A role for glial-associated kynurenine pathway activation in modulating neuronal outgrowth and complexity

The kynurenine pathway (KP) is the main pathway by which the amino acid tryptophan is metabolised. Within the central nervous system, the neurotoxic arm of the KP is located in microglia, while the neuro-protective arm is found in astrocytes. Both microglia and astrocytes possess indoleamine 2,3-dioxygenase (IDO) which catalyses the conversion of tryptophan to kynurenine, however unlike astrocytes, microglia also express kynurenine monooxygenase (KMO) and kynureninase (KYNU) which catalyse the production of the free radical producing metabolites, 3-hydroxykynurenine and 3-hydroxyanthranillic acid respectively and subsequently the production of the NMDA receptor agonist quinolinic acid. IDO and the two neurotoxic enzymes are induced by inflammatory stimuli, and as such, microglial activation as a consequence of inflammation, may lead to altered neuronal complexity due to the excessive production of neurotoxic KP metabolites. The aims of these studies were to identify the effect that IFNg-stimulated glial cultures have on neuronal complexity, and to delineate the effects that activation of each arm of the pathway has on neuronal complexity. Moreover, these studies aspired to determine a role for microglial kynurenine pathway activation in driving IFNg-induced changes in neuronal complexity. Primary neuronal cultures were prepared from 1 day old Wistar rats and treatments were performed at days in vitro (DIV) 3, mixed glia and astrocytes were prepared from 2−3 day old Wistar rats and were treated at DIV 10, and BV-2 microglia were used as a microglial model. Conditioned media (CM) was harvested 24 hr following treatment and was placed on neurons for 48 hr. Neurons were fixed and sholl analysis was carried out to assess neurite morphology in response to treatments. BV-2 cells were harvested for mRNA analysis by qPCR and the concentrations of KP metabolites in the BV-2 CM were assessed by HPLC or GCMS. Results from these studies show that CM from IFNg-stimulated mixed glia (P<0.05), BV-2 microglia (P<0.01), and astrocytes (P<0.01) significantly reduced neuronal outgrowth and complexity. IFNg induced the expression of IDO, KMO and KYNU (P<0.001), and non-significantly increased the concentration of quinolinic acid (P = 0.06) in the CM. Furthermore, the reductions in various parameters of neurite outgrowth as a result of CM from IFNg-stimulated BV-2 microglia were prevented when BV-2 microglia were pre-treated with inhibitors of KP activation. Inhibition of KMO using Ro 61–8048 (1mM; 30 min) was found to have the most beneficial effects in terms of attenuating IFNg-induced BV-2-mediated reductions in neuronal complexity (P