h treatment with 5 μM Aβ-AChEf (b, d and f). (B) The figure shows a representative SDS-PAGE of AChE immunoblot to detect Aβ-AChEo complexes on neuronal surface by biotinylation. The graph shows a quantification of two independent experiments, results are the mean ± S.E.M. (C) Hippocampal neurons were loaded with Fluo-3 AM and treated with increasing Aβ-AChEf concentrations (μM), indicated by the black bars on top of the graph. The complexes were not washed out in between increasing concentrations. Fluorescence changes were recorded at 1 min intervals and are shown as ratio ΔF/Fo. (D) Final normalized fluorescence intensities reached at the end of experiments are expressed over the control situation for the following treatments: 5 μM Aβ-AChEf; 2 mM EGTA; 30 μM BAPTA-AM; * p ≤ 0.001. (E) Cell viability was evaluated by MTT assay. Hippocampal neurons were treated for 24 h as follow: 5 μM Aβ-AChEf; 2 mM EGTA. Mean MTT reduction value was expressed as % of control situation; *p ≤ 0.001 compared with the control condition; #p ≤ 0.005 compared to the Aβ-AChEf treatment.
Mentions: Given the effect of the Aβ-AChE on intracellular calcium homeostasis, we examined whether the Aβ-AChEf alter the integrity of the plasma membrane. We treated hippocampal neurons with 5 μM Aβ-AChEf and then loaded with calcein-AM and Fura-red. After 1 h treatment the fluorescence of Fura-red in neurons treated with Aβ-AChEf (Fig. 2Ad) decreased respect to the control (Fig. 2Ac), whereas the calcein fluorescence was not affected respect to the control (Fig. 2Aa and 2b), suggesting that the Aβ-AChEf treatment did not alters the integrity of the plasma membrane. We also evaluated whether Aβ-AChEo preparation interacts with the neuronal membrane through surface biotinyl assay. Fig. 2B shows that Aβ-AChEo interacts with the cell membrane, suggesting that the effects observed are because the complex is acting at the neuronal membrane level. Then, we evaluated the effect of different concentrations of Aβ-AChEf and the source of calcium responsible for the intracellular calcium increase. We determined the behaviour of calcium levels in neurons loaded with Fluo-3 AM and then treated with increasing Aβ-AChEf concentrations. We observed that the increase in the cytoplasmic calcium levels correlated with increasing Aβ-AChEf concentrations (Fig. 2C). In order to determine the source of the intracellular calcium increase induced by the complex, we treated neurons with the complex (5 μM Aβ-AChEf) in the presence of the extracellular calcium chelator, EGTA (2 mM), or the intracellular calcium chelator, BAPTA-AM (30 μM). We observed that treatment with EGTA prevented the increase of intracellular calcium induced by the complex (Fig. 2D). Additionally in neurons pre-incubated with BAPTA-AM and treated with the complex, we observed a ~ 2.8-fold increase in the intracellular calcium (Fig. 2D). These results suggest that intracellular calcium deregulation induced by the complex is dependent on extracellular calcium and that massive entrance of the ion could release intracellular calcium reservoirs. In order to study the role of the intracellular calcium deregulation in the neurotoxic properties of Aβ-AChE, we performed MTT cell viability assays in the presence of 2 mM EGTA, as indicated in Fig. 2E. Five μM Aβ-AChEf induced ~ 55% cell death, while EGTA partially reduced neuronal death triggered by the Aβ-AChEf complex (~ 25% reduction of viability), suggesting that extracellular calcium has an important role, but is not the only player in the mechanism of toxicity induced by the complex.

Bottom Line: The Abeta-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Abeta-AChE fibrillar complexes.Our results indicate that the Abeta-AChE complexes enhance Abeta-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Abeta 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 Abeta-AChE attack.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Regulación Celular y Patología "Joaquín V, Luco" (CRCP), Instituto Milenio MIFAB, Centro de Envejecimiento y Regeneración (CARE), Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, 8331010 Santiago, Chile. ninestrosa@bio.puc.cl.

ABSTRACT
The presence of amyloid-beta (Abeta) 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 Abeta-acetylcholinesterase (Abeta-AChE) complexes induced neurite network dystrophia and apoptosis. Moreover, the Abeta-AChE complexes induced a sustained increase in intracellular Ca2+ as well as a loss of mitochondrial membrane potential. The Abeta-AChE oligomers complex also induced higher alteration of Ca2+ homeostasis compared with Abeta-AChE fibrillar complexes. These alterations in calcium homeostasis were reversed when the neurons were treated previously with lithium, a GSK-3beta 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 Abeta-AChE complexes enhance Abeta-dependent deregulation of intracellular Ca2+ as well as mitochondrial dysfunction in hippocampal neurons, triggering an enhanced damage than Abeta 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 Abeta-AChE attack.

No MeSH data available.


Related in: MedlinePlus