PMC

(A-D) MEFs derived from either wild-type or ATG5 −/− mice were incubated for 24 hours in the absence or presence of 50μM latrepirdine. Cell lysates were analyzed for levels of APP metabolites, as well as markers of autophagy including LC3, P62, S6K and pS6K by Western blot. (E-K) N2a cells stably over-expressing Swedish APP (N2a SweAPP) were incubated for 24 hours in the absence or presence of 50μM latrepirdine. Cell lysates and conditioned media were analyzed for levels of APP metabolites, as well as markers of autophagy including LC3, P62, S6K and pS6K by Western blot. All figures are representative of three or more independent experiments, performed in duplicate or triplicate. Graphs are mean ± SEM; *p<0.05; **p<0.01; ***p<0.001.

(A) HeLa cells stably expressing eGFP-LC3 were treated in the absence or presence of 50μM latrepirdine for 6 hours. (B-F) N2a cells were treated in the absence (Vehicle; Ctrl) or presence of 5nM, 500nM, or 50μM latrepirdine for 3 or 6 hours. (B) Western blot analysis of lysates for levels of endogenous LC3-I/–II, p62, (p)mTOR, (p)S6K, and total mTOR or S6K. (C-F) Quantification of Western blot densitometry for experiments summarized in A and B. All figures are representative of three or more independent experiments, performed in duplicate or triplicate. Graphs are mean ± SEM; *p<0.05; **p<0.01; ***p<0.001.

(A-E) Mouse embryonic fibroblasts (MEFs) derived from wild-type or ATG5 −/− mice were treated for 3 hours in the absence (VEH; Ctrl) or presence of 50 μM latrepirdine (LAT). Lysates were analyzed by Western blot for levels of p-S6K, total S6K, p62, and LC3 and α/β-CTFs (using the pan-species pAb 369). (F-L) N2a cells stably over-expressing Swedish APP were treated for 3 hours in the absence (VEH; Ctrl) or presence of 50 μM latrepirdine (LAT). Lysates were analyzed by Western blot for levels of p-S6K, total S6K, p62, and LC3. Lysates and conditioned medium were also analyzed by Western blot for levels of intracellular (IC Aβ, holoAPP, C99-CTF) or secreted (sAPPa and sAβ) APP metabolites, respectively, with the human APP-specific mAb 6E10. All figures are representative of three or more independent experiments, performed in duplicate or triplicate. Graphs are mean ± SEM; ^nsp<0.15; ^#p<0.10; *p<0.05; **p<0.01; ***p<0.001.

90-day-old and 120-day-old male TgCRND8 mice or their non-transgenic littermates (nTg) were sacrificed and brains were analyzed for levels of soluble and insoluble APP metabolites, α-syn, LC3, and p62. (A, B) Composite micrograph of 90-day-old nTg (A) or TgCRND8 (B) brain immunostaining with LC3 and Aβ42 and DAPI as counter-stain (as labeled). Aβ42-immunoreactive amyloid plaques are visible in the cortex and hippocampus of TgCRND8 (B) mice, but not nTg (A) littermates. Scale bar is 1.0mm. (C) Western blot analysis of soluble and insoluble (formic acid) APP metabolites, LC3, and p62 from the gross cerebral cortex of 90- or 120-day old mice (genotype as indicated). (D-I) Analysis of Western blot integrated density following normalization to Actin of p62 (D-90-days; E-120-days), LC3 (F-90-days; G-120-days), and α-syn (H-90-days; I-120-days). Only 120-day-old TgCRND8 mice accumulated soluble LC3-I, LC3-II, p62, and α-syn by comparison to nTg littermates (C, E, G). Graphs are mean % nTg expression ± SEM; *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001.

90-day-old male TgCRND8 mice and their wild-type littermates received 31 consecutive once daily i.p. injections of either 3.5 mg/kg latrepirdine (n=10; LAT) or 0.9% saline (n=10; VEH). Mice were tested on both contextual (A) and cued (B) fear conditioning tasks, as described in Methods. (C,D) Low magnification representative coronal sections comparing the CA1 region of the hippocampus, and cortex from vehicle-treated (C) or latrepirdine-treated (D) animals. Green punctae were determined to be Aβ42-immunoreactive amyloid plaques (Pan1G6 anti-Aβ42 antibody), red staining is LC3, DAPI was used as counter-stain. Scale Bars = 1.0mm (E-H) Western blot analysis of soluble and insoluble fractions from TgCRND8 mouse brains for p62, LC3, α-syn and APP metabolites. Western blots and brain sections are representative littermates from each treatment group. The noted decrease in absolute concentration of insoluble Aβ42 (I) is in agreement with our observation of decreased Aβ42-immunoreactive plaques among latrepirdine-treated mice (C-Veh vs D-Lat). Graphs are mean ± SEM; n.s. p=0.099 (non-significant trend); *p<0.05; **p<0.01; ***p<0.001.