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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

Aβ and Aβ-AChE complexes induce morphological changes, apoptosis and intracellular Ca2+ increase in rat hippocampal neurons. (A) Immunofluorescence for MAP-2 protein of 14 DIV hippocampal neurons treated with: (a) control, (b) 5 μM Aβ-AChEo, (c) 5 μM Aβo, (d) 5 μM Aβ-AChEf, (e) 5 μM Aβf, (f) 5 nM AChE and (g) 5 μM Aβ42-1 for 1 h. Scale bar, 10 μm. (B) Cell viability was performed with the MTT reduction assay in hippocampal neurons treated for 12 h with the indicated μM concentrations. *p ≤ 0.01 and **p ≤ 0.001 compared with the control condition. (C) Hippoccampal neurons in culture were loaded with Fluo-3 AM (5 μM for 30 min at 37°C) to measure changes in free intracellular Ca2+. The graph shows normalized fluorescence intensities according to the ratio ΔF/Fo (arbitrary units) in function of time. Black bar indicates onset of treatment. 5 μM Aβf (White circle); 5 μM Aβ-AChEf (Black circle); 5 nM AChE (Grey circle). Inset shows the final normalized fluorescence intensities reached at the end of 1 h of recording. *p ≤ 0.001 compared to control; #p ≤ 0.001 compared to the Aβ-AChEf treatment. (D) Detection of Caspase-3 activity in hippocampal neurons. The graph shows the activity of Caspase-3 under effect of different preparation of Aβ and Aβ-AChE peptide. The cells were treated by 1 h at 37°C. Results are the mean ± S.E.M; *p < 0.05.
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Figure 1: Aβ and Aβ-AChE complexes induce morphological changes, apoptosis and intracellular Ca2+ increase in rat hippocampal neurons. (A) Immunofluorescence for MAP-2 protein of 14 DIV hippocampal neurons treated with: (a) control, (b) 5 μM Aβ-AChEo, (c) 5 μM Aβo, (d) 5 μM Aβ-AChEf, (e) 5 μM Aβf, (f) 5 nM AChE and (g) 5 μM Aβ42-1 for 1 h. Scale bar, 10 μm. (B) Cell viability was performed with the MTT reduction assay in hippocampal neurons treated for 12 h with the indicated μM concentrations. *p ≤ 0.01 and **p ≤ 0.001 compared with the control condition. (C) Hippoccampal neurons in culture were loaded with Fluo-3 AM (5 μM for 30 min at 37°C) to measure changes in free intracellular Ca2+. The graph shows normalized fluorescence intensities according to the ratio ΔF/Fo (arbitrary units) in function of time. Black bar indicates onset of treatment. 5 μM Aβf (White circle); 5 μM Aβ-AChEf (Black circle); 5 nM AChE (Grey circle). Inset shows the final normalized fluorescence intensities reached at the end of 1 h of recording. *p ≤ 0.001 compared to control; #p ≤ 0.001 compared to the Aβ-AChEf treatment. (D) Detection of Caspase-3 activity in hippocampal neurons. The graph shows the activity of Caspase-3 under effect of different preparation of Aβ and Aβ-AChE peptide. The cells were treated by 1 h at 37°C. Results are the mean ± S.E.M; *p < 0.05.

Mentions: In order to evaluate the morphological changes induced by Aβ-AChE complexes in hippocampal neurons, the following immunofluorescence studies were performed. Hippocampal neurons were treated with 5 μM of Aβ preparations: Aβ fibrils (Aβf), Aβ oligomers (Aβo), Aβ-AChE mostly fibers (Aβ-AChEf) and Aβ-AChE mostly oligomers (Aβ-AChEo) (see additional file 1). We used an Aβ reverse sequence (5 μM) and AChE (5 nM), for 1 hr as controls. Neurons were stained for MAP-1B (Fig. 1A) or NF-200 (Table 1). Neurons treated with Aβo (Fig. 1Ac) were observed to have a higher loss of their neurite network compared to neurons treated with Aβf (fig 1Ae). Furthermore, while neurons treated with Aβ-AChEo (Fig. 1Ab) appeared to have a small, loss of their neuritic network, neurons treated with Aβ-AChEf (Fig. 1Ad) were observed to have more damage on their neurite network than neurons treated with Aβf (Fig. 1Ae). Complementary immunofluorescence studies with a NF-200 antibody showed that Aβ-AChEf significantly decreased the length of the neurites by 40%, in comparison with Aβf-treated neurons (see Table 1). Additionally, we evaluated the effect of different Aβ and Aβ-AChE preparations on synaptic proteins (Additional file 2). We performed an immunofluorescence assay for presynaptic protein synapsin-1 and postsynaptic protein PSD-95. In agreement with previous studies, Aβo treatment decreased the immunostaining for PSD-95 whereas Aβf had no effect [17]. Also, Aβ-AChEo treatment induced a critical decrease in PSD-95 immunofluorescence similar to Aβo preparations, whereas Aβ-AChEf had no effect (Additional file 2), suggesting that Aβ oligomers formed in the presence of AChE have a similar synaptotoxicity to those formed in its absence.


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)

Aβ and Aβ-AChE complexes induce morphological changes, apoptosis and intracellular Ca2+ increase in rat hippocampal neurons. (A) Immunofluorescence for MAP-2 protein of 14 DIV hippocampal neurons treated with: (a) control, (b) 5 μM Aβ-AChEo, (c) 5 μM Aβo, (d) 5 μM Aβ-AChEf, (e) 5 μM Aβf, (f) 5 nM AChE and (g) 5 μM Aβ42-1 for 1 h. Scale bar, 10 μm. (B) Cell viability was performed with the MTT reduction assay in hippocampal neurons treated for 12 h with the indicated μM concentrations. *p ≤ 0.01 and **p ≤ 0.001 compared with the control condition. (C) Hippoccampal neurons in culture were loaded with Fluo-3 AM (5 μM for 30 min at 37°C) to measure changes in free intracellular Ca2+. The graph shows normalized fluorescence intensities according to the ratio ΔF/Fo (arbitrary units) in function of time. Black bar indicates onset of treatment. 5 μM Aβf (White circle); 5 μM Aβ-AChEf (Black circle); 5 nM AChE (Grey circle). Inset shows the final normalized fluorescence intensities reached at the end of 1 h of recording. *p ≤ 0.001 compared to control; #p ≤ 0.001 compared to the Aβ-AChEf treatment. (D) Detection of Caspase-3 activity in hippocampal neurons. The graph shows the activity of Caspase-3 under effect of different preparation of Aβ and Aβ-AChE peptide. The cells were treated by 1 h at 37°C. Results are the mean ± S.E.M; *p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Aβ and Aβ-AChE complexes induce morphological changes, apoptosis and intracellular Ca2+ increase in rat hippocampal neurons. (A) Immunofluorescence for MAP-2 protein of 14 DIV hippocampal neurons treated with: (a) control, (b) 5 μM Aβ-AChEo, (c) 5 μM Aβo, (d) 5 μM Aβ-AChEf, (e) 5 μM Aβf, (f) 5 nM AChE and (g) 5 μM Aβ42-1 for 1 h. Scale bar, 10 μm. (B) Cell viability was performed with the MTT reduction assay in hippocampal neurons treated for 12 h with the indicated μM concentrations. *p ≤ 0.01 and **p ≤ 0.001 compared with the control condition. (C) Hippoccampal neurons in culture were loaded with Fluo-3 AM (5 μM for 30 min at 37°C) to measure changes in free intracellular Ca2+. The graph shows normalized fluorescence intensities according to the ratio ΔF/Fo (arbitrary units) in function of time. Black bar indicates onset of treatment. 5 μM Aβf (White circle); 5 μM Aβ-AChEf (Black circle); 5 nM AChE (Grey circle). Inset shows the final normalized fluorescence intensities reached at the end of 1 h of recording. *p ≤ 0.001 compared to control; #p ≤ 0.001 compared to the Aβ-AChEf treatment. (D) Detection of Caspase-3 activity in hippocampal neurons. The graph shows the activity of Caspase-3 under effect of different preparation of Aβ and Aβ-AChE peptide. The cells were treated by 1 h at 37°C. Results are the mean ± S.E.M; *p < 0.05.
Mentions: In order to evaluate the morphological changes induced by Aβ-AChE complexes in hippocampal neurons, the following immunofluorescence studies were performed. Hippocampal neurons were treated with 5 μM of Aβ preparations: Aβ fibrils (Aβf), Aβ oligomers (Aβo), Aβ-AChE mostly fibers (Aβ-AChEf) and Aβ-AChE mostly oligomers (Aβ-AChEo) (see additional file 1). We used an Aβ reverse sequence (5 μM) and AChE (5 nM), for 1 hr as controls. Neurons were stained for MAP-1B (Fig. 1A) or NF-200 (Table 1). Neurons treated with Aβo (Fig. 1Ac) were observed to have a higher loss of their neurite network compared to neurons treated with Aβf (fig 1Ae). Furthermore, while neurons treated with Aβ-AChEo (Fig. 1Ab) appeared to have a small, loss of their neuritic network, neurons treated with Aβ-AChEf (Fig. 1Ad) were observed to have more damage on their neurite network than neurons treated with Aβf (Fig. 1Ae). Complementary immunofluorescence studies with a NF-200 antibody showed that Aβ-AChEf significantly decreased the length of the neurites by 40%, in comparison with Aβf-treated neurons (see Table 1). Additionally, we evaluated the effect of different Aβ and Aβ-AChE preparations on synaptic proteins (Additional file 2). We performed an immunofluorescence assay for presynaptic protein synapsin-1 and postsynaptic protein PSD-95. In agreement with previous studies, Aβo treatment decreased the immunostaining for PSD-95 whereas Aβf had no effect [17]. Also, Aβ-AChEo treatment induced a critical decrease in PSD-95 immunofluorescence similar to Aβo preparations, whereas Aβ-AChEf had no effect (Additional file 2), suggesting that Aβ oligomers formed in the presence of AChE have a similar synaptotoxicity to those formed in its absence.

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