Kluss, Jillian H., Mazza, Melissa Conti, Li, Yan, Manzoni, Claudia, Lewis, Patrick A., Cookson, Mark R., and Mamais, Adamantios
Additional file 2 S2. Characterization of pT73 Rab10 [MJF-R21 and MJF-R21–22-5] antibodies. (A, B) HEK293FT cells transiently expressing LRRK2 mutant constructs were analyzed for endogenous Rab10 phosphorylation [MJF-R21]. The LRRK2 genetic variants R1441G, Y1699C and I2020T invoked the highest increase in Rab10 phosphorylation compared to WT LRRK2, while the kinase-dead K1906M LRRK2 construct conferred a significantly lower pRab10 levels compared to WT. (C, D) HEK293FT cells transiently expressing LRRK2 genetic variants following Rab10 siRNA knock-down were probed for Rab10. Rab10 levels revealed successful knock-down of endogenous Rab10 compared to non-targeting controls. The pT73 Rab10 [MJF-R21] antibody detected a band at ~ 24 kDa, which was not detected in the Rab10 siRNA groups, suggesting specificity for Rab10. (E, F) In a similar experiment to (C), Rab10 expression was knocked-down in primary astrocytes from WT and homozygous G2019S LRRK2 knock-in mice. Rab10 knock-down was followed by a significant decrease in pT73 Rab10 signal using the MJF-R21 antibody (E, F). (G, H) Primary astrocytes treated with 1 μM of MLi-2 for 90 min showed that both pT73 Rab10 antibodies (MJF-R21 and MJF-R21–22-5) show significantly decreased levels of phosphorylation compared to control cells. Quantitation of phosphorylation levels in D and F are presented as raw pT73 intensity normalized to loading while B and H represent T73 phosphorylation signal over total Rab10 levels (B: one-way ANOVA with Tukey’s post hoc, ****P 1.4x log fold change, red = 1.4x log fold change. S6. Kidney hits are tissue-specific with proteins identified in the 10-week cohort and do not show differential change in the 10-day cohort. Proteins identified in the kidney proteomics and validated via Western blot were also investigated in the brain and lung tissues of the 10-week cohort animals. We found that these proteins (Jip4, Lamp1, HGS/Vps27, Sfxn3 and legumain) were not statistically altered in the G2019S KI animals, neither MLi-2 treated nor untreated, in brain and lung (A-D). This implies a kidney-specific phenotype of these altered lysosomal and other membrane proteins. Additionally, we explored two of the mitochondrial proteins found in the brain total proteomics, Atp5mc1 and Cytochrome C. No difference was observed in G2019S MLi-2 treated mice compared to any other group (C-D). Additionally, proteins Cyp1a1 and pS163 Marcks identified in the lung proteomics were validated via Western blot analysis (C-D). Prosurfactant C was measured in both 10-week (C) and 10-day (E) cohorts in lung tissue of which Lrrk2 KO animals showed a significant elevation while G2019S KI animals receiving MLi-2 treatment were not affected in either cohort (C-F). Proteins identified as statistically significant in the 10-week cohort were also investigated in the 10-day cohort in order to determine if the effects observed occur after continued use of LRRK2 inhibitor long-term as opposed to more immediate effects (after 10 days). Western blot analysis confirmed that both therapeutic and dysregulatory effects were not observed in the 10-day cohort of treated animals (G-I). This suggests that the endolysosomal, trafficking, and mitochondrial changes we observed in our proteomics screen are effects of long-term treatment with MLi-2 in G2019S KI mice. Interestingly, the increase in protein expression in our untreated G2019S KI that was observed in our 10-week cohort, specifically non-glycosylated Lamp1, Hgs, and Sfxn3 was not seen in our 10-day cohort (H). Our current hypothesis is that these changes in protein expression are age-dependent in the context of the G2019S mutation in the mouse model, as the 10-week animals are double the age of the 10-day cohort at time of sacrifice. One-way ANOVA with Tukey’s post hoc; n = 6; ****P