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

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. CooksonElisa Greggio, Sabine Hilfiker

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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.

Original languageEnglish
Article number3
Pages (from-to)1-22
Number of pages22
JournalMolecular Neurodegeneration
Volume13
Issue number1
DOIs
Publication statusPublished - 23 Jan 2018

Fingerprint

Parkinson Disease
Phosphorylation
Mutation
Centrosome
Phosphotransferases
RNA Interference
Guanosine Triphosphate
Neuroblastoma
Small Interfering RNA
Transfection
Proteins

Keywords

  • Centrosome
  • LRRK2
  • Parkinson's disease
  • Phosphorylation
  • Rab8a

Cite this

Madero-Pérez, J., Fdez, E., Fernández, B., Lara Ordóñez, A. J., Blanca Ramírez, M., Gómez-Suaga, P., ... Hilfiker, S. (2018). Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. Molecular Neurodegeneration, 13(1), 1-22. [3]. https://doi.org/10.1186/s13024-018-0235-y
Madero-Pérez, Jesús ; Fdez, Elena ; Fernández, Belén ; Lara Ordóñez, Antonio J. ; Blanca Ramírez, Marian ; Gómez-Suaga, Patricia ; Waschbüsch, Dieter ; Lobbestael, Evy ; Baekelandt, Veerle ; Nairn, Angus C. ; Ruiz-Martínez, Javier ; Aiastui, Ana ; López de Munain, Adolfo ; Lis, Pawel ; Comptdaer, Thomas ; Taymans, Jean-Marc ; Chartier-Harlin, Marie-Christine ; Beilina, Alexandria ; Gonnelli, Adriano ; Cookson, Mark R. ; Greggio, Elisa ; Hilfiker, Sabine. / Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. In: Molecular Neurodegeneration. 2018 ; Vol. 13, No. 1. pp. 1-22.
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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.",
keywords = "Centrosome, LRRK2, Parkinson's disease, Phosphorylation, Rab8a",
author = "Jes{\'u}s Madero-P{\'e}rez and Elena Fdez and Bel{\'e}n Fern{\'a}ndez and {Lara Ord{\'o}{\~n}ez}, {Antonio J.} and {Blanca Ram{\'i}rez}, Marian and Patricia G{\'o}mez-Suaga and Dieter Waschb{\"u}sch and Evy Lobbestael and Veerle Baekelandt and Nairn, {Angus C.} and Javier Ruiz-Mart{\'i}nez and Ana Aiastui and {L{\'o}pez de Munain}, Adolfo and Pawel Lis and Thomas Comptdaer and Jean-Marc Taymans and Marie-Christine Chartier-Harlin and Alexandria Beilina and Adriano Gonnelli and Cookson, {Mark R.} and Elisa Greggio and Sabine Hilfiker",
note = "S.H. was supported by the Michael J. Fox Foundation, the BBVA Foundation, FEDER, and the Spanish Ministry of Economy and Competitiveness (SAF2014–58653-R). E.G. was funded by the Michael J. Fox Foundation. J-M.T. and M.-C. C.-H. were funded by the Michael J. Fox Foundation, and M.-C.C.-H. was supported by Inserm, CHU de Lille, Lille University and the Minist{\`e}re de la Recherche et de la Sant{\'e} (PHRC Convergence). We gratefully acknowledge funding from the European Union’s Horizon 2020 research and innovation programme (Marie Sklodowska-Curie Action Individual Fellowship to J.-M.T.). A.C.N. was supported by the Dept. of the Army (USAMRAA W23RYX-9049-N610) and NIH (DA10044). This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (to M.R.C.).",
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Madero-Pérez, J, Fdez, E, Fernández, B, Lara Ordóñez, AJ, Blanca Ramírez, M, Gómez-Suaga, P, Waschbüsch, D, Lobbestael, E, Baekelandt, V, Nairn, AC, Ruiz-Martínez, J, Aiastui, A, López de Munain, A, Lis, P, Comptdaer, T, Taymans, J-M, Chartier-Harlin, M-C, Beilina, A, Gonnelli, A, Cookson, MR, Greggio, E & Hilfiker, S 2018, 'Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation', Molecular Neurodegeneration, vol. 13, no. 1, 3, pp. 1-22. https://doi.org/10.1186/s13024-018-0235-y

Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. / Madero-Pérez, Jesús; Fdez, Elena; Fernández, Belén; Lara Ordóñez, Antonio J.; Blanca Ramírez, Marian; Gómez-Suaga, Patricia; Waschbüsch, Dieter; Lobbestael, Evy; Baekelandt, Veerle; Nairn, Angus C.; Ruiz-Martínez, Javier; Aiastui, Ana; López de Munain, Adolfo; Lis, Pawel; Comptdaer, Thomas; Taymans, Jean-Marc; Chartier-Harlin, Marie-Christine; Beilina, Alexandria; Gonnelli, Adriano; Cookson, Mark R.; Greggio, Elisa; Hilfiker, Sabine (Lead / Corresponding author).

In: Molecular Neurodegeneration, Vol. 13, No. 1, 3, 23.01.2018, p. 1-22.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

AU - Madero-Pérez, Jesús

AU - Fdez, Elena

AU - Fernández, Belén

AU - Lara Ordóñez, Antonio J.

AU - Blanca Ramírez, Marian

AU - Gómez-Suaga, Patricia

AU - Waschbüsch, Dieter

AU - Lobbestael, Evy

AU - Baekelandt, Veerle

AU - Nairn, Angus C.

AU - Ruiz-Martínez, Javier

AU - Aiastui, Ana

AU - López de Munain, Adolfo

AU - Lis, Pawel

AU - Comptdaer, Thomas

AU - Taymans, Jean-Marc

AU - Chartier-Harlin, Marie-Christine

AU - Beilina, Alexandria

AU - Gonnelli, Adriano

AU - Cookson, Mark R.

AU - Greggio, Elisa

AU - Hilfiker, Sabine

N1 - S.H. was supported by the Michael J. Fox Foundation, the BBVA Foundation, FEDER, and the Spanish Ministry of Economy and Competitiveness (SAF2014–58653-R). E.G. was funded by the Michael J. Fox Foundation. J-M.T. and M.-C. C.-H. were funded by the Michael J. Fox Foundation, and M.-C.C.-H. was supported by Inserm, CHU de Lille, Lille University and the Ministère de la Recherche et de la Santé (PHRC Convergence). We gratefully acknowledge funding from the European Union’s Horizon 2020 research and innovation programme (Marie Sklodowska-Curie Action Individual Fellowship to J.-M.T.). A.C.N. was supported by the Dept. of the Army (USAMRAA W23RYX-9049-N610) and NIH (DA10044). This research was supported in part by the Intramural Research Program of the NIH, National Institute on Aging (to M.R.C.).

PY - 2018/1/23

Y1 - 2018/1/23

N2 - 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.

AB - 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.

KW - Centrosome

KW - LRRK2

KW - Parkinson's disease

KW - Phosphorylation

KW - Rab8a

U2 - 10.1186/s13024-018-0235-y

DO - 10.1186/s13024-018-0235-y

M3 - Article

C2 - 29357897

VL - 13

SP - 1

EP - 22

JO - Molecular Neurodegeneration

JF - Molecular Neurodegeneration

SN - 1750-1326

IS - 1

M1 - 3

ER -

Madero-Pérez J, Fdez E, Fernández B, Lara Ordóñez AJ, Blanca Ramírez M, Gómez-Suaga P et al. Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation. Molecular Neurodegeneration. 2018 Jan 23;13(1):1-22. 3. https://doi.org/10.1186/s13024-018-0235-y