Maternal immune activation results in complex microglial transcriptome signature in the adult offspring that is reversed by minocycline treatment.

Maternal immune activation (MIA) during pregnancy has been linked to an increased risk of developing psychiatric pathologies in later life. This link may be bridged by a defective microglial phenotype in the offspring induced by MIA, as microglia have key roles in the development and maintenance of neuronal signaling in the central nervous system. The beneficial effects of the immunomodulatory treatment with minocycline on schizophrenic patients are consistent with this hypothesis. Using the MIA mouse model, we found an altered microglial transcriptome and phagocytic function in the adult offspring accompanied by behavioral abnormalities. The changes in microglial phagocytosis on a functional and transcriptional level were similar to those observed in a mouse model of Alzheimer's disease hinting to a related microglial phenotype in neurodegenerative and psychiatric disorders. Minocycline treatment of adult MIA offspring reverted completely the transcriptional, functional and behavioral deficits, highlighting the potential benefits of therapeutic targeting of microglia in psychiatric disorders.

Long-lasting pro-inflammatory suppression of microglia by LPS-preconditioning is mediated by RelB-dependent epigenetic silencing.

Microglia, the innate immune cells of the central nervous system (CNS), react to endotoxins like bacterial lipopolysaccharides (LPS) with a pronounced inflammatory response. To avoid excess damage to the CNS, the microglia inflammatory response needs to be tightly regulated. Here we report that a single LPS challenge results in a prolonged blunted pro-inflammatory response to a subsequent LPS stimulation, both in primary microglia cultures (100 ng/ml) and in vivo after intraperitoneal (0.25 and 1mg/kg) or intracerebroventricular (5 µg) LPS administration. Chromatin immunoprecipitation (ChIP) experiments with primary microglia and microglia acutely isolated from mice showed that LPS preconditioning was accompanied by a reduction in active histone modifications AcH3 and H3K4me3 in the promoters of the IL-1ß and TNF-α genes. Furthermore, LPS preconditioning resulted in an increase in the amount of repressive histone modification H3K9me2 in the IL-1ß promoter. ChIP and knock-down experiments showed that NF-κB subunit RelB was bound to the IL-1ß promoter in preconditioned microglia and that RelB is required for the attenuated LPS response. In addition to a suppressed pro-inflammatory response, preconditioned primary microglia displayed enhanced phagocytic activity, increased outward potassium currents and nitric oxide production in response to a second LPS challenge. In vivo, a single i.p. LPS injection resulted in reduced performance in a spatial learning task 4 weeks later, indicating that a single inflammatory episode affected memory formation in these mice. Summarizing, we show that LPS-preconditioned microglia acquire an epigenetically regulated, immune-suppressed phenotype, possibly to prevent excessive damage to the central nervous system in case of recurrent (peripheral) inflammation.

[Neuroglia--living nerve glue]. / Neuroglia, der lebende Nervenkitt.

The brain is composed of two major cell types - neurons and glial cells. While neurons have been extensively studied, research on glia cells has picked up only in the last decades. There are three types of glia cells in the central nervous system: astrocytes, oligodendrocytes and microglia cells. In the peripheral nervous system the glia cells are called Schwann cells. Astrocytes are a very heterogeneous population of cells which interact with neurons and blood vessels. These cells detect neuronal activity and can modulate neuronal networks. Oligodendrocytes in the central and Schwann cells in the peripheral nervous system form myelin and therefore are prerequisites for the high conduction velocity of axons in vertebrates. Microglia cells are the immune cells of the central nervous system and respond by a process called activation to any change in the environment. They are therefore considered as pathological sensors of the brain. They migrate to the site of injury, can proliferate and phagocytose and interact with the peripheral immune system by antigen presentation. Today, we view the brain as an organ which fulfils its function by the interaction of all these cell types. This is also particularly relevant for brain diseases.

Minocycline reduces glioma expansion and invasion by attenuating microglial MT1-MMP expression.

Glioma cells release soluble factors, which induce the expression of membrane type 1 matrix metalloprotease (MT1-MMP) in tumor associated microglia and then exploit MT1-MMP mediated matrix degradation for invasion. Here, we show that minocycline blocked the increase in MT1-MMP expression and activity in cultivated microglia stimulated with glioma conditioned medium. Glioma growth within an organotypic brain slice preparation was reduced by minocycline and this reduction depended on the presence of microglia. Glioma growth in an experimental mouse model was strongly reduced by the addition of minocycline to drinking water, compared to untreated controls. Coherently, we observed in our orthotopic glioma implantation model, that MT1-MMP was abundantly expressed in glioma associated microglia in controls, but was strongly attenuated in tumors of minocycline treated animals. Overall, our study indicates that the clinically approved antibiotic minocycline is a promising new candidate for adjuvant therapy against malignant gliomas.

Astrocytes control GABAergic inhibition of neurons in the mouse barrel cortex.

Astrocytes in the barrel cortex respond with a transient Ca2+ increase to neuronal stimulation and this response is restricted to the stimulated barrel field. In the present study we suppressed the astrocyte response by dialysing these cells with the Ca2+ chelator BAPTA. Electrical stimulation triggered a depolarization in stellate or pyramidal 'regular spiking' neurons from cortex layer 4 and 2/3 and this response was augmented in amplitude and duration after astrocytes were dialysed with BAPTA. Combined blockade of GABAA and GABAB receptors mimicked the effect of BAPTA dialysis, while glutamate receptor blockers had no effect. Moreover, the frequency of spontaneous postsynaptic currents was increased after BAPTA dialysis. Outside the range of BAPTA dialysis astrocytes responded with a Ca2+ increase, but in contrast to control, the response was no longer restricted to one barrel field. Our findings indicate that astrocytes control neuronal inhibition in the barrel cortex.

A mesenchymal-like ZEB1(+) niche harbors dorsal radial glial fibrillary acidic protein-positive stem cells in the spinal cord.

In humans and rodents the adult spinal cord harbors neural stem cells located around the central canal. Their identity, precise location, and specific signaling are still ill-defined and controversial. We report here on a detailed analysis of this niche. Using microdissection and glial fibrillary acidic protein (GFAP)-green fluorescent protein (GFP) transgenic mice, we demonstrate that neural stem cells are mostly dorsally located GFAP(+) cells lying ependymally and subependymally that extend radial processes toward the pial surface. The niche also harbors doublecortin protein (Dcx)(+) Nkx6.1(+) neurons sending processes into the lumen. Cervical and lumbar spinal cord neural stem cells maintain expression of specific rostro-caudal Hox gene combinations and the niche shows high levels of signaling proteins (CD15, Jagged1, Hes1, differential screening-selected gene aberrative in neuroblastoma [DAN]). More surprisingly, the niche displays mesenchymal traits such as expression of epithelial-mesenchymal-transition zinc finger E-box-binding protein 1 (ZEB1) transcription factor and smooth muscle actin. We found ZEB1 to be essential for neural stem cell survival in vitro. Proliferation within the niche progressively ceases around 13 weeks when the spinal cord reaches its final size, suggesting an active role in postnatal development. In addition to hippocampus and subventricular zone niches, adult spinal cord constitutes a third central nervous system stem cell niche with specific signaling, cellular, and structural characteristics that could possibly be manipulated to alleviate spinal cord traumatic and degenerative diseases.

Gliomas induce and exploit microglial MT1-MMP expression for tumor expansion.

Diffuse infiltration of glioma cells into normal brain tissue is considered to be a main reason for the unfavorable outcomes of patients with malignant gliomas. Invasion of glioma cells into the brain parenchyma is facilitated by metalloprotease-mediated degradation of the extracellular matrix. Metalloproteases are released as inactive pro-forms and get activated upon cleavage by membrane bound metalloproteases. Here, we show that membrane type 1 metalloprotease (MT1-MMP) is up-regulated in glioma-associated microglia, but not in the glioma cells. Overexpression of MT1-MMP is even lethal for glioma cells. Glioma-released factors trigger the expression and activity of MT1-MMP via microglial toll-like receptors and the p38 MAPK pathway, as deletion of the toll-like receptor adapter protein MyD88 or p38 inhibition prevented MT1-MMP expression and activity in cultured microglial cells. Microglial MT1-MMP in turn activates glioma-derived pro-MMP-2 and promotes glioma expansion, as shown in an ex vivo model using MT1-MMP-deficient brain tissue and a microglia depletion paradigm. Finally, MyD88 deficiency or microglia depletion largely attenuated glioma expansion in 2 independent in vivo models.

Transferrin-receptor-mediated iron accumulation controls proliferation and glutamate release in glioma cells.

Transferrin receptors (TfR) are overexpressed in brain tumors, but the pathological relevance has not been fully explored. Here, we show that TfR is an important downstream effector of ets transcription factors that promotes glioma proliferation and increases glioma-evoked neuronal death. TfR mediates iron accumulation and reactive oxygen formation and thereby enhanced proliferation in clonal human glioma lines, as shown by the following experiments: (1) downregulating TfR expression reduced proliferation in vitro and in vivo; (2) forced TfR expression in low-grade glioma accelerated proliferation to the level of high-grade glioma; (3) iron and oxidant chelators attenuated tumor proliferation in vitro and tumor size in vivo. TfR-induced oxidant accumulation modified cellular signaling by inactivating a protein tyrosine phosphatase (low-molecular-weight protein tyrosine phosphatase), activating mitogen-activated protein kinase and Akt and by inactivating p21/cdkn1a and pRB. Inactivation of these cell cycle regulators facilitated S-phase entry. Besides its effect on proliferation, TfR also boosted glutamate release, which caused N-methyl-D-aspartate-receptor-mediated reduction of neuron cell mass. Our results indicate that TfR promotes glioma progression by two mechanisms, an increase in proliferation rate and glutamate production, the latter mechanism providing space for the progressing tumor mass.

Nitric oxide signals parallel fiber activity to Bergmann glial cells in the mouse cerebellar slice.

Stimulation of parallel fibers in the cerebellar cortex triggers a transient calcium increase in Bergmann glial cells, a special form of astrocytes. Using patch-clamping and imaging techniques we have found that this form of neuron-glia interaction is mediated by nitric oxide (NO) since the response is blocked by the NO-synthase inhibitor N omega-nitro-l-arginine and mimicked by NO donors. None of the neurotransmitter receptors of Bergmann glia identified so far participates in or interferes with this signaling cascade. The NO-triggered increases in [Ca(2+)](i), as studied in Bergmann glial cells in the slice or in cultured astrocytes, are due to Ca(2+) influx and not to release from cytoplasmic stores. Thus, NO released from parallel fibers serves as a signaling substance to the neighboring glial elements.

GABA(A)-receptor expression in glioma cells is triggered by contact with neuronal cells.

The expression of functional GABA(A)-receptors in glioma cells correlates with low malignancy of tumours and cell lines from glioma lack these receptors. Here we show that contact with neurons induces the expression of functional GABA(A)-receptors. C6 and F98 glioma cell lines were labelled by recombinant expression of enhanced green fluorescent protein injected into rat brain and studied in acute slices after two to three weeks of tumour growth. The cells responded to GABA or the specific agonist, muscimol with a current typical for GABA(A)-receptors, as studied with the patch-clamp technique. To get insight into the mechanism of GABA(A) receptor induction, the C6 or F98 cells were co-cultured with neurons, astrocytes, oligodendrocytes and microglia. Glioma cells expressed functional GABA(A) receptors within 24 h only in cultures where physical contact to neurons occurred. Activation of GABA(A)-receptors in the co-cultures attenuated glioma cell metabolism while blockade of the receptors increased metabolism. We conclude that with this form of interaction, neurons can influence tumour behaviour in the brain.

The protein tyrosine kinase inhibitor AG126 prevents the massive microglial cytokine induction by pneumococcal cell walls.

Central nervous system (CNS) infections caused by Streptococcus pneumoniae still have a disastrous outcome. Underlying immunological and CNS cellular events are largely enigmatic. We used pneumococcal cells walls (PCW) to investigate microglial responses as these cells are prominent sensors and effectors during neuropathological changes. PCW stimulation of mouse microglia in vitro evoked the release of the cyto- and chemokines, TNF-alpha, IL-6, IL-12, KC, MCP-1, MIP-1alpha, MIP-2 and RANTES as well as soluble TNF receptor II, a potential TNF-alpha antagonist. The release induction followed extremely steep dose-response relations, and short exposure periods (15 min) were already sufficient to trigger substantial responses. PCW signaling controlling the release depended on both p38 and p42/p44 (ERK2/ERK1) MAP kinase activities. The kinase inhibitor, tyrphostin AG126 prevented the PCW-inducible phosphorylation of p42/p44(MAPK), potently blocked cytokine release and drastically reduced the bioavailable TNF-alpha, since it only marginally affected the release of soluble TNF receptors. Moreover, in an in vivo model of pneumococcal meningitis, AG126 significantly attenuated the PCW-induced leukocyte influx to the cerebrospinal fluid. The findings imply that pneumococcal CNS infection can cause a rapid and massive microglial activation and that ERK/MAPK pathway(s) are potential targets for pharmacological interventions.

AN2/NG2 protein-expressing glial progenitor cells in the murine CNS: isolation, differentiation, and association with radial glia.

During early neural development, the lineage specification of initially pluripotent progenitor cells is associated with proliferation, differentiation, and migration. Oligodendroglial progenitor cells migrate from their sites of origin to reach the axons that they will myelinate. We have described a cell-surface protein, AN2, expressed by oligodendroglial progenitor cells in vitro and showed that antibodies against AN2 inhibited the migration of cultured primary oligodendroglial progenitor cells, suggesting that the AN2 antigen plays a role in their migration. Recently, results from MALDI mass spectroscopy showed that AN2 is the mouse homologue of the rat NG2 protein. In this study, we have analyzed cells staining with AN2 antibodies during development and in the adult murine central nervous system (CNS), carried out double stainings with antibodies against NG2, and investigated the differentiation potential of cells in vitro after isolation from early postnatal brain using AN2 antibodies. AN2 and NG2 antibodies stained totally overlapping populations of cells in the CNS. AN2/NG2 expressing cells in embryonic and postnatal brain expressed the PDGF-alpha-receptor and in postnatal brain exhibited electrophysiological properties typical of glial progenitor cells. Cells isolated from early postnatal brain using AN2 monoclonal antibody developed into oligodendrocytes in low serum medium or into astrocytes in the presence of fetal calf serum. In the embryonic spinal cord, cells staining with AN2 antibodies were found closely apposed to radial glial cells, suggesting that glial precursors, like neurons, may use radial glia as scaffolds for migration.

beta-adrenergic receptor stimulation selectively inhibits IL-12p40 release in microglia.

The cytokine interleukin-12 (IL-12) is mainly produced in response to bacterial or parasitic infections. We examined the capacity of mouse brain microglia to release IL-12 forms upon challenge with bacterial lipopolysaccharide (LPS) and studied its modulation by sympathomimetics. LPS evoked the release of IL-12p40 whereas the heterodimeric form, IL-12p70 was virtually undetectable. Sympathomimetics such as salbutamol dose-dependently inhibited IL-12p40 release, whereas the production of IL-6, TNFalpha and MIP-1alpha was only marginally influenced. The inhibitory effect of salbutamol could be abolished by beta-antagonists, such as oxprenolol. The cAMP-elevating agent forskolin could mimic the effects of beta-agonists, indicating that IL-12p40 release inhibition involves intracellular cAMP accumulation. While microglial IL-12p40 may play a role in the regulation of IL-12p70 bioactivity, microglial release is itself modulated by IL-12p70. Recombinant IL-12p70 was found to enhance the LPS-evoked release of MIP-1alpha and to have a biphasic effect on both TNFalpha and MIP-1alpha with release augmentation at lower and attenuation at higher doses. Finally, no functional correlation was found between the release of IL-12p40 and the induction of Kv1.3 potassium channels, another marker of microglial activation. Taken together, beta(2)-adrenoreceptor-mediated effects on microglial cyto- and chemokine release via cAMP accumulation could modulate inflammatory cascades during bacterial infections.

GFAP promoter-controlled EGFP-expressing transgenic mice: a tool to visualize astrocytes and astrogliosis in living brain tissue.

We have generated transgenic mice in which astrocytes are labeled by the enhanced green fluorescent protein (EGFP) under the control of the human glial fibrillary acidic protein (GFAP) promoter. In all regions of the CNS, such as cortex, cerebellum, striatum, corpus callosum, hippocampus, retina, and spinal cord, EGFP-positive cells with morphological properties of astrocytes could be readily visualized by direct fluorescence microscopy in living brain slices or whole mounts. Also in the PNS, nonmyelinating Schwann cells from the sciatic nerve could be identified by their bright green fluorescence. Highest EGFP expression was found in the cerebellum. Already in acutely prepared whole brain, the cerebellum appeared green-yellowish under normal daylight. Colabeling with GFAP antibodies revealed an overlap with EGFP in the majority of cells. Some brain areas, however, such as retina or hypothalamus, showed only low levels of EGFP expression, although the astrocytes were rich in GFAP. In contrast, some areas that were poor in immunoreactive GFAP were conspicuous for their EGFP expression. Applying the patch clamp technique in brain slices, EGFP-positive cells exhibited two types of membrane properties, a passive membrane conductance as described for astrocytes and voltage-gated channels as described for glial precursor cells. Electron microscopical investigation of ultrastructural properties revealed EGFP-positive cells enwrapping synapses by their fine membrane processes. EGFP-positive cells were negative for oligodendrocyte (MAG) and neuronal markers (NeuN). As response to injury, i.e., by cortical stab wounds, enhanced levels of EGFP expression delineated the lesion site and could thus be used as a live marker for pathology.

Characterization of microglial cells and their response to stimulation in an organotypic retinal culture system.

An organotypic culture system of the early postnatal rat retina was developed to study microglial activation within a tissue environment. One day after tissue preparation, microglial cells of the ganglion cell/nerve fiber layer revealed features of activation. Cells acquired an ameboid morphology as revealed by Bandeiraea simplicifolia lectin staining. Proliferation-as revealed by Ki67 immunocytochemistry-resulted in higher cell densities. In the supernatant, tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and monocyte chemoattractant factor-1 (MCP-1) were detected by using specific enzyme-linked immunosorbent assay systems, activated microglia being the most likely source of their release. After 6 days in vitro (div), microglial cells regained their resting morphology, and cell counts returned to control levels. Concomitantly, the release activity decreased to undetectable levels. When slices were treated at this later stage of cultivation (>6 div) with bacterial lipopolysaccharide (LPS; 100 ng/ml for 24 hours), microglial cells became activated, as revealed by a change in morphology. In parallel, the LPS treatment also resulted in high levels of TNF-alpha, IL-6, and MCP-1 in the culture medium. Both the release from the tissue and the morphological changes of the microglia were reversible. Seventy-two hours after LPS removal, only microglia with ramified morphology were found, and release activities returned to baseline. These data suggest that the organotypic culture of the retina is a useful model for studying microglial activation from its resting form.

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

BACKGROUND: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. METHODS: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. RESULTS: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. CONCLUSION: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.