Fig. 4
Enhancing mCa2+ efflux rescues mitochondrial dysfunction in APPswe cells. a Experimental protocol timeline for N2a maturation and adenovirus encoding NCLX (Ad-NCLX) transduction. b Oxygen consumption rate (OCR) at baseline and following: oligomycin (oligo; Complex V inhibitor; to uncover ATP-linked respiration), FCCP (protonophore to induce maximum respiration), and rotenone + antimycin A (Rot/AA; complex I and III inhibitor for complete ETC inhibition). c Quantification of basal respiration (base OCR – non-mito respiration (post-Rot/AA). d Quantification of ATP-linked respiration (post-oligo OCR−base OCR). e Maximum respiratory capacity (post-FCCP OCR−post-Rot/AA). f Spare respiratory capacity (post-FCCP OCR−basal OCR). g Proton leak (post-Oligo OCR−post-Rot/AA OCR). h Quantification of Cell Rox green fluorescent intensity (total cellular ROS production); fold change vs. N2a controls, n = 8 for all groups. i Quantification of DHE fluorescent intensity; fold change vs. N2a controls. j Quantification of MitoSOX fluorescent intensity; fold change vs. N2a controls, n = 52 for N2a control, n = 59 APPswe, and n = 59 for APPswe + Ad-NCLX. k Western blots of full-length APP, ADAM-10 (α-secretase) BACE1 (β-secretase), PS1, Nicastrin, APH (γ-secretase), and tubulin (loading con). l Quantification of β-secretase activity, n = 3 for all groups. m Quantification of extracellular Aβ1–40 and Aβ1–42 levels. n Representative images of intracellular protein aggregates in N2a control, APPswe and APPswe + Ad-NCLX cells stained with proteostat aggresome detection reagent (red) and Hoechst 33342 nuclear stain (blue), scale bars = 20 μm. o Total aggregates per cell, n = 41 for N2a control, n = 62 APPswe and n = 69 APPswe + Ad-NCLX. (P-R) Control, APPswe and APPswe transduced with Ad-NCLX for 48 h were assessed for plasma membrane rupture, Sytox Green, after treatment with: p Ionomycin (Ca2+ ionophore, 1–5 µM), q glutamate (-NMDAR agonist, 10–50 µM), r tert-Butyl hydroperoxide (TBH, oxidizing agent, 10–30 µM), n = 4 experiments for each reagent. (n = individual dots shown for each group in all graphs. All data presented as mean ± SEM; ****p < 0.001, **p < 0.01, *p < 0.05; one-way ANOVA with Sidak's multiple comparisons test.) Source data are available as a Source Data file
