Fig 2: (A) MLi‐2 concentration response curves (range, 0.038 to 10 μM) for inhibition of pSer935 leucine‐rich repeat kinase 2 (LRRK2) are shown as the geometric mean for each genotype as indicated; correspondingly colored droplines show aggregate IC50 values by genotype; error bars show geometric standard deviation (SD). (B) Similar representation for pThr73 Rab10. (C) Individual patient IC50 values for pSer935 LRRK2 ranged from 0.8 to 1.7 nM in G2019S LRRK2 noncarriers (NC) (open green circles, orange fill indicates presence of L119P LRRK2 variant), 0.7 to 2.1 nM in heterozygous (HET) (open blue circles, turquoise and magenta fills indicate presence of additional HET and homozygous (HOM) GBA1 variants, respectively), and 1.4 to 1.8 nM in HOM G2019S LRRK2 (open red circles). (D) Individual patient IC50 values for pThr73 Rab10 ranged from 2.7 to 8.5 nM in G2019S LRRK2 NC, 3.1 to 4.9 nM in HET, and 2.5 to 4.0 nM in HOM G2019S LRRK2 (colors of circles represent similar genotypes as in C).

Fig 3: Rab10 knock-down rescues LRRK2-G2019S -induced bradykinesia. a) Optogenetic stimulation of the Proboscis Extension Response (PER). ai) To reach for food or liquid, the fly extends its proboscis in response to an optogenetic stimulus to sensory neurons on the legs. The full extension response is shown in Supplementary Movie M1. aii) Expression of LRRK2-G2019S in dopaminergic neurons (THG2) reduces the proportion of flies that respond to a single flash of light, and this is rescued by Rab10 reduction with Rab10RNAi. aiii. Dopaminergic reduction of Rab10 rescues the bradykinesia (slower response) of flies expressing G2019S in their dopaminergic neurons. aiii). Raw traces; aiv). Mean data. To respond to the optogenetic stimuli all flies carry LexA/Op Gr5a> ReachR. b) Sucrose stimulation of the PER. bi) Flies expressing G2019S in their dopaminergic neurons (THG2, magenta bars) respond less frequently to sucrose than wild-type flies (green), i.e., they show akinesia. This is rescued in THG2 flies with dopaminergic reduction in Rab10 using Rab10RNAi (THG2 > Rab10RNAi). bii) Reduction of dopaminergic Rab10 with the deGradFP technique (Rab10 YFP; THG2 > vhhGFP, yellow bars) also rescues the G2019S-induced akinesia. biii) The Rab10 null (Rab10−, orange bar) also reverts the G2019S deficit, while expression of Rab10 in the null background again induces akinesia (Rab10−; TH > Rab10, light blue bar). biv) By itself, Rab10 overexpression phenocopies G2019S expression. Exact genotypes and full statistical data in Supplementary Table 3.

Fig 4: Memory depends on dopaminergic Rab10 but not LRRK2-G2019S. A) Depletion of Rab10 in the control background (TH > Rab10RNAi) increases the proportion of flies with low memory index (MI) compared to flies with no transgene expression (TH/+). Expression of LRRK2-G2019S has no effect on performance; either for the THG2/+v TH/+control flies, nor for the flies with Rab10RNAi. a) Raw scores, b) summary data. Exact genotypes and statistical results in Supplementary Table 6.

Fig 5: Rab10 and RILPL1 do not contribute to LRRK2-mediated, enhanced cilia loss upon serum readdition. (A) Timeline of this imaging experiment. (B) Percentage of cells with primary cilia at time = 0. Values represent the mean ± SEM from four independent experiments, each containing >40 cells. Significance was determined by repeated measures one-way ANOVA with Dunnett post hoc test (control versus shRab10: P = 0.047, control versus shRILPL1: P = 0.159). (C and D) Percentage of remaining cilia compared with time = 0 (100%) after serum readdition. Line plot (C) and detailed bar graph for each time point (D) are shown. Significance was determined by repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test (at 4 h, control +MLi-2 versus shRab10 [P = 0.037]; at 7 h, control +MLi-2 versus control −MLi-2 [P = 0.009], control +MLi+2 versus shRab10 [P = 0.024]; at 10 h, control +MLi-2 versus control −MLi-2 [P = 0.037], control +MLi-2 versus shRab10 [P = 0.022], and control +MLi+2 versus shRILPL1 [P = 0.029]).