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.