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01.12.2018 | Research article | Ausgabe 1/2018 Open Access

Molecular Neurodegeneration 1/2018

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

Zeitschrift:
Molecular Neurodegeneration > Ausgabe 1/2018
Autoren:
Jesús Madero-Pérez, Elena Fdez, Belén Fernández, Antonio J. Lara Ordóñez, Marian Blanca Ramírez, Patricia Gómez-Suaga, Dieter Waschbüsch, Evy Lobbestael, Veerle Baekelandt, Angus C. Nairn, Javier Ruiz-Martínez, Ana Aiastui, Adolfo López de Munain, Pawel Lis, Thomas Comptdaer, Jean-Marc Taymans, Marie-Christine Chartier-Harlin, Alexandria Beilina, Adriano Gonnelli, Mark R. Cookson, Elisa Greggio, Sabine Hilfiker
Wichtige Hinweise

Electronic supplementary material

The online version of this article (https://​doi.​org/​10.​1186/​s13024-018-0235-y) contains supplementary material, which is available to authorized users.

Abstract

Background

Mutations in LRRK2 are a common genetic cause of Parkinson’s disease (PD). LRRK2 interacts with and phosphorylates a subset of Rab proteins including Rab8a, a protein which has been implicated in various centrosome-related events. However, the cellular consequences of such phosphorylation remain elusive.

Methods

Human neuroblastoma SH-SY5Y cells stably expressing wildtype or pathogenic LRRK2 were used to test for polarity defects in the context of centrosomal positioning. Centrosomal cohesion deficits were analyzed from transiently transfected HEK293T cells, as well as from two distinct peripheral cell types derived from LRRK2-PD patients. Kinase assays, coimmunoprecipitation and GTP binding/retention assays were used to address Rab8a phosphorylation by LRRK2 and its effects in vitro. Transient transfections and siRNA experiments were performed to probe for the implication of Rab8a and its phosphorylated form in the centrosomal deficits caused by pathogenic LRRK2.

Results

Here, we show that pathogenic LRRK2 causes deficits in centrosomal positioning with effects on neurite outgrowth, cell polarization and directed migration. Pathogenic LRRK2 also causes deficits in centrosome cohesion which can be detected in peripheral cells derived from LRRK2-PD patients as compared to healthy controls, and which are reversed upon LRRK2 kinase inhibition. The centrosomal cohesion and polarity deficits can be mimicked when co-expressing wildtype LRRK2 with wildtype but not phospho-deficient Rab8a. The centrosomal defects induced by pathogenic LRRK2 are associated with a kinase activity-dependent increase in the centrosomal localization of phosphorylated Rab8a, and are prominently reduced upon RNAi of Rab8a.

Conclusions

Our findings reveal a new function of LRRK2 mediated by Rab8a phosphorylation and related to various centrosomal defects.
Zusatzmaterial
Additional file 1: Figure S1. Distinct pathogenic LRRK2 mutants cause deficits in centrosome cohesion in transfected HEK293T cells. (DOCX 1160 kb)
13024_2018_235_MOESM1_ESM.docx
Additional file 2: Figure S2. Pathogenic LRRK2 disturbs centrosome cohesion in a kinase-dependent manner. (DOCX 1155 kb)
13024_2018_235_MOESM2_ESM.docx
Additional file 3: Figure S3. LRRK2 phosphorylates Rab8a at T72, and phosphomimetic mutants do not display altered nucleotide binding or retention. (DOCX 1005 kb)
13024_2018_235_MOESM3_ESM.docx
Additional file 4: Figure S4. Differential interactions of wildtype and phospho-mimetic Rab8a mutants with GDI1/2 and Rabin8, effects on centrosome splitting and subcellular localization. (DOCX 824 kb)
13024_2018_235_MOESM4_ESM.docx
Additional file 5: Figure S5. Golgi dispersal/disruption has no effect on LRRK2-mediated pericentrosomal/centrosomal accumulation of Rab8a. (DOCX 1670 kb)
13024_2018_235_MOESM5_ESM.docx
Additional file 6: Figure S6. Rab8a protein levels and pericentrosomal/centrosomal accumulation of phosphorylated Rab8a in lymphoblasts from control and G2019S mutant LRRK2 PD patients. (DOCX 636 kb)
13024_2018_235_MOESM6_ESM.docx
Additional file 7: Figure S7. Detection of phospho-Rab8a in pathogenic LRRK2-expressing cells as well as in cells co-transfected with wildtype LRRK2 and wildtype Rab8a, but not phospho-deficient Rab8a. (DOCX 958 kb)
13024_2018_235_MOESM7_ESM.docx
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