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The deubiquitinating enzyme CYLD negatively regulates NF-κB signaling by removing activating ubiquitin chains from several members of the NF-κB pathway. Thereby, CYLD is critical for the maintenance and differentiation of various immune cells. Despite the importance of the NF-κB pathway in microglia regulation, the role of CYLD in microglia has not been investigated so far. In this study, we investigated whether CYLD in microglia can protect against neuroinflammation using a newly generated conditional mouse strain (Rosa26-Cyld-tdTomato) that allows cell type-specific CYLD overexpression. Here, we show that overexpression of CYLD in microglia did not alter microglia numbers or microglia morphology in different brain regions. Additionally, CYLD overexpression did not modify the microglial response to LPS-induced neuroinflammation or the disease severity in experimental autoimmune encephalomyelitis (EAE). Finally, also immune cell infiltration into the CNS during EAE and under steady state conditions remained unaffected by microglial CYLD overexpression. Our findings suggest that CYLD overexpression does not alter microglial function, and thus does not represent a viable therapeutic strategy in neuroinflammatory conditions. This study highlights the complexity of ubiquitin-mediated signaling in neuroinflammation and the need for cell-type-specific investigations. The Rosa26-Cyld-tdTomato mouse model offers a valuable tool for studying CYLD's role across various tissues and cell types. KEY MESSAGES: Novel mouse strain for cell type-specific overexpression of the deubiquitinating enzyme CYLD. CYLD overexpression in microglia did not alter microglia numbers or morphology in the steady state. CYLD overexpression in microglia did not protect mice from LPS-induced neuroinflammation or EAE. CYLD overexpression in microglia did not influence their gene expression during neuroinflammation., (© 2024. The Author(s).)

A20 is a ubiquitin-modifying protein that negatively regulates NF-κB signaling. Mutations in A20/TNFAIP3 are associated with a variety of autoimmune diseases, including multiple sclerosis (MS). We found that deletion of A20 in central nervous system (CNS) endothelial cells (ECs) enhances experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. A20ΔCNS-EC mice showed increased numbers of CNS-infiltrating immune cells during neuroinflammation and in the steady state. While the integrity of the blood-brain barrier (BBB) was not impaired, we observed a strong activation of CNS-ECs in these mice, with dramatically increased levels of the adhesion molecules ICAM-1 and VCAM-1. We discovered ICOSL to be expressed by A20-deficient CNS-ECs, which we found to function as adhesion molecules. Silencing of ICOSL in CNS microvascular ECs partly reversed the phenotype of A20ΔCNS-EC mice without reaching statistical significance and delayed the onset of EAE symptoms in WT mice. In addition, blocking of ICOSL on primary mouse brain microvascular ECs impaired the adhesion of T cells in vitro. Taken together, we propose that CNS EC-ICOSL contributes to the firm adhesion of T cells to the BBB, promoting their entry into the CNS and eventually driving neuroinflammation.

Heterodimeric β2 integrin surface receptors (CD11a-d/CD18) are specifically expressed by leukocytes that contribute to pathogen uptake, cell migration, immunological synapse formation and cell signaling. In humans, the loss of CD18 expression results in leukocyte adhesion deficiency syndrome (LAD-)1, largely characterized by recurrent severe infections. All available mouse models display the constitutive and ubiquitous knockout of either α or the common β2 (CD18) subunit, which hampers the analysis of the cell type-specific role of β2 integrins in vivo. To overcome this limitation, we generated a CD18 gene floxed mouse strain. Offspring generated from crossing with CD11c-Cre mice displayed the efficient knockdown of β2 integrins, specifically in dendritic cells (DCs). Stimulated β2-integrin-deficient splenic DCs showed enhanced cytokine production and the concomitantly elevated activity of signal transducers and activators of transcription (STAT) 1, 3 and 5, as well as the impaired expression of suppressor of cytokine signaling (SOCS) 2-6 as assessed in bone marrow-derived (BM) DCs. Paradoxically, these BMDCs also showed the attenuated expression of genes involved in inflammatory signaling. In line, in experimental autoimmune encephalomyelitis mice with a conditional DC-specific β2 integrin knockdown presented with a delayed onset and milder course of disease, associated with lower frequencies of T helper cell populations (Th)1/Th17 in the inflamed spinal cord. Altogether, our mouse model may prove to be a valuable tool to study the leukocyte-specific functions of β2 integrins in vivo.

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood-brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.

Tumor-necrosis-factor-alpha-induced protein 3 (TNFAIP3), or A20, is a ubiquitin-modifying protein and negative regulator of canonical nuclear factor κB (NF-κB) signaling. Several single-nucleotide polymorphisms in TNFAIP3 are associated with autoimmune diseases, suggesting a role in tissue inflammation. While the role of A20 in peripheral immune cells has been well investigated, less is known about its role in the central nervous system (CNS). Here, we show that microglial A20 is crucial for maintaining brain homeostasis. Without microglial A20, CD8 + T cells spontaneously infiltrate the CNS and acquire a viral response signature. The combination of infiltrating CD8 + T cells and activated A20-deficient microglia leads to an increase in VGLUT1 + terminals and frequency of spontaneous excitatory currents. Ultimately, A20-deficient microglia upregulate genes associated with the antiviral response and neurodegenerative diseases. Together, our data suggest that microglial A20 acts as a sensor for viral infection and a master regulator of CNS homeostasis., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cell adhesion molecule E-cadherin is a major component of adherens junctions and marks Langerhans cells (LC), the only dendritic cell (DC) population of the epidermis. LC form a dense network and attach themselves to the surrounding keratinocytes via homophilic E-cadherin binding. LC activation, mobilization, and migration require a reduction in LC E-cadherin expression. To determine whether E-cadherin plays a role in regulating LC homeostasis and function, we generated CD11c-specific E-cadherin knockout mice (CD11c-Ecad del ). In the absence of E-cadherin-mediated cell adhesion, LC numbers remained stable and similar as in control mice, even in aged animals. Intriguingly, E-cadherin-deficient LC displayed a dramatically changed morphology characterized by a more rounded cell body and fewer dendrites than wild-type cells. Nevertheless, maturation and migration of LC lacking E-cadherin was not altered, neither under steady-state nor inflammatory conditions. Accordingly, CD11c-Ecad del and control mice developed comparable contact hypersensitivity reactions and imiquimod-triggered psoriatic skin inflammation, indicating that E-cadherin on LC does not influence their ability to orchestrate T cell-mediated immunity. In conclusion, our data demonstrate that E-cadherin is dispensable to maintain LC in the epidermis and does not regulate LC maturation, migration, and function., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background & Aims: The CYLD lysine 63 deubiquitinase gene (CYLD) encodes tumor suppressor protein that is mutated in familial cylindromatosus, and variants have been associated with Crohn disease (CD). Splice forms of CYLD that lack exons 7 and 8 regulate transcription factors and functions of immune cells. We examined the expression of splice forms of CYLD in colon tissues from patients with CD and their effects in mice., Methods: We performed immunohistochemical analyses of colon tissues from patients with untreated CD and patients without inflammatory bowel diseases (controls). We obtained mice that expressed splice forms of CYLD (sCYLD mice) without or with SMAD7 (sCYLD/SMAD7 mice) from transgenes and CYLD-knockout mice (with or without transgenic expression of SMAD7) and performed endoscopic analyses. Colitis was induced in Rag1 -/- mice by transfer of CD4 + CD62L + T cells from C57/Bl6 or transgenic mice. T cells were isolated from mice and analyzed by flow cytometry and quantitative real-time polymerase chain reaction and intestinal tissues were analyzed by histology and immunohistochemistry. CYLD forms were expressed in mouse embryonic fibroblasts, primary T cells, and HEK293T cells, which were analyzed by immunoblot, mobility shift, and immunoprecipitation assays., Results: The colonic lamina propria from patients with CD was infiltrated by T cells and had higher levels of sCYLD (but not full-length CYLD) and SMAD7 than tissues from controls. Incubation of mouse embryonic fibroblasts and T cells with transforming growth factor β increased their production of sCYLD and decreased full-length CYLD. Transgenic expression of sCYLD and SMAD7 in T cells prevented the differentiation of regulatory T cells and T-helper type 17 cells and increased the differentiation of T-helper type 1 cells. The same effects were observed in colon tissues from sCYLD/SMAD7 mice but not in those from CYLD-knockout SMAD7 mice. The sCYLD mice had significant increases in the numbers of T-helper type 1 cells and CD44 high CD62L low memory-effector CD4 + T cells in the spleen and mesenteric lymph nodes compared with wild-type mice; sCYLD/SMAD7 mice had even larger increases. The sCYLD/SMAD7 mice spontaneously developed severe colitis, with infiltration of the colon by dendritic cells, neutrophils, macrophages, and CD4 + T cells and increased levels of Ifng, Il6, Il12a, Il23a, and Tnf mRNAs. Co-transfer of regulatory T cells from wild-type, but not from sCYLD/SMAD7, mice prevented the induction of colitis in Rag1 -/- mice by CD4 + T cells. We found increased levels of poly-ubiquitinated SMAD7 in sCYLD CD4 + T cells. CYLD formed a nuclear complex with SMAD3, whereas sCYLD recruited SMAD7 to the nucleus, which inhibited the expression of genes regulated by SMAD3 and SMAD4. We found that sCYLD mediated lysine 63-linked ubiquitination of SMAD7. The sCYLD-SMAD7 complex inhibited transforming growth factor β signaling in CD4 + T cells., Conclusions: Levels of the spliced form of CYLD are increased in colon tissues from patients with CD. sCYLD mediates ubiquitination and nuclear translocation of SMAD7 and thereby decreases transforming growth factor β signaling in T cells. This prevents immune regulatory mechanisms and leads to colitis in mice., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)

The pleiotropic cytokine IL-1 mediates its biological functions via association with the signaling receptor IL-1R1. Despite an apparent simplicity in IL-1 signaling activation, multiple negative regulators have been identified. The decoy receptor IL-1R2 (also known as CD121b) can suppress IL-1 maturation, sequester its active forms or hinder the signaling complex assembly. IL-1R2 is differentially expressed among numerous cell types and displays cis- and trans- modes of action. In this review, we link different forms of IL-1R2 (membrane-bound (mIL-1R2), secreted (sIL-1R2), shedded (shIL-1R2), cytoplasmic, and intracellular domain (IL-1R2 ICD ) restricted) with their ability to interfere with IL-1, thereby regulating immune responses. We also discuss the intriguing possible function of IL-1R2 as a transcriptional regulator. Finally, we summarize the known impact of IL-1R2 in disease pathogenesis and discuss its potential role in treatment of inflammatory conditions.

The neurofibromatosis type 2 (NF2) tumor suppressor protein Merlin functions as a negative regulator of cell growth and actin dynamics in different cell types amongst which Schwann cells have been extensively studied. In contrast, the presence and the role of Merlin in oligodendrocytes, the myelin forming cells within the CNS, have not been elucidated. In this work, we demonstrate that Merlin immunoreactivity was broadly distributed in the white matter throughout the central nervous system. Following Merlin expression during development in the cerebellum, Merlin could be detected in the cerebellar white matter tract at early postnatal stages as shown by its co-localization with Olig2-positive cells as well as in adult brain sections where it was aligned with myelin basic protein containing fibers. This suggests that Merlin is expressed in immature and mature oligodendrocytes. Expression levels of Merlin were low in oligodendrocytes as compared to astrocytes and neurons throughout development. Expression of Merlin in oligodendroglia was further supported by its identification in either immortalized cell lines of oligodendroglial origin or in primary oligodendrocyte cultures. In these cultures, the two main splice variants of Nf2 could be detected. Merlin was localized in clusters within the nuclei and in the cytoplasm. Overexpressing Merlin in oligodendrocyte cell lines strengthened reduced impedance in XCELLigence measurements and Ki67 stainings in cultures over time. In addition, the initiation and elongation of cellular projections were reduced by Merlin overexpression. Consistently, cell migration was retarded in scratch assays done on Nf2-transfected oligodendrocyte cell lines. These data suggest that Merlin actively modulates process outgrowth and migration in oligodendrocytes.

Microglia are resident macrophages of the central nervous system that contribute to homeostasis and neuroinflammation. Although known to play an important role in brain development, their exact function has not been fully described. Here, we show that in contrast to healthy adult and inflammation-activated cells, neonatal microglia show a unique myelinogenic and neurogenic phenotype. A CD11c + microglial subset that predominates in primary myelinating areas of the developing brain expresses genes for neuronal and glial survival, migration, and differentiation. These cells are the major source of insulin-like growth factor 1, and its selective depletion from CD11c + microglia leads to impairment of primary myelination. CD11c-targeted toxin regimens induced a selective transcriptional response in neonates, distinct from adult microglia. CD11c + microglia are also found in clusters of repopulating microglia after experimental ablation and in neuroinflammation in adult mice, but despite some similarities, they do not recapitulate neonatal microglial characteristics. We therefore identify a unique phenotype of neonatal microglia that deliver signals necessary for myelination and neurogenesis., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Isolation of neurons from the adult mouse CNS is important in order to study their gene expression during development or the course of different diseases., New Methods: Here we present two different methods for the enrichment or isolation of neurons from adult mouse CNS. These methods: are either based on flow cytometry sorting of eYFP expressing neurons, or by depletion of non-neuronal cells by sorting with magnetic-beads., Results: Enrichment by FACS sorting of eYFP positive neurons results in a population of 62.4% NeuN positive living neurons. qPCR data shows a 3-5fold upregulation of neuronal markers. The isolation of neurons based on depletion of non-neuronal cells using the Miltenyi Neuron Isolation Kit, reaches a purity of up to 86.5%. qPCR data of these isolated neurons shows an increase in neuronal markers and an absence of glial markers, proving pure neuronal RNA isolation., Comparison With Existing Methods: Former data related to neuronal gene expression are mainly based on histology, which does not allow for high-throughput transcriptome analysis to examine differential gene expression., Conclusion: These protocols can be used to study cell type specific gene expression of neurons to unravel their function in the process of damage to the CNS., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Arrival of encephalitogenic T cells at inflammatory foci represents a critical step in development of experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. EBI2 and its ligand, 7α,25-OHC, direct immune cell localization in secondary lymphoid organs. CH25H and CYP7B1 hydroxylate cholesterol to 7α,25-OHC. During EAE, we found increased expression of CH25H by microglia and CYP7B1 by CNS-infiltrating immune cells elevating the ligand concentration in the CNS. Two critical pro-inflammatory cytokines, interleukin-23 (IL-23) and interleukin-1 beta (IL-1β), maintained expression of EBI2 in differentiating Th17 cells. In line with this, EBI2 enhanced early migration of encephalitogenic T cells into the CNS in a transfer EAE model. Nonetheless, EBI2 was dispensable in active EAE. Human Th17 cells do also express EBI2, and EBI2 expressing cells are abundant within multiple sclerosis (MS) white matter lesions. These findings implicate EBI2 as a mediator of CNS autoimmunity and describe mechanistically its contribution to the migration of autoreactive T cells into inflamed organs., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

The molecular trigger of CNS myelination is unknown. By targeting Pten in cerebellar granule cells and activating the AKT1-mTOR pathway, we increased the caliber of normally unmyelinated axons and the expression of numerous genes encoding regulatory proteins. This led to the expansion of genetically wild-type oligodendrocyte progenitor cells, oligodendrocyte differentiation and de novo myelination of parallel fibers. Thus, a neuronal program dependent on the phosphoinositide PI(3,4,5)P 3 is sufficient to trigger all steps of myelination.

Traumatic brain injury (TBI) is a major cause of death and disability. The underlying pathophysiology is characterized by secondary processes including neuronal death and gliosis. To elucidate the role of the NG2 proteoglycan we investigated the response of NG2-knockout mice (NG2-KO) to TBI. Seven days after TBI behavioral analysis, brain damage volumetry and assessment of blood brain barrier integrity demonstrated an exacerbated response of NG2-KO compared to wild-type (WT) mice. Reactive astrocytes and expression of the reactive astrocyte and neurotoxicity marker Lcn2 (Lipocalin-2) were increased in the perilesional brain tissue of NG2-KO mice. In addition, microglia/macrophages with activated morphology were increased in number and mRNA expression of the M2 marker Arg1 (Arginase 1) was enhanced in NG2-KO mice. While TBI-induced expression of pro-inflammatory cytokine genes was unchanged between genotypes, PCR array screening revealed a marked TBI-induced up-regulation of the C-X-C motif chemokine 13 gene Cxcl13 in NG2-KO mice. CXCL13, known to attract immune cells to the inflamed brain, was expressed by activated perilesional microglia/macrophages seven days after TBI. Thirty days after TBI, NG2-KO mice still exhibited more pronounced neurological deficits than WT mice, up-regulation of Cxcl13, enhanced CD45+ leukocyte infiltration and a relative increase of activated Iba-1+/CD45+ microglia/macrophages. Our study demonstrates that lack of NG2 exacerbates the neurological outcome after TBI and associates with abnormal activation of astrocytes, microglia/macrophages and increased leukocyte recruitment to the injured brain. These findings suggest that NG2 may counteract neurological deficits and adverse glial responses in TBI., (© 2015 Wiley Periodicals, Inc.)

The NG2 proteoglycan is characteristically expressed by oligodendrocyte progenitor cells (OPC) and also by aggressive brain tumours highly resistant to chemo- and radiation therapy. Oligodendrocyte-lineage cells are particularly sensitive to stress resulting in cell death in white matter after hypoxic or ischemic insults of premature infants and destruction of OPC in some types of Multiple Sclerosis lesions. Here we show that the NG2 proteoglycan binds OMI/HtrA2, a mitochondrial serine protease which is released from damaged mitochondria into the cytosol in response to stress. In the cytosol, OMI/HtrA2 initiates apoptosis by proteolytic degradation of anti-apoptotic factors. OPC in which NG2 has been downregulated by siRNA, or OPC from the NG2-knockout mouse show an increased sensitivity to oxidative stress evidenced by increased cell death. The proapoptotic protease activity of OMI/HtrA2 in the cytosol can be reduced by the interaction with NG2. Human glioma expressing high levels of NG2 are less sensitive to oxidative stress than those with lower NG2 expression and reducing NG2 expression by siRNA increases cell death in response to oxidative stress. Binding of NG2 to OMI/HtrA2 may thus help protect cells against oxidative stress-induced cell death. This interaction is likely to contribute to the high chemo- and radioresistance of glioma.

During early embryogenesis, microglia arise from yolk sac progenitors that populate the developing central nervous system (CNS), but how the tissue-resident macrophages are maintained throughout the organism's lifespan still remains unclear. Here, we describe a system that allows specific, conditional ablation of microglia in adult mice. We found that the microglial compartment was reconstituted within 1 week of depletion. Microglia repopulation relied on CNS-resident cells, independent from bone-marrow-derived precursors. During repopulation, microglia formed clusters of highly proliferative cells that migrated apart once steady state was achieved. Proliferating microglia expressed high amounts of the interleukin-1 receptor (IL-1R), and treatment with an IL-1R antagonist during the repopulation phase impaired microglia proliferation. Hence, microglia have the potential for efficient self-renewal without the contribution of peripheral myeloid cells, and IL-1R signaling participates in this restorative proliferation process., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Ectopic gene expression studies in primary immune cells have been notoriously difficult to perform due to the limitations in conventional transfection and viral transduction methods. Although replication-defective adenoviruses provide an attractive alternative for gene delivery, their use has been hampered by the limited susceptibility of murine leukocytes to adenoviral infection, due to insufficient expression of the human coxsackie/adenovirus receptor (CAR). In this issue of the European Journal of Immunology, Heger et al. [Eur. J. Immunol. 2015. 45: XXXX-XXXX] report the generation of transgenic mice that enable conditional Cre/loxP-mediated expression of human CAR. The authors demonstrate that this R26/CAG-CAR∆1(StopF) mouse strain facilitates the faithful monitoring of Cre activity in situ as well as the specific and efficient adenoviral transduction of primary immune cell populations in vitro. Further tweaking of the system towards more efficient gene transfer in vivo remains a future challenge., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Mechanisms underlying central neuropathic pain are poorly understood. Although glial dysfunction has been functionally linked with neuropathic pain, very little is known about modulation of pain by oligodendrocytes. Here we report that genetic ablation of oligodendrocytes rapidly triggers a pattern of sensory changes that closely resemble central neuropathic pain, which are manifest before overt demyelination. Primary oligodendrocyte loss is not associated with autoreactive T- and B-cell infiltration in the spinal cord and neither activation of microglia nor reactive astrogliosis contribute functionally to central pain evoked by ablation of oligodendrocytes. Instead, light and electron microscopic analyses reveal axonal pathology in the spinal dorsal horn and spinothalamic tract concurrent with the induction and maintenance of nociceptive hypersensitivity. These data reveal a role for oligodendrocytes in modulating pain and suggest that perturbation of oligodendrocyte functions that maintain axonal integrity can lead to central neuropathic pain independent of immune contributions.

NG2 (nerve/glia antigen-2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2-expressing (NG2(+) ) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2(+) cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter(+) astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen-sensitive and NG2 gene locus-dependent gene recombination could be detected in a small, but persistent population of cortical NeuN(+) neurons starting from the second postnatal week., (Copyright © 2014 Wiley Periodicals, Inc.)

Multiple sclerosis is a demyelinating autoimmune disease of the CNS. Its animal model experimental autoimmune encephalomyelitis is commonly induced by active immunization with myelin antigens. To investigate human immune responses against myelin antigens in vivo we established a new subclinical experimental autoimmune encephalomyelitis model in humanized mice. NOD/Scidγc⁻/⁻ animals were transferred with peripheral blood mononuclear cells from healthy human donors and immunized with myelin antigens in complete Freund's adjuvant and antigen-pulsed autologous dendritic cells. Human T cells recovered from these animals reacted specifically to the soluble domain of myelin oligodendrocyte glycoprotein and secreted proinflammatory cytokines. Furthermore, immunized animals developed subclinical CNS inflammation with infiltrating CD4⁺ and CD8⁺ T cells and production of encephalitogenic cytokines. Thus, this model of myelin-induced CNS inflammation by human T cells may allow testing of new human-specific therapeuticals for multiple sclerosis.

Topical application of imiquimod (IMQ) on the skin of mice induces inflammation with common features found in psoriatic skin. Recently, it was postulated that IL-17 has an important role both in psoriasis and in the IMQ model. To further investigate the impact of IL-17RA signaling in psoriasis, we generated IL-17 receptor A (IL-17RA)-deficient mice (IL-17RA(del)) and challenged these mice with IMQ. Interestingly, the disease was only partially reduced and delayed but not abolished when compared with controls. In the absence of IL-17RA, we found persisting signs of inflammation such as neutrophil and macrophage infiltration within the skin. Surprisingly, already in the naive state, the skin of IL-17RA(del) mice contained significantly elevated numbers of Th17- and IL-17-producing γδ T cells, assuming that IL-17RA signaling regulates the population size of Th17 and γδ T cells. Upon IMQ treatment of IL-17RA(del) mice, these cells secreted elevated amounts of tumor necrosis factor-α, IL-6, and IL-22, accompanied by increased levels of the chemokine CXCL2, suggesting an alternative pathway of neutrophil and macrophage skin infiltration. Hence, our findings have major implications in the potential long-term treatment of psoriasis by IL-17-targeting drugs.

Gene inactivation reporters are powerful tools to circumvent limitations of the widely used Cre/loxP system of conditional mutagenesis. With new conditional transgenic mouse lines expressing the enhanced cyan fluorescent protein (ECFP) instead of connexin43 (Cx43) after Cre-mediated recombination, we demonstrate dual reporter approaches to simultaneously examine astrocyte subpopulations expressing different connexins, identify compensatory up-regulation within gene families, and quantify Cre-mediated deletion at the allelic level. Analysis of a newly generated Cx43 knock-in ECFP mouse revealed an unexpected heterogeneity of Cx43-expressing astrocytes across brain areas.

Mature dendritic cells (DCs) are established as unrivaled antigen-presenting cells (APCs) in the initiation of immune responses, whereas steady-state DCs induce peripheral T cell tolerance. Using various genetic approaches, we depleted CD11c(+) DCs in mice and induced autoimmune CNS inflammation. Unexpectedly, mice lacking DCs developed aggravated disease compared to control mice. Furthermore, when we engineered DCs to present a CNS-associated autoantigen in an induced manner, we found robust tolerance that prevented disease, which coincided with an upregulation of the PD-1 receptor on antigen-specific T cells. Additionally, we showed that PD-1 was necessary for DC-mediated induction of regulatory T cells. Our results show that a reduction of DCs interferes with tolerance, resulting in a stronger inflammatory response, and that other APC populations could compensate for the loss of immunogenic APC function in DC-depleted mice., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Anti-myelin immunity is commonly thought to drive multiple sclerosis, yet the initial trigger of this autoreactivity remains elusive. One of the proposed factors for initiating this disease is the primary death of oligodendrocytes. To specifically test such oligodendrocyte death as a trigger for anti-CNS immunity, we inducibly killed oligodendrocytes in an in vivo mouse model. Strong microglia-macrophage activation followed oligodendrocyte death, and myelin components in draining lymph nodes made CNS antigens available to lymphocytes. However, even conditions favoring autoimmunity-bystander activation, removal of regulatory T cells, presence of myelin-reactive T cells and application of demyelinating antibodies-did not result in the development of CNS inflammation after oligodendrocyte death. In addition, this lack of reactivity was not mediated by enhanced myelin-specific tolerance. Thus, in contrast with previously reported impairments of oligodendrocyte physiology, diffuse oligodendrocyte death alone or in conjunction with immune activation does not trigger anti-CNS immunity.

NG2-expressing glia are precursors to oligodendrocytes and subpopulations of astrocytes. They are unique among glial cells in that they enter into synaptic specialisations with neurons throughout all areas of grey and white matter and at all ages. To date, the NG2 cells appear to represent a postsynaptic compartment, and synapses are formed with axons. With differentiation to oligodendrocytes, NG2 is downregulated and myelin antigens upregulated: this coincides with a loss of the synaptic contacts between neurons and NG2 glial cells. The functional roles of this glial-neuron synapse in regulation of differentiation into myelinating oligodendrocytes or additionally responding to and modulating neuronal network activity remain to be elucidated., (© 2011 The Authors. Journal of Anatomy © 2011 Anatomical Society of Great Britain and Ireland.)

NG2 cells, the fourth type of glia in the mammalian CNS, receive synaptic input from neurons. The function of this innervation is unknown yet. Postsynaptic changes in intracellular Ca(2+)-concentration ([Ca(2+)](i)) might be a possible consequence. We employed transgenic mice with fluorescently labeled NG2 cells to address this issue. To identify Ca(2+)-signaling pathways we combined patch-clamp recordings, Ca(2+)-imaging, mRNA-transcript analysis and focal pressure-application of various substances to identified NG2-cells in acute hippocampal slices. We show that activation of voltage-gated Ca(2+)-channels, Ca(2+)-permeable AMPA-receptors, and group I metabotropic glutamate-receptors provoke [Ca(2+)](i)-elevations in NG2 cells. The Ca(2+)-influx is amplified by Ca(2+)-induced Ca(2+)-release. Minimal electrical stimulation of presynaptic neurons caused postsynaptic currents but no somatic [Ca(2+)](i) elevations, suggesting that [Ca(2+)](i) elevations in NG2 cells might be restricted to their processes. Local Ca(2+)-signaling might provoke transmitter release or changes in cell motility. To identify structural prerequisites for such a scenario, we used electron microscopy, immunostaining, mRNA-transcript analysis, and time lapse imaging. We found that NG2 cells form symmetric and asymmetric synapses with presynaptic neurons and show immunoreactivity for vesicular glutamate transporter 1. The processes are actin-based, contain ezrin but not glial filaments, microtubules or endoplasmic reticulum. Furthermore, we demonstrate that NG2 cell processes in situ are highly motile. Our findings demonstrate that gray matter NG2 cells are endowed with the cellular machinery for two-way communication with neighboring cells.

According to previously published ultrastructural studies, oligodendrocytes in white matter exhibit gap junctions with astrocytes, but not among each other, while in vitro oligodendrocytes form functional gap junctions. We have studied functional coupling among oligodendrocytes in acute slices of postnatal mouse corpus callosum. By whole-cell patch clamp we dialyzed oligodendrocytes with biocytin, a gap junction-permeable tracer. On average 61 cells were positive for biocytin detected by labeling with streptavidin-Cy3. About 77% of the coupled cells stained positively for the oligodendrocyte marker protein CNPase, 9% for the astrocyte marker GFAP and 14% were negative for both CNPase and GFAP. In the latter population, the majority expressed Olig2 and some NG2, markers for oligodendrocyte precursors. Oligodendrocytes are known to express Cx47, Cx32 and Cx29, astrocytes Cx43 and Cx30. In Cx47-deficient mice, the number of coupled cells was reduced by 80%. Deletion of Cx32 or Cx29 alone did not significantly reduce the number of coupled cells, but coupling was absent in Cx32/Cx47-double-deficient mice. Cx47-ablation completely abolished coupling of oligodendrocytes to astrocytes. In Cx43-deficient animals, oligodendrocyte-astrocyte coupling was still present, but coupling to oligodendrocyte precursors was not observed. In Cx43/Cx30-double deficient mice, oligodendrocyte-to-astrocyte coupling was almost absent. Uncoupled oligodendrocytes showed a higher input resistance. We conclude that oligodendrocytes in white matter form a functional syncytium predominantly among each other dependent on Cx47 and Cx32 expression, while astrocytic connexins expression can promote the size of this network., (Copyright 2010 Wiley-Liss, Inc.)

The NG2 proteoglycan is a type 1-transmembrane protein expressed by a range of cell types within and outside the mammalian nervous system. NG2-expressing (NG2) cells are found in grey and white matter tracts of the developing and adult CNS and have previously been assumed to represent oligodendrocyte precursor cells: new work using transgenic mice has shown that NG2 cells generate oligodendrocytes, protoplasmic astrocytes and in some instances neurons in vivo. NG2 cells express GABAA receptors and the AMPA subtype of glutamate receptors. They make intimate contact to neurons prior to myelinating axons and also form electron-dense synaptic specialisations with axons in the cerebellum, cortex and hippocampus and with non-myelinated axons in the corpus callosum. These synaptic NG2 cells respond to neuronal release of glutamate and GABA. This neuron-glia interaction may thus regulate the differentiation and proliferation of NG2 cells. The C-terminal PDZ-binding motif of the NG2 protein binds several PDZ proteins including Mupp1, Syntenin and the Glutamate Receptor Interacting Protein (GRIP). Since GRIP can bind subunits of the AMPA receptors expressed by NG2 cells, the interaction between GRIP and NG2 may orientate the glial AMPA receptors towards sites of neuronal glutamate release. The origin, heterogeneity and function of NG2 cells as modulators of the neuronal network are important incompletely resolved questions., (Copyright 2009 Elsevier B.V. All rights reserved.)

Semaphorins and their receptors, plexins, have emerged as important regulators of a multitude of biological processes. Plexin-B3 has been shown to be selectively expressed in postnatal oligodendrocytes. In contrast to the well-characterized Plexin-A family and the Plexin-B family members Plexin-B1 and -B2, no data are available on the functional role of Plexin-B3 in the central nervous system in vivo. Here we have elucidated the functional significance of Plexin-B3 by generating and analyzing constitutive knock-out mice. Plexin-B3-deficient mice were found to be viable and fertile. A systematic histological analysis revealed no morphological defects in the brain or spinal cord of mutant animals. In detailed behavioural analyses of locomotor activity, motor coordination, motor learning, and anxiety levels Plexin-B3-deficient mice were indistinguishable from wild-type controls. Thus we conclude that under physiological conditions Plexin-B3 is not essential for the development and function of the central nervous system.

The NG2 glycoprotein is a type I membrane protein expressed by immature cells in the developing and adult mouse. NG2+ cells of the embryonic and adult brain have been principally viewed as oligodendrocyte precursor cells but have additionally been considered a fourth glial class. They are likely to be a heterogeneous population. In order to facilitate studies on the function of NG2+ cells and to characterize these cells in situ, we generated an enhanced yellow fluorescent protein (EYFP) "knockin mouse." EYFP-expressing cells in heterozygous knockin mice expressed the NG2 protein in all regions and at all ages studied. The EYFP+ cells did not express markers of mature glia, developing or mature neurons or microglia, but expressed markers typical for immature oligodendrocyte-lineage cells. Examination of the hippocampus showed heterogeneity in the population with regard to expression of S100ss and glutamine synthetase. Furthermore, different subpopulations of NG2+ cells in the hippocampus could be recognized by their electrophysiological properties., ((c) 2008 Wiley-Liss, Inc.)

Migration of oligodendrocyte precursors along axons is a necessary prerequisite for myelination, but little is known about underlying mechanisms. NG2 is a large membrane proteoglycan implicated in oligodendrocyte migration. Here we show that a PDZ domain protein termed syntenin-1 interacts with NG2 and that syntenin-1 is necessary for normal rates of migration. The association of syntenin-1 with NG2, identified in a yeast two-hybrid screen, was confirmed by colocalization of both proteins within processes of oligodendroglial precursor cells and by coimmunoprecipitation from cell extracts. Syntenin-1 also colocalizes with NG2 in "co-capping" assays, demonstrating a lateral association of both proteins in live oligodendrocytes. RNA interference-mediated down-regulation of syntenin-1 in glial cells results in a significant reduction of migration in vitro, as does the presence of polyclonal antibody against NG2. Thus syntenin plays a role in the migration of oligodendroglial precursors, and we suggest that NG2-syntenin-1 interactions contribute to this.

The NG2 glycoprotein is a type I membrane protein expressed in the developing and adult central nervous system (CNS) by subpopulations of glia including oligodendroglial precursor cells (OPCs), and in the developing CNS additionally by pericytes. In the mouse CNS, expression of NG2 protein is already observed at embryonic day 13 and peaks between postnatal days 8 and 12. NG2+ cells persist in grey and white matter in adult mouse brain: cells in the developing and adult brain show clear differences in migration, cell-cycle length and lineage restriction. Several groups have provided evidence that subpopulations of NG2+ cells can generate neurons in vivo. Neuronal stimulation in the developing and adult hippocampus leads to Ca2+ signals in apposing NG2+ glia, suggesting that these cells may modulate synaptic activity, and NG2+ cells often ensheath synapses. The structure of the protein with two N-terminal LamininG/Neurexin/Sex-hormone-binding globulin domains suggests a role in adhesion. The C-terminal PSD-95/DiscsLarge/Zona Occludens-1 (PDZ)-binding motif has been found to associate with several PDZ proteins including the Glutamate Receptor Interacting Protein GRIP: NG2 may thus act to position AMPA receptors on glia towards sites of neuronal glutamate release. Furthermore, the NG2 proteoglycan plays a role in cell migration and spreading and associates with actin-containing cytoskeletal structures.

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.

Self-renewing, totipotent embryonic stem (ES) cells may provide a virtually unlimited donor source for transplantation. A protocol that permits the in vitro generation of precursors for oligodendrocytes and astrocytes from ES cells was devised. Transplantation in a rat model of a human myelin disease shows that these ES cell-derived precursors interact with host neurons and efficiently myelinate axons in brain and spinal cord. Thus, ES cells can serve as a valuable source of cell type-specific somatic precursors for neural transplantation.

Limited experimental access to the central nervous system (CNS) is a key problem in the study of human neural development, disease, and regeneration. We have addressed this problem by generating neural chimeras composed of human and rodent cells. Fetal human brain cells implanted into the cerebral ventricles of embryonic rats incorporate individually into all major compartments of the brain, generating widespread CNS chimerism. The human cells differentiate into neurons, astrocytes, and oligodendrocytes, which populate the host fore-, mid-, and hindbrain. These chimeras provide a unique model to study human neural cell migration and differentiation in a functional nervous system.

During embryogenesis, pluripotent stem cells segregate into daughter lineages of progressively restricted developmental potential. In vitro, this process has been mimicked by the controlled differentiation of embryonic stem cells into neural precursors. To explore the developmental potential of these cell-culture-derived precursors in vivo, we have implanted them into the ventricles of embryonic rats. The transplanted cells formed intraventricular neuroepithelial structures and migrated in large numbers into the brain tissue. Embryonic-stem-cell-derived neurons, astrocytes, and oligodendrocytes incorporated into telencephalic, diencephalic, and mesencephalic regions and assumed phenotypes indistinguishable from neighboring host cells. These observations indicate that entirely in vitro-generated neural precursors are able to respond to environmental signals guiding cell migration and differentiation and have the potential to reconstitute neuronal and glial lineages in the central nervous system.