Suchergebnisse

Transcriptomic data of dermal cell fractions are deposited in NCBI's Gene Expression Omnibus (Edgar et al., 2002) under accession number GEO: GSE67693. ; The dermal Panniculus carnosus (PC) muscle is important for wound contraction in lower mammals and represents an interesting model of muscle regeneration due to its high cell turnover. The resident satellite cells (the bona fide muscle stem cells) remain poorly characterized. Here we analyzed PC satellite cells with regard to developmental origin and purported function. Lineage tracing shows that they originate in Myf5(+), Pax3/Pax7(+) cell populations. Skin and muscle wounding increased PC myofiber turnover, with the satellite cell progeny being involved in muscle regeneration but with no detectable contribution to the wound-bed myofibroblasts. Since hematopoietic stem cells fuse to PC myofibers in the absence of injury, we also studied the contribution of bone marrow-derived cells to the PC satellite cell compartment, demonstrating that cells of donor origin are capable of repopulating the PC muscle stem cell niche after irradiation and bone marrow transplantation but may not fully acquire the relevant myogenic commitment. ; We thank investigators for monoclonal antibodies A4.1025 (H.M. Blau) and F5D (W.E. Wright), which were obtained from the Developmental Studies Hybridoma Bank (developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA). Special thanks to G. Cossu for critical reading of the manuscript. We are also grateful to F. Costantini, C.-M. Fan, C. Lepper, M.J. Sánchez-Sanz, H. Sakai, and S. Tajbakhsh for kindly providing study materials; S. Lamarre of the GeT-Biochip facility for help in the microarray data; D. Ortiz de Urbina, J.C. Mazabuel, and A. Guisasola for help with irradiation protocol; and A. Aduriz, A. Pavón, and M. P. López-Mato for help with FACS analyses. This work was supported by grants from Instituto de Salud Carlos III (ISCIII; PS09/00660, PI13/02172, and PI14/7436), Gobierno Vasco (SAIO12-PE12BN008) from Spain and the European Union (POCTEFA-INTERREG IV A program; REFBIO13/BIOD/006 and REFBIO13/BIOD/009). N.N.G. received a studentship from the Department of Education, University and Research of the Basque Government (PRE2013-1-1168). P.G.P. received fellowships from the Department of Health of the Basque government (2013011016), EMBO (Short-Term; ASTF 542–2013), and Boehringer Ingelheim Fonds. M.L.M. and J.J.C. were supported by a Marie Curie Career Integration Grant from the European Commission (PEOPLE-CIG/1590). A.I. was supported by the Programa I3SNS (CES09/015) from ISCIII and by Osakidetza-Servicio Vasco de Salud (Spain). M.G. and S.A.M. contributed equally to this work. ; Peer-reviewed ; Publisher Version

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. ; 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.).