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Opposite microglial activation stages upon loss of PGRN or TREM 2 result in reduced cerebral glucose metabolism

2019; Springer Nature; Volume: 11; Issue: 6 Linguagem: Inglês

10.15252/emmm.201809711

1757-4684

Julia K Götzl, Matthias Brendel, Georg Werner, Samira Parhizkar, Laura Sebastián Monasor, Gernot Kleinberger, Alessio-Vittorio Colombo, Maximilian Deußing, Matias Wagner, Juliane Winkelmann, Janine Diehl‐Schmid, Johannes Levin, Katrin Fellerer, Anika Reifschneider, Sebastian Bultmann, Peter Bartenstein, Axel Rominger, Sabina Tahirović, Scott T. Smith, Charlotte Madore, Oleg Butovsky, Anja Capell, Christian Haass,

Inflammation biomarkers and pathways

Research Article23 May 2019Open Access Source DataTransparent process Opposite microglial activation stages upon loss of PGRN or TREM2 result in reduced cerebral glucose metabolism Julia K Götzl Julia K Götzl Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Matthias Brendel Matthias Brendel Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Georg Werner Georg Werner Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Samira Parhizkar Samira Parhizkar Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Laura Sebastian Monasor Laura Sebastian Monasor German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Gernot Kleinberger Gernot Kleinberger orcid.org/0000-0002-5811-8226 Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Alessio-Vittorio Colombo Alessio-Vittorio Colombo German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Maximilian Deussing Maximilian Deussing Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Matias Wagner Matias Wagner Institut für Neurogenomik, Helmholtz Zentrum München, Munich, Germany Institut of Human Genetics, Technische Universität München, Munich, Germany Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Juliane Winkelmann Juliane Winkelmann Institut für Neurogenomik, Helmholtz Zentrum München, Munich, Germany Institut of Human Genetics, Technische Universität München, Munich, Germany Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Janine Diehl-Schmid Janine Diehl-Schmid Department of Psychiatry, Technische Universität München, Munich, Germany Search for more papers by this author Johannes Levin Johannes Levin German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Department of Neurology, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Katrin Fellerer Katrin Fellerer Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Anika Reifschneider Anika Reifschneider Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Sebastian Bultmann Sebastian Bultmann Department of Biology and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians-Universität München, Munich, Germany Search for more papers by this author Peter Bartenstein Peter Bartenstein Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Axel Rominger Axel Rominger Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Sabina Tahirovic Sabina Tahirovic orcid.org/0000-0003-4403-9559 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Scott T Smith Scott T Smith Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Charlotte Madore Charlotte Madore Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Oleg Butovsky Oleg Butovsky Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA Evergrande Center for Immunologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Anja Capell Corresponding Author Anja Capell [email protected] orcid.org/0000-0003-3118-911X Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Christian Haass Corresponding Author Christian Haass [email protected] orcid.org/0000-0002-4869-1627 Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Julia K Götzl Julia K Götzl Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Matthias Brendel Matthias Brendel Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Georg Werner Georg Werner Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Samira Parhizkar Samira Parhizkar Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Laura Sebastian Monasor Laura Sebastian Monasor German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Gernot Kleinberger Gernot Kleinberger orcid.org/0000-0002-5811-8226 Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Alessio-Vittorio Colombo Alessio-Vittorio Colombo German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Maximilian Deussing Maximilian Deussing Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Matias Wagner Matias Wagner Institut für Neurogenomik, Helmholtz Zentrum München, Munich, Germany Institut of Human Genetics, Technische Universität München, Munich, Germany Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Juliane Winkelmann Juliane Winkelmann Institut für Neurogenomik, Helmholtz Zentrum München, Munich, Germany Institut of Human Genetics, Technische Universität München, Munich, Germany Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Janine Diehl-Schmid Janine Diehl-Schmid Department of Psychiatry, Technische Universität München, Munich, Germany Search for more papers by this author Johannes Levin Johannes Levin German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Department of Neurology, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Katrin Fellerer Katrin Fellerer Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Anika Reifschneider Anika Reifschneider Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Sebastian Bultmann Sebastian Bultmann Department of Biology and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians-Universität München, Munich, Germany Search for more papers by this author Peter Bartenstein Peter Bartenstein Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Axel Rominger Axel Rominger Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Sabina Tahirovic Sabina Tahirovic orcid.org/0000-0003-4403-9559 German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Scott T Smith Scott T Smith Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Charlotte Madore Charlotte Madore Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Oleg Butovsky Oleg Butovsky Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA Evergrande Center for Immunologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Search for more papers by this author Anja Capell Corresponding Author Anja Capell [email protected] orcid.org/0000-0003-3118-911X Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany Search for more papers by this author Christian Haass Corresponding Author Christian Haass [email protected] orcid.org/0000-0002-4869-1627 Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Munich Cluster for Systems Neurology (SyNergy), Munich, Germany Search for more papers by this author Author Information Julia K Götzl1,‡, Matthias Brendel2,‡, Georg Werner1,‡, Samira Parhizkar1, Laura Sebastian Monasor3, Gernot Kleinberger1,4, Alessio-Vittorio Colombo3, Maximilian Deussing2, Matias Wagner5,6,7, Juliane Winkelmann5,6,7, Janine Diehl-Schmid8, Johannes Levin3,9, Katrin Fellerer1, Anika Reifschneider1, Sebastian Bultmann10, Peter Bartenstein2,4, Axel Rominger2,4, Sabina Tahirovic3, Scott T Smith11, Charlotte Madore11, Oleg Butovsky11,12, Anja Capell *,1 and Christian Haass *,1,3,4 1Chair of Metabolic Biochemistry, Biomedical Center (BMC), Faculty of Medicine, Ludwig-Maximilians-Universität München, Munich, Germany 2Department of Nuclear Medicine, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany 3German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 4Munich Cluster for Systems Neurology (SyNergy), Munich, Germany 5Institut für Neurogenomik, Helmholtz Zentrum München, Munich, Germany 6Institut of Human Genetics, Technische Universität München, Munich, Germany 7Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany 8Department of Psychiatry, Technische Universität München, Munich, Germany 9Department of Neurology, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany 10Department of Biology and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians-Universität München, Munich, Germany 11Ann Romney Center for Neurologic Diseases, Department of Neurology, Brigham and Women′s Hospital, Harvard Medical School, Boston, MA, USA 12Evergrande Center for Immunologic Diseases, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Tel: +4989440046535; E-mail: [email protected] *Corresponding author. Tel: +4989440046550; E-mail: [email protected] EMBO Mol Med (2019)11:e9711https://doi.org/10.15252/emmm.201809711 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 Microglia adopt numerous fates with homeostatic microglia (HM) and a microglial neurodegenerative phenotype (MGnD) representing two opposite ends. A number of variants in genes selectively expressed in microglia are associated with an increased risk for neurodegenerative diseases such as Alzheimer's disease (AD) and frontotemporal lobar degeneration (FTLD). Among these genes are progranulin (GRN) and the triggering receptor expressed on myeloid cells 2 (TREM2). Both cause neurodegeneration by mechanisms involving loss of function. We have now isolated microglia from Grn−/− mice and compared their transcriptomes to those of Trem2−/− mice. Surprisingly, while loss of Trem2 enhances the expression of genes associated with a homeostatic state, microglia derived from Grn−/− mice showed a reciprocal activation of the MGnD molecular signature and suppression of gene characteristic for HM. The opposite mRNA expression profiles are associated with divergent functional phenotypes. Although loss of TREM2 and progranulin resulted in opposite activation states and functional phenotypes of microglia, FDG (fluoro-2-deoxy-d-glucose)-μPET of brain revealed reduced glucose metabolism in both conditions, suggesting that opposite microglial phenotypes result in similar wide spread brain dysfunction. Synopsis Microglia from Grn−/− & Trem2−/− mice display opposite molecular signatures. While microglia are either locked in a hyperactivated or homeostatic state, Grn−/− & Trem2−/− mice both show reduced glucose metabolism, suggesting that opposite microglial phenotypes result in similar brain dysfunction. First demonstration that microglia from both extremes of their functional stages cause brain wide dysfunctions. This study indicates that the therapeutic window for microglial modulation is rather narrow and care must be taken to balance microglial activity. Introduction While for a long time researchers distinguished only two distinct stages of microglia, the M1 and the M2 phenotype, recent evidence strongly indicates that a multitude of functionally diverse microglial populations exists in a dynamic equilibrium (Ransohoff, 2016; Keren-Shaul et al, 2017; Krasemann et al, 2017). This becomes very apparent when one compares mRNA signatures of microglia isolated from various mouse models for neurodegeneration and compares them to controls (Abduljaleel et al, 2014; Butovsky et al, 2015; Holtman et al, 2015; Keren-Shaul et al, 2017; Krasemann et al, 2017). In mouse models for neurodegenerative diseases, mRNA signatures were identified which are characteristic for a disease-associated microglia (DAM)/a microglial neurodegenerative phenotype (MGnD) whereas in controls a homeostatic microglial (HM) signature was observed (Butovsky et al, 2014, 2015; Holtman et al, 2015; Keren-Shaul et al, 2017; Krasemann et al, 2017). MGnD upregulates a characteristic set of genes, which may initially allow microglia to respond to neuronal injury in a defensive manner. This includes the induction of pathways triggering phagocytosis, chemotaxis/migration, and cytokine release. The upregulation of genes involved in those pathways goes along with a suppression of homeostatic genes (Butovsky et al, 2015; Krasemann et al, 2017). Key genes involved in the switch of HM to MGnD are regulated by the TREM2 (triggering receptor expressed on myeloid cells 2) ApoE (apolipoprotein E) pathway (Krasemann et al, 2017). A pivotal role of microglia in neurodegeneration is strongly supported by the identification of sequence variants found in a number of genes robustly or even selectively expressed within microglia in the brain, among them GRN (encoding the progranulin (PGRN) protein; Baker et al, 2006; Cruts & Van Broeckhoven, 2008; Zhang et al, 2014; Gotzl et al, 2016; Lui et al, 2016) and TREM2 (Guerreiro et al, 2013; Jonsson & Stefansson, 2013; Jonsson et al, 2013; Rayaprolu et al, 2013; Borroni et al, 2014; Cuyvers et al, 2014; Ulrich et al, 2017). Mutations in the GRN gene (Baker et al, 2006; Cruts et al, 2006) cause frontotemporal lobar degeneration (FTLD) with TDP-43 (TAR-DNA binding protein 43)-positive inclusions due to haploinsufficiency. PGRN is a growth factor-like protein with neurotrophic properties in the brain (Van Damme et al, 2008). PGRN is also transported to lysosomes (Hu et al, 2010; Zhou et al, 2015) where it appears to regulate expression and activity of lysosomal proteins (Ahmed et al, 2010; Hu et al, 2010; Wils et al, 2012; Tanaka et al, 2013a,b; Gotzl et al, 2014, 2016, 2018; Beel et al, 2017; Chang et al, 2017; Ward et al, 2017; Zhou et al, 2017). PGRN is proteolytically processed into granulin peptides, which can be found in biological fluids (Bateman et al, 1990; Shoyab et al, 1990; Belcourt et al, 1993; Cenik et al, 2012). TREM2 is produced as a membrane-bound type-1 protein (Kleinberger et al, 2014), which traffics to the cell surface where it mediates signaling via binding to its co-receptor, the DNAX activation protein of 12 kDa (DAP12; Ulrich & Holtzman, 2016; Yeh et al, 2017). Signaling is terminated by proteolytic shedding of the TREM2 ectodomain (Kleinberger et al, 2014; Schlepckow et al, 2017). Several sequence variants associated with TREM2 cause neurodegeneration via a loss of function (Kleinberger et al, 2014, 2017; Schlepckow et al, 2017; Ulland et al, 2017; Song et al, 2018). Sequence variants of TREM2 affect a multitude of functions including chemotaxis, migration, survival, binding of phospholipids and ApoE, proliferation, survival, and others (Kleinberger et al, 2014, 2017; Atagi et al, 2015; Bailey et al, 2015; Wang et al, 2015; Yeh et al, 2016; Mazaheri et al, 2017; Ulland et al, 2017). Strikingly, a loss of TREM2 function locks microglia in a homeostatic state (Krasemann et al, 2017; Mazaheri et al, 2017). Instead of suppressing their homeostatic mRNA signature like mouse models of neurodegenerative disorders, in the absence of TREM2 microglia even enhance expression of homeostatic genes and fail to express the disease-associated signature (Krasemann et al, 2017; Mazaheri et al, 2017). As a result, TREM2 deficiency decreases chemotaxis, phagocytosis, and barrier function (Kleinberger et al, 2017; Mazaheri et al, 2017; Ulland et al, 2017). TREM2 therefore appears to play a key role as a central hub gene in the regulation of microglial homeostasis. We now investigated microglial gene expression and function in the absence of PGRN and made the surprising observation that loss of TREM2 or PGRN leads to opposite microglial activity phenotypes, which, however, both cause wide spread brain dysfunction. Results Opposite molecular signatures of microglia in Grn−/− and Trem2−/− mice Loss-of-function mutations in TREM2 are associated with various types of neurodegeneration, including a FTLD-like syndrome (Ulrich & Holtzman, 2016). Similarly, haploinsufficiency of GRN is associated with TDP-43-positive FTLD (Baker et al, 2006; Cruts et al, 2006; Cruts & Van Broeckhoven, 2008). At least some of the GRN-dependent FTLD-associated phenotypes can be mimicked in a mouse model entirely lacking PGRN (Ahmed et al, 2010; Yin et al, 2010; Wils et al, 2012; Gotzl et al, 2014). Furthermore, both, a Trem2 knockout and the knockin of the p.T66M mutation, mimic features of a FTD-like syndrome (Kleinberger et al, 2017; Mazaheri et al, 2017). Since both proteins are preferentially expressed in microglia, we compared loss-of-PGRN-associated microglial phenotypes with those known for TREM2 deficiency (Krasemann et al, 2017; Mazaheri et al, 2017). To do so, we first purified microglia from brains of adult Grn−/− mice by fluorescence-associated cell sorting (FACS) using microglia-specific anti-FCRLS and anti-CD11b antibodies. We then investigated the expression pattern of gene characteristic for MGnD and HM using NanoString gene expression profiling (MG534; Butovsky et al, 2014; Krasemann et al, 2017; Mazaheri et al, 2017; Dataset EV1). Gene expression levels in each sample were normalized against the geometric mean of five housekeeping genes including Cltc, Gapdh, Gusb, Hprt1, and Tubb5. Out of 529 genes analyzed, 58 genes were significantly upregulated and 58 genes were downregulated. Strikingly, genes most strongly upregulated in Grn−/− microglia are those previously described for MGnD (Fig 1A; Butovsky et al, 2015; Holtman et al, 2015; Keren-Shaul et al, 2017; Krasemann et al, 2017). These include ApoE, as the most upregulated gene, Ly9, Clec7a, Dnajb4, Cccl4, and many others suggesting that PGRN-deficient microglia adopt the MGnD state. We then compared the Grn−/− microglial signature to the previously analyzed molecular signature of Trem2−/− microglia (Fig 1B and C; Mazaheri et al, 2017). Both NanoString gene expression panels overlapped in 418 genes. Out these, 359 mRNAs could be detected in both screens (Dataset EV1). In the Grn−/− microglia, 40 mRNAs were upregulated and 55 mRNAs were downregulated, whereas in the Trem2−/− microglia, 87 mRNAs were increased and 27 mRNAs were decreased (Fig 1C; left two panels). Interestingly, while only six mRNAs were equally up/downregulated in both phenotypes, 32 mRNAs showed an opposite regulation (Fig 1C; right panel). Strikingly, while in Grn−/− mice the neurodegenerative disease-associated signature is massively upregulated, this set of genes is suppressed in the Trem2−/− microglia (Fig 1B and D; Mazaheri et al, 2017). Similarly, the homeostatic mRNA signature is slightly but significantly upregulated in Trem2−/− microglia (Mazaheri et al, 2017) but severely suppressed in Grn−/− microglia (Fig 1B and D). Figure 1. Opposite mRNA signatures of Grn−/−/Trem2+/+ and Trem2−/−/Grn+/+ microglia A. Volcano blot representation of the differently expressed genes in FCRLS- and CD11b-positive Grn−/− microglia in comparison with wt microglia isolated from 5.5-month-old mice (male, n = 5). 116 genes out of 529 genes analyzed are significantly changed, and from these, 58 genes are upregulated and 58 genes are downregulated. Eight up- and down regulated genes with the highest fold change are indicated. B. Heatmap of significantly affected genes (P < 0.05) in FCRLS- and CD11b-positive Grn−/− microglia in comparison with Grn+/+ microglia isolated from 5.5-month-old mice (n = 5 per genotype). For the significantly affected genes of the Grn−/− microglia, mRNA expression data of the Trem2−/− microglia in comparison with the corresponding wt microglia were taken from previously published data (Mazaheri et al, 2017). The RNA counts for each gene and sample were normalized to the mean value of wt followed by a log2 transformation (n ≥ 5 per genotype). *labeled genes were not analyzed or below detection limit in Trem2−/− microglial mRNA expression dataset (Mazaheri et al, 2017). C. Changes in gene expression of 359 detected genes in Grn−/− microglia and Trem2−/− microglia. Note that from the 38 genes significantly altered in both genotypes, 32 genes are regulated in opposite direction. D. Expression levels of significantly altered homeostatic and MGnD genes of Grn−/− and Trem2−/− microglia from the data set in (B). Gene expression is normalized to the mean of the wt cohort in comparisons with the published normalized dataset of Trem2−/− microglia (Mazaheri et al, 2017). Data represent the mean +/− SD. Data information: For statistical analysis, unpaired two-tailed Student's t-test was performed between Grn−/− microglia in comparison with Grn+/+ microglia or Trem2−/− microglia in comparison with Trem2+/+ microglia: not significant P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001. Download figure Download PowerPoint Confirmation of molecular microglial signatures on protein level Expression of proteins associated with MGnDs such as ApoE, CLEC7A, TREM2, and CD68 was also increased in acutely isolated microglia (Fig 2A and B). sTREM2 was also found to be increased in brains and serum of Grn−/− mice (Fig 2C and D), although no increase in Trem2 mRNA levels was observed in the NanoString analysis (Fig 1B), suggesting posttranscriptional regulation. Furthermore, protein expression of the homeostatic P2ry12 gene is downregulated in cortical Grn−/− microglia (Fig 2E and F), while cortical microglia from Trem2−/− mice show strongly elevated P2RY12 levels (Fig 2E and F), consistent with the finding that microglia from both mouse models are in opposite activation states. These findings were further confirmed in genetically modified BV2 microglia-like cells lacking PGRN expression, where ApoE, CLEC7A, and TREM2 were upregulated, whereas P2RY12 was downregulated (Fig 2G–I). In line with the protein expression, in PGRN-deficient BV2 cells, transcripts encoding Apoe, Clec7a, and Trem2 were also significantly upregulated, whereas P2ry12 was downregulated (Fig 2J) further confirming that loss of PGRN results in a microglial hyperactivation. Figure 2. Expression of microglial marker protein characteristic for the homeostatic or disease-associated state A. Immunoblot analysis of ApoE, CLEC7A, TREM2, and CD68 in lysates of acutely isolated microglia from Grn−/− and Grn+/+ mice (9 months of age, n = 3 per genotype, female). IBA1 was used as loading control. The asterisk indicates an unspecific band. B. Quantification of protein expression normalized to levels of Grn+/+ microglia (n = 3). Data represent the mean ± SD. C. ELISA-mediated quantification of sTREM2 in brain homogenates of Grn−/− and Grn+/+ mice. Data are shown as mean ± SEM (n = 3–6). D. ELISA-mediated quantification of sTREM2 in serum of Grn−/− and Grn+/+ mice. Data are shown as mean ± SEM (n = 4–13). E. Microglial expression of P2RY12 in cortical sections of wt, Grn−/− and Trem2−/− mice. (9 months of age, female). Scale bar indicates 10 μm. F. Quantification of P2RY12-positive microglia. Data are shown as mean ± SD (n = 3 per genotype, female, except one male for Trem2−/−). G. Immunoblot analysis of secreted PGRN and ApoE (ApoEmed) in conditioned media and PGRN, ApoE, CLEC7A, P2RY12, and TREM2 in lysates of BV2 wild-type (Grnwt) and PGRN-deficient (Grnmut) cells. Soluble amyloid precursor protein (APPs) and calnexin were used as a loading control. The asterisk indicates an unspecific band. H. Quantification of immunoblots normalized to BV2 Grnwt levels (n = 3–5). Data represent the mean ± SD. I. ELISA-mediated quantification of sTREM2 in conditioned media of BV2 Grnwt and Grnmut cells. Data represent the mean ± SD (n = 4). J. Quantification of relative mRNA levels of Apoe, Clec7a, P2ry12, and Trem2 in BV2 Grnwt and Grnmut cells. Data represent the mean ± SD (n = 3). Data information: For statistical analysis, unpaired two-tailed Student's t-test was performed between Grnmut or Grn−/− in comparison with Grnwt or Grn+/+: n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. For (F), the one-way ANOVA with Tukey post hoc test was used. Source data are available online for this figure. Source Data for Figure 2 [emmm201809711-sup-0003-SDataFig2.zip] Download figure Download PowerPoint Increased phagocytic capacity, chemotaxis, and clustering around amyloid plaques upon loss of PGRN Next, we investigated a series of functional phenotypes in Grn loss-of-function mutants, which may be differentially affected by dysregulated Trem2 or Grn gene expression. First, we investigated the phagocytic capacity of PGRN-deficient BV2 cells, which recapitulate expression profile changes found in isolated Grn−/− microglia such as enhanced expression of ApoE, CLEC7A, and TREM2 (Fig 2). We observed a significantly increased uptake of pHrodo-labeled Escherichia coli (E. coli) in PGRN-deficient BV2 cells compared to wt (Fig 3A). Enhanced uptake of pHrodo-labeled bacteria is in strong contrast to the reduced uptake detected in Trem2 loss-of-function mutations (Fig EV1A; Kleinberger et al, 2014, 2017; Schlepckow et al, 2017), thus demonstrating that differentially regulated mRNA signatures translate into opposite phagocytic phenotypes. In acutely isolated microglia, uptake of bacteria was also reduced upon loss of TREM2 (Fig EV1B). Grn−/− microglia displayed only a slight but significant increase in phagocytosis (Fig EV1B). The rather minor effect of the Grn knockout on increased microglial phagocytosis is most likely due to isolation-induced activation of microglia (Gosselin et al, 2017), which as a consequence already show a rather high uptake capacity. Figure 3. Enhanced phagocytosis, migration, and clustering around amyloid plaques upon PGRN deficiency A. Flow cytometric analysis of phagocytic capacity in BV2wt and BV2mut cells using pHrodo green E. coli as target particles. Phagocytosis was terminated after 30 and 60 min (min) of incubation. Data are presented as mean percentage of cells positive for pHrodo u