Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated activation of murine N9 microglial cell line

Authors

  • Luisa Franco,

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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  • Nicoletta Bodrato,

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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  • Iliana Moreschi,

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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  • Cesare Usai,

    1. Institute of Biophysics, CNR, Genova, Italy
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  • Santina Bruzzone,

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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  • Sonia Scarf ì,

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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  • Elena Zocchi,

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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  • Antonio De Flora

    1. Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Genova, Italy
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Address correspondence and reprint requests to Antonio De Flora, Department of Experimental Medicine, Section of Biochemistry, and Center of Excellence for Biomedical Research, University of Genova, Viale Benedetto XV/1–16132 Genova, Italy.
E-mail: toninodf@unige.it

Abstract

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.

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