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. Author manuscript; available in PMC: 2019 Jan 15.
Published in final edited form as: Neuroscience. 2017 Nov 8;369:1–14. doi: 10.1016/j.neuroscience.2017.11.001

Quantitative analysis of kynurenine aminotransferase II in the adult rat brain reveals high expression in proliferative zones and corpus callosum

Chang Song 1, Sarah M Clark 1, Chloe N Vaughn 1, James D Nicholson 1, Kelley J Murphy 2, Ta-Chung M Mou 1, Robert Schwarcz 3, Gloria E Hoffman 2, Leonardo H Tonelli 1,*
PMCID: PMC5766363  NIHMSID: NIHMS922058  PMID: 29126954

Abstract

Kynurenic acid, a metabolite of the kynurenine pathway of tryptophan degradation, acts as an endogenous antagonist of alpha7 nicotinic and NMDA receptors and is implicated in a number of neurophysiological and neuropathological processes including cognition and neurodegenerative events. Therefore, kynurenine aminotransferase II (KAT II/AADAT), the enzyme responsible for the formation of the majority of neuroactive kynurenic acid in the brain, has prompted significant interest. Using immunohistochemistry, this enzyme was localized primarily in astrocytes throughout the adult rat brain, but detailed neuroanatomical studies are lacking. Here, we employed quantitative in situ hybridization to analyze the relative expression of KAT II mRNA in the brain of rats under normal conditions and 6 hours after the administration of lipopolysaccharides (LPS). Specific hybridization signals for KAT II were detected, with the highest expression in the subventricular zone (SVZ), the rostral migratory stream and the floor of the third ventricle followed by the corpus callosum and the hippocampus. This pattern of mRNA expression was paralleled by differential protein expression, determined by serial dilutions of antibodies (up to 1:1 million), and was confirmed to be primarily astrocytic in nature. The mRNA signal in the SVZ and the hippocampus was substantially increased by the LPS treatment without detectable changes elsewhere. These results demonstrate that KAT II is expressed in the rat brain in a region-specific manner and that gene expression is sensitive to inflammatory processes. This suggests an unrecognized role for kynurenic acid in the brain’s germinal zones.

Keywords: subventricular zone, astrocytes, doublecortin, lipopolysaccharides, in situ hybridization, tanycytes

INTRODUCTION

Kynurenic acid (KYNA), a metabolite of the kynurenine pathway (KP) of tryptophan degradation, acts as an endogenous antagonist of alpha7 nicotinic and NMDA receptors in the brain (Ganong et al. 1983; Hilmas et al. 2001; Kessler et al. 1989; Perkins and Stone 1982) and may also target additional recognition sites (Stone et al. 2013). Of special interest to neuroscience, and neural immune function, the compound is implicated in a number of neurophysiological and neuropathological processes (see, (Schwarcz and Stone 2017) for review). For example, KYNA may be neuroprotective in disorders in which excitotoxicity is likely to play a causal role. This includes cerebral ischemia, epilepsy and neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease (Schwarcz et al. 2012; Stone et al. 2012; Szalardy et al. 2012). On the other hand, elevated brain KYNA levels lead to cognitive impairments and an array of neurotransmitter abnormalities (Pershing et al. 2016; Schwarcz et al. 2012). This may explain the cognitive deficits seen in people with schizophrenia who show increased levels of KYNA in brain and cerebrospinal fluid (Erhardt et al. 2001; Miller et al. 2008; Schwarcz et al. 2001).

Because of these links to brain physiology, the biosynthesis of KYNA in the mammalian brain has been examined in considerable detail. So far, four aminotransferases have been shown to catalyze the irreversible transamination of the pivotal KP metabolite L-kynurenine (“kynurenine”) to KYNA (Guidetti et al. 2007; Guidetti et al. 1997; Han et al. 2010). Of these, kynurenine aminotransferase II [KAT II; = α-aminoadipate aminotransferase (AADAT); (Okuno et al. 1991; Tobes and Mason 1975; Tobes and Mason 1977)] is primarily responsible for the rapid “de novo” synthesis of KYNA in the brain (Amori et al. 2009); see (Schwarcz et al. 2012), for review, with biochemical studies revealing a wide distribution of enzymatic activity across rat brain regions (Guidetti et al. 1997). Of note, genetic elimination of KAT II (Potter et al. 2010; Yu et al. 2004) or selective pharmacological inhibition of KAT II (Kozak et al. 2014; Wu et al. 2010) improves cognitive functions in rodents. Due to this critical role of KAT II in regulating the levels and effects of KYNA in the brain, the structure, function and cellular distribution of this enzyme became a focus in neuroscience research (Goh et al. 2002; Guidetti et al. 2007; Guidetti et al. 1997; Potter et al. 2010).

The transcript for KAT II was initially identified in liver and kidney and later confirmed to be identical to the gene coding for the protein KAT II (Buchli et al. 1995; Han et al. 2010; Yu et al. 1999). Analyses of the mRNA sequence by our laboratory revealed an 88% homology between rats and mice and 76% conserved sequences between human and rat mRNA. Both rodent and human mRNAs contain mitochondrial leader cleavage signals and a conserved pyridoxal phosphate binding site, highlighting the evolutionary relevance of this protein. Using immunohistochemistry, KAT II was localized primarily in astrocytes throughout the adult rat brain (Guidetti et al. 2007), and KYNA formation via KAT II was confirmed using cultured human astrocytes (Kiss et al. 2003). However, studies with human cell cultures, as well as a recent study in mice (Heredi et al. 2017), have also indicated that KAT II is expressed in other cells, including neurons (Rzeski et al. 2005; Wejksza et al. 2005).

Due to regional differences in the production of KYNA in the brain (Turski et al. 1989), the present study was designed to fill a void by mapping the cellular distribution of KAT II/AADAT gene expression in the adult rat brain. To this end, we performed in situ hybridization histochemistry with radioactive riboprobes both under normal conditions and in animals receiving lipopolysaccharides (LPS), an established experimental approach to stimulate an immune response as well as KP metabolism in the mammalian brain (Larsson et al. 2016; Walker et al. 2013). In complementary experiments, we examined the cellular expression of KAT II by immunohistochemistry. Results of these studies converged to demonstrate high levels of KAT II in the brain’s neurogenic and gliogenic niches, pointing to a potential novel role of KYNA in adult brain cellular homeostasis.

EXPERIMENTAL PROCEDURES

Animals and treatments

Two-month old male and female Wistar rats were obtained from Charles River Laboratories (Cambridge, MA, USA) and kept at the animal facility of the University of Maryland School of Medicine. Animals were housed in groups of three in Plexiglas cages with standard food pellets and water available ad libitum. All animals were maintained on a 12:12 light:dark cycle (lights on at 07:00 AM) at a constant temperature of 23°C. The rats were left undisturbed for one week and then handled daily for an additional week before starting the experimental procedures. Male rats were injected intraperitoneally (i.p) with 2 mg/kg LPS (Sigma-Aldrich, St. Louis, MO, USA; serotype 055:B5) (n = 17) or 0.9 % saline (n = 17) between 09:00 and 10:00 AM and monitored 3 and 6 h later for sickness behavior and temperature. Female rats (n = 6) were injected with saline only. At the later time point, all animals were killed and the tissue was processed for in situ hybridization or immunohistochemistry. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore.

Riboprobe generation

Liver tissue was processed for mRNA extraction using TRIzol (Invitrogen, Carlsband, CA, USA) as described previously (Gunsolly et al. 2010; Tonelli et al. 2004). Total RNA was treated with DNAse I (Invitrogen) for 15 minutes at room temperature according to the manufacturer’s instruction. Specific sequences were produced to target regions 411 to 1151 or 69 to 1145 (Fig. 1A) corresponding to the rat KAT II mRNA (aminoadipate aminotransferase, Aadat, Genebank accession number NM_017193.1). The specific fragments were produced by PCR amplification using the PCR SuperMix High Fidelity enzyme mixture (ThermoFisher Scientific, USA). Both sequences produced a single band of expected size of 741 bp (Fig. 1B) or 1077 bp (Fig. 1C). These fragments were cloned into the TA Dual Promoter pCR™ II vector (ThermoFisher Scientific) and sequenced to confirm the length, identity and orientation of the template sequence. Anti-sense and sense (control) riboprobes were labeled by in vitro transcription in the presence of 10 μM [35S]-UTPα S (PerkinElmer, Waltham, MA, USA; > 1000 Ci/mmol), 1 μg of linearized plasmid, and 20 units of T3 or T7 RNA polymerase using the RNA labeling kit (ThermoFisher Scientific) according to the manufacturer’s protocol. After transcription, the template DNA was digested with DNAse I for 15 minutes at 37° C. Unincorporated [35S]UTPαS was removed by centrifugation through ProbeQuant G-50 micro columns (ThermoFisher Scientific).

Figure 1.

Figure 1

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A) Template design for in vitro transcription of riboprobes of 741 or 1077 base pairs (b.p) targeting the rat KAT II mRNA Genebank accession number NM_017193.1. Specific PCR amplification products from RNA purified from different tissues for the regions generating a sequence of 741 bp (B) or 1077 bp (C) corresponding to KAT II mRNA. D) Film autoradiographic images of specific hybridization signals in different tissues generated by the riboprobe of 741 bp. E) Identical hybridization signals were obtained with the 2 riboprobes. Addition of purified KAT II to diluted antibody (1:30,000) completely blocks all staining. F–G: Specificity of the antibody, micrographs showing the staining in the corpus callosum (cc) and subventricular zone (SVZ) in rat sections processed normally (F) or preadsorbed with purified KAT II (B). LV: lateral ventricle. Scale bar = 100μm.

In Situ Hybridization histochemistry

The in situ hybridization procedure was carried out as described previously (Gunsolly et al. 2010; Tonelli et al. 2004). Briefly, fresh frozen serial consecutive coronal brain sections (n = 6 male, 6 female saline, 6 male LPS) were cut in a cryostat at 20 μm of thickness through the entire rat brain and collected onto silanated slides and stored at −80° C until processed. Sections were fixed for 10 minutes with a 4% paraformaldehyde solution in phosphate buffered saline (PBS), rinsed twice in PBS, acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) for 15 minutes and dehydrated through graded ethanols. Each slide was treated with 150 μl of hybridization buffer containing 40,000 cpm/μl of labeled sense or anti-sense riboprobe, 50% formamide, 0.3 M NaCl, 2 mM EDTA, 20 mM Tris ph 8.00, 1× Denhardt’s solution, 10% dextran sulfate, 100 μg/ml salmon sperm DNA, 250 μg/ml yeast RNA, 250 μg/ml yeast tRNA, 100 mM dithiothreitol and 0.1% sodium dodecyl sulfate. After hybridization for 16–18 h at 54° C, sections were rinsed 4 times in 4× saline citrate buffer (SSC) to remove coverslips and excess of riboprobes. Non-hybridized riboprobes were digested by incubation with 40 μg/ml Rnase A (Sigma-Aldrich) for 30 minutes at room temperature. After a final high stringency wash in 0.1× SSC at 65° C for 60 minutes, sections were dehydrated in graded ethanol containing 0.3 M ammonium acetate and air-dried. Sections were exposed to BioMax MR film (Sigma) along with 14C standards (Amersham Pharmacia, USA) for 3, 10 and 21 days and developed in an automatic film developer (AFP Imaging, GA, USA). The mRNA expression was analyzed by measuring optical film densities using the public domain Fiji program. Values were transformed to nCi/g of gray matter after calibration with standards. Background signal from sense control slides was subtracted to obtain the specific signal with antisense riboprobe. Anatomical localization of the signals was determined by Nissl staining of adjacent sections. For evaluation of mRNA expression at the cellular level, slides were dipped in NTB photo emulsion (Eastman Kodak, NY, USA), exposed for 2 months and developed in D-19 developer substitute (Photographers’ Formulary Inc, MT, USA) for 4 minutes at 15° C, fixed for 4 minutes and counterstained with toluidine blue. Positive hybridization signal was evaluated with the use of a Zeiss Axioscop and the ZEN 2011 microimaging software (Carl Zeiss AG, Oberkochen, Germany). Sections were analyzed at 100 times magnification with a Nikon Plan-Apo lens with a 0.45 numerical aperture and a dark field condenser with a 0.95 to 0.8 numerical aperture and then analyzed at 400 times magnification to confirm that silver grains were deposited over cell bodies.

Real-Time RT-PCR

Brains from saline control and LPS-treated rats (n = 5 each) were dissected and the hippocampus was processed for RNA extraction as described above. The remaining portions of the brain, minus the cerebellum, were also processed for mRNA extraction. Five hundred ng of total RNA per sample were reverse transcribed into cDNA in a 20 μl reaction volume using an iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to manufacturer’s instructions and then diluted 1:1 with ultrapure water. Real-time RT-PCR was conducted using the iQ SYBR Green Supermix (Bio-Rad) in a 25 μl reaction using the following set of primers: Forward 5′-TGTCAACTACTCACCCAACG-3′; Reverse 3′-GCAAAAAGGGCTCCTGAATAC-5′; corresponding to nucleotides 409 to 555 of the rat KAT II mRNA and the set of primers published in Tonelli et al. (2009) for the control genes 18s, GAPDH and Actin-beta, which were used for normalization. Relative expression was determined using the 2- ΔΔCt method (Schmittgen and Livak 2008).

Immunohistochemistry

Control and LPS-treated rats (n = 6 each) were transcardially perfused with 30 ml of saline followed by 150 ml of 4% buffered paraformaldehyde. The brains were removed and immersion post-fixed for 24 h in the same solution and transferred to 30% sucrose for cryoprotection. Free-floating sections (30 μm) were collected in a cryostat and stored in cryoprotectant at −80°C until processed. Immunohistochemistry for KAT II was performed as described in (Hoffman et al. 2016) using a Pelco BioWave® Pro histological microwave and its associated SteadyTemp™ (Ted Pella, Redding, CA, USA). Briefly, the cryoprotectant was removed from the sections with PBS (5 one-minute washes) and sections were treated with 1% hydrogen peroxide, followed by three 1 minute washes with PBS (BioWave® programs 1, 2, and 3 in Hoffman et al). Sections were incubated at room temperature with rabbit anti-KAT II antibody (Guidetti et al. 2007) at dilutions of 1:100,000; 1:300,000 and 1:1,000,000 for 1 hour while on a rotator (55 rpm) followed by incubation at 4°C for 48–72 h. Specificity of this antibody was determined in our previous study (Guidetti et al. 2007) and by the addition of purified KAT II from rat kidney to the diluted antibody, which completely blocked all staining (Fig. 1F–G). All steps other than primary antibody incubation and substrate reaction at the end of the procedure were performed in the microwave. After incubation with the primary antibody, sections were washed 3 times in PBS for 1 minute followed by incubation with biotin-conjugated goat anti-rabbit IgG (Cat #BA-1000, Vector Laboratories, Burlingame, CA, USA) for 19 minutes, washed 3 times in PBS for one minute and then incubated in ABC-HRP reagent (Elite Cat #PK-6100, Vector Laboratories) for 19 minutes (BioWave® program 4). The sections were washed 3 times in PBS for 1 minute followed by three 1 minute washes in 0.175 M sodium acetate solution. They were then reacted in 3,3′-diaminobenzidine (DAB) (Sigma-Aldrich) and nickel sulfate heptahydrate (Sigma-Aldrich, Fluka) and hydrogen peroxide in 0.175 M sodium acetate solution. Oxidized Ni-DAB produces an insoluble dark gray signal that is readily detectable over background. Systematic dilution of the primary antibody allows the differentiation of cells with high and low levels of the antigen since higher amounts of antigen can be detected using greater dilutions of the primary antibody. For double labeling of KAT II and glial fibrillary acidic protein (GFAP) or doublecortin (DCX), a marker of newly differentiating neurons, sections were sequentially stained using a modification of a method that permits use of two primary antibodies generated in the same species (Shindler and Roth 1996). Briefly, sections were first incubated with rabbit anti-KAT II antibody (1:100,000 dilution) for 48–72 h followed by a tyramine signal amplification step (BioWave® program 5) and detected with streptavidin-conjugated Alexa Fluor® 546 (Molecular Probes, S11225; 1:200 dilution; BioWave® program 7). Sections were then incubated with rabbit anti-GFAP (DAKO, Z-0334; 1:3000 dilution) or rabbit anti-Doublecortin (Cell Signaling, #4604, 1:3,000 dilution) for 48–72 h at 4°C followed by incubation with Alexa Fluor™ 488 goat anti-rabbit IgG (Cat#A11034, Life Technologies Corporation, Eugene, OR) or Alexa Fluor™ 488 goat anti-mouse IgG (Cat# A28175, both antibodies 1:200 dilution, BioWave® program 7). After three 1-minute washes the sections were counterstained with 0.0001% bisbenzimide (Cat#H 33258, Sigma-Aldrich) for 30 minutes at room temperature, washed 5 times for 10 minutes each, mounted onto subbed SuperFrost® slides (Cat#4951-001, Thermo Fisher Scientific), and air dried. Once dry, the sections were cleared in xylenes and mounted with Entellan® mounting medium (Cat#107960, Millipore Sigma, Billerica, MA, USA). Controls for separation of the two antigens include verification that single-labeled amplified signals do not bleed into the channels for the second fluorophores, and verification that the direct-tagged secondary antibody will not produce a signal when reacted with the dilute KAT II antibody used in the amplification reaction (Shindler and Roth 1996). Addition of purified KAT II completely blocks staining (Guidetti et al. 2007). Elimination of the primary antibodies or use of a secondary antibody directed towards a species other than rabbit IgG results in no staining.

Microscopy and image analysis

Sections treated with Ni-DAB as a chromogen were analyzed with a Zeiss Axioscop with ZEN 2011 microimaging software (Carl Zeiss AG) or a Nikon 90i microscope with Image Pro Plus software (Rockville MD). Images were taken at 20, 100 and 200 times magnification under constant illumination and processed with Adobe Photoshop (Adobe Systems Inc., San Jose CA) for building composite images. Low resolution fluorescence imaging of brain sections were performed on a Zeiss LSM 780 (Carl Zeiss Microscopes) using tiled z-stacks in either lambda scan mode (10 nm bins) or two channel imaging (488 nm and 561 nm), with image stitching, projection, and linear unmixing performed with Zen software (Carl Zeiss AG). Super resolution fluorescent images were collected on a Zeiss Airyscan (Zeiss LSM 880 base, 63× Oil, N.A. 1.4, optimal resolution determined by Zen Software) and processed to adjust brightness and contrast using Zen Software, Fiji image processing software (Schindelin et al. 2012), and Adobe Photoshop. Custom color lookup tables were used for rendering of signals in the SVZ.

Image processing for figure presentation

All digital images corresponding to a single experiment were acquired during the same session under constant illumination for each magnification. They were combined in a single file using Adobe Photoshop and linked prior to adjusting brightness for better visualization. No further processing was applied to the original images.

Statistics

A one-way ANOVA with Fisher’s LSD post hoc analysis was used to compare mean intensity values of in situ hybridization signals across brain regions. A Student’s t test was used to compare male vs female and control vs LPS data by brain region. Data are presented as the mean ± standard error of the mean (SEM); a p value of ≤ 0.05 was considered significant. Statistical analysis was conducted with GraphPad Prism 6 (GraphPad Software, Inc., La Jolla CA, USA).

RESULTS

KAT II mRNA is highly expressed in the subventricular zone, rostral migratory stream and floor of the 3rd ventricle

Strong and specific hybridization signals were detected in several peripheral organs including the liver, kidney and spleen but not in the thymus (Fig. 1D). The two probes used produced the same signal with regard to both distribution pattern and intensity (Fig. 1E). Specific hybridization signals were detected across the rat brain with a defined neuroanatomical distribution. Particularly robust signals were observed in the subventricular zone (SVZ) and the rostral migratory stream (RMS) extending as a continuum from the olfactory bulbs to the hippocampal formation (Fig. 2). A strong defined signal was also observed in the floor of the 3rd ventricle (3V), especially in regions covering the rostrocaudal portions of the median eminence (ME) (Fig. 2). Well-defined mRNA signals were observed across the entire regions of the corpus callosum (cc) (Fig. 2). In the hippocampal formation, expression of KAT II mRNA signal was highest in the stratum lacunosum moleculare (SLM) with very low or no signal in the granule and pyramidal cell layers (Fig. 2, 3C–D). Of note, no clear signals were detected by film autoradiography in several other brain regions including the cortical mantle, thalamus, hypothalamus, cerebellum and brainstem (Fig. 2).

Figure 2.

Figure 2

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Film autoradiographic digital images of coronal sections of the brain of adult male rats hybridized with 35S labeled riboprobes directed against KAT II mRNA (antisense) or sense control probes. Left panel: The annotated Nissl stained images provided the anatomical coordinates with respect to bregma for the adjacent sections showing autoradiographic signals. 3V: third ventricle, cc: corpus callosum, Hipp (SLM): stratum lacunosum moleculare of the hippocampus, RMS: rostral migratory stream, SVZ: subventricular zone. The scale bar applies to all panels, and the gray bar refers to 14C standards used for quantification showing the linear range of the film according to the exposure time.

Figure 3.

Figure 3

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Film autoradiographic digital images (A, C, E) and Nissl stained adjacent sections (B, D, F) of representative signals used for quantification by densitometry in different brain regions. The image in panel A shows the hybridization signal in the subventricular zone (SVZ) and corpus callosum (cc) and the shaded areas in panel B depict the “mask” that was applied to measure mean intensity levels. Similarly, panels C and D show representative sections used for quantification in the hippocampal formation (Hipp) and corresponding level of the cc. Panels E and F correspond to the floor of the 3rd ventricle. G: Quantification of mean intensity values of KAT II expression in the brain regions shown above of adult male rats (n= 6). 3V: 3rd ventricle; CA1: pyramidal cell layer of the hippocampal CA1 region; SLM: stratum lacunosum moleculare; DG: granule cell layer of the dentate gyrus; ME: median eminence. (*): p < 0.05; (***): p < 0.0001.

Quantification of the mRNA signal revealed that the SVZ/RMS and the 3V had the highest levels of intensity, followed by the cc and the SLM, respectively (Fig. 3A–E). Analysis of emulsion-coated slides not only confirmed high intensity signals in these regions but also revealed scattered positive cells in the septum and the caudate putamen (CPu), as well as a few scattered positive cells in the cortex. Again, no signal could be detected in the cerebellum, brainstem, thalamus and the rest of the hypothalamus. Notably, no sex differences were observed in either distribution or intensity of KAT II expression. Thus, in the adult rat brain, KAT II mRNA is expressed in a region-specific manner, with a previously unrecognized and remarkably high expression in the brain’s germinal zones and the cc.

Immunohistochemistry confirms high expression of KAT II in the SVZ, corpus callosum and tanycytes of the 3rd ventricle

Immunohistochemical visualization of KAT II was carried out with the same antibody that was used in the study of Guidetti et al. (2007). However, in contrast to the earlier work where the antibody was diluted 1:30,000 prior to use, we now employed higher antibody dilutions (1:100,000, 1:300,000 and 1:1,000,000). The staining pattern seen at a dilution of 1:100,000 confirmed the results of our previous work (Guidetti et al. 2007), showing specific staining of astrocyte-like KAT II-positive cells in both gray and white matter throughout the brain (Fig. 4). Guided by the results of the mRNA expression data (see above), we next focused specifically on the SVZ, the RMS and tanycytes at the base of the 3V where the high intensity of staining suggested saturation of the reactants (Fig. 4A). After antibody dilution to 1:300,000, cellular staining was lost in the striatum and cerebral cortex, but remained strong in astrocytes of the SVZ, RMS, cc, and SLM, as well as in tanycytes at the base of the 3V (Fig. 4B). At a dilution of 1:1,000,000, KAT II-positive cells were found only in the SVZ and in tanycytes (Fig. 4C). Thus, these differential immunocytochemical KAT II signals paralleled the regionally heterogeneous KAT II mRNA expression data described above.

Figure 4.

Figure 4

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Digital microscopic images of sections showing immunoreactivity against KAT II incubated 48–72 h with primary antibody dilutions of 1:100,000 (1/100K), 1:300,000 (1/300K) or 1:1,000,000 (1/1M). SVZ: subventricular zone; 3V: 3rd ventricle; cc: corpus callosum; Hipp: hippocampus; CPu: caudate putamen; Ctx: cerebral cortex. Scale bar applied to all panels.

KAT-II immunoreactivity in astrocytes of the SVZ/RMS

KAT II-expressing cells in the SVZ and RMS were studied further by double immunohistochemistry using antibodies against the astrocyte marker GFAP or against doublecortin (DCX), a marker of neuroblasts. Extensive co-expression of KAT II with GFAP was found in the SVZ and the RMS (Fig. 5A–D). Co-localization of expression in the fine cellular processes in these regions was assessed using super resolution Airyscan imaging. Orthogonal x–z and y–z projections confirmed co-localization of KAT II and GFAP (Fig. 5D). Co-localization of the two proteins in the cc and the hippocampus, as described in Guidetti et al. (2007), was also confirmed by confocal microscopy.

Figure 5.

Figure 5

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Digital images of double fluorescent immunohistochemistry of KAT II (red) and glial fibrillary acidic protein (GFAP, green) in sagittal sections of the brain of adult male rats. A: low power micrograph at the level of the subventricular zone (SVZ) and the rostral migratory stream (RMS) indicates significant overlap of the two signals (yellow). Co-localization of KAT II and GFAP in the SVZ and the RMS, respectively, is shown at the cellular level in B and C, and with high resolution imaging in D. Orthogonal projections of z-stack images confirm co-localization of KAT II and GFAP in the RMS (D). cc: corpus callosum; CPu: caudate putamen; Ctx: cerebral cortex; LV: lateral ventricle; OB: olfactory bulb

Close anatomical relationships between KAT II- and DCX-positive cells were found in the SVZ and the RMS (Fig. 6A–C). Super resolution imaging revealed that, while there is a significant degree of cellular contact between DCX-positive cells and KAT II-positive cells, these proteins are expressed in different processes and do not appear to be co-expressed in the same cell (Fig. 6D). Similarly, examination of KAT II immunoreactivity in the subgranular zone (SGZ) of the dentate gyrus (DG), a region known to express DCX, revealed few scattered cells with GFAP-positive radial processes (Fig. 7C, D), but no DCX co-labeling (Fig. 7D, E).

Figure 6.

Figure 6

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Digital images of double fluorescent immunohistochemistry of KAT II (red) and doublecortin (DCX, green) in sagittal sections of the brain of adult male rats. A: low power micrographs at the level of the subventricular zone (SVZ) and the rostral migratory stream (RMS) illustrate the close apposition of the two signals. B and C: higher magnification of the areas denoted in A indicates a close association of KAT II with DCX but not co-localization. This is shown at the cellular level with high-resolution imaging (D). Orthogonal projections of z-stack images confirm that KAT II and DCX signals are not present in the same cellular structures. cc: corpus callosum; CPu: caudate putamen; Ctx: cerebral cortex; OB: olfactory bulb.

Figure 7.

Figure 7

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KAT II immunolabeling in the hippocampal formation. A) Low magnification image of KAT II Ni-DAB labeling (1:100K) in the hippocampus. Densely labeled KAT II positive cells are located in the stratum lacunosum moleculare (LM). Scale bar = 500 μm. B) High magnification image of the area outlined in (A) showing KAT II positive cells in the dentate gyrus, restricted primarily to the subgranular zone (SGZ). Scale bar = 50 μm. C) Low magnification (10×) image of double immunofluorescence of KAT II (red) and GFAP (green). Scale bar = 100 μm. D) High magnification (400×) image of area outlined in (C) showing KAT II positive cells associated with GFAP positive astrocytes (arrows) and radial glial cells (arrowheads). Scale bar = 10 μm. E) KAT II (red) and doublecortin (DCX, green) immunofluorescence in the dentate gyrus at low magnification. Scale bar = 100 μm. F) High magnification of region in (E) showing that KAT II (arrowheads) and DCX (arrows) are not expressed in the same cells. Scale bars = 10 μm. cc: corpus callosum, GL: granule cell layer; H: hilus; Mol: molecular layer; Py: pyramidal cell layer.

KAT-II immunoreactivity in tanycytes of the 3rd ventricle

Analysis of KAT II immunoreactivity in the hypothalamus revealed a cellular morphology corresponding to tanycytes, with densely stained cell bodies in the ependymal layer of the 3V and long radial processes extending into the hypothalamic parenchyma and median eminence (ME) (Fig. 8). Staining was observed from the rostral to the caudal portions of the floor of the hypothalamus lateral to the 3V, with minimal to weak staining in the ME (Fig. 8A–C). KAT II immunoreactivity was also seen in astrocyte-like cells in the hypothalamic parenchyma, but was comparatively weak with respect to tanycytes. Fluorescent double labeling showed that a proportion of the KAT II signal in tanycyte-like cells co-localized with GFAP (Fig. 8 D–F). The overlap of the KAT II and GFAP signals was seen at the level of the ependymal cell layer and in radial processes (Fig. 8G–I).

Figure 8.

Figure 8

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Expression of KAT II immunoreactivity in tanycytes of the 3rd ventricle (3V). A–C: Nickel DAB immunoreactivity (1:300,000 antibody dilution) along rostrocaudal levels (A: −1.8 mm, B: −2.8 mm and C: −4.3 mm from bregma). D–I: Double fluorescent immunohistochemistry of KAT II (red) and GFAP (green) at the levels of the median eminence (ME, −1.8 mm from bregma) showing co-localization of the signals in tanycytes (arrows).

KAT II mRNA is increased by peripheral LPS administration in the SVZ and SLM

In a first attempt to determine if KAT II mRNA expression in the brain is responsive to inflammatory processes, the expression of this gene was examined 6 h after peripheral administration of 2 mg/kg LPS. Quantification of the mRNA signal revealed an increase in the SVZ (unpaired t-test: t = 5.35; p < 0.001) and SLM of the hippocampus (unpaired t-test: t = 5.98; p = 0.001) (Fig. 9 A–D), without significant changes in the 3V, the cc and the cerebral cortex (Fig. 9E). No differences from saline-treated control animals were observed by film autoradiography in other regions of the brain. To confirm the LPS-induced changes, relative expression was determined by real-time RT-PCR in the hippocampus and in the rest of the brain (excluding the cerebellum and brainstem). An increase in KAT II mRNA was observed in dissected hippocampal tissue (unpaired t-test: t = 4.43; p = 0.01) as well as in the remaining forebrain (unpaired t-test: t = 7.46; p = 0.0001) (Fig. 8F). Results from quantitative PCR confirmed the increase in cerebral KAT II mRNA expression following the immune challenge. Finally, analyses of emulsion-coated slides confirmed that KAT II mRNA in the cerebral cortex was not responsive to LPS challenge (data not shown). These results indicate that increases in KAT II mRNA expression in response to a peripheral LPS challenge are brain region-specific, involving the SVZ and SLM in the hippocampus.

Figure 9.

Figure 9

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Digital images of Nissl stained coronal sections (A, C) and film autoradiographic images of adjacent sections (B, D) hybridized with 35S labeled riboprobes directed against KAT II mRNA at the level of the subventricular zone (SVZ) in rats in control saline (A, B) or after 6 h of LPS injection (C, D) (2mg/kg). E: Quantification of the mRNA signal in different brain regions by film autoradiography. F: Quantification of KAT II mRNA expression by real time RT-PCR in forebrain and hippocampus. 3V: 3rd ventricle; cc: corpus callosum; Hipp/SLM: stratum lacunosum moleculare of the hippocampus; CPu: caudate putamen; Ctx: cerebral cortex. Data are the mean ± SEM of 6 rats per group. (**): p < 0.01; (***): p < 0.001.

DISCUSSION

The present quantitative neuroanatomical analysis of the expression of the gene coding for KAT II revealed that this enzyme is highly expressed in the germinal zones in the adult rat brain. Expression of this gene extends from the most caudal portions of the SVZ through the RMS. Relatively high expression was also found in the floor of the 3V and, to a lesser extent, throughout the cc. This expression pattern, which was fully supported in immunocytochemical studies, not only indicates that KYNA, the product of KAT II, might play different roles in various parts of the brain, but also suggests a previously unrecognized function of KYNA in the adult brain’s germinal zones. Of considerable interest in this context, KAT II expression was up-regulated in some of these neurogenic areas following immune stimulation by systemically applied LPS.

Recent work by Sohn et al. (2015) showed that the SVZ continues to generate astrocytes in the adult brain as a part of a homeostatic mechanism maintaining normal astrocyte populations in the adult cc (Sohn et al. 2015). These newly formed astrocytes are recruited into the cc and the RMS, do not retain immature astrocytic markers, express glutamate transporters and aquaporin-4, and form perivascular contacts. Remarkably, SVZ-derived astrocytes in the adult brain do not populate other adjacent brain structures, including grey matter in the cerebral cortex and the striatum. Notably, the location of these newly formed astrocytes overlaps with the sites of highest expression of KAT II, as determined in the present study both by quantitative in situ hybridization and by immunohistochemistry using serial antibody dilutions. Of interest, KAT II in the SVZ and RMS co-localized with GFAP, but not with DCX, excluding neuroblasts as a source of the enzyme and suggesting that KAT II may constitute a useful marker of early astrocytic differentiation in the adult SVZ.

The cellular composition and function of the cc, and especially its critical role in cortical interhemispheric communication, have been extensively documented (Innocenti et al. 1995; Keshavan et al. 2002; Schulte and Muller-Oehring 2010; Schulte et al. 2010). Of importance in the context of the present studies, the cc contains a high density of astrocytes (Reyes-Haro et al. 2013), which express substantial levels of KAT II. It is therefore tempting to speculate that newly formed KYNA readily liberated into the extracellular compartment (Turski et al. 1989), may serve physiological functions in this white matter region in adulthood. Although α7 nicotinic acetylcholine receptors (α7nACh) (Severance and Yolken 2008), and possibly other targets of the metabolite (Moroni et al. 2012; Stone et al. 2013), are also present in the region, local effects of KYNA are most likely mediated through the inhibition of NMDA receptors, which are present both on myelinated fibers and on cell bodies of oligodendrocytes in the adult cc (Zhang et al. 2013). Notably, oligodendrocytes in the cc also express AMPA receptors, another possible target of endogenous KYNA (Perkins and Stone 1982; Prescott et al. 2006), which may not only be of physiological significance but also account for the vulnerability of these cells to excessive glutamate (Bradl and Lassmann 2010). The heterogeneity and intricate cytoarchitecture of oligodendrocytes in the cc (Bradl and Lassmann 2010; Osanai et al. 2017), as well as the realization that a single oligodendrocyte can myelinate axons that originate from different neurons (Osanai et al. 2017), suggest that locally produced KYNA, through direct influence of these cells, may actively participate in a wide range of functions involving this white matter structure.

The present results may be relevant to the pathophysiology of schizophrenia, since the cognitive deficits seen in patients are increasingly believed to be causally related to white matter abnormalities (Cropley et al. 2017; Eastwood and Harrison 2005; Joshi et al. 2012; Kochunov and Hong 2014). While KYNA in the white matter, as discussed above, may be part of a physiological mechanism under normal conditions, an excess of KYNA, as seen in schizophrenia, may contribute to disease pathology. Thus, chronic infusion of KYNA into the spinal cord of rats results in myelin damage without infiltration of peripheral immune cells or apparent death of oligodendrocytes (Dabrowski et al. 2015). Moreover, high concentrations of KYNA impair the viability of oligodendrocytes in vitro (Langner et al. 2017), further suggesting a potential causal role of KYNA in white matter abnormalities in schizophrenia. It is noteworthy that significant white matter irregularities have also been observed in several animal models that are relevant to the pathophysiology of the disease. In line with the NMDA receptor hypofunction hypothesis of schizophrenia (Coyle et al. 2003; Javitt 2007), such abnormalities are seen, for example, after chronic treatment of adult mice with an NMDA receptor antagonist (Xiu et al. 2015). Of special interest in the present context, Duchatel et al. (2016) reported an increased density of interstitial white matter neurons (IWMN) in the cc in adult offspring of rat dams that had received an injection of polyinosinic:polycytidylic acid (Poly I:C) during pregnancy (Duchatel et al. 2016). This study, which implies that an abnormal function of IWMN is associated with inflammation, not only supports the widely assumed role of maternal immune activation in the etiology of schizophrenia (Brown and Patterson 2011; Meyer and Feldon 2010; Shi et al. 2003), but more generally, is in line with the hypothesis that KYNA-producing cells in the cc affect white matter function (see above). Our finding that the mRNA expression of KAT II in the SVZ increases rapidly following LPS-induced immune activation could turn out to be especially important in this regard since KYNA levels are elevated in the brain of persons with schizophrenia (Sathyasaikumar et al. 2011), and increased brain KYNA levels cause cognitive deficits in adult rats (Alexander et al. 2012; Pocivavsek et al. 2011).

The results of the present study immediately suggest a role of endogenous KYNA in neuronal and glial development. Experimental evidence supporting such a role of KYNA was recently provided by Bagasrawala et al. (2016), who demonstrated that KYNA concentrations in the low nanomolar, i.e. endogenous, range affect the development of human cortical cells in culture (Bagasrawala et al. 2016). Specifically, the investigators showed that KYNA-induced inhibition of NMDA receptors caused a reduction in the number of GABAergic cells and an increased number of astrocytes. As glutamate may trigger these effects in undifferentiated neurons (Balazs 2006), KAT II-containing astrocytes in the SVZ and the RMS may provide a “niche” of protection from this neurotransmitter to ensure migration of newborn neurons before differentiation. In other words, it is tempting to conclude that fluctuating levels of SVZ/RMS-derived KYNA are part of a homeostatic mechanism, which maintains adequate levels of neuronal proliferation and differentiation in the adult brain by locally modulating the function of NMDA, and possibly also α7nACh receptors (Narla et al. 2013). Notably, this concept also applies to DCX-containing cells, which express NMDA receptors (Platel et al. 2010) do not contain KAT II, and are in close contact with KAT II-positive astrocytes (Fig. 6).

A role for KYNA on glial proliferation is also suggested by studies using cell lines in culture. Thus, mouse hypothalamic microglial N11 cells proliferate dose-dependently in the presence of 1 and 10 micromolar KYNA and KYNA also causes proliferation of U-343 human glioblastoma cells, albeit with lower potency and without clear dose-dependence (Di Serio et al. 2005). In contrast, a recent study reported anti-proliferative effects of KYNA in human glioblastoma T98G cells (Walczak et al. 2014). While the cellular specificity of these effects remains to be elaborated further, these studies, taken together, indicate that endogenous KYNA may influence the proliferation, and perhaps the differentiation, of several glial cell types and may therefore affect newly generated neurons.

The high levels of KAT II expression in tanycytes of the 3V further support of a role for KYNA in cell proliferation and/or differentiation in the adult brain. The involvement of these cells in modulating hypothalamic physiology including neuroendocrine function has been extensively studied (Bolborea and Dale 2013; Goodman and Hajihosseini 2015; Hofmann et al. 2017), and the existence of a proliferative zone in the floor of the 3V has been demonstrated in several studies (Rizzoti and Lovell-Badge 2017; Robins et al. 2013; Rojczyk-Golebiewska et al. 2014; Xu et al. 2005). Fate mapping experiments have shown the proliferative properties of the wall of the 3V and the capacity to generate neuronal progenitors that migrate and integrate in the hypothalamic parenchyma (Lee et al. 2012). Importantly, these cells become functional and influence hypothalamic physiology in response to environmental signals (Bolborea and Dale 2013; Lee et al. 2012; Recabal et al. 2017; Rizzoti and Lovell-Badge 2017). Long-term tracing with recombinant transgenes in vivo has revealed that a defined set of tanycytes, mainly GFAP-positive alpha-tanycytes, express markers of neuroprogenitors, display the ability to self-renew and give rise to other tanycyte subtypes (Robins et al. 2013). The pattern of KAT II expression in the region containing alpha-tanycytes, and the observed co-localization of KAT II and GFAP in these cells, suggest that at least a proportion of tanycytes have the potential to generate neurons and glial cells by producing – and hence releasing – KYNA. As tanycytes display unique cellular characteristics, including the ability to sense glucose and to undergo significant morphological changes in response to environmental signals (Goodman and Hajihosseini 2015; Hofmann et al. 2017; Lee et al. 2012; Rizzoti and Lovell-Badge 2017) the effect of KYNA formed by these cells requires careful further examination and may possibly involve mechanisms other than the inhibition of NMDA and/or α7nACh receptors on surrounding cells.

Finally, although the level of expression of KAT II in the SGZ is significantly lower than in the SVZ and 3V, KAT II expression was also found in GFAP-positive radial glial cells in this proliferative region of the hippocampus, further supporting a role of KYNA in neurogenesis. These cells are the putative progenitor cell in this region, undergoing asymmetric cell division to give rise to new neurons and glial cells (Seri et al. 2004; Seri et al. 2001; Steiner et al. 2004). Unlike in the SVZ, however, newborn cells are required to migrate only a short distance, with a majority of neuroblasts remaining within the granule cell layer, while newborn glial cells move into the hilus and the molecular cell layer (Brunne et al. 2010; Steiner et al. 2004). This migration pattern parallels our immunocytochemistry results, showing relatively low KAT II expression in GFAP-positive radial glial cells and high expression and colocalization with GFAP in astrocytes of the SLM, with a lack of KAT II+, DCX positive cells.

In summary, the present study reports high expression of KAT II in astrocytes of the SVZ and RMS as well as the 3V, canonical regions of neuronal and glial proliferation indicating a previously unconsidered role for KYNA in adult brain germinal zones.

HIGHLIGHTS.

  • -

    The enzyme responsible for the majority of kynurenic acid production in the brain is expressed with a region specific pattern.

  • -

    In the adult rat brain, KAT II is expressed with the highest levels in germinal zones including the subventricular zone.

  • -

    KAT II in these regions is specific for astrocytes

  • -

    KAT II mRNA in the subventricular zone is responsive to peripheral immune challenge

Acknowledgments

This work was supported by NIMH grant Silvio O. Conte Centers for Basic Neuroscience or Translational Mental Health Research P50 MH103222. The authors would like to thank the Biological Imaging Core Facility at the National Institutes of Health, Bethesda, MD, for their generous contribution in the use of the super resolution microscope. CS and LHT generated the probes and performed ISH. CNV and SMC did animal treatment and processed the tissue. KJM and GEH performed IHC. JDN, SMC, TCM and LHT performed image and data analyses. LHT, SMC, GEH and RS wrote and edited the manuscript.

Footnotes

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All the authors declare no conflict of interest.

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