Inhibition of the kynurenine pathway protects against reactive microglial-associated reductions in the complexity of primary cortical neurons

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Abstract

Brain glia possess the rate limiting enzyme indoleamine 2, 3-dioxygenase (IDO) which catalyses the conversion of tryptophan to kynurenine. Microglia also express kynurenine monooxygenase (KMO) and kynureninase (KYNU) which lead to the production of the free radical producing metabolites, 3-hydroxykynurenine and 3-hydroxyanthranillic acid respectively and subsequently production of the NMDA receptor agonist quinolinic acid. The aim of this study was to examine the effect of IFNγ-stimulated kynurenine pathway (KP) induction in microglia on neurite outgrowth and complexity, and to determine whether alterations could be abrogated using pharmacological inhibitors of the KP. BV-2 microglia were treated with IFNγ (5 ng/ml) for 24 h and conditioned media (CM) was placed on primary cortical neurons 3 days in vitro (DIV) for 48 h. Neurons were fixed and neurite outgrowth and complexity was assessed using fluorescent immunocytochemistry followed by Sholl analysis. Results show increased mRNA expression of IDO, KMO and KYNU, and increased concentrations of tryptophan, kynurenine, and 3-hydroxykynurenine in the CM of IFNγ-stimulated BV-2 microglia. The IFNγ-stimulated BV-2 microglial CM reduced neurite outgrowth and complexity with reductions in various parameters of neurite outgrowth prevented when BV-2 microglia were pre-treated with either the IDO inhibitor, 1-methyltryptophan (1-MT) (L) (0.5 mM; 30 min), the KMO inhibitor, Ro 61–8048 (1 μM; 30 min), the synthetic glucocorticoid, dexamethasone (1 μM; 2 h) -which suppresses IFNγ-induced IDO - and the N-methyl-D-aspartate (NMDA) receptor antagonist, MK801 (0.1 μM; 30 min). Overall this study indicates that inhibition of the KP in microglia may be targeted to protect against reactive microglial-associated neuronal atrophy.

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