No abstract

Brain microglia are a major source of inflammatory cytokines, such as tumor necrosis factor-␣ (TNF-␣), which have been implicated in the progression of neurodegenerative diseases. Recently, microglia were revealed to be highly responsive to ATP, which is released from nerve terminals, activated immune cells, or damaged cells. It is not clear, however, whether released ATP can regulate TNF-␣ secretion from microglia. Here we demonstrate that ATP potently stimulates TNF-␣ release, resulting from TNF-␣ mRNA expression in rat cultured brain microglia. The TNF-␣ release was maximally elicited by 1 mM ATP and also induced by a P2X 7 receptorselective agonist, 2Ј-and 3Ј-O-(4-benzoylbenzoyl)adenosine 5Ј-triphosphate, suggesting the involvement of P2X 7 receptor. ATP-induced TNF-␣ release was Ca 2ϩ -dependent, and a sustained Ca 2ϩ influx correlated with the TNF-␣ release in ATP-stimulated microglia. ATP-induced TNF-␣ release was inhibited by PD 098059, an inhibitor of extracellular signal-regulated protein kinase (ERK) kinase 1 (MEK1), which activates ERK, and also by SB 203580, an inhibitor of p38 mitogen-activated protein kinase. ATP rapidly activated both ERK and p38 even in the absence of extracellular Ca 2ϩ . These results indicate that extracellular ATP triggers TNF-␣ release in rat microglia via a P2 receptor, likely to be the P2X 7 subtype, by a mechanism that is dependent on both the sustained Ca 2ϩ influx and ERK/p38 cascade, regulated independently of Ca 2ϩ influx. Key Words: Microglia-Tumor necrosis factor-␣-ATP-P2X 7 receptor-Ca 2ϩ -Mitogenactivated protein kinase. J. Neurochem. 75, 965-972 (2000).

Microglia are resident monocyte-lineaged cells in the brain. Their characteristic feature is that they react to injury and diseases of the brain and become morphologically and functionally activated. Although some trigger molecules which activate microglia are predicted to be released from injured or affected cells, such molecules have not yet been identified. The main role of activated microglia is believed to be in brain defense, as scavengers of dead cells, and as immune or immunoeffector cells. Recent biochemical and neurobiological studies have further indicated that they significantly affect the pathological state and/or regulate the regenerative state and remodeling of the brain by producing a variety of biologically active molecules including cytotoxic and neurotrophic molecules.

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain-derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin-3 (NT-3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol-12-myristate-13-acetate, enhanced the secretion of BDNF. Pre-treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS-stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCalpha probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCalpha-associated signal transduction mechanism.