Regulation of LRRK2 Expression Points to a Functional Role in Human Monocyte Maturation

Sagot YJ (2011) Regulation of LRRK2 Expression Points to a Functional Role in Human Monocyte Maturation. PLoS ONE 6(6): e21519. doi:10.1371/journal.pone.0021519 Regulation of LRRK2 Expression Points to a Functional Role in Human Monocyte Maturation Jonathan The venet 0 Rosanna Pescini Gobert 0 Robertus Hooft van Huijsduijnen 0 Christoph Wiessner 0 Yves Jean Sagot 0 Silvano Sozzani, University of Brescia, Italy 0 1 Pharmacology Parkinson's Disease, TA-NDD Research , Merck-Serono SA, Geneva , Switzerland , 2 Multiple Sclerosis Drug Discovery, TA-NDD Research , Merck-Serono SA, Geneva , Switzerland Genetic variants of Leucine-Rich Repeat Kinase 2 (LRRK2) are associated with a significantly enhanced risk for Parkinson disease, the second most common human neurodegenerative disorder. Despite major efforts, our understanding of LRRK2 biological function and regulation remains rudimentary. In the present study we analyze LRRK2 mRNA and protein expression in sub-populations of human peripheral blood mononuclear cells (PBMCs). LRRK2 mRNA and protein was found in circulating CD19+ B cells and in CD14+ monocytes, whereas CD4+ and CD8+ T cells were devoid of LRRK2 mRNA. Within CD14+ cells the CD14+CD16+ sub-population of monocytes exhibited high levels of LRRK2 protein, in contrast to CD14+CD16- cells. However both populations expressed LRRK2 mRNA. As CD14+CD16+ cells represent a more mature subset of monocytes, we monitored LRRK2 expression after in vitro treatment with various stress factors known to induce monocyte activation. We found that IFN-c in particular robustly increased LRRK2 mRNA and protein levels in monocytes concomitant with a shift of CD14+CD162 cells towards CD14+CD16+cells. Interestingly, the recently described LRRK2 inhibitor IN-1 attenuated this shift towards CD14+CD16+ after IFN-c stimulation. Based on these findings we speculate that LRRK2 might have a role in monocyte maturation. Our results provide further evidence for the emerging role of LRRK2 in immune cells and regulation at the transcriptional and translational level. Our data might also reflect an involvement of peripheral and brain immune cells in the disease course of PD, in line with increasing awareness of the role of the immune system in PD. - Competing Interests: All authors are employees of Merck Serono SA. This does not alter the authors adherence to all the PLoS ONE policies on sharing data and materials. Parkinsons disease (PD) is the second most common neurodegenerative disease affecting 1.5% of the population over 50 years [1]. Recent studies have linked several genes with PD [1], although the majority of PD cases is sporadic. Among associated genes, Leucine-Rich Repeat Kinase 2 (LRRK2 alias Dardarin) stands out since in some populations up to 30% of all PD patients carry the G2019S mutation [2]. LRRK2 is a large and complex 2,527 aminoacid protein that contains a ROC-COR domain with GTPase activity and a kinase domain with homology to MAPKKKs. Overall, biological functions of LRRK2 remain largely unknown, and the identification of physiological substrates remains controversial [3]. Nevertheless, there is consensus that LRRK2 multimerizes, auto-phosphorylates, and exists predominantly in a dimeric conformation when active [4]. Disease-associated mutations are localized in the ROC-COR and kinase domains, but not all result in modification of GTPase or kinase activities, leaving the pathogenic mechanism of such mutations unresolved [3]. It has been reported that the LRRK2 I2020T mutation is associated with enhanced intracellular degradation [5]. Studies performed in Caenorhabditis elegans or Drosophila, as well as recent studies using bacterial artificial chromosome (BAC) transgenic mice suggest that mutations in LRRK2 disturb the dopaminergic system, i.e. decrease dopamine release and cause motor deficits [67]. Since LRRK2 deficient [8] or LRRK2 wt over-expressing [9] transgenic mice do not present severe clinical neurological symptoms it seems likely that pathological mutations are not associated with a simple gain or loss of kinase or GTPase activity (for review [10]). In general, these studies rely on artificially over-expressing or knocking down LRRK2 expression. Since LRRK2 is thought to be a stress kinase [11], and therefore can be expected to be tightly regulated, we believe it is important to study its function and regulation at endogenous levels. Due to its pathophysiological role in PD, the major focus to date was to study LRRK2 function in the brain. However, mRNA analysis revealed that LRRK2 is also highly expressed in peripheral organs such as kidney, lung, spleen and peripheral blood mononuclear cells [1213]. The expression of LRRK2 in immune cells [1415] supports the idea that LRRK2 could play a role in B cell development. In contrast to neuronal cells, human PBMCs are easily accessible and present a valuable source for studying LRRK2 biology. The current work describes the characterization of a human PBMC sub-population expressing LRRK2 and the regulation and stabilization of LRRK2 by IFN-c at the mRNA and protein level. Finally, experiments using LRRK2 kinase inhibitors suggest that LRRK2 may play an important role in monocyte responses to IFN-c. From a clinical perspective our data suggest that hPBMC and more specifically monocytes derived from hPBMCs might yield biomarkers for therapeutic LRRK2 inhibitors. Materials and Methods Reagents Different inducers of cellular stress and cytokines were used in this study: recombinant Human IFN-c, TNF-a, IL-1b, IGF-1 (R&D Systems GmbH, Minneapolis, MN), LPS (from E. coli O111:B4, Sigma-Aldrich, St. Louis, MO), and H2O2 (Sigma-Aldrich, St. Louis, MO). Several LRRK2 inhibitors were used: H1152 (Toronto Research Chemicals Inc., Ontario Canada), Sunitinib (Sellek Chemicals, Texas, USA), K252a (Sigma-Aldrich, St. Louis, MO), Y27632 (Tocris Bioscience, Bristol, UK) and IN-1 (Generous gift from Dr. D. Alessi, College of Life Science, University of Dundee, Dundee, UK). Antibodies Three different antibodies against LRRK2 were used in this study. Rabbit polyclonal antibody to LRRK2 (ref. ab60937) was purchased from Abcam (Cambridge, UK), rabbit polyclonal antibody to LRRK2 (AT106) from Alexis Biochemicals (Enzo Life Sciences Inc., Plymouth Meeting, PA) and rabbit monoclonal antibody to LRRK2 (clone MJFF3-c69-6) from Epitomics Inc.(Burlingame, CA). Mouse monoclonal antibody anti-Actin (clone C4) was from Merck-Millipore (DE). Mouse monoclonal antibody anti-GAPDH (6C5) was from HyTest Ltd (Turku, FI). Mouse monoclonal antibody anti-Hsp70 (Hsp72, C92F3A-5) was from StressgenH (Enzo Life Sciences Inc., Plymouth Meeting, PA). For chemiluminescence Western blotting, goat polyclonal antibody anti-Rabbit IgG/HRP was from Bio-Rad Laboratories (Hercules, CA) and goat polyclonal antibody anti-Mouse IgG/ HRP from DakoCytomation (Carpinteria, CA). IRDyeH 680 donkey polyclonal antibody anti-mouse IgG and IRDyeH 800CW donkey polyclonal a (...truncated)