Fig 2: Drug duration of action of GNE-7915 on LRRK2 kinase inhibition.To determine efficacy and duration of GNE-7915 on LRRK2 inhibition, a pThr73-Rab10 and c pSer106-Rab12 levels in 3-month old WT and LRRK2R1441G mutant mouse lungs were determined by western blotting at 1, 6, 24, 48 and 72 h after single GNE-7915 dose (100 mg/kg; s.c.). b pRab10 and d pRab12 levels were reduced after injection which reached maximum inhibition within 24 h. Kinase inhibition dissipated gradually with pRab10 and pRab12 levels returning to basal levels by 72 h. Data are expressed as mean ± SEM (N = 4). **p < 0.01 and *p < 0.05 represent statistical significance between the designated time point to its corresponding basal levels (time 0) by one-way ANOVA with post hoc Bonferroni’s multiple comparison correction. e Design of long-term GNE-7915 injection protocol to achieve intermittent LRRK2 inhibition over 18 weeks. f Bioavailability of GNE-7915 in serum and brain. GNE-7915 concentration in mouse brain and serum was quantified at different time points using LC-MS-MS after single subcutaneous injection of GNE-7915 at 100 mg/kg. Data are expressed as mean ± SD (N = 3 per time point). **p < 0.01 and ##p < 0.01 represent statistical significance between the designated time point to its corresponding basal levels (time 0) in serum and brain, respectively, using unpaired Student’s t-test.

Fig 3: Knock-out of Rab29 does not reduce elevated Rab10 phosphorylation in pathogenic LRRK2[R1441C] knock-in mice.(A) The indicated matched primary MEFs were treated with vehicle (DMSO) or 100 nM LRRK2 inhibitor MLi-2 for 90 min prior to harvest. Twenty micrograms of whole-cell extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. Technical replicates represent cell extract obtained from a different dish of cells. Cell extract derived from a wildtype MEF cell line (WT*) was added to each gel in order to accurately compare the Rab10 pThr73/Rab10 total ratios between genotypes. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the ratios of phospho-Rab10/total Rab10, calculated using the Image Studio software. Values were normalized to the average of wildtype MEFs treated with DMSO. Similar results were obtained in two separate experiments. Quantifications are presented as mean ± SD. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test and there was a statistically significant difference between wildtype and LRRK2[R1441C] knock-in MEFs (****P < 0.0001) but not between wildtype and Rab29 knock-out MEFs (P = 0.8351) or between LRRK2[R1441C] knock-in and Rab29 knock-out LRRK2[R1441C] knock-in MEFs (P = 0.9452). (B–D) The indicated 6-month-old matched mice were administered with vehicle (40% (w/v) (2-hydroxypropyl)-ß-cyclodextrin) or 30 mg/kg MLi-2 dissolved in vehicle by subcutaneous injection 2 h prior to tissue collection. Forty micrograms of tissue extracts were subjected to quantitative immunoblot analysis with the indicated antibodies. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the ratios of phospho-Rab10/total Rab10 and phospho-Rab12/total Rab12, which were calculated using the Image Studio software. Quantifications are presented as mean ± SD, normalized to vehicle treated, wildtype animals. Each lane represents a tissue sample from a different animal. Data were analyzed by one-way ANOVA with Tukey's multiple comparisons test and there was a statistically significant difference in Rab10 phosphorylation between wildtype and LRRK2[R1441C] spleen samples (**P = 0.0093 (E)). All other phospho-Rab10 comparisons were not statistically significant. Wildtype vs LRRK2[R1441C]: P = 0.7169 (B), P = 0.2792 (C), P = 0.1555 (D). Wildtype vs Rab29 knock-out: P = 0.2594 (B), P = 0.8942 (C), P = 0.9372 (D), P = 0.3964 (E). LRRK2[R1441C] vs Rab29 knock-out LRRK2[R1441C]: P = 0.6629 (B), P = 0.9955 (C), P = 0.9962 (D), P = 0.4184 (E). There was a statistically significant difference in Rab12 phosphorylation between wildtype and LRRK2[R1441C] tissue samples: brain ****P < 0.0001 (B), lungs *P = 0.0438 (C), kidneys **P = 0.0041 (D) and spleen *P = 0.0390 (E). All other phospho-Rab12 comparisons were not statistically significant. Wildtype vs Rab29 knock-out: P = 0.3057 (B), P = 0.8915 (C), P = 0.9595 (D), P = 0.2078 (E). LRRK2[R1441C] vs Rab29 knock-out LRRK2[R1441C]: P = 0.7791 (B), P = 0.9747 (C), 0.6681 (D), P = 0.4093 (E).

Fig 4: Cation ionophores nigericin and monensin, and lysosomal stressors chloroquine and LLOMe enhance LRRK2-mediated Rab10 and Rab12 phosphorylation in wildtype and Rab29 knock-out MEFs.(A–D) Littermate-matched wildtype and Rab29 knock-out MEFs were treated with vehicle or (A) 2 µM nigericin, (B) 10 µM monensin, (C) 50 µM chloroquine, or (D) 1 mM LLOMe for the indicated periods of time. Cells were treated with DMSO or 100 nM LRRK2 inhibitor MLi-2 for 2 h prior to harvest. 15–20 µg of cell extract was subjected to quantitative immunoblot analysis with the indicated antibodies. The membranes were developed using the LI-COR Odyssey CLx Western Blot imaging system. Quantified data are presented as the ratios of phospho-Rab10/total Rab10 and phospho-Rab12/total Rab12, which were calculated using the Image Studio software. Values were normalized to the average of the vehicle-treated cells for each respective genotype. Similar results were obtained in a second independent trial for each stimulus, and data for this including a 100 µM chloroquine experiment is shown in Supplementary Figure S8.

Fig 5: 23 LRRK2 variants with mutations spanning multiple domains significantly augment LRRK2-mediated Rab10Thr73 phosphorylation.(A) FLAG-tagged LRRK2 wildtype, kinase dead (KD = D2017A) and the indicated variants were transiently expressed in HEK293 cells. Twenty-four hours post-transfection, cells were lysed and analysed by quantitative immunoblotting using the indicated antibodies. Each lane represents a different dish of cells. Data quantification is shown in (B–E). (B–E) Quantified immunoblotting data are presented as ratios of pRab10 Thr73/total Rab10 (B), pRab12 Ser106/total Rab12 (C), phospho-LRRK2 Ser1292/total LRRK2 (D), phospho-LRRK2 Ser935/total LRRK2, phospho-LRRK2 Ser955/total LRRK2, or phospho-LRRK2 Ser973/total LRRK2 (E), normalized to the average of LRRK2 wildtype values for each replicate (mean ± SD). Combined immunoblotting data from two independent biological replicates (each performed in duplicate) are shown. Data were analysed using one-way ANOVA with Dunnett's multiple comparisons test. Statistical significance was determined from four replicate values for each variant, and represented with P-values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (F) FLAG LRRK2 WT or R1628P was expressed in HEK293 cells. Each lane represents a different dish of cells. One hour prior to lysis, cells were treated with vehicle (0.1% v/v DMSO) or 100 nM MLi-2. Cell lysates were analysed by quantitative immunoblotting and quantified data are analysed and presented as in (A–E). Quantified data are representative of three independent experiments, each performed in triplicate. (G) Domain schematic of LRRK2 highlighting the position of the 23 LRRK2 variants selected for further analysis.