Glycogen synthase kinase 3 inhibition promotes lysosomal biogenesis and autophagic degradation of the amyloid-β precursor protein

Abstract

Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function.

Fig 1

GSK3 inhibition reduces Aβ generation independently of α-secretase. (A) Representative Western blot and quantification of secreted Aβ (4 kDa) in media from N2asw cells treated overnight with vehicle (DMSO) or various GSK3 inhibitors, including inhibitors VIII (5 μM) and XI (10 μM; Calbiochem) and lithium chloride (20 mM) (n = 12). Cntrl, control. (B) Representative Western blot of GSK3α/β showing reductions in GSK3 expression after transfection with GSK3α/β siRNA (10 nM). (C) Secreted Aβ was also reduced in media from cells transfected with GSK3α/β siRNA (n = 9). (D) Total sAPP in media from cells treated with GSK3 inhibitor VIII (5 μM) (n = 9). (E) Levels of the secreted sAPPα (96 kDa) fragment were determined in media from cells treated with GSK3 inhibitors, including inhibitors VIII (5 μM) and LiCl (5 mM) (n = 18). (F) Enzymatic activity of α-secretase was monitored by a commercial fluorometric assay in membrane preparations from N2asw cells using GSK3 inhibitor VIII (5 μM) (n = 9). Bars represent means ± standard errors of the means (SEM). Asterisks represent significant differences between control and treated cells (determined by one-way ANOVA, Tukey's post hoc test, or Student's t test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 2

Inhibition of GSK3 affects the stability of full-length APP. (A) Representative Western blots and quantification of APP protein expression levels in N2asw cells treated with GSK3 inhibitor VIII (5 μM) (n = 9). (B) Representative Western blots and quantification of APP protein expression in N2asw cells transfected with GSK3α/β siRNA (n = 9). (C) Representative Western blots and quantification of APP protein expression in the presence of actinomycin D (1 μg/ml) shows a decrease in the presence of inhibitor VIII (n = 3). (D) Pulse-chase analysis of N2asw cells transfected with GSK3α/β siRNA. Cells were labeled with [³⁵S]methionine for 10 min and chased for the indicated time periods. APP was immunoprecipitated from cell lysates using 6E10 antibody (n = 4). (E) Representative Western blots and quantification of APP protein expression levels in N2asw cells treated with GSK3 inhibitor VIII (5 μM) with or without the proteasomal inhibitor MG132 (50 μg/ml) overnight (n = 9). (F) Representative Western blots and quantification of APP protein expression levels in N2asw cells treated with GSK3 inhibitor VIII (5 μM) with or without the proteasomal inhibitor PS1 (10 μg/ml) overnight (n = 6). Bars represent means ± SEM. Asterisks represent significant differences between control and treated cells (determined by one-way ANOVA, Tukey's post hoc test, or Student's t test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 3

GSK3 inhibition induces autophagy pathway. (A) GSK3 inhibition induces autophagy. N2asw cells transfected with LC3-GFP cDNA were incubated for 18 h in serum-free media containing GSK3 inhibitor VIII (5 μM) and visualized with an epifluorescence microscope, and images were captured with a digital camera (×20 magnification). (B) HEK-293 cells stably transfected with LC3-GFP cDNA were incubated for 18 h in serum-free media containing GSK3 inhibitor VIII (5 and 10 μM) and visualized with Z-stacks from a Leica LAS AF SP5 confocal microscope (×63 optical magnification and 2.5× digital zoom). Quantification of punctum LC3 vesicles in those cells shows increased numbers with specific GSK3 inhibition. (C) Representative Western blot and quantification of LC3 in N2asw cells. Cells were incubated in serum-free media containing different concentrations of GSK3 inhibitor VIII, with or without the autophagy inhibitor 3-MA (500 μM) or the autophagy inducer rapamycin (20 μM) for 16 h. The intensity of the LC3-I and LC3-II bands was measured, and results are expressed as the ratio of LC3-II to total LC3 and LC3-II with respect to β-actin values (n = 9). (D) Autophagy flux was determined in N2asw cells by incubation with or without the inhibitors of lysosomal proteolysis, leupeptin (200 μM) and NH₄Cl (20 mM), for 4 h. The LC3-II/β-actin ratio was quantified and is represented (n = 4). (E) Representative Western blot and quantification of APP, p62, and β-actin of cell lysates from N2asw cells incubated for 18 h in serum-free media containing GSK3 inhibitor VIII (5 μM) or DMSO. Bars represent means ± SEM. Asterisks represent significant differences between control and treated cells (determined by one-way ANOVA, Tukey's post hoc test, or Student's t test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 4

GSK3 inhibition induces APP and CTF degradation via autophagy/lysosomal pathway. (A) Representative Western blot and quantification of APP expression in N2asw cells incubated with the autophagy inhibitor 3-MA (500 μM) (n = 4). FL, full length. (B) Representative Western blot and quantification of full-length APP expression of N2asw cells transfected with the control or siRNA for BEC1 in the presence or absence of GSK3 inhibitor VIII (5 μM) (n = 9). (C) Representative Western blot and quantification of APP full-length expression of N2asw cells transfected with control siRNA or siRNA for ATG5 in the presence or absence of GSK3 inhibitor VIII (5 μM) (n = 8). (D) Representative Western blot and quantification of total CTFs of APP in N2asw cells transfected with control siRNA or siRNA for ATG5 treated with GSK3 inhibitor VIII (5 μM) (n = 3). (E) Representative Western blot and quantification of CTF (12 kDa) levels in CHO cells stably transfected with APP-C99 and treated with GSK3 inhibitor VIII (5 μM) (n = 8). (D) Inhibition of lysosomal proteases with 100 μM leupeptin resulted in increased stability of APP (n = 4). Bars represent means ± SEM. Asterisks represent significant differences between control and treated cells (determined by one-way ANOVA, Tukey's post hoc test, or Student's t test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Fig 5

GSK3 inhibition increases lysosomal biogenesis. (A) Staining with Lysotracker in N2asw cells in the presence or absence of GSK3 inhibitor VIII (5 μM). The quantification of the number of vesicles per cell shows a clear increase in organelles with low internal pH after GSK3 inhibition. As an internal control, bafilomycin A1 (10 nM) was used. (B) Electron micrographs ×6500 of N2asw cells incubated for 18 h in deprived medium in the absence or presence of GSK3 inhibitor VIII (5 μM). Micrographs show representative pictures of primary lysosomes (PL), mature lysosomes (ML), and increased numbers of primary lysosomes in cells treated with GSK3 inhibitor. The total number of lysosomes per cell was counted for at least 30 cells/condition, and the average number of vesicles per cell is represented in the graphs. (C) Double immunostaining of APP (with antibody against its C terminus) and LAMP-1 in cells treated with DMSO or GSK3 inhibitor. Bars represent means ± SEM. Asterisks represent significant differences between control and treated cells (Student's t test). *, P ≤ 0.05; ***, P ≤ 0.001.

Fig 6

GSK3 regulates TFEB nuclear translocation. (A) Representative images and quantification of N2asw cells overexpressing TFEB cultured in DMSO or GSK3 inhibitor VIII (5 μM). TFEB nuclear localization was analyzed by Flag antibody and nuclear staining with 4′,6′-diamidino-2-phenylindole (DAPI) (n = 4). (B) Cells were subjected to subcellular fractionation, and the nucleus was blotted with an antibody against Flag. H4 was used as a nuclear marker (n = 9). UT, untransfected. (C) Endogenous TFEB staining shows increased TFEB nuclear trafficking in cells treated with GSK3 inhibitor (n = 9). (D) Representative Western blot and quantification of APP full-length expression of N2asw cells transfected with control siRNA or siRNA for TFEB in the presence or absence of GSK3 inhibitor VIII (5 μM) (n = 9) (determined by one-way ANOVA, Tukey's post hoc test, or Student's t test). *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.