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 inducedby 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 usedas 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<0.05). By contrast the NMDA receptor antagonist, MK801 protected, but did not reverse these microglial-associated reductions in neuronal complexity. Overall the studies outlined in this thesis show that inhibition of the KP in microglia protects against inflammation-associated reductions in neuronal complexity providing an independent pathway to target in order to protect against the deleterious effects that these metabolites have on neuronal outgrowth. In addition, it appears that it is more so the generation of free radicals rather than NMDA receptor over-activation that provokes these reductions in neuronal complexity.
|Number of pages||2|
|Publication status||Published - Sep 2015|
|Event||Congress of the European College of Neuropsychopharmacology (28th : 2015) - Amsterdam, Netherlands|
Duration: 29 Aug 2015 → 1 Sep 2015
O'Farrell, K., Lim, E., Guillemin, G. J., & Harkin, A. (2015). A role for glial-associated kynurenine pathway activation in modulating neuronal outgrowth and complexity. European Neuropsychopharmacology, 25, S206-S207. https://doi.org/10.1016/S0924-977X(15)30211-X