Activated microglia are thought to undergo apoptosis as a self-regulatory mechanism. To better understand molecular mechanisms of the microglial apoptosis, apoptosis-resistant variants of microglial cells were selected and characterized. The expression of lipocalin 2 (lcn2) was significantly down-regulated in the microglial cells that were resistant to NO-induced apoptosis. lcn2 expression was increased by inflammatory stimuli in microglia. The stable expression of lcn2 as well as the addition of rLCN2 protein augmented the sensitivity of microglia to the NO-induced apoptosis, while knockdown of lcn2 expression using short hairpin RNA attenuated the cell death. Microglial cells with increased lcn2 expression were more sensitive to other cytotoxic agents as well. Thus, inflammatory activation of microglia may lead to up-regulation of lcn2 expression, which sensitizes microglia to the self-regulatory apoptosis. Additionally, the stable expression of lcn2 in BV-2 microglia cells induced a morphological change of the cells into the round shape with a loss of processes. Treatment of primary microglia cultures with the rLCN2 protein also induced the deramification of microglia. The deramification of microglia was closely related with the apoptosis-prone phenotype, because other deramification-inducing agents such as cAMP-elevating agent forskolin, ATP, and calcium ionophore also rendered microglia more sensitive to cell death. Taken together, our results suggest that activated microglia may secrete LCN2 protein, which act in an autocrine manner to sensitize microglia to the self-regulatory apoptosis and to endow microglia with an amoeboid form, a canonical morphology of activated microglia in vivo.

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Paraquat (1,1'-dimethyl-4,4'-bipyridinium) is structurally similar to the neurotoxin 1-methyl-4-phenyl-4-phenylpyridium ion (MPP+), the active metabolite of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which can induce the parkinsonism property in rodents, nonhuman primates, and human. In contrast to the neurotoxic effects of paraquat, little is known about its effects on glial cells. Here, we examined the mechanisms of paraquat toxicity in glial cells in culture. Paraquat treatment also reduced the viability of C6 glial cells in primary astrocyte cultures, and cell death was mostly apoptotic in nature. PKCdelta played a central role in the paraquat-induced glial cell death: (1) the PKCdelta-specific inhibitor rottlerin blocked paraquat-induced glial cell death; (2) paraquat induced tyrosine and threonine phosphorylation of PKCdelta; and (3) transfection of the dominant-negative mutant of PKCdelta attenuated paraquat toxicity. PKCdelta was also involved in the generation of reactive oxygen species (ROS), which mediated the paraquat toxicity. The nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (NADPH oxidase) inhibitor diphenyleneiodonium blocked the paraquat-induced ROS production and subsequent cell death, indicating the involvement of NADPH oxidase in the cytotoxic action of paraquat in glia. PKCdelta was also important in glial cell death induced by MPP+ but not in that induced by rotenone. Last, Rac1 appeared to antagonize paraquat toxicity in glia. These results indicate a gliotoxic effect of paraquat and an opposing role of PKCdelta and Rac1 in paraquat-induced glial cell death.

Microglia-driven inflammatory responses have both neuroprotective and neurotoxic effects in the CNS. The excessive and chronic activation of microglia, however, may shift the balance towards neurotoxic effects. In this regard, proteins secreted from activated microglia likely play a key role in the neurotoxic effects. To characterize secreted proteins of activated microglia, conditioned media obtained from BV-2 mouse microglia cells were analyzed by two-dimensional gel electrophoresis or liquid chromatography coupled with tandem mass spectrometry. Among many proteins identified in the secretome of activated microglia, an aspartic endoprotease cathepsin D has been found to mediate microglial neurotoxicity based on the following results: (i) the expression of cathepsin D protein was markedly increased in lipopolysaccharide/interferon-c-stimulated microglia compared with resting microglia as determined by western blot analysis of conditioned media; (ii) knockdown of cathepsin D expression in microglia using short hairpin RNA diminished the neurotoxicity in the coculture of microglia and neuroblastoma cells and (iii) recombinant procathepsin D protein exerted cytotoxic effects toward cultured neurons. In conclusion, cathepsin D appears to play a central role in the microglial neurotoxicity, and could be a potential biomarker or drug target for the diagnosis and treatment of neurodegenerative diseases that are associated with excessive microglial activation and subsequent neurotoxic inflammation.

lator of G protein signaling (RGS) family members, such as RGS2, interact with G␣ subunits of heterotrimeric G proteins, accelerating the rate of GTP hydrolysis and attenuating the intracellular signaling triggered by the G protein-coupled receptor-ligand interaction. They are also reported to regulate G protein-effector interactions and form multiprotein signaling complexes. Ischemic stress-induced changes in RGS2 expression have been described in astrocytes, and these changes are associated with intracellular signaling cascades, suggesting that RGS2 upregulation may be an important mechanism by which astrocytes may regulate RGS2 function in response to physiological stress. However, information on the functional roles of stress-induced modulation of RGS2 protein expression in astrocyte function is limited. We report the role of ischemic stress in RGS2 protein expression in rat C6 astrocytoma cells and primary mouse astrocytes. A marked increase in RGS2 occurred after ischemic stress induced by chemicals (sodium azide and 2-deoxyglucose) or oxygen-glucose deprivation (OGD, real ischemia). RGS2 mRNA expression was markedly enhanced by 1 h of exposure to chemical ischemia or 6 h of OGD followed by 2 or 6 h of recovery, respectively. This enhanced expression in primary astrocytes and C6 cells was restored to baseline levels after 12 h of recovery from chemically induced ischemic stress or 4 -6 h of recovery from OGD. RGS2 protein was also significantly expressed at 12-24 h of recovery from ischemic insult. Ischemiainduced RGS2 upregulation was associated with enhanced apoptosis. It significantly increased annexin V-positive cells, cleaved caspase-3, and enhanced DNA ladder formation and cell cycle arrest. However, a small interfering RNA (siRNA)-mediated RGS2 knockdown reversed the apoptotic cell death associated with ischemia-induced RGS2 upregulation. Upregulated RGS2 was significantly inhibited by SB-203580, a p38 MAPK inhibitor. Rottlerin, a potent inhibitor of PKC␦, completely abrogated the increased RGS2 expression. We also examine whether ischemia-induced RGS2-mediated apoptosis is affected by siRNA-targeted endogenous PKC␦ downregulation or its phosphorylation. Although RGS2 upregulation was not affected, siRNA transfection significantly suppressed endogenous PKC␦ mRNA and protein expressions. Ischemia-induced PKC␦ phosphorylation and caspase-3 cleavage were dose dependently inhibited by PKC␦ knockdown, and this endogenous PKC␦ suppression reversed ischemia-induced annexin V-positive cells. This study suggests that ischemic stress increases RGS2 expression and that this condition contributes to enhanced apoptosis in C6 cells and primary astrocytes. The signaling it follows may involve PKC␦ and p38 MAPK pathways. stress; protein kinase C; p38 MAPK; gliocytoma ASTROCYTES, THE MOST ABUNDANT glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity (52). It has been reported that astrocytes play an important role in the metabolism of neurotransmitters and in ...

Neuropeptides are short-chain peptides found in brain tissue, some of which function as neurotransmitters and others as hormones. Neuropeptides may directly or indirectly modulate glial functions in the CNS. In the present study, effects of various neuropeptides on the viability and inflammatory activation of cultured microglia were investigated. Vasoactive intestinal peptide, substance P, cholecystokinin and neuropeptide Y did not affect microglial cell viability, whereas corticotropin-releasing hormone (CRH) induced a classical apoptosis of mouse microglia in culture as shown by nuclear condensation and fragmentation, terminal deoxynucleotidyl transferase dUTP nick-end labeling, and cleavage of caspase 3 and poly(ADP-ribose) polymerase protein. CRH, however, did not influence nitric oxide production or expression of inflammatory genes including those encoding cytokines and chemokines, indicating that CRH did not affect the inflammatory activation of microglia. The CRH-induced microglial apoptosis appeared to involve a mitochondrial pathway and reactive oxygen species, based on the mitochondrial membrane potential change, caspase 9 activation and sensitivity to antioxidants. Taken together, our results indicate that the stress neuropeptide CRH may regulate neuroinflammation by inducing the apoptosis of microglia, the major cellular source of inflammatory mediators in the CNS.

Discoidin domain receptor 1 (DDR1) is a nonintegrin collagen receptor tyrosine kinase with an extracellular domain homologous to discoidin 1 of a soil-living amoeba Dictyostelium discoideum. We have previously demonstrated that DDR1 mediates collagen-induced nitric oxide production in J774A.1 murine macrophages. Because collagen is one of the main components of extracellular matrix in the central nervous system, we hypothesized that collagen also induces inflammatory activation of brain microglia, and DDR1 may mediate collagen-induced microglial activation. Using BV-2 mouse microglial cells and mouse primary microglial cultures, we have demonstrated that (1) collagen induces inflammatory activation of microglia as evidenced by production of nitric oxide, expression of inducible nitric oxide synthase, COX-2, CD40, and matrix metalloproteinase-9; (2) DDR1 is expressed in microglia and is phosphorylated by collagen treatment; and (3) collagen-induced microglial activation is abrogated by DDR1 blockade but not by integrin neutralization. We have further shown that p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-kappa B are involved in the collagen-DDR1-induced microglial activation. Our results suggest that collagen can induce inflammatory activation of brain microglia and that DDR1 mediates this effect of collagen in an integrin-independent manner.

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