The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson's disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.

Fig. 1

Detection of autophagy in the control and G2019S fibroblast groups. a, b Control and mutant fibroblasts were transfected with pDest-mCherry-GFP-LC3B. The number of yellow (autophagosomes) or red vesicles (autophagolysosomes) was quantified. The frequency of the measured changes is presented in a, and representative images are shown in b [obtained from the C1 (control) and GS1 (G2019S) cell lines]. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3), counted in 200 cells per condition (***p ≤ 0.001 between the untreated control and G2019S groups). c Electron microscopy images of the representative cells are shown in Fig. S4. Data represent the mean ± SEM values of the number of autophagosomes per cross-sectioned cell counted in ten cells per condition, control (C1–C4) and G2019S (GS1–GS3) (**p ≤ 0.01 between the control and G2019S groups). d, e G2019S LRRK2 mutants yield a greater number of cells with an accumulation of acidic compartments. Fibroblasts were stained with CMFDA to observe the presence of acidic compartments as indicated in the “Materials and methods” section. Acidic compartments are visualized as holes. Representative microphotographs are depicted in Fig. S5. The data represent the mean ± SEM values of area occupied by acidic compartments per cell counted in 200 cells per condition, control (C1–C4) and G2019S (GS1–GS3) (d) and % of cells with acidic compartments, counted in 200 cells per condition (e) (***p ≤ 0.001, **p ≤ 0.01 between the control and G2019S groups). f Study of protein degradation in control and mutant fibroblasts. The protein half-life was determined by pulse-chasing with radioactive valine as indicated in the “Materials and methods” section. The data represent the mean ± SEM of the individuals within each group, control (C1–C4) and G2019S (GS1–GS3), in the presence or absence of Baf A1 (***p ≤ 0.001 between the untreated control and G2019S groups)

Fig. 2

Western-blot analyses of autophagy-related proteins. Western-blot analysis showing differences in autophagy proteins in the control and G2019S LRRK2 mutant fibroblasts. Western blots were probed for all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3), with antibodies against Beclin-1 (a), p-mTOR and mTOR (c), p-p70^S6K and p70^S6K (e), LC3-I/II (g), p62 (i), LAMP-2 (k), and GAPDH (used as a loading control under the same conditions). b (Beclin-1), d (p-mTOR), f (p-p70^S6K), h (LC3), j (p62) and l (LAMP-2) show the densitometry of each band, expressed in arbitrary units for each Western-blot analysis. Horizontal bars represent the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3), and individual values are represented by points (***p ≤ 0.001, **p ≤ 0.01 between the control and G2019S groups)

Fig. 3

Effects of starvation in fibroblasts with or without the G2019S LRRK2 mutation. a, b Detection of apoptotic events by immunofluorescence. Fibroblasts cultured on poly-l-lysine-treated coverslips were exposed to nutrient deprivation with EBSS medium for 24 h followed by fixation and immunostaining for active caspase-3 (green) and staining with Hoechst 33342 to visualize nuclear chromatin condensation (blue). The frequency of the measured changes is presented in b, and representative images are shown in a [obtained from the C1 (control) and GS1 (G2019S) cell lines]. The scale bar represents 10 μm. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 between the treated and untreated cells). c Detection of PI-positive cells. Fibroblasts were treated as in a, followed by staining with PI to determine the viable cells. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001, *p ≤ 0.05 between the treated and untreated cells). d–j Autophagy exacerbation mediated by EBSS in fibroblasts with or without the G2019S mutation. d Detection of acidic compartments and lysosomal accumulation after starvation. Fibroblasts cultured on poly-l-lysine-treated coverslips were exposed to nutrient deprivation for 24 h followed by staining with CMFDA (green) to observe the presence of acidic compartments, and LTR to observe lysosomal swelling, as indicated in the “Materials and methods” section. Acidic compartments are visualized as holes. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001, *p ≤ 0.05 between the treated and untreated cells). e–j Western-blot analysis showing the effects of starvation on autophagy in both fibroblast groups. e Representative blot of the C1 and GS1 cell lines probed with antibodies against p-mTOR, mTOR, LC3-I/II, p62, LAMP-2, and cathepsin B. GAPDH expression was used as a loading control under the same conditions. f (mTOR), g (LC3), h(p62), i (LAMP-2), and j (cathepsin B) show the densitometry of each band, expressed in arbitrary units for each Western-blot analysis (***p ≤ 0.001, *p ≤ 0.05 between the treated and untreated cells)

Fig. 4

Effects of treatment with bafilomycin A1 in fibroblasts with or without the G2019S LRRK2 mutation. a, b Apoptotic events were detected by immunofluorescence. Fibroblasts cultured on poly-l-lysine-treated coverslips were treated with Baf A1 (100 nM) for 24 h followed by fixation and immunostaining for active caspase-3 (green) and Ho staining for nuclear chromatin condensation (blue). The frequency of the measured changes is presented in b, and representative images are shown in a [obtained from the C3 (control) and GS1 (G2019S) cell lines]. The scale bar represents 10 μm. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001 between the treated and untreated cells). c Detection of PI-positive cells. Fibroblasts were treated as in a, followed by staining with PI to determine the viable cells. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001 between the treated and untreated cells)

Fig. 5

Autophagy inhibition mediated by bafilomycin A1 in fibroblasts with or without the G2019S LRRK2 mutation. a, b Detection of acidic compartments and lysosomal accumulation after Baf A1 treatment. Fibroblasts cultured on poly-l-lysine-treated coverslips were treated with Baf A1 (100 nM) for 24 h, followed by staining with CMFDA (green) to observe the presence of cytoplasmic vacuoles, LTR to observe lysosome swelling, as indicated in the “Materials and methods” section. Acidic compartments are visualized as holes. Representative microphotographs are depicted in a [obtained from the C1 (control) and GS2 (G2019S) cell lines], and quantitative data are analyzed in b. The scale bar represents 10 μm. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001 between the treated and untreated cells). c, d Detection of the puncta formation of LC3B and determination of the expression levels of LAMP-2 by immunostaining after Baf A1 treatment. Fibroblasts cultured on poly-l-lysine-treated coverslips were treated as in a, followed by fixation and immunostaining for LC3B (green), LAMP-2 (red). Representative microphotographs are depicted in d [obtained from the C1 (control) and GS1 (G2019S) cell lines], and quantitative data are analyzed in c. The scale bar represents 10 μm. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001 between the treated and untreated cells). e–i Western-blot analysis showing the effects of Baf A1 on autophagic proteins. e Representative blots probed in C1 and GS1 cell lines, with antibodies against LC3, p62, LAMP-2, cathepsin B, and GAPDH (used as a loading control under the same conditions). f (LC3), g (p62), h (LAMP-2), and i (Cathepsin B) show the densitometry of each band, expressed in arbitrary units for each Western-blot analysis (***p ≤ 0.001 between the treated and untreated cells)

Fig. 6

MEK/ERK inhibition reduces autophagy and sensitivity observed in cells with the G2019S LRRK2 mutation. a, b Western-blot analyses of the phosphorylation levels of the MAPK/ERK protein (p-ERK 1/2) under basal conditions. a Western blots probed in all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3), with antibodies against p-ERK1/2, ERK1/2 and GAPDH (used as loading controls under the same conditions). b The densitometry of each band, expressed in arbitrary units for Western-blot analysis of a. Horizontal bars represent the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3), and individual values are represented by points (***p ≤ 0.001 between control and G2019S groups). c, d Detection of acidic compartments after U0126 treatment (20 μM). Fibroblasts cultured on poly-l-lysine-treated coverslips were exposed to U0126 for 24 h followed by staining with CMFDA (green) to observe the presence of acidic compartments, as indicated in the “Materials and methods” section. Acidic compartments are visualized as holes. Representative microphotographs are depicted in d [obtained from the C1 (control) and GS2 (G2019S) cell lines]. The scale bar represents 10 μm. Quantitative data are presented in c. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3) (***p ≤ 0.001 compared to the untreated cells). e–j Western-blot analyses of autophagy-related protein LC3-II in fibroblasts treated with U0126 (e, f), Atg5 siRNA (g, h) or LRRK2 siRNA (i, j). e, g, i Representative blots probed in the C1 and GS2 cell lines, with antibodies against LC3-II and GAPDH (used as a loading control under the same conditions). f, h, j The densitometry of each band, expressed in arbitrary units for each Western-blot analysis (***p ≤ 0.001 compared treated and untreated cells). k The detection of PI-positive cells. Fibroblasts were treated with 20 μM U0126 for 24 h, transiently transfected with Atg5 siRNA or LRRK2 siRNA, followed by staining with PI to determine the viable cells. Data are expressed as the mean ± SEM of all cell lines within each group, control (C1–C4) and G2019S (GS1–GS3). (***p ≤ 0.001, **p ≤ 0.01 between the treated and untreated cells)