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Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is known to activate microglial cells following its interaction with the CD14/Toll-like receptor complex (TLR-4). The activation pathway triggered by lipopolysaccharide in microglia involves enhanced basal levels of intracellular calcium ([Ca2+]i) and terminates with increased generation of cytokines/chemokines and nitric oxide. Here we demonstrate that in lipopolysaccharide-stimulated murine N9 microglial cells, cyclic ADP-ribose, a universal and potent Ca2+ mobiliser generated from NAD+ by ADP-ribosyl cyclases (ADPRC), behaves as a second messenger in the cell activation pathway. Lipopolysaccharide induced phosphorylation, mediated by multiple protein kinases, of the mammalian ADPRC CD38, which resulted in significantly enhanced ADPRC activity and in a 1.7-fold increase in the concentration of intracellular cyclic ADP-ribose. This event was paralleled by doubling of the basal [Ca2+]i levels, which was largely prevented by the cyclic ADP-ribose antagonists 8-Br-cyclic ADP-ribose and ryanodine (by 75% and 88%, respectively). Both antagonists inhibited, although incompletely, functional events downstream of the lipopolysaccharide-induced microglia-activating pathway, i.e. expression of inducible nitric oxide synthase, overproduction and release of nitric oxide and of tumor necrosis factor α. The identification of cyclic ADP-ribose as a key signal metabolite in the complex cascade of events triggered by lipopolysaccharide and eventually leading to enhanced generation of pro-inflammatory molecules may suggest a new therapeutic target for treatment of neurodegenerative diseases related to microglia activation.
Microglial cells, the monocyte/macrophage equivalent of the CNS, represent the first line of defence in the brain, capable of removing infectious agents and damaged cells (Farber and Kettenmann 2005). In response to pathological events, the normally resting microglia gradually transforms into motile, secretory and potentially cytotoxic phagocytes (Hoffmann et al. 2003). Activated microglial cells then participate in mechanisms of innate and immune defence, tissue repair and neuroprotection (Town et al. 2005). However, under conditions of chronic inflammation, excessive activation of microglia can contribute to the neurodegenerative process by producing and releasing a number of potentially cytotoxic substances, which include pro-inflammatory cytokines and nitric oxide (Aschner et al. 1999; Gonzalez-Scarano and Baltuch 1999). Recently, microglial cells have been implicated in the development of ischaemia-induced damage and neurodegenerative diseases such as multiple sclerosis, Alzheimer's disease and Parkinson's disease (Gebicke-Haerter 2001; Town et al. 2005). Accordingly, the identification of the molecular mechanisms of microglial activation might provide new therapeutic targets, representing an important step towards the successful treatment of these diseases.
Lipopolysaccharide, the main component of the cell wall of Gram-negative bacteria, is widely known to activate microglial cells ‘in vitro’ upon interacting with the CD14/Toll-like receptor (TLR-4) complex (Raetz and Whitfield 2001). Lipopolysaccharide activation of murine microglia leads to an elevation of the intracellular free calcium concentration ([Ca2+]i). This event is necessary to allow the overproduction and the release of inflammatory cytokines and nitric oxide to occur (Hoffmann et al. 2003). Despite its importance in physiology and pathology, the mechanisms and the chemical mediators of this lipopolysaccharide-induced signalling pathway seem to be multiple and are still incompletely known.
Cyclic ADP-ribose (cADPR) is a potent and universal Ca2+ mobiliser from ryanodine-sensitive stores/channels which is generated by a family of multifunctional enzymes designated ADP-ribosyl cyclases (Lee et al. 1989; Mehta and Malavasi 2000; Lee 2002; Schuber and Lund 2004). CD38, the most represented mammalian member of this family, is a multifunctional type II glycoprotein known to catalyse, at the surface of several mammalian cells but also intracellularly, the production of a number of signal molecules involved in the regulation of intracellular calcium concentration ([Ca2+]i) levels. Specifically, CD38 generates cADPR and ADP-ribose (ADPR) from the substrate NAD+ via ADP-ribosyl cyclase and cADPR hydrolase activities, respectively (Lee et al. 1989; Lee 2002; Guse 2005).
The lack of information about the CD38/cADPR system in microglial cells, and the recent finding that cADPR is a second messenger in the lipopolysaccharide-stimulated proliferation of human peripheral blood mononuclear cells (Bruzzone et al. 2003), prompted us to investigate whether cADPR plays a role in the basal [Ca2+]i elevation during the lipopolysaccharide-induced microglial activation process (Hoffmann et al. 2003). The results obtained in N9 cells, a murine microglial cell line, demonstrate that CD38 is constitutively expressed in these cells and that lipopolysaccharide stimulates its phosphorylation, resulting in increased ADP-ribosyl cyclase activity and enhanced generation of intracellular cADPR. This signal metabolite elicits a basal [Ca2+]i increase, which triggers production and release of nitric oxide and the pro-inflammatory cytokine tumor necrosis factor α (TNF-α). Therefore, cADPR behaves as a second messenger in the lipopolysaccharide-stimulated generation of nitric oxide and TNF-α in N9 microglial cells.
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Inflammatory events in the CNS are associated with infections and injuries, as well as with chronic degenerative diseases, such as multiple sclerosis, Parkinson's or Alzheimer's (Nakamura et al. 1999). For this reason, it is of great importance to obtain insights into the molecular mechanisms that underlie immune reactions in brain tissue. Given the particularly daunting technical difficulties of the in vivo study of the CNS, this task is best tackled by the in vitro study of the activation pathways of the major cell type responsible for brain inflammation – the microglia.
The results obtained in this study on the microglial murine cell line N9 show that cell activation by lipopolysaccharide, leading to nitric oxide and TNF-α release, occurs through the intracellular production of cADPR, which behaves as a second messenger in this signalling cascade. Lipopolysaccharide induces a doubling of the GDP-ribosyl cyclase activity of CD38 (Fig. 1) and a 70% increase of the intracellular concentration of cADPR (see Results). Lipopolysaccharide-induced cyclase activation occurs via protein kinase-mediated phosphorylation of CD38, as demonstrated by prevention of this activation by broad (staurosporine) and specific (I-PKA, I-PKC and AG126, Fig. 3) protein kinase inhibitors. In microglial cells, lipopolysaccharide is indeed known to bind to a TLR-4 and to induce activation of PKC (Kim et al. 2005) and a protein tyrosine kinase sensitive to AG126 (Kann et al. 2004). PKA-mediated activation of ADP-ribosyl cyclases has been also reported in several other cell types (Morita et al. 1997; Zocchi et al. 2001; Boittin et al. 2003; Xie et al. 2005; Bruzzone et al. 2006). The cytosolic amino-terminal region of murine CD38 harbours three serine residues, with Ser19 scoring the highest phosphorylation potential (0.992), and one tyrosine at position 4 scoring 0.55 (NetPhos 2.0 Server, Center for Biological Sequence Analysis, CBS, Technical University of Denmark, Copenhagen, Denmark). Our data strongly implicate Ser19 and Tyr4 as being involved in the lipopolysaccharide-triggered double phosphorylation of CD38 (by PKA + PKC and by the AG126-inhibitable protein tyrosine kinase, respectively) that results in stimulation of CD38 ADPRC activity and hence in overproduction of cADPR. The fact that several protein kinases are involved in the lipopolysaccharide-induced CD38 activation may be part of a redundant mechanism for ADP-ribosyl cyclase activation in microglia, possibly resulting in the fine tuning of the cyclase activity and of the [cADPR]i in response to extracellular stimuli.
It has already been reported that incubation with lipopolysaccharide induces an increase of the basal [Ca2+]i in primary cultures of microglial cells (Hoffmann et al. 2003). Here, we demonstrate that the elevation of the [cADPR]i in N9 cells, due to lipopolysaccharide-induced CD38 activation, is responsible for the sustained [Ca2+]i elevation. Indeed, pretreatment of N9 cells with either of the cADPR antagonists 8-Br-cADPR or ryanodine greatly down-regulated the lipopolysaccharide-induced [Ca2+]i increase (Table 1).
Elevation of the [Ca2+]i following lipopolysaccharide exposure is in turn fundamental to promote the microglial activation process, leading to induction of the pro-inflammatory activity typical of these cells, which includes nitric oxide production and TNF-α release (Hoffmann et al. 2003). Specifically, nitric oxide generation induced by lipopolysaccharide in microglial cultures was demonstrated to be significantly reduced in a concentration-dependent manner by the intracellular Ca2+ chelator BAPTA-AM (Hoffmann et al. 2003). Results obtained in the present study also demonstrate a causal role of cADPR in nitric oxide and TNF-α release from lipopolysaccharide-stimulated N9 cells. Exogenously added cADPR, which has been shown to be transported through the plasma membrane of different cell types by the nucleoside transporters ENT and CNT (Guida et al. 2002, 2004; Podestàet al. 2005), induced progressive release of both nitric oxide (Fig. 4a) and TNF-α (Fig. 6a). Pretreatment of N9 cells with either of the two cADPR antagonists 8-Br-cADPR or ryanodine or with the cyclase inhibitor nicotinamide resulted in the partial or total abrogation of the lipopolysaccharide-induced release of nitric oxide (Fig. 4b). A Ca2+-dependent, inducible nitric oxide synthase (iNOS) is known to be responsible for nitric oxide production in N9 microglial cells (Chang and Liu 1999; Possel et al. 2000). The results obtained in our study indicate that iNOS expression is up-regulated by the [cADPR]i (Figs 4b and 5b).
The signalling cascade that links lipopolysaccharide stimulation to enhanced TNF-α release also seems to involve cADPR, similar to what was observed for nitric oxide overproduction (Fig. 6b). This view is supported by the significant inhibition of TNF-α release elicited by the cADPR antagonist 8-Br-cADPR, the cyclase inhibitor nicotinamide and ryanodine, as well as by the various protein kinase inhibitors, with staurosporine being the most effective.
In conclusion, the present results indicate that lipopolysaccharide-induced stimulation of N9 microglial cells is dependent on CD38 phosphorylation, ADP-ribosyl cyclase activation and the consequent increase of the [cADPR]i, resulting in a sustained [Ca2+]i elevation and enhanced nitric oxide and TNF-α release. This signalling pathway has three main regulatory modules. The first one is related to activation of CD38, which takes place via cyclase phosphorylation involving several protein kinases (PKA, PKC and protein tyrosine kinase). The second regulatory module downstream of the cADPR overproduction is the increase of the [Ca2+]i. The substantial inhibition of the [Ca2+]i increase afforded by 8-Br-cADPR and ryanodine (Table 1) argues for a major role of cADPR in enhancing the [Ca2+]i levels in lipopolysaccharide-stimulated N9 cells. Failure of 8-Br-cADPR to completely abrogate this increase could imply a limited role for Ca2+-regulating signal molecules other than cADPR. Pointedly, ADPR has been recently shown to induce Ca2+ influx in lipopolysaccharide-stimulated microglial cells (Kraft et al. 2004). Indeed, cADPR seems to be a potent coregulator of Ca2+ entry across ADPR-gated TRPM2 channels (Kolisek et al. 2005; Gasser et al. 2006).
The third and last regulatory module in the lipopolysaccharide-induced activation of N9 cells ultimately leads to enhanced nitric oxide and TNF-α release and is dependent on the [Ca2+]i increase, although the molecular mechanisms leading to cytokine release are probably manifold. Indeed, 8-Br-cADPR, nicotinamide and ryanodine fail to afford comparable extents of inhibition on nitric oxide and TNF-α release: 50 μm ryanodine quenched nitric oxide release (Fig. 4B) and iNOS expression (Fig. 5B) almost completely, while inhibiting TNF-α release only by 30% (Fig. 6B). In addition, there are quantitatively poor correlations between the fractional extents of inhibition of CD38 double phosphorylation and those elicited by protein kinase inhibitors on TNF-α release, respectively (see Figs 3 and 6 for comparison). A relevant example is the sharp inhibiting effect on CD38 serine phosphorylation and a low inhibition of TNF-α release by the PKC inhibitor. These findings are consistent with the involvement of other molecular mechanisms or systems, which in fact have been implicated in lipopolysaccharide-induced cytokine release from glial cells: these include the (MAPK)/arachidonate/cyclooxygenase 2 cascade (Paris et al. 2000), the p38 MAPK subfamily, the AG126-inhibitable extracellular signal-regulated protein kinase (ERK) (Bhat et al. 1998; Hanisch et al. 2001; Kann et al. 2004) and nuclear factor-kB (Possel et al. 2000; Quin et al. 2005).
Demonstration of the role of cADPR as a second messenger responsible for the functional activation of microglial cells may suggest a new target for therapeutic strategies. cADPR antagonists (Guse 2005) hold promise for the prevention or reduction of microglial cell activation whenever the excessive pro-inflammatory effects induced by the prolonged activation of this cell population concur with brain tissue damage (Gebicke-Haerter et al. 1996; Tan et al. 1999).