Endogenous kynurenate controls the vulnerability of striatal neurons to quinolinate: Implications for Huntington's disease

Experimental Neurology 197 (2006) 31 – 40 www.elsevier.com/locate/yexnr

Regular Article

Endogenous kynurenate controls the vulnerability of striatal neurons to quinolinate: Implications for Huntington’s disease Michael T. Sapko a, Paolo Guidetti a, Ping Yu b,1, Danilo A. Tagle b,1, Roberto Pellicciari c, Robert Schwarcz a,* a Maryland Psychiatric Research Center, University of Maryland School of Medicine, P.O. Box 21247, Baltimore, MD 21228, USA Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institute of Health, Bethesda, MD 20892, USA c Dipartimento di Chimica e Tecnologia del Farmaco, University of Perugia, Perugia, Italy

b

Received 2 June 2005; revised 5 July 2005; accepted 7 July 2005 Available online 15 August 2005

Abstract Excessive activation of NMDA receptors results in excitotoxic nerve cell loss, which is believed to play a critical role in the pathophysiology of Huntington’s disease (HD) and several other catastrophic neurodegenerative diseases. Kynurenic acid (KYNA), a neuroinhibitory tryptophan metabolite, has neuroprotective properties and may serve as an endogenous anti-excitotoxic agent. This hypothesis was tested in the striatum, using mice with a targeted deletion of kynurenine aminotransferase II (KAT II), a major biosynthetic enzyme of KYNA in the mammalian brain. On post-natal day (PND) 14, the striatum of mkat-2 / mice showed a reduction in KYNA levels but contained normal concentrations of the metabolically related neurotoxins 3-hydroxykynurenine and quinolinic acid (QUIN). Intrastriatal injections of QUIN, a NMDA receptor agonist, caused significantly larger lesions in these immature mutant mice than in age-matched wildtype animals. This lesion enlargement was not observed when mkat-2 / mice were acutely pre-treated with the kynurenine 3-hydroxylase inhibitor UPF 648, which counteracted the striatal KYNA deficit. Moreover, no increased vulnerability to QUIN was observed in 2-monthold mkat-2 / mice, which present with normal brain KYNA levels. Intrastriatal injections of the non-NMDA receptor agonist kainate caused similar lesion sizes in both genotypes regardless of age. These results indicate that endogenous KYNA preferentially controls the vulnerability of striatal neurons to QUIN. Our data suggest that timely pharmacological interventions resulting in an up-regulation of brain KYNA levels may benefit patients suffering from HD or other neurodegenerative diseases. D 2005 Elsevier Inc. All rights reserved. Keywords: Excitotoxicity; Kainic acid; Kynurenine 3-hydroxylase; Kynurenines; Neuroprotection; NMDA receptor

Introduction Excessive stimulation of ionotropic excitatory amino acid (EAA) receptors triggers excitotoxicity, a mechanism that is believed to play a role in the pathophysiology of several acute and chronic human brain diseases (Albin and Greenamyre, 1992; Bowling and Beal, 1995; Schwarcz et al., 1984). The intracellular cascade of biochemical events leading to excitotoxic cell death has been * Corresponding author. Fax: +1 410 747 2434. E-mail address: [email protected] (R. Schwarcz). 1 Current addresses: National Cancer Institute (PY) and National Institute of Neurological Disorders and Stroke (DAT). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.07.004

elaborated in considerable detail, and can be exploited for neuroprotective strategies (Choi, 1995; Murphy et al., 1999; Nicholls, 2004). In addition, a number of chemically diverse endogenous factors present in the brain’s extracellular milieu influence EAA receptor function and thereby control the susceptibility of neurons to an excitotoxic insult. Abnormal functioning of any of these endogenous agents therefore constitutes a risk factor in excitotoxic brain disorders. One of these potential pathogens, quinolinic acid (QUIN), a metabolite of the kynurenine pathway of tryptophan degradation (Fig. 1), is a relatively weak yet specific NMDA receptor agonist (Stone and Perkins, 1981). Intrastriatal injection of QUIN in experimental animals creates a local

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A third metabolite of the kynurenine pathway, kynurenic acid (KYNA), has neuroinhibitory properties (Perkins and Stone, 1982). In the high micromolar range, KYNA blocks all ionotropic EAA receptors non-specifically but it inhibits the glycine co-agonist site of the NMDA receptor preferentially at lower concentrations (Parsons et al., 1997). More recently, KYNA was also shown to be a potent inhibitor of the a7 nicotinic acetylcholine receptor (Hilmas et al., 2001). These properties may be jointly responsible for KYNA’s efficacy as an anti-excitotoxic agent (Foster et al., 1984). In the brain, two aminotransferases are responsible for the irreversible transamination of kynurenine to KYNA (Fig. 1). Of these, kynurenine aminotransferase II (KAT II; EC 2.5.1.7.) accounts for the majority of KYNA formation in the rat forebrain (Guidetti et al., 1997). Recently, we generated mice with a genomic disruption of KAT II (mkat-2 / ) and described their biochemical, behavioral, and structural abnormalities (Yu et al., 2004). These mutant mice show a transient reduction in cerebral KYNA levels during the first month of life but present with normal brain KYNA thereafter. In the present study, we have taken advantage of this temporary decrease in brain KYNA to test the hypothesis that endogenous KYNA controls the vulnerability of striatal neurons to excitotoxic insults. Our results, which have been communicated in abstract form (Sapko et al., 2003), demonstrate that QUIN-induced neurotoxicity is selectively enhanced in the striatum of young mkat-2 / mice.

Materials and methods Animals

Fig. 1. The kynurenine pathway of tryptophan degradation. UPF 648 is an inhibitor of kynurenine 3-hydroxylase.

lesion that closely duplicates the neuropathological characteristics of the neostriatum of Huntington’s disease (HD) patients (Beal et al., 1986; Schwarcz et al., 1983). QUINinduced striatal neurodegeneration is qualitatively similar to, but not identical with, lesions caused by an intrastriatal injection of kainic acid, an exogenous non-NMDA receptor agonist that was originally used to create an animal model of HD (Coyle and Schwarcz, 1976). Notably, elevations in brain QUIN, which are observed in the early stages of HD, have been speculatively linked to the pathophysiology of the disease (Guidetti et al., 2004). Another kynurenine pathway metabolite, the free radical generator 3-hydroxykynurenine (3-HK), is toxic to cultured striatal neurons at concentrations close to those found in brain tissue homogenates (Okuda et al., 1996). 3-HK also potentiates striatal QUIN neurotoxicity and is therefore viewed as a potential co-pathogen in HD (Chiarugi et al., 2001; Guidetti and Schwarcz, 1999).

mkat-2 / mice (generated on a 129SvEv background; Yu et al., 2004) and 129SvEVwild-type (WT) mice were bred and housed in an AAALAC-approved animal facility at the University of Maryland School of Medicine. The animals were kept on a 12 h:12 h light/dark cycle and had free access to food and water. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Maryland, Baltimore. Materials dl-3-HK, kainic acid, l-kynurenine (sulfate salt), KYNA, QUIN, and all other biochemicals were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The enantiopure kynurenine 3-hydroxylase inhibitor (+)-(1S,2S)2-(3,4-dichlorobenzoyl)-cyclopropyl-1-carboxylic acid (DBCC) (UPF 648; IC50: 40 nM) (Pellicciari et al., 2003) was synthesized by the Friedel – Craft coupling of odichlorobenzene with (S,S)-cyclopropyl-1,2-dicarboxylic acid monomethyl ester, obtained by using l-menthol as chiral inductor (Pellicciari et al., manuscript in preparation). All other chemicals were purchased from various commercial suppliers and were of the highest available purity.

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UPF 648 was dissolved in a small volume of 1 M NaOH, titrated to approximately neutral pH with 1 M HCl, and brought to the appropriate volume with phosphate-buffered saline (pH 7.4). 3 H-Kynurenine (specific activity: 9.3 Ci/mmol) was custom-synthesized by Amersham Corp. (Arlington Heights, IL, USA). Prior to use, 3H-kynurenine was purified by HPLC as described by Guidetti et al. (1995). Kynurenine aminotransferase assay Total KAT activity was measured as described previously (Guidetti et al., 1997), with slight modifications. Briefly, mice were deeply anesthetized and decapitated, and striata were rapidly dissected out and placed on ice. The tissues were suspended in ultrapure water (1:10, w/v) and sonicated. Incubation was initiated by adding 100 Al of 150 mM Tris –acetate (pH 7.0), containing 2 AM (2.5 nCi) 3 H-kynurenine, 1 mM pyruvate, and 80 AM pyridoxal-5Vphosphate, to 100 Al of the tissue homogenate, and incubation proceeded for 20 h at 37-C. The reaction was terminated by the addition of 20 Al of 50% (w/v) trichloroacetic acid. Subsequently, 1 ml of 0.1 M HCl was added, and denatured protein was removed by centrifugation in a microfuge. One milliliter of the resulting supernatant was applied to a Dowex 50 W H+ cation exchange resin (BioRad, Hercules, CA), washed sequentially with 1 ml of 0.1 M HCl and 1 ml of ultrapure water. 3H-KYNA was then eluted with 2  1 ml of ultrapure water, and radioactivity was quantified by liquid scintillation spectrometry. Measurement of kynurenine pathway metabolites Animals were deeply anesthetized and decapitated, and striata were rapidly dissected out, frozen on dry ice, and either used immediately or stored at 80-C. 3-Hydroxykynurenine and kynurenic acid Tissues were sonicated (1:10, w/v) in ultrapure water. Thirty microliters of 6% perchloric acid were added to 120 Al of the homogenate, and the suspension was thoroughly mixed and centrifuged (10 min, 12,000  g). Using a refrigerated auto-injector, 20 Al of the supernatant was then applied to a reversed-phase HPLC column (HR-80, ESA, Chelmsford, MA) perfused isocratically at 1 ml/min with a mobile phase consisting of 1.5% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM sodium EDTA, and 8.9 mM heptane sulfonic acid. In the eluate, 3HK was detected electrochemically (Coulochem, ESA) at an oxidation potential of +0.2 V (Heyes and Quearry, 1988). Another 500 Al of the original tissue homogenate was thoroughly mixed with 125 Al of 5 N HCl and 500 Al of chloroform, and centrifuged (10 min, 12,000  g). A separate sample received 20 Al of 50 nM KYNA as an internal standard. Two hundred microliters of the supernatant was applied to a HPLC column (HR-80, ESA), perfused

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isocratically at 1 ml/min with a mobile phase consisting of 5% acetonitrile, 250 mM zinc acetate, and 50 mM sodium acetate (pH 6.2). In the eluate, KYNA was detected fluorimetrically (Wu et al., 1992) using an excitation wavelength of 344 nm and an emission wavelength of 398 nm. Kynurenine and quinolinic acid Tissues were sonicated (1:25, w/v) in ultrapure water. Fifty microliters of the homogenate was combined with 50 Al of an internal standard solution containing 100 nM of 3,5pyridinedicarboxylic acid and 200 nM homophenylalanine. After addition of 25 Al of 5 N HCl and 100 Al of chloroform, the samples were thoroughly mixed and centrifuged (10 min, 12,000  g). Fifty microliters of 62.5 mM tert-butylammonium hydrogen sulfate (TBAS) was then added to 100 Al of the supernatant. The mixture was lyophilized and resuspended in 25 Al of methylene chloride containing 7.5% diisopropylethylamine and 3% pentafluorobenzylbromide (PFB). The samples were incubated in sealed tubes for 15 min at 60-C. Fifty microliters of decane and 750 Al of ultrapure water were then added, and the samples were vigorously mixed and centrifuged (15 min, 7000  g). One microliter of the organic phase was injected into a gas chromatograph/mass spectrometer (GC/MS). A Trace GC gas chromatograph was used in series with a Trace MS quadrupole mass spectrometer (ThermoFinnigan, West Palm Beach, FL). Chromatographic separation was achieved using a 30-m DB-5 ms capillary column (internal diameter: 0.25 mm, film thickness: 0.25 Am; Alltech, Deerfield, IL) and helium as the carrier gas. A split/splitless injection port (1 Al injection volume) was used with a port temperature of 228-C. The temperature was programmed as follows: 155-C for 1.25 min, 40-C/min to 270-C, 10-C/min to 320-C, 1 min at 320-C. Analysis was performed using electron capture negative-ion chemical ionization mass spectrometry with methane as the reagent gas. The ion source temperature was 250-C, and selected ion monitoring analysis was performed by recording the signals of characteristic (M-PFB) ions. For QUIN and kynurenine, the characteristic (M-PFB) ions are di-PFB derivatives, with m/z of 346 and 387, respectively (Naritsin et al., 1995). Intrastriatal injections Fourteen-day (PND 14)- and 2-month-old mice were anesthetized with i.p. Avertin (14 days: 0.3 mg/g; 2 months: 0.8 mg/g) and mounted in a David Kopf stereotaxic apparatus (Tujunga, CA). An incision in the scalp was made along the midline, and the skin was retracted. The tip of a 33-gauge injection cannula was centered on the midline at the level of bregma and then moved laterally (PND 14: 1.7 mm; 2 months: 2.0 mm) and rostrally (PND 14: 0.5 mm; 2 months: 1.0 mm). Through a small burr hole, the needle was then lowered to the center of the striatum (PND 14: 2.5 mm; 2 months: 3.0 mm below dura). QUIN (7.5 – 45 nmoles) or

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kainate (0.5 – 5 nmoles) (solutions adjusted to pH 7.4 using phosphate-buffered saline) were infused (0.5 Al over 5 min) using a CMA/100 microinjection pump (Carnegie Medicin, Stockholm, Sweden). The animals remained in the stereotaxic frame for an additional 2.5 min to minimize retrograde leakage. Animals were killed 4 days later, and their brains were quickly removed, frozen on dry ice, and stored at 80-C until processed.

between wild-type and mutant mice were observed during the first week of post-natal life. In agreement with previous results (Yu et al., 2004), mkat-2 / mice exhibited increased locomotor activity by PND 14, but the hyperactive behavior subsided after the first month, and no obvious phenotypic differences were noted in animals 2 months of age and older (data not shown). KAT activity

Histology Thirty micrometer tissue sections were cut coronally through the entire rostro-caudal extent of the striatum using a cryostat (Zeiss, Thornwood, NY). Every fourth section was mounted on a poly-l-lysine-coated slide for cytochrome oxidase staining, and consecutive sections were used for Nissl staining. Cytochrome oxidase staining was performed essentially as described by Poeggeler et al. (1998). Briefly, the tissue sections were warmed to room temperature and incubated for 75 min in 100 ml of 0.1 M HEPES buffer, pH 7.5, containing 22.4 mg cytochrome c, 115 mg diaminobenzadine, 4.5 g sucrose, and 12.5 ml of a 1% nickel ammonium sulfate solution. The slides were then post-fixed in neutral buffered formaldehyde, dehydrated in graded alcohols, and cover-slipped with Permount. Nissl-staining was performed according to standard procedures using thionine as the chromogen. Lesion volume quantification Quantification of lesion volume was performed blindly using cytochrome oxidase-stained tissue sections and a video-based image analysis system with Loats Image Analyzer software (Silver Spring, MD). Area measurements were multiplied by the intersection distance (120 Am) and summed to determine the lesion volume. Lesions were verified in adjacent Nissl-stained sections.

0.18 T 0.03 pmol KYNA/h/mg tissue was formed in striatal tissue homogenates from PND 14 wild-type mice, compared to 0.10 T 0.01 pmol/h/mg tissue in age-matched mkat-2 / mice, i.e., enzyme activity was reduced by 42% in the mutant animals (Fig. 2; P < 0.001, F = 12.15, oneway ANOVA). No difference in striatal enzyme activity was observed between 2-month-old wild-type and mkat-2 / mice (0.23 T 0.00 and 0.20 T 0.01 pmol KYNA/h/mg tissue, respectively; P > 0.05). This suggests that KAT II contributes substantially to total striatal KAT activity in PND 14 but not in 2-month-old mice. Striatal kynurenine, 3-HK, QUIN, and KYNA levels and effect of UPF 648 The tissue levels of four kynurenine pathway metabolites were compared in the striata of PND 14- and 2-monthold mice of the two genotypes. Paralleling the decrease in KAT activity (see above), endogenous KYNA was 45% lower in mkat-2 / than in wild-type mice (1.6 T 0.2 and 3.0 T 0.2 fmol/mg tissue, respectively; P < 0.001, F = 42.53, two-way ANOVA). No differences in striatal KYNA levels were observed between genotypes in 2-month-old animals (Fig. 3). Striatal concentrations of kynurenine, 3-HK and QUIN were not significantly different between genotypes at either PND 14 or 2 months of age (Fig. 3).

Statistics An unpaired Student’s t test was used for two-group comparisons. One-way or two-way analysis of variance (ANOVA) with Bonferroni’s post-hoc analysis was used to compare three or more groups. In cases where large dose ranges were tested, the logarithm of each data point was analyzed by ANOVA in order to normalize the variance. A P value of <0.05 was considered significant in all analyses.

Results mkat-2

/

mice

As described (Yu et al., 2004), mkat-2 / mice bred normally and had a normal life span. No differences

Fig. 2. Striatal KAT activity in wild-type (open bars) and mkat-2 / (solid bars) mice of different ages. Data are from four mice per group and are expressed as the mean T SEM. *P < 0.05 vs. wild-type (one-way ANOVA with Bonferroni’s post-hoc analysis).

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Fig. 3. Kynurenine pathway metabolites in the striatum of wild-type (WT) and mkat-2 / (KO) mice of different ages: effect of acute treatment with UPF 648. Data (mean T SEM) are from 5 to 7 mice per group. Kynurenine, KYNA, 3-HK, and QUIN were measured in the same striatal tissue as described in the text. UPF 648 (30 mg/kg, i.p.) was administered 2 h prior to sacrifice. *P < 0.05 vs. drug-naive; .P < 0.05 vs. WT (two-way ANOVA with Bonferroni’s post-hoc analysis).

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shown in Fig. 5A, the dose-effect curve was shifted to the left in PND 14 mkat-2 / mice, i.e., the striatal lesion size was larger in mutant than in wild-type mice (Figs. 4 and 5A) (genotype, P < 0.001, F = 51.07; dose, P < 0.001, F = 33.12). Notably, the difference in lesion volume was not due to differences in total striatal volume in the two genotypes (7.11 T 0.06 mm3 in wild-type and 7.20 T 0.12 mm3 in mkat-2 / mice; n = 4 per group, P = 0.44, t = 0.829, df = 6, Student’s t test). In agreement with studies in rats (Foster et al., 1983), QUIN was more potent in 2-month-old than in PND 14 wild-type mice, with 15–30 nmoles resulting in the destruction of approximately half of the striatum. In these animals, QUIN was equally neurotoxic in mkat-2 / and wild-type mice over a broad dose range (Fig. 5B) (genotype, P = 0.77, F = 0.08; dose, P < 0.001, F = 78.12). As in the younger animals, total striatal volumes in 2-month-old mice did not differ by genotype (wild-type: 8.22 T 0.32 mm3; mkat-2 / : 8.53 T 0.20 mm3; n = 4 per group, P = 0.50, t = 0.711, df = 6, Student’s t test). Fig. 4. Pseudocolor images of unilateral excitotoxic lesions caused by a focal intrastriatal injection of 25 nmoles QUIN in PND 14 mice. Representative examples of lesions, delineated by manually drawn black lines, are illustrated for wild-type (A) and mkat-2 / (B) mice. Lesion size was analyzed as described in the text.

Kainate-induced striatal lesions To determine if mkat-2 / mice show increased vulnerability to a non-NMDA receptor agonist, separate animals

Two hours after the administration of the kynurenine hydroxylase inhibitor UPF 648 (30 mg/kg, i.p.), striatal kynurenine and KYNA levels were elevated in all test animals. As illustrated in Fig. 3, UPF 648 raised kynurenine levels 4.3- and 7.4-fold in PND 14 wild-type and mkat-2 / mice, respectively (each P < 0.001 vs. control, F = 138.45). In 2-month-old mice, the inhibitor increased striatal kynurenine levels 5.2- and 14-fold, respectively, in wildtype and mutant animals (each P < 0.001 vs. control, F = 477.07). After UPF 648 treatment, KYNA levels increased from 3.0 T 0.3 to 43.0 T 6.3 pmol/mg tissue (i.e. 14.5-fold) in wild-type mice, and from 1.6 T 0.6 to 16.5 T 1.8 pmol/mg tissue (i.e. 10-fold) in mkat-2 / mice (Fig. 3) (each P < 0.001 vs. control, F = 435.93). At 2 months of age, UPF 648 raised striatal KYNA levels from 3.3 T 0.9 to 30.8 T 8.9 pmol/mg tissue (i.e. 9.2-fold) in wild-type mice, and from 2.8 T 1.0 to 33.9 T 4.5 pmol/mg tissue (i.e. 12.2-fold) in mkat-2 / mice (each P < 0.001 vs. control, F = 97.15). Neither 3-HK nor QUIN levels were significantly altered by UPF 648 in PND 14- (3-HK: P = 0.78, F = 0.08; QUIN: P = 0.43, F = 0.64) or 2-month-old (3-HK: P = 0.87, F = 0.03; QUIN: P = 0.84, F = 0.04) mice of either genotype (Fig. 3). QUIN-induced striatal lesions Focal, intrastriatal infusions of QUIN resulted in dosedependent lesions, which were readily quantifiable using cytochrome oxidase-stained tissue sections (Fig. 4). As

Fig. 5. Dose-dependency of QUIN-induced striatal lesions in PND 14 (A) and 2-month-old (B) wild-type (open bars) and mkat-2 / (solid bars) mice. QUIN was infused over 5 min in a volume of 0.5 Al. Lesion volume was determined by image analysis of cytochrome oxidase-stained tissue sections as described in the text. Data are from 5 to 13 animals per group and are expressed as the mean T SEM. *P < 0.05 vs. wild-type controls (two-way ANOVA with Bonferroni’s post-hoc analysis).

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received intrastriatal injections of kainate. Intrastriatal injections of 0.5 – 2.0 nmoles of this potent excitotoxin in either PND 14 or 2-month-old mice of either genotype caused quantitatively very similar, dose-dependent lesions (Fig. 6) (PND 14, genotype, P = 0.97, F = 0.00; dose, P < 0.001, F = 30.83; 2 months, genotype, P = 0.86, F = 0.03; dose, P < 0.001, F = 36.73). QUIN-induced striatal lesions in PND 14 mutant mice: attenuation by UPF 648 The final study was designed to test the hypothesis that increased vulnerability to QUIN in PND 14 mkat-2 / mice is due to a deficit in brain KYNA in these animals. To this end, brain KYNA levels were acutely raised in mutant mice by administering 30 mg/kg UPF 648 2 h prior to the QUIN injection. As described above (cf. Fig. 3), this treatment raised KYNA in the brain of mutant mice beyond wild-type levels. As shown in Fig. 7, UPF 648 treatment reduced QUIN toxicity in mkat-2 / mouse striatum to wild-type levels. The same treatment failed to affect the size of QUINinduced striatal lesions in PND 14 wild-type mice (geno-

Fig. 7. Effect of UPF 648 on QUIN-induced striatal lesions in PND 14 wild-type (open bar) and mkat-2 / (solid bar) mice. UPF 648 (30 mg/kg, i.p.) was administered 2 h prior to an intrastriatal injection of QUIN (25 nmoles/0.5 Al). UPF 648 (hatched bars) reduced the lesion volume in mkat-2 / but not in wild-type mice. Data are from 13 to 18 animals per group and are expressed as the mean T SEM. *P < 0.05 (two-way ANOVA analysis with Bonferroni’s post-hoc test).

type, P = 0.002, F = 11.00; treatment, P = 0.06, F = 3.72; genotype  treatment, P = 0.03, F = 5.22).

Discussion

Fig. 6. Dose-dependency of kainate-induced striatal lesions in PND 14 (A) and 2-month-old (B) wild-type (open bars) and mkat-2 / (solid bars) mice. Kainate was infused over 5 min in a volume of 0.5 Al. Lesion volume was determined by image analysis of cytochrome oxidase-stained tissue sections as described in the text. Data are from 4 to 6 animals per group and are expressed as the mean T SEM. Two-way ANOVA revealed no significant genotype differences.

The present study demonstrated that young mkat-2 / mice, which produce and contain significantly less KYNA than age-matched wild-type animals, are disproportionately susceptible to an intrastriatal injection of QUIN. No difference in neuronal vulnerability between genotypes was noted in 2-month-old mice, i.e., once brain KYNA levels in mutant animals had normalized. A causal link between reduced endogenous KYNA and enhanced neuronal vulnerability was further supported by the fact that the potentiation in lesion size was not observed when striatal KYNA levels in PND 14 mkat-2 / mice were raised pharmacologically using UPF 648. Finally, the finding that striatal kainate toxicity was not affected by genotype in PND 14 mice demonstrated that the enhanced neuronal vulnerability described here was NMDA receptor-specific and did not extend to all EAA receptors. Previous studies with mkat-2 / mice have shown that KAT II is an important determinant of brain KYNA formation in the immature mouse brain but that its role diminishes as the animal ages (Alkondon et al., 2004; Yu et al., 2004). In this regard, the mouse brain differs qualitatively from the rat brain, where KAT II remains the dominant KYNA-producing enzyme in adulthood (Guidetti et al., 1997). Due to this age-dependent role of KAT II in the normal mouse brain, mkat-2 / mice provided a fortuitous opportunity to test the functional significance of reduced brain KYNA levels simply by contrasting immature and adult animals. This approach was also facilitated by the fact that the brain tissue levels of 3-HK and QUIN, the other two neuroactive kynurenine pathway metabolites, were not compromised in mutant mice.

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PND 14 mkat-2 / mice were disproportionately susceptible to an intrastriatal infusion of QUIN, and the enlarged lesions were not seen in adult mutant animals, i.e., at an age when cerebral KYNA levels had essentially normalized. To test whether a causal relationship existed between diminished endogenous KYNA and increased neuronal vulnerability, the KYNA deficit in young mkat-2 / mice was acutely reversed using the kynurenine 3-hydroxylase inhibitor UPF 648 as a pharmacological tool. This treatment counteracted the enhanced vulnerability seen in mutant mice. Notably, and in line with the properties of other kynurenine 3-hydroxylase inhibitors (Chiarugi and Moroni, 1999), this effect of UPF 648 was not due to a reduction in the levels of the toxic metabolites 3-HK and QUIN. Our results therefore support the idea that a decline in the brain levels of KYNA enhances the susceptibility to QUIN toxicity in the striatum in vivo, and that this effect can be abolished by pharmacological up-regulation of KYNA. In other words, our data provide experimental evidence for a functional control of striatal QUIN toxicity by endogenous KYNA. Importantly, the reduced KYNA levels in PND 14 mkat-2 / mice failed to potentiate the excitotoxic effects of the non-NMDA receptor agonist kainic acid. This is in agreement with the preferential ability of exogenously applied KYNA to inhibit QUIN-induced neurodegeneration (Foster et al., 1984) and indicates that non-NMDA receptors are not affected by the removal of endogenous KYNA. Experiments in rats in which striatal KYNA levels were acutely reduced by a peripheral injection of dopaminergic agents such as d-amphetamine (Poeggeler et al., 1998; Rassoulpour et al., 1998) support the proposed role of KYNA as a gatekeeper of striatal QUIN neurotoxicity. These treatments cause a rapid and transient 30– 50% decrease in brain KYNA levels, and QUIN-induced excitotoxicity is potentiated if—and only if—the toxin is applied at a time when tissue KYNA content is low. QUIN reverts to its original potency a few hours later, i.e. once KYNA levels have returned to normal. As in the present study, these pharmacologically generated KYNA reductions do not influence striatal kainate toxicity, and the lesion enlargement is prevented by pre-treatment with kynurenine 3-hydroxylase inhibitors (B. Poeggeler, A. Rassoulpour and R. Schwarcz, unpublished observations). Taken together, these studies suggest that endogenous KYNA normally occupies and functionally inhibits a receptor or recognition site that mediates the neurotoxic actions of QUIN. Since the levels of KYNA in the rodent brain are in the low to mid-nanomolar range (Moroni et al., 1988), the NMDA receptor (IC50 of KYNA at the glycine co-agonist site: ¨10 AM) (Parsons et al., 1997) is an improbable primary target. More likely, endogenous KYNA preferentially inhibits the a7 nicotinic acetylcholine receptor (Hilmas et al., 2001), which, in fact, is up-regulated in the brain of immature mkat-2 / mice (Alkondon et al., 2004). However, it is also possible that another, as yet unidentified, high affinity site recognizes KYNA specifically, and that

this novel site plays a critical role in cerebral KYNA function (Schwarcz et al., 1999). Ongoing work in our laboratory is designed to further delineate and characterize the natural targets of KYNA in the brain and to evaluate the possibility that the free radical-scavenging properties of KYNA, too, play a functional role in the brain (Giorgini et al., 2005; Hardeland et al., 1999). Eventually, these studies should not only explain the complex neurophysiological and pharmacological properties of KYNA but should also clarify the mechanism(s) by which the relatively modest KYNA reductions seen in PND 14 mkat-2 / mice (Fig. 3) and in d-amphetaminetreated rats (Poeggeler et al., 1998; Rassoulpour et al., 1998) enhance QUIN neurotoxicity. The present study also addressed a largely unresolved issue in kynurenine neurobiology, namely the respective roles of kynurenine aminotransferases in brain KYNA synthesis in vivo. In agreement with previous studies (Alkondon et al., 2004; Yu et al., 2004), the targeted genetic deletion of KAT II caused an approximately 50% decline in brain KYNA in immature mutant animals, and cerebral KYNA levels normalized in adult mkat-2 / mice. These results demonstrated that KATs other than KAT II, such as KAT I or mitochondrial glutamic acid oxaloacid transaminase (Amori et al., 2005), participate in KYNA formation in the mouse brain, and that the respective roles of enzymes other than KAT II increase with age. In the present study, systemic administration of the kynurenine 3-hydroxylase inhibitor UPF 648 raised brain kynurenine and KYNA levels in both mutant and wild-type animals. This effect was less pronounced in PND 14 mkat-2 / than in wild-type mice of the same age, supporting the idea that KAT II is important for cerebral KYNA synthesis but that alternative enzymes can catalyze the irreversible transamination of kynurenine in the absence of KAT II. Notably, the relative contribution of KAT II and other aminotransferases to KYNA formation in vivo is not only age-dependent, as shown here and elsewhere (Ceresoli-Borroni and Schwarcz, 2000; Csillik et al., 2002; Rejdak et al., 2004), but also varies between species (Amori et al., 2005; Guidetti et al., 1997; Kiss et al., 2003; Zarnowski et al., 2004) and under physiological and pathological conditions (Baran et al., 1999; Ceresoli-Borroni et al., 1999; Ceresoli-Borroni and Schwarcz, 2000; Cooper, 2004; Kocki et al., 2003; Luchowska et al., 2005; Saran et al., 2004). Our demonstration that endogenous KYNA controls neuronal vulnerability to QUIN in the striatum could have implications for the early clinical management of HD. Probably as a consequence of abnormal microglial function (Sapp et al., 2001), HD presents with an increased neostriatal QUIN/KYNA ratio, caused by a rise in QUIN levels, in the first stages of the disease (Guidetti et al., 2004). Thus, prolonged treatment with KYNA-reducing agents such as dopaminergic agonists (Poeggeler et al., 1998; Rassoulpour et al., 1998) could conceivably exacerbate the pathophysiological process, whereas elevations of

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brain KYNA, for example by kynurenine administration or kynurenine 3-hydroxylase inhibition, could be beneficial (Cozzi et al., 1999; Harris et al., 1998; Nozaki and Beal, 1992; Speciale et al., 1996). In other situations, however, a reduction in brain KYNA levels could be physiologically and therapeutically desirable (Coyle and Tsai, 2004; Nakazawa et al., 2004), and a chronic increase in cerebral KYNA levels may be deleterious by causing side effects associated with malfunctioning a7 nicotinic and NMDA receptors (Levin et al., 2005; Olney et al., 1991). Pharmacological agents capable of specifically targeting kynurenine pathway enzymes in vivo are now available (Schwarcz and Pellicciari, 2002) and can be used to further explore the functional consequences of altering the balance between QUIN and KYNA metabolism in the brain acutely and chronically.

Acknowledgments This work was conducted in partial fulfillment of the PhD dissertation of MTS. The study was supported by USPHS grants HD 16596 and NS 42487.

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