Publications by authors named "Hendrikus W G M Boddeke"

42 Publications

Identification of highly connected hub genes in the protective response program of human macrophages and microglia activated by alpha B-crystallin.

Glia 2017 03 7;65(3):460-473. Epub 2017 Jan 7.

Department of Pathology, VU University Medical Center, Amsterdam, HV, the Netherlands.

The glial stress protein alpha B-crystallin (HSPB5) is an endogenous agonist for Toll-like receptor 2 in CD14 cells. Following systemic administration, HSPB5 acts as a potent inhibitor of neuroinflammation in animal models and reduces lesion development in multiple sclerosis patients. Here, we show that systemically administered HSPB5 rapidly crosses the blood-brain barrier, implicating microglia as additional targets for HSPB5 along with peripheral monocytes and macrophages. To compare key players in the HSPB5-induced protective response of human macrophages and microglia, we applied weighted gene co-expression network analysis on transcript expression data obtained 1 and 4 h after activation. This approach identified networks of genes that are co-expressed in all datasets, thus reducing the complexity of the nonsynchronous waves of transcripts that appear after activation by HSPB5. In both cell types, HSPB5 activates a network of highly connected genes that appear to be functionally equivalent and consistent with the therapeutic effects of HSPB5 in vivo, since both networks include factors that suppress apoptosis, the production of proinflammatory factors, and the development of adaptive immunity. Yet, hub genes at the core of the network in either cell type were strikingly different. They prominently feature the well-known tolerance-promoting programmed-death ligand 1 as a key player in the macrophage response to HSPB5, and the immune-regulatory enzyme cyclooxygenase-2 (COX-2) in that of microglia. This latter finding indicates that despite its reputation as a potential target for nonsteroidal anti-inflammatory drugs, microglial COX-2 plays a central role in the therapeutic effects of HSPB5 during neuroinflammation. GLIA 2017;65:460-473.
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http://dx.doi.org/10.1002/glia.23104DOI Listing
March 2017

Aging, microglia and cytoskeletal regulation are key factors in the pathological evolution of the APP23 mouse model for Alzheimer's disease.

Biochim Biophys Acta Mol Basis Dis 2017 02 10;1863(2):395-405. Epub 2016 Nov 10.

Laboratory of Neurochemistry and Behavior, Institute Born-Bunge, University of Antwerp, Antwerp, Belgium; Department of Neurology and Memory Clinic, Hospital Network Antwerp (ZNA) Middelheim and Hoge Beuken, Antwerp, Belgium. Electronic address:

Aging is the key risk factor for Alzheimer's disease (AD). In addition, the amyloid-beta (Aβ) peptide is considered a critical neurotoxic agent in AD pathology. However, the connection between these factors is unclear. We aimed to provide an extensive characterization of the gene expression profiles of the amyloidosis APP23 model for AD and control mice and to evaluate the effect of aging on these profiles. We also correlated our findings to changes in soluble Aβ-levels and other pathological and symptomatic features of the model. We observed a clear biphasic expression profile. The first phase displayed a maturation profile, which resembled features found in young carriers of familial AD mutations. The second phase reflected aging processes and showed similarities to the progression of human AD pathology. During this phase, the model displayed a clear upregulation of microglial activation and lysosomal pathways and downregulation of neuron differentiation and axon guidance pathways. Interestingly, the changes in expression were all correlated to aging in general, but appeared more extensive/accelerated in APP23 mice.
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http://dx.doi.org/10.1016/j.bbadis.2016.11.014DOI Listing
February 2017

Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus.

Glia 2016 08 1;64(8):1350-62. Epub 2016 Jun 1.

Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, via G. Campi, 287, Modena, Italy.

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4(pos) T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350-1362.
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http://dx.doi.org/10.1002/glia.23008DOI Listing
August 2016

Critical data-based re-evaluation of minocycline as a putative specific microglia inhibitor.

Glia 2016 10 1;64(10):1788-94. Epub 2016 Jun 1.

Department of Neuroscience, University Medical Center Groningen, Groningen, The Netherlands.

Minocycline, a second generation broad-spectrum antibiotic, has been frequently postulated to be a "microglia inhibitor." A considerable number of publications have used minocycline as a tool and concluded, after achieving a pharmacological effect, that the effect must be due to "inhibition" of microglia. It is, however, unclear how this "inhibition" is achieved at the molecular and cellular levels. Here, we weigh the evidence whether minocycline is indeed a bona fide microglia inhibitor and discuss how data generated with minocycline should be interpreted. GLIA 2016;64:1788-1794.
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http://dx.doi.org/10.1002/glia.23007DOI Listing
October 2016

Single-cell whole genome sequencing reveals no evidence for common aneuploidy in normal and Alzheimer's disease neurons.

Genome Biol 2016 05 31;17(1):116. Epub 2016 May 31.

European Research Institute for the Biology of Ageing (ERIBA), University of Groningen, University Medical Center Groningen, 9713 AV, Groningen, The Netherlands.

Background: Alzheimer's disease (AD) is a neurodegenerative disease of the brain and the most common form of dementia in the elderly. Aneuploidy, a state in which cells have an abnormal number of chromosomes, has been proposed to play a role in neurodegeneration in AD patients. Several studies using fluorescence in situ hybridization have shown that the brains of AD patients contain an increased number of aneuploid cells. However, because the reported rate of aneuploidy in neurons ranges widely, a more sensitive method is needed to establish a possible role of aneuploidy in AD pathology.

Results: In the current study, we used a novel single-cell whole genome sequencing (scWGS) approach to assess aneuploidy in isolated neurons from the frontal cortex of normal control individuals (n = 6) and patients with AD (n = 10). The sensitivity and specificity of our method was shown by the presence of three copies of chromosome 21 in all analyzed neuronal nuclei of a Down's syndrome sample (n = 36). Very low levels of aneuploidy were found in the brains from control individuals (n = 589) and AD patients (n = 893). In contrast to other studies, we observe no selective gain of chromosomes 17 or 21 in neurons of AD patients.

Conclusion: scWGS showed no evidence for common aneuploidy in normal and AD neurons. Therefore, our results do not support an important role for aneuploidy in neuronal cells in the pathogenesis of AD. This will need to be confirmed by future studies in larger cohorts.
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http://dx.doi.org/10.1186/s13059-016-0976-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4888403PMC
May 2016

Glial cells as drug targets: What does it take?

Glia 2016 10 28;64(10):1742-54. Epub 2016 Apr 28.

Department of Neuroscience, Section of Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.

The last two decades have brought a significant increase in our understanding of glial biology and glial contribution to CNS disease. Yet, despite the fact that glial cells make up the majority of CNS cells, no drug specifically targeting glial cells is on the market. Given the long development times of CNS drugs, on average over 12 years, this is not completely surprising. However, there is increasing interest from academia and industry to exploit glial targets to develop drugs for the benefit of patients with currently limited or no therapeutic options. CNS drug development has a high attrition rate and has encountered many challenges. It seems unlikely that developing drugs against glial targets would be any less demanding. However, the knowledge generated in traditional CNS drug discovery teaches valuable lessons, which could enable the glial community to accelerate the cycle time from basic discovery to drug development. In this review we will discuss steps necessary to bring a "glial target idea" to a clinical development program. GLIA 2016;64:1742-1754.
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http://dx.doi.org/10.1002/glia.22993DOI Listing
October 2016

CD14 is a key organizer of microglial responses to CNS infection and injury.

Glia 2016 Apr 18;64(4):635-49. Epub 2015 Dec 18.

Institute of Neuropathology, University of Göttingen, Göttingen, 37075, Germany.

Microglia, innate immune cells of the CNS, sense infection and damage through overlapping receptor sets. Toll-like receptor (TLR) 4 recognizes bacterial lipopolysaccharide (LPS) and multiple injury-associated factors. We show that its co-receptor CD14 serves three non-redundant functions in microglia. First, it confers an up to 100-fold higher LPS sensitivity compared to peripheral macrophages to enable efficient proinflammatory cytokine induction. Second, CD14 prevents excessive responses to massive LPS challenges via an interferon β-mediated feedback. Third, CD14 is mandatory for microglial reactions to tissue damage-associated signals. In mice, these functions are essential for balanced CNS responses to bacterial infection, traumatic and ischemic injuries, since CD14 deficiency causes either hypo- or hyperinflammation, insufficient or exaggerated immune cell recruitment or worsened stroke outcomes. While CD14 orchestrates functions of TLR4 and related immune receptors, it is itself regulated by TLR and non-TLR systems to thereby fine-tune microglial damage-sensing capacity upon infectious and non-infectious CNS challenges.
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http://dx.doi.org/10.1002/glia.22955DOI Listing
April 2016

Enhanced microglial pro-inflammatory response to lipopolysaccharide correlates with brain infiltration and blood-brain barrier dysregulation in a mouse model of telomere shortening.

Aging Cell 2015 Dec 3;14(6):1003-13. Epub 2015 Aug 3.

Section Medical Physiology, Department of Neuroscience, University of Groningen, University Medical Center Groningen, Groningen, 9713 AV, The Netherlands.

Microglia are a proliferative population of resident brain macrophages that under physiological conditions self-renew independent of hematopoiesis. Microglia are innate immune cells actively surveying the brain and are the earliest responders to injury. During aging, microglia elicit an enhanced innate immune response also referred to as 'priming'. To date, it remains unknown whether telomere shortening affects the proliferative capacity and induces priming of microglia. We addressed this issue using early (first-generation G1 mTerc(-/-) )- and late-generation (third-generation G3 and G4 mTerc(-/-) ) telomerase-deficient mice, which carry a homozygous deletion for the telomerase RNA component gene (mTerc). Late-generation mTerc(-/-) microglia show telomere shortening and decreased proliferation efficiency. Under physiological conditions, gene expression and functionality of G3 mTerc(-/-) microglia are comparable with microglia derived from G1 mTerc(-/-) mice despite changes in morphology. However, after intraperitoneal injection of bacterial lipopolysaccharide (LPS), G3 mTerc(-/-) microglia mice show an enhanced pro-inflammatory response. Nevertheless, this enhanced inflammatory response was not accompanied by an increased expression of genes known to be associated with age-associated microglia priming. The increased inflammatory response in microglia correlates closely with increased peripheral inflammation, a loss of blood-brain barrier integrity, and infiltration of immune cells in the brain parenchyma in this mouse model of telomere shortening.
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http://dx.doi.org/10.1111/acel.12370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4693462PMC
December 2015

Glia Open Access Database (GOAD): A comprehensive gene expression encyclopedia of glia cells in health and disease.

Glia 2015 Sep 25;63(9):1495-506. Epub 2015 Mar 25.

Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Recently, the number of genome-wide transcriptome profiles of pure populations of glia cells has drastically increased, resulting in an unprecedented amount of data that offer opportunities to study glia phenotypes and functions in health and disease. To make genome-wide transcriptome data easily accessible, we developed the Glia Open Access Database (GOAD), available via www.goad.education. GOAD contains a collection of previously published and unpublished transcriptome data, including datasets from isolated microglia, astrocytes and oligodendrocytes both at homeostatic and pathological conditions. It contains an intuitive web-based interface that consists of three features that enable searching, browsing, analyzing, and downloading of the data. The first feature is differential gene expression (DE) analysis that provides genes that are significantly up and down-regulated with the associated fold changes and p-values between two conditions of interest. In addition, an interactive Venn diagram is generated to illustrate the overlap and differences between several DE gene lists. The second feature is quantitative gene expression (QE) analysis, to investigate which genes are expressed in a particular glial cell type and to what degree. The third feature is a search utility, which can be used to find a gene of interest and depict its expression in all available expression data sets by generating a gene card. In addition, quality guidelines and relevant concepts for transcriptome analysis are discussed. Finally, GOAD is discussed in relation to several online transcriptome tools developed in neuroscience and immunology. In conclusion, GOAD is a unique platform to facilitate integration of bioinformatics in glia biology.
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http://dx.doi.org/10.1002/glia.22810DOI Listing
September 2015

Glioma-associated microglia/macrophages display an expression profile different from M1 and M2 polarization and highly express Gpnmb and Spp1.

PLoS One 2015 6;10(2):e0116644. Epub 2015 Feb 6.

Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany.

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new potential targets for future anti-glioma therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116644PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320099PMC
January 2016

Priming of microglia in a DNA-repair deficient model of accelerated aging.

Neurobiol Aging 2014 Sep 28;35(9):2147-60. Epub 2014 Mar 28.

Department of Neuroscience, Section Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. Electronic address:

Aging is associated with reduced function, degenerative changes, and increased neuroinflammation of the central nervous system (CNS). Increasing evidence suggests that changes in microglia cells contribute to the age-related deterioration of the CNS. The most prominent age-related change of microglia is enhanced sensitivity to inflammatory stimuli, referred to as priming. It is unclear if priming is due to intrinsic microglia ageing or induced by the ageing neural environment. We have studied this in Ercc1 mutant mice, a DNA repair-deficient mouse model that displays features of accelerated aging in multiple tissues including the CNS. In Ercc1 mutant mice, microglia showed hallmark features of priming such as an exaggerated response to peripheral lipopolysaccharide exposure in terms of cytokine expression and phagocytosis. Specific targeting of the Ercc1 deletion to forebrain neurons resulted in a progressive priming response in microglia exemplified by phenotypic alterations. Summarizing, these data show that neuronal genotoxic stress is sufficient to switch microglia from a resting to a primed state.
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http://dx.doi.org/10.1016/j.neurobiolaging.2014.03.025DOI Listing
September 2014

Mesenchymal differentiation mediated by NF-κB promotes radiation resistance in glioblastoma.

Cancer Cell 2013 Sep 29;24(3):331-46. Epub 2013 Aug 29.

Department of Neurosurgery, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.

Despite extensive study, few therapeutic targets have been identified for glioblastoma (GBM). Here we show that patient-derived glioma sphere cultures (GSCs) that resemble either the proneural (PN) or mesenchymal (MES) transcriptomal subtypes differ significantly in their biological characteristics. Moreover, we found that a subset of the PN GSCs undergoes differentiation to a MES state in a TNF-α/NF-κB-dependent manner with an associated enrichment of CD44 subpopulations and radioresistant phenotypes. We present data to suggest that the tumor microenvironment cell types such as macrophages/microglia may play an integral role in this process. We further show that the MES signature, CD44 expression, and NF-κB activation correlate with poor radiation response and shorter survival in patients with GBM.
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http://dx.doi.org/10.1016/j.ccr.2013.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3817560PMC
September 2013

Histone deacetylase inhibitors suppress immune activation in primary mouse microglia.

J Neurosci Res 2013 Sep 17;91(9):1133-42. Epub 2013 May 17.

Department of Neuroscience, Section of Medical Physiology, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands.

Neuroinflammation is required for tissue clearance and repair after infections or insults. To prevent excessive damage, it is crucial to limit the extent of neuroinflammation and thereby the activation of its principal effector cell, microglia. The two main major innate immune cell types in the CNS are astrocytes and microglia. Histone deacetylases (HDACs) have been implicated in regulating the innate inflammatory response, and here we addressed their role in pure astrocyte and microglia cultures. Endogenous HDAC expression levels were determined in microglia and astrocytes and after treatment with lipopolysaccharide (LPS) or LPS and interferon γ (IFNγ). The relative expression level of HDACs was reduced in LPS- or LPS/IFNγ (with the exception of HDAC1 and -7)-stimulated astrocytes and increased in microglia after LPS treatment both in primary cultures and in microglia acutely isolated from LPS-treated mice, so we focused on the inflammatory response in microglia. Primary microglia cultures were treated with LPS in the presence or absence of HDAC inhibitors (HDACi). Expression and release of inflammatory cytokines was determined by quantitative RT-PCR, flow cytometry, and ELISA. HDACi strongly suppressed LPS-induced cytokine expression and release by microglia. Furthermore, expression of M1- and M2-associated activation markers was suppressed, and the migratory behavior of microglia was attenuated. Our findings strongly suggest that HDACi suppress innate immune activation in microglia.
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http://dx.doi.org/10.1002/jnr.23221DOI Listing
September 2013

Toll-like receptor activation reveals developmental reorganization and unmasks responder subsets of microglia.

Glia 2012 Dec 21;60(12):1930-43. Epub 2012 Aug 21.

Institute of Neuropathology, University of Göttingen, Göttingen, Germany.

The sentinel and immune functions of microglia require rapid and appropriate reactions to infection and damage. Their Toll-like receptors (TLRs) sense both as threats. However, whether activated microglia mount uniform responses or whether subsets conduct selective tasks is unknown. We demonstrate that murine microglia reorganize their responses to TLR activations postnatally and that this process comes with a maturation of TLR4-organized functions. Although induction of MHCI for antigen presentation remains as a pan-populational feature, synthesis of TNFα becomes restricted to a subset, even within adult central nervous system regions. Response heterogeneity is evident ex vivo, in situ, and in vivo, but is not limited to TNFα production or to TLR-triggered functions. Also, clearance activities for myelin under physiological and pathophysiological conditions, IFNγ-enforced upregulation of MHCII, or challenged inductions of other proinflammatory factors reveal dissimilar microglial contributions. Notably, response heterogeneity is also confirmed in human brain tissue. Our findings suggest that microglia divide by constitutive and inducible capacities. Privileged production of inflammatory mediators assigns a master control to subsets. Sequestration of clearance of endogenous material versus antigen presentation in exclusive compartments can separate potentially interfering functions. Finally, subsets rather than a uniform population of microglia may assemble the reactive phenotypes in responses during infection, injury, and rebuilding, warranting consideration in experimental manipulation and therapeutic strategies.
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http://dx.doi.org/10.1002/glia.22409DOI Listing
December 2012

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

J Neuroinflammation 2012 Aug 16;9:198. Epub 2012 Aug 16.

Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen, University of Groningen, A. Deusinglaan 1, 9713 AV, Groningen, The Netherlands.

Background: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling.

Methods: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A(2B) receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis.

Results: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A(2B) receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody.

Conclusions: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.
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http://dx.doi.org/10.1186/1742-2094-9-198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3458985PMC
August 2012

Neuroprotective function for ramified microglia in hippocampal excitotoxicity.

J Neuroinflammation 2012 Jan 31;9:27. Epub 2012 Jan 31.

Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen (UMCG), Rijksuniversiteit Groningen (RUG), Groningen, The Netherlands.

Background: Most of the known functions of microglia, including neurotoxic and neuroprotective properties, are attributed to morphologically-activated microglia. Resting, ramified microglia are suggested to primarily monitor their environment including synapses. Here, we show an active protective role of ramified microglia in excitotoxicity-induced neurodegeneration.

Methods: Mouse organotypic hippocampal slice cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic neuronal cell death. This procedure was performed in slices containing resting microglia or slices that were chemically or genetically depleted of their endogenous microglia.

Results: Treatment of mouse organotypic hippocampal slice cultures with 10-50 μM N-methyl-D-aspartic acid (NMDA) induced region-specific excitotoxic neuronal cell death with CA1 neurons being most vulnerable, whereas CA3 and DG neurons were affected less. Ablation of ramified microglia severely enhanced NMDA-induced neuronal cell death in the CA3 and DG region rendering them almost as sensitive as CA1 neurons. Replenishment of microglia-free slices with microglia restored the original resistance of CA3 and DG neurons towards NMDA.

Conclusions: Our data strongly suggest that ramified microglia not only screen their microenvironment but additionally protect hippocampal neurons under pathological conditions. Morphological activation of ramified microglia is thus not required to influence neuronal survival.
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http://dx.doi.org/10.1186/1742-2094-9-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292937PMC
January 2012

Activation of serotonin receptors promotes microglial injury-induced motility but attenuates phagocytic activity.

Brain Behav Immun 2012 Mar 17;26(3):419-28. Epub 2011 Dec 17.

Max Delbrueck Center for Molecular Medicine, Robert-Roessle-Str. 10, Berlin, Germany.

Microglia, the brain immune cell, express several neurotransmitter receptors which modulate microglial functions. In this project we studied the impact of serotonin receptor activation on distinct microglial properties as serotonin deficiency not only has been linked to a number of psychiatric disease like depression and anxiety but may also permeate from the periphery through blood-brain barrier openings seen in neurodegenerative disease. First, we tested the impact of serotonin on the microglial response to an insult caused by a laser lesion in the cortex of acute slices from Cx3Cr1-GFP-/+ mice. In the presence of serotonin the microglial processes moved more rapidly towards the laser lesion which is considered to be a chemotactic response to ATP. Similarly, the chemotactic response of cultured microglia to ATP was also enhanced by serotonin. Quantification of phagocytic activity by determining the uptake of microspheres showed that the amoeboid microglia in slices from early postnatal animals or microglia in culture respond to serotonin application with a decreased phagocytic activity whereas we could not detect any significant change in ramified microglia in situ. The presence of microglial serotonin receptors was confirmed by patch-clamp experiments in culture and amoeboid microglia and by qPCR analysis of RNA isolated from primary cultured and acutely isolated adult microglia. These data suggest that microglia express functional serotonin receptors linked to distinct microglial properties.
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http://dx.doi.org/10.1016/j.bbi.2011.12.002DOI Listing
March 2012

Identification of a microglia phenotype supportive of remyelination.

Glia 2012 Feb 9;60(2):306-21. Epub 2011 Nov 9.

Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen, Groningen, The Netherlands.

In multiple sclerosis, endogenous oligodendrocyte precursor cells (OPCs) attempt to remyelinate areas of myelin damage. During disease progression, however, these attempts fail. It has been suggested that modulating the inflammatory environment of the lesion might provide a promising therapeutic approach to promote endogenous remyelination. Microglia are known to play a central role in neuroinflammatory processes. To investigate the microglia phenotype that supports remyelination, we performed genome-wide gene expression analysis of microglia from the corpus callosum during demyelination and remyelination in the mouse cuprizone model, in which remyelination spontaneously occurs after an episode of toxin-induced primary demyelination. We provide evidence for the existence of a microglia phenotype that supports remyelination already at the onset of demyelination and persists throughout the remyelination process. Our data show that microglia are involved in the phagocytosis of myelin debris and apoptotic cells during demyelination. Furthermore, they express a cytokine and chemokine repertoire enabling them to activate and recruit endogenous OPCs to the lesion site and deliver trophic support during remyelination. This study not only provides a detailed transcriptomic analysis of the remyelination-supportive microglia phenotype but also reinforces the notion that the primary function of microglia is the maintenance of tissue homeostasis and the support of regeneration already at the earliest stages in the development of demyelinating lesions.
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http://dx.doi.org/10.1002/glia.21266DOI Listing
February 2012

An optimized protocol for the acute isolation of human microglia from autopsy brain samples.

Glia 2012 Jan 11;60(1):96-111. Epub 2011 Oct 11.

Section Medical Physiology, Department of Neuroscience, UMCG-RuG, Groningen, The Netherlands.

Microglia are increasingly recognized to be crucially involved in the maintenance of tissue homeostasis of the brain and spinal cord. Not surprisingly is therefore the growing scientific interest in the microglia phenotypes associated with various physiological and pathological processes of the central nervous system. Until recently the investigation of these phenotypes was hindered by the lack of an isolation protocol that (without an extended culturing period) would offer a microglia population of high purity and yield. Thus, our objective was to establish a rapid and efficient method for the isolation of human microglia from postmortem brain samples. We tested multiple elements of already existing protocols (e.g., density separation, immunomagnetic bead separation) and combined them to minimize preparation time and maximize yield and purity. The procedure presented in this article enables acute isolation of human microglia from autopsy (and biopsy) samples with a purity and yield that is suitable for downstream applications, such as protein and gene expression analysis and functional assays. Moreover, the present protocol is appropriate for the isolation of microglia from autopsy samples irrespective of the neurological state of the brain or specific brain regions and (with minor modification) could be even used for the isolation of microglia from human glioma tissue.
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http://dx.doi.org/10.1002/glia.21251DOI Listing
January 2012

Interleukin-6-type cytokines in neuroprotection and neuromodulation: oncostatin M, but not leukemia inhibitory factor, requires neuronal adenosine A1 receptor function.

J Neurochem 2010 Sep 19;114(6):1667-77. Epub 2010 Aug 19.

Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen (UMCG), Rijksuniversiteit Groningen (RUG), Groningen, the Netherlands.

Neuroprotection is one of the prominent functions of the interleukin (IL)-6-type cytokine family, for which the underlying mechanism(s) are not fully understood. We have previously shown that neuroprotection and neuromodulation mediated by IL-6 require neuronal adenosine A(1) receptor (A(1) R) function. We now have investigated whether two other IL-6-type cytokines [oncostatin M (OSM) and leukemia inhibitory factor (LIF)] use a similar mechanism. It is presented here that OSM but not LIF, enhanced the expression of A(1) Rs (both mRNA and protein levels) in cultured neurons. Whereas the neuroprotective effect of LIF was unchanged in A(1) R deficient neurons, OSM failed to protect neurons in the absence of A(1) R. In addition, OSM pre-treatment for 4 h potentiated the A(1) R-mediated inhibition of electrically evoked excitatory post-synaptic currents recorded from hippocampal slices either under normal or hypoxic conditions. No such effect was observed after LIF pre-treatment. Our findings thus strongly suggest that, despite known structural and functional similarities, OSM and LIF use different mechanisms to achieve neuroprotection and neuromodulation.
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http://dx.doi.org/10.1111/j.1471-4159.2010.06881.xDOI Listing
September 2010

CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus.

Hippocampus 2011 Feb;21(2):220-32

Department of Neuroscience, Section Medical Physiology, University Medical Center Groningen (UMCG), Rijksuniversiteit Groningen (RUG), Groningen, The Netherlands.

The chemokine CXCL10 and its receptor CXCR3 are implicated in various CNS pathologies since interference with CXCL10/CXCR3 signaling alters the onset and progression in various CNS disease models. However, the mechanism and cell-types involved in CXCL10/CXCR3 signaling under pathological conditions are far from understood. Here, we investigated the potential role for CXCL10/CXCR3 signaling in neuronal cell death and glia activation in response to N-methyl-D-aspartic acid (NMDA)-induced excitotoxicity in mouse organotypic hippocampal slice cultures (OHSCs). Our findings demonstrate that astrocytes express CXCL10 in response to excitotoxicity. Experiments in OHSCs derived from CXCL10-deficient (CXCL10(-/-) ) and CXCR3-deficient (CXCR3(-/-) ) revealed that in the absence of CXCL10 or CXCR3, neuronal cell death in the CA1 and CA3 regions was diminished after NMDA-treatment when compared to wild type OHSCs. In contrast, neuronal cell death in the DG region was enhanced in both CXCL10(-/-) and CXCR3(-/-) OHSCs in response to a high (50 μM) NMDA-concentration. Moreover, we show that in the absence of microglia the differential changes in neuronal vulnerability between CXCR3(-/-) and wild type OHSCs are fully abrogated and therefore a prominent role for microglia in this process is suggested. Taken together, our results identify a region-specific role for CXCL10/CXCR3 signaling in neuron-glia and glia-glia interactions under pathological conditions.
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http://dx.doi.org/10.1002/hipo.20742DOI Listing
February 2011

Expression of CXCL10 in cultured cortical neurons.

J Neurochem 2010 Feb 16;112(3):703-14. Epub 2009 Nov 16.

Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Chemokines expressed in neurons are important mediators in neuron-neuron and neuron-glia signaling. One of these chemokines is CCL21 that activates microglia via the chemokine receptor CXCR3. As neurons also express CXCL10, a main ligand for CXCR3, we have thus investigated in detail the expression pattern of CXCL10 in neurons. We show that CXCL10 is constitutively expressed by neurons, is stored in large dense-core vesicles and is not regulated by neuronal injury or stress. Neuronal CXCL10 release occurred constitutively at low level. In vivo CXCL10 expression was found in the developing brain at various embryonic stages and its peak expression correlates with the presence of CD11b- and GFAP-positive cells expressing CXCR3. These results suggest a possible role of neuronal CXCL10 in recruitment and homing of glial cells during embryogenesis.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06495.xDOI Listing
February 2010

CCL21-induced calcium transients and proliferation in primary mouse astrocytes: CXCR3-dependent and independent responses.

Brain Behav Immun 2010 Jul 4;24(5):768-75. Epub 2009 May 4.

Department of Neuroscience, University Medical Center Groningen (UMCG), Rijksuniversiteit Groningen (RUG), University of Groningen, Groningen, The Netherlands.

CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10(-9) M and 10(-8) M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10(-7) M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations.
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http://dx.doi.org/10.1016/j.bbi.2009.04.007DOI Listing
July 2010

Enhanced hippocampal neurogenesis in the absence of microglia T cell interaction and microglia activation in the murine running wheel model.

Glia 2009 Aug;57(10):1046-61

Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Recently, activated microglia have been shown to be involved in the regulation of several aspects of neurogenesis under certain experimental conditions both in vitro and in vivo. A neurogenesis supportive microglia phenotype has been suggested to arise from the interaction of microglia with homing encephalitogenic T cells. However, a unified hypothesis regarding the exact nature of microglia activity that is supportive of neurogenesis is yet missing from the field. Our aim was to investigate the connection between microglia activity and adult hippocampal neurogenesis under physiological conditions. To address this question we compared the level of microglia activation in the hippocampus of mice, which had access to a running wheel for 10 days and that of sedentary controls. Surprisingly, despite elevated levels of proliferation of neural precursors and survival of newborn neurons in the dentate gyrus microglia remained in a "resting" state morphologically, antigenically, and at the transcriptional level. Moreover, neither T cells nor MHCII expressing microglia were present in the hippocampal brain parenchyma. Though microglia in the dentate gyrus of the runners proliferated at a higher level than in the sedentary controls, this difference was also present in non-neurogenic sites. Therefore, our findings suggest that classical signs of microglia activation and microglia activation arising from interaction with T cells in particular are not a prerequisite for the activity-induced increase in adult hippocampal neurogenesis in C57Bl/6 mice. Thus, our results draw attention on the species and model differences that might exist regarding the regulation of adult hippocampal neurogenesis.
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http://dx.doi.org/10.1002/glia.20828DOI Listing
August 2009

Brain antigens in functionally distinct antigen-presenting cell populations in cervical lymph nodes in MS and EAE.

J Mol Med (Berl) 2009 Mar 3;87(3):273-86. Epub 2008 Dec 3.

Department of Immunology, Erasmus MC, CA, Rotterdam, The Netherlands.

Drainage of central nervous system (CNS) antigens to the brain-draining cervical lymph nodes (CLN) is likely crucial in the initiation and control of autoimmune responses during multiple sclerosis (MS). We demonstrate neuronal antigens within CLN of MS patients. In monkeys and mice with experimental autoimmune encephalomyelitis (EAE) and in mouse models with non-inflammatory CNS damage, the type and extent of CNS damage was associated with the frequencies of CNS antigens within the cervical lymph nodes. In addition, CNS antigens drained to the spinal-cord-draining lumbar lymph nodes. In human MS CLN, neuronal antigens were present in pro-inflammatory antigen-presenting cells (APC), whereas the majority of myelin-containing cells were anti-inflammatory. This may reflect a different origin of the cells or different drainage mechanisms. Indeed, neuronal antigen-containing cells in human CLN did not express the lymph node homing receptor CCR7, whereas myelin antigen-containing cells in situ and in vitro did. Nevertheless, CLN from EAE-affected CCR7-deficient mice contained equal amounts of myelin and neuronal antigens as wild-type mice. We conclude that the type and frequencies of CNS antigens within the CLN are determined by the type and extent of CNS damage. Furthermore, the presence of myelin and neuronal antigens in functionally distinct APC populations within MS CLN suggests that differential immune responses can be evoked.
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http://dx.doi.org/10.1007/s00109-008-0421-4DOI Listing
March 2009

Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles.

FASEB J 2008 Dec 12;22(12):4136-45. Epub 2008 Aug 12.

Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Ant. Deusinglaan 1, 9713AV Groningen, The Netherlands.

Neurons are highly polarized cells, and neuron-neuron communication is based on directed transport and release of neurotransmitters, neuropeptides, and neurotrophins. Directed communication may also be attributed to neuron-microglia signaling, since neuronal damage can induce a microglia reaction at specific sites only. However, the mechanism underlying this site-specific microglia reaction is not yet understood. Neuronal CCL21 is a microglia-activating chemokine, which in brain is solely found in endangered neurons and is therefore a candidate for neuron-microglia signaling. Here we present that neuronal CCL21 is sorted into large dense-core vesicles, the secretory granules of the regulated release pathway of neurons. Live-cell imaging studies show preferential sorting of CCL21-containing vesicles into axons, indicating its directed transport. Thus, mouse neurons express and transport a microglia activating factor very similar to signaling molecules used in neuron-neuron communication. These data show for the first time the directed transport of a microglia activating factor in neurons and corroborate the function of neuronal CCL21 in directed neuron-microglia communication.
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http://dx.doi.org/10.1096/fj.07-101907DOI Listing
December 2008

Region-specific expression of immunoregulatory proteins on microglia in the healthy CNS.

Glia 2008 Jun;56(8):888-94

Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

In accordance with a high degree of spatial organization in the central nervous system (CNS), most CNS diseases display a regional distribution. Although microglia have been established as key players in various CNS diseases, it is not yet clear whether microglia display region-specific properties. Therefore, this study aimed to evaluate the existence of distinct microglia phenotypes in various regions of the healthy, adult mouse CNS. Using ex vivo flow cytometric analysis surface expression of CD11b, CD40, CD45, CD80, CD86, F4/80, TREM-2b, MHCII, CXCR3, CCR9, and CCR7 were analyzed. Most of these immunoregulatory markers were found on microglia and showed significant region-specific differences in expression levels. These findings considerably corroborate the existence of immunological diversity among microglia in the healthy, unchallenged CNS of adult mice.
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http://dx.doi.org/10.1002/glia.20663DOI Listing
June 2008

Expression of CXCL4 in microglia in vitro and in vivo and its possible signaling through CXCR3.

J Neurochem 2008 Jun 1;105(5):1726-36. Epub 2008 Feb 1.

Department of Medical Physiology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Signaling through chemokine receptor CXCR3 in the brain has been implicated in various brain diseases, as CXCR3 and its ligands are found under these conditions. Recently, a new chemokine ligand for CXCR3 was reported. In humans, an alternatively spliced variant of CXCR3 expressed on microvascular endothelial cells, named CXCR3b, was shown to bind CXCL4. In the periphery, the cellular expression and functions of CXCL4 are well described but in the brain its expression and function are unknown. Here, we show that brain microglia are a cellular source of CXCL4 in vitro and in vivo under neurodegenerating conditions. Microglial migration induced by CXCL4 is absent in CXCR3-deficient microglia, indicating a role of CXCR3. CXCL4 furthermore attenuates lipopolysaccharide-induced microglial phagocytosis and nitric oxide production in microglia and BV-2 cells. Based on these findings, it is proposed that locally released CXCL4 may control microglia responses.
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http://dx.doi.org/10.1111/j.1471-4159.2008.05267.xDOI Listing
June 2008

Neuronal 'On' and 'Off' signals control microglia.

Trends Neurosci 2007 Nov 24;30(11):596-602. Epub 2007 Oct 24.

Department of Medical Physiology, University Medical Center Groningen (UMCG), University of Groningen, Groningen, The Netherlands.

Recent findings indicate that neurons are not merely passive targets of microglia but rather control microglial activity. The variety of different signals that neurons use to control microglia can be divided into two categories: 'Off' signals constitutively keep microglia in their resting state and antagonize proinflammatory activity. 'On' signals are inducible and include purines, chemokines, glutamate. They instruct microglia activation under pathological conditions towards a beneficial or detrimental phenotype. Various neuronal signaling molecules thus actively control microglia function, thereby contribute to the inflammatory milieu of the central nervous system. Thus, neurons should be envisaged as key immune modulators in the brain.
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http://dx.doi.org/10.1016/j.tins.2007.08.007DOI Listing
November 2007
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