Fig 1: Effect of LRRK2 overexpression on mitochondria morphology in LUHMES cells. (A) Naïve, WT (L10WT) and G2019S (L14GS) LUHMES clones were differentiated for 3 days and then fixed and immunostained with the anti-TOM20 antibody to identify mitochondria. Green, TOM20; blue, Hoechst 33342. Insets show enlargements of boxed areas. Fragmented mitochondria are clearly visible in the G2019S (L14GS) clone. Scale bar: 10 µm. The displayed experiment is representative of three independent experiments. (B) Representative pre-processed IF images (I,III) of WT and G2019S LUHMES clones showing mitochondria morphology (green, TOM20; blue, Hoechst 33342) and segmentation (II,IV). Scale bar: 10 µm. (C) Quantification of mitochondrial morphology in L14GS clone revealed a significant decrease in the aspect ratio (left and center), and an increase in the percentage of cells displaying rounder mitochondria (right) compared to naïve and L10WT LUHMES. Experiments were repeated three times. Data are presented as mean±s.d. n=6 per cell line. Statistical significance was examined by ANOVA and Tukey's multiple comparisons test with a statistical criterion of 0.05. *P<0.05, **P<0.01, ***P<0.001; n.s., not significant.
Fig 2: LRRK2 inhibitor prevented further progression of PD caused by neuron-released αSyn. (A) We schematized the plan of injection of VtCM or αS-CM and the administration of the LRRK2 inhibitor, MLi-2. We recorded the mobility of mice in terms of distance (B) and mean speed (C) on days 14 (pre-Treatment, circle) and 28 (Treatment, square), respectively. (D,E) The number of microglia that exhibited reactive morphology were analyzed. n = 4, approximately 10 sections for ICC. * p < 0.05, ** p < 0.01, and *** p < 0.001. All data are represented as the mean ± SEM.
Fig 3: Pathogenic LRRK2 mutants co‐localize with Rab29 and disperse Golgi membranesHeLa cells were transfected with LRRK2‐G2019S or Myc‐Rab29 or LRRK2‐G2019S and 24 h later transfected with Myc‐Rab29. After 48 h, cells were permeabilized by liquid nitrogen freeze–thaw to deplete cytosol and then fixed and stained with mouse anti‐p115, mouse anti‐Myc, and rabbit anti‐GFP or rabbit anti‐LRRK2 antibodies. Myc‐Rab29 (green) and p115 (red) show co‐localization at the Golgi. Left and right panels were treated with or without MLI2 (200 nM, 4 h) as indicated.Myc‐Rab29 (red) and eGFP‐LRRK2‐G2019S (green) show co‐localization and dispersed Rab29‐labeled Golgi membranes.Cells treated with MLI‐2 (200 nM, 4 h) show compact, Rab29‐positive Golgi, and associated LRRK2‐G2019S (green).Percent of cells with compact Rab29 staining; ***P = 0.0002; **P = 0.002 with Student's unpaired, two‐tailed t‐test. Error bars represent SEM for three experiments with > 30 cells per condition in each experiment.LRRK2‐G2019S alone showing punctate staining throughout the cell.Data information: Scale bars, 10 μm.
Fig 4: Rab29 increases membrane association of LRRK2‐R1441GHEK293T Rab29−/− (KO) and WT cells were transfected with LRRK2‐R1441G and 24 h later transfected with Myc‐Rab29. After 48 h, cells were harvested and fractionated into cytosol and membrane fractions. Immunoblot of membrane protein (75 μg) and the 50% of equivalent volume of cytosolic proteins, ±Rab29 as indicated. Numbers at left indicate mobility of marker proteins in kDa; proteins were detected with rabbit anti‐pS1292, rabbit anti‐LRRK2 UDD3, mouse anti‐LRRK2, mouse anti‐LAMP2, mouse anti‐tubulin, and mouse anti‐Myc antibodies.Quantitation of the fraction of total LRRK2‐R1441G on membranes ± Rab29 (transfected and endogenous).Amount of active (pS1292) LRRK2 in membrane and cytosol fractions ± Rab29 expression normalized to the amount of total LRRK2 in the fraction.Data information: Error bars represent SEM from two experiments. **P = 0.0025; ****P = 0.00013; ns = not significant (P = 0.1968) by Student's unpaired, two‐tailed t‐test. Differences between Rab29 KO and (minus) Myc‐Rab29 are not significant. Source data are available online for this figure.
Fig 5: Rab29 selectively activates LRRK2 A, BHEK293 cells were transfected with the wild‐type LRRK2 (A) or LRRK2[R1441G] (B) with either HA‐empty vector (−) or the indicated HA‐tagged Rab proteins. 24 h post‐transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. Blots were quantified by LiCor and presented as average ± SEM, and analyzed using one‐way ANOVA with Dunnett's multiple comparison test comparing all groups to the HA‐empty vector (−). For (A) WT LRRK2, there was a statistically significant difference between groups (P = 0.0135, one‐way ANOVA, F(11, 12) = 3.911). ns P > 0.05, **P < 0.01 by one‐way ANOVA with Dunnett's multiple comparison test with mean difference 95% confidence intervals of groups compared to HA‐empty vector control: Rab1B −356.9 to 332.6; Rab5B −342.0 to 347.6; Rab7A −332.4 to 357.2; Rab8A −378.6 to 311.0; Rab8B −346.7 to 342.9; Rab10 −372.3 to 317.3; Rab12 −499.8 to 189.8; Rab29 −865.3 to −175.7; Rab32 −372.7 to 316.8; Rab38 −367.4 to 322.2; Rab39B −346.5 to 343.1. For (B) LRRK2[R1441G], there was also a statistically significant difference between groups (P = 0.0007, one‐way ANOVA, F(11, 12) = 7.642). ns P > 0.05, ***P < 0.001 by one‐way ANOVA with Dunnett's multiple comparison test with mean difference 95% confidence intervals of groups compared to HA‐empty vector control: Rab1B −386.5 to 411.2; Rab5B −410.2 to 387.5; Rab7A −377.0 to 420.7; Rab8A −668.2 to 129.6; Rab8B −517.5 to 280.3; Rab10 −421.1 to 376.7; Rab12 −503.2 to 294.6; Rab29 −1,243 to −445.7; Rab32 −433.5 to 364.3; Rab38 −548.2 to 249.5; Rab39B −390.4 to 407.4. Results were obtained in two separate experiments, each performed in duplicate.
Supplier Page from Abcam for Anti-LRRK2 (phospho S1292) antibody [MJFR-19-7-8]