To test whether mitogen-activated protein kinases (MAPKs) are involved in microglial activation, pure microglia prepared from 1- to 3-day-old rat brains were activated with either 100 ng/ml lipopolysaccharide (LPS) or 5 nM synthetic beta-amyloid (Abeta) (25-35). The patterns of MAPK activation following LPS and Abeta treatment were very similar. Three MAPK subtypes, p38, extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) were activated within 15 min and the activities of p38 and ERK were rapidly reduced to background level within 30 min while that of JNK was maintained for over 1 h. Both inhibitors of p38 (SB203580) and ERK pathway (PD098059) reduced LPS-induced nitric oxide (NO) release and Abeta-induced tumor necrosis factor-alpha (TNF-alpha) release. Furthermore, co-treatment of SB203580 and PD098059 additively reduced NO and TNF-alpha release. These results suggest that MAPK, at least p38 and ERK, mediate LPS-, and Abeta-induced microglial activation.

Microglia, brain resident macrophages, become activated in brains injured due to trauma, ischemia, or neurodegenerative diseases. In this study, we found that thrombin treatment of microglia induced NO release/ inducible nitric-oxide synthase expression, a prominent marker of activation. The effect of thrombin on NO release increased dose-dependently within the range of 5-20 units/ml. In immunoblot analyses, inducible nitricoxide synthase expression was detected within 9 h after thrombin treatment. This effect of thrombin was significantly reduced by protein kinase C inhibitors, such as Go6976, bisindolylmaleimide, and Ro31-8220. Within 15 min, thrombin activated three subtypes of mitogen-activated protein kinases: extracellular signal-regulated kinase, p38, and c-Jun N-terminal kinase/stress-activated protein kinase. Inhibition of the extracellular signalregulated kinase pathway and p38 reduced the NO release of thrombin-treated microglia. Thrombin also activated nuclear factor B (NF-B) within 5 min, and N-acetyl cysteine, an inhibitor of NF-B, reduced NO release. However, thrombin receptor agonist peptide (an agonist of protease activated receptor-1 (PAR-1)), could not mimic the effect of thrombin, and cathepsin G, a PAR-1 inhibitor, did not reduce the effect of thrombin. These results suggest that thrombin can activate microglia via protein kinase C, mitogen-activated protein kinases, and NF-B but that this occurs independently of PAR-1.

Microglia, brain resident macrophages, are activated in brain injuries and several neurodegenerative diseases. However, microglial activators that are produced in the brain are not yet defined. In this study, we showed that gangliosides, sialic acid-containing glycosphingolipids, could be a microglial activator. Gangliosides induced production of nitric oxide (NO) and tumor necrosis factor-␣ (TNF-␣) and expression of cyclooxygenase-2 (COX-2). The effect of gangliosides on NO release increased dose-dependently in the range of 10 -100 g/ml; however, the effect decreased at concentrations higher than 200 g/ml.

Microglia, the major immune effector cells in the CNS, become activated when the brain suffers injury. In this study, we observed that prothrombin, a zymogen of thrombin, induced NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α in rat brain microglia. The effect of prothrombin was independent of the protease activity of thrombin since hirudin, a specific inhibitor of thrombin, did not inhibit prothrombin-induced NO release. Furthermore, factor Xa enhanced the effect of prothrombin on microglial NO release. Kringle-2, a domain of prothrombin distinct from thrombin, mimicked the effect of prothrombin in inducing NO release and mRNA expression of inducible NO synthase, IL-1β, and TNF-α. Prothrombin and kringle-2 both triggered the same intracellular signaling pathways. They both activated mitogen-activated protein kinases and NF-κB in a similar pattern. NO release stimulated by either was similarly reduced by inhibitors of the extracellular signal-regulated kinase pathway (PD98059), p38 (SB203580), NF-κB (N-acetylcysteine), protein kinase C (Go6976, bisindolylmaleimide, and Ro31-8220), and phospholipase C (D609 and U73122). These results suggest that prothrombin can activate microglia, and that, in addition to thrombin, kringle-2 is a domain of prothrombin independently capable of activating microglia.

No abstract