Fig 2: Structure-guided mutations in the N-terminus of LRRK2 stimulate LRRK2-mediated Rab10 phosphorylation.(A,B) Cartoon representation of LRRK2 [558–2527] with detailed views of ANK (blue):CT α-helix (magenta) (A) and LRR (green):CORA (yellow) (B) interactions. Distance measurements in Å are indicated by dark gray dashed lines. (Right panel) HEK293 cells were transfected with wildtype, kinase dead (KD = D2017A), and the indicated LRRK2 variants. Each lane represents a different dish of cells. Cells were harvested 24 h post-transfection and subjected to quantitative immunoblot analysis with the indicated antibodies. Each lane represents a different dish of cells. The ratios of phospho-Rab10 Thr73/total Rab10 and phospho-LRRK2 Ser935/total LRRK2 were normalized to wildtype LRRK2 values. Quantified data are presented as mean ± SD and are representative of two independent experiments.

Fig 3: Exposure to low doses of LRRK2 kinase inhibitor also restored mtDNA damage to basal levels. Healthy control or LRRK2 G2019S patient derived LCLs were treated with either RA334 or RA283 with doses ranging from 1 to 100 nM for 24 h. (A) Treatment with RA334 reversed mtDNA damage in LRRK2 G2019S-patient derived LCLs, with (B) no effect on mtDNA copy number. (C) Similarly, exposure to RA283 reduced LRRK2 G2019S-induced mtDNA damage to healthy control levels. (D) Treatment with RA283 did not change mtDNA copy number. The PCR-based assay was performed in technical triplicate for each biological replicate. (*p < 0.001, determined by one-way ANOVA with a Tukey’s post-hoc comparison). n = 3 biological replicates (6 cell lines total), each performed in technical replicate. Data are presented as mean ± SEM.

Fig 4: Dose–response curve of RA283 on LRRK2 Ser935 dephosphorylation in control and LRRK2 G2019S PD patient LCLs. (A) Representative western blots of healthy control and LRRK2 G2019S patient-derived LCLs treated for 24 h treatment with Compound RA283. (B) Quantification of western blots demonstrated that Compound RA283 decreased LRRK2 pSer935 levels at all doses tested. Data are mean ± SEM. (*p < 0.001, determined by one-way ANOVA with a Tukey’s post-hoc comparison). n = 3 biological replicates (6 cell lines total), each performed in technical replicate. Full blots available in Supplemental Figs. S9 and S10.

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.