The Parkinson's disease–associated kinase LRRK2 regulates genes required for cell adhesion, polarization, and chemotaxis in activated murine macrophages
2020; Elsevier BV; Volume: 295; Issue: 31 Linguagem: Inglês
10.1074/jbc.ra119.011842
ISSN1083-351X
AutoresDaniel Robert Siegfried Levy, Atul Udgata, Panagiotis Tourlomousis, Martyn F. Symmons, Lee Hopkins, Clare Bryant, Nicholas J. Gay,
Tópico(s)Nuclear Receptors and Signaling
ResumoLeucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq–based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis. Leucine-rich repeat kinase 2 (LRRK2) encodes a complex protein that includes kinase and GTPase domains. Genome-wide association studies have identified dominant LRRK2 alleles that predispose their carriers to late-onset idiotypic Parkinson's disease (PD) and also to autoimmune disorders such as Crohn's disease. Considerable evidence indicates that PD initiation and progression involve activation of innate immune functions in microglia, which are brain-resident macrophages. Here we asked whether LRRK2 modifies inflammatory signaling and how this modification might contribute to PD and Crohn's disease. We used RNA-Seq–based high-resolution transcriptomics to compare gene expression in activated primary macrophages derived from WT and Lrrk2 knockout mice. Remarkably, expression of a single gene, Rap guanine nucleotide exchange factor 3 (Rapgef3), was strongly up-regulated in the absence of LRRK2 and down-regulated in its presence. We observed similar regulation of Rapgef3 expression in cells treated with a highly specific inhibitor of LRRK2 protein kinase activity. Rapgef3 encodes an exchange protein, activated by cAMP 1 (EPAC-1), a guanine nucleotide exchange factor that activates the small GTPase Rap-1. Rap-1 mediates cell adhesion, polarization, and directional motility, and our results indicate that LRRK2 modulates chemotaxis of microglia and macrophages. Dominant PD-associated LRRK2 alleles may suppress EPAC-1 activity, further restricting motility and preventing efficient migration of microglia to sites of neuronal damage. Functional analysis in vivo in a subclinical infection model also indicated that Lrrk2 subtly modifies the inflammatory response. These results indicate that LRRK2 modulates the expression of genes involved in murine immune cell chemotaxis. LRRK2 is a large protein of 286 kDa consisting of a complex and unique arrangement of protein–protein interaction and functional domains. This arrangement consists of N-terminal repeats, including ankyrin repeats, a leucine-rich repeat (LRR) 3The abbreviations used are: LRRleucine-rich repeatPDParkinson's diseaseTLRToll-like receptorLPSlipopolysaccharideMDPmuramyl dipeptideqRT-PCRquantitative RT-PCRqPCRquantitative PCRGEFguanine nucleotide exchange factorpBMDMprimary bone marrow–derived macrophageDAPI4′,6-diamidino-2-phenylindole. 3The abbreviations used are: LRRleucine-rich repeatPDParkinson's diseaseTLRToll-like receptorLPSlipopolysaccharideMDPmuramyl dipeptideqRT-PCRquantitative RT-PCRqPCRquantitative PCRGEFguanine nucleotide exchange factorpBMDMprimary bone marrow–derived macrophageDAPI4′,6-diamidino-2-phenylindole. domain, a Ras of complex proteins (Roc) GTPase with an associated C-terminal of Roc domain, a Ser/Thr protein kinase, and a WD40 domain at the C terminus of the protein (1Mills R.D. Mulhern T.D. Liu F. Culvenor J.G. Cheng H.C. Prediction of the repeat domain structures and impact of parkinsonism-associated variations on structure and function of all functional domains of leucine-rich repeat kinase 2 (LRRK2).Hum. Mutat. 2014; 35 (24470158): 395-41210.1002/humu.22515Crossref PubMed Scopus (25) Google Scholar). The presence of a Roc-C–terminal of Roc tandem domain defines LRRK2 as a member of the Roco protein family, a family first detected in the slime mold Dictyostelium discoideum (2Marín I. van Egmond W.N. van Haastert P.J. The Roco protein family: a functional perspective.FASEB J. 2008; 22 (18523161): 3103-311010.1096/fj.08-111310Crossref PubMed Scopus (97) Google Scholar, 3Russo I. Berti G. Plotegher N. Bernardo G. Filograna R. Bubacco L. Greggio E. Leucine-rich repeat kinase 2 positively regulates inflammation and down-regulates NF-κB p50 signaling in cultured microglia cells.J. Neuroinflammation. 2015; 12 (26646749): 23010.1186/s12974-015-0449-7Crossref PubMed Scopus (82) Google Scholar). leucine-rich repeat Parkinson's disease Toll-like receptor lipopolysaccharide muramyl dipeptide quantitative RT-PCR quantitative PCR guanine nucleotide exchange factor primary bone marrow–derived macrophage 4′,6-diamidino-2-phenylindole. leucine-rich repeat Parkinson's disease Toll-like receptor lipopolysaccharide muramyl dipeptide quantitative RT-PCR quantitative PCR guanine nucleotide exchange factor primary bone marrow–derived macrophage 4′,6-diamidino-2-phenylindole. There is great interest in all aspects of LRRK2 biology because genome-wide association studies have identified many variants in this protein that predispose to late-onset Parkinson's disease. The most common mutation in the LRRK2 gene results in a change from glycine to serine at amino acid 2019 (G2019S). This SNP is the highest known risk factor for development of Parkinson's disease, accounting for 5%–7% of autosomal dominant familial cases (4Di Fonzo A. Rohé C.F. Ferreira J. Chien H.F. Vacca L. Stocchi F. Guedes L. Fabrizio E. Manfredi M. Vanacore N. Goldwurm S. Breedveld G. Sampaio C. Meco G. Barbosa E. et al.A frequent LRRK2 gene mutation associated with autosomal dominant Parkinson's disease.Lancet. 2005; 365 (15680456): 412-41510.1016/S0140-6736(05)17829-5Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 5Nichols W.C. Pankratz N. Hernandez D. Paisán-Ruíz C. Jain S. Halter C.A. Michaels V.E. Reed T. Rudolph A. Shults C.W. Singleton A. Foroud T. Parkinson Study Group-PROGENI Investigators Genetic screening for a single common LRRK2 mutation in familial Parkinson's disease.Lancet. 2005; 365 (15680455): 410-41210.1016/S0140-6736(05)17828-3Abstract Full Text Full Text PDF PubMed Scopus (400) Google Scholar) and 1%–2% of sporadic cases in Western populations (6Gilks W.P. Abou-Sleiman P.M. Gandhi S. Jain S. Singleton A. Lees A.J. Shaw K. Bhatia K.P. Bonifati V. Quinn N.P. Lynch J. Healy D.G. Holton J.L. Revesz T. Wood N.W. A common LRRK2 mutation in idiopathic Parkinson's disease.Lancet. 2005; 365 (15680457): 415-41610.1016/S0140-6736(05)17830-1Abstract Full Text Full Text PDF PubMed Scopus (631) Google Scholar). Genome-wide association studies have also revealed a link with Crohn's disease and leprosy. Genetic links with these diseases demonstrate a nonneuronal but clearly innate immune component to LRRK2 biology (7Greggio E. Civiero L. Bisaglia M. Bubacco L. Parkinson's disease and immune system: is the culprit LRRKing in the periphery?.J. Neuroinflammation. 2012; 9 (22594666): 94Crossref PubMed Scopus (30) Google Scholar). LRRK2 expression is enriched in macrophages, B cells, and dendritic cells (8Gardet A. Benita Y. Li C. Sands B.E. Ballester I. Stevens C. Korzenik J.R. Rioux J.D. Daly M.J. Xavier R.J. Podolsky D.K. LRRK2 is involved in the IFN-γ response and host response to pathogens.J. Immunol. 2010; 185 (20921534): 5577-558510.4049/jimmunol.1000548Crossref PubMed Scopus (280) Google Scholar). Innate immune stimuli, such as IFN-γ, stimulate LRRK2 expression, revealing responsiveness to activation of innate immune signaling pathways mediated by pattern recognition receptors (9Thévenet J. Pescini Gobert R. Hooft van Huijsduijnen R. Wiessner C. Sagot Y.J. Regulation of LRRK2 expression points to a functional role in human monocyte maturation.PLoS ONE. 2011; 6 (21738687): e2151910.1371/journal.pone.0021519Crossref PubMed Scopus (130) Google Scholar). Furthermore, activation of Toll-like receptors (TLRs) by pathogen-associated molecules such as bacterial lipopolysaccharide (LPS) leads to phosphorylation of LRRK2 by IκB kinase at two serine residues (Ser-910 and Ser-935) (10Dzamko N. Inesta-Vaquera F. Zhang J. Xie C. Cai H. Arthur S. Tan L. Choi H. Gray N. Cohen P. Pedrioli P. Clark K. Alessi D.R. The IκB kinase family phosphorylates the Parkinson's disease kinase LRRK2 at Ser935 and Ser910 during Toll-like receptor signaling.PLoS ONE. 2012; 7 (22723946): e3913210.1371/journal.pone.0039132Crossref PubMed Scopus (158) Google Scholar). The IκB kinase family is normally associated with phosphorylation of IκB proteins that sequester NF-κB in the cytoplasm. Phosphorylation and ubiquitination of IκB proteins leads to proteolysis and subsequent transfer of NF-κB into the nucleus (11Karin M. How NF-κB is activated: the role of the IκB kinase (IKK) complex.Oncogene. 1999; 18 (10602462): 6867-687410.1038/sj.onc.1203219Crossref PubMed Scopus (1006) Google Scholar). LRRK2 phosphorylation depends on the TLR adaptor (Myd88), an innate immune signal transducer that mediates signaling from cell-surface TLRs, as well as TLR7, TLR8, and TLR9, which signal from the endosomal compartment. The role of inflammatory processes in the etiology of Parkinson's disease is further illustrated by administration of LPS systemically and directly into the substantia nigra. In the latter case, LPS causes irreversible degeneration of dopaminergic neurons of the pars compacta, observed a week after injection (12Castaño A. Herrera A.J. Cano J. Machado A. The degenerative effect of a single intranigral injection of LPS on the dopaminergic system is prevented by dexamethasone, and not mimicked by rh-TNF-α, IL-1β and IFN-γ.J. Neurochem. 2002; 81 (12067227): 150-15710.1046/j.1471-4159.2002.00799.xCrossref PubMed Scopus (212) Google Scholar, 13Iravani M.M. Leung C.C. Sadeghian M. Haddon C.O. Rose S. Jenner P. The acute and the long-term effects of nigral lipopolysaccharide administration on dopaminergic dysfunction and glial cell activation.Eur. J. Neurosci. 2005; 22 (16045485): 317-33010.1111/j.1460-9568.2005.04220.xCrossref PubMed Scopus (101) Google Scholar). Notably nondopaminergic neurons of the nigrostriatal system, as well as proximal dopaminergic neurons not associated with the nigrostriatal pathway, remain unaffected by direct LPS injection. Therefore, LPS injection and the resulting inflammatory insult demonstrate remarkable sensitivity and specificity to the dopaminergic circuitry associated with Parkinson's disease. In another study, the same pattern of Parkinson's disease–like microglial activation, followed by neurodegeneration over 10 months, was observed when LPS or tumor necrosis factor α were administered systemically in mice via intraperitoneal injection (14Qin L. Wu X. Block M.L. Liu Y. Breese G.R. Hong J.S. Knapp D.J. Crews F.T. Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration.Glia. 2007; 55 (17203472): 453-46210.1002/glia.20467Crossref PubMed Scopus (1555) Google Scholar). In humans, a laboratory worker accidentally exposed to Salmonella-derived LPS developed many symptoms of Parkinson's disease, including bradykinesia, rigidity, and tremor at rest, as well as other neurological problems resulting from damage to the substantia nigra as well as the cerebral cortex (15Niehaus I. Lange J.H. Endotoxin: is it an environmental factor in the cause of Parkinson's disease?.Occup. Environ. Med. 2003; 60 (12709528): 37810.1136/oem.60.5.378Crossref PubMed Scopus (44) Google Scholar). Human parkinsonism has been further linked to immune activation through neurotrophic viral infection and, in particular, infection by the human influenza virus. Individual cases of viral infection leading to neuropathology and death have been reported, as well as increased incidence of Parkinson's disease following pandemic flu, such as experienced in 1918 (16Jang H. Boltz D.A. Webster R.G. Smeyne R.J. Viral parkinsonism.Biochim. Biophys. Acta. 2009; 1792 (18760350): 714-72110.1016/j.bbadis.2008.08.001Crossref PubMed Scopus (215) Google Scholar). At present, little is known about how Lrrk2 modifies the gene expression program induced by innate signaling pathways and how this might contribute to Parkinson's disease initiation and progression. In this study, we used high-resolution transcriptomics to compare gene expression in primary macrophages derived from WT and Lrrk2-deficient mice. This analysis reveals that LRRK2 modulates the expression of a small subset of genes that are involved in chemotaxis and membrane remodeling. We used RNA-Seq to determine how LRRK2 modifies gene expression in primary bone marrow–derived macrophages from WT and Lrrk2−/− mice. If valid comparisons are to be made, then it is necessary to confirm the similarity in the nature and purity of macrophage cultures before RNA extraction and RNA-Seq. One day prior to RNA extraction, a portion of differentiated macrophages was prepared for flow cytometry analysis and stained for various cell surface markers: CD11b for cells of the myeloid lineage, F4/80 for mouse macrophages, and CD11c for monocyte-derived cells, including macrophages (17Murray P.J. Wynn T.A. Protective and pathogenic functions of macrophage subsets.Nat. Rev. Immunol. 2011; 11 (21997792): 723-73710.1038/nri3073Crossref PubMed Scopus (3349) Google Scholar). These markers revealed no significant differences in the differentiation state of the cells, with uniform expression of CD11b and highly similar expression levels of F4/80 and CD11c. CD11c surface expression in Lrrk2 KO macrophages displayed a slightly higher level of variability between cultures than equivalent WT cells (Fig. S1). Overall, cultures were considered similar enough to proceed with differential gene expression analysis. We then treated WT and Lrrk2−/− macrophages with either LPS or muramyl dipeptide (MDP), activators of the TLR4- and NOD2-mediated innate responses, respectively. After 2 h of stimulation, RNA was extracted for mRNA sequencing. A mean read depth of over 22.2 × 106 reads/sample was achieved with a range of 16.0 × 106 to 24.9 × 106 reads/sample (Table S1). Reads were of high quality, requiring a mean of less than 0.1% of reads to be trimmed during quality control. A mean of 87.5% of reads could be unambiguously mapped to gene-encoding regions of the genome. Therefore, by comparing the frequency of reads per gene between samples, relative levels of expression could be determined. These datasets were then analyzed for differential gene expression. Datasets of mapped counts were interrogated for differences in each Lrrk2 genotype upon innate immune stimulation as well as for underlying differences between genotypes in unstimulated cells (Fig. 1a). DEseq2 (18Love M.I. Huber W. Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol. 2014; 15 (25516281): 55010.1186/s13059-014-0550-8Crossref PubMed Scopus (32857) Google Scholar) determines a statistical model accounting for variance in counts per gene and base mean of counts, allowing the statistical significance of apparent differences in gene expression to be estimated. This analysis revealed that LPS treatment caused differential expression of 4985 and 5354 genes in WT and Lrrk2−/− macrophages, respectively. In contrast, MDP treatment resulted in 1483 significantly differentially expressed genes in WT macrophages compared with 1478 genes in Lrrk2 KO macrophages (Fig. 1b). In the absence of stimulation, only eight genes were significantly differentially expressed (Table 1). One of these is Lrrk2, a result confirmed by qPCR. The lesion in the Lrrk2 KO mouse deletes part of exon 1 and exon 2, leading to termination at an out-of-frame stop codon in exon 3. As the Lrrk2 transcription unit has 51 exons, this should lead to nonsense-mediated decay of the transcript. The level of transcript measured is about 50% of the WT, which indicates that nonsense-mediated decay is inefficient in this case (19Linde L. Boelz S. Neu-Yilik G. Kulozik A.E. Kerem B. The efficiency of nonsense-mediated mRNA decay is an inherent character and varies among different cells.Eur. J. Hum. Genet. 2007; 15 (17625509): 1156-116210.1038/sj.ejhg.5201889Crossref PubMed Scopus (87) Google Scholar). Other genes regulated include kif21a, a member of the kinesin family of motor proteins; camk2b, a calcium/calmodulin-responsive protein kinase; cd59a, a regulator of the membrane attack complex in mice; and nnt, a NAD(P) transhydrogenase with implications in defense against oxidative stress. Very little is known about the lrmda gene except that it consists of a region of LRRs. The remaining results are not represented at the protein level and so are unlikely to have relevance to the current study. The gene detected as being of the highest significance, gm14150, is described as a pseudogene, produced by incorporation of reverse-transcribed mRNA into the genome, whereas gm44305 is a retained intron. These are likely not differentially expressed genes but pre-existing genomic differences between strains (20Akagi K. Li J. Stephens R.M. Volfovsky N. Symer D.E. Extensive variation between inbred mouse strains due to endogenous L1 retrotransposition.Genome Res. 2008; 18 (18381897): 869-88010.1101/gr.075770.107Crossref PubMed Scopus (68) Google Scholar).Table 1Differentially expressed genes between unstimulated macrophagesEnsembl Gene IDBaseMean-Fold Change (KO/WT)PadjGene SymbolENSMUSG00000082809177.985.652.52E − 92Pseudogene Gm14150ENSMUSG0000006345883.020.443.99E − 20LrmdaENSMUSG0000002262931.471.931.07E − 14Kif21aENSMUSG0000010570389.252.011.94E − 13Gm43305ENSMUSG00000036273137.370.548.10E − 10Lrrk2ENSMUSG0000005789758.281.661.17E − 07Camk2bENSMUSG00000032679297.271.551.02E − 04Cd59aENSMUSG00000025453784.611.439.56E − 04Nnt Open table in a new tab The broad characteristics of the MDP and LPS responses as well as similarities and differences between WT and Lrrk2-deficient macrophages in their response to innate immune activation were visualized with volcano plots (Fig. 2). In LPS and MDP experiments, a greater number of genes were up-regulated than down-regulated. LPS treatment led to 3192 genes being up-regulated and 2696 down-regulated; MDP treatment caused 1020 genes to be up-regulated and 676 down-regulated (Fig. 1). Furthermore, quantification confirmed that a greater number of genes was differentially expressed upon LPS treatment in Lrrk2-deficient macrophages than WT macrophages. Perhaps the most interesting observation from this analysis is that a single gene was found to be down-regulated in WT macrophages and up-regulated in Lrrk2−/− macrophages upon treatment with LPS. Transcription of this gene, Rapgef3, is almost halved upon LPS treatment in WT macrophages while being increased just over 7-fold in Lrrk2-null macrophages, a complete reversal in transcriptional regulation upon loss of LRRK2. In contrast to conventional differential gene expression analysis, two-parameter analysis provides an alternative method to identify differentially responding genes between genotypes (21Robinson M.D. McCarthy D.J. Smyth G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics. 2010; 26 (19910308): 139-14010.1093/bioinformatics/btp616Crossref PubMed Scopus (21040) Google Scholar) (RRID: SCR_012802). This method revealed 11 genes with significantly different responses to LPS as between WT and Lrrk2−/− macrophages (adjusted P-value (Padj) < 0.1) (Fig. 3 and Table 2). All differentially responding genes showed an increased level of transcription upon LPS stimulation in Lrrk2 KO cells compared with WT cells. Rapgef3 with a Padj value of 5 × 10−37 and a -fold change of about 11 was the gene most strongly regulated by the presence of Lrrk2. Four other genes identified encode either chemokine ligands (Ccl3, Ccl4, and Ccl5) or receptors (Ccrl2) that mediate chemotactic responses, suggesting a common theme of regulated motility (see "Discussion").Table 2Differentially responding genes in LPS stimulated macrophagesEnsembl Gene IDBaseMean-Fold Change (KO/WT)PadjGene SymbolENSMUSG00000022469176.3410.974.98E − 37Rapgef3ENSMUSG0000001893011796.253.261.94E − 12Ccl4ENSMUSG000000266282039.032.725.58E − 07Atf3ENSMUSG000000009824095.932.241.02E − 03Ccl3ENSMUSG000000325151117.611.731.07E − 03Csrnp1ENSMUSG00000032724417.682.805.72E − 03Abtb2ENSMUSG000000350423664.772.022.75E − 02Ccl5ENSMUSG000000439531652.793.392.75E − 02Ccrl2ENSMUSG00000045502249.853.554.19E − 02Hcar2ENSMUSG00000001156477.832.099.07E − 02Mxd1ENSMUSG000000002758075.681.409.83E − 02Trim25 Open table in a new tab To confirm the results of the RNA-Seq experiments, qRT-PCR was used to directly measure the level of six of the 11 genes identified in WT and Lrrk2−/− macrophages (Fig. 4A). This confirms the results of the RNA-Seq analysis for Rapgef3, chemokines, and the transcription factor atf3. We next asked whether chemical inhibition of the LRRK2 kinase also induces expression of Rapgef3. We treated WT and Lrrk2−/− macrophages with the highly specific inhibitor GSK2578215A (22Reith A.D. Bamborough P. Jandu K. Andreotti D. Mensah L. Dossang P. Choi H.G. Deng X. Zhang J. Alessi D.R. Gray N.S. GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.Bioorg. Med. Chem. Lett. 2012; 22 (22863203): 5625-562910.1016/j.bmcl.2012.06.104Crossref PubMed Scopus (122) Google Scholar). Rapgef3 was induced about 6-fold in treated WT macrophages compared with untreated controls. In contrast, no differences in expression level were detected when Lrrk2 mutant macrophages were treated with GSK2578215A relative to the untreated controls (Fig. 4B). Thus, Lrrk2 kinase activity is required for the observed regulation of Rapgef3 gene expression, consistent with the results of the RNA-Seq analysis. In contrast to LPS, MDP treatment identified no significant differentially responding genes (Fig. 3, c and d). This aligns with the less immunogenic nature of MDP compared with LPS stimulation and demonstrates the high stringency of the two-parameter method. To determine whether Rapgef3 mRNA and Epac-1 protein levels are correlated, we stained Lrrk2-deficient and WT macrophages with a fluorescent mAb specific for Epac-1. As shown in Fig. 5a, Epac-1 is ubiquitous and, in many cells, distributed in the expected punctate, perinuclear pattern (Fig. S2). We then quantified protein levels. 6 h after treatment with LPS, Lrrk2-deficient cells had significantly higher levels of Epac-1, consistent with the RNA-Seq and qPCR results (Fig. 5b). We were unable to further validate these observations using Western blotting, as the available antibodies are insufficiently specific or sensitive in this assay. To explore whether LRRK2 affects innate immune function, we used a model of subclinical bacterial infection. WT and Lrrk2-deficient mice were infected with Salmonella enterica serovar Typhimurium, and three markers of inflammation were measured: IL-18, IFN-γ, and splenomegaly. As shown in Fig. 6 Lrrk2−/− mice had elevated levels of IL-18 and IFN-γ compared with controls 14 days after challenge. These results are statistically significant (p < 0.05, with the exception IFN-γ at 14 days, p = 0.06; see legend for Fig. 6). This indicates that absence of Lrrk2 causes an enhanced inflammatory response. In this study, we investigated how Lrrk2 modifies inflammatory signaling mediated by LPS and MDP. We identified a small subset of genes that are activated by LPS in macrophages that lack Lrrk2 but not in WT control cells. About half of these molecules are involved in cell migration, motility, and chemotaxis. Of particular note is the guanine nucleotide exchange factor (GEF) Rapgef3, by far the most strongly induced gene identified, with an 11- to 15-fold increase in transcription in the absence of Lrrk2. Rapgef3 is also the only gene that is down-regulated in WT macrophages but up-regulated in Lrrk2−/− cells as compared with unstimulated control cells. Rapgef 3 is also derepressed by treatment of macrophages with the highly specific kinase inhibitor GSK2578215A (22Reith A.D. Bamborough P. Jandu K. Andreotti D. Mensah L. Dossang P. Choi H.G. Deng X. Zhang J. Alessi D.R. Gray N.S. GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor.Bioorg. Med. Chem. Lett. 2012; 22 (22863203): 5625-562910.1016/j.bmcl.2012.06.104Crossref PubMed Scopus (122) Google Scholar), indicating that inter- or intramolecular phosphorylation is required. Rapgef3 encodes Epac-1, a GEF that mediates cAMP-dependent activation of the small G-protein Rap1. Epac-1 promotes Rap-1 GDP–GTP exchange, leading to cell adhesion, cell polarization, and enhanced leukocyte chemotaxis (23Robichaux 3rd, W.G. Cheng X. Intracellular cAMP sensor EPAC: physiology, pathophysiology, and therapeutics development.Physiol. Rev. 2018; 98 (29537337): 919-105310.1152/physrev.00025.2017Crossref PubMed Scopus (103) Google Scholar, 24Lorenowicz M.J. van Gils J. de Boer M. Hordijk P.L. Fernandez-Borja M. Epac1-Rap1 signaling regulates monocyte adhesion and chemotaxis.J. Leukoc. Biol. 2006; 80 (16940330): 1542-155210.1189/jlb.0506357Crossref PubMed Scopus (90) Google Scholar). On the other hand, another study found that LPS treatment paralyzes monocyte chemotaxis, an effect that requires activated Rap-1 (25Yi L. Chandrasekaran P. Venkatesan S. TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.PLoS ONE. 2012; 7 (22347375): e3040410.1371/journal.pone.0030404Crossref PubMed Scopus (22) Google Scholar). It is thus likely that LRRK2 indirectly regulates the mobility of macrophages and microglia that have been activated by innate stimuli such as LPS. An attractive hypothesis is that dominant PD-associated LRRK2 alleles, such as G2019S, that have higher constitutive kinase activity may regulate the motility of macrophages by further suppressing EPAC-1 levels. In that regard, a recent study found that G2019S microglia and LRRK2−/− cells, when activated by ADP, have retarded and enhanced motility, respectively, compared with WT control cells. G2019S microglia also have an impaired ability to isolate brain injury (26Choi I. Kim B. Byun J.W. Baik S.H. Huh Y.H. Kim J.H. Mook-Jung I. Song W.K. Shin J.H. Seo H. Suh Y.H. Jou I. Park S.M. Kang H.C. Joe E.H. LRRK2 G2019S mutation attenuates microglial motility by inhibiting focal adhesion kinase.Nat. Commun. 2015; 6 (26365310): 825510.1038/ncomms9255Crossref PubMed Scopus (66) Google Scholar). These authors present evidence of involvement of focal adhesion kinases; however, this may be indirect, and a possible role of the LRRK2/EPAC-1/RAP-1 axis should be investigated. LRRK2 is part of an ancient and highly conserved pathway of directional motility (2Marín I. van Egmond W.N. van Haastert P.J. The Roco protein family: a functional perspective.FASEB J. 2008; 22 (18523161): 3103-311010.1096/fj.08-111310Crossref PubMed Scopus (97) Google Scholar). In the slime mold Dictyostelium, gbpC is one of 11 paralogs of LRRK2. GbpC-null cells are severely defective in chemotaxis because they cannot polarize cells effectively and have altered patterns of myosin phosphorylation that are probably mediated by activation of Rap1. It is interesting to also note that, in Dictyostelium, many LRRK2 paralogues encode GEFs within their modular structure. As well as Epac1, four other messages that encode proteins with functions in chemotaxis are differentially expressed. Three are chemokines, and one is a chemokine receptor–like protein. CCL3 is also known as macrophage inflammatory protein 1, and CCL4 is also known as MIP-1α and MIP-1β. CCL5 is also known as regulated on activation, normal T cell expressed and secreted (RANTES). These chemokines are all members of the CC chemokine/receptor family and share a common receptor in CCR5. CCL3 and CCL5 may also bind CCR1, whereas CCL5 binds a further receptor, CCR3 (27Zlotnik A. Yoshie O. Chemokines: a new classification system and their role in immunity.Immunity. 2000; 12 (10714678): 121-12710.1016/S1074-7613(00)80165-XAbstract Full Text Full Text PDF PubMed Scopus (3280) Google Scholar). These chemokines are all classified as pro-inflammatory, meaning they are induced by inflammatory stimuli to recruit inflammatory cells to a site of inflammation, as opposed to homeostatic chemokines, which are constitutively expressed in certain tissues (28Turner M.D. Nedjai B. Hurst T. Pennington D.J. Cytokines and chemokines: at the crossroads of cell signalling and inflammatory disease.Biochim. Biophys. Acta. 2014; 1843 (24892271): 2563-258210.1016/j.bbamcr.2014.05.014Cro
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