Alterations in antibody-accessible BMP and endolysosomal morphology in R1441G LRRK2 MEF cells.

(A) Confocal microscopy of endogenous BMP (green) and LAMP2 (red) immunofluorescence in WT and R1441G LRRK2 MEFs. Scale bar: 20 µm. (B-C) Quantification of vesicular BMP intensity (B) and LAMP2 relative intensity (C) per cell area. Colored dots represent mean value from 4 independent experiments and violin plots show the distribution of individual cell data. Significance determined by two-tailed paired t test **p<0.01, ***p<0.001. (D) Representative transmission electron microscopy (TEM) images of Multivesicular endosomes (MVE) from WT and R1441G LRRK2 MEFs. MVB periphery highlighted in yellow. Scale bar: 250 nm. (E) MVE area (µm2) quantification in WT and R1441G LRRK2 mutant cells. Colored dots represent mean values from 3 independent experiments and violin plots show the distribution of individual cell data (35-45 cells/group). (F) Quantification of Intraluminal Vesicles (ILVs) per MVE in WT and R1441G LRRK2 MEF cells. The number of ILVs per MVE are binned in three groups and plotted as a percentage of MVE from the total population of each experiment independently. Data from 3 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t test (E) and ordinary two-way ANOVA, uncorrected Fisher’s LSD (F) *p<0.05, ****p<0.0001.

LRRK2 and GCase activities modulate extracellular vesicle production.

(A) Whole cell lysates from WT and R1441G LRRK2 MEF cells treated with 200nM MLi-2 for 24h were analyzed by immunoblotting. Representative images of LAMP2, phospho-Rab10 and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. (B) Flow cytometry measurement of GCase activity using PFB-FDGlu fluorescent GCase substrate in WT and R1441G LRRK2 mutant MEF cells treated with 300µM CBE for 24h. (C and G) Whole cell lysates (WCL) and isolated extracellular vesicles (EVs) from WT and R1441G LRRK2 mutant MEF cells treated with 200nM MLi-2 (C) or 300µM CBE (G) for 48h were analyzed by immunoblotting. Representative images of LAMP2, Flotillin-1 and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa. Immunoblots for LAMP2 and Flotillin-1 in EV fractions required longer exposure times to visualize clear signals across all conditions. (D-F and H-J) Quantification of LAMP2 and Flotillin-1 levels relative to R1441G LRRK2 MEF cells in whole cell lysates (WCL). (D and H) and isolated EVs (E,F,I and J). Data from 6-8 independent experiments (mean ± SEM). Significance determined by Kruskal-Wallis test followed by an uncorrected Dunn’s post hoc test compared to R1441G LRRK2 control *p<0.05, **p<0.01, ****p<0.0001; ns, not significant.

Targeted lipid pathway analysis of BMP abundance in cellular and isolated EV fractions.

(A-D) UPLC-MS/MS determination of BMP isoforms normalized to protein content from cells treated with 200nM MLi-2 (A-B) or 300µM CBE (C-D) for 48h. Data shown as fold change relative to untreated R1441G LRRK2 MEF cells. Only BMP isoforms that were detected are shown. (E) UPLC-MS/MS determination of BMP isoforms normalized to protein content in EVs isolated from cells treated with 200nM MLi-2 or 300µM CBE for 48h. Only BMP isoforms that were detected are shown. Data from 3-6 independent experiments (mean ± SEM). Significance determined by ordinary one-way ANOVA, uncorrected Fisher’s LSD (A-D) and one-way ANOVA with the Geisser-Greenhouse correction, uncorrected Fisher’s LSD (E) *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Inhibition of LRRK2 or GCase activities does not significantly impact BMP biosynthetic and catabolic rates.

(A) Schematic representation of the BMP metabolic labeling protocol with deuterated docosahexaenoic acid and 13C-labeled oleic acid. WT and R1441G mutant MEF cells were incubated with a pulse of DHA-d5 / OA-13C for 20min, followed by a chase for different times. Cells were then collected for subsequent BMP lipidomic analysis. Structures of unlabeled (L) and isotope-labeled (H) fatty acids are shown in black or red, respectively. (B-D) UPLC-MS/MS determination of BMP isoforms normalized to protein content from WT MEF cells and R1441G LRRK2 mutant MEF cells ± MLi-2 (200nM) or CBE (300µM). Long (B-C) and short (D) chase time points shown as fold change relative to WT control MEF cells time 0. Only BMP isoforms that were detected are shown. Data from 3 replicated experiments (mean ± SEM).

LRRK2 activity modulates CLN5 expression levels.

(A) Whole cell lysates from WT and R1441G LRRK2 MEF cells were analyzed by immunoblotting. Representative immunoblots of CLN5, phospho-Rab10 (pRab10) and α-Tubulin are shown from two (#1 and #2) out of six independent experiments. Molecular weight marker mobility is shown in kDa. Plot at the bottom shows quantification of CLN5 immunoblot levels relative to WT and R1441G LRRK2 MEF cells. (B) Whole cell lysates from R1441G LRRK2 MEF cells treated with 200nM MLi-2 for 24h were analyzed by immunoblotting. Representative immunoblots of phosphor-LRRK2 (pLRRRK2), CLN5 and α-Tubulin levels are shown from two (#1 and #2) out of three independent experiments. Molecular weight marker mobility is shown in kDa. Plot at the bottom shows quantification of CLN5 immunoblot levels in whole cell lysates of R1441G LRRK2 MEF cells untreated or MLi-2-treated. (C) Whole cell lysates from G2019S LRRK2 patient-derived fibroblasts treated with indicated increasing MLi-2 concentrations for 24h were analyzed by immunoblotting. Immunoblots of CLN5, phospho-Rab10 (pRab10) and α-Tubulin levels are shown from one representative experiment (n=6 per condition, obtained from two independent replicate experiments using fibroblast cell lines derived from three different patients). Molecular weight marker mobility is shown in kDa. Plot at the bottom shows quantification of CLN5 immunoblot levels relative to G2019S LRRK2 patient-derived fibroblasts treated with MLi-2 at the indicated concentrations. (D) Whole cell lysates from R1441G LRRK2 patient-derived fibroblasts treated with indicated increasing MLi-2 concentrations for 24h were analyzed by immunoblotting. Immunoblots of CLN5, phospho-Rab10 (pRab10) and α-Tubulin levels are shown from one representative experiment. Significance in (A) and (B) determined by two-tailed, unpaired t test; significance in (C) determined by Dunnett’s One-way ANOVA test; *p<0.05, ***p<0.001.

Pharmacological modulation of EV-mediated BMP exocytosis.

(A) Whole cell lysates (WCL) and isolated EVs from WT MEF cells treated with 10µM GW4869 or 10nM bafilomycin-A1 (B-A1) for 24h were analyzed by immunoblotting. Representative immunoblots of LAMP2, Flotillin-1 and α-Tubulin are shown. Molecular weight marker mobility is shown in kDa. (B-C) UPLC-MS/MS determination of BMP isoforms normalized to protein content from WT MEF cells treated with 10nM B-A1 (B) or 10µM GW4869 (C) for 24h. Data shown as fold change relative to WT control MEF cells. Only BMP isoforms that were detected are shown. Data from 6 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. (D-E) Quantitation of BMP isoforms normalized to protein content in EVs isolated from MEF WT cells treated with 10nM B-A1 (D) or 10µM GW4869 (E) for 24h. Data shown as fold change relative to WT control MEF cells. Only BMP isoforms that were detected are shown. Data from 3 independent experiments (mean ± SEM). Significance determined by two-tailed unpaired t test *p<0.05, **p<0.01, ***p<0.001.

Patient-derived G2019S LRRK2 fibroblasts exhibit alterations of antibody-accessible BMP and increased endolysosomal exocytosis.

(A) Confocal microscopy of endogenous BMP (yellow) and LAMP1 (magenta) immunofluorescence in control (CTRL) and LRRK2-G2019S mutant–derived fibroblasts. Scale bar: 40 µm. (B–C) Quantification of vesicular BMP intensity (B) and LAMP1 relative intensity (C) per cell area. Colored dots represent the mean value of three independent experiments (n=3 CTRL and n=3 G2019S LRRK2 fibroblast cell lines); violin plots show the distribution of individual cell data (60 cells per independent experiment). Significance determined by two-tailed paired t test; ****p < 0.0001. (D) Confocal microscopy of endogenous BMP (yellow) and CD63 (magenta) immunofluorescence in CTRL and G2019S LRRK2 fibroblasts. Scale bar: 10 µm. (E–F) Quantification of the number of CD63 vesicles in the peripheral cell region (E) and the percentage of peripheral CD63 vesicles positive for BMP (F). Colored dots represent mean values of three independent experiments (n=3 CTRL and n=3 G2019S LRRK2 fibroblast cell lines); violin plots show the distribution of individual cell data (60 cells per independent experiment). Significance determined by two-tailed paired t test; **p < 0.01. (G) A representative confocal microscopy image of endogenous BMP (yellow) and transduced CD63-pHluorin (magenta) in a CTRL human fibroblast cell line used for total internal reflection fluorescence (TIRF) microscopy experiments. Inset image shows co-localization between BMP and CD63-pHluorin in vesicular structures (a similar degree of co-localization was observed in G2019S LRRK2 cells; data not shown). Scale bar: 10 µm. (H) Representative epifluorescence (EPI) and TIRF microscopy images of the same cell from a G2019S LRRK2 patient-derived fibroblast cell line treated with vehicle (DMSO) overnight. Inset images shows a TIRF microscopy time-lapse sequence (0.3 s per frame) from a single pHluorin-CD63–positive fusion event at the plasma membrane. Scale bar: 10 µm. (I) Quantification of pHluorin-CD63–positive fusion events in stably expressing control (CTRL) and G2019S LRRK2 fibroblasts treated with vehicle (Ø) or 200 nM MLi-2 for 16 h. Each dot represents one cell (n=20 cells quantified from four CTRL and four G2019S LRRK2 cell lines). Significance determined by Tukey’s multiple comparisons test, ordinary one-way ANOVA; *p < 0.05, **p < 0.001.

Further characterization of MEF-derived EV fractions.

(A-C) No significant differences in EV release between MLi-2/CBE-treated and untreated WT MEF cells. Quantification of LAMP2 and Flotillin-1 levels relative to WT control MEF cells in WCL (A) and isolated EVs (B-C). Data from 7-8 independent experiments (mean ± SEM). Significance determined by ordinary one-way ANOVA, uncorrected Fisher’s LSD. (D) and (E), Characterization of WT or R1441G LRRK2 MEF-derived purified EVs by Nanoparticle Tracking Analysis (NTA). (D) Representative plots of EV size distribution in each indicated condition. (E) Yield comparison of MEF-derived EVs from each indicated condition determined by NTA. Each colored dot represents an independent experiment. (F,G) No significant differences in cellular BMP levels between MLi-2/CBE-treated and untreated WT MEFs, as measured by UPLC-MS/MS. (H) Table showing absolute values of UPLC-MS/MS measurements of cellular docosahexaenoyl (22:6)-, arachidonyl (20:4)-, oleoyl (18:1)-, and linoleyl (18:2)-BMP species from 3 representative independent experiments. (I) No significant differences in EV-associated BMP levels between MLi-2/CBE-treated and untreated WT MEF cells, as measured by UPLC-MS/MS. Lipidomics measurements of cellular (F,G) and EV-associated (I) BMP isoforms normalized to protein content from cells treated with 200nM MLi-2 or 300µM CBE for 48h, or left untreated (ctrl). Data shown as fold change relative to WT control MEF cells. Only BMP isoforms that were detected are shown. Data from 6-7 independent experiments (mean ± SEM). Significance determined by ordinary two-tailed paired t test; ns, not significant.

Quantitative analysis of GCase substrates in MEF cells and EV fractions.

(A-B) Lipidomic determination of Glucosylceramide (GlcCer), Galactosylceramide (GalCer) and Glucosylsphingosine (GlcSph) isoforms normalized to protein content from WT and R1441G LRRK2 mutant MEF cells (A) and isolated EVs (B) treated with 300µM CBE or 200nM MLi-2 for 48h. Heatmap showing values as fold change relative to R1441G LRRK2 mutant MEF cell control. Only GlcCer, GalCer and GlcSph isoforms that were detected are shown. Data from 3-9 independent experiments (mean ± SEM). Significance determined by ordinary two-way ANOVA, uncorrected Fisher’s LSD *p<0.05, **p<0.01, ***p<0.001 ****p<0.0001.

Pharmacological modulation of EV-mediated BMP exocytosis in mutant LRRK2 MEF cells.

Whole cell lysates (WCL) and isolated EVs from R1441G LRRK2 MEF cells treated with 10µM GW4869 or 10nM bafilomycin-A1 for 24h were analyzed by immunoblotting. Representative images of LAMP2, Flotillin-1 and α-Tubulin levels are shown. Molecular weight marker mobility is shown in kDa.

Analysis of endolysosomal fractions in control and G2019S LRRK2 human- derived skin fibroblasts.

(A) Immunoblots showing fractions recovered after ultacentrifugation of sucrose gradients of cell extracts from control (CTRL) and G2019S LRRK2 mutant-derived fibroblasts. Endolysosomes fractions correspond to fractions 8-10. Representative immunoblots for LRRK2, CLN5, CD63 and α-tubulin are shown. Plots on the right show quantification of LRRK2 and CLN5 band intensities. n=3 CTRL and n=3 G2019S LRRK2 fibroblast cell lines. Molecular weight marker mobility is shown in kDa. (B) Whole cell lysates from CTRL individuals and G2019S LRRK2 mutant-derived fibroblasts analyzed by immunoblotting. Representative blots of CD63 and calnexin are shown. Plot on the right shows quantification of relative CD63 protein levels in whole-cell lysates normalized to Calnexin (used as a loading control). Molecular weight marker mobility is shown in kDa. Significance determined by ordinary two-tailed paired t test; ns, not significant.