Figure 6.

Annexin A5 restores mitochondrial depolarisation in Aβ-treated choroid plexus cultures in a Ca²⁺-dependent manner. (a) Representative images of tetramethyl-rhodamine methylester (TMRM) fluorescence used in redistribution mode (40 nM) in choroid plexus cultures incubated with and without oligomerised Aβ₄₂ (10 µM), in absence and presence of annexin A5 (15 µl/ml). (b) Annexin A5 restored the Aβ-induced mitochondrial depolarisation. Data were normalised to untreated cells and are represented as mean ± SEM from at least three independent experiments. (*p < 0.05 versus untreated cells; ^##p < 0.01 versus Aβ₄₂-treated cells; n = 4). (c) Traces showing changes-over time in fura-2 (upper panels) and simultaneous rhodamine 123 (Rh123) (bottom panels) fluorescence in choroid plexus epithelial cells upon physiological Ca²⁺ stimuli. Physiological Ca²⁺ was induced by addition of ATP (100 μM). Following Ca²⁺ release from ER, FCCP (1 μM) was added to obtain the maximal mitochondrial depolarisation allowing mitochondrial Ca²⁺ release. Upon stimulation of choroid plexus cells with ATP, Ca²⁺ stored in the ER was released and profound mitochondrial depolarisation was found in Aβ-incubated cells as shown by the increase in the Rh123 signal (iii). This effect was not observed in untreated cells (i) or annexin A5-treated cells only (ii). Annexin A5 treatment prevented the mitochondrial depolarisation when Ca²⁺ stimulus was added (iv). (d) Histograms showing ER Ca²⁺ (left panel) and mitochondrial Ca²⁺ levels (right panel) after addition of ATP and FCCP respectively as explained above. ER Ca²⁺ in choroid plexus cells incubated with Aβ were higher compared with untreated cells or annexin A5-treated cells only. Annexin A5 co-treatment restored the Aβ-induced increase of ER Ca²⁺ levels. Mitochondrial Ca²⁺ in Aβ-incubated choroid plexus cells was significantly higher compared with untreated cells or annexin A5-treated cells only. These levels were restored upon co-incubation with annexin A5. Data were normalised to untreated cells and are represented as mean ± SEM from at least three independent experiments. (****p < 0.0001 versus untreated cells; ^####p < 0.0001 versus Aβ₄₂-treated cells; n = 4).