Lysosomal ATPase and acidity in neurons derived from Alzheimer’s disease patients were less than in control cells. (A) Colocalization of vacuolar-type H⁺-ATPase (V-ATPase; red) was measured using immunostaining with specific markers targeting lysosomes (LAMP-2, green), endosomes (EEA, green), and endoplasmic reticulum (calnexin, green), in induced pluripotent stem cells of healthy human subjects (CON), sporadic Alzheimer’s disease (SAD), or familial Alzheimer’s disease (FAD) patients. (B) Cell acidity was measured by lysotracker-positive acidic vehicles (red) in control, sporadic Alzheimer’s disease, and familial Alzheimer’s disease cells (4′,6-diamidino-2-phenylindole [DAPI], blue). (C) Vacuolar-type H⁺-ATPase in lysosomes (LAMP-2) was significantly lower in sporadic Alzheimer’s disease (P = 0.001) and familial Alzheimer’s disease cells (P = 0.010) than controls. There was significant source of variation for interaction (F[4,23] = 4.35, P = 0.008) and organelle type (F[2,23] = 29.15, P < 0.0001). (D) With the addition of dantrolene (DAN, 30 µM), V-ATPase in lysosomes (LAMP-2) in familial Alzheimer’s disease cells was no longer significantly reduced (P = 0.965) but remained significantly lower in sporadic Alzheimer’s disease cells (P = 0.007) compared with controls. In addition, vacuolar-type H⁺-ATPase in the endoplasmic reticulum (calnexin) of the controls was significantly reduced compared with sporadic Alzheimer’s disease (P = 0.001) and familial Alzheimer’s disease (P < 0.0001) cells. There was significant source of variation for interaction (F[4,27] = 8.66, P = 0.0001), organelle type (F[2,27] = 79.49, P < 0.0001), and cell type (F[2,27] = 5.96, P = 0.007). (E) Lysotracker-positive acidic vesicles were significantly lower in sporadic Alzheimer’s disease (P < 0.0001) and familial Alzheimer’s disease (P = 0.0004) compared with control cells. Dantrolene also significantly increased tracker-positive acidic vesicles in both sporadic Alzheimer’s disease (P = 0.025) and familial Alzheimer’s disease (P = 0.036) cells compared with dimethyl sulfoxide (DMSO). Cell type and dantrolene were significant sources of variation (F[2,19] = 29.88, P < 0.0001; and F[1,19] = 23.16, P = 0.0001, respectively). All data are expressed as the means ± SD from four independents (n = 4 replicates for all groups) and were analyzed by two-way analysis of variance followed by Sidak’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.