Elucidating the role of 3-hydroxykynurenine on neuronal physiology

The kynurenine pathway (KP) is the primary route for dietary tryptophan catabolism and culminates in NAD production. Several KP metabolites have neuromodulator functions and dysregulation of the KP is observed in a range of neurological disorders. Previous studies have suggested that the KP metabolite 3-hydroxykynurenine (3-HK) is a neurotoxin at supraphysiological levels in vitro (>100 μM). However, physiological levels of this metabolite in humans are much lower than these neurotoxic levels (~ 0.1 μM in healthy individuals and ~ 1 μM in disease). The present study examines the effects of 3-HK treatment on neuronal cells to better understand its role in disease by examining a range of cellular outcomes including mRNA expression profiles, cell viability, mitochondrial function and the release of extracellular vesicles (EVs).The neuroblastoma cell line SH-SY5Y was treated with 3-HK (0.1 M, 1 μM and 10 M) for 24 hours. Transcriptomic analysis revealed altered gene expression which was associated with RNA stability and protein modifications. Cell viability assays revealed that neuronal death occurred at concentrations one hundred times higher than that required for significant caspase-3/7 activation (1 μM). Mitochondria were imaged in live cells using confocal imaging and mitochondrial networks were analysed in FIJI. Mitochondrial fragmentation was observed at 10 μM 3-HK, alongside a significant increase in mitochondria number relative to other physiological concentrations. Interestingly, mitochondrial morphology was significantly altered by 0.1 μM 3-HK compared with the drug-naïve control. This suggested a dual, concentration dependent role for 3-HK in mitochondrial dynamics. Finally, EVs were isolated from serum-free conditioned media and analysed by nanoparticle tracking analysis which found increased EV diameter in cells treated with 3-HK.This study highlights the multifaceted role of 3-HK in neuronal function as treatment with physiological levels of 3-HK induced a variety of cellular responses that may be relevant for disease.