Abstract Background Alzheimer’s Disease (AD) is the most common cause of dementia in the elderly and affects over 35 million people worldwide, imposing increasing social and economic burden as the population ages. While it is widely known that the most prominent genetic risk factor for AD is the presence of the Apolipoprotein E (APOE) ε4 allele, the effects of APOE in the development of AD is still poorly understood. As part of the IMI ADAPTED consortium, we aim to clarify the role of APOE as a risk factor in the development of AD. Here we present an in‐depth analysis of the effect of the APOE genotype on the transcriptome of brain cells derived from human‐induced pluripotent stem cells (hiPSCs). Method Isogenic hiPSC lines were modified to carry different APOE genotypes: ε3/ε3, ε4/ε4, ε3/ε4, ε2/ε2, as well as an APOE knock‐out (KO) cell line. Lines carrying each of these genotypes were differentiated into distinct cell types. Differential gene expression (DGE) and protein expression (DPE) was calculated, followed by gene set enrichment. Clustering approaches were used to identify shared and differing gene signatures across genotypes. We further applied upstream regulator and network analysis on the individual cell‐type results and integrated these results across cell types. The results were compared with DGE and DEP results from an APOE mouse model. Finally, the identified genes and mechanisms were combined with the results of data from postmortem human brain samples of AD cases and controls. Results The observed transcriptional changes confirmed phenotypic observations made for the hiPSCs and refined the insight of genes identified human brain OMICS data. Several genes and pathways were identified, which showed consistent gene expression on transcriptome and proteome level. Further, shared patterns of expressions of genes across genotypes and potential mechanisms involved in this were detected. Conclusions In depth transcriptomics and proteomics analysis of APOE modified hiPSCs enabled to study cell type specific effects and contributed with this to the understanding of mechanisms affected by different APOE genotypes.
To examine the pathogenic role of α-synuclein (αS) in Parkinson's Disease, we have generated induced Pluripotent Stem Cell lines from early onset Parkinson's Disease patients with SNCA A53T and SNCA Triplication mutations, and in this study have differentiated them to PSC-macrophages (pMac), which recapitulate many features of their brain-resident cousins, microglia. We show that SNCA Triplication pMac, but not A53T pMac, have significantly increased intracellular αS versus controls and release significantly more αS to the medium. SNCA Triplication pMac, but not A53T pMac, show significantly reduced phagocytosis capability and this can be phenocopied by adding monomeric αS to the cell culture medium of control pMac. Fibrillar αS is taken up by pMac by actin-rearrangement-dependent pathways, and monomeric αS by actin-independent pathways. Finally, pMac degrade αS and this can be arrested by blocking lysosomal and proteasomal pathways. Together, these results show that macrophages are capable of clearing αS, but that high levels of exogenous or endogenous αS compromise this ability, likely a vicious cycle scenario faced by microglia in Parkinson's disease.
Article22 May 2018Open Access Transparent process LRRK2 is a negative regulator of Mycobacterium tuberculosis phagosome maturation in macrophages Anetta Härtlova Anetta Härtlova MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Susanne Herbst Susanne Herbst Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Crick-GSK Biomedical LinkLabs, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK Search for more papers by this author Julien Peltier Julien Peltier MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Angela Rodgers Angela Rodgers Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Orsolya Bilkei-Gorzo Orsolya Bilkei-Gorzo MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Antony Fearns Antony Fearns Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Brian D Dill Brian D Dill MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Heyne Lee Heyne Lee Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Rowan Flynn Rowan Flynn Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Sally A Cowley Sally A Cowley orcid.org/0000-0003-0297-6675 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Paul Davies Paul Davies MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Patrick A Lewis Patrick A Lewis University of Reading, Reading, UK UCL Institute of Neurology, Queen Square, London, UK Search for more papers by this author Ian G Ganley Ian G Ganley orcid.org/0000-0003-1481-9407 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Jennifer Martinez Jennifer Martinez NIEHS, Research Triangle Park, NC, USA Search for more papers by this author Dario R Alessi Dario R Alessi orcid.org/0000-0002-2140-9185 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Alastair D Reith Alastair D Reith Neurodegeneration Discovery Performance Unit, RD Neurosciences, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK Search for more papers by this author Matthias Trost Corresponding Author Matthias Trost [email protected] orcid.org/0000-0002-5732-700X MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Maximiliano G Gutierrez Corresponding Author Maximiliano G Gutierrez [email protected] orcid.org/0000-0003-3199-0337 Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Anetta Härtlova Anetta Härtlova MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Susanne Herbst Susanne Herbst Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Crick-GSK Biomedical LinkLabs, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK Search for more papers by this author Julien Peltier Julien Peltier MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Angela Rodgers Angela Rodgers Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Orsolya Bilkei-Gorzo Orsolya Bilkei-Gorzo MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Antony Fearns Antony Fearns Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Brian D Dill Brian D Dill MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Heyne Lee Heyne Lee Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Rowan Flynn Rowan Flynn Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Sally A Cowley Sally A Cowley orcid.org/0000-0003-0297-6675 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Search for more papers by this author Paul Davies Paul Davies MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Patrick A Lewis Patrick A Lewis University of Reading, Reading, UK UCL Institute of Neurology, Queen Square, London, UK Search for more papers by this author Ian G Ganley Ian G Ganley orcid.org/0000-0003-1481-9407 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Jennifer Martinez Jennifer Martinez NIEHS, Research Triangle Park, NC, USA Search for more papers by this author Dario R Alessi Dario R Alessi orcid.org/0000-0002-2140-9185 MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Search for more papers by this author Alastair D Reith Alastair D Reith Neurodegeneration Discovery Performance Unit, RD Neurosciences, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK Search for more papers by this author Matthias Trost Corresponding Author Matthias Trost [email protected] orcid.org/0000-0002-5732-700X MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK Newcastle University, Newcastle-upon-Tyne, UK Search for more papers by this author Maximiliano G Gutierrez Corresponding Author Maximiliano G Gutierrez [email protected] orcid.org/0000-0003-3199-0337 Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK Search for more papers by this author Author Information Anetta Härtlova1,2,‡, Susanne Herbst3,4,‡, Julien Peltier1,2, Angela Rodgers3, Orsolya Bilkei-Gorzo1, Antony Fearns3, Brian D Dill1, Heyne Lee5, Rowan Flynn5, Sally A Cowley5, Paul Davies1, Patrick A Lewis6,7, Ian G Ganley1, Jennifer Martinez8, Dario R Alessi1, Alastair D Reith9, Matthias Trost *,1,2 and Maximiliano G Gutierrez *,3 1MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, UK 2Newcastle University, Newcastle-upon-Tyne, UK 3Host-Pathogen Interactions in Tuberculosis Laboratory, The Francis Crick Institute, London, UK 4Crick-GSK Biomedical LinkLabs, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK 5Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 6University of Reading, Reading, UK 7UCL Institute of Neurology, Queen Square, London, UK 8NIEHS, Research Triangle Park, NC, USA 9Neurodegeneration Discovery Performance Unit, RD Neurosciences, GlaxoSmithKline Pharmaceuticals R&D, Stevenage, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 191 2087009; E-mail: [email protected] *Corresponding author. Tel: +44 203 7961460; E-mail: [email protected] The EMBO Journal (2018)37:e98694https://doi.org/10.15252/embj.201798694 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutations in the leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol-3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2-dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation. Synopsis Possible immune functions of LRRK2, a kinase frequently mutated in Parkinson's disease, have remained ill-defined. Genetic, pharmacological and proteomics approaches now reveal it as a negative regulator of phagosome maturation in macrophages, thereby affecting the control of Mycobacterium tuberculosis (Mtb) infection. LRRK2 loss targets Mtb to phagolysosomes and limits Mtb replication. LRRK2 inhibition enhances phagosome maturation in macrophages. LRRK2 activity is required for the recruitment of class III PI3K/Rubicon into phagosomes. Loss of LRRK2 enhances innate immunity to Mtb in mice. LRRK2 KO alters inflammatory profiles after Mtb infection in vitro and in vivo. Introduction Tuberculosis (TB) is an infectious disease caused by the intracellular pathogen Mycobacterium tuberculosis (Mtb), which is characterised by chronic inflammatory responses (Russell, 2011; Kaufmann & Dorhoi, 2013). TB is one of the most pernicious infectious diseases borne by mankind with an estimated 1.6 million deaths in 2016 (WHO, 2017). Mtb infects mostly macrophages and within these cells establishes a replicative niche by subverting the host cell and the normal process of phagosome maturation. This cellular pathway, that represents the cornerstone of the innate immune system, targets Mtb to phagolysosomes where they are eventually eliminated (Levin et al, 2016). Mutations in the multi-domain leucine-rich repeat kinase 2 (LRRK2) are associated with inflammatory diseases such as Crohn's disease and ulcerative colitis (Zhang et al, 2009; Franke et al, 2010; Liu et al, 2011; Umeno et al, 2011; Marcinek et al, 2013). Additionally, genomewide association studies have implicated LRRK2 in mycobacterial immunopathology and identified LRRK2 as a risk factor for inflammatory responses in leprosy, an infection by the intracellular pathogen Mycobacterium leprae (Zhang et al, 2009; Wang et al, 2015; Fava et al, 2016) as well as Mtb (Wang et al, 2018). However, the implicated cellular pathways linking LRRK2 function and immunity are poorly characterised. Mutations in LRRK2 represent a genetic risk associated with dominantly inherited and sporadic Parkinson's disease (PD; Funayama et al, 2002; Paisan-Ruiz et al, 2004; Zimprich et al, 2004; Ross et al, 2011). In PD, mutations in LRRK2 are distributed over the kinase and Ras of complex proteins (ROC) domains. Most of these mutations, including the most frequent mutation G2019S, are characterised by enhanced kinase activity (West et al, 2005). Despite evidence showing a strong association between LRRK2 activities and the regulation of intracellular trafficking and lysosomal degradation pathways (Alegre-Abarrategui et al, 2009; Tong et al, 2010; MacLeod et al, 2013; Manzoni et al, 2013; Orenstein et al, 2013; Steger et al, 2016), how pathogenic mutations regulate LRRK2 function remains elusive (Cookson, 2010). Compelling evidence supports the idea that LRRK2 may have an important immune function (Dzamko & Halliday, 2012; Greggio et al, 2012). In fact, myeloid cells such as monocytes and macrophages express LRRK2 at high levels (Gardet et al, 2010; Hakimi et al, 2011) and several immune stimuli induce LRRK2 expression (Gardet et al, 2010; Hakimi et al, 2011). In this work, we examined the function of LRRK2 in macrophages and show that LRRK2 negatively regulates phagosome maturation and that this contributes to mycobacterial replication and impaired innate immune responses. Using several independent experimental approaches, we show that phagosomal function is regulated by a LRRK2 kinase-dependent recruitment of the Class III phosphatidylinositol-3 kinase (PI3K) complex and its negative regulator Rubicon (RUN domain protein as Beclin-1 interacting and cysteine-rich containing). Strikingly, macrophages lacking LRRK2 or treated with an inhibitor of LRRK2 kinase activity showed more efficient control of Mtb replication by macrophages and an enhanced early immune response in LRRK2 KO mice. This study provides a cellular function underlying human genetic studies linking LRRK2 to mycobacterial infections and reveals an unexpected function for LRRK2 in macrophages and infectious diseases control. Results Loss of LRRK2 activity targets Mtb to phagolysosomes and limits Mtb replication Given that LRRK2 is highly expressed in macrophages and several human genetic studies linked LRRK2 and mycobacterial diseases (Zhang et al, 2009; Wang et al, 2015, 2018; Fava et al, 2016), we investigated the effect of LRRK2 on Mtb infection using bone marrow-derived mouse macrophages (BMDMs) from LRRK2 KO mice. LRRK2 KO macrophages were able to control Mtb replication significantly better (Fig 1A). Confirming this result in human cells, LRRK2 KO human-induced pluripotent stem cell-derived macrophages (iPSDM) also significantly restricted Mtb replication (Fig 1A). Notably, treatment of both BMDM and iPSDM with the LRRK2 kinase inhibitor GSK2578215A (Fig EV1A) significantly restricted Mtb replication (Fig 1B), indicating that inhibition of the LRRK2 kinase activity enhanced Mtb control by macrophages. The improved control of Mtb was not due to enhanced macrophage toxicity in LRRK2 KO or GSK2578215A-treated macrophages (Fig EV1B and C). Interestingly, the secretion of the pro-inflammatory cytokines TNF-α and IL-6 was not altered in LRRK2 KO or GSK2578215A-treated BMDMs infected with Mtb (Fig 1C and D), whilst IL-10 secretion was significantly down-regulated (Fig 1C and D). In agreement with an enhanced Mtb control in LRRK2 KO macrophages, the percentage of Mtb positive for the late endocytic/lysosomal marker LAMP-1 was at least twofold higher in LRRK2 KO than in WT macrophages (Fig 1E and F). Moreover, the fraction of Mtb in proteolytic phagosomes was higher in LRRK2 KO macrophages as measured by the activity of the lysosomal enzyme cathepsin L (Fig 1G and H). Consistently, GSK2578215A treatment also resulted in a remarkable increase of co-localisation between LAMP-1 as well as active cathepsin L with Mtb (Fig 1I–L). Similar results were obtained in the mouse macrophage cell line RAW264.7 (Fig EV1D–F). In contrast, macrophages harbouring the LRRK2 gain-of-function mutation G2019S were more susceptible to Mtb replication (Fig 1M) and showed a reduction in lysosomal targeting of Mtb to phagolysosomes as measured by LAMP-1 recruitment (Fig 1N and O). An image-based approach (Schnettger et al, 2017) confirmed reduced Mtb growth in LRRK2 KO macrophages and enhanced growth in G2019S KI macrophages (Fig EV1G and H). Moreover, IFN-γ activation and control of Mtb was not synergistic (Fig EV1I) and enhanced Mtb phagosome maturation was not due to a general defect in late endosomal morphology (LAMP-1) or CtsL activity (Fig EV2). In contrast to LRRK2 KO BMDM, G2019S KI BMDM showed reduced secretion of both TNF-α and IL-6 with increased secretion of IL-10 (Fig 1O). Altogether, these results indicate that LRRK2 and its kinase activity affect not only Mtb replication in both human and mouse macrophages by regulating phagosome maturation but also cytokine responses. Figure 1. Loss of LRRK2 activity targets Mtb to phagolysosomes and limits Mtb replication CFU from WT and LRRK2 KO mouse bone marrow-derived macrophages (BMDMs) and WT and LRRK2 KO human-induced pluripotent stem cell-derived macrophages (iPSDM). One representative experiment out of four, data show mean ± SEM of technical replicates. CFU from WT BMDM and WT iPSDM treated with 1 μM GSK2578215A. For clarity, controls are the same as for panel (A). One representative experiment out of two, data show mean ± SEM of technical replicates. Cytokine secretion levels in BMDM measured by ELISA at the indicated MOI. One representative experiment out of three shown. Cytokine secretion levels in BMDM infected with Mtb (MOI = 5) and treated with 0.5; 1 and 3 μM GSK2578215A as measured by ELISA. One representative experiment out of two shown. Representative images of WT and LRRK2 KO BMDM infected with Mtb-eGFP at 24 h after infection and stained for LAMP-1. Nuclei were labelled with DAPI. Scale bar = 10 μm. Quantification of LAMP-1 co-localisation with Mtb as in panel (E). Data show three independent experiments. Representative images of WT and LRRK2 KO BMDM infected with Mtb-eGFP at 24 h after infection and incubated with a substrate specific for cathepsin L as described in methods. Nuclei were labelled with DAPI. Scale bar = 10 μm. Quantification of cathepsin L co-localisation with Mtb from panel (G). Data show four independent experiments. Representative images of WT BMDM infected with Mtb-eGFP, treated with either DMSO (control) or 1 μM GSK2578215A at 24 h after infection and stained for LAMP-1. Nuclei were labelled with DAPI. Scale bar = 5 μm. Quantification of LAMP-1 co-localisation with Mtb as in panel (I). Data show three independent experiments. Representative images of cathepsin L-stained WT BMDM as in panel (I). Scale bar = 10 μm. Quantification of cathepsin L co-localisation with Mtb from panel (K). Data show two independent experiments. CFU from WT and G2019S LRRK2 KI BMDM. One representative experiment out of two shown. LAMP-1 co-localisation with Mtb was quantified as in panels (F and J). Cytokine secretion levels in BMDM measured by ELISA at the indicated MOI. One representative experiment out of two shown. Data information: All data show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ns not significant. Panels (A, B, M) t-test adjusted for multiple comparison; panels (C, D, O) one-way ANOVA with Holm-Sidak post-test, panels (F, H, J, L, N) t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Loss of LRRK2 activity targets Mtb to phagolysosomes and limits Mtb replication A. Confirmation of efficient LRRK2 kinase inhibition by GSK2578215A 24 h after infection of BMDM or iPSDM with Mtb. Representative image of whole cell lysate Western blotted for LRRK2 pS935, total LRRK2 and α-tubulin. B, C. LDH assay performed at 72 h post-infection as toxicity control for CFU experiments in Fig 1. Data show mean ± SD from three technical replicates. LDH assays were routinely performed for CFU and ELISA experiments. D. CFUs in RAW264.7 cells pre-treated with GSK2578215A (1 μM) or DMSO control for 2 h and infected with Mtb-eGFP (MOI = 1). *P < 0.5, ***P < 0.001 by Student's t-test corrected for multiple comparison. Data show mean ± SD. One out of two experiments shown. E. RAW264.7 cells were pre-treated with 1 μM GSK2578215A or DMSO (Control) for 2 h, and LAMP-1 recruitment at 24 h post-infection was assessed using confocal microscopy. F. Quantification of panel (E). Data show mean ± SEM from three independent experiments. **P < 0.01 by Student's t-test. G. Mtb growth analysed by single cell imaging in WT, LRRK2 KO and LRRK2 G2019S KI BMDM. Data show mean ± SEM from three independent experiments. ***P < 0.001 by Student's t-test corrected for multiple comparisons. H. Mtb growth analysed by single cell imaging in WT, LRRK2 G2019S KI BMDM and LRRK2 G2019S KI BMDM treated with GSK2578215A for 2 h. Data show mean ± SEM from three independent experiments. **P < 0.001 by Student's t-test corrected for multiple comparisons. I. CFU in WT BMDM, WT BMDM treated with GSK2578215A and LRRK2 KO BMDM left untreated or pre-activated with IFN-γ (100 U/ml over night). Data show mean ± SD from technical replicates. One representative experiment out of three experiments shown. ***P < 0.001 by Student's t-test corrected for multiple comparison. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. LRRK2 KO has no major impact on lysosomal morphology Representative images of WT and LRRK2 KO BMDM incubated with cathepsin L probe for 30 min. Quantification of (A), showing average lysosomal size and numbers. Each dot represents a single cell. Representative images of WT and LRRK2 KO BMDMs stained for LAMP-1. Quantification of (C), showing average lysosomal size and numbers. Each dot represents a single cell. Data information: Data show mean ± SEM from three independent experiments. ns = not significant by Student's t-test. Download figure Download PowerPoint LRRK2 does not alter autophagic targeting of Mycobacterium tuberculosis LRRK2 inhibition has been described to induce autophagy (Manzoni et al, 2013), a process which targets Mtb to phagolysosomes (Gutierrez et al, 2004). Therefore, we investigated whether LRRK2 KO resulted in Mtb growth restriction via the induction of autophagy. As reported previously, LRRK2 kinase inhibition induced LC3BII accumulation and reduced p62 levels over time (Fig EV3A and B), indicative for autophagy induction. Additionally, we saw a minor but reproducible increase in LC3BII levels in LRRK2 KO macrophages (Fig EV3B and C). However, this increase seemed to be independent of autophagic flux as there were no differences in LC3BII accumulation after Bafilomycin A1 treatment between WT BMDMs, BMDMs treated with GSK2578215A or LRRK2 KO BMDMs (Fig 2A and B). In agreement with these data, there were no differences between WT and LRRK2 KO BMDM in LC3B processing and p62 degradation after induction of autophagy with IFN-γ or nutrient starvation (Fig EV3D and E). Mtb itself is able to block autophagic flux in macrophages. Concordantly, we detected LC3BII and p62 accumulation in infected WT macrophages over time. However, we did not observe any differences between WT and LRRK2 KO macrophages, indicating that LRRK2 KO is not able to overcome the Mtb-induced block in autophagic flux (Fig 2C and D). Given that we observed increased autophagy in infected macrophages, we analysed the targeting of Mtb to LC3B- or p62-positive compartments. There was targeting of Mtb to LC3B- and p62-positive compartments; however, there were no significant differences in the number of Mtb positive for LC3B and p62 across WT, LRRK2 KO and G2019S KI macrophages (Fig 2E and H). We concluded that although LRRK2 function alters autophagy, it does not regulate Mtb targeting to autophagosomes in macrophages. Click here to expand this figure. Figure EV3. Role of LRRK2 in autophagy regulation WT BMDMs were treated with 1 μM GSK2578215A, and p62 and LC3B levels were monitored by Western blotting over time. α-tubulin was used as a loading control. WT and LRRK2 KO BMDMs were treated or not with 1 μM GSK2578215A and p62 and LC3B levels were monitored by Western blotting at 24 h after treatment. α-Tubulin was used as a loading control. Quantification of (B). Each dot represents an independent experiment. Data show mean + SEM analysed by Student's t-test. WT and LRRK2 KO BMDMs were treated with 100 U/ml IFN-γ, and p62 and LC3B levels were monitored by Western blotting over time. α-Tubulin was used as a loading control. WT and LRRK2 KO BMDMs were left in full medium or starved in HBSS for 4 h, and p62 and LC3B levels were monitored by Western blotting. α-Tubulin was used as a loading control. Download figure Download PowerPoint Figure 2. LRRK2 does not affect xenophagic targeting of Mycobacterium tuberculosis WT and LRRK2 KO BMDM treated with DMSO (control) or 1 μM GSK2578215A for 2 h were treated with 100 nM Bafilomycin A1 for 4 h. Whole cell lysates were analysed by Western blotting for LC3B and α-tubulin. Densitometry quantification of panel (A). WT and LRRK2 KO BMDMs were infected with Mtb at MOI = 1 and whole cell lysates were analysed by Western blotting for p62, LC3BII and α-tubulin levels. Quantification of panel (C). Data show mean ± SEM of three independent experiments. WT, LRRK2 KO and G2019S KI BMDMs were infected with Mtb-GFP at MOI of 1 for 24 h. Recruitment of LC3B was analysed by immunofluorescence. Scale bars = 10 μm. Quantitative analysis of panel (E). WT, LRRK2 KO and G2019S KI BMDMs were infected with Mtb-GFP at MOI of 1 for 24 h. Recruitment of p62 was analysed by immunofluorescence. Quantitative analysis of panel (G). Data information: Data show mean ± SEM. Each dot represents an independent experiment. Data in panels (F and H) were analysed using a one-way ANOVA. ns: not significant. Download figure Download PowerPoint Loss of LRRK2 activity enhances phagosome maturation in macrophages Because the loss of LRRK2 activity targeted Mtb to phagolysosomes independently of autophagy, we next investigated the functional role of LRRK2 in phagosome maturation. For this, we measured the intra-phagosomal proteolysis by fluorescence-based assays with latex beads in real time (Yates & Russell, 2008) in both WT and LRRK2 KO BMDM. Notably, LRRK2 KO macrophages showed an enhanced phagosome proteolysis compared to WT macrophages (Fig 3A) without affecting phagocytic uptake (Fig EV4). In order to identify the LRRK2-dependent cellular pathways that enhanced phagosome maturation, we performed a proteomics analysis of isolated latex bead phagosomes (Trost et al, 2009) from WT and LRRK2 KO macrophages (Fig 3B, Table EV1). Strikingly, the gene ontology analysis of significantly up-regulated proteins from the LRRK2 KO phagosomes revealed a strong enrichment for proteins associated with late endocytic/lysosomal compartments and hydrolytic activity (Fig 3B, Table EV2). This analysis revealed an increase in the content of lysosomal hydrolases such as cathepsins and lysozyme-C in LRRK2 KO phagosomes when compared to WT phagosomes (Fig 3C). In agreement with the proteomic data, Western blot analysis showed that phagosomes recruited LRRK2 and LRRK2 KO phagosomes were associated with high levels of active cathepsin D (Fig 3D). We next analysed whether LRRK2 kinase activity was required for phagosomal function by using four structurally diverse, highly specific LRRK2 kinase inhibitors, namely HG10-102-01 (Choi et al, 2012), GSK2578215A (Reith et al, 2012), LRRK2-IN-1 (Deng et al, 2011) and MLI-2 (Fell et al, 2015). All inhibitors significantly enhanced the proteolytic activity of phagosomes at different levels, indicating that the kinase activity negatively regulates phagosome maturation (Fig 3E). As expected, in macrophages from LRRK2 A2016T KI, a mutant that is active, but resistant to the inhibitors (Nichols et al, 2009), MLI-2 did not enhance phagosome proteolysis (Fig 3F). Next, we tested whether enhanced kinase activity affects phagosome function by using macrophages harbouring the PD pathogenic mutation G2019S, which enhances LRRK2 kinase activity about fourfold (West et al, 2005). Indeed, phagosomes from LRRK2 G2019S knock-in (KI) macrophages displayed reduced proteolytic activity when compared to WT macrophages (Fig 3G). This reduction in proteolytic activity observed in phagosomes from LRRK2 G2019S KI macrophages was reverted by the LRRK2 kinase inhibitor GSK2578215A in a dose-dependent manner (Fig 3H). Taken together, our data show that LRRK2 kinase activity acts as a negative regulator of phagosomal function in macrophages. Figure 3. Loss of LRRK2 activity enhances phagosome maturation in macrophages Intra-phagosomal proteolysis in WT and LRRK2 KO BMDM. Cells pre-treated with 100 nM leupeptin for 1 h were used as a negative control of proteolysis. One representative experiment out of three shown. Gene Ontology (GO) enrichment of cellular components and molecular functions of significantly up-regulated proteins in the proteome of LRRK2 KO-derived phagosomes compared to WT. Mass spectrometry analysis of LRRK2 KO phagosomes compared to WT. Data show mean ± SEM of three biological replicates. Isolated phagosomes and total cell lysates (TLC) from WT and LRRK2 KO BMDM were blotted for LRRK2, cathepsin D and Rab7 as a loading control. Data are representative of two independent experiments. Intra-phagosomal proteolysis of WT BMDM pre-treated or not with 1 μM of the LRRK2 protein kinase inhibitors HG10-102-01, GSK2578215A or LRRK2-IN1. One representative experiment out of three shown. Intra-phagosomal proteolysis of WT or LRRK2 A2016T KI BMDM pre-treated or not (DMSO control) with 1 μM of the LRRK2 protein kinase inhibitor MLI-2. One representative experiment out of three shown. Intra-phagosomal proteolysis of WT, LRRK2 KO and LRRK2 G2019S KI BMDM. One representative experiment out of three shown. Intra-phagosomal proteolysis of WT and LRRK2 G2019S KI BMDM pre-treated or not with 0.3, 1.0 and 3.0 μM GSK2578215A LRRK2 kinase inhibitor. Data show mean ± SEM and are representative of three independent biological replicates. Data information: Shaded areas represent standard error of mean (SEM). Download
The vertebrate blood-brain barrier (BBB) is critical for ensuring the maintenance of brain homeostasis, whilst protecting the brain against toxic insults. Various pathological events disrupt BBB integrity, holding several important clinical implications. In instances where the normal mechanisms controlling passage of substances into the brain are compromised, these could sensitize or even worsen endogenous pathological conditions. Recognition has grown recently that patients diagnosed with Parkinson's disease (PD) present with concurrent medical problems, including cerebrovascular lesions. However, cerebrovascular disturbances may also result from PD-related disease processes; the pathological mechanisms which could entail interaction between environment-derived and genetic factors. The current review addresses the accumulation of studies aimed at better understanding the series of processes affecting the neurovascular unit in human Parkinsonism, due in part to the BBB presenting as a formidable opponent in the effective delivery of therapeutics that have shown promise as therapeutic strategies for treating aspects of PD when tested in vitro.
Phagocytosis of pathogens, apoptotic cells and debris is a key feature of macrophage function in host defense and tissue homeostasis. Quantification of macrophage phagocytosis in vitro has traditionally been technically challenging. Here we report the optimization and validation of the IncuCyte ZOOM® real time imaging platform for macrophage phagocytosis based on pHrodo® pathogen bioparticles, which only fluoresce when localized in the acidic environment of the phagolysosome. Image analysis and fluorescence quantification were performed with the automated IncuCyte™ Basic Software. Titration of the bioparticle number showed that the system is more sensitive than a spectrofluorometer, as it can detect phagocytosis when using 20× less E. coli bioparticles. We exemplified the power of this real time imaging platform by studying phagocytosis of murine alveolar, bone marrow and peritoneal macrophages. We further demonstrate the ability of this platform to study modulation of the phagocytic process, as pharmacological inhibitors of phagocytosis suppressed bioparticle uptake in a concentration-dependent manner, whereas opsonins augmented phagocytosis. We also investigated the effects of macrophage polarization on E. coli phagocytosis. Bone marrow-derived macrophage (BMDM) priming with M2 stimuli, such as IL-4 and IL-10 resulted in higher engulfment of bioparticles in comparison with M1 polarization. Moreover, we demonstrated that tolerization of BMDMs with lipopolysaccharide (LPS) results in impaired E. coli bioparticle phagocytosis. This novel real time assay will enable researchers to quantify macrophage phagocytosis with a higher degree of accuracy and sensitivity and will allow investigation of limited populations of primary phagocytes in vitro.
We utilized a mice model of Parkinsonism: (1) to evaluate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity; and (2) to evaluate whether manganese (Mn) exposure can affect MPTP-induced neurotoxicity. A 2 × 3 experimental design (MPTP ×± Mn) was as follows: SS, MPTP(-) × Mn(-); SLMn, MPTP(-) × low Mn(+); SHMn, MPTP(-) × high Mn(+); MpS, MPTP(+) × Mn(-); MpLMn, MPTP(+) × low Mn(+); MpHMn, MPTP(+) × high Mn(+). We administered MPTP (30 mg/kg per day) to male C57BL/6 mice intraperitoneally, once a day for 5 days. Subsequently, mice were treated with either 2 or 8 mg/kg of MnCl 2 .4H 2 O intraperitoneally, once a day for 3 weeks. Blood and striatal Mn levels were elevated in the Mnexposed groups. The number of tyrosine hydroxylase (TH)-immunoreactive (ir) neurons in the substantia nigra pars compacta were decreased significantly in the MPTP-exposed groups. The densities of TH-ir axon terminals in caudate-putamen (CPU) were significantly decreased in the MPTP-treated groups. However, Mn treatment did not affect MPTP neurotoxicity. The densities of glial fibrillary acidic protein (GFAP)-ir astrocytes in the CPU or globus pallidus were significantly increased in the MPTP-treated groups. Concentrations of dopamine in the striatum were decreased significantly in the MPTP-exposed groups only, but Mn had no effect.
Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are found in familial and idiopathic cases of Parkinson's disease (PD), but are also associated with immune-related disorders, notably Crohn's disease and leprosy. Although the physiological function of LRRK2 protein remains largely elusive, increasing evidence suggests that it plays a role in innate immunity, a process that also has been implicated in neurodegenerative diseases, including PD. Innate immunity involves macrophages and microglia, in which endogenous LRRK2 expression is precisely regulated and expression is strongly up-regulated upon cell activation. This brief report discusses the current understanding of the involvement of LRRK2 in innate immunity particularly in relation to PD, critically examining its role in myeloid cells, particularly macrophages and microglia.
To investigate the potential benefits which may arise from pseudotyping the HIV-1 lentiviral vector with its homologous gp41 envelope glycoprotein (GP) cytoplasmic tail (CT), we created chimeric RVG/HIV-1gp41 GPs composed of the extracellular and transmembrane sequences of RVG and either the full-length gp41 CT or C terminus gp41 truncations sequentially removing existing conserved motifs. Lentiviruses (LVs) pseudotyped with the chimeric GPs were evaluated in terms of particle release (physical titer), biological titers, infectivity, and in vivo central nervous system (CNS) transduction. We report here that LVs carrying shorter CTs expressed higher levels of envelope GP and showed a higher average infectivity than those bearing full-length GPs. Interestingly, complete removal of GP CT led to vectors with the highest transduction efficiency. Removal of all C-terminal gp41 CT conserved motifs, leaving just 17 amino acids (aa), appeared to preserve infectivity and resulted in a significantly increased physical titer. Furthermore, incorporation of these 17 aa in the RVG CT notably enhanced the physical titer. In vivo stereotaxic delivery of LV vectors exhibiting the best in vitro titers into rodent striatum facilitated efficient transduction of the CNS at the site of injection. A particular observation was the improved retrograde transduction of neurons in connected distal sites that resulted from the chimeric envelope R5 which included the "Kennedy" sequence (Ken) and lentivirus lytic peptide 2 (LLP2) conserved motifs in the CT, and although it did not exhibit a comparable high titer upon pseudotyping, it led to a significant increase in distal retrograde transduction of neurons.In this study, we have produced novel chimeric envelopes bearing the extracellular domain of rabies fused to the cytoplasmic tail (CT) of gp41 and pseudotyped lentiviral vectors with them. Here we report novel effects on the transduction efficiency and physical titer of these vectors, depending on CT length and context. We also managed to achieve increased neuronal transduction in vivo in the rodent CNS, thus demonstrating that the efficiency of these vectors can be enhanced following merely CT manipulation. We believe that this paper is a novel contribution to the field and opens the way for further attempts to surface engineer lentiviral vectors and make them more amenable for applications in human disease.
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