Figure 3.

EVs generated by human neural cells expressing mutant PS1 impair cellular Ca²⁺ handling and mitochondrial function in cerebral cortical neurons. A. Preincubation of rat cortical neurons with H4Psn1Δ9-derived EVs for 24 hours resulted in a greater sustained Ca²⁺ response to glutamate (10 μM) stimulation (arrow indicates the time of glutamate application) compared to neurons preincubated with EVs from H4Psn1WT cells. Values are the mean and SEM of measurements made in 20 neurons per culture in 7 different cultures. B and C. Quantification of peak amplitude (B) and time to reach 50% recovery (B) following glutamate stimulation in cortical neurons that had been pretreated for 24 hours with EVs (H4D9 cells with either WT or Δ9 PS1; H4(PS1Δ9)+AβAb was a condition in which the EVs were incubated with an Aβ antibody (6E10). Values are the mean and SEM of measurements made in neurons from 7 different cultures. D. AD patient iPSC-derived neuronal EVs induce Ca²⁺ dysregulation following stimulation with glutamate (10 μM; arrow indicates the time of glutamate application). Values are the mean and SEM of measurements made in 3 different cultures (20 neurons per culture). E and F. Peak amplitude (E), and time to reach 50% recovery (F) following glutamate stimulation in rat cortical neurons pretreated for 24 hours with EVs from AD patient or WT iPSC-derived neurons (Aβ condition is rat cortical neurons incubated with 10 μM synthetic Aβ1-42). G. Analysis of the percentage of cortical neurons unresponsive to glutamate following incubation with mutant or WT EVs derived from H4 cells and IPSC-derived neurons (n =7 for H4 EVs treated neurons and n=3 for IPC EVs treated neurons). H. Representative Seahorse oxygen consumption assay showing mitochondrial impairment 24 hours following treatment with H4Psn1Δ9-derived EVs or 20 μM Aβ1-42. I. Basal oxygen consumption rate, maximal consumption rate and ATP production in cortical neurons that had been treated for 24 hours with H4Psn1Δ9-derived EVs or 20 μM Aβ1-42. Values are the mean and SEM from measurements made in 5 independent cultures. *p<0.05, **p<0.01.