wild type lrrk2  (Addgene inc)

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW , and LRRK2 KW . ( B and C ) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2 RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. ( D ) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Construct, Binding Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Activation Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) The co-crystal structure of RN129 ( 28 ) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). ( B ) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. ( C and D ) Close-ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 (C) and LRRK2 RCKW :GZD824 (PDB: 8TZE) (D). ( E ) Superposition of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2 RCKW :MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆ T m shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆ T m shift, as indicated in the legend. ( B ) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆ T m shift of LRRK2 KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆ T m shifts, as indicated in the legend (as in A). Kinases with ∆ T m > 6 K are labeled. ( C ) Waterfall plots of the ReactionBiology 33 PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. ( D ) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC 50 (JNK2) = 2.7 μM, EC 50 (STK10) = 1.5 μM, EC 50 (MAPK14) = 1.7 μM, EC 50 (TTK) = 3.2 μM, EC 50 (CDKL1) = 17 μM, EC 50 (CLK1) = 6.0 μM, EC 50 (JNK3) = 15 μM, EC 50 (DYRK2) ≥ 20 μM, EC 50 (SLK) > 20 μM, EC 50 (DDR2) > 20 μM, and EC 50 (STK17B) ≥ 20 μM. ( E ) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. ( F ) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic of the single-molecule in vitro motility assay. ( B ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( C ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0007, DMSO condition; *** P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. ( D ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( E ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.

Article Snippet: For analysis of inhibitor activity against wild-type and G2019S mutant LRRK2, 293T cells were transfected with 500 ng of GFP-Rab8a and either 1000 ng of GFP-11 tagged wild-type LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2, RRID: Addgene_231174) or GFP-11 tagged G2019S LRRK2 (pcDNA5-FRT-TO-GFP11-LRRK2-G2019S, RRID: Addgene_231175).

Techniques: In Vitro, Motility Assay, Concentration Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW , and LRRK2 KW . ( B and C ) Close-up of the inhibitor binding pocket from cryo–electron microscopy (cryo-EM) maps and models of LRRK2 RCKW bound to the type I inhibitor MLi-2 [Protein Data Bank (PDB): 8TXZ] (B) and type II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange, C-lobe; light orange, N-lobe; black, DYG motif; gray, G-loop; green, activation loop. ( D ) Scheme depicting our hybrid design strategy to develop potent type II inhibitors targeting LRRK2.

Article Snippet: For Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

Techniques: Construct, Binding Assay, Cryo-Electron Microscopy, Cryo-EM Sample Prep, Activation Assay

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) The co-crystal structure of RN129 ( 28 ) with CLK3 highlighting the type II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3). ( B ) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI) built into the cryo-EM map. ( C and D ) Close-ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 (C) and LRRK2 RCKW :GZD824 (PDB: 8TZE) (D). ( E ) Superposition of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (in lighter shades) and our previously published structure of a LRRK2 RCKW :MLi-2:E11 DARPin complex (PDB: 8TXZ) (in darker shades). Only the kinase domains, which were aligned on their C-lobes, are shown. Major features of the kinase, including those that are indicators of type I and type II inhibitor binding, are shown.

Article Snippet: For Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue or light blue. The 18.5 K ∆ T m shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size and color correlates with the degree of ∆ T m shift, as indicated in the legend. ( B ) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20-K ∆ T m shift of LRRK2 KW is highlighted in red. The bubble size or color for each kinase correlates with the ∆ T m shifts, as indicated in the legend (as in A). Kinases with ∆ T m > 6 K are labeled. ( C ) Waterfall plots of the ReactionBiology 33 PanQinase screen of RN341 at 1 and 10 μM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to <22% of the control value are labeled. ( D ) Off-target validation from both screens via in cellulo nanoBRET assay in two biological replicates, error bars ± SD, EC 50 (JNK2) = 2.7 μM, EC 50 (STK10) = 1.5 μM, EC 50 (MAPK14) = 1.7 μM, EC 50 (TTK) = 3.2 μM, EC 50 (CDKL1) = 17 μM, EC 50 (CLK1) = 6.0 μM, EC 50 (JNK3) = 15 μM, EC 50 (DYRK2) ≥ 20 μM, EC 50 (SLK) > 20 μM, EC 50 (DDR2) > 20 μM, and EC 50 (STK17B) ≥ 20 μM. ( E ) Representative immunoblot from 293T cells transiently co-transfected with LRRK1 and its substrate GFP-Rab7 before treatment with a dilution series of RN277 and RN341. Lysed cells were immunoblotted for LRRK1, GFP-Rab7, phospho-Rab7 (pS72), and GAPDH. ( F ) Quantification of the GFP-pRab7/GFP-Rab7/LRRK1 ratio from three independent Western blots. Statistical analysis performed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons of means. P < 0.0001 for all inhibitor concentrations versus DMSO; error bars ± SEM.

Article Snippet: For Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A and B ) Dose-response curve of RN277 (A) and RN341 (B) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a versus non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341. ( C ) Representative immunoblot from 293T cells transiently co-transfected with LRRK2 and GFP-Rab8a, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( D ) Sample from (C) run separately under identical conditions and immunoblotted for phospho-S935 LRRK2 and GAPDH. ( E ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from three independent immunoblots (C). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0049, DMSO versus MLi-2; *** P = 0.0004, DMSO versus Ponatinib; *** P = 0.0006, DMSO versus 5 μM RN277; *** P = 0.0003, DMSO versus 10 μM RN277; * P = 0.0406, DMSO versus 5 μM RN341; ** P = 0.0065, DMSO versus 10 μM RN341; error bars ± SEM. ( F ) Quantification of the pS935 LRRK2/LRRK2 ratio (run under identical conditions on separate blots) from three independent immunoblots (D). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. **** P < 0.0001 for all conditions versus MLi-2; error bars ± SEM. ( G ) Representative immunoblot from 293T cells transiently co-transfected with GFP-Rab8a and either GFP-11 tagged wild-type (WT) or GFP-11 tagged G2019S LRRK2, treated with the indicated inhibitors. Lysed cells were immunoblotted for LRRK2, GFP-Rab8a, phospho-Rab8a (pT72), and GAPDH. ( H ) Quantification of the GFP-pRab8a/GFP-Rab8a/LRRK2 ratio from four independent immunoblots (G). Statistical analysis performed using one-way ANOVA with Tukey’s multiple comparisons of means. ** P = 0.0077, WT LRRK2 DMSO versus MLi-2; * P = 0.0324, WT LRRK2 DMSO versus 5 μM RN277; * P = 0.0461, WT LRRK2 DMSO versus 5 μM RN341; **** P < 0.0001 for all inhibitor treatments versus G2019S LRRK2 DMSO; error bars ± SEM.

Article Snippet: For Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection

Journal: Science Advances

Article Title: Type II kinase inhibitors that target Parkinson’s disease–associated LRRK2

doi: 10.1126/sciadv.adt2050

Figure Lengend Snippet: ( A ) Schematic of the single-molecule in vitro motility assay. ( B ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type I inhibitor MLi-2 (5 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( C ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0007, DMSO condition; *** P = 0.0003, MLi-2 condition, one-way ANOVA with Šidák’s multiple comparisons test within drug only. ( D ) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type II inhibitors Ponatinib, RN277, and RN341 (10 μM) in the presence or absence of LRRK2 RCKW . Scale bars, 5 μm ( x ) and 30 s ( y ). ( E ) Quantification of the percentage (means ± SEM) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type II inhibitors Ponatinib, RN277, and RN341 (10 μM). Three technical replicates were collected per condition, with data points represented as circles, triangles, and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; *** P = 0.0003, one-way ANOVA with Šidák’s multiple comparisons test within drug only.

Article Snippet: For Western blot analysis of LRRK2 kinase activity in cells, 293T cells (ATCC, CRL-3216; RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2, RRID: Addgene_229019) and 500 ng of GFP-Rab8A (RRID: Addgene_49543) using polyethylenimine (Polysciences).

Techniques: In Vitro, Motility Assay, Concentration Assay

Journal: International journal of molecular sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson's Disease.

doi: 10.3390/ijms26073229

Figure Lengend Snippet: Figure 1. Heterologous expression of LRRK2 and its increased activity mutant LRRK2G2019S. (a) Confocal images of HEK-293 cells expressing the two kinase variants fused to GFP. The nuclei are stained with DAPI (blue). The upper panels show overexpression of the LRRK2WT protein, while the lower panels illustrate the expression of the LRRK2G2019S variant. The far-right panels display the merged images for both constructs. The data shown are representative of three separate experiments. Scale bar = 10 µm. (b) Protein expression levels were evaluated in extracts from untransfected (UT) HEK-293 cells or cells co-expressing the variants of the LRRK2 kinase. Protein extracts were subjected to Western blot analysis using the antibodies listed in Supplementary Table S1. The molecular weight markers are shown on the left. Actin served as a loading control.

Article Snippet: The wild-type LRRK2 (LRRK2WT) cDNA clone and its LRRK2G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Expressing, Activity Assay, Mutagenesis, Staining, Over Expression, Variant Assay, Construct, Western Blot, Molecular Weight, Control

Journal: International journal of molecular sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson's Disease.

doi: 10.3390/ijms26073229

Figure Lengend Snippet: Figure 2. LRRK2 interacts with the CaV1.3 (L-type) channel complex. (a) Proteins from HEK-293 cells transiently transfected with the two kinase variants (WT and G2019S), along with the CaV1.3α1, CaVβ3, or CaVα2δ-1 channel subunits, were subjected to immunoprecipitation (IP) assays using anti- GFP antibodies. The anti-EspC antibody, which is directed against a serine protease autotransporter protein secreted by enteropathogenic E. coli, was utilized as an irrelevant control antibody (IgG 0). The immunoprecipitated proteins were subsequently analyzed using Western blot with specific antibodies as indicated. The auxiliary subunit CaVβ3 was IP using the anti-GFF antibody (bottom panel). In contrast, the pore-forming subunit CaV1.3α1 did not show interaction with the kinase (middle panel). (b) Western blot analysis was conducted to investigate the actions of the two LRRK2 variants on the expression levels of proteins that form the L-type Ca2+ channel complex, CaV1.3. Actin served as a loading control. The data shown represent findings from three independent experiments.

Article Snippet: The wild-type LRRK2 (LRRK2WT) cDNA clone and its LRRK2G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Transfection, Immunoprecipitation, Control, Western Blot, Expressing

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet: ( a ) Schematic representation of gene editing strategy to knock-in the G2019S mutation in the LRRK2 locus. ( b ) Sanger sequencing of a single gene edited clone showing successful homozygous editing of the indicated nucleotide. ( c ) KOLF2-1J wild-type (LRRK2 WT ) control iPSCs and KOLF2-1J LRRK2 G2019S/G2019S (LRRK2 G2019S ) iPSCs show normal expression of pluripotency markers OCT4 (yellow), SOX2 (purple), NANOG (purple), and TRA-1–81 (green). Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. ( d ) KOLF2-1J wild-type (LRRK2 WT ) control and KOLF2-1J LRRK2 G2019S/G2019S (LRRK2 G2019S ) mutant ventral midbrain neural progenitor cells show normal expression of FOXA2 (magenta), LMX1A (green), and OTX2 (green), confirming that the neural progenitor cells are ventral midbrain-specific and capable of differentiating into DAN. Nuclei are counterstained with DAPI (blue). Scale bar: 100 μm. ( e ) Quantification of the frequency of TH+/MAP2+DAN. Statistical significance calculated with an ordinary t-test: ns, not significant. Error bars represent mean ± SEM. Data were collected in three independent vmDAN differentiations. ( f ) Differentiation protocol used.

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques: Knock-In, Mutagenesis, Sequencing, Control, Expressing

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet: ( a–a” ) Schematic of the sunburst plot indicating Gene Ontology (GO) terms for each sector ( a ) and the mapping of the DEGs in nucleus basalis of Meynert (NBM), nucleus accumbens, and putamen brain samples idiopathic PD patients (with LRRK2 risk mutations) and controls ( a’ ) and mapping of the DEGs found commonly in fly and human samples ( a” ). Inner rings represent the different GO categories (indicated in a), with their subcategories in the outer rings, rings (in a’–a”) are color-coded according to enrichment Q-value. ( b ) GO analysis of DEG in cholinergic neurons of young PD fly models and NBM neurons of PD patients. Redundant terms were removed. Color: adjusted p-value. ( c ) Schematic of the DEGs found commonly in fly PD models (blue) and human PD samples (black) manually sorted according to their previously described synaptic functions. contains the summarized results of the DEG analysis of fly brains and postmortem human brain samples and the SynGO analysis.

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques:

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet: ( a , left) Synaptic area of dopaminergic neuron (DAN) innervating the mushroom body in aged PD models (Aux R927G , synj R258Q , or LRRK2 G2019S ) and with or without GH146-Gal4-driven expression of the wild-type PD gene ( aux or synj ) or EndoA S75D , respectively. Bars: mean ± SEM. n≥5, *p<0.05 in ANOVA, Dunnett’s test. ( a , right) Startle-induced negative geotaxis (SING) of the PD models with or without GH146-Gal4-driven expression of wild-type gene or endoA S75D . Points: mean ± SEM. n≥5, *p<0.05 in two-way ANOVA. Gray zone: variance of controls. ( b ) Odor choice performance, stimulus-induced changes in synaptic Ca 2+ signal and olfactory projection neuron (OPN) synapse area of young controls and hLRRK2 G2019S flies with or without chronic nicotine (Nic) feeding (up to 1 day before testing). Bars: mean ± SEM. n≥5 assays, *p<0.05 in ANOVA, Dunnett’s test. ( c ) SING, stimulus-induced changes in synaptic Ca 2+ and DAN synapse area of aged controls and hLRRK G2019S flies with or without chronic application of nicotine. Bars: mean ± SEM. n≥5 assays, *p<0.05 in ANOVA, Dunnett’s test. ( d ) Confocal images of differentiated (60 days) wild-type and LRRK2 G2019S ventral midbrain DAN labeled with the ventral midbrain marker FOXA2, dopaminergic marker TH, and neuronal marker MAP2. Scale bar: 20 µm. ( e ) Scheme of the treatment protocol and spontaneous Ca 2+ activity ( e’ ) and amplitude ( e’’ ) of human induced DAN, 2 days after 20 days of no treatment (Ctrl), nicotine (Nic) treatment or nicotine+mecamylamine (Nic+Meca) treatment. Bars: mean ± SEM. n≥60 DAN from three independent differentiations, *p<0.05 in ANOVA, Dunnett’s test.

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques: Expressing, Labeling, Marker, Activity Assay

Journal: eLife

Article Title: Synaptic deregulation of cholinergic projection neurons causes olfactory dysfunction across five fly Parkinsonism models

doi: 10.7554/eLife.98348

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapiens ) , KOLF2-1J wild-type (LRRK2 WT ) control iPSCs , ; from the Jackson Laboratory under the iPSC Neurodegenerative Disease Initiative , Product code: JIPSC001000 , .

Techniques: Knock-Out, Knock-In, Control, Mutagenesis, Transfection, Construct, Plasmid Preparation, Expressing, Imaging, Recombinant, Sequencing, Multiplex Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: Heterologous expression of LRRK2 and its increased activity mutant LRRK2G2019S. ( a ) Confocal images of HEK-293 cells expressing the two kinase variants fused to GFP. The nuclei are stained with DAPI (blue). The upper panels show overexpression of the LRRK2 WT protein, while the lower panels illustrate the expression of the LRRK2 G2019S variant. The far-right panels display the merged images for both constructs. The data shown are representative of three separate experiments. Scale bar = 10 μm. ( b ) Protein expression levels were evaluated in extracts from untransfected (UT) HEK-293 cells or cells co-expressing the variants of the LRRK2 kinase. Protein extracts were subjected to Western blot analysis using the antibodies listed in . The molecular weight markers are shown on the left. Actin served as a loading control.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Expressing, Activity Assay, Mutagenesis, Staining, Over Expression, Variant Assay, Construct, Western Blot, Molecular Weight, Control

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: LRRK2 interacts with the Ca V 1.3 (L-type) channel complex. ( a ) Proteins from HEK-293 cells transiently transfected with the two kinase variants (WT and G2019S), along with the Ca V 1.3α 1 , Ca V β 3 , or Ca V α 2 δ-1 channel subunits, were subjected to immunoprecipitation (IP) assays using anti-GFP antibodies. The anti-EspC antibody, which is directed against a serine protease autotransporter protein secreted by enteropathogenic E. coli, was utilized as an irrelevant control antibody (IgG 0). The immunoprecipitated proteins were subsequently analyzed using Western blot with specific antibodies as indicated. The auxiliary subunit Ca V β 3 was IP using the anti-GFF antibody (bottom panel). In contrast, the pore-forming subunit Ca V 1.3α 1 did not show interaction with the kinase (middle panel). ( b ) Western blot analysis was conducted to investigate the actions of the two LRRK2 variants on the expression levels of proteins that form the L-type Ca 2+ channel complex, Ca V 1.3. Actin served as a loading control. The data shown represent findings from three independent experiments.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Transfection, Immunoprecipitation, Control, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: Regulation of the functional expression of L-type channels (Ca V 1.3) by LRRK2 G2019S ( a ) Typical whole-cell patch-clamp I Ca recordings obtained in HEK-293 cells expressing the Ca V 1.3α 1 /Ca V β 3 /Ca V α 2 δ-1 channel subunits in the presence of both kinase variants (WT and G2019S). The currents were generated by depolarizing pulses to −15 mV from a V h of −80 mV. ( b ) Average current density measured in HEK-293 cells expressing the Ca V 1.3 channels in the presence of the LRRK2 variants as indicated. The asterisk denotes a significant difference ( p < 0.05). ( c ) Average I - V curves for I Ca obtained from cells expressing the Ca V 1.3α 1 , Ca V β 3 , and Ca V α 2 δ-1 channels with LRRK2 WT and LRRK2 G2019S ( n = 22–28 cells). The currents were generated by applying 5 mV activating steps from a V h of −80 mV to potentials from −70 to +70 mV. ( d ) The voltage dependence of channel activation was assessed in control cells and cells co-transfected with both variants of the LRRK2 kinase. Conductance values were obtained from ( c ), as detailed in the Methods section, and the fitting parameters of the curves were similar across the three experimental conditions: V 1/2 = −31.9, −29.9, and −31.9; k = 4.2, 4.9, and 4.7 for the control condition, LRRK2 WT , and LRRK2 G2019S , respectively.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Functional Assay, Expressing, Patch Clamp, Generated, Activation Assay, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: The regulatory effect of LRRK2 G2019S on the channels is absent without the auxiliary subunit Ca V β 3 . ( a ) Typical traces of I Ba obtained from HEK-293 cells transfected with cDNAs encoding L-type channels Ca V 1.3 (Ca V 1.3α 1 /Ca V α 2 δ-1) without the auxiliary subunit Ca V β 3 . The currents were evoked by activating pulses to −10 mV from a V h of −80 mV. ( b ) Average I Ba density measured in control cells and in the presence of the LRRK variants, as in panel ( a ). ( c ) Average maximum currents graphed against the test potential in the experimental conditions, as indicated. The currents were evoked by applying voltage-activating pulses ranging from −70 to +70 mV, starting from a V h of −80 mV. The number of recorded cells is indicated in parentheses. ( d ) Voltage dependence of activation for L-type channels (Ca V 1.3α 1 /Ca V α 2 δ-1) in control cells and those expressing different variants of LRRK2. Conductance data were derived from panel ( c ), and the fitting parameters of the curves were similar across the three experimental conditions: G max = 0.97, 0.96, and 0.96; V 1/2 = −28.7, −29, and −29; k = 7.4, 7.0, and 7.4 for the control condition, LRRK2 WT , and LRRK2 G2019S , respectively. The error bars are hidden within the symbols.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Transfection, Control, Activation Assay, Expressing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: Molecular determinants of the Ca V 1.3 channel regulation by LRRK2 G2019S . ( a ) Amino acid sequence alignment of selected regions within the Ca V β 3 subunit, illustrating the putative LRRK2 phosphorylation sites that are conserved across three species as indicated. The red black boxes indicate the sequence corresponding to the consensus phosphorylation site for the kinase. Automated sequencing of the PCR products verified that the constructs produced through site-directed mutagenesis of the LRRK2 phosphorylation site incorporated the intended mutant. ( b ) Localization of serine residues potentially phosphorylatable by LRRK2 across the different domains of the Ca V β 3 protein sequence. ( c ) Western blot of protein extracts from HEK-293 cells co-expressing the wild-type or the mutant variants of Ca V β 3 (S152A, S245A, T283A, and T316A) showing robust expression of all mutations in the heterologous system. ( d ) Representative Ca 2+ currents through recombinant Ca V 1.3α 1 /Ca V β 3 /Ca V α 2 δ-1 channels recorded in HEK-293 cells co-expressing the wild-type Ca V β3 or its phosphorylation variants in the presence of LRRK2 WT or its variant LRRK2 G2019S . The currents were generated by 500 ms pulses at −15 mV, starting from a V h of −80 mV. ( e ) Average I Ca density in HEK-293 cells expressing Ca V 1.3α 1 /α 2 δ-1 channels along with the wild-type Ca V β 3 subunit or its phosphorylation mutants (filled bars). I Ca density was determined from the current amplitude after the application of a depolarizing pulse of −15 mV from a V h of −80 mV, normalized to C m , in the presence of LRRK2 or LRRK2 G20219S , as indicated ( n = 13–20 cells). The open bar shows the average I Ca amplitude through recombinant Ca V 1.3 channels containing the wild-type Ca V β 3 subunit and in the presence of LRRK2 WT, and it is identical to what is presented in b for the purposes of comparison. The asterisk indicates a statistically significant difference ( p < 0.05).

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Sequencing, Phospho-proteomics, Construct, Produced, Mutagenesis, Western Blot, Expressing, Recombinant, Variant Assay, Generated, Comparison

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: Effects of the phosphorylation mutants of the Ca V β 3 subunit on current voltage dependence and density. ( a ) Superimposed average ± SEM I - V curves for I Ca obtained from HEK-293 cells expressing Ca V 1.3 channels comprising the wild-type auxiliary Ca V β 3 subunit and its phosphorylation mutants (S152A, S245A, T283A, and T316A) in the presence of LRRK2 G2019S , as indicated. I Ca density was estimated from a series of activating pulses between −70 and +70 mV from a V h of −80 mV, in 5 mV steps ( n = 14–20 cells). ( b ) The amplitude of I Ca was then normalized to their corresponding C m values, and activation curves were constructed with the data derived, shown in panel ( a ). The fitting parameters of the curves are as follows: G max = 0.95, 0.88, 0.86, 0.92, and 0.91; V 1/2 = −30.9, −40.1, −32.6, −36.8, and −33.1; k = 4.8, 4.4, 3.5, 4.0, and 4.5 for Ca V β 3 WT , Ca V β 3 S152A , Ca V β 3 S245A , Ca V β 3 T283A , Ca V β 3 T319A , respectively.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Phospho-proteomics, Expressing, Activation Assay, Construct, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: The regulatory effect of LRRK2 G2019S on Ca V 1.3 channels may not involve the main Ca V 1.3α 1 subunit. ( a ) The schematic representation of the Ca V 1.3α 1 ion-conducting subunit highlights a serine residue at position 793, which LRRK2 may phosphorylate. This residue is situated in the intracellular loop connecting the repeated domains II and III of the channels. ( b ) Western blot analysis was conducted on proteins from untransfected HEK-293 cells (UT; lane 1) and cells expressing Ca V 1.3α 1 /Ca V β 3 /Ca V α 2 δ-1 channels (lanes 2 and 3), using an antibody that recognizes the Ca V 1.3α 1 protein. The blot reveals two bands above the 150 kDa marker; one of these bands, likely the upper one, has not been observed previously and may indicate a nonspecific interaction with the antibody. This band is present in both lanes corresponding to the WT protein and the S793A mutant, suggesting that it might not impact te interpretation of the data. ( c ) Typical whole-cell currents obtained in HEK-293 cells co-expressing the Ca V 1.3α 1 /Ca V β 3 /Ca V α 2 δ-1 channel subunits in the presence of both kinase variants (WT and G2019S). The currents were elicited by depolarizing pulses from a V h of −80 to −15 mV, as indicated. ( d ) Average I - V curves for I Ca were obtained from cells expressing Ca V 1.3 channels in conjunction with the two variants of the LRRK2 kinase ( n = 16–23 cells). The currents were elicited by applying 5 mV activating steps from a V h of −80 mV to potentials from −70 to +70 mV.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Residue, Western Blot, Expressing, Marker, Mutagenesis

Journal: International Journal of Molecular Sciences

Article Title: The Leucine-Rich Repeat Kinase 2 Variant LRRK2 G2019S Up-Regulates L-Type (CaV1.3) Calcium Channel via the Ca V β 3 Subunit: Possible Role in the Pathogenesis of Parkinson’s Disease

doi: 10.3390/ijms26073229

Figure Lengend Snippet: The mutant protein LRRK2 G2019S can alter the function of Ca V 1.3 channels because the auxiliary subunit Ca V β 3 is essential for their expression and regulation. ( a ) Phosphorylation of Ca V β 3 enhances the intracellular transport of the channel complex to the cell membrane, stabilizes the channel complex, and prevents degradation. This modification is crucial for proper calcium signaling in neurons. The G2019S mutation in LRRK2 increases its activity approximately twofold compared to the wild-type kinase, which is key to its pathogenic effects. The hyperactivity of this protein may lead to an increase in the number of Ca V 1.3 channels at the neuronal membrane. ( b ) Since Ca V 1.3 channels control the rhythmic firing of dopaminergic neurons in the substantia nigra, the increased expression at the membrane could affect pacemaker frequency and elevate intracellular calcium levels. Even more, the hypothesis that the progression of PD could be associated with a dysregulation of Ca V 1.3 channels due to increased LRRK2 activity is consistent with the idea that, during the development of the disease, there is an increase in the intracellular calcium concentration, which demands extra effort from the calcium reuptake and extrusion mechanisms. Likewise, it is also recognized that, in PD, the functionality of certain calcium buffering proteins is disrupted. This, in conjunction with an increase in energy demand and oxidative stress, results in mitochondrial damage and ultimately in the loss of dopaminergic cells in the SNc.

Article Snippet: The wild-type LRRK2 (LRRK2 WT ) cDNA clone and its LRRK2 G2019S mutant were purchased from Addgene (Watertown, MA, USA, catalogs #25044 and 25045, respectively).

Techniques: Mutagenesis, Expressing, Phospho-proteomics, Membrane, Modification, Activity Assay, Control, Concentration Assay

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) Schematic domain structure of LRRK2. The three constructs used in this study are indicated: full-length LRRK2, LRRK2 RCKW and LRRK2 KW ; (B) and (C) Close up of the inhibitor binding pocket from cryo-EM maps and models of LRRK2 RCKW bound to the type-I inhibitor MLi-2 (PDB: 8TXZ) (B) and type-II inhibitor GZD-824 (PDB: 8TZE) (C). Key residues and features are labelled. Both structures are shown in the same view, aligned through the C-lobe of the kinase. Dark orange: C-lobe; light orange: N-lobe; black: DYG motif; grey: G-loop; green: activation loop. (D) Scheme depicting our hybrid design strategy to develop potent and selective type-II inhibitors for LRRK2.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Construct, Binding Assay, Cryo-EM Sample Prep, Activation Assay

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) The co-crystal structure of RN129 (28) with CLK3 highlighting the type-II binding mode and interactions between the protein and inhibitor (PDB: 9EZ3); (B) Ribbon diagram of the atomic model of LRRK2 RCKW :RN277:E11 DARPin complex (PDB: 9DMI); (C) and ( D) Close ups of the active sites of the cryo-EM structures of LRRK2 RCKW :RN277 ( C ) and LRRK2 RCKW :GZD824 (PDB: 8TZE) ( D ).

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Binding Assay, Cryo-EM Sample Prep

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) Kinome phylogenetic tree, with 96 kinases screened in the DSF assay against Rebastinib highlighted in blue. The 18.5 K ΔTm shift of LRRK2 KW is highlighted in red. For all screened kinases, the bubble size correlates with the degree of ΔTm shift, as indicated in the legend; (B) Kinome phylogenetic tree, with 103 kinases screened in the DSF assay against RN341 highlighted in blue. The 20 K ΔTm shift of LRRK2 KW is highlighted in red. The bubble size for each kinase correlates with the ΔTm shifts, as indicated in the legend (as in A ). Kinases with ΔTm > 6 K are labeled; (C) Waterfall plots of the ReactionBiology 33 PanQinase™ screen of RN341 at 1 µM and 10 µM against 350 wild-type kinases. Kinases with decreased activity in the presence of RN341 to < 22 % of the control value are labeled; (D) Off-target validation from both screens via in cellulo nanoBRET assay in 2 biological replicates, error bars ± sd, EC 50 (JNK2) = 2.7 µM, EC 50 (STK10) = 1.5 µM, EC 50 (MAPK14) = 1.7 µM, EC 50 (TTK) = 3.2 µM, EC 50 (CDKL1) = 17 µM, EC 50 (CLK1) = 6.0 µM, EC 50 (JNK3) = 15 µM, EC 50 (DYRK2) = >20 µM, EC 50 (SLK) >20 µM, EC 50 (DDR2) >20 µM, EC 50 (STK17B) = >20 µM.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Labeling, Activity Assay, Control, Biomarker Discovery

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) and (B) Dose response curve of RN277 ( 30 ) and RN341 ( 32 ) inhibiting LRRK2 RCKW -mediated phosphorylation of Rab8a. Activity was calculated as the percentage (%) of phosphorylated Rab8a vs. non-phosphorylated Rab8a detected in the presence of different concentrations of RN277/RN341; (C) Western blots from 293T cells transiently co-transfected with LRRK2 (full-length) and GFP-Rab8a for 48h prior to treatment with a dilution series of RN277 ( 30 ) and RN341 ( 32 ) for 4h. DMSO and MLi-2 (500 nM) treatment for 4h were used as negative and positive controls, respectively. Lysed cells were immunoblotted for LRRK2, total GFP-Rab8a, phospho-Rab8a (pT72) and GAPDH as a loading control; (D) Quantification from four independent western blots showing the ratio of GFP-pRab8a to total GFP-Rab8a upon treatment with RN277 ( 30 ) and RN341 ( 32 ) at the indicated concentrations. Statistical analysis was performed using one-way ANOVA analysis with Tukey’s multiple comparison of means. ****p<0.0001, error bars ± s.e.m. (E) Western blots from 293T cells transiently co-transfected with LRRK2 (full-length) and GFP-Rab8a, treated with DMSO (control), 500 nM MLi-2, 5 µM RN277 or 5 µM RN341 for 4h, 48h post-transfection. Lysed cells were immunoblotted for LRRK2, phospho-LRRK2 (pS935), total GFP-Rab8a, phospho-Rab8a (pT72) and GAPDH as a loading control; (F) Quantification from four independent western blots showing the ratio of GFP-pRab8a to total GFP-Rab8a upon treatment with the indicated inhibitors (as in D ). Statistical analysis was performed using a one-way ANOVA analysis with Tukey’s multiple comparison of means. ****p<0.0001, error bars ± s.e.m.; (G) Quantification from four independent western blots showing the ratio of pLRRK2 to total LRRK2 upon treatment with the indicated inhibitors. Statistical analysis was performed using a one-way ANOVA analysis with Tukey’s multiple comparison of means. *p 0.0469, error bars ± s.e.m.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: Phospho-proteomics, Activity Assay, Western Blot, Transfection, Control, Comparison

Journal: bioRxiv

Article Title: Type-II kinase inhibitors that target Parkinson’s Disease-associated LRRK2

doi: 10.1101/2024.09.17.613365

Figure Lengend Snippet: (A) Schematic of the single-molecule in vitro motility assay; (B) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type-I inhibitor MLi-2 (5 µM) in the presence or absence of LRRK2 RCKW . Scale bars 5 µM (x) and 30 s (y); (C) Quantification of the percentage (mean ± s.e.m) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of MLi-2 (5 µM). Three technical replicates were collected per condition, with data points represented as circles, triangles and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; DMSO condition ***p 0.0007, MLi-2 condition ***p 0.0003, One-way ANOVA with Holm-Sidaks multiple comparison test within drug only; (D) Example kymographs from single-molecule motility assays showing kinesin motility with DMSO or the type-II inhibitors Ponatinib, RN277 and RN341 (5 µM) in the presence or absence of LRRK2 RCKW . Scale bars 5 µM (x) and 30 s (y); (E) Quantification of the percentage (mean ± s.e.m) of motile events per microtubule as a function of LRRK2 RCKW concentration in the absence (DMSO) or presence of type-II inhibitors Ponatinib, RN277 and RN341 (5 µM). Three technical replicates were collected per condition, with data points represented as circles, triangles and squares corresponding to single data points (microtubules) within each replicate. Statistical analysis was performed using the mean of each technical replicate; ***p 0.0003, One-way ANOVA with Holm-Sid-aks multiple comparison test within drug only.

Article Snippet: 293T cells (American Type Culture Collection, ATCC cat. no. CRL-3216, RRID: CVCL_0063) were transfected with 1000 ng of wild-type full-length LRRK2 (pcDNA5-LRRK2) and 500 ng GFP-Rab8A (Addgene, RRID: Addgene_49543) using PEI (Polyethylenimine, Polysciences) (Supporting Information Table S4 ).

Techniques: In Vitro, Motility Assay, Concentration Assay, Comparison