Fig 2: Rab10 is a mediator of GCase activity by LRRK2 in fibroblasts and DA neurons.Western blot analysis of fibroblasts, from healthy controls or from patients with the LRRK2 G2019S mutation treated with lentivirus encoding Rab8 and Rab10 shRNA, probed for Rab8, Rab10, and GAPDH (loading control) a. Examination of relative lysosomal GCase activity in fibroblasts upon Rab8 and Rab10 knock-down b–e. Western blot analysis of fibroblasts from 3 patients with LRRK2 G2019S and from 3 healthy controls were probed for phospho-Rab10 (p-Rab10), Rab10, and GAPDH (loading control). The data is presented as the average p-Rab10 signal for the 3 G2019S samples relative to the 3 controls f. Western blot analysis of fibroblasts from 3 controls treated with MLi-2 were probed for phospho-Rab10 (p-Rab10), Rab10, GCase, and tubulin (loading control). Data is presented as the ratio of p-Rab10 to total Rab10 for the treated relative to untreated cells g. Representative western blots of lysates from LRRK2 G2019S and R1441C DA neurons relative to the corresponding isogenic controls h or relative to MLi-2 treated neurons i were probed for p-Rab10, Rab10 and β-3-tubulin (loading control). The data are presented as the mean ± SEM, n = 3; *p < 0.05, **p < 0.01, using one-way ANOVA followed by Tukey’s multiple comparison post hoc test b–e, or paired two-tailed t-test f, g. Source data are provided as a Source Data file.

Fig 3: Overexpression of Rab10 WT increases GCase activity in fibroblasts and DA neurons.Relative GCase activity in control fibroblasts transduced with lentivirus expressing GFP, Rab10 WT or Rab10 T72E a–c. Representative western blots showing overexpression of Rab10 and Rab10 T72E in control (CTRL) fibroblasts (a right). Relative GCase activity in DA neurons containing LRRK2 R1441C d, LRRK2 G2019S e, GBA1 N370S f, or GBA1 E326K g mutations transduced with CTRL or Rab10 lentivirus for 10 days, with representative western blots showing overexpression of Rab10 in DA neurons (f right). The data are presented as the mean ± SEM, n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, using one-way ANOVA followed by Tukey’s multiple comparison post hoc test a–c, or paired two-tailed t-test d–g. Source data are provided as a Source Data file.

Fig 4: LRRK2 RC mutation increases synaptic glutamate receptor content in the striatum.(A) Schematic diagram of LRRK2 protein highlighting the armadillo repeats (ARM), ankyrin (ANK) repeats, Ras of complex (ROC), C-terminal of ROC (COR), kin (KIN), and WD40 domains. Knock- in mice expressing the R1441C and G2019S mutations found in the ROC and kinase domains respectively, crossed with either Drd1-Tomato or Drd2-eGFP mouse lines. (B) Workflow schematic for subcellular fractionation of striatal homogenate for the enrichment of postsynaptic density fraction (PSD). (C) Representative western blot analysis of the subcellular fractionation results, showing supernatant (S1), crude synaptosomal preparation (P2), PSD, and Triton soluble fractions (TSF). (D) Western blot analysis of +/+, +/RC, and +/GS P2 striatal fractions probed for p-PKA substrates, pS845 GluA1, total GluA1, pT72Rab8A, total Rab8A, pT73Rab10, total Rab10, and PSD95. (E) Western blot analysis of +/+, +/RC, and +/GS mice probed for GluA1, p-PKA, and PSD95. S1 and PSD fractions are shown. (F-G) Quantification of GluA1 and p-PKA proteins in PSD fractions normalized to PSD95. Summary graphs reflect the mean, error bars reflect SEM. *p<0.05, Tukey post-hoc test following one-way ANOVA.Figure 1—source data 1.Numerical data of the graphs in Figure 1.

Fig 5: Cdk9 was identified as a target gene of microRNA‐483‐3p (miR‐483‐3p).The miR‐483‐3p mimic and negative control were transfected into cardiomyocytes to analyze the target genes of miR‐483‐3p. A, Volcano plot analysis showing the differentially expressed proteins with the miR‐483‐3p mimic compared with the negative control mimic by mass spectrometry analysis. B, Venn diagram depicting the intersection between the differentially expressed proteins and the predicted target genes of miR‐483‐3p. The characteristics of the 4 proteins in the intersection included the AccID, log2‐fold change, P value, and false discovery rate (FDR). C, The protein levels of Cdk9, Rab10, U2af2, and Lypla2 were measured after the introduction of miR‐483‐3p into primary murine cardiomyocytes. β‐Actin was used as an internal control, and the results are expressed as the mean±SD, n=3, **P<0.01 with Student t‐test. D, Sequence alignment of miR‐483‐3p and its targets in wild‐type or mutant 3′UTRs of Cdk9. E, Changes in the luciferase activity following addition of miR‐483‐3p mimic to the 3′UTR of Cdk9. Results are expressed as the mean±SD, n=3; **P<0.01 with 2‐way ANOVA. Cdk9 indicates cyclin‐dependent kinase 9; FC, fold change; FDR, false discovery rate; Lypla2, lysophospholipase 2; miR‐483‐3P, microRNA‐483‐3p; NC, negative control; Rab10, ras‐related protein rab‐10; UTR, untranslated region; U2af2, U2 small nuclear RNA auxiliary factor 2; and WT, wild‐type.