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• W3014129098 abstract "•Human iPSC macrophages and microglia for studying endogenous LRRK2 function•LRRK2 is not involved in initial phagocytic uptake of bioparticles•LRRK2 is recruited to LAMP1+/RAB9+ “maturing” phagosomes•LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1+/RAB9+ “maturing” phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes. The Parkinson's disease-associated gene, LRRK2, is also associated with immune disorders and infectious disease and is expressed in immune subsets. Here, we characterize a platform for interrogating the expression and function of endogenous LRRK2 in authentic human phagocytes using human induced pluripotent stem cell-derived macrophages and microglia. Endogenous LRRK2 is expressed and upregulated by interferon-γ in these cells, including a 187-kDa cleavage product. Using LRRK2 knockout and G2019S isogenic repair lines, we find that LRRK2 is not involved in initial phagocytic uptake of bioparticles but is recruited to LAMP1+/RAB9+ “maturing” phagosomes, and LRRK2 kinase inhibition enhances its residency at the phagosome. Importantly, LRRK2 is required for RAB8a and RAB10 recruitment to phagosomes, implying that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in these professional phagocytes. LRRK2 (leucine-rich repeat kinase 2) encodes a large (286 kDa), multi-domain cytoplasmic protein, with both guanosine triphosphatase (GTPase) and kinase domains, flanked by several protein-protein interaction domains. Mutations in LRRK2 account for approximately 4% of familial and 1% of idiopathic cases of the progressive neurodegenerative disorder, Parkinson's disease (PD), forming an important genetic risk factor for PD. Most PD-causing mutations cluster within the two enzymatic sites, notably G2019S and R1441C/G (leading to a modest 2- to 3-fold increase in kinase activity and decreased GTPase activity, respectively) (Ferreira and Massano, 2017Ferreira M. Massano J. An updated review of Parkinson's disease genetics and clinicopathological correlations.Acta Neurol. Scand. 2017; 135: 273-284Crossref PubMed Scopus (116) Google Scholar). LRRK2 variants are also associated with autoimmune disorders (Witoelar et al., 2017Witoelar A. Jansen I. Wang Y. Desikan R. Gibbs R. Blauwendraat C. Thompson W. Hernandez D. Djurovic S. Schork A. et al.Genome-wide pleiotropy between Parkinson disease and autoimmune diseases.JAMA Neurol. 2017; 74: 780-792Crossref PubMed Scopus (163) Google Scholar), particularly Crohn's disease, and with infectious diseases, notably Mycobacterium leprae (Wang et al., 2015Wang D. Xu L. Lv L. Su L.Y. Fan Y. Zhang D.F. Bi R. Yu D. Zhang W. Li X.A. et al.Association of the LRRK2 genetic polymorphisms with leprosy in Han Chinese from Southwest China.Genes Immun. 2015; 16: 112-119Crossref PubMed Scopus (47) Google Scholar, Zhang et al., 2009Zhang F.-R. Huang W. Chen S.-M. Sun L.-D. Liu H. Li Y. Cui Y. Yan X.-X. Yang H.-T. Yang R.-D. et al.Genomewide association study of leprosy.New Engl. J. Med. 2009; 361: 2609-2618Crossref PubMed Scopus (537) Google Scholar). LRRK2 expression has also been linked with Mycobacterium tuberculosis infection (Härtlova et al., 2018Härtlova A. Herbst S. Peltier J. Rodgers A. Bilkei-Gorzo O. Fearns A. Dill B. Lee H. Flynn R. Cowley S. et al.LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.EMBO J. 2018; 37: e98694Crossref PubMed Scopus (98) Google Scholar, Wang et al., 2018Wang Z. Arat S. Magid-Slav M. Brown J. Meta-analysis of human gene expression in response to Mycobacterium tuberculosis infection reveals potential therapeutic targets.BMC Syst. Biol. 2018; 12: 3Crossref PubMed Scopus (41) Google Scholar). LRRK2 is expressed in a variety of cell lineages, including several immune subsets, notably B cells, neutrophils, monocytes, macrophages, and microglia (Atashrazm et al., 2019Atashrazm F. Hammond D. Perera G. Bolliger M.F. Matar E. Halliday G.M. Schüle B. Lewis S.J.G. Nichols R.J. Dzamko N. LRRK2-mediated Rab10 phosphorylation in immune cells from Parkinson's disease patients.Movement Disord. 2019; 34: 406-415Crossref PubMed Scopus (51) Google Scholar, Fan et al., 2018Fan Y. Howden A. Sarhan A. Lis P. Ito G. Martinez T. Brockmann K. Gasser T. Alessi D. Sammler E. Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.Biochem. J. 2018; 475: 23-44Crossref PubMed Scopus (83) Google Scholar, Gardet et al., 2010Gardet A. Benita Y. Li C. Sands B. Ballester I. Stevens C. Korzenik J. Rioux J. Daly M. Xavier R. et al.LRRK2 is involved in the IFN-gamma response and host response to pathogens.J. Immunol. 2010; 185: 5577-5585Crossref PubMed Scopus (280) Google Scholar, Hakimi et al., 2011Hakimi M. Selvanantham T. Swinton E. Padmore R. Tong Y. Kabbach G. Venderova K. Girardin S. Bulman D. Scherzer C. et al.Parkinson's disease-linked LRRK2 is expressed in circulating and tissue immune cells and upregulated following recognition of microbial structures.J. Neural Transm. 2011; 118: 795-808Crossref PubMed Scopus (187) Google Scholar, Kim et al., 2012Kim B. Yang M.-S. Choi D. Kim J.-H. Kim H.-S. Seol W. Choi S. Jou I. Kim E.-Y. Joe E.-H. Impaired inflammatory responses in murine Lrrk2-knockdown brain microglia.PloS one. 2012; 7: e34693Crossref PubMed Scopus (146) Google Scholar, Marker et al., 2012Marker D. Puccini J. Mockus T. Barbieri J. Lu S.-M. Gelbard H. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein.J. neuroinflammation. 2012; 9: 261Crossref PubMed Scopus (66) Google Scholar, Moehle et al., 2012Moehle M. Webber P. Tse T. Sukar N. Standaert D. DeSilva T. Cowell R. West A. LRRK2 inhibition attenuates microglial inflammatory responses.J. Neurosci. 2012; 32: 1602-1611Crossref PubMed Scopus (303) Google Scholar, Thévenet et al., 2011Thévenet 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: e21519Crossref PubMed Scopus (130) Google Scholar; reviewed in Lee et al., 2017Lee H. James W. Cowley S. LRRK2 in peripheral and central nervous system innate immunity: its link to Parkinson's disease.Biochem. Soc. Trans. 2017; 45: 131-139Crossref PubMed Scopus (59) Google Scholar). Macrophages populate most tissues of the body, deriving initially from primitive macrophages that migrate in during embryogenesis, and are replenished as necessary during the lifespan by either local proliferation and/or replacement by blood monocyte-derived macrophages, depending on the tissue (Hoeffel and Ginhoux, 2015Hoeffel G. Ginhoux F. Ontogeny of tissue-resident macrophages.Front. Immunol. 2015; 6: 486Crossref PubMed Scopus (200) Google Scholar). Macrophages perform tissue homeostatic functions and are also a first-line defense against pathogens, armed with a plethora of pattern-recognition and opsonin receptors. They rapidly phagocytose and kill incoming bacteria, fungi, and protoctists, and have strong antiviral defenses. Nonetheless, various pathogens can overcome these defenses to survive and proliferate in macrophages, including M. leprae and M. tuberculosis, Interestingly, LRRK2 has recently been shown to be required for survival of M. tuberculosis in macrophages (Härtlova et al., 2018Härtlova A. Herbst S. Peltier J. Rodgers A. Bilkei-Gorzo O. Fearns A. Dill B. Lee H. Flynn R. Cowley S. et al.LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.EMBO J. 2018; 37: e98694Crossref PubMed Scopus (98) Google Scholar). Microglia are a resident, primitive macrophage-derived population in the central nervous system, performing homoeostatic functions (phagocytosing cell debris, extracellular protein aggregates, and incompetent synapses) to maintain a healthy environment for neurons. However, they can also secrete inflammatory mediators when activated, notably tumor necrosis factor α, and a myriad of cytotoxic factors, especially reactive oxygen species and nitric oxide, which can instigate a feedforward cycle of chronic inflammation and neurodegeneration. Therefore, microglia are not only involved in preventing neurodegenerative disease by phagocytosing potentially harmful materials but also can contribute to disease progression by initiating exaggerated inflammatory responses (reviewed in Wolf et al., 2017Wolf S. Boddeke H.W. Kettenmann H. Microglia in physiology and disease.Annu. Rev. Physiol. 2017; 79: 619-643Crossref PubMed Scopus (746) Google Scholar). Due to the difficulty in obtaining primary patient material, most studies of LRRK2 have used animal models, in vitro biochemical assays, or transformed cell lines, often involving non-physiological exogenous overexpression of LRRK2 in irrelevant lineages. Studies of LRRK2 using transformed myeloid cell lines have progressed this field (Gardet et al., 2010Gardet A. Benita Y. Li C. Sands B. Ballester I. Stevens C. Korzenik J. Rioux J. Daly M. Xavier R. et al.LRRK2 is involved in the IFN-gamma response and host response to pathogens.J. Immunol. 2010; 185: 5577-5585Crossref PubMed Scopus (280) Google Scholar, Marker et al., 2012Marker D. Puccini J. Mockus T. Barbieri J. Lu S.-M. Gelbard H. LRRK2 kinase inhibition prevents pathological microglial phagocytosis in response to HIV-1 Tat protein.J. neuroinflammation. 2012; 9: 261Crossref PubMed Scopus (66) Google Scholar), and Eguchi et al., 2018Eguchi T. Kuwahara T. Sakurai M. Komori T. Fujimoto T. Ito G. Yoshimura S.-I. Harada A. Fukuda M. Koike M. et al.LRRK2 and its substrate Rab GTPases are sequentially targeted onto stressed lysosomes and maintain their homeostasis.Proc. Natl. Acad. Sci. United States America. 2018; 115: E9115-E9124Crossref PubMed Scopus (121) Google Scholar have reported that LRRK2 recruits and phosphorylates RABs 8 and 10 to chloroquine-induced overload-stressed lysosomes in mouse RAW264.7 cells, leading to release of lysosomal contents. Yet such observations need to be subsequently assessed in a karyotypically normal human cellular system at physiologically relevant expression levels to validate their applicability to normal human physiology and disease. We have previously developed methods for efficient differentiation of human induced pluripotent stem cells (hiPSCs) to macrophages, which exhibit authentic phagocytic properties and cytokine-profiles (Flynn et al., 2015Flynn R. Grundmann A. Renz P. Hänseler W. James W. Cowley S. Moore M. CRISPR-mediated genotypic and phenotypic correction of a chronic granulomatous disease mutation in human iPS cells.Exp. Hematol. 2015; 43: 838-848Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, Haenseler et al., 2017bHaenseler W. Zambon F. Lee H. Vowles J. Rinaldi F. Duggal G. Houlden H. Gwinn K. Wray S. Luk K. et al.Excess α-synuclein compromises phagocytosis in iPSC-derived macrophages.Scientific Rep. 2017; 7: 9003Crossref PubMed Scopus (60) Google Scholar, Karlsson et al., 2008Karlsson K. Cowley S. Martinez F. Shaw M. Minger S. James W. Homogeneous monocytes and macrophages from human embryonic stem cells following coculture-free differentiation in M-CSF and IL-3.Exp. Hematol. 2008; 36: 1167-1175Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, van Wilgenburg et al., 2013van Wilgenburg B. Browne C. Vowles J. Cowley S. Efficient, long term production of monocyte-derived macrophages from human pluripotent stem cells under partly-defined and fully-defined conditions.PloS One. 2013; 8: e71098Crossref PubMed Scopus (170) Google Scholar). The differentiation pathway is demonstrably independent of c-Myb expression (Buchrieser et al., 2017Buchrieser J. James W. Moore M. Human induced pluripotent stem cell-derived macrophages share ontogeny with MYB-independent tissue-resident macrophages.Stem Cel. Rep. 2017; 8: 334-345Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), indicating that they represent an embryonic/primitive ontogeny and are therefore also suitable as a precursor for differentiation to microglia. We have shown that they can be further differentiated to microglia by co-culture with hiPSC neurons, whereupon they acquire a ramified morphology and associated neuronal surveillance activity (Haenseler et al., 2017aHaenseler W. Sansom S. Buchrieser J. Newey S. Moore C. Nicholls F. Chintawar S. Schnell C. Antel J. Allen N. et al.A highly efficient human pluripotent stem cell microglia model displays a neuronal-co-culture-specific expression profile and inflammatory response.Stem Cel. Rep. 2017; 8: 1727-1742Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar). In this study, we have used hiPSC macrophages and microglia from patient, control, and gene-edited lines to explore the expression of LRRK2 protein from the endogenous locus and the role of LRRK2 in this lineage. We show that LRRK2 is expressed in hiPSC macrophages and microglia, with expression significantly upregulated by interferon-γ (IFN-γ) and identify the cleavage region of a truncated LRRK2 product found in this lineage. In this system, LRRK2 is not involved in the initial phagocytic uptake of particles but is recruited to maturing phagosomes, and this is exacerbated by inhibition of LRRK2 kinase activity. Importantly, we show that LRRK2 is required for recruitment to phagosomes of RAB8a and RAB10 (members of the membrane trafficking regulator family of RAB GTPases and substrates of LRRK2 kinase activity). This demonstrates that LRRK2 operates at the intersection between phagosome maturation and recycling pathways in the myeloid lineage. The hiPSC lines used in this study are listed in Table S1, with quality control information in Figure S1. Wild-type lines (WT.1 to WT.6) were from six healthy control donors. LRRK2 knockout (KO) was generated in a control hiPSC (line WT.1) by a double nickase CRISPR/Cas9 (Ran et al., 2013Ran A. Hsu P. Lin C.-Y. Gootenberg J. Konermann S. Trevino A. Scott D. Inoue A. Matoba S. Zhang Y. et al.Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity.Cell. 2013; 154: 1380-1389Abstract Full Text Full Text PDF PubMed Scopus (2347) Google Scholar) strategy, using a pair of guide RNAs (gRNAs) targeting exon 3 of LRRK2 (Figures 1A and S2A). A patient line containing a heterozygous LRRK2 mutation G2019S (GS) was successfully repaired to WT (GS-Repair) as shown by sequence analysis (Figure 1B). Two KO clones (KO.1 and KO.2) displayed out-of-frame homozygous deletion of LRRK2 (Figure S1A) and showed complete absence of LRRK2 protein when differentiated to macrophages (Figure 1C). There was no significant difference in the production of macrophage precursors in edited versus parental lines (Figures S2B and S2C). Western blot of hiPSC macrophages using LRRK2 monoclonal antibody N241A/34 (binding site amino acids [aa] 1,836–1,845) consistently showed multiple faint bands and a major band (approximately 170 kDa relative to size markers) in addition to full-length LRRK2 (286 kDa). This was particularly evident upon IFN-γ stimulation. The staining pattern was confirmed to be LRRK2 specific by its absence in LRRK2 KO hiPSC macrophages (Figure 1C). Since LRRK2 transcripts that would correspond to this product have not been reported, we reasoned that this is likely a proteolytically cleaved product. It is not a result of technical post-lysis proteolysis, as spiking recombinant full-length LRRK2 into LRRK2-KO macrophage lysate did not lead to its proteolytic degradation under our standard lysis conditions (Figure S2D). Addition of protease inhibitors to live macrophages reduced the proportion of the truncated versus full-length protein, implying that it is a natural cleavage generated within intact macrophages (Figure S2E). Immunoprecipitation using the N-terminal antibody revealed that the cleaved product can heterodimerize with full-length LRRK2 (Figures S2F and S2G). To identify the cleavage site, we isolated endogenous LRRK2 protein from hiPSC macrophages by immunoprecipitation with N241A/34 antibody, ran it on a denaturing gel, and analyzed the cleaved product by mass spectrometry (Figure 1D). MaxQuant analysis of trypsin-digested peptide fragments revealed that cleavage occurs within the ANK-LRR interdomain region (aa 861–983), generating a C-terminal predicted product of ∼170–187 kDa (Figure 1E). Western blot of hiPSC macrophage whole-cell lysate using an antibody against LRRK2 pSer935, did not co-localize with the C-terminal cleavage product band but did co-localize with a ∼110-kDa band detected by the N-terminal antibody, and treatment of the cells with LRRK2 kinase inhibitor GNE-7915 (GNE) reduced the intensity of the pSer935 band (Figure S2H). Together, these results indicate that S935 can be present on the N-terminal cleavage product, predicting the major cleavage site to be downstream of S935. IFN-γ has been shown to upregulate LRRK2 protein expression in myeloid cells (Gardet et al., 2010Gardet A. Benita Y. Li C. Sands B. Ballester I. Stevens C. Korzenik J. Rioux J. Daly M. Xavier R. et al.LRRK2 is involved in the IFN-gamma response and host response to pathogens.J. Immunol. 2010; 185: 5577-5585Crossref PubMed Scopus (280) Google Scholar). Similarly, in hiPSC macrophages, LRRK2 protein expression increased significantly (up to 10-fold) upon IFN-γ treatment (Figures 2A and S3A). Phosphorylation of LRRK2 at S935 was observed, significantly decreasing in the presence of LRRK2 kinase inhibitor GNE, in accordance with the published literature (Hatcher et al., 2017Hatcher J.M. Choi H.G. Alessi D.R. Gray N.S. Small-molecule inhibitors of LRRK2.in: Rideout H.J. Leucine-Rich Repeat Kinase 2 (LRRK2). Springer International Publishing, 2017: 241-264Crossref Scopus (21) Google Scholar) (Figure 2B). hiPSC macrophages with the heterozygous G2019S mutation showed the same pattern, with no significant difference in either the basal phosphorylation level at S935 or the degree of dephosphorylation upon treatment with LRRK2 kinase inhibitors compared with its isogenic pair (Figure S3B), likely because G2019S only produces a modest 2-fold increase in kinase activity. We next examined LRRK2 expression in hiPSC microglia, co-cultured with hiPSC cortical neurons. LRRK2 protein was clearly expressed in hiPSC microglia while its expression level was not detectable in hiPSC cortical neurons (Figure 2C). The specificity of LRRK2 staining in microglia was confirmed by co-culturing microglia differentiated from LRRK2 KO hiPSCs with cortical neurons differentiated from LRRK2 WT hiPSCs (Figure 2C). To test whether IFN-γ upregulates LRRK2 protein in hiPSC microglia or neurons, we treated hiPSC microglia/cortical neuron co-cultures with IFN-γ for 16 h, 48 h, or 72 h. IFN-γ treatment significantly upregulated the percentage of LRRK2 expressing hiPSC microglia to 86%, its expression level plateauing by 48 h post IFN-γ treatment (Figure 2D). Together, these results demonstrate the validity of the hiPSC macrophage and microglia models for investigating endogenous LRRK2 function. We next investigated whether LRRK2 is involved in phagocytosis using hiPSC macrophages. hiPSC macrophages readily phagocytose a wide variety of “meals,” including killed yeast bioparticles (“zymosan”), a process ablated by inhibiting actin polymerization with cytochalasin D. Complete absence of LRRK2 in hiPSC macrophages did not alter their ability to take up fluorescent zymosan, with or without IFN-γ induction (Figures 3A and 3B ). Similarly, zymosan uptake by G2019S patient-derived hiPSC macrophages was not significantly different from that of its isogenic pair (Figure 3C). Lastly, pharmacological inhibition of LRRK2 kinase activity with two structurally distinct LRRK2 kinase inhibitors, GSK2578215A (GSK) or GNE, had no significant effect on zymosan uptake (Figure 3D). Acidification of the phagosomes, as assessed by uptake of pH-sensitive fluorescent (pHrodo) zymosan particles, was also not significantly altered by manipulating LRRK2 in hiPSC macrophages (Figure S4). While no functional difference in initial phagocytic uptake was observed across LRRK2 lines, confocal imaging clearly demonstrated the presence of LRRK2 on a subset of zymosan-containing phagosomes (in IFN-γ-treated cells to enable visualization of LRRK2) (Figure 4A). This was also observable with Escherichia coli bioparticles, with and without opsonization, and Salmonella typhimurium (Figure 4A). LRRK2 was not observed on phagosomes containing αsyn fibrils, even when opsonized (Figure S5), indicating that LRRK2 recruitment is context dependent. The number of LRRK2-positive (LRRK2+) zymosan-containing phagosomes was time dependent, peaking at 1–2 h after addition of the meal to cells (mean 14.7%, range 6.8%–25.2% at 2 h), so 2-h zymosan incubation was used for all subsequent experiments (Figure 4B). LRRK2+ phagosomes were found to also be positive for the late phagosomal markers lysosome-associated protein LAMP-1 and RAB9, with significantly more LRRK2+LAMP-1+ or LRRK2+RAB9+ phagosomes than LRRK2+RAB5+ phagosomes (an early phagosome marker) (Figure 4C). Together, these data show that LRRK2 is recruited during later stages of phagosome maturation in hiPSC macrophages, around the time when lysosomes are recruited to phagosomes. RAB GTPases regulate various fission and fusion events during phagocytosis, and in LRRK2-overexpression systems and non-human systems LRRK2 has been shown to associate physically with subsets of this family of proteins (Dodson et al., 2011Dodson M.W. Zhang T. Jiang C. Chen S. Guo M. Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.Hum. Mol. Genet. 2011; 21: 1350-1363Crossref PubMed Scopus (162) Google Scholar, Gómez-Suaga et al., 2014Gómez-Suaga P. Rivero-Ríos P. Fdez E. Blanca Ramírez M. Ferrer I. Aiastui A. López De Munain A. Hilfiker S. LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity.Hum. Mol. Genet. 2014; 23: 6779-6796Crossref PubMed Scopus (120) Google Scholar, Steger et al., 2016Steger M. Tonelli F. Ito G. Davies P. Trost M. Vetter M. Wachter S. Lorentzen E. Duddy G. Wilson S. et al.Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.eLife. 2016; 5: e12813Crossref PubMed Google Scholar, Waschbüsch et al., 2014Waschbüsch D. Michels H. Strassheim S. Ossendorf E. Kessler D. Gloeckner C.J. Barnekow A. LRRK2 transport is regulated by its novel interacting partner Rab32.PLOS ONE. 2014; 9: e111632Crossref PubMed Scopus (67) Google Scholar, Yun et al., 2015Yun H.J. Kim H. Ga I. Oh H. Ho D.H. Kim J. Seo H. Son I. Seol W. An early endosome regulator, Rab5b, is an LRRK2 kinase substrate.J. Biochem. 2015; 157: 485-495Crossref PubMed Scopus (54) Google Scholar). Importantly, several RABs, particularly RAB8a and RAB10, have been identified as physiological substrates of LRRK2, able to be phosphorylated by LRRK2 (on Thr72/73, respectively) (Fan et al., 2018Fan Y. Howden A. Sarhan A. Lis P. Ito G. Martinez T. Brockmann K. Gasser T. Alessi D. Sammler E. Interrogating Parkinson's disease LRRK2 kinase pathway activity by assessing Rab10 phosphorylation in human neutrophils.Biochem. J. 2018; 475: 23-44Crossref PubMed Scopus (83) Google Scholar, Steger et al., 2016Steger M. Tonelli F. Ito G. Davies P. Trost M. Vetter M. Wachter S. Lorentzen E. Duddy G. Wilson S. et al.Phosphoproteomics reveals that Parkinson's disease kinase LRRK2 regulates a subset of Rab GTPases.eLife. 2016; 5: e12813Crossref PubMed Google Scholar). We therefore investigated whether these RAB GTPases are involved during LRRK2 recruitment to phagosomes. RAB8a and RAB10 could be observed coating the same phagosomes as LRRK2, while RAB7 had no significant association with LRRK2+ phagosomes (Figures 5A and 5B ). 14-3-3 proteins, which associate with pS935-LRRK2 (Dzamko et al., 2010Dzamko N. Deak M. Hentati F. Reith A. Prescott A. Alessi D. Nichols J. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.Biochem. J. 2010; 430: 405-413Crossref PubMed Scopus (287) Google Scholar), did not appear closely co-localized with LRRK2 but could be observed just peripheral to LRRK2-coated clusters of phagosomes (not quantified). Importantly, the number of RAB8+ and RAB10+ phagosomes was significantly reduced (to background levels) in LRRK2 KO hiPSC macrophages (Figures 5A and 5C), and the number of phosphoT73 RAB10+ phagosomes was also significantly reduced (Figures 5D and 5E), demonstrating that the presence of LRRK2 is necessary for recruitment of RAB8a and RAB10 to phagosomes. In LRRK2-overexpressing HEK 293T cells, it has been reported that LRRK2 kinase inhibitors affect localization of LRRK2 within the cell, from diffused cytosolic distribution to more discrete cytosolic pools (Dzamko et al., 2010Dzamko N. Deak M. Hentati F. Reith A. Prescott A. Alessi D. Nichols J. Inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser(910)/Ser(935), disruption of 14-3-3 binding and altered cytoplasmic localization.Biochem. J. 2010; 430: 405-413Crossref PubMed Scopus (287) Google Scholar). Therefore, we investigated whether the application of LRRK2 kinase inhibitors would affect LRRK2 recruitment to phagosomes during phagocytosis. Pre-treating hiPSC macrophages with LRRK2 kinase inhibitors did not change the total number of LRRK2+ phagosomes. However, we noticed that significantly more (4-fold) of these LRRK2+ phagosomes displayed enhanced LRRK2 signal (referred to as “supercoated” LRRK2 phagosomes, LRRK2++, Figures 6A–6C ). This was also observable with the LRRK2 G2019S patient line and its isogenic control (Figures S6A and S6B). There was no significant difference between the isogenic pair of lines, suggesting that monoallelic G2019S LRRK2 kinase enhancement is not potent enough to give a detectable difference in ”supercoating” compared with strong drug-induced kinase inhibition. Although LRRK2 kinase inhibition led to an increase in LRRK2 presence at phagosomes, RAB8a and RAB10 co-localization at LRRK2+ phagosomes was reduced (Figures 6D, 6E, S6C, and S6D). This observation was also replicated in the G2019S isogenic pair (Figure S6E). Note that while kinase inhibition significantly reduced RAB8a andRAB10 recruitment, there was no significant difference between the G2019S line and its isogenic corrected line, implying again that this monoallelic mutation is subtle in relation to the effects of potent chemical inhibitors. Importantly, LRRK2 kinase inhibition also significantly reduced detection of phosphoRAB10 at phagosomes (Figures 6D and 6E). Together, these results show that although kinase inhibition causes LRRK2 to accumulate on phagosomes, without kinase activity LRRK2 cannot recruit RAB8a or RAB10. Here, we have characterized a platform for understanding LRRK2 function using hiPSC-derived macrophages and microglia, including gene-edited lines, which has enabled us to interrogate the involvement of LRRK2 in phagocytic processes in authentic, professional phagocytes. While numerous studies have used hiPSC-derived neurons and astrocytes to study LRRK2 function (reviewed in Booth, 2017Booth H.D.E. Modelling and Analysis of LRRK2 Mutations in iPSC-Derived Dopaminergic Neurons and Astrocytes. University of Oxford, 2017Google Scholar), the advantages of iPSC technology have barely begun to be applied to studying its function in the myeloid lineage. Our hiPSC macrophages have been used in one previous study as a validation of observations in mouse that M. tuberculosis survival inside macrophages requires LRRK2 (Härtlova et al., 2018Härtlova A. Herbst S. Peltier J. Rodgers A. Bilkei-Gorzo O. Fearns A. Dill B. Lee H. Flynn R. Cowley S. et al.LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages.EMBO J. 2018; 37: e98694Crossref PubMed Scopus (98) Google Scholar). To our knowledge, only one other group has previously looked at LRRK2 in hiPSC-derived myeloid cells, describing an impact of the G2019S mutation on differentiation capacity (Speidel et al., 2016Speidel A. Felk S. Reinhardt P. Sterneckert J. Gillardon F. Leucine-rich repeat kinase 2 influences fate decision of human monocytes differentiated from induced pluripotent stem cells.PLoS ONE. 2016; 11: e0165949Crossref PubMed Scopus (13) Google Scholar); however, we did not find this in our system over multiple differentiations with our G2019S isogenic pair or our LRRK2 KO isogenic pair. We show that endogenous LRRK2 in hiPSC macrophages and microglia is strongly induced by immune signals, particularly IFN-γ, as reported in other systems (Gardet et al., 2010Gardet A. Benita Y. Li C. Sands B. Ballester I. Stevens C. Korzenik J. Rioux J. Daly M. Xavier R. et al.LRRK2 is involved in the IFN-gamma response and host response to pathogens.J. Immunol. 2010; 185: 5577-5585Crossref PubMed Scopus (280) Google Scholar, Lee et" @default.
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