Characterization of the kynurenine pathway in human neurons - PubMed (original) (raw)

Comparative Study

Characterization of the kynurenine pathway in human neurons

Gilles J Guillemin et al. J Neurosci. 2007.

Abstract

The kynurenine pathway is a major route of L-tryptophan catabolism producing neuroactive metabolites implicated in neurodegeneration and immune tolerance. We characterized the kynurenine pathway in human neurons and the human SK-N-SH neuroblastoma cell line and found that the kynurenine pathway enzymes were variably expressed. Picolinic carboxylase was expressed only in primary and some adult neurons but not in SK-N-SH cells. Because of this difference, SK-N-SH cells were able to produce the excitotoxin quinolinic acid, whereas human neurons produced the neuroprotectant picolinic acid. The net result of kynurenine pathway induction in human neurons is therefore predicted to result in neuroprotection, immune regulation, and tumor inhibition, whereas in SK-N-SH cells, it may result in neurotoxicity, immune tolerance, and tumor promotion. This study represents the first comprehensive characterization of the kynurenine pathway in neurons and the first description of the involvement of the kynurenine pathway as a mechanism for controlling both tumor cell neurotoxicity and persistence.

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Figures

Figure 1.

Figure 1.

Simplified diagram of the kynurenine pathway.

Figure 2.

Figure 2.

Immunodetection of kynurenine pathway enzymes and products in human neurons and brain. A, B, Cultures of human primary neurons (A) and of SK-N-SH cells (B) were immunostained for the enzymes IDO, TDO, KYNU, KAT-II, KMO, QPRT, and ACMSD, as well as for QUIN and MAP2 as indicated. C, Control preparations of cultures of human primary neurons: irrelevant IgG and nonimmune rabbit serum staining of human primary neurons. D, Human hippocampal sections were also immunostained for IDO, KMO, KYNU, ACMSD, TDO, KAT-II, and QUIN as indicated. Neuronal labeling for all markers was predominantly cytosolic, although occasional staining of proximal processes was seen (e.g., for KMO). Neurons of the hilus (CA4) were the most consistently labeled. Non-neuronal cells were also labeled. Punctate/vesicular labeling of probable microglial cells was widespread in all areas of cortex and hippocampus. QUIN labeling was distinctly granular, as shown in the pyramidal cells of CA1 (D).

Figure 3.

Figure 3.

Detection of kynurenine pathway enzyme expression using RT-PCR. A, Ethidium bromide-stained gels showing RT-PCR for (from top to bottom) IDO, TDO, KAT-I, KAT-II, KYNU, KMO, HAAO, QPRT, ACMSD, and GAPDH expression. The first column corresponds to unstimulated SK-N-SH cells, the second to IFN-γ-stimulated SK-N-SH cells, the third to unstimulated human primary neurons, the fourth to IFN-γ-stimulated human primary neurons, and the fifth to human primary macrophages stimulated with IFN-γ and used as a positive control for kynurenine pathway enzyme expression. B, The histograms indicate the ratio of the expression of the nine kynurenine pathway enzyme mRNA relative to the GAPDH mRNA expression. Individual samples were analyzed in triplicate, and the SEM for all data was determined to be <5%.

Figure 4.

Figure 4.

Quantification of kynurenine pathway products by HPLC and GC/MS. A–H, Media samples derived from cultures of SK-N-SH cells and primary neurons either untreated or treated for 24 h with 100 IU/ml IFN-γ (A–D) or at the times indicated (E–H) were analyzed by HPLC for TRP (A), KYN (B), KYNA (C), and 3-HK (D) and by using GC/MS for QUIN (E) and PIC (F, H). Statistical differences comparing the untreated and IFN-γ-treated conditions (A–D) or the change in concentration detected at the 48 or 72 h compared with the 24 h time point are indicated by the asterisks. **p < 0.02 and ***p < 0.01 (Mann–Whitney test).

Figure 5.

Figure 5.

Effects of PIC and QUIN on SK-H-SH proliferation. A, B, Phase-contrast microscopy of SK-N-SH cell cultures (A) and histogram indicating the number of SK-N-SH cells (from left to right) in control untreated cells and cells treated with either 1 or 5 μ

m

PIC or with 5 μ

m

QUIN 5 for 24 h (B). p values for the comparisons with control condition were determined (Mann–Whitney test): *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 6.

Figure 6.

Scheme indicating differences between human primary neurons and neuroblastoma cells with respect to the production of kynurenine pathway metabolites and their effects in tumor persistence and the immune response.

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