Effects of Kynurenine Pathway Inhibition on NAD Metabolism and Cell Viability in Human Primary Astrocytes and Neurons - PubMed (original) (raw)

Effects of Kynurenine Pathway Inhibition on NAD Metabolism and Cell Viability in Human Primary Astrocytes and Neurons

Nady Braidy et al. Int J Tryptophan Res. 2011.

Abstract

The kynurenine pathway (KP) is the principle route of L-Tryptophan (TRP) metabolism, producing several neurotoxic and neuroprotective metabolic precursors before complete oxidation to the essential pyridine nucleotide nicotinamide adenine dinucleotide (NAD(+)). KP inhibition may prove therapeutic in central nervous system (CNS) inflammation by reducing the production of excitotoxins such as quinolinic acid (QUIN). However, KP metabolism may also be cytoprotective through the de novo synthesis of intracellular NAD(+). We tested the hypothesis that the KP is directly involved in the maintenance of intracellular NAD(+) levels and SIRT1 function in primary astrocytes and neurons through regulation of NAD(+) synthesis. Competitive inhibition of indoleamine 2,3 dioxygenase (IDO), and quinolinic acid phosphoribosyltransferase (QPRT) activities with 1-methyl-L-Tryptophan (1-MT), and phthalic acid (PA) respectively, resulted in a dose-dependent decrease in intracellular NAD(+) levels and sirtuin deacetylase-1 (SIRT1) activity, and correlated directly with reduced cell viability. These results support the hypothesis that the primary role of KP activation during neuroinflammation is to maintain NAD(+) levels through de novo synthesis from TRP. Inhibition of KP metabolism under these conditions can compromise cell viability, NAD-dependent SIRT1 activity and CNS function, unless alternative precursors for NAD(+) synthesis are made available.

Keywords: IDO; NAD+; astrocytes neurons 1-MT; sirtuins.

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Figures

Figure 1

Figure 1

A) Effect of 1-MT on IDO activity in human astrocytes and neurons. A dose-dependent inhibition of IDO activity was observed following treatment with 1-MT in human astrocytes and neurons. For astrocytes, no 1-MT (control) = 35.86 nmol kynurenine/hr/mg protein; 10 μM 1-MT = 31.15 ± 5.61 nmol kynurenine/hr/mg protein; 100 μM 1-MT = 14.70 ± 4.85 nmol kynurenine/hr/mg protein; 1000 μM 1-MT = 4.55 ± 1.93 nmol kynurenine/hr/mg protein; Significance *P < 0.05 compared to previous dose (n = 4 for each treatment group). For neurons, no 1-MT (control) = 27.22 ± 7.28 nmol kynurenine/hr/mg protein; 10 μM 1-MT = 24.77 ± 6.74 nmol kynurenine/hr/mg protein; 100 μM 1-MT = 14.15 ± 2.94 nmol kynurenine/hr/mg protein; 1000 μM 1-MT = 2.99 ± 1.42 nmol kynurenine/hr/mg protein; Significance *P < 0.05 compared to previous dose (n = 4 for each treatment group). B) PA on QPRT activity in human astrocytes and neurons. A dose-dependent inhibition of QPRT activity was observed following treatment with PA in human astrocytes and neurons. For astrocytes, no PA (control) = 41.33 ± 8.32 nmol kynurenine/hr/mg protein; 10 μM PA = 35.54 ± 3.22 nmol kynurenine/hr/mg protein; 100 μM PA = 21.08 ± 7.39 nmol kynurenine/hr/mg protein; 1000 μM PA = 3.31 ± 1.32 nmol kynurenine/hr/mg protein; Significance *P < 0.05 compared to previous dose (n = 4 for each treatment group). For neurons, no PA (control) = 21.55 ± 3.62 nmol kynurenine/hr/mg protein; 10 μM PA = 17.46 ± 3.49 nmol kynurenine/hr/mg protein; 100 μM PA = 11.42 ± 3.11 nmol kynurenine/hr/mg protein; 1000 μM PA = 2.59 ± 0.81 nmol kynurenine/hr/mg protein; Significance *P < 0.05 compared to previous dose (n = 4 for each treatment group).

Figure 2

Figure 2

Effect of (A) 1-MT and (B) PA on intracellular NAD+ levels in human astrocytes and neurons. NAD+ levels significantly declined in a dose-dependent manner with increasing concentrations of (A) 1-MT and (B) PA respectively following 24 hours incubation with the selected inhibitor. Significance *P < 0.05 compared to previous dose (n = 4 for each treatment group).

Figure 3

Figure 3

Effect of (A) 1-MT and (B) PA on extracellular LDH activity in human astrocyte and neuron cultures. Extracellular LDH activities were significantly elevated in a dose-dependent manner with increasing concentrations of (A) 1-MT and (B) PA respectively, following 24 hours incubation with the selected inhibitor. Significance *P < 0.05 compared to previous dose (n = 4 for each treatment group).

Figure 4

Figure 4

Effect of 1-MT and PA on SIRT1 activity in human astrocytes and neurons. Treatment with 1-MT or PA at 100 μM significantly reduced SIRT1 activity in human astrocytes and neurons. Significance *P < 0.05 compared to control (n = 4 for each treatment group).

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