Xanthurenic acid is localized in neurons in the central nervous system

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XANTHURENIC ACID IS LOCALIZED IN NEURONS IN THE CENTRAL NERVOUS SYSTEM

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GUY ROUSSEL, b ALBAN BESSEDE, c CHRISTIAN KLEIN, a MICHEL MAITRE a* AND AYIKOE GUY MENSAH-NYAGAN a

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a Biopathologie de la Mye´line, Neuroprotection et Strate´gies The´rapeutiques, INSERM U1119, Fe´de´ration de Me´decine Translationnelle de Strasbourg (FMTS), Universite´ de Strasbourg, Baˆtiment 3 de la Faculte´ de Me´decine, 11 rue Humann, 67000 Strasbourg, France

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b

INSERM 1109 – MN3t Lab, Strasbourg, France

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c

IMS Lab, EPHE UMR 5218 CNRS, Bordeaux, France

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Abstract—Kynurenine pathway metabolites (KPM) are thought to be synthesized mainly by non-neuronal cells in the mammalian brain. KPM are of particular interest because several studies demonstrated their implication in various disorders of the nervous system. Among KPM is xanthurenic acid (XA) deriving from the catabolism of 3-hydroxykynurenine. Based on its chemical structure, XA appears as a close analog of kynurenic acid which has been extensively investigated and is considered as a potent neuroprotective compound. Contrary to kynurenine acid, XA has received little attention and its role in the brain remains not elucidated. We have previously described several characteristics of XA, suggesting its possible involvement in neurotransmission. XA is also proposed as a potential modulator at glutamatergic synapses. Here, we used a selective antibody against XA and various neuronal, glial and synaptic markers to show that XA is essentially localized in the soma and dendrites of brain neurons, but is absent from axonal compartments and terminal endings. Our results also reveal that XA-like immunoreactivity is not expressed by glial cells. To double-check our findings, we have also used another XA antibody obtained from a commercial source to confirm the neuronal expression of XA. Together, our results suggest that, differently to several other KPM produced by glial cells, XA exhibits a neuronal distribution in the mouse brain. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: xanthurenic acid, mouse brain, immunocytochemistry, neuronal localization, kynurenine pathway. *Corresponding author. Address: INSERM U1119, Universite´ de Strasbourg, Faculty of Medicine, 11 rue Humann, 67085 Strasbourg, France. Tel: +33-3-68-85-30-96; fax: +33-3-68-85-35-70. E-mail address: [email protected] (M. Maitre). Abbreviations: BSA, bovine serum albumin; EGTA, ethylene glycol tetraacetic acid; ELISA, enzyme-linked immunosorbent assay; KATS, kynurenine aminotransferases; KLH, keyhole limpet hemocyanin; KMO, kynurenine monooxygenase; KPM, kynurenine pathway metabolites; KYNA, kynurenic acid; PBST, phosphate-buffered saline-tween; TH, tyrosine hydroxylase; XA, xanthurenic acid. http://dx.doi.org/10.1016/j.neuroscience.2016.05.006 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 1

INTRODUCTION

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The essential amino acid tryptophan is the precursor of many neuroactive substances, several of them belonging to the kynurenine pathway (Stone and Darlington, 2013; Vecsei et al., 2013). Among the most studied molecules of this pathway are kynurenic acid (KYNA) and quinolinic acid (QUIN) (Schwarcz et al., 2012). In the brain, tryptophan metabolism occurs via the protohemoprotein enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO), the latter action of which has a number of effects in the body including both antimicrobial defense and immune regulation. These enzymes convert tryptophan to N-formylkynurenine which gives L-kynurenine. This compound is the main precursor for the production of KYNA via kynurenine aminotransferases (KATS) or for xanthurenic acid (XA) after hydroxylation of L-kynurenine by kynurenine monooxygenase (KMO), followed by its transformation by KATS (Fig. 1). XA is generally considered as the final compound of a dead-end branch with no functional role. However, several recent indications contradict these views. In the rat brain, distribution, transport and Ca2+-dependent release of XA suggest a role for this compound in neurotransmission (Gobaille et al., 2008). Additionally, several lines of evidence support the idea that, in the brain, XA acts via a family of GPCR (G-protein coupled receptor) whose stimulation in NCB-20 cells (mouse neuroblastoma Chinese hamster brain hybrid cell line) elicited specific activation of cationic channels (Taleb et al., 2012). It has also been reported that XA is a vesicular glutamate transport inhibitor and an mGlu 2/3 receptor agonist (Copeland et al., 2013; Neale et al., 2013a,b). These findings suggest a specific role of XA in the central nervous system but its cellular distribution in the brain has never been determined. The present study reveals that, while most part of the cerebral metabolism of kynurenine occurs in glia, particularly in astrocytes (Schwarcz and Pellicciari, 2002; Guillemin et al., 2007), XA is localized in neurons. This demonstration was made possible by the performance of several series of single- and doubleimmunostaining studies using a specific and very well characterized antibody against XA and various efficient markers for neurons, glial cells and synapses. Independently from these experiments, another specific antibody directed against XA (anti-XA II, from commercial source)

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Fig. 1. Tryptophan metabolism leading to XA. The pivotal metabolite Kynurenine is formed from the amino acid Tryptophan by the successive action of indoleamine 2,3-dioxygenases and tryptophan 2,3-dioxygenases to yield N-formylkynurenine (not shown) then degraded into Kynurenine by formamidase. Kynurenine 3-hydroxylase (3-HK) catalyzes the formation of 3-hydroxykynurenine. Isoforms of Kynurenine aminotransferases (KATs) transform 3-HK into XA, the hydroxylated analog of Kynurenic acid. No degradative enzyme of XA has yet been described in the mammalian brain.

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has been used to confirm the neuronal localization of XA-immunoreactivity in several regions of the mouse brain.

EXPERIMENTAL PROCEDURES

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Animal care

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Procedures involving animals and their care were conducted in compliance with a European Communities Council Directive (86/609/EEC) and under the supervision of authorized investigators. A total of five

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adult male Swiss Albinos mice were used. Anesthesia was induced with I.P. administration of 20 mg/kg pentobarbital sodium.

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Xanthurenic antibody development and characterization

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XA was conjugated to bovine serum albumin (BSA) according to the protocol described previously (Duleu et al., 2010) and rabbits were immunized three times over a 2-month period with 100 lg of immunogen per injection. Serum samples were collected 10 days after the last immunization and were by means of Enzyme-linked immunosorbent assay (ELISA) for the presence and characteristics of anti-XA antibodies. Maxisorp 96-well plates (Nunc) were coated overnight with conjugated XA in carbonate buffer (pH 9.6) at 4 °C. Plates were rinsed with PBS-Tween (PBST) and blocked with BSA 2.5 g/L diluted in PBST for 1 h at 37 °C. All sera were pre-adsorbed against an excess amount of unmodified carrier protein (s) (BSA in the case of anti-XA I and keyhole limpet hemocyanin (KLH) in the case of anti-XA II) and the immunoprecipitates were removed. To assess the quality of these immunoprecipitations, competing ELISA has been performed using free BSA or KLH. No competitions were observed as for the others conjugates. Rabbit sera were first incubated with increasing concentrations of conjugated-XA or its analogs. Plates were then washed and incubated with horseradish peroxidase (HRP)conjugated goat anti-rabbit serum for 1 h at 37 °C. Plates were finally exposed to tetramethylbenzidine for 10 min. The detection reaction was stopped by the addition of 1 N HCl. Optical density was measured at 450 nm using a Thermo Multiskan EX.

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Brain perfusion and tissue preparation

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Mice were deeply anesthetized with sodium pentobarbital before transcardial perfusion with a fixative solution (carbodiimide 0.1 M, 4% formaldehyde, phosphate buffer 0.1 M pH 7.4). The brain tissue was taken from animals prepared as follows. The perfusion apparatus (peristaltic pump) was filled with the chilled fixative solution and circulated with both tubing ends in the reservoir to exclude all air bubbles. After anesthetization, mice were immediately perfused transcardially with the fixative solution during 5 min, then five other min with chilled 4% formaldehyde in phosphate buffer 0.1 M pH 7.4. The brain was removed after the end of the perfusion and post-fixed 3 to 4 h in 4% formaldehyde in phosphate buffer 0.1 M pH 7.4. Following fixation, brains were rinsed in PBS pH 7.4.

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Immunohistochemistry

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Brain sections were dissected according to the Paxinos and Watson coordinates (Paxinos, 1986). Seventy-micrometer sections were cut with a vibratome (Leica VT 1000 S) and collected in PBS pH 7.4. Floating sections were immunostained as follows: Tissue slides were rinsed with PBS then blocked 15 min at room temperature with 5% (v/ v) fetal bovine serum in PBS. Sections were stirred over-

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night at 4 °C with the antibody, at optimal dilution for each of them. Sections were then washed 9 to 12 times during 45 min in PBS pH 7.4. Then the sections were stirred with species-specific secondary antibodies coupled to Dylight fluoroProbe-488 or Dylight fluoroProbe-594 (1/500, Interchim) for 3 h at room temperature in the dark. Sections were again washed (9 to 12 times during 45 min) with PBS pH 7.4. After this second period of washing with PBS, the sections were mounted in glycerol/PBS (v/v) before microscopic analysis using a microscope (Zeiss AxioImager Z2) equipped with a digital camera (Axiocam MRc, Zeiss, Germany). Confocal microscopy was performed using a Leica sp2 confocal microscope fitted with

serum (Roussel et al., 1981). After 10 days of culture, the cells were fixed in carbodiimide 0.1 M pH 5.4 in PBS for 10 min, followed by a second fixative containing formaldehyde (4%) in PBS for 10 min. After washing in PBS, the cells were permeabilized in 0.1% Triton X-100 in PBS and then incubated in the presence of the primary antibodies (Anti-XA 2, commercial source and monoclonal anti-MAP2 from chicken). All antibodies dilutions were identical to those used for tissue studies.

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Antibodies

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1-Primary antibodies:

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Target

Host

Source – Ref

Dilution

Anti-glial fibrillary acidic protein (GFAP)

Mice

Chemicon – Ref MAB360

1/500

Anti-tyrosine hydroxylase

Mice

Sigma-Aldrich – Ref T1299

1/1000

Anti-MAP2

Chicken

Covance – Ref PCK554P

1/1000

Anti pan-axonal neurofilament marker

Mice

Covance – Ref SMI 312-R

1/500

Anti-synaptophysin

Mice

Sigma-Aldrich – Ref S5768

1/500

Anti-XA-1 Anti-XA-2

Rabbit Rabbit

Obtained from IMS Lab Immusmol – Ref IS1014

1/800 1/400

an argon ion laser for Alexa 488 excitation, equipped also with a diode DPSS 561for Alexa 594 excitation and a Helium Neon 633 for Cyanine 5 excitation. An oil immersion plan apo (NA 1.4) objective was used. Specificity control experiments were performed as follows: (i) Incubation with secondary antibody alone (ii) replacement of primary antibodies by non-immune mouse or rabbit serum

Host

Ref

Dilution

Anti-rabbit F (ab0 ) 2 IgG Anti-mouse F (ab0 ) 2 IgG Anti-chicken (IgG)

Donkey Donkey Donkey

FP-SD5120 FP-SA4120 FP-SA1110

1/500 1/500 1/500

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Primary cell cultures of mouse brain

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The dissociated cells obtained from new born rat hemispheres were seeded in culture dish containing Dulbecco (NaHCO3 2.1 g/l) supplemented with penicillin (50 units/ml), streptomycin (50 mg/ml) and 10% calf

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2-Secondary Interchim

Target

and (iii) preincubation/pre-adsorption of primaries antibodies with the respective purified immunogens.

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Monoclonal Unpurified IgG1 Clone GA5 Monoclonal IgG1 Clone TH-2 Monoclonal Purified IgY Clone Poly28225 Monoclonal IgG1/IgM Clone SMI 312 Monoclonal IgG1 Clone SVP-38 Purified polyclonal antibody Purified polyclonal antibody Batch 140801

antibodies:

Jackson

Immunoresearch

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DyLight fluoroprobe 594 Immunoglobulin Dylight fluoroprobe 488 Immunoglobulin Dylight fluoroprobe 488 Immunoglobulin

The compound DAPI (40 ,6-siamidino-2-phenylindole dihydrochloride) was used for nuclei labeling (1 lg/ml).

RESULTS

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XA antisera specificity

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The specificity of XA antiserum was established following two methods (Fig. 2). A sample of XA-antibody was preadsorbed during 8 h at 4 °C using a mixture of the

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Fig. 2. Anti XA I antisera specificity. Images of cerebellum demi-folium positive Purkinje cells and their dendrites in the molecular layers labeled with the XA-antiserum I (1/800) (A) or treated with the pre-adsorbed XA-antiserum I (B). Results of ELISA competition between XA and different analogus (C). Only kynurenic acid exhibits a very slight activity but not significant under the present experimental conditions.

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immunogen (conjugated XA, 20 lg/ml) and the diluted antibody (1/500). Competitive ELISA technic was performed using several XA analogs and only KYNA exhibits a very slight activity, about 10,000 fold less than XA (Fig. 2C). Finally, the incubation of cerebellar brain tissues with the immune serum (1/800) revealed a strong labeling of the cytoplasm and dendrites of Purkinje cells and granules cells (Fig. 2A) while no staining was seen with the pre-adsorbed antisera (Fig. 2B). Also, no immunoreactive materials were detected when anti-XA I or anti-XA II antibodies were pre-adsorbed or replaced by non-immune serum (data not shown). XA immunoreactivity is present in neurons but not in astrocytes The experiments were performed using slices from the cerebellum, parietal and frontal cortex, corpus callosum and striatum. Numerous fibrous astrocytes and glial cell processes were stained with the antibody against GFAP. Double labeling with anti-GFAP + anti-XA revealed the absence of XA in astrocytes as no colocalization of GFAP and XA was seen (Fig. 3A–C). By contrast, XA and MAP-2, the specific cytoskeletal protein abundantly expressed in neuronal dendrites (Shafit-Zagardo and Kalcheva, 1998), were co-localized in neurons, including pyramidal hippocampal cells (Fig. 3D–F).

Absence of XA immunoreactivity in axonal compartments

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As shown in Fig. 4, neurofilaments were detected in several axons of the cerebellum (Fig. 4A, B), corpus callosum-striatum (Fig. 4E, F) and hippocampus (Fig. 4C, D) while XA was only found in dendritic processes and in neuronal soma (Fig. 4A–F).

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XA is not co-localized with synaptophysin

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Synaptophysin immunostaining is conventionally used to identify synapses. Therefore, we performed double labeling with anti-synaptophysin and anti-XA to confirm the absence of XA from synaptic endings. As shown in Figs. 5 and 6, a strong labeling of XA was detected in the pallidum (Figs. 5 and 6A, B), the Islands of Calleja (Figs. 5 and 6C), the dendrites and soma of Purkinje cells (Fig. 6D, E) and in several dendrites of the striatum (Fig. 5D, E). The cell bodies of cortical (Fig. 6F) and striatal neurons (Fig. 6G) are also labeled. Synaptophysin-positive terminals are widely expressed in all cortical and striatal synapses (Figs. 5F–H, 6). Also, the cerebellar glomerulus (granular cell layer) contained numerous synaptophysin-positive neurons (Fig. 6D, E). In the deep nuclei of the cerebellum, various neurons were surrounded by positive synaptophysin contacts (Fig. 6D). In the hippocampus, neuronal cell bodies and dendrites of the CA3 strongly expressed XA-

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Fig. 3. Representative images of double labeling of xanthurenic acid (A–C red) and GFAP (A–C green) in vibratome sections of adult mouse cerebellum (A, B) cortex, corpus callosum and striatum (C). Xanthurenic acid was visualized in granular and Purkinje neuronal cell bodies and dendritic arbors but not in glia (arrows B and D). In contrast, GFAP was found in fibrous astrocyte and radial glia processes but not in neuronal elements. Representative images of double labeling of xanthurenic acid (D–F red) and MAP-2 (D–F green) in vibratome sections of adult mice cerebellum (D), cortex (E) and hippocampus (F). Co-expression of xanthurenic acid and MAP-2 was detected in dendritic poles of neurons (double arrow in D and E as an example) Bar: A and D = 50 lm, B and E, F = 20 lm, C = 100 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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immunoreactivity (Fig. 5F–H) and the immunolabeled dendrites were surrounded by synaptophysin-positive synapses.

However, in all brain areas investigated, we did not observe a co-localization of synaptophysin and XA labeling in synaptic-like structures.

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Fig. 4. Images of double labeling of xanthurenic acid (A–F red) and phosphorylated neurofilaments (A–F green) in vibratome sections of adult mice cerebellum (A, B), hippocampus (C, D) and corpus callosum-striatum (E, F). Neurofilaments were visualized in axons (arrow C as an example for hippocampus) while xanthurenic acid was found in neuronal cell bodies and dendritic arbors. These labelings are juxtaposed. Note that the myelinated axons in striatum (E, F) are only positive to phosphorylated neurofilaments and negative to xanthurenic acid. Bar: A, C and E = 50 lm, B, D and F = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) 236 237 238 239 240

Dopaminergic terminals are juxtaposed with XAcontaining neurons in the ventral frontal cortex and the striatum We analyzed a possible relationship between tyrosine hydroxylase (TH), the rate-limiting enzyme of dopamine

synthesis, and the cerebral distribution of XA, because the mesencephalic dopaminergic circuits A9-A10 (involving the striatum and the prefrontal cortex) strongly expressed XA-immunoreactivity (Gobaille et al., 2008). In addition, XA binding sites are well distributed in these

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Fig. 5. Images of double labeling of xanthurenic acid (A–H red), tyrosine hydroxylase (A–E green), synaptophysin (F–H green) in vibratome sections of adult mice ventral frontal cortex (A–C), striatum (D, E) and hippocampus (F–H). Tyrosine hydroxylase was detected in forebrain and striatal axons. Xanthurenic acid is intensively expressed in the pallidum (A, B), the Islands of Calleja (C) and in several dendrites of the striatum (D, E). These labelings are juxtaposed. Myelinated axons of the corpus callosum (D) and fasciculated axons of the striatum (D, E) were devoid of XA and tyrosine hydroxylase immunoreactivities. Hippocampal neurons (cell bodies and dendrites) of CA3 were intensively labeled by the anti-XA antibody (single arrow F). At higher magnification, positive synapses and terminal axons labeled with synaptophysin can be visualized around XAimmunoreactive dendrites (F-H) (single arrow H as an example). Bar: F = 100 lm, A and G = 50 lm, B, E and H = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 6. Representative images of double labeling of xanthurenic acid (A–G red) and synaptophysin (A–G green) in vibratome sections of adult mouse ventral frontal cortex (A–C) cerebellum (D, E). Xanthurenic acid was detected in the pallidum (A, B), the Islands of Calleja (C), Purkinje cell bodies’ dendrites (D, E), cortical (F) and striatal (G) neurons. Synaptophysin punctuates around Purkinje cells in (E). Synaptophysin was found in several synapses of the cortex (A–C, F) and striatum (G). The glomerules (double arrow in E for example) of the granular cell layer were intensively labeled (D, E). Neurons of the cerebellar deep nuclei were surrounded by positive synaptophysin contacts (D) (single arrow). Bar: A = 500 lm, B, C = 20 lm, D = 100 lm, E–G = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 7. Representative images showing the neuronal localization of xanthurenic acid by the use of an anti-XA antibody obtained from a commercial source (xanthurenic acid polyclonal antibody, IS 1014, ImmuSmol SAS, Pessac, France). Xanthurenic labeling appears in red (B) in vibratome sections of adult mouse cortex and is present in the cell bodies of cortical neurons and in their dendritic portions (arrows) identified by MAP2 labeling (A). Xanthurenic acid is absent from axons identified with anti-neurofilament antibody (C). Note the superposition of labeling between the MAP2 and XA in the dendritic portions of neurons. Bar: A–D = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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areas. Our experiments revealed a strong XA labeling in clusters of granule neuronal cells of the basal forebrain. These collections of neurons are well-known as neuronal cells of the Islands of Calleja (Hsieh and Puche, 2013). Consistently, a close juxtaposition of TH and XA was evidenced in the ventral frontal cortex and striatum (Fig. 6A–C and F–G).

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Labeling with another anti-XA antibody

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To confirm our results obtained with anti-XA I additional immunohistochemical experiments were carried out using another antibody (anti-XA II), commercially available. Multiple labeling with anti-XA II confirmed the presence of XA in neurons and dendrites of different areas of the mouse brain (Figs. 7 and 8). These last figures have been obtained using a higher magnification

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to clearly shown the different subcellular localization of the different antigens.

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Localization of XA in primary cell cultures of the mouse brain

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In 10-day old cultures, a layer of glial cells was present in the plates (mainly astrocytes) together with a small number of neurons detected with MAP-2 antibody. As shown in Fig. 8, MAP-2 labeling was co-localized with XA. The remaining cells, identified by their DAPI-labeled nuclei, were not fluorescent (see Fig. 9).

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DISCUSSION

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By using an immunocytochemical approach double checked with two specific anti-XA (obtained from two

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Fig. 8. Confocal microscopy images showing the neuronal localization of xanthurenic acid by the use of an anti-XA antibody obtained from a commercial source (xanthurenic acid polyclonal antibody, IS 1014, ImmuSmol SAS, Pessac, France). Xanthurenic labeling appears in red (B) in vibratome sections of adult mouse cortex. Xanthurenic acid is present in the cell bodies of hippocampal neurons and in their dendritic portions (arrows) identified by MAP2 labeling (A). Xanthurenic acid is absent from axons labeled with anti-neurofilament antibody (C). Note the superposition of XA and MAP2 labeling in dendritic portions of hippocampal neurons (D). Bar: A–D = 10 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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independent sources) and different key markers of nerve cells, the present study provides the sub-cellular distribution of XA in the mouse brain. Anti-XA I or anti-XA II immunoreactive materials detected in the mouse brain completely disappeared when both antisera used were pre-adsorbed with their respective immunogens. In addition, a series of tryptophan derivatives of the kynurenine pathway were conjugated to BSA or KLH, using the same protocol as for XA. The ELISA method was applied to further confirm the specificity of XA anti-sera and to assess eventual cross-reactivity with structurally close analogs of XA (Duleu et al., 2010). Clearly, the results indicated a high affinity for XA-immunogens while no interaction was detected with the analogs, except a weak reaction with KYNA. This weak affinity for KYNA has no chance to interfere with the specific detection of XA using high dilutions of anti-XA,

Neurofilament proteins (NFPs) are highly phosphorylated molecules in the axonal compartment of the adult nervous system (Grant and Pant, 2000). We used this specific marker to identify axonal processes and to visualize the possible presence of XA in axons and presynaptic ending, in order to determine whether XA may be involved in synaptic organization. In another set of experiments, we examined the possible colocalization of XA with Synaptophysin, a synaptic vesicle glycoprotein with four transmembrane domains that is present in neuroendocrine cells and virtually in all brain neurons involved in synaptic transmission (Valtorta et al., 2004). The results of these experiments clearly demonstrated the somato-dendritic localization of XA which is absent from axons and synaptic endings. Another interesting point is the strong expression of XA-immunostaining in the Islands of Calleja which are innervated by neuronal fibers arising from A9-A10 and

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Fig. 9. Representative images of xanthurenic acid labeling (Rabbit Anti-XA-2 (Immusmol IS 1014, batch 140801) diluted 1/400, (C-red) in primary culture of glial cells (10 days in vitro) obtained after seeding dissociated cells obtained from newborn rat hemispheres in culture dishes. The nuclei of all the cells were labeled with DAPI (A); some cells of neuronal origin, present beside the glial cells, were labeled with MAP2 antibody (B-green) and express xanthurenic acid (C-red). In D, the superposed labeling (yellow) was only seen in the neuronal cells present in the primary culture. Bar: A– D = 20 lm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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accumbens nuclei. This observation suggests that XA may eventually interact with the dopaminergic system. However, it appears important to specify that the labeling of TH is juxtaposed to XA-immunoreactive material but never co-localized with it. Up to now, most of the enzymes belonging to the kynurenine pathway have been localized in macrophages, microglial cells and also in astrocytes (Du et al., 1992; Guillemin et al., 2001; Guillemin et al., 2005, 2007; Guidetti et al., 2007). However, KATS, which are present in astrocytes and catalyze the synthesis of KYNA have also been detected (KAT I and III) in neurons (Du et al., 1992). KAT II was also expressed, after stimulation by a specific cytokine, in a human neuroblastoma cell line (Guillemin et al., 2007). KAT I and II have been detected in cerebral cortical neurons (Rzeski et al., 2005). Moreover, KAT-I was also found in dopaminergic neurons of the substantia nigra (Knyihar-Csillik et al., 2004). Interestingly, it has been shown that 3-HK, the precursor of XA, is a substrate of brain KAT II, III and IV in human and rodents (Han et al., 2010). Furthermore, mRNAs encoding for KMO (a key enzyme involved in

3-HK synthesis), were evidenced in neuronal cultures and 3-HK was detected in the cell culture medium (Schwarcz et al., 2012). Also, the occurrence of 3-HK has been demonstrated in human hippocampal neurons (Bonda et al., 2010). Therefore, it appears that the required substrates and enzymatic materials are all present in brain neurons to produce XA from 3-HK. Thus, XA occurring in neurons may result from the local or endogenous production in the intra-neuronal compartment. XA accumulated in the extracellular space may also be internalized by neuronal cells thanks to the activity of a specific transporter (Gobaille et al., 2008). Previous data reported XA release in the extracellular spaces of the rat prefrontal cortex, a process induced by electrical stimulation and blocked by EGTA (Gobaille et al., 2008). Therefore, since XA-immunoreactivity was not detected in synaptic endings, it may be hypothesized that XA could be released from neuron somatic and/or dendritic sites via an extra-synaptic exocytosis mechanism (Trueta and De-Miguel, 2012). Thus, XA released by exocytotic mechanisms outside the synaptic cleft may interact with XA-metabotropic receptors expressed

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by NCB-20 cell cultures which do not exhibit synaptic contacts in our experimental conditions (Taleb et al., 2012). For many signaling molecules, including low molecular weight transmitters, this release appears to be calciumdependent from small clear or large dense core vesicles, acting in a paracrine way. Taken together, our studies, which used various specific markers, indicate that XA is distributed in the somato-dendritic part of neurons and absent from the axo-synaptic endings, at least in brain structures investigated herein. Our data do not support the existence of significant amount of XA in nonneuronal cells, at least in the mouse brain. Recently, the administration of XA in a mouse model of schizophrenia supports a role for this compound in the mechanism of psychotic behavior, possibly through the interaction with metabotropic glutamate receptor(s). This result has been correlated with the finding of low XA levels in the serum of schizophrenic patients (Fazio et al., 2015). Several studies have also pointed out the possible involvement of kynurenine pathway metabolites (KPM) in auto-immune diseases and in psychiatric disorders. It has also been shown that the upregulation of XA in the brain may decrease the synthesis of tetrahydrobiopterin, serotonin, dopamine and various other neurotransmitters (Haruki et al., 2015). Altogether, these data suggest that the biosynthesis and regulatory mechanisms of XA release in the brain may be an interesting piece of the puzzle implicating KPM in several important pathophysiological processes. The role of XA neuronal system may however fluctuate according to animal species since some differences between species may occur in the biological activities of certain enzymes involved in the kynurenine pathway (Murakami and Saito, 2013).

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CONFLICT OF INTEREST

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The authors declare no conflict of interest.

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Acknowledgments—This work was supported by the University of Strasbourg, Inserm and the European Union through the European Regional Development Fund, Interreg IV and Offensive Sciences Grant attributed to AGMN.

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(Accepted 3 May 2016) (Available online xxxx)

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