Cellular Localization of Kynurenine 3-Monooxygenase in the Brain: Challenging the Dogma

Abstract

Kynurenine 3-monooxygenase (KMO), a key player in the kynurenine pathway (KP) of tryptophan degradation, regulates the synthesis of the neuroactive metabolites 3-hydroxykynurenine (3-HK) and kynurenic acid (KYNA). KMO activity has been implicated in several major brain diseases including Huntington's disease (HD) and schizophrenia. In the brain, KMO is widely believed to be predominantly localized in microglial cells, but verification in vivo has not been provided so far. Here, we examined KP metabolism in the brain after depleting microglial cells pharmacologically with the colony stimulating factor 1 receptor inhibitor PLX5622. Young adult mice were fed PLX5622 for 21 days and were euthanized either on the next day or after receiving normal chow for an additional 21 days. Expression of microglial marker genes was dramatically reduced on day 22 but had fully recovered by day 43. In both groups, PLX5622 treatment failed to affect Kmo expression, KMO activity or tissue levels of 3-HK and KYNA in the brain. In a parallel experiment, PLX5622 treatment also did not reduce KMO activity, 3-HK and KYNA in the brain of R6/2 mice (a model of HD with activated microglia). Finally, using freshly isolated mouse cells ex vivo, we found KMO only in microglia and neurons but not in astrocytes. Taken together, these data unexpectedly revealed that neurons contain a large proportion of functional KMO in the adult mouse brain under both physiological and pathological conditions.

Keywords: Huntington’s disease; astrocyte; kynurenine pathway; microglia; schizophrenia.

Figure 1

Conventional “glio-centric” view of the distinct de novo formation of 3-hydroxykynurenine and kynurenic acid in microglia and astrocytes, respectively.

Figure 2

Effect of PLX5622 (PLX) treatment on microglial marker genes. Wild-type controls and PLX5622: n = 6 each; 22-day recovery (R): n = 3. Data (mean ± SEM) are expressed as a percentage of control values. See text for experimental details. *** p < 0.01 vs. control, ⁺⁺ p < 0.05 vs. 22-day recovery (Kruskal–Wallis test with Dunn’s test for multiple pairwise comparisons).

Figure 3

Effect of PLX5622 (PLX) on Kmo expression (A), KMO activity (B), 3-HK levels (C) and KYNA levels (D) in the forebrain of the same wild-type mice used to examine microglial marker genes (same n/group as in Figure 2). Data are the mean ± SEM. See text for experimental details. Kruskal–Wallis test, with Dunn’s test for multiple pairwise comparisons, revealed no significant group differences (p > 0.05).

Figure 4

Effect of PLX5622 (PLX) on KMO activity (A), 3-HK (B) and KYNA (C) levels in the forebrain of wild-type (WT) and R6/2 mice. Data are the mean ± SEM (n = 10 per group). See text for experimental details. *** p < 0.001 (Kruskal–Wallis test with Dunn’s test for multiple pairwise comparisons).

Figure 5

(A–C) Representative dot plots of side scatter height (SSC-H) vs. its corresponding marker, illustrating the purity of brain cells that were isolated from 6 separate mouse brains. After magnetic cell separation, the purity of astrocytes (GLAST-PE+), microglia (CD11b-PE+) and neurons (CD24-APC+) was evaluated by flow cytometry; (D) Kmo mRNA, determined by RT-qPCR. Data are the mean ± SEM. See text for experimental details.

Figure 6

Dot plots illustrating intracellular staining of KMO protein in purified brain cells, assessed by FACS. The percentage of KMO+ cells in (A) astrocytes (GLAST-PE), (B) microglia (CD11b-PE) and (C) neurons (CD24-APC) is shown in Q2. See text for experimental details.

Figure 7

Representative images illustrating the presence of KMO protein in freshly isolated microglial cells (CD11b-PE: red), neurons (NeuN-Alexa 488: green) and astrocytes (GFAP-Cy3; red). Nuclei were stained with DAPI (blue). KMO was identified using anti-rabbit Alexa 488 in microglia and astrocytes, and anti-rabbit Alexa 549 in neurons, as secondary antibodies. See text for experimental details. Images were acquired at 40× magnification. Scale bars: 20 µm.

Figure 8

(A) Proportion of astrocytes, microglia, and neurons, assessed in whole mouse brain homogenate (n = 4–6). Cell suspensions were incubated with markers for astrocytes (GFAP and GLAST), microglia (CD11b and Iba1) and neurons (MAP2), respectively. See text for experimental details; (B) KMO activity in isolated cells (>90% purity). Data are the mean ± SEM. * p < 0.01 (Mann–Whitney test).