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BackgroundLRRK2 mutations and risk variants increase susceptibility to inherited and idiopathic Parkinson’s disease, while recent studies have identified potential protective variants. This, and the fact that LRRK2 mutation carriers develop symptoms and brain pathology almost indistinguishable from idiopathic Parkinson’s disease, has led to enormous interest in this protein. LRRK2 has been implicated in a range of cellular events, but key among them is canonical Wnt signalling, which results in increased levels of transcriptionally active β-catenin. This pathway is critical for the development and survival of the midbrain dopaminergic neurones typically lost in Parkinson’s disease.\ud MethodsHere we use Lrrk2 knockout mice and fibroblasts to investigate the effect of loss of Lrrk2 on canonical Wnt signalling in vitro and in vivo. Micro-computed tomography was used to study predicted tibial strength, while pulldown assays were employed to measure brain β-catenin levels. A combination of luciferase assays, immunofluorescence and co-immunoprecipitation were performed to measure canonical Wnt activity and investigate the relationship between LRRK2 and β-catenin. TOPflash assays are also used to study the effects of LRRK2 kinase inhibition and pathogenic and protective LRRK2 mutations on Wnt signalling. Data were tested by Analysis of Variance.\ud \ud ResultsLoss of Lrrk2 causes a dose-dependent increase in the levels of transcriptionally active β-catenin in the brain, and alters tibial bone architecture, decreasing the predicted risk of fracture. Lrrk2 knockout cells display increased TOPflash and Axin2 promoter activities, both basally and following Wnt activation. Consistently, over-expressed LRRK2 was found to bind β-catenin and repress TOPflash activation. Some pathogenic LRRK2 mutations and risk variants further suppressed TOPflash, whereas the protective R1398H variant increased Wnt signalling activity. LRRK2 kinase inhibitors affected canonical Wnt signalling differently due to off-targeting; however, specific LRRK2 inhibition reduced canonical Wnt signalling similarly to pathogenic mutations.\ud \ud ConclusionsLoss of LRRK2 causes increased canonical Wnt activity in vitro and in vivo. In agreement, over-expressed LRRK2 binds and represses β-catenin, suggesting LRRK2 may act as part of the β-catenin destruction complex. Since some pathogenic LRRK2 mutations enhance this effect while the protective R1398H variant relieves it, our data strengthen the notion that decreased canonical Wnt activity is central to Parkinson’s disease pathogenesis.

Increased canonical Wnt activity in Lrrk2 knockout cells. A, B) Cells were transfected with TK-renilla and TOPflash or FOPflash in the presence of A) FLAG-DVl1 or C) GFP-FZD5 and HA-LRP6, or appropriate vector controls. A) 1-way ANOVA (n = 9; F = 146.199, p

Association of mycLRRK2 and FLAG-β-catenin in HEK293 cells 24 h post transfection, cells expressing myc-tagged LRRK2 and FLAG-tagged β-catenin were treated with MG132 for 1 h to allow β-catenin accumulation. The cells were subsequently fixed and stained with antibodies for A) myc (green) and FLAG (red), or B) myc (green) and β-catenin (red). DAPI staining (blue) was also performed to show cell nuclei. The right-hand panel shows an overlay of all channels – note the considerable co-localisation between LRRK2 and β-catenin in the cytoplasm. (PDF 102 kb)

Effects of loss of Lrrk2 on tibial cortical Zmax and Zmin. Images in Figures A and B show i) values for Zmax and Zmin, predicted resistance to fracture along the shortest and longest cross-sectional axes, respectively in female wild-type (WT) and Lrrk2 knockout (KO) mice; together with ii) the same data expressed as a graphical heat map along the tibial length; and iii) the points at which differences between genotypes for these parameters become statistically significant. (blue = n/s, yellow, p

Effects of loss of Lrrk2 on tibial cortical Cross Sectional Area, Mean Cortical Thickness, and Ellipticity. Images in Figures A, B and C show i) values for cross-sectional area, mean cortical thickness and ellipticity, respectively in female wild-type (WT) and Lrrk2 knockout (KO) mice; together with ii) the same data expressed as a graphical heat map along the tibial length; and iii) the points at which differences between genotypes for these parameters become statistically significant. (blue = n/s, yellow, p

LRRK2 interacts with β-catenin and represses β-catenin-driven TOPflash activity. A) HEK293 cells were co-transfected with FLAG-tagged β-catenin (lanes 1 and 4), myc-tagged LRRK2 (lanes 2 and 5), or myc-tagged LRRK2 and FLAG-tagged β-catenin (lanes 3 and 6) for 24 h prior to lysis. Lysates were immunoprecipitated with anti-FLAG antibodies and bound protein was resolved by Western blot (lanes 4–6), with the original lysates run alongside to confirm expression of transfected protein (lanes 1–3). Myc-tagged LRRK2 can clearly be seen in anti-FLAG immunoprecipitates from co-transfected cells (lane 6, upper panel). Full images of all blots are also shown. B) HEK293 cells were transfected with the TOPflash or FOPflash reporter plasmids with the indicated combinations of mycLRRK2, FLAG-β-catenin or appropriate control vectors. After 24 h lysates were taken and luciferase activity measured. 1-way ANOVA of TOPflash values revealed a significant effect of transfection on canonical Wnt activity (n = 9, F = 44.893, p

Entire images of Western Blots shown in Fig. 4D and E and total β-catenin levels in aged female mouse brains A) Full blot from the image shown in Fig. 4d, plus a reprobe of the same membrane for calnexin to show that pulldowns are clean and this protein is only present in cell lysates. B) Western blotting shows no discernible differences between levels of total β-catenin in brains from aged female mice. C) Full blot from the image shown in Fig. 4e. (PDF 102 kb)

Co-localisation between mycLRRK2 and HA-AXIN1, and mycLRRK2 and HA-GSK3β in HEK293 cells. A) Recruitment of mycLRRK2 (green) into polymers formed by HA-AXIN1 (red). Magnified images of a selected region are included. B) shows cytoplasmic co-localisation between mycLRRK2 (green) and HA-GSK3β (red), also with magnified images included. Note that in both experiments, counterstaining with phalloidin (blue) and DAPI (grey) were performed to visualise filamentous actin and chromosomal DNA respectively. The scale bar = 10 μm. (PDF 92 kb)

Entire images of Western Blots shown in Fig. 4a. (PDF 70 kb)

Entire images of Western Blots shown in Fig. 5b. (PDF 80 kb)

Background: Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) cause Parkinson disease. Results: LRRK2 binds directly to three β-tubulin isoforms at the luminal face of microtubules and suppresses α-tubulin acetylation. Interaction is weakened by the R1441G LRRK2 GTPase domain mutant. Conclusion: LRRK2 modulates microtubule stability. Significance: Deregulation of microtubule-dependent processes likely contribute to neurodegeneration in Parkinson disease., Mutations in LRRK2, encoding the multifunctional protein leucine-rich repeat kinase 2 (LRRK2), are a common cause of Parkinson disease. LRRK2 has been suggested to influence the cytoskeleton as LRRK2 mutants reduce neurite outgrowth and cause an accumulation of hyperphosphorylated Tau. This might cause alterations in the dynamic instability of microtubules suggested to contribute to the pathogenesis of Parkinson disease. Here, we describe a direct interaction between LRRK2 and β-tubulin. This interaction is conferred by the LRRK2 Roc domain and is disrupted by the familial R1441G mutation and artificial Roc domain mutations that mimic autophosphorylation. LRRK2 selectively interacts with three β-tubulin isoforms: TUBB, TUBB4, and TUBB6, one of which (TUBB4) is mutated in the movement disorder dystonia type 4 (DYT4). Binding specificity is determined by lysine 362 and alanine 364 of β-tubulin. Molecular modeling was used to map the interaction surface to the luminal face of microtubule protofibrils in close proximity to the lysine 40 acetylation site in α-tubulin. This location is predicted to be poorly accessible within mature stabilized microtubules, but exposed in dynamic microtubule populations. Consistent with this finding, endogenous LRRK2 displays a preferential localization to dynamic microtubules within growth cones, rather than adjacent axonal microtubule bundles. This interaction is functionally relevant to microtubule dynamics, as mouse embryonic fibroblasts derived from LRRK2 knock-out mice display increased microtubule acetylation. Taken together, our data shed light on the nature of the LRRK2-tubulin interaction, and indicate that alterations in microtubule stability caused by changes in LRRK2 might contribute to the pathogenesis of Parkinson disease.

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/β-catenin pathway in the hippocampus of adult DS individuals with Alzheimer's disease and the 'Tc1' DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/β-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/β-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer's disease treatment.

Pathological mechanisms underlying Down syndrome (DS)/Trisomy 21, including dysregulation of essential signalling processes remain poorly understood. Combining bioinformatics with RNA and protein analysis, we identified downregulation of the Wnt/β-catenin pathway in the hippocampus of adult DS individuals with Alzheimer's disease and the 'Tc1' DS mouse model. Providing a potential underlying molecular pathway, we demonstrate that the chromosome 21 kinase DYRK1A regulates Wnt signalling via a novel bimodal mechanism. Under basal conditions, DYRK1A is a negative regulator of Wnt/β-catenin. Following pathway activation, however, DYRK1A exerts the opposite effect, increasing signalling activity. In summary, we identified downregulation of hippocampal Wnt/β-catenin signalling in DS, possibly mediated by a dose dependent effect of the chromosome 21-encoded kinase DYRK1A. Overall, we propose that dosage imbalance of the Hsa21 gene DYRK1A affects downstream Wnt target genes. Therefore, modulation of Wnt signalling may open unexplored avenues for DS and Alzheimer's disease treatment.

Kinase-dead but not GTP-non-binding mutations weaken basal Wnt signalling. Lrrk2 knockout (KO) cells were transfected with TOPflash or FOPflash plus wild-type LRRK2 or the indicated LRRK2 mutant. 1-way ANOVA (n = 12–15, F = 7.449, p

Entire images of Western Blots shown in Fig. 4D and E and total β-catenin levels in aged female mouse brains A) Full blot from the image shown in Fig. 4d, plus a reprobe of the same membrane for calnexin to show that pulldowns are clean and this protein is only present in cell lysates. B) Western blotting shows no discernible differences between levels of total β-catenin in brains from aged female mice. C) Full blot from the image shown in Fig. 4e. (PDF 102 kb)