Fig. 4

PSEN1 abnormally high cell metabolic activity is coupled with impaired mitochondrial function. a MTS assay in iPSC derived BMECs. Cells were incubated in presence of MTS for 2 h. The MTS-conditioned medium was recovered and measured by spectrophotometry. Note the higher cell metabolic activity observed in PSEN1-BMECs compared to control-BMECs and PSEN2-BMECs. b JC-1 live imaging micrograph picture. Note the difference in JC-1 aggregation (as seen by the change in the emission fluorescence wavelength) with control cells showing predominantly JC-1 aggregates (red). In contrast, PSEN1-BMECs showed mostly JC-1 monomers (green) indicative of impaired mitochondrial membrane potential. Scale bar = 200 µm. c JC-1 flow cytometry analysis. The physiological mitochondrial membrane potential (MMP) is reflected as a “high-red”/low-green” JC-1 dye fluorescence. Inhibition of such membrane potential using an uncoupling agent (CCCP) results in the disruption of the proton gradient, resulting in the loss of the “red” fluorescence and a shift of the JC-1 fluorescence towards the “green” fluorescence. Note the absence of population shift occurring in PSEN1 (and to a lesser extent in PSEN2) BMECs following treatment with CCCP compared to the untreated cell group, suggesting an impaired mitochondrial membrane potential impairment. d Representative micrograph pictures of Lysosensor-Green(R) in iPSC-derived BMECs, counterstained with DAPI (nuclei stain, blue). Note the decreased Lysosensor-positive nuclei, indicative of impaired lysosomal acidification. Scale bar = 10 µm. e Flow cytometry profile of iPSC-BMECs following autophagy induction (by serum-starvation) for 24 h, followed by incubation with acridine orange. Note the cell events distribution in the control group (high FITC/high PerCP) indicative of acidic lysosomes. Such distribution is drastically changed in PSEN1-BMECs. f Representative CellRox(R) staining in iPSC-BMECs monolayers using live imaging. Note the significant increase in CellRox(R) fluorescence in PSEN1-BMECs indicative of an elevated ROS production in such cells. Scale bar = 200 µm. f Quantitative analysis of ROS levels using DCFDA fluorometric assay. Note the exacerbated ROS levels in PSEN1-BMECs compared to the two other cell types. N = 3/group, * and ** denote P < 0.05 and P < 0.01 versus the control group