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Kynurenine 3-Monooxygenase Activity in Human Primary Neurons and Effect on Cellular Bioenergetics Identifies New Neurotoxic Mechanisms

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Abstract

Upregulation of the kynurenine pathway (KP) of tryptophan metabolism is commonly observed in neurodegenerative disease. When activated, L-kynurenine (KYN) increases in the periphery and central nervous system where it is further metabolised to other neuroactive metabolites including 3-hydroxykynurenine (3-HK), kynurenic acid (KYNA) and quinolinic acid (QUIN). Particularly biologically relevant metabolites are 3-HK and QUIN, formed downstream of the enzyme kynurenine 3-monooxygenase (KMO) which plays a pivotal role in maintaining KP homeostasis. Indeed, excessive production of 3-HK and QUIN has been described in neurodegenerative disease including Alzheimer’s disease and Huntington’s disease. In this study, we characterise KMO activity in human primary neurons and identified new mechanisms by which KMO activation mediates neurotoxicity. We show that while transient activation of the KP promotes synthesis of the essential co-enzyme nicotinamide adenine dinucleotide (NAD+), allowing cells to meet short-term increased energy demands, chronic KMO activation induces production of reactive oxygen species (ROS), mitochondrial damage and decreases spare-respiratory capacity (SRC). We further found that these events generate a vicious-cycle, as mitochondrial dysfunction further shunts the KP towards the KMO branch of the KP to presumably enhance QUIN production. These mechanisms may be especially relevant in neurodegenerative disease as neurons are highly sensitive to oxidative stress and mitochondrial impairment.

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Abbreviations

3-HK:

3-Hydroxykynurenine

AA:

Anthranilic acid

AD:

Alzheimer’s disease

CNS:

Central nervous system

DAPI:

4,6-Diamindino-2-phenylindole

DCF:

2-,7-Dichlorofluorescin

ETS:

Electron transport system

FAD:

Flavin adenine dinucleotide

FCCP:

Carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone

H2O2 :

Hydrogen peroxide

HD:

Huntington’s Disease

HPLC:

High-performance liquid chromatography

IDO1:

Indoleamine 2,3-dioxygenase 1

IDO2:

Indoleamine 2,3-dioxygenase 2

INF-γ:

Interferon gamma

KAT:

Kynurenine aminotransferase

KMO:

Kynurenine 3-monooxygenase

KP:

Kynurenine pathway

KYN:

Kynurenine

KYNA:

Kynurenic acid

KYNU:

Kynureninase

MR:

Maximal respiration

NAD:

Nicotinamide adenine dinucleotide

NADPH:

Nicotinamide adenine dinucleotide phosphate

NMDA:

N-methyl-D-aspartate

OCR:

Oxygen consumption rate

PBS:

Phosphate-buffered saline

QUIN:

Quinolinic acid

R123:

Rhodamine 123

ROS:

Reactive oxygen species

ROX :

Residual oxygen consumption/non-mitochondrial respiration

RT:

Room temperature

SRC:

Spare respiratory capacity

TDO:

Tryptophan 2,3-dioxygenase

TRP:

Tryptophan

UHPLC:

Ultra-high performance liquid chromatography

α7nAChR:

α-7 nicotinic acetyl choline receptor

ΔΨm :

Mitochondrial membrane potential

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Acknowledgements

This work was supported by the National Health and Medical Research Council (NHMRC), the Australian Research Council (ARC), Macquarie University, The Snow Foundation and the Ramaciotti Perpetual Foundation (Australia). Dr. Gloria Castellano-Gonzalez was a recipient of the Macquarie University international scholarship.

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  1. Gloria Castellano-Gonzalez and Kelly R. Jacobs contributed equally as co-first author

Authors and Affiliations

  1. Neuroinflammation Group, Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences (FMHS), Macquarie University, 2, Technology Place, North Ryde, NSW, 2109, Australia

    Gloria Castellano-Gonzalez, Kelly R. Jacobs, Seray Adams, Chai K. Lim, David B. Lovejoy & Gilles J. Guillemin

  2. Centre for Motor Neuron Disease Research, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences (FMHS), Macquarie University, North Ryde, NSW, 2109, Australia

    Emily Don & Nicholas J. Cole

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Correspondence to David B. Lovejoy or Gilles J. Guillemin.

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Castellano-Gonzalez, G., Jacobs, K.R., Don, E. et al. Kynurenine 3-Monooxygenase Activity in Human Primary Neurons and Effect on Cellular Bioenergetics Identifies New Neurotoxic Mechanisms. Neurotox Res 35, 530–541 (2019). https://doi.org/10.1007/s12640-019-9997-4

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