Protective Effect of the LRRK2 Kinase Inhibition in Human Fibroblasts Bearing the Genetic Variant GBA1 K198E: Implications for Parkinson's Disease

Abstract

Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder for which there are currently no curative therapies. Therefore, the need for innovative treatments for this illness is critical. The glucosylceramidase beta 1 (GBA1) and leucine-rich repeated kinase 2 (LRRK2) genes have been postulated as potential genetically defined drug targets. We report for the first time that the LRRK2 inhibitor PF-06447475 (PF-475) not only restores GCase enzyme activity, but also increases mitochondrial membrane potential, significantly decreases DJ-1 Cys106-SO₃, reduces lysosome accumulation, and diminishes cleaved caspase-3 (CC3) in GBA1 K198E fibroblasts. Furthermore, in addition to a significant reduction in p-Ser935 LRRK2 kinase, we found that PF-475 reduced p-Thr73 RAB 10 and p-Ser129 α-Syn in mutant skin fibroblasts. In addition, we found that the GCase activator GCA (NCGC00188758) increased GCase activity and decreased lysosomal accumulation, but did not affect p-Ser935 LRRK2, ∆Ψm, p-Ser129 α-Syn, DJ-1 Cys106-SO₃, or CC3 in K198E GBA1 fibroblasts. The GCase inhibitor conduritol-β-epoxide (CBE), used as an internal control, significantly reduced GCase and left the other pathological markers largely unaltered in GBA1 K198E, but reduced GCase and increased the accumulation of lysosomes only in WT GBA1 fibroblasts. Taken together, these results suggest that LRRK2 is a critical signaling kinase in the pathogenic mechanism associated with the lysosomal GBA1/GCase K198E variant. Our findings suggest that the use of LRRK2 inhibitors in PD patients with GBA1 mutations, such as K198E, may be effective in reversing GBA1/GCase deficiency, autophagy impairment, oxidative stress, and neuronal death.

Keywords: Fibroblasts; GBA; LRRK2; PF-06447475; Parkinson’s disease; RAB10.

Fig. 1

GBA1 K198E mutation alters glucocerebrosidase (GCase) substrate affinity, reduces enzymatic activity, and increases GCase levels and phosphorylation of LRRK2 (Ser935) and RAB10 (Thr73). A Enzyme activity of GCase in WT GBA1 (blue dots) and GBA1 K198E fibroblasts (red dots). B Protein expression levels of glucocerebrosidase (GCase) in WT GBA1 (blue curve) and GBA1 K198E fibroblasts (red curve) were analyzed via flow cytometry. C Quantification of GCase expression levels. Histogram numbers indicate the percentage of positive cellular populations for the tested marker. D Representative flow cytometry histogram analysis showing phosphorylated LRRK2 at Ser935 protein in WT (blue curve) and GBA1 K198E fibroblasts (red curve). E Quantitative analysis of p-Ser935 LRRK2 protein. F-G Immunofluorescence images showing p-Ser935 LRRK2 protein (green fluorescence) in WT GBA1 (F) and GBA1 K198E fibroblasts (G). H Quantitative analysis of p-Ser935 LRRK2 protein in WT GBA1 (blue curve) and GBA1 K198E fibroblasts (red curve). GBA1 K198E fibroblasts show an endogenously high percentage of phosphorylated RAB10 at Thr73. I Representative flow cytometry histogram showing phosphorylated RAB10 at Thr73 in WT GBA1 (blue curve) and GBA1 K198E fibroblasts (red curve). J Quantitative analysis of p-Thr73 RAB10 levels. K-L Immunofluorescence images showing p-Thr73 RAB10 protein (green fluorescence) in WT GBA1 fibroblasts (K) and GBA1 K198E fibroblasts (L). M Quantitative analysis of phosphorylated RAB10 at Thr73 in WT GBA1 (blue) and GBA1 K198E fibroblasts (red). Numbers in histograms represent positive cellular population for the tested marker. The histograms and photomicrographs represent one out of three independent experiments (n = 3). The data are presented as mean ± SD of three independent experiments (dots in bar). One-way ANOVA followed by Tukey’s test. Statistically significant differences: ***p < 0.001. Image magnification 400 ×

Fig. 2

PF-06447475 (PF-475) LRRK2 kinase inhibitor reduced levels of phosphorylated (Ser935) LRRK2 in fibroblasts bearing GBA1 K198E mutation. A Representative flow cytometry histograms showing p-Ser935 LRRK2 in WT GBA1 fibroblasts untreated or treated with the LRRK2 inhibitor PF-475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM), or conduritol-B-epoxide (CBE, 10 µM) for 24 h. B Quantitative analysis of p-Ser935 LRRK2 levels in WT GBA1 fibroblasts. C Representative flow cytometry histograms showing p-Ser935 LRRK2 in GBA1 K198E fibroblasts untreated or treated with PF-475, GCA, or CBE for 24 h. D Quantitative analysis of p-Ser935 LRRK2 levels in GBA1 K198E fibroblasts. Immunofluorescence image of p-Ser935 LRRK2 (green fluorescence) and nuclei (blue fluorescence) in WT GBA1 (E–H) and GBA1 K198E (I–L) fibroblasts untreated (E, I) or treated with PF-475 (F, J), GCA (G, K), or CBE (H, L) for 24 h. M Quantitative analysis of p-Ser935 LRRK2 in WT fibroblasts. N Quantitative analysis of p-Ser935 LRRK2 in GBA1 K198E fibroblasts. Numbers in histograms represent the percentage of the positive cell population for the tested marker. Results are from three independent experiments (n = 3) presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test; ***p < 0.001; ns = no significant. Image magnification 400 ×

Fig. 3

LRRK2 kinase inhibition reduced levels of phosphorylated (Thr73) RAB10 in fibroblasts bearing the GBA1 K198E mutation. A Representative flow cytometry histograms showing p-Thr73 RAB10 in WT and K198E GBA1 fibroblasts untreated or treated with LRRK2 inhibitor PF-475 (1 µM), B Quantitative analysis of p-Thr73 RAB10 levels in WT and K198E GBA1 fibroblasts. Immunofluorescence image of p-Thr73 RAB10 (green fluorescence) and nuclei (blue fluorescence) in WT GBA1 (C-D) and GBA1 K198E (E-F) fibroblasts untreated (C, E) or treated with PF-475 (D, F) for 24 h. G Quantitative analysis of p-Thr73 RAB10. Numbers in histograms represent the percentage of the positive cell population for the tested marker. Results are from three independent experiments (n = 3) presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test; ***p < 0.001; ns no significant. Image magnification 400 ×

Fig. 4

LRRK2 kinase inhibition increases glucocerebrosidase enzymatic activity in fibroblasts bearing GBA1 K198E mutation. A GCase enzyme activity in WT GBA1 fibroblasts (blue bars) treated for 24 h with the LRRK2 inhibitor PF-06447475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM, CID: 46,907,762) and conduritol-B-epoxide (CBE, 10 µM, CID: 136,345). B GCase enzyme activity in GBA1 K198E fibroblasts (red bar) under the same treatment conditions. Data are presented as mean ± SD of three independent experiments (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s test; **p < 0.01 and ***p < 0.001

Fig. 5

LRRK2 kinase inhibition prevents lysosome accumulation and protects mitochondrial membrane potential (∆Ψm) in fibroblasts bearing the GBA1 K198E mutation. A Representative flow cytometry histograms showing Lysotracker® staining in WT GBA1 fibroblasts (blue curve) untreated or treated with LRRK2 inhibitor PF-475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM), or conduritol-B-epoxide (CBE, 10 µM) for 24 h. B Quantitative analysis of Lysotracker® in WT GBA1 fibroblasts. C Representative flow cytometry histograms showing Lysotracker® staining in GBA1 K198E fibroblasts untreated or treated with PF-475, GCA, or CBE for 24 h. D Quantitative analysis of Mitotracker® in GBA1 K198E fibroblasts. E Representative flow cytometry histograms showing Mitotracker® staining in WT GBA1 fibroblasts (blue curve) untreated or treated with the LRRK2 inhibitor PF-475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM), or conduritol-B-epoxide (CBE, 10 µM) for 24 h. F Quantitative analysis of Mitotracker® in WT GBA1 fibroblasts. G Representative flow cytometry histograms showing Mitotracker® staining in GBA1 K198E fibroblasts untreated or treated with PF-475, GCA, or CBE for 24 h. H Quantitative analysis of Lysotracker® in GBA1 K198E fibroblasts. Representative fluorescence microscopy images showing Lysotracker® (green fluorescence), Mitotracker® (red fluorescence), and Hoechst (blue fluorescence) in WT GBA1 (I-J) and GBA1 K198E (K-L) fibroblasts untreated (I, K) or treated with PF-475 (J, L) for 24 h. M Quantification of the mean fluorescence intensity (MFI) for Lysotracker®. N Quantification of the mean fluorescence intensity (MFI) for Mitotracker®. Numbers in histograms represent the percentage of the positive cell population for the tested marker. Results are from three independent experiments (n = 3) presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test; *p < 0.05, **p < 0.01, ***p < 0.001; ns = no significant. Image magnification 400 ×

Fig. 6

LRRK2 kinase inhibition reduced levels of phosphorylated (Ser129) α-synuclein (α-Syn) in fibroblasts bearing the GBA1 K198E mutation. A Representative flow cytometry histograms showing p-Ser129 α-Syn in WT GBA1 fibroblasts untreated or treated with the LRRK2 inhibitor PF-475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM), or conduritol-B-epoxide (CBE, 10 µM) for 24 h. B Quantitative analysis of p-Ser129 α-Syn levels in WT GBA1 fibroblasts. C Representative flow cytometry histograms showing p-Ser129 α-Syn in GBA1 K198E fibroblasts untreated or treated with PF-475, GCA, or CBE for 24 h. D Quantitative analysis of p-Ser129 α-synuclein levels in GBA1 K198E fibroblasts. Immunofluorescence image of p-Ser129 α-Syn (green fluorescence) and nuclei (blue fluorescence) in WT GBA1 (E–H) and GBA1 K198E (I-L) fibroblasts untreated (E, I) or treated with PF-475 (F, J), GCA (G, K), or CBE (H, L) for 24 h. M Quantitative analysis of p-Ser129 α-Syn in WT fibroblasts. N Quantitative analysis of p-Ser129 α-Syn in GBA1 K198E fibroblasts. Numbers in histograms represent the percentage of the positive cell population for the tested marker. Results are from three independent experiments (n = 3) presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test; **p < 0.01, ns = no significant). Image magnification 400 ×

Fig. 7

LRRK2 kinase inhibition reduced levels of oxidized (Cys106) DJ-1 (ox(Cys106)DJ-1) in fibroblasts bearing GBA1 K198E mutation. A Representative flow cytometry histograms showing ox(Cys106)DJ-1 in WT GBA1 fibroblasts untreated or treated with the LRRK2 inhibitor PF-475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM), or conduritol-B-epoxide (CBE, 10 µM) for 24 h. B Quantitative analysis of ox(Cys106)DJ-1 levels in WT GBA1 fibroblasts. C Representative flow cytometry histograms showing ox(Cys106)DJ-1 in GBA1 K198E fibroblasts untreated or treated with PF-475, GCA, or CBE for 24 h. D Quantitative analysis of ox(Cys106)DJ-1 levels in GBA1 K198E fibroblasts. Immunofluorescence image of ox(Cys106)DJ-1 (green fluorescence) and nuclei (blue fluorescence) in WT GBA1 (E–H) and GBA1 K198E (I-L) fibroblasts untreated (E, I) or treated with PF-475 (F, J), GCA (G, K), or CBE (H, L) for 24 h. M Quantitative analysis of ox(Cys106)DJ-1 in WT fibroblasts. N Quantitative analysis of ox(Cys106)DJ-1 in GBA1 K198E fibroblasts. Numbers in histograms represent the percentage of the positive cell population for the tested marker. Results are from three independent experiments (n = 3) presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test; *p < 0.05, ns no significant. Image magnification 400 ×

Fig. 8

LRRK2 kinase inhibition reduced levels of cleaved (active) caspase-3 (CC3) in fibroblasts bearing the GBA1 K198E mutation. A Representative flow cytometry histograms showing CC3 in WT GBA1 fibroblasts untreated or treated with the LRRK2 inhibitor PF-475 (1 µM), the GCase stabilizer NCGC00188758 (GCA, 10 µM), or conduritol-B-epoxide (CBE, 10 µM) for 24 h. B Quantitative analysis of CC3 levels in WT GBA1 fibroblasts. C Representative flow cytometry histograms showing CC3 in GBA1 K198E fibroblasts untreated or treated with PF-475, GCA, or CBE for 24 h. D Quantitative analysis of CC3 levels in GBA1 K198E fibroblasts. Immunofluorescence image of CC3 (green fluorescence) and nuclei (blue fluorescence) in WT GBA1 (E–H) and GBA1 K198E (I-L) fibroblasts untreated (E, I) or treated with PF-475 (F, J), GCA (G, K), or CBE (H, L) for 24 h. M Quantitative analysis of CC3 in WT fibroblasts. N Quantitative analysis of CC3 in GBA1 K198E fibroblasts. Numbers in histograms represent the percentage of the positive cell population for the tested marker. Results are from three independent experiments (n = 3) presented as mean ± SD. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test; *p < 0.05, ***p < 0.001; ns no significant. Image magnification 400 ×

Fig. 9

Schematic representation of the cellular effects of GBA1 K198E variant in skin fibroblasts: A pathogenic phenotype reverted by the inhibitor LRRK2 PF-06447475 (PF-475). For explanation, see text