Additional file 2 of Preclinical modeling of chronic inhibition of the Parkinson’s disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo
Creators: name: Kluss, Jillian H.
name: Mazza, Melissa Conti
name: Li, Yan
name: Manzoni, Claudia
name: Lewis, Patrick A.
name: Cookson, Mark R.
name: Mamais, Adamantios
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Chemical Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Cancer

description: 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 &lt; 0.0001, ***P &lt; 0.0002, n = 3, F (8, 18)= 45.45. D: two-way ANOVA with Sidak’s multiple comparisons test; LRRK2 construct, p &lt; 0.0001, F (2, 12)=30.73; siRNA, p &lt; 0.0001, F (1, 16)=550.0, n = 3. F: two-way ANOVA with Sidak’s multiple comparisons test; LRRK2 construct, p = 0.7361, F (1, 8)=0.1218; siRNA, p &lt; 0.0001, F (1, 8)=56.29, n = 3. H: one-way ANOVA with Tukey’s post hoc, ****P &lt; 0.0001, n = 3; F (3, 15)= 160.3). S3. Characterization of pS106 Rab12 [MJF-25-9] and pT71 Rab29 [MJF-R24–17-1] antibodies. (A) HEK293FT cells transiently expressing LRRK2 genetic variants following siRNA knock-down of Rab12 were analyzed for Rab12 phosphorylation. The pS106 Rab12 antibody showed a strong band at ~ 25 kDa with LRRK2 transfection, which was significantly decreased by Rab12 siRNA compared to non-targeting control (A, B). (C, D) HEK293FT cells transiently expressing LRRK2 mutant constructs were analyzed for endogenous pT71 Rab29. Endogenous Rab29 was found hyperphosphorylated in R1441C, R1441G, Y1699C, G2019S and I2020T LRRK2 expressing cells, compared to WT LRRK2. (E, F) siRNA Rab29 knockdown resulted in a significant decrease in pT71 Rab29 signal in HEK293FT cells over-expressing LRRK2 mutants compared to non-targeting controls. (G) The T71 Rab29 antibody tested did not detect a band in cells expressing the T71A Rab29 phospho-null variant while a strong band was observed in cells expressing WT Rab29 construct, in co-expression with LRRK2 constructs. (H) Lung tissue collected from Rab29 KO mice tissue showed no pT71 Rab29 signal, whereas G2019S Lrrk2 knock-in mice showed increase in Rab29 phosphorylation, compared to WT mice. Quantitation of phosphorylation levels is presented as S106 normalized to loading (cyclophilin B levels; in B), T71 over total Rab29 (in D) or normalized to loading (F). (B: two-way ANOVA with Sidak’s multiple comparisons test; LRRK2 construct, p = 0.0083, F (2, 12)=7.329; siRNA, p &lt; 0.0001, F (1, 16)=367.8, n = 3. D: one-way ANOVA with Tukey’s post hoc, ****P &lt; 0.0001, ***P &lt; 0.0002, ** &lt; 0.004, F (8, 18)= 528.0, n = 3. F: two-way ANOVA with Sidak’s multiple comparisons test; LRRK2 construct, p &lt; 0.0001 F (2, 12)=31.83; siRNA, p &lt; 0.0001, F (1, 16)=191.1, n = 3. Mean values with SD bars shown). S4. pT73 Rab10 and pT71 Rab29 phosphorylation following chronic MLi-2 treatment. Full blots from the 10-day and 10-week chronic studies are shown to account for animal-to-animal variability (A, B). pT73 Rab10 across tissues and treatment groups show large variability with no discernable trend after LRRK2 inhibition (C, E). Rab29 T71 phosphorylation levels do not respond to treatment across tissues and time points, with exception to brain tissue of the short-term cohort (D, F). (G) Pearson’s correlation coefficients were conducted to test confounding variables within the acute and chronic paradigms of this study with regard to pT73 Rab10 variability, of which relationships with dose of MLi-2, length of experiment, age of mice, gender, and batch of tissue processing were not found. One-way ANOVA with Tukey’s post hoc; n = 6; ****P &lt; 0.0001, ***P &lt; 0.0002, **P &lt; 0.005, *P &lt; 0.05; (C) F (11, 60) = 28.56, (D) F (11, 60) = 43.20, (E) F (11, 59) = 24.70, (F) F (11, 60) = 8.279. S5. Proteomic analysis of changes in brain, kidney, and lung between untreated G2019S LRRK2 and wildtype mouse cohorts. Volcano plots depicting differences in levels of total proteins in kidney (A), brain (B), and lung (C) between untreated G2019S KI and wildtype mice. A number of proteins show subtle changes involved in iron transport and mitochondrial homeostasis are highlighted. Lung hits were not preferentially enriched for any particular cellular component. Volcano plots key: grey = non-significant, blue = &lt; 0.05 p-value, green = &gt; 1.4x log fold change, red = &lt; 0.05 p-value and &gt; 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 &lt; 0.0001, ***P &lt; 0.0002, **P &lt; 0.005, *P &lt; 0.05; (B) Jip4: F (3, 20)= 0.8688, glycosylated Lamp1: F (3, 20)= 0.4520, HGS: F (3, 20)= 0.1410, Legumain: F (3, 20)= 0.2312, Atp5mc1: F (3, 20)= 1.953, Sfxn3: F (3, 20)= 0.2285, Cytochrome C: F (3, 20)= 1.698, (D) F (3, 20)= Jip4: F (3, 20)= 1.772, HGS: F (3, 20)= 0.1636, Legumain: F (3, 20)= 0.1724, Sfxn3: F (3, 20)= 1.156, pS163 Marcks: F (3, 20)= 4.739, Cyp1a1: F (3, 20)= 208.2, ProSP-C: F (3, 20)= 4.495, (F) ProSP-C: F (3, 20)= 4.185, (H) non-glycosylated Lamp1: F (3, 20)= 2.802, Hgs: F (3, 20)= 5.173, Sfxn3: F (3, 20)= 3.002, (I) glycosylated Lamp1: 4.702, Legumain: F (3, 20)= 4.409, Jip4: F (3, 20)= 12.35. S7. LRRK2 protein interactome from long-term chronic kidney proteomics. The 149 mouse kidney protein hits from chronically treated G2019S KI mice were converted to their human orthologues for LRRK2 interactome analyses using PINOT online tool. Seventy-six matches were identified within the LRRK2 interactome. Five of the matching proteins (AHCYL1, EEF2, HSP90AA1, HSP90AB1 and RANBP2) were present in the first layer while 71 in the second layer of LRRK2 interactions (one step from direct LRRK2 interactors). Two visualizations are shown: (A) highlights the proteins from total or phospho-proteomics runs, (B) depicts primary and secondary interactome layers.