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. 2022 Sep 16;479(17):1759-1783.
doi: 10.1042/BCJ20220161.

Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding

Alexia F Kalogeropulou #  1   2 , Elena Purlyte #  1 , Francesca Tonelli #  1   2 , Sven M Lange  1 , Melanie Wightman  1 , Alan R Prescott  3 , Shalini Padmanabhan  4 , Esther Sammler  1   2   5 , Dario R Alessi  1   2
Affiliations

Impact of 100 LRRK2 variants linked to Parkinson's disease on kinase activity and microtubule binding

Alexia F Kalogeropulou et al. Biochem J. .

Abstract

Mutations enhancing the kinase activity of leucine-rich repeat kinase-2 (LRRK2) cause Parkinson's disease (PD) and therapies that reduce LRRK2 kinase activity are being tested in clinical trials. Numerous rare variants of unknown clinical significance have been reported, but how the vast majority impact on LRRK2 function is unknown. Here, we investigate 100 LRRK2 variants linked to PD, including previously described pathogenic mutations. We identify 23 LRRK2 variants that robustly stimulate kinase activity, including variants within the N-terminal non-catalytic regions (ARM (E334K, A419V), ANK (R767H), LRR (R1067Q, R1325Q)), as well as variants predicted to destabilize the ROC:CORB interface (ROC (A1442P, V1447M), CORA (R1628P) CORB (S1761R, L1795F)) and COR:COR dimer interface (CORB (R1728H/L)). Most activating variants decrease LRRK2 biomarker site phosphorylation (pSer935/pSer955/pSer973), consistent with the notion that the active kinase conformation blocks their phosphorylation. We conclude that the impact of variants on kinase activity is best evaluated by deploying a cellular assay of LRRK2-dependent Rab10 substrate phosphorylation, compared with a biochemical kinase assay, as only a minority of activating variants (CORB (Y1699C, R1728H/L, S1761R) and kinase (G2019S, I2020T, T2031S)), enhance in vitro kinase activity of immunoprecipitated LRRK2. Twelve variants including several that activate LRRK2 and have been linked to PD, suppress microtubule association in the presence of a Type I kinase inhibitor (ARM (M712V), LRR (R1320S), ROC (A1442P, K1468E, S1508R), CORA (A1589S), CORB (Y1699C, R1728H/L) and WD40 (R2143M, S2350I, G2385R)). Our findings will stimulate work to better understand the mechanisms by which variants impact biology and provide rationale for variant carrier inclusion or exclusion in ongoing and future LRRK2 inhibitor clinical trials.

Keywords: G-proteins; Parkinson's disease; leucine-rich repeat kinase; signaling.

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Conflict of interest statement

The authors declare that there are no competing interests associated with the manuscript.

Figures

Figure 1.
Figure 1.. Domain location of 100 LRRK2 variants and experimental workflow to assess LRRK2 variant activity by quantitative immunoblotting.
(A) LRRK2 domain structure highlighting 100 PD and CD-associated variants within the armadillo (ARM), ankyrin (ANK), leucine rich repeats (LRR), Ras of complex proteins (ROC), C-terminal of ROC A and B (CORA, CORB), kinase (KIN), and WD40 domains. The LRRK2 variants located in the linker region between the HH and LRR domain are listed in black. (B) Workflow schematic outlining the characterization of the selected LRRK2 variants in a HEK293 overexpression system, followed by quantitative immunoblotting and quantitation of LRRK2 activity relative to wildtype LRRK2. (C) FLAG-tagged LRRK2 wildtype, kinase dead (KD = D2017A), and the indicated variants were transiently expressed in HEK293 cells. Twenty-four hours post-transfection, cells were lysed and analyzed by quantitative immunoblotting (as in Supplementary Figure S2A). Quantified immunoblotting data are presented as ratios of pRab10Thr73/total Rab10, normalized to the average of LRRK2 wildtype values for each replicate (mean ± SD). Combined immunoblotting data from up to six independent biological replicates are shown. Dashed lines segment the graphs into corresponding regions of LRRK2 as listed in the domain schematic.
Figure 2.
Figure 2.. Quantitative analysis of phosphorylation and expression of selected PD and CD-associated LRRK2 variants assessed in primary screens.
FLAG-tagged LRRK2 wildtype, kinase dead (KD = D2017A), and the indicated variants were transiently expressed in HEK293 cells. Twenty-four hours post-transfection, cells were lysed and analysed by quantitative immunoblotting (as in Supplementary Figure S2A). Quantified immunoblotting data are presented as ratios of phospho-LRRK2 Ser1292/total LRRK2 (A), phospho-LRRK2 Ser935 (B), and total LRRK2/Tubulin (C), normalized to the average of LRRK2 wildtype values for each replicate (mean ± SD). Combined immunoblotting data from six independent biological replicates are shown. Dashed lines segment the graphs into corresponding regions of LRRK2 as listed in the domain schematic.
Figure 3.
Figure 3.. 23 LRRK2 variants with mutations spanning multiple domains significantly augment LRRK2-mediated Rab10Thr73 phosphorylation.
(A) FLAG-tagged LRRK2 wildtype, kinase dead (KD = D2017A) and the indicated variants were transiently expressed in HEK293 cells. Twenty-four hours post-transfection, cells were lysed and analysed by quantitative immunoblotting using the indicated antibodies. Each lane represents a different dish of cells. Data quantification is shown in (B–E). (B–E) Quantified immunoblotting data are presented as ratios of pRab10 Thr73/total Rab10 (B), pRab12 Ser106/total Rab12 (C), phospho-LRRK2 Ser1292/total LRRK2 (D), phospho-LRRK2 Ser935/total LRRK2, phospho-LRRK2 Ser955/total LRRK2, or phospho-LRRK2 Ser973/total LRRK2 (E), normalized to the average of LRRK2 wildtype values for each replicate (mean ± SD). Combined immunoblotting data from two independent biological replicates (each performed in duplicate) are shown. Data were analysed using one-way ANOVA with Dunnett's multiple comparisons test. Statistical significance was determined from four replicate values for each variant, and represented with P-values (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). (F) FLAG LRRK2 WT or R1628P was expressed in HEK293 cells. Each lane represents a different dish of cells. One hour prior to lysis, cells were treated with vehicle (0.1% v/v DMSO) or 100 nM MLi-2. Cell lysates were analysed by quantitative immunoblotting and quantified data are analysed and presented as in (A–E). Quantified data are representative of three independent experiments, each performed in triplicate. (G) Domain schematic of LRRK2 highlighting the position of the 23 LRRK2 variants selected for further analysis.
Figure 4.
Figure 4.. CORB and kinase domain LRRK2 variants enhance in vitro LRRK2 kinase activity against recombinant Rab8A.
(A) Workflow schematic outlining the immunoprecipitation kinase assay method employed to assess in vitro kinase activity of LRRK2 variants against recombinant Rab8A. Kinase reaction products were analysed by quantitative immunoblotting (as in Supplementary Figure S6–S8). (B–D) Data obtained from quantitative immunoblotting analysis of FLAG-LRRK2 immunoprecipitation kinase reactions for the indicates variants are presented as ratios of pRab8AThr72/total Rab8A (B), phospho-LRRK2 Ser1292/total LRRK2 (C), and phospho-LRRK2 Thr1357/total LRRK2 (D) normalized to the average of LRRK2 wildtype values (mean ± SD). (E) Data obtained from quantitative immunoblotting analysis of FLAG-LRRK2 immunoprecipitation kinase reactions for the indicates variants are presented as ratios of pRab8AThr72/total Rab8A, phospho-LRRK2 Ser1292/total LRRK2, relative to the average of LRRK2 wildtype values (mean ± SD).
Figure 5.
Figure 5.. Selected LRRK2 variants are activated by Rab29.
FLAG-tagged LRRK2 wildtype, kinase dead (KD = D2017A) and the indicated variants were transiently expressed in HEK293 cells with HA empty vector or HA-Rab29. Twenty-four hours post-transfection, cells were lysed and analysed by quantitative immunoblotting (as in Supplementary Figure S9). (A–C) Quantified immunoblotting data are presented as ratios of phospho-Rab10/total Rab10 (A), phospho-LRRK2 Ser1292/total LRRK2 (B), phospho-LRRK2 Ser935/total LRRK2 (C), normalized to the average of LRRK2 wildtype values for each replicate (mean ± SD). Combined immunoblotting data from two independent biological replicates (each performed in duplicate) are shown.
Figure 6.
Figure 6.. Impact of 98 LRRK2 variants on Type I inhibitor-induced microtubule association.
(A) HEK293 cells transiently transfected with Flag-tagged LRRK2 wildtype, kinase dead (KD = D2017A) or the indicated variants were treated with 100 nM MLi-2 (or DMSO, control vehicle) for 3 h to induce microtubule association. Cells were then fixed and subjected to immunofluorescent microscopy imaging of Flag-tagged LRRK2. Data are presented as % of LRRK2 signal-positive cells that show filamentous LRRK2. Bars represent mean ± SD and each circle represents a data point from an independent experiment with at least 50 Flag-LRRK2 staining-positive cells evaluated. The full experiment with 98 variants was performed twice and select few variants with lower expression levels were tested again separately in a third smaller scale experiment. Two-way ANOVA with the Dunnett's multiple comparisons test was used to evaluate the statistical significance of the results (P values marked on the graph comparing the variant MLi-2-treated group to the WT MLi-2 treated group: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. None of the DMSO treated groups showed statistically significant differences from the WT group). Data are arranged by % of cells with filamentous LRRK2 signal upon MLi-2 treatment (low to high). (B) Sample images of the Flag-LRRK2 staining of selected variants. Scale bar — 10 μm. Cells with filamentous LRRK2 are marked with white arrowheads.
Figure 7.
Figure 7.. Structural analysis of identified activating LRRK2 variants.
(A) Schematic domain overview of LRRK2 with domain boundaries. (B) Cartoon representation of LRRK2 [558–2527] with domains colored as in (A). Domain interfaces harboring activating mutations and kinase domain are indicated by black arrows. (C) Schematic representation of LRRK2 domains as viewed in (B). (D–L) Detailed views of LRRK2 variants in kinase active site (D), domain interfaces (E–L), coloring as in (A) and variants highlighted in magenta. Second LRRK2 molecule of dimer shown in gray (G,L). (G) R1728 side chain modeled in PyMOL shown as semi-transparent stick model. Distance measurements in Å are indicated by dark gray dashed lines. (M) Alphafold model of LRRK2 ARM domain colored by local confidence score (pLDDT) with variant residues shown as stick models. LRRK2 structures used are PDB 7LI4 (B,D,E,H–K), PDB 7LHT (F,G), PDB 6DLO (L), AFDB AF-Q5S007-F1_v1 (M).
Figure 8.
Figure 8.. Structure-guided mutations in the N-terminus of LRRK2 stimulate LRRK2-mediated Rab10 phosphorylation.
(A,B) Cartoon representation of LRRK2 [558–2527] with detailed views of ANK (blue):CT α-helix (magenta) (A) and LRR (green):CORA (yellow) (B) interactions. Distance measurements in Å are indicated by dark gray dashed lines. (Right panel) HEK293 cells were transfected with wildtype, kinase dead (KD = D2017A), and the indicated LRRK2 variants. Each lane represents a different dish of cells. Cells were harvested 24 h post-transfection and subjected to quantitative immunoblot analysis with the indicated antibodies. Each lane represents a different dish of cells. The ratios of phospho-Rab10 Thr73/total Rab10 and phospho-LRRK2 Ser935/total LRRK2 were normalized to wildtype LRRK2 values. Quantified data are presented as mean ± SD and are representative of two independent experiments.
Figure 9.
Figure 9.. Correlation between activating LRRK2 mutations and high REVEL pathogenicity prediction or high evolutionary amino acid conservation scores.
(A) Schematic summarizing biochemical data of 100 LRRK2 variants and categorization of variants based on Rab10 phosphorylation, LRRK2 Ser1292 phosphorylation, and biomarker phosphorylation. Variants that enhance in vitro LRRK2 kinase activity or block MLi-2 induced microtubule binding are marked with a superscript highlighted in red. (B) REVEL scores for LRRK2 variants were acquired from Bryant et al. [41] or through the online pathogenicity prediction tool http://database.liulab.science/dbNSFP. REVEL scores were plotted against phospho-Rab10/total Rab10 ratios acquired for each LRRK2 variant that were normalized to wildtype. High activity variants (pRab10Thr73 > 1.5-fold relative to wildtype) are marked in green, low activity variants (similar to kinase-inactive LRRK2) are marked in red, protective variants are marked in yellow, and variants that block MLi-2 induced microtubule binding are represented with an open circle. The REVEL pathogenicity threshold is marked with a dashed line (0.6). Above this line are variants predicted to be ‘likely pathogenic or damaging,' and variants below this line are predicted to be ‘likely benign.' (C) LRRK2 orthologue sequences were acquired from OrthoDB. Orthologue sequences were aligned using MAFFT. The multiple sequence alignment of LRRK2 orthologues was submitted to the ConSurf server to determine evolutionary conservation scores for LRRK2 amino acids (1 is low conservation and 9 is high conservation). Conservation scores were plotted against pRab10Thr73/total Rab10 ratios acquired for each LRRK2 variant that were normalized to wildtype. Activating variants (pRab10Thr73 > 1.5-fold relative to wildtype) are marked in green, low activity variants (similar to kinase-inactive LRRK2) are marked in red, protective variants are marked in yellow, and variants that block MLi-2 induced microtubule binding are represented with an open circle.
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