Bijay Parajuli

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.

Oligomeric amyloid β induces IL-1β processing via production of ROS: implication in Alzheimer's disease.

Alzheimer’s disease (AD) is a chronic neurodegenerative disease characterized by progressive neuronal loss and cognitive decline. Oligomeric amyloid β (oAβ) is involved in the pathogenesis of AD by affecting synaptic plasticity and inhibiting long-term potentiation. Although several lines of evidence suggests that microglia, the resident immune cells in the central nervous system (CNS), are neurotoxic in the development of AD, the mechanism whether or how oAβ induces microglial neurotoxicity remains unknown. Here, we show that oAβ promotes the processing of pro-interleukin (IL)-1β into mature IL-1β in microglia, which then enhances microglial neurotoxicity. The processing is induced by an increase in activity of caspase-1 and NOD-like receptor family, pyrin domain containing 3 (NLRP3) via mitochondrial reactive oxygen species (ROS) and partially via NADPH oxidase-induced ROS. The caspase-1 inhibitor Z-YVAD-FMK inhibits the processing of IL-1β, and attenuates microglial neurotoxicity. Our results indicate that microglia can be activated by oAβ to induce neuroinflammation through processing of IL-1β, a pro-inflammatory cytokine, in AD.

Interleukin-1β Induces Blood-Brain Barrier Disruption by Downregulating Sonic Hedgehog in Astrocytes

The blood–brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.

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.

Fingolimod Phosphate Attenuates Oligomeric Amyloid β–Induced Neurotoxicity via Increased Brain-Derived Neurotrophic Factor Expression in Neurons

The neurodegenerative processes that underlie Alzheimers disease are mediated, in part, by soluble oligomeric amyloid β, a neurotoxic protein that inhibits hippocampal long-term potentiation, disrupts synaptic plasticity, and induces the production of reactive oxygen species. Here we show that the sphingosine-1-phosphate (S1P) receptor (S1PR) agonist fingolimod phosphate (FTY720-P)-a new oral drug for multiple sclerosis-protects neurons against oligomeric amyloid β-induced neurotoxicity. We confirmed that primary mouse cortical neurons express all of the S1P receptor subtypes and FTY720-P directly affects the neurons. Treatment with FTY720-P enhanced the expression of brain-derived neurotrophic factor (BDNF) in neurons. Moreover, blocking BDNF-TrkB signaling with a BDNF scavenger, TrkB inhibitor, or ERK1/2 inhibitor almost completely ablated these neuroprotective effects. These results suggested that the neuroprotective effects of FTY720-P are mediated by upregulated neuronal BDNF levels. Therefore, FTY720-P may be a promising therapeutic agent for neurodegenerative diseases, such as Alzheimers disease.

Sirtuin 1 attenuates oxidative stress via upregulation of superoxide dismutase 2 and catalase in astrocytes.

Sirtuin 1 (SIRT1) exerts a neuroprotective effect on various neurologic diseases. Here we investigated the protective functions of SIRT1 in astrocytes, which are the most abundant cells in the central nervous system. Upregulation of SIRT1 suppressed the expression levels of pro-inflammatory cytokines and increased the expression levels of superoxide dismutase 2 and catalase. Inversely, inhibition of SIRT1 significantly increased the acetylation of forkhead box protein O4, decreased the expression levels of superoxide dismutase 2 and catalase, and increased the production of reactive oxygen species. Our data suggest that astrocytic SIRT1 may elicit neuroprotective effects through its anti-oxidative and anti-inflammatory functions.

CCL11 enhances excitotoxic neuronal death by producing reactive oxygen species in microglia.

The chemokine CCL11 (also known as eotaxin‐1) is a potent eosinophil chemoattractant that mediates allergic diseases such as asthma, atopic dermatitis, and inflammatory bowel diseases. Previous studies demonstrated that concentrations of CCL11 are elevated in the sera and cerebrospinal fluids (CSF) of patients with neuroinflammatory disorders, including multiple sclerosis. Moreover, the levels of CCL11 in plasma and CSF increase with age, and CCL11 suppresses adult neurogenesis in the central nervous system (CNS), resulting in memory impairment. However, the precise source and function of CCL11 in the CNS are not fully understood. In this study, we found that activated astrocytes release CCL11, whereas microglia predominantly express the CCL11 receptor. CCL11 significantly promoted the migration of microglia, and induced microglial production of reactive oxygen species by upregulating nicotinamide adenine dinucleotide phosphate‐oxidase 1 (NOX1), thereby promoting excitotoxic neuronal death. These effects were reversed by inhibition of NOX1. Our findings suggest that CCL11 released from activated astrocytes triggers oxidative stress via microglial NOX1 activation and potentiates glutamate‐mediated neurotoxicity, which may be involved in the pathogenesis of various neurological disorders. GLIA 2015;63:2274–2284

FGF-2 released from degenerating neurons exerts microglial-induced neuroprotection via FGFR3-ERK signaling pathway

BackgroundThe accumulation of activated microglia is a hallmark of various neurodegenerative diseases. Microglia may have both protective and toxic effects on neurons through the production of various soluble factors, such as chemokines. Indeed, various chemokines mediate the rapid and accurate migration of microglia to lesions. In the zebra fish, another well-known cellular migrating factor is fibroblast growth factor-2 (FGF-2). Although FGF-2 does exist in the mammalian central nervous system (CNS), it is unclear whether FGF-2 influences microglial function.MethodsThe extent of FGF-2 release was determined by ELISA, and the expression of its receptors was examined by immunocytochemistry. The effect of several drug treatments on a neuron and microglia co-culture system was estimated by immunocytochemistry, and the neuronal survival rate was quantified. Microglial phagocytosis was evaluated by immunocytochemistry and quantification, and microglial migration was estimated by fluorescence-activated cell sorting (FACS). Molecular biological analyses, such as Western blotting and promoter assay, were performed to clarify the FGF-2 downstream signaling pathway in microglia.ResultsFibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid β 1-42. FGF-2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia.ConclusionsFGF-2 secreted from degenerating neurons may act as a ‘help-me’ signal toward microglia by inducing migration and phagocytosis of unwanted debris.

The neuroprotective effects of milk fat globule-EGF factor 8 against oligomeric amyloid β toxicity

BackgroundPhosphatidylserine receptor is a key molecule that mediates the phagocytosis of apoptotic cells. Milk fat globule-EGF factor 8 (MFG-E8) is a phosphatidylserine receptor that is expressed on various macrophage lineage cells, including microglia in the central nervous system (CNS). Targeted clearance of degenerated neurons by microglia is essential to maintain healthy neural networks. We previously showed that the CX3C chemokine fractalkine is secreted from degenerated neurons and accelerates microglial clearance of neuronal debris via inducing the release of MFG-E8. However, the mechanisms by which microglia produce MFG-E8 and the precise functions of MFG-E8 are unknown.MethodsThe release of MFG-E8 from microglia treated with conditioned medium from neurons exposed to neurotoxic substances, glutamate or oligomeric amyloid β (oAβ) was measured by ELISA. The neuroprotective effects of MFG-E8 and MFG-E8 − induced microglial phagocytosis of oAβ were assessed by immunocytochemistry. The effects of MFG-E8 on the production of the anti-oxidative enzyme hemeoxygenase-1 (HO-1) were determined by ELISA and immunocytochemisty.ResultsMFG-E8 was induced in microglia treated with conditioned medium from neurons that had been exposed to neurotoxicants, glutamate or oAβ. MFG-E8 significantly attenuated oAβ-induced neuronal cell death in a primary neuron − microglia coculture system. Microglial phagocytosis of oAβ was accelerated by MFG-E8 treatment due to increased CD47 expression in the absence of neurotoxic molecule production, such as tumor necrosis factor-α, nitric oxide, and glutamate. MFG-E8 − treated microglia induced nuclear factor E(2) − related factor 2 (Nrf2) − mediated HO-1 production, which also contributed to neuroprotection.ConclusionsThese results suggest that microglia release MFG-E8 in response to signals from degenerated neurons and that MFG-E8 protects oAβ-induced neuronal cell death by promoting microglial phagocytic activity and activating the Nrf2-HO-1 pathway. Thus, MFG-E8 may have novel roles as a neuroprotectant in neurodegenerative conditions.