A potential patient stratification biomarker for Parkinson´s disease based on LRRK2 kinase-mediated centrosomal alterations in peripheral blood-derived cells

Abstract

Parkinson´s disease (PD) is a common neurodegenerative movement disorder and leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for disease intervention. However, the ability to stratify patients who will benefit from such treatment modalities based on shared etiology is critical for the success of disease-modifying therapies. Ciliary and centrosomal alterations are commonly associated with pathogenic LRRK2 kinase activity and can be detected in many cell types. We previously found centrosomal deficits in immortalized lymphocytes from G2019S-LRRK2 PD patients. Here, to investigate whether such deficits may serve as a potential blood biomarker for PD which is susceptible to LRKK2 inhibitor treatment, we characterized patient-derived cells from distinct PD cohorts. We report centrosomal alterations in peripheral cells from a subset of early-stage idiopathic PD patients which is mitigated by LRRK2 kinase inhibition, supporting a role for aberrant LRRK2 activity in idiopathic PD. Centrosomal defects are detected in R1441G-LRRK2 and G2019S-LRRK2 PD patients and in non-manifesting LRRK2 mutation carriers, indicating that they accumulate prior to a clinical PD diagnosis. They are present in immortalized cells as well as in primary lymphocytes from peripheral blood. These findings indicate that analysis of centrosomal defects as a blood-based patient stratification biomarker may help nominate idiopathic PD patients who will benefit from LRRK2-related therapeutics.

Fig. 1. A subset of PD patient LCLs display centrosomal cohesion deficits reverted by short-term treatment with the LRRK2 kinase inhibitor MLi2.

a Example of one healthy control and two PD LCL lines stained for two centrosomal markers (γ-tubulin and pericentrin) and DAPI. Arrows point to centrosomes co-stained with both markers. Scale bar, 10 μm. b The centrosome phenotype was quantified from 100–150 cells per line from 3 control and 35 PD lines. c In parallel experiments, the centrosome phenotype was quantified from 5 previously described control and 5 G2019S-LRRK2 PD lines. Based on this comparison, idiopathic PD LCL lines were considered to have a cohesion deficit when displaying ≥20% splitting, with 10/35 lines (28%) found to display a centrosomal cohesion deficit reverted by MLi2 (50 nM, 2 h). Bars represent mean ± s.e.m.; control versus PD (split) (p = 0.001); PD (split) versus PD (split) + MLi2, (p < 0.001); ctrl versus G2019S-LRRK2 PD (p < 0.001); G2019S-LRRK2 PD versus G2019S-LRRK2 PD + MLi2 (p < 0.001). ***p < 0.005; ****p < 0.001. d Paired t-test analysis of centrosomal cohesion deficits from each cell line in the absence or presence of MLi2 as indicated. Note that differences in the values between 0 and 15% are not significant given the small number of cells displaying a duplicated split centrosome phenotype. e Quantification of the percent of cells displaying two centrosomes (positive for both pericentrin and γ-tubulin) from a total of 100–150 cells per LCL line.

Fig. 2. Analysis of LRRK2, S935-LRRK2, Rab10 and pT73-Rab10 levels in PD LCLs with or without a centrosome cohesion phenotype.

a Example of two control and 12 PD LCL lines. Cells were lysed and extracts subjected to quantitative immunoblot analysis with the indicated antibodies and membranes were developed using Odyssey CLx scan Western Blot imaging system. pT73-Rab10 and total Rab10, as well as pS935-LRRK2 and total LRRK2 were multiplexed, and the same control line (S001) was run on every gel to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin, pS935/tubulin, pS935/LRRK2, Rab10/tubulin, pT73-Rab10/tubulin and pT73-Rab10/Rab10 as indicated, with no differences observed between PD LCL lines with or without a centrosome splitting phenotype. Bars represent mean ± s.e.m. c Spearman correlation analysis between levels of LRRK2/tubulin and pT73-Rab10/tubulin (top) or pS935/tubulin and pT73-Rab10/tubulin (bottom). A significant association is observed between LRRK2 or S935-LRRK2 levels and pT73-Rab10 levels in PD LCLs. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p-values are indicated for each correlation analysis.

Fig. 3. Effects of LLOMe treatment on LRRK2 kinase-mediated pT73-Rab10 levels.

a Example of three PD LCL lines with or without treatment with LLOMe (1 mM) and MLi2 (50 nM) for 2 h as indicated. Cells were lysed and extracts subjected to multiplexed immunoblotting with the indicated antibodies. b The percentage of LLOMe-triggered increase in pT73-Rab10/Rab10 levels in the absence or presence of MLi2 was calculated for each LCL line. LLOMe triggers similar increases in pT73-Rab10/Rab10 levels in control and PD LCLs with or without a cohesion phenotype. Bars represent mean ± s.e.m.; ctrl versus ctrl + MLi2 (p = 0.004); PD (split) versus PD (split) + MLi2 (p = 0.006); PD (non-split) versus PD (non-split) + MLi2 (p < 0.001): ****p < 0.001; ***p < 0.005; **p < 0.01. c Paired t-test analysis of LLOMe-triggered increase in pT73-Rab10/Rab10 levels from each cell line in the absence or presence of MLi2. Note that the LLOMe-triggered increase in pT73-Rab10/Rab10 levels is reduced by MLi2 treatment in most cell lines. d Spearman correlation analysis between the percentage of LLOMe-triggered increase in pT73-Rab10/Rab10 levels versus basal pT73-Rab10/Rab10 levels in the absence of LLOMe treatment. There is a negative correlation between basal pT73-Rab10/Rab10 levels and the efficacy of the LLOMe-mediated increase in pT73-Rab10/Rab10 levels. Red datapoints indicate the ten PD samples which display a centrosomal cohesion deficit. Rho and p-values are indicated (in italics values without the two outliers).

Fig. 4. C/C cohesion deficits in R1441G-LRRK2 and G2019S-LRRK2 LCLs.

a Example of a healthy control (ctrl) and an R1441G-LRRK2 NMC LCL line stained for the centrosomal marker pericentrin and DAPI. Arrows point to pericentrin-positive dots, yellow arrows to duplicated split pericentrin-positive dots. Scale bar, 5 μm. b The cohesion phenotype was quantified from 150–200 cells per line from 10 control, 12 R1441G-LRRK2 PD, 9 R1441G-LRRK2 NMC, 7 G2019S-LRRK2 PD, 6 G2019S-LRRK2 NMC and 4 idiopathic PD patient LCLs in either the absence or presence of MLi2 (50 nM, 2 h) as indicated. Bars represent mean ± s.e.m.; ctrl versus R1441G mutation (p < 0.0001); ctrl versus R1441G NMC (p = 0.0039); ctrl versus G2019S mutation (p < 0.0001); ctrl versus G2019S NMC (p = 0.012); ctrl versus idiopathic PD (p < 0.0001); R1441G mutation versus R1441G mutation + MLi2 (p < 0.0001); R1441G NMC versus R1441G NMC + MLi2 (p = 0.002); G2019S mutation versus G2019S mutation + MLi2 (p < 0.0001); idiopathic PD versus idiopathic PD + MLi2 (p = 0.033). ****p < 0.001; ***p < 0.005; **p < 0.01; *p < 0.05. c Paired t-test analysis of cohesion deficits from each cell line in the absence or presence of MLi2 as indicated.

Fig. 5. Analysis of LRRK2, Rab10 and pT73-Rab10 levels in R1441G-LRRK2 and G2019S-LRRK2 LCLs.

a Example of control, R1441G-LRRK2 PD and R1441G-LRRK2 NMC LCL lines in the absence or presence of MLi2 (50 nM, 2 h) as indicated. Cell extracts were subjected to multiplexed quantitative immunoblot analysis with the indicated antibodies, and membranes were developed using Odyssey CLx scan Western Blot imaging system. The same control line (002 M) was run on every gel as an internal standard to compare samples run on different gels. b Immunoblots were quantified for LRRK2/tubulin levels (left) and Rab10/tubulin levels (right). Note large variability in the total LRRK2 levels amongst different LCL lines. Scatter plots represent mean ± s.e.m. c Immunoblots were quantified for pT73-Rab10/Rab10 levels, with no significant differences observed between control and PD LCL lines. Scatter plots represent mean ± s.e.m.; ctrl versus ctrl + MLi2 (p = 0.004); R1441G-LRRK2 PD versus R1441G-LRRK2 PD + MLi2 (p = 0.0002). ****p < 0.001; ***p < 0.005. d Spearman correlation analysis between levels of pT73-Rab10/Rab10 and LRRK2/tubulin from all LCL lines analyzed. Rho and p-values are indicated. A significant association is observed between the total levels of LRRK2/tubulin and the levels of pT73-Rab10/Rab10.

Fig. 6. C/C cohesion deficits in lymphocytes from R1441G-LRRK2 and G2019S-LRRK2 mutation carriers.

a Example of healthy control (ctrl) and R1441G-LRRK2 PD lymphocytes from PBMC preparations stained for pericentrin and DAPI. White arrows point to pericentrin-positive structures and yellow arrows to two pericentrin-positive structures displaying a split phenotype. Scale bar, 5 μm. b The C/C splitting phenotype was quantified from 150–200 cells from 10 control, 12 R1441G-LRRK2 PD, 9 R1441G-LRRK2 NMC, 7 G2019S-LRRK2 PD, 6 G2019S-LRRK2 NMC and 4 idiopathic PD patients in either the absence or presence of MLi2 (200 nM, 30 min) as indicated. Bars represent mean ± s.e.m.; ctrl versus R1441G mutation (p < 0.0001); ctrl versus R1441G NMC (p < 0.0001); ctrl versus G2019S mutation (p < 0.0001); ctrl vrsus G2019S NMC (p < 0.0001); ctrl versus idiopathic PD (p < 0.0001); R1441G mutation versus R1441G mutation + MLi2 (p < 0.0001); R1441G NMC versus R1441G NMC + MLi2 (p < 0.0001); G2019S mutation versus G2019S mutation + MLi2 (p < 0.0001); G2019S NMC versus G2019S NMC + MLi2 (p = 0.0001); idiopathic PD versus idiopathic PD + MLi2 (p = 0.006). ****p < 0.001; **p < 0.01. c Paired t-test analysis of C/C cohesion deficits from each cell line in the absence or presence of MLi2 as indicated.