Mariko Noda

Production and functions of IL-33 in the central nervous system

Interleukin-33 (IL-33) is a novel multifunctional IL-1 family cytokine. IL-33 signals via a heterodimer composed of IL-1 receptor-related protein ST2 and IL-1 receptor accessory protein (IL-1RAcP). IL-33 has been shown to activate T helper 2 cells (Th2), mast cells and basophils to produce a variety of Th2 cytokines and mediate allergic-type immune responses. Recent studies have revealed that glial cells are induced to express IL-33 mRNA and protein. However, the functions of IL-33 and its producing cells in the central nervous system (CNS) are still uncertain. In this study, we investigated the expression and function of IL-33 in the CNS. IL-33 is produced by endothelial cells and astrocytes but not by microglia or neurons. The IL-33 receptors are expressed mainly in microglia and astrocytes. IL-33 dose-dependently induces the proliferation of microglia and enhances the production of pro-inflammatory cytokines, such as IL-1β and TNFα, as well as the anti-inflammatory cytokine IL-10. It also enhances chemokines and nitric oxide production and phagocytosis by microglia. Thus, IL-33 produced in the CNS activates microglia and may function as a pro-inflammatory mediator in the pathophysiology of the CNS.

Blockade of Gap Junction Hemichannel Suppresses Disease Progression in Mouse Models of Amyotrophic Lateral Sclerosis and Alzheimer's Disease

BACKGROUND Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimers disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases. METHODS AND FINDINGS In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimers disease mouse model. CONCLUSIONS Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.

Fractalkine Attenuates Excito-neurotoxicity via Microglial Clearance of Damaged Neurons and Antioxidant Enzyme Heme Oxygenase-1 Expression

Glutamate-induced excito-neurotoxicity likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases. Microglial clearance of dying neurons and associated debris is essential to maintain healthy neural networks in the central nervous system. In fact, the functions of microglia are regulated by various signaling molecules that are produced as neurons degenerate. Here, we show that the soluble CX3C chemokine fractalkine (sFKN), which is secreted from neurons that have been damaged by glutamate, promotes microglial phagocytosis of neuronal debris through release of milk fat globule-EGF factor 8, a mediator of apoptotic cell clearance. In addition, sFKN induces the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in microglia in the absence of neurotoxic molecule production, including NO, TNF, and glutamate. sFKN treatment of primary neuron-microglia co-cultures significantly attenuated glutamate-induced neuronal cell death. Using several specific MAPK inhibitors, we found that sFKN-induced heme oxygenase-1 expression was primarily mediated by activation of JNK and nuclear factor erythroid 2-related factor 2. These results suggest that sFKN secreted from glutamate-damaged neurons provides both phagocytotic and neuroprotective signals.

IL-9 Promotes Th17 Cell Migration into the Central Nervous System via CC Chemokine Ligand-20 Produced by Astrocytes

Newly discovered IL-9–producing helper T cells (Th9) reportedly exert both aggravating and suppressive roles on experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. However, it is still unclear whether Th9 is a distinct Th cell subset and how IL-9 functions in the CNS. In this study, we show that IL-9 is produced by naive CD4+ T cells that were stimulated with anti-CD3 and anti-CD28 Abs under the conditions of Th2-, inducible regulatory T cell-, Th17-, and Th9-polarizing conditions and that IL-9 production is significantly suppressed in the absence of IL-4, suggesting that IL-4 is critical for the induction of IL-9 by each producing cell. The IL-9 receptor complex, IL-9R and IL-2Rγ, is constitutively expressed on astrocytes. IL-9 induces astrocytes to produce CCL-20 but not other chemokines, including CCL-2, CCL-3, and CXCL-2 by astrocytes. The conditioned medium of IL-9–stimulated astrocytes induces Th17 cell migration in vitro, which is cancelled by adding anti–CCL-20 neutralizing Abs. Treating with anti–IL-9 neutralizing Abs attenuates experimental autoimmune encephalomyelitis, decreases the number of infiltrating Th17 cells, and reduces CCL-20 expression in astrocytes. These results suggest that IL-9 is produced by several Th cell subsets in the presence of IL-4 and induces CCL-20 production by astrocytes to induce the migration of Th17 cells into the CNS.

Characterization of the dipeptide repeat protein in the molecular pathogenesis of c9FTD/ALS

The expansion of the GGGGCC hexanucleotide repeat in the non-coding region of the chromosome 9 open-reading frame 72 (C9orf72) gene is the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) (c9FTD/ALS). Recently, it was reported that an unconventional mechanism of repeat-associated non-ATG (RAN) translation arises from C9orf72 expansion. Sense and anti-sense transcripts of the expanded C9orf72 repeat, i.e. the dipeptide repeat protein (DRP) of glycine-alanine (poly-GA), glycine-proline (poly-GP), glycine-arginine (poly-GR), proline-arginine (poly-PR) and proline-alanine (poly-PA), are deposited in the brains of patients with c9FTD/ALS. However, the pathological significance of RAN-translated peptides remains unknown. We generated synthetic cDNAs encoding 100 repeats of DRP without a GGGGCC repeat and evaluated the effects of these proteins on cultured cells and cortical neurons in vivo. Our results revealed that the poly-GA protein formed highly aggregated ubiquitin/p62-positive inclusion bodies in neuronal cells. In contrast, the highly basic proteins poly-GR and PR also formed unique ubiquitin/p62-negative cytoplasmic inclusions, which co-localized with the components of RNA granules. The evaluation of cytotoxicity revealed that overexpressed poly-GA, poly-GP and poly-GR increased the substrates of the ubiquitin-proteasome system (UPS), including TDP-43, and enhanced the sensitivity to a proteasome inhibitor, indicating that these DRPs are cytotoxic, possibly via UPS dysfunction. The present data indicate that a gain-of-function mechanism of toxic DRPs possibly contributes to pathogenesis in c9FTD/ALS and that DRPs may serve as novel therapeutic targets in c9FTD/ALS.

Interleukin-34 Selectively Enhances the Neuroprotective Effects of Microglia to Attenuate Oligomeric Amyloid-β Neurotoxicity

Microglia, macrophage-like resident immune cells in the brain, possess both neurotoxic and neuroprotective properties and have a critical role in the development of Alzheimers disease (AD). We examined the function of Interleukin-34 (IL-34), a newly discovered cytokine, on microglia because it reportedly induces proliferation of monocytes and macrophages. We observed that the neuronal cells primarily produce IL-34 and that microglia express its receptor, colony-stimulating factor 1 receptor. IL-34 promoted microglial proliferation and clearance of soluble oligomeric amyloid-β (oAβ), which mediates synaptic dysfunction and neuronal damage in AD. IL-34 increased the expression of insulin-degrading enzyme, aiding the clearance of oAβ, and induced the antioxidant enzyme heme oxygenase-1 in microglia to reduce oxidative stress, without producing neurotoxic molecules. Consequently, microglia treated with IL-34 attenuated oAβ neurotoxicity in primary neuron-microglia co-cultures. In vivo, intracerebroventricular administration of IL-34 ameliorated impairment of associative learning and reduced oAβ levels through up-regulation of insulin-degrading enzyme and heme oxygenase-1 in an APP/PS1 transgenic mouse model of AD. These findings support the idea that enhancement of the neuroprotective property of microglia by IL-34 may be an effective approach against oAβ neurotoxicity in AD.

GM-CSF increases LPS-induced production of proinflammatory mediators via upregulation of TLR4 and CD14 in murine microglia

BackgroundMicroglia are resident macrophage-like cells in the central nervous system (CNS) and cause innate immune responses via the LPS receptors, Toll-like receptor (TLR) 4 and CD14, in a variety of neuroinflammatory disorders including bacterial infection, Alzheimer’s disease, and amyotrophic lateral sclerosis. Granulocyte macrophage-colony stimulating factor (GM-CSF) activates microglia and induces inflammatory responses via binding to GM-CSF receptor complex composed of two different subunit GM-CSF receptor α (GM-CSFRα) and common β chain (βc). GM-CSF has been shown to be associated with neuroinflammatory responses in multiple sclerosis and Alzheimer’s disease. However, the mechanisms how GM-CSF promotes neuroinflammation still remain unclear.MethodsMicroglia were stimulated with 20 ng/ml GM-CSF and the levels of TLR4 and CD14 expression were evaluated by RT-PCR and flowcytometry. LPS binding was analyzed by flowcytometry. GM-CSF receptor complex was analyzed by immunocytechemistry. The levels of IL-1β, IL-6 and TNF-α in culture supernatant of GM-CSF-stimulated microglia and NF-κB nuclear translocation were determined by ELISA. Production of nitric oxide (NO) was measured by the Griess method. The levels of p-ERK1/2, ERK1/2, p-p38 and p38 were assessed by Western blotting. Statistically significant differences between experimental groups were determined by one-way ANOVA followed by Tukey test for multiple comparisons.ResultsGM-CSF receptor complex was expressed in microglia. GM-CSF enhanced TLR4 and CD14 expressions in microglia and subsequent LPS-binding to the cell surface. In addition, GM-CSF priming increased LPS-induced NF-κB nuclear translocation and production of IL-1β, IL-6, TNF-α and NO by microglia. GM-CSF upregulated the levels of p-ERK1/2 and p-p38, suggesting that induction of TLR4 and CD14 expression by GM-CSF was mediated through ERK1/2 and p38, respectively.ConclusionsThese results suggest that GM-CSF upregulates TLR4 and CD14 expression in microglia through ERK1/2 and p38, respectively, and thus promotes the LPS receptor-mediated inflammation in the CNS.

Glutamate induces neurotrophic factor production from microglia via protein kinase C pathway

Microglia are intrinsic immune cells in the central nervous system and play key roles in the pathogenesis of various central nervous system disorders. Microglia have been shown to attack damaged neurons by secreting a variety of neurotoxic factors including inflammatory cytokines, reactive oxygen species and glutamate. On the other hand, they can produce neurotrophic factors (NTFs) which support neuronal survival and growth. However, the precise mechanism that regulates microglial NTF production is not fully understood, and the relation between glutamate and NTFs remains unclear. In the present study, we show that glutamate significantly induces microglial NTF production by the activation of N-methyl-d-aspartate (NMDA) receptors, group III metabotropic glutamate receptors, and glutamate transporters. Activation of NMDA receptors and group III metabotropic glutamate receptors induces intracellular Ca(2+) release from the endoplasmic reticulum. Further, stimulation of glutamate transporters leads to influx of extracellular Ca(2+) in a Na(+)-dependent manner. This intracellular Ca(2+) elevation activates the protein kinase C pathway which induces microglial NTF expression and production. These results suggest that microglia play a neuroprotective role during the excitotoxic state in neurodegenerative diseases.

Secreted Ectodomain of Sialic Acid-Binding Ig-Like Lectin-9 and Monocyte Chemoattractant Protein-1 Promote Recovery after Rat Spinal Cord Injury by Altering Macrophage Polarity

Engrafted mesenchymal stem cells from human deciduous dental pulp (SHEDs) support recovery from neural insults via paracrine mechanisms that are poorly understood. Here we show that the conditioned serum-free medium (CM) from SHEDs, administered intrathecally into rat injured spinal cord during the acute postinjury period, caused remarkable functional recovery. The ability of SHED-CM to induce recovery was associated with an immunoregulatory activity that induced anti-inflammatory M2-like macrophages. Secretome analysis of the SHED-CM revealed a previously unrecognized set of inducers for anti-inflammatory M2-like macrophages: monocyte chemoattractant protein-1 (MCP-1) and the secreted ectodomain of sialic acid-binding Ig-like lectin-9 (ED-Siglec-9). Depleting MCP-1 and ED-Siglec-9 from the SHED-CM prominently reduced its ability to induce M2-like macrophages and to promote functional recovery after spinal cord injury (SCI). The combination of MCP-1 and ED-Siglec-9 synergistically promoted the M2-like differentiation of bone marrow-derived macrophages in vitro, and this effect was abolished by a selective antagonist for CC chemokine receptor 2 (CCR2) or by the genetic knock-out of CCR2. Furthermore, MCP-1 and ED-Siglec-9 administration into the injured spinal cord induced M2-like macrophages and led to a marked recovery of hindlimb locomotor function after SCI. The inhibition of this M2 induction through the inactivation of CCR2 function abolished the therapeutic effects of both SHED-CM and MCP-1/ED-Siglec-9. Macrophages activated by MCP-1 and ED-Siglec-9 extended neurite and suppressed apoptosis of primary cerebellar granule neurons against the neurotoxic effects of chondroitin sulfate proteoglycans. Our data suggest that the unique combination of MCP-1 and ED-Siglec-9 repairs the SCI through anti-inflammatory M2-like macrophage induction.

The neurotoxic effect of astrocytes activated with toll-like receptor ligands

Toll-like receptors (TLRs) are key molecules in the innate immune system in the central nervous system. Although astrocytes are believed to play physiological roles in regulating neuronal activity and synaptic transmission, activated astrocytes may also be toxic to neurons. Here, we show that the ligands for TLRs 2, 4, 5 and 6 induce neuronal cell death in neuron-astrocytes co-cultures through the production of reactive oxygen species (ROS). Inhibition of ROS production by NADPH oxidase inhibitor apocynin significantly suppresses neuronal cell death. ROS induced in astrocytes via TLRs may be involved in neuroinflammation and a therapeutic target for neurotoxicity by activated astrocytes.