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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 mitochondria in the calcium increase induced by Aβ-AChE. (A) Hippocampal neurons were treated with (a) control medium, (b) 2 μM Aβ-AChEf, (c) 5 μM Aβ-AChEf, (d) 2 μM Aβf, (e) 5 μM Aβf, (f) 2 μM Aβo, (g) 5 μM Aβo and (h) 5 nM AChE for 1 h and stained with MitoTracker (red stain in the insets) and Phalloidin (blue stain in the inset) to observe neuronal morphology. Bar = 10 μm. (B) Quantification of MitoTracker fluorescence intensity representative of mitochondrial membrane potential of the different treatments. Results are the mean ± S.E.M, in duplicate experiments, n = 2 independent experiments; *p < 0.05 (C) Hippocampal neurons were loaded with 5 μM Rhod-2 AM for 40 min and mitochondrial calcium uptake were determined. Mitotracker Green™ (MTG) was used to estimate Rhod-2 signal in the mitochondria. Treatment with 3 μM Aβf gradually increased mitochondrial calcium uptake. Control experiments with reverse peptide Aβ42-1 did not show significant changes in mitochondrial calcium levels. However, 3 μM Aβ-AChEf complexes induced a rapid and acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels.
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Figure 4: Role of mitochondria in the calcium increase induced by Aβ-AChE. (A) Hippocampal neurons were treated with (a) control medium, (b) 2 μM Aβ-AChEf, (c) 5 μM Aβ-AChEf, (d) 2 μM Aβf, (e) 5 μM Aβf, (f) 2 μM Aβo, (g) 5 μM Aβo and (h) 5 nM AChE for 1 h and stained with MitoTracker (red stain in the insets) and Phalloidin (blue stain in the inset) to observe neuronal morphology. Bar = 10 μm. (B) Quantification of MitoTracker fluorescence intensity representative of mitochondrial membrane potential of the different treatments. Results are the mean ± S.E.M, in duplicate experiments, n = 2 independent experiments; *p < 0.05 (C) Hippocampal neurons were loaded with 5 μM Rhod-2 AM for 40 min and mitochondrial calcium uptake were determined. Mitotracker Green™ (MTG) was used to estimate Rhod-2 signal in the mitochondria. Treatment with 3 μM Aβf gradually increased mitochondrial calcium uptake. Control experiments with reverse peptide Aβ42-1 did not show significant changes in mitochondrial calcium levels. However, 3 μM Aβ-AChEf complexes induced a rapid and acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels.

Mentions: Treatment with Aβ-AChE complexes caused a calcium influx in hippocampal neurons and a severe reduction in mitochondrial membrane potential. In order to study the role of mitochondria in the calcium deregulation induced by Aβ-AChE, we treated hippocampal neurons with Aβ and Aβ-AChE for 1 h and stained with MitoTracker. In control neurons without treatment we observed that most of the active mitochondria were localized through neurites (Fig. 4Aa). In the case of neurons treated with Aβ-AChEf, the fluorescence of active mitochondria decreased proportional to increasing Aβ-AChEf concentrations (Fig. 4Ab, c). Aβf (Fig. 4Ad, e) reduced the fluorescence of active mitochondria more than Aβo (Fig. 4Af, g), however both showed lower effect than Aβ-AChEf (Fig. 4B). AChE treatment had no effect (Fig. 4Ah). The quantification of MitoTracker fluorescence intensity indicated that Aβf and Aβo significant decreased the mitochondrial potential respect to control neurons. However, Aβ-AChEf treatment produced around 50% decrease in the mitochondrial potential. Then, we evaluated the mitochondrial calcium uptake [20,21]. Hippocampal neurons were loaded with Rhod-2 and then treated with Aβf and Aβ-AChEf. Aβf treatment produced an increase in the calcium uptake whereas Aβ-AChEf induced an acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels (Fig. 4C). Interestingly, decrease in mitochondrial calcium uptake correlates with severe mitochondrial potential loss induced by Aβ-AChE complexes in neurons. These results suggest that Aβ-AChEf produces the release of mitochondrial calcium, according with our previous result with the intracellular calcium chelator BAPTA-AM (Fig. 2D).


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 mitochondria in the calcium increase induced by Aβ-AChE. (A) Hippocampal neurons were treated with (a) control medium, (b) 2 μM Aβ-AChEf, (c) 5 μM Aβ-AChEf, (d) 2 μM Aβf, (e) 5 μM Aβf, (f) 2 μM Aβo, (g) 5 μM Aβo and (h) 5 nM AChE for 1 h and stained with MitoTracker (red stain in the insets) and Phalloidin (blue stain in the inset) to observe neuronal morphology. Bar = 10 μm. (B) Quantification of MitoTracker fluorescence intensity representative of mitochondrial membrane potential of the different treatments. Results are the mean ± S.E.M, in duplicate experiments, n = 2 independent experiments; *p < 0.05 (C) Hippocampal neurons were loaded with 5 μM Rhod-2 AM for 40 min and mitochondrial calcium uptake were determined. Mitotracker Green™ (MTG) was used to estimate Rhod-2 signal in the mitochondria. Treatment with 3 μM Aβf gradually increased mitochondrial calcium uptake. Control experiments with reverse peptide Aβ42-1 did not show significant changes in mitochondrial calcium levels. However, 3 μM Aβ-AChEf complexes induced a rapid and acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Role of mitochondria in the calcium increase induced by Aβ-AChE. (A) Hippocampal neurons were treated with (a) control medium, (b) 2 μM Aβ-AChEf, (c) 5 μM Aβ-AChEf, (d) 2 μM Aβf, (e) 5 μM Aβf, (f) 2 μM Aβo, (g) 5 μM Aβo and (h) 5 nM AChE for 1 h and stained with MitoTracker (red stain in the insets) and Phalloidin (blue stain in the inset) to observe neuronal morphology. Bar = 10 μm. (B) Quantification of MitoTracker fluorescence intensity representative of mitochondrial membrane potential of the different treatments. Results are the mean ± S.E.M, in duplicate experiments, n = 2 independent experiments; *p < 0.05 (C) Hippocampal neurons were loaded with 5 μM Rhod-2 AM for 40 min and mitochondrial calcium uptake were determined. Mitotracker Green™ (MTG) was used to estimate Rhod-2 signal in the mitochondria. Treatment with 3 μM Aβf gradually increased mitochondrial calcium uptake. Control experiments with reverse peptide Aβ42-1 did not show significant changes in mitochondrial calcium levels. However, 3 μM Aβ-AChEf complexes induced a rapid and acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels.
Mentions: Treatment with Aβ-AChE complexes caused a calcium influx in hippocampal neurons and a severe reduction in mitochondrial membrane potential. In order to study the role of mitochondria in the calcium deregulation induced by Aβ-AChE, we treated hippocampal neurons with Aβ and Aβ-AChE for 1 h and stained with MitoTracker. In control neurons without treatment we observed that most of the active mitochondria were localized through neurites (Fig. 4Aa). In the case of neurons treated with Aβ-AChEf, the fluorescence of active mitochondria decreased proportional to increasing Aβ-AChEf concentrations (Fig. 4Ab, c). Aβf (Fig. 4Ad, e) reduced the fluorescence of active mitochondria more than Aβo (Fig. 4Af, g), however both showed lower effect than Aβ-AChEf (Fig. 4B). AChE treatment had no effect (Fig. 4Ah). The quantification of MitoTracker fluorescence intensity indicated that Aβf and Aβo significant decreased the mitochondrial potential respect to control neurons. However, Aβ-AChEf treatment produced around 50% decrease in the mitochondrial potential. Then, we evaluated the mitochondrial calcium uptake [20,21]. Hippocampal neurons were loaded with Rhod-2 and then treated with Aβf and Aβ-AChEf. Aβf treatment produced an increase in the calcium uptake whereas Aβ-AChEf induced an acute mitochondrial calcium increase, with a subsequent decrease in mitochondrial calcium levels (Fig. 4C). Interestingly, decrease in mitochondrial calcium uptake correlates with severe mitochondrial potential loss induced by Aβ-AChE complexes in neurons. These results suggest that Aβ-AChEf produces the release of mitochondrial calcium, according with our previous result with the intracellular calcium chelator BAPTA-AM (Fig. 2D).

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