Fibroblasts from idiopathic Parkinson's disease exhibit deficiency of lysosomal glucocerebrosidase activity associated with reduced levels of the trafficking receptor LIMP2

Abstract

Lysosomal dysfunction is a central pathway associated with Parkinson's disease (PD) pathogenesis. Haploinsufficiency of the lysosomal hydrolase GBA (encoding glucocerebrosidase (GCase)) is one of the largest genetic risk factors for developing PD. Deficiencies in the activity of the GCase enzyme have been observed in human tissues from both genetic (harboring mutations in the GBA gene) and idiopathic forms of the disease. To understand the mechanisms behind the deficits of lysosomal GCase enzyme activity in idiopathic PD, this study utilized a large cohort of fibroblast cells from control subjects and PD patients with and without mutations in the GBA gene (N370S mutation) (control, n = 15; idiopathic PD, n = 31; PD with GBA N370S mutation, n = 6). The current data demonstrates that idiopathic PD fibroblasts devoid of any mutations in the GBA gene also exhibit reduction in lysosomal GCase activity, similar to those with the GBA N370S mutation. This reduced GCase enzyme activity in idiopathic PD cells was accompanied by decreased expression of the GBA trafficking receptor, LIMP2, and increased ER retention of the GBA protein in these cells. Importantly, in idiopathic PD fibroblasts LIMP2 protein levels correlated significantly with GCase activity, which was not the case in control subjects or in genetic PD GBA N370S cells. In conclusion, idiopathic PD fibroblasts have decreased GCase activity primarily driven by altered LIMP2-mediated transport of GBA to lysosome and the reduced GCase activity exhibited by the genetic GBA N370S derived PD fibroblasts occurs through a different mechanism.

Keywords: GBA; Idiopathic PD fibroblasts; LIMP2; Lysosomal dysfunction.

Fig. 1

Idiopathic PD fibroblasts exhibit reduced basal lysosomal GCase activity. a Basal level of lysosomal GCase activity was measured in fibroblasts derived from idiopathic PD patients (PD), those harboring GBA N370S mutation (gPD-GBA N370S) and age-matched healthy subject controls (HS) using 4-MU glucopyranoside substrate (n = 14, HS; n = 31, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F_(2,48) = 12.25, p < 0.0001). b GBA transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S) and c, d protein level (normalized to GAPDH) measured across HS, PD and gPD-GBA N370S group of cells (n = 13, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F_(2,45) = 5.203, p = 0.0093). Data represented as mean ± SEM. * = p < 0.05; ** = p < 0.01; *** = p < 0.001

Fig. 2

Idiopathic PD fibroblasts exhibit altered levels of PGRN, LIMP2 and localization of the GBA protein. a Representative image and b quantification of PGRN protein (normalized to GAPDH) in HS, PD and gPD-GBA N370S group of cells (n = 10, HS; n = 29, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F_(2,42) = 6.577, p = 0.0033). c LIMP2 transcript (n = 14, HS; n = 28, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F_(2,45) = 6.147, p = 0.0044) and d ELISA-based LIMP2 protein expression across the three groups of cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F_(2,39) = 11.32, p = 0.0001). e Ratio of the post ER to ER fraction of GBA protein was measured in cell lysates using Endo-H and PNGase F digestion (n = 7, HS; n = 25, PD; n = 3, gPD-GBA N370S; One-way ANOVA with Tukey’s multiple comparison test, F_(2,32) = 8.084, p = 0.0014). f Correlation analysis between LIMP2 protein and GCase activity was performed and Pearson’s correlation coefficient was determined between the two variables across HS, PD and gPD-GBA N370S cells (n = 13, HS; n = 23, PD; n = 6, gPD-GBA N370S). Data represented as mean ± SEM. * = p < 0.05; ** = p < 0.01; **** = p < 0.0001