Kyoung-Jin Min

Gangliosides activate microglia via protein kinase C and NADPH oxidase

Microglia, the major immune effector cells in the central nervous system, are activated when the brain suffers injury. A number of studies indicate that gangliosides activate microglia. However, the signaling mechanisms involved in microglial activation are not yet to be elucidated. Our results show that gangliosides induce the expression of interleukin (IL)‐1β, tumor necrosis factor‐α (TNF‐α), and inducible nitric oxide synthase (iNOS) in rat brain microglia and BV2 murine microglia via protein kinase C (PKC) and NADPH oxidase. Expression of IL‐1β, TNF‐α, and iNOS in ganglioside‐treated cells was significantly reduced in the presence of inhibitors of PKC (GF109203X, Gö6976, Ro31‐8220, and rottlerin) and NADPH oxidase (diphenyleneiodonium chloride [DPI]). In response to gangliosides, PKC‐α, βII, and δ and NADPH oxidase p67phox translocated from the cytosol to the membrane. ROS generation was also activated within 5 min of ganglioside treatment. Ganglioside‐induced ROS generation was blocked by PKC inhibitors. Furthermore, ganglioside‐induced activation of NF‐κB, an essential transcription factor that mediates the expression of IL‐1β, TNF‐α, and iNOS, was reduced in the presence of GF109203X and DPI. Our results collectively suggest that gangliosides activate microglia via PKC and NADPH oxidase, which regulate activation of NF‐κB.

Resident microglia die and infiltrated neutrophils and monocytes become major inflammatory cells in lipopolysaccharide-injected brain

Generally, it has been accepted that microglia play important roles in brain inflammation. However, recently several studies suggested possible infiltration of blood neutrophils and monocytes into the brain. To understand contribution of microglia and blood inflammatory cells to brain inflammation, the behavior of microglia, neutrophils, and monocytes was investigated in LPS (lipopolysaccharide)‐injected substantia nigra pars compacta, cortex, and hippocampus of normal and/or leukopenic rats using specific markers of neutrophils (myeloperoxidase, MPO), and microglia and monocytes (ionized calcium binding adaptor molecule‐1, Iba‐1), as well as a general marker for these inflammatory cells (CD11b). CD11b‐immunopositive (CD11b+) cells and Iba‐1+ cells displayed similar behavior in intact and LPS‐injected brain at 6 h after the injection. Interestingly, however, CD11b+ cells and Iba‐1+ cells displayed significantly different behavior at 12 h: Iba‐1+ cells disappeared while CD11b+ cells became round in shape. We found that CD11b/Iba‐1‐double positive (CD11b+/Iba‐1+) ramified microglia died within 6 h after LPS injection. The round CD11b+ cells detected at 12 h were MPO+. These CD11b+/MPO+ cells were not found in leukopenic rats, suggestive of neutrophil infiltration. MPO+ neutrophils expressed inducible nitric oxide synthase, interleukin‐1β, cyclooxygenase‐2, and monocyte chemoattractant protein‐1, but died within 18 h. CD11b+ cells detected at 24 h appeared to be infiltrated monocytes, since these cells were once labeled with Iba‐1 and were not found in leukopenic rats. Furthermore, transplanted monocytes were detectable in LPS‐injected brain. These results suggest that at least a part of neutrophils and monocytes could have been misinterpreted as activated microglia in inflamed brain.

Astrocytes in injury states rapidly produce anti‐inflammatory factors and attenuate microglial inflammatory responses

J. Neurochem. (2010) 115, 1161–1171.

Adenosine induces hemeoxygenase‐1 expression in microglia through the activation of phosphatidylinositol 3‐kinase and nuclear factor E2‐related factor 2

Adenosine, a purine nucleoside, has been reported to suppress the inflammatory responses of microglia in the brain. However, the underlying mechanisms of its anti‐inflammatory action are unclear at present. Here we show that adenosine reduces the increase in intracellular reactive oxygen species (ROS) through expression of an antioxidant enzyme, hemeoxygenase‐1 (HO‐1). The H2O2‐induced intracellular ROS level was significantly low in microglia pretreated with adenosine for 3–6 h, compared with that in untreated cells. Adenosine induced HO‐1 mRNA and protein expression within 3 h, which was maintained for up to 12 h. Nuclear factor E2‐related factor 2 (Nrf2), a transcription factor, and phosphatidylinositol 3‐kinase (PI3K) and Akt pathways appear to mediate HO‐1 expression. In response to adenosine, Nrf2 translocated from the cytosol to nuclei, and bound to the antioxidant response element (ARE). Adenosine enhanced HO‐1 promoter activity in an ARE‐dependent manner. Moreover, the nucleoside stimulated Akt phosphorylation, and suppressors of PI3K (LY294002 and wortmannin) reduced adenosine‐induced HO‐1 expression. However, we propose that the effects of adenosine are independent of adenosine receptors, since agonists and antagonists of A1, A2a, and A3 had little effect on the regulation of intracellular ROS and HO‐1 expression. Our results collectively suggest that adenosine acts as an endogenous regulator of brain inflammation via modulation of microglial ROS production.

Multiple mechanisms that prevent excessive brain inflammation

Inflammation of the injured brain has a double‐edged effect. Inflammation protects the brain from infection, but it aggravates injury. Furthermore, brain inflammation is considered a risk factor for neurodegenerative disorders, such as Alzheimers and Parkinsons diseases. Emerging evidence supports the activation of negative regulatory mechanisms during this process to prevent prolonged and extensive inflammation. The inflammatory stimulators themselves or products of inflammatory cells may induce the expression of negative feedback regulators, such as suppressor of cytokine signaling (SOCS)‐family proteins, antioxidant enzymes, and antiinflammatory cytokines. Furthermore, death of activated microglia (major inflammatory cells in the brain) may regulate brain inflammation. Astrocytes, the most abundant cells in the brain, may also act in preventing microglial overactivation. Therefore, we propose that the extent and duration of brain inflammation is tightly regulated through the cooperation of multiple mechanisms to maximize antipathogenic effects and minimize tissue damage.

Spatial and temporal correlation in progressive degeneration of neurons and astrocytes in contusion-induced spinal cord injury

BackgroundTraumatic spinal cord injury (SCI) causes acute neuronal death followed by delayed secondary neuronal damage. However, little is known about how microenvironment regulating cells such as microglia, astrocytes, and blood inflammatory cells behave in early SCI states and how they contribute to delayed neuronal death.MethodsWe analyzed the behavior of neurons and microenvironment regulating cells using a contusion-induced SCI model, examining early (3–6 h) to late times (14 d) after the injury.ResultsAt the penumbra region close to the damaged core (P1) neurons and astrocytes underwent death in a similar spatial and temporal pattern: both neurons and astrocytes died in the medial and ventral regions of the gray matter between 12 to 24 h after SCI. Furthermore, mRNA and protein levels of transporters of glutamate (GLT-1) and potassium (Kir4.1), functional markers of astrocytes, decreased at about the times that delayed neuronal death occurred. However, at P1 region, ramified Iba-1+ resident microglia died earlier (3 to 6 h) than neurons (12 to 24 h), and at the penumbra region farther from the damaged core (P2), neurons were healthy where microglia were morphologically activated. In addition, round Iba-1/CD45-double positive monocyte-like cells appeared after neurons had died, and expressed phagocytic markers, including mannose receptors, but rarely expressed proinflammatory mediators.ConclusionLoss of astrocyte function may be more critical for delayed neuronal death than microglial activation and monocyte infiltration.

Astrogliosis Is a Possible Player in Preventing Delayed Neuronal Death

Mitigating secondary delayed neuronal injury has been a therapeutic strategy for minimizing neurological symptoms after several types of brain injury. Interestingly, secondary neuronal loss appeared to be closely related to functional loss and/or death of astrocytes. In the brain damage induced by agonists of two glutamate receptors, N-ethyl-D-aspartic acid (NMDA) and kainic acid (KA), NMDA induced neuronal death within 3 h, but did not increase further thereafter. However, in the KA-injected brain, neuronal death was not obviously detectable even at injection sites at 3 h, but extensively increased to encompass the entire hemisphere at 7 days. Brain inflammation, a possible cause of secondary neuronal damage, showed little differences between the two models. Importantly, however, astrocyte behavior was completely different. In the NMDA-injected cortex, the loss of glial fibrillary acidic protein-expressing (GFAP+) astrocytes was confined to the injection site until 7 days after the injection, and astrocytes around the damage sites showed extensive gliosis and appeared to isolate the damage sites. In contrast, in the KA-injected brain, GFAP+ astrocytes, like neurons, slowly, but progressively, disappeared across the entire hemisphere. Other markers of astrocytes, including S100β, glutamate transporter EAAT2, the potassium channel Kir4.1 and glutamine synthase, showed patterns similar to that of GFAP in both NMDA- and KA-injected cortexes. More importantly, astrocyte disappearance and/or functional loss preceded neuronal death in the KA-injected brain. Taken together, these results suggest that loss of astrocyte support to neurons may be a critical cause of delayed neuronal death in the injured brain.

Enhanced phosphatidylinositol 4-phosphate 5-kinase α expression and PI(4,5)P2 production in LPS-stimulated microglia

Microglia are the major glial cells responsible for immune responses against harmful substances in the central nervous system. Type I phosphatidylinositol 4-phosphate 5-kinase alpha (PIP5Kalpha) and its lipid product, phosphatidylinositol 4,5-bisphosphate (PI[4,5]P(2)), regulate important cell surface functions. Here, we report that lipopolysaccharide (LPS) significantly enhanced PIP5Kalpha mRNA and protein expression levels in a time- and concentration-dependent manner in microglia. Furthermore, LPS stimulation led to a robust increase in PI(4,5)P(2) in the plasma membrane, demonstrated by PI(4,5)P(2) immunostaining or PI(4,5)P(2) imaging using a PI(4,5)P(2)-specific probe, tubby (R332H), fused to yellow fluorescent protein. Phosphatidylinositol 3-kinase, p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and c-Jun N-terminal kinase signaling pathway inhibitors clearly reduced PIP5Kalpha expression, indicating that these pathways are necessary for LPS-induced PIP5Kalpha expression. In addition, inhibition of nuclear factor-kappaB and Sp1 transcription factors interfered with the LPS-induced upregulation of PIP5Kalpha. Delivery of PI(4,5)P(2) into microglia increased the expression of interleukin-1beta and tumor necrosis factor alpha. These findings indicate that PIP5Kalpha upregulation and the subsequent rise in PI(4,5)P(2) in LPS-stimulated microglia may positively regulate microglial inflammatory responses.