Effect of latrepirdine on the levels of APP metabolites from cell lysates and conditioned media of mouse N2a cells. Cells were treated for 6 hours in the presence of vehicle or increasing concentrations of latrepirdine (as labeled). (a) Representative western blot of total intracellular and secreted metabolites of APP from at least 3 independent experiments, each performed in duplicate. (b) Quantification of western blot band densitometry represented as mean percent of vehicle +/- S.E.M. 5 nM latrepirdine produced an approximately half-maximal ~37% increase (SD = 0.33, t(10) = 2.75, p = 0.02), and 500 nM produced a maximal significant ~64% increase (SD = 0.49, t(10) = 3.16, p = 0.01) in extracellular Aβ compared to vehicle. The highest concentration (5 μM) also caused a significant ~59% increase (SD = 0.52, t(10) = 2.78, p = 0.02) in extracellular Aβ levels, and an increase in extracellular Aβ levels was observed with 50 nM latrepirdine, which approached significance on a two-tailed test (SD = 0.54, t(4) = 2.62, p = 0.059). Incubation with 5 nM or 500 nM latrepirdine resulted in a significant ~34% (SD = 0.35, t(10) = 2.33, p = 0.04) and ~27% (SD = 0.14, t(10) = 4.87, p = 0.0006) increase in sAPPα accumulation, respectively, in conditioned media compared to vehicle. An increase in extracellular sAPPα levels was observed with 50 nM latrepirdine, which approached significance on a two-tailed test (SD = 0.53, t(4) = 2.31, p = 0.081). No significant increases in sAPPα levels were distinguished between vehicle and either 500 pM or 5 μM latrepirdine. No significant accumulation of holoAPP of APP-CTFs (C83-CTF or C99-CTF) was observed following 6 h incubation in the absence (vehicle) or presence of varying concentrations of latrepirdine (as indicated). (c) Vehicle and all latrepirdine concentrations were indistinguishable by mean Aβx-42/Aβx-40 ratio, quantified by Aβ species-specific sandwich ELISA (mean Aβx-42/Aβx-40, n = 3 independent experiments, each performed in duplicate). Absolute Aβx-40 and Aβx-42 levels in the media were ~380-490 pM and ~14-20 pM, respectively. *Value represents a significant mean difference from vehicle by independent samples t-test, two-tailed α = 0.05, #p < 0.10; *p < 0.05; **p < 0.01.