Abstract Background Administration of exogenous interferon-γ (IFNγ) aggravates the symptoms of multiple sclerosis (MS), whereas interferon-β (IFNβ) is used for treatment of MS patients. We previously demonstrated that IFNγ induces apoptosis of oligodendroglial progenitor cells (OPCs), suggesting that IFNγ is more toxic to OPCs than IFNβ. Thus we hypothesized that a difference in expression profiles between IFNγ-inducible and IFNβ-inducible genes in OPCs would predict the genes responsible for IFNγ-mediated cytotoxic effects on OPCs. We have tested this hypothesis particularly focusing on the interferon regulatory factors (IRFs) well-known transcription factors up-regulated by IFNs. Methods Highly pure primary rat OPC cultures were treated with IFNγ and IFNβ. Cell death and proliferation were assessed by MTT reduction, caspse-3-like proteinase activity, Annexin-V binding, mitochondrial membrane potential, and BrdU-incorporation. Induction of all nine IRFs was comprehensively compared by quantitative PCR between IFNγ-treated and IFNβ-treated OPCs. IRFs more strongly induced by IFNγ than by IFNβ were selected, and tested for their ability to induce OPC apoptosis by overexpression and by inhibition by dominant-negative proteins or small interference RNA either in the presence or absence of IFNγ. Results Unlike IFNγ, IFNβ did not induce apoptosis of OPCs. Among nine IRFs, IRF1 and IRF8 were preferentially up-regulated by IFNγ. In contrast, IRF7 was more robustly induced by IFNβ than by IFNγ. Overexpressed IRF1 elicited apoptosis of OPCs, and a dominant negative IRF1 protein partially protected OPCs from IFNγ-induced apoptosis, indicating a substantial contribution of IRF1 to IFNγ-induced OPC apoptosis. On the other hand, overexpression of IRF8 itself had only marginal proapoptotic effects. However, overexpressed IRF8 enhanced the IFNγ-induced cytotoxicity and the proapoptotic effect of overexpressed IRF1, and down-regulation of IRF8 by siRNA partially but significantly reduced preapoptotic cells after treatment with IFNγ, suggesting that IRF8 cooperatively enhances IFNγ-induced OPC apoptosis. Conclusions This study has identified that IRF1 and IRF8 mediate IFNγ-signaling leading to OPC apoptosis. Therapies targeting at these transcription factors and their target genes could reduce IFNγ-induced OPC loss and thereby enhance remyelination in MS patients.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously we and others reported a role of microglia in the pathophysiology of RTT. Because microglia in the Mecp2 knockout (Mecp2KO) mouse model of RTT over-produce neurotoxic mediators glutamate and reactive oxygen species, we hypothesize that blocking neuron-microglia interaction by ablation of CX₃CR1, a chemokine receptor expressed in microglia/myeloid cells mediating such interaction by pairing with its neuronal ligand CX₃CL1, would ameliorate the RTT-like phenotype in Mecp2KO mice. Here we report that CX₃CR1 ablation prolonged the lifespan of Mecp2KO mice from a median survival of 54.5-74days, and significantly improved the body weight gain, symptomatic scores, major respiratory parameters, and motor coordination and performance. CX₃CR1 ablation rectified previously identified histological abnormalities in the Mecp2KO brain such as neuronal soma size in hippocampal CA2, and the number, soma size, and process complexity of microglia. Moreover, CX₃CR1 ablation enhanced the neurotrophic action of microglia in Mecp2KO mice by producing higher amount of insulin-like growth factor 1. Our data support a role of myeloid cells/microglia in RTT and suggest a novel therapeutic approach for RTT by targeting CX₃CR1 with specific antagonists or genetic downregulation.

Differentiation of oligodendroglial progenitor cells (OPCs) into myelinating oligodendrocytes is known to be regulated by the microenvironment where they differentiate. However, current research has not verified whether or not oligodendroglial lineage cells (OLCs) derived from different anatomical regions of the central nervous system (CNS) respond to microenvironmental cues in the same manner. Here, we isolated pure OPCs from rat neonatal forebrain (FB) and spinal cord (SC) and compared their phenotypes in the same in vitro conditions. We found that although FB and SC OLCs responded differently to the same external factors; they were distinct in proliferation response to mitogens, oligodendrocyte phenotype after differentiation, and cytotoxic responses to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate-type glutamate receptor-mediated excitotoxicity at immature stages of differentiation in a cell-intrinsic manner. Moreover, transcriptome analysis identified genes differentially expressed between these OPC populations, including those encoding transcription factors (TFs), cell surface molecules, and signaling molecules. Particularly, FB and SC OPCs retained the expression of FB- or SC-specific TFs, such as Foxg1 and Hoxc8, respectively, even after serial passaging in vitro. Given the essential role of these TFs in the regional identities of CNS cells along the rostrocaudal axis, our results suggest that CNS region-specific gene regulation by these TFs may cause cell-intrinsic differences in cellular responses between FB and SC OLCs to extracellular molecules. Further understanding of the regional differences among OPC populations will help to improve treatments for demyelination in different CNS regions and to facilitate the development of stem cell-derived OPCs for cell transplantation therapies for demyelination. Cover Image for this issue: doi. 10.1111/jnc.13809.

Abstract Background Recent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia. Methods The CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model. Results Our analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice. Conclusions This study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.

Abstract Background Endogenous retroviruses (ERVs), including murine leukemia virus (MuLV) type-ERVs (MuLV-ERVs), are presumed to occupy ~10% of the mouse genome. In this study, following the identification of a full-length MuLV-ERV by in silico survey of the C57BL/6J mouse genome, its distribution in different mouse strains and expression characteristics were investigated. Results Application of a set of ERV mining protocols identified a MuLV-ERV locus with full coding potential on chromosome 8 (named ERVmch8). It appears that ERVmch8 shares the same genomic locus with a replication-incompetent MuLV-ERV, called Emv2; however, it was not confirmed due to a lack of relevant annotation and Emv2 sequence information. The ERVmch8 sequence was more prevalent in laboratory strains compared to wild-derived strains. Among 16 different tissues of ~12 week-old female C57BL/6J mice, brain homogenate was the only tissue with evident expression of ERVmch8. Further ERVmch8 expression analysis in six different brain compartments and four peripheral neuronal tissues of C57BL/6J mice revealed no significant expression except for the cerebellum in which the ERVmch8 locus' low methylation status was unique compared to the other brain compartments. The ERVmch8 locus was found to be surrounded by genes associated with neuronal development and/or inflammation. Interestingly, cerebellum-specific ERVmch8 expression was age-dependent with almost no expression at 2 weeks and a plateau at 6 weeks. Conclusions The ecotropic ERVmch8 locus on the C57BL/6J mouse genome was relatively undermethylated in the cerebellum, and its expression was cerebellum-specific and age-dependent.

Genetic or pharmacological activation of canonical Wnt/β-catenin signaling inhibits oligodendrocyte differentiation. Transcription factor 7-like 2 (TCF7l2), also known as TCF4, is a Wnt effector induced transiently in the oligodendroglial lineage. A well accepted dogma is that TCF7l2 inhibits oligodendrocyte differentiation through activation of Wnt/β-catenin signaling. We report that TCF7l2 is upregulated transiently in postmitotic, newly differentiated oligodendrocytes. Using in vivo gene conditional ablation, we found surprisingly that TCF7l2 positively regulates neonatal and postnatal mouse oligodendrocyte differentiation during developmental myelination and remyelination in a manner independent of the Wnt/β-catenin signaling pathway. We also reveal a novel role of TCF7l2 in repressing a bone morphogenetic protein signaling pathway that is known to inhibit oligodendrocyte differentiation. Thus, our study provides novel data justifying therapeutic attempts to enhance, rather than inhibit, TCF7l2 signaling to overcome arrested oligodendroglial differentiation in multiple sclerosis and other demyelinating diseases.

Rett syndrome (RTT) is an autism spectrum disorder caused by loss-of-function mutations in the gene encoding MeCP2, an epigenetic modulator that binds the methyl CpG dinucleotide in target genes to regulate transcription. Previously, we and others reported a role of microglia in the pathophysiology of RTT. To understand the mechanism of microglia dysfunction in RTT, we identified a MeCP2 target gene, SLC38A1, which encodes a major glutamine transporter (SNAT1), and characterized its role in microglia. We found that MeCP2 acts as a microglia-specific transcriptional repressor of SNAT1. Because glutamine is mainly metabolized in the mitochondria, where it is used as an energy substrate and a precursor for glutamate production, we hypothesize that SNAT1 overexpression in MeCP2-deficient microglia would impair the glutamine homeostasis, resulting in mitochondrial dysfunction as well as microglial neurotoxicity because of glutamate overproduction. Supporting this hypothesis, we found that MeCP2 downregulation or SNAT1 overexpression in microglia resulted in (1) glutamine-dependent decrease in microglial viability, which was corroborated by reduced microglia counts in the brains of MECP2 knock-out mice; (2) proliferation of mitochondria and enhanced mitochondrial production of reactive oxygen species; (3) increased oxygen consumption but decreased ATP production (an energy-wasting state); and (4) overproduction of glutamate that caused NMDA receptor-dependent neurotoxicity. The abnormalities could be rectified by mitochondria-targeted expression of catalase and a mitochondria-targeted peptide antioxidant, Szeto-Schiller 31. Our results reveal a novel mechanism via which MeCP2 regulates bioenergetic pathways in microglia and suggest a therapeutic potential of mitochondria-targeted antioxidants for RTT.

Motoneuron death after transection of the axons (axotomy) in neonates is believed to share the same mechanistic bases as naturally occurring programmed cell death during development. The c-Jun N-terminal kinase pathway is activated in both forms of motoneuron death, but it remains unknown to what extent these two forms of motoneuron death depend on this pathway and which upstream kinases are involved. We found that numbers of facial motoneurons are doubled in neonatal mice deficient in either ZPK/DLK (zipper protein kinase, also known as dual leucine zipper kinase), a mitogen-activated protein kinase kinase kinase, or in MKK4/MAP2K4, a mitogen-activated protein kinase kinase directly downstream of ZPK/DLK, and that the facial motoneurons in those mutant mice are completely resistant to axotomy-induced death. Conditional deletion of MKK4/MAP2K4 in neurons further suggested that ZPK/DLK and MKK4/MAP2K4-dependent mechanisms underlying axotomy-induced death are motoneuron autonomous. Nevertheless, quantitative analysis of facial motoneurons during embryogenesis revealed that both ZPK/DLK and MKK4/MAP2K4-dependent and -independent mechanisms contribute to developmental elimination of excess motoneurons. In contrast to MKK4/MAP2K4, mice lacking MKK7/MAP2K7, another mitogen-activated protein kinase kinase directly downstream of ZPK/DLK, conditionally in neurons did not have excess facial motoneurons. However, some MKK7/MAP2K7-deficient facial motoneurons were resistant to axotomy-induced death, indicating a synergistic effect of MKK7/MAP2K7 on axotomy-induced death of these facial motoneurons. Together, our study provides compelling evidence for the pivotal roles of the ZPK/DLK and MKK4/MAP2K4-dependent mechanism in axotomy-induced motoneuron death in neonates and also demonstrates that axotomy-induced motoneuron death is not identical to developmental motoneuron death with respect to the involvement of ZPK/DLK, MKK4/MAP2K4 and MKK7/MAP2K7.

Microglia are highly plastic cells that can assume different phenotypes in response to microenvironmental signals. Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) promote differentiation into classically activated M1-like microglia, which produce high levels of pro-inflammatory cytokines and nitric oxide and are thought to contribute to neurological damage in ischemic stroke and Alzheimer's disease. IL-4 in contrast induces a phenotype associated with anti-inflammatory effects and tissue repair. We here investigated whether these microglia subsets vary in their K⁺ channel expression by differentiating neonatal mouse microglia into M(LPS) and M(IL-4) microglia and studying their K⁺ channel expression by whole-cell patch-clamp, quantitative PCR and immunohistochemistry. We identified three major types of K⁺ channels based on their biophysical and pharmacological fingerprints: a use-dependent, outwardly rectifying current sensitive to the K_V 1.3 blockers PAP-1 and ShK-186, an inwardly rectifying Ba²⁺ -sensitive K_ir 2.1 current, and a Ca²⁺ -activated, TRAM-34-sensitive K_Ca 3.1 current. Both K_V 1.3 and K_Ca 3.1 blockers inhibited pro-inflammatory cytokine production and iNOS and COX2 expression demonstrating that K_V 1.3 and K_Ca 3.1 play important roles in microglia activation. Following differentiation with LPS or a combination of LPS and IFN-γ microglia exhibited high K_V 1.3 current densities (∼50 pA/pF at 40 mV) and virtually no K_Ca 3.1 and K_ir currents, while microglia differentiated with IL-4 exhibited large K_ir 2.1 currents (∼ 10 pA/pF at -120 mV). K_Ca 3.1 currents were generally low but moderately increased following stimulation with IFN-γ or ATP (∼10 pS/pF). This differential K⁺ channel expression pattern suggests that K_V 1.3 and K_Ca 3.1 inhibitors could be used to inhibit detrimental neuroinflammatory microglia functions. GLIA 2016;65:106-121.