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Immunohistochemical localization of d-serine dehydratase in chicken tissues Yoshihiro Nishimura a , Hiroyuki Tanaka b,∗ , Tetsuo Ishida b , Shinji Imai a , Yoshitaka Matsusue a , Yasutoshi Agata b , Kihachiro Horiike b a b
Department of Orthopaedic Surgery, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan
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Article history: Received 13 November 2013 Received in revised form 21 December 2013 Accepted 22 December 2013 Keywords: d-Serine dehydratase d-Serine Immunohistochemistry Chicken brain Astrocyte
a b s t r a c t Chicken d-serine dehydratase (DSD) degrades d-serine to pyruvate and ammonia. The enzyme requires both pyridoxal 5 -phosphate and Zn2+ for its activity. d-Serine is a physiological coagonist that regulates the activity of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate. We have recently found in chickens that d-serine is degraded only by DSD in the brain, whereas it is also degraded to 3hydroxypyruvate by d-amino acid oxidase (DAO) in the kidney and liver. In mammalian brains, d-serine is degraded only by DAO. It has not been clarified why chickens selectively use DSD for the control of d-serine concentrations in the brain. In the present study, we measured DSD activity in chicken tissues, and examined the cellular localization of DSD using a specific anti-chicken DSD antibody. The highest activity was found in kidney. Skeletal muscles and heart showed no activity. In chicken brain, cerebellum showed about 6-fold-higher activity (1.1 ± 0.3 U/g protein) than cerebrum (0.19 ± 0.03 U/g protein). At the cellular level DSD was demonstrated in proximal tubule cells of the kidney, in hepatocytes, in Bergmann-glia cells of the cerebellum and in astrocytes. The finding of DSD in glial cells seems to be important because d-serine is involved in NMDAR-dependent brain functions. © 2014 Elsevier GmbH. All rights reserved.
Introduction Chicken d-serine dehydratase (DSD) is one of the founding members of the recently discovered pyridoxal 5 -phosphate (PLP)dependent enzyme family that requires a catalytic Zn2+ ion in the active site (Ito et al., 2008; Tanaka et al., 2011). DSD catalyzes the dehydration of d-serine to produce pyruvate and ammonia. When O2 molecules are available, d-serine can also be degraded by damino acid oxidase (DAO), a flavin-dependent enzyme, through oxidative deamination. In the case of DAO, 3-hydroxypyruvate, ammonia, and hydrogen peroxide are produced. We have developed a method to measure DSD- and DAO-dependent degradation of d-serine simultaneously by quantifying the respective products of pyruvate and 3-hydroxypyruvate (Tanaka et al., 2007). By using this method, we have found that in chicken brain d-serine is
Abbreviations: DAB, 3,3 -diaminobenzidine; DAO, d-amino acid oxidase; DSD, d-serine dehydratase; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; NMDAR, N-methyl-d-aspartate receptor; PBS, phosphate-buffered saline; PLP, pyridoxal 5 -phosphate. ∗ Corresponding author. E-mail address: [email protected] (H. Tanaka).
degraded only by DSD, whereas in chicken kidney and liver, both DSD and DAO are responsible for d-serine degradation. d-Serine and glycine are physiological allosteric activators of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate (Schell, 2004; Wolosker et al., 2008; Papouin et al., 2012). Therefore, the control of regional concentrations of d-serine in the central nervous system is important. In mammalian brains, lower levels of d-serine are found where DAO activity is higher (Nagata et al., 1994; Horiike et al., 2001) suggesting the importance of DAO in the regulation of NMDAR activities (Pollegioni and Sacchi, 2010). Because mammals lack DSD, DAO is the only enzyme to degrade d-serine in mammals. However, avian and many other non-mammalian vertebrate species have DSD in addition to DAO (Tanaka et al., 2009). DAO can degrade various d-amino acids and glycine (Dixon and Kleppe, 1965; Marchi and Johnston, 1969). In fact, d-serine is not the best substrate for DAO. However, it is the best substrate for DSD, which shows very narrow substrate specificity (Tanaka et al., 2008). These different properties between DAO and DSD seem to have implications for the loss of DSD in mammals and the exclusive expression of DSD in chicken brains. However, it has not been clarified why mammals have lost DSD and why avian species utilize only DSD in the brain. To elucidate the physiological role of DSD further, it is important not only to understand its enzymatic properties (Tanaka et al., 2008,
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2011; Ito et al., 2012) but also to reveal its cellular localization in various tissues. We have developed a specific polyclonal antibody against chicken DSD (Tanaka et al., 2008). In the present study, using this antibody, we identified DSD-expressing cells in various chicken tissues. Materials and methods Tissue collection Small organ parts (1–5 g) were removed from four adult male chickens (White Leghorn) killed at a local slaughterhouse. Blocks 5 mm3 were made from the individual parts and immediately fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 4 h at room temperature. The remaining portions were immediately frozen in liquid nitrogen, and then stored at −80 ◦ C until use. Assay of DSD activity Each chicken tissue (1–2 g) was homogenized on ice with 3 volumes of 10 mM potassium phosphate, pH 7.2, containing 50 M pyridoxal 5 -phosphate (PLP), using a glass homogenizer. The standard assay mixture (100 L) contained 50 mM potassium phosphate, pH 7.8, 50 mM d-serine, and 10 M PLP. The reaction was initiated by the addition of a 5-L aliquot of each homogenate, run at 37 ◦ C for 30 min, and then stopped with the addition of 40 L of 12.5% trichloroacetic acid. After the addition of 5 nmol of 2-oxoglutaric acid (5 L of 1 mM stock solution in H2 O) as an internal standard, the mixture was centrifuged at 10,000 × g for 10 min at 4 ◦ C. As we described previously (Tanaka et al., 2007), an aliquot of the supernatant (100 L) was treated with 3-methyl-2benzothiazoline hydrazone hydrochloride to convert 2-oxo-acids into the corresponding azine derivatives, which were then quantified by HPLC on a Cosmosil 3C18 column (Nacalai Tesque, Japan). One unit of DSD activity was defined as the amount of enzyme producing 1 mol of pyruvate per min at 37 ◦ C under the standard assay conditions. Western blot analysis Aliquots of the total tissue homogenates prepared for the DSD assay were incubated in the presence of 2% SDS and 5% 2mercaptoethanol at about 80 ◦ C for 3 min, and then separated by SDS-PAGE using a 12.5% gel (ATTO, Tokyo, Japan). The protein contents of the tissue homogenates were determined using a BioRad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were transferred to Clear Blot Membrane-P using a semidry blotter (ATTO). All subsequent procedures were carried out at room temperature. The membrane was rinsed twice by incubation with 100 mL of PBS containing 0.1% Tween 20 (Buffer A) for 5 min, and then incubated with PBS containing 5% (w/v) skim milk and 0.1% Tween 20 for 1 h. The anti-chicken DSD antibody (3 mg/mL stock) (Tanaka et al., 2008) was diluted 10,000-fold with Buffer A containing 1% bovine serum albumin. The blocked membrane was incubated with the diluted antibody for 1 h. The membrane was washed three times with Buffer A, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (1:5000, diluted with Buffer A) (Amersham ECL Plus Western Blotting Reagent Pack; GE Healthcare, UK) for 1 h at room temperature. After five washes with Buffer A, the antibodies were detected using Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Amersham, UK). The specificity of the anti-chicken DSD antibody was confirmed using negative controls with the diluted antibody (1:10,000) pre-incubated overnight with 2 mg/mL of the DSD purified from chicken kidney (Tanaka et al., 2008) at 4 ◦ C.
Table 1 Distribution of DSD activity in chicken tissues. Tissues
DSD activity (U/g protein)
Kidney Liver Whole brain Cerebrum Cerebellum Brain stem Midbrain Heart Skeletal muscle
4.4 ± 1.4 0.40 ± 0.10 0.40 ± 0.20 0.19 ± 0.03 1.1 ± 0.3 0.40 ± 0.10 0.25 ± 0.10 ND ND
Values are the mean ± SD determined for 4–5 male chickens. No DSD activity was detected for heart and skeletal muscles (ND, not detected).
Immunohistochemistry The fixed tissues were embedded in paraffin wax. Three-mthick paraffin sections were cut and placed on silane-coated glass slides, and then deparaffinized by incubation for 10 min in xylene. The sections were rehydrated through consecutive incubations in 100%, 90%, 80%, and 70% ethanol, and finally washed with PBS. The sections were first incubated in 3% H2 O2 in methanol for 20 min at room temperature to inhibit endogenous peroxidase. Then, the sections were washed with PBS and incubated overnight at 4 ◦ C with the anti-DSD antibody diluted in PBS containing 1% BSA (1:500). After removing unbound antibodies by washing with PBS, the sections were incubated with an HRP-labeled goat antibody against rabbit IgG (Histofine Simple Stain Max PO (Multi); Nichirei Bioscience, Tokyo, Japan) twofold diluted with PBS containing 1% BSA. The sections were washed with PBS, and then peroxidase activity was developed by incubation with 3,3 -diaminobenzidine (DAB) using a Simple Stain DAB Solution (Nichirei Bioscience). Counterstaining was performed with hematoxylin. Then, the sections were dehydrated in ethanol, cleared with xylene, coverslipped with Crystal/Mount (Biomeda, Pittsburgh, PA, USA), and observed with a Nikon Microphoto-FXA (Nikon Co., Tokyo, Japan). The specificity of the immunohistochemical procedures was evaluated by omitting the primary anti-DSD antibody or the secondary antibody in the above procedures. Immunofluorescence microscopy Three-m-thick deparaffinized sections were incubated overnight at 4 ◦ C with the anti-DSD antibody (1:1000). The sections were washed three times with PBS, incubated with
Fig. 1. Western blot detection of DSD in whole tissue homogenates with the antichicken DSD antibody. Aliquots of whole tissue homogenates (containing 20 g of protein) were loaded on a 12.5% SDS-PAGE gel and consequently transferred to a PVDF membrane. The immunoblotting was done using the antibody (1:10,000, final concentration of 0.3 g/mL). The lane of purified DSD is the DSD (2 g) purified from chicken kidney.
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Fig. 2. Immunohistochemical detection of DSD in chicken kidney cortex. (a) The collecting tubules were visualized by Alcian blue staining (Díaz et al., 1996). Strong staining was found only in the renal proximal tubules. (b) Higher magnification shows intense staining in the cytoplasm of the epithelial cells. G, glomerulus; PT, proximal tubule; DT, distal tubule; CT, collecting tubule. Scale bar = 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Alexa-Fluor® 594-labeled donkey anti-rabbit antibodies (1:500, Invitrogen, Eugene, OR, USA) for 1 h at room temperature, and rinsed in PBS three times. The sections were coverslipped using Gel/Mount (Biomeda) and examined with an LSM 510 META (Carl Zeiss Corporation, Oberkochen, Germany). For double staining of DSD and glial fibrillary acidic protein (GFAP) or NeuN (Mullen et al., 1992) or Pax-7 (Shin et al., 2003), the sections were incubated overnight at 4 ◦ C with the anti-DSD antibody (1:1000) and mouse monoclonal anti-GFAP antibody (1:1000; Chemicon, Temecula, CA, USA) or mouse monoclonal anti-NeuN antibody (1:1000; Chemicon) or mouse monoclonal anti-Pax-7 antibody (1:1000; R&D Systems, Minneapolis, MN, USA), respectively, rinsed three
times in PBS, and then incubated with Alexa-Fluor 594-labeled donkey anti-rabbit antibodies (1:500) and Alexa-Fluor® 488labeled donkey anti-mouse IgG antibodies (1:500) for 1 h at room temperature. Results Distribution of DSD in chicken tissues Table 1 summarizes DSD activity in chicken tissues. The highest DSD activity was found in the kidney (4.4 ± 1.4 U/g protein). The liver and brain showed DSD activity of 0.4 U/g protein, about 10%
Fig. 3. Immunohistochemical detection of DSD in chicken liver. (a) Strong cytoplasmic staining was found only in hepatocytes. Higher magnification shows the interlobular bile duct (panel b) and artery (panel c). PV, portal vein; BD, interlobular bile duct; A, interlobular artery. Scale bar = 50 m.
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Fig. 4. DSD immunolocalization in chicken cerebellum. (a) The cytoplasm of the cell body and long processes of Bergmann glial cells was stained for DSD. The astrocytes in the granular layer were stained in the cytoplasm. (b) Confocal microscopy analysis. Merged image of DSD staining (red) and GFAP (green). DSD-positive cells (red) were also positive for GFAP (green) in the granular layer, located immediately beneath the Purkinje cell layer. (c) Merged image of DSD (green) and Pax-7 (red). The nuclei of the DSD-positive cells were stained for Pax-7. PL, Purkinje cell layer; ML, molecular layer; GL, granular layer; P, the soma of the Purkinje cell. Scale bar = 25 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of that in the kidney. In the chicken brain regions, the cerebellum showed about 6-fold-higher DSD activity than the cerebrum. We could not detect DSD activity in the skeletal muscles and heart. Specificity of DSD antibody The specificity and sensitivity of the anti-DSD antibody were examined by Western blot analysis of the total homogenate of chicken tissues. Except for the skeletal muscles and heart, a distinct signal was observed for all the tissues examined only at a molecular weight corresponding to that of purified DSD (40 kDa) (Fig. 1). The strength of the signal correlated well with the tissue levels of DSD activity (Table 1 and Fig. 1). These results demonstrated that the antibody is highly specific for DSD. Immunohistochemical localization of DSD The cellular distribution of DSD in the following tissues was revealed by using DAB as a substrate for the final HRP reaction, which resulted in a brown stain. Tissues were counterstained with hematoxylin (blue stain). No DSD immunoreactivity was detected in parallel incubations of control sections when the anti-DSD antibody was omitted or pre-absorbed with 2 mg/mL of DSD purified from chicken kidney.
Kidney cortex Strong staining for DSD was found only in the renal proximal tubules (Fig. 2a). No significant staining was found in the glomeruli, renal distal tubules, and collecting tubes. Higher magnification revealed non-granular staining in the cytoplasm of the epithelial cells of the proximal tubules (Fig. 2b), consistent with the cytoplasmic localization of DSD (Tanaka et al., 2008). Liver All of the hepatocytes exhibited intense and homogeneous staining in the cytoplasm (Fig. 3a). The intensity was evenly distributed between the different liver lobules. In the interlobular regions, no DSD staining was found for arterioles, bile ducts, and veins (Fig. 3b and c). Cerebellar cortex In the granular layer, granule cells were unstained for DSD (Fig. 4a). A group of cells with many short processes exhibited intense cytosolic staining for DSD. The cytoplasm of the processes of these cells was also stained for DSD, and the DSD-positive processes wrapped the granule cells. Purkinje cells were unstained for DSD (Fig. 4). In the vicinity of the cell body of the Purkinje cells, a small group of cells showed intense staining for DSD in their cell body. These cells had long
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Fig. 5. DSD immunolocalization in chicken cerebrum. (a) Strong cytoplasmic staining was found in the cells with short processes. Higher magnification shows the staining of the fine processes (panel b). (c) Double labeling of DSD (red) and NeuN (green). The nuclei of the DSD-positive cells (red cytoplasm) showed no staining for NeuN. Scale bar = 25 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fiber-like processes running across the molecular layer, and the cytoplasm of the processes was stained for DSD. As shown in Fig. 4b, in the granular layer DSD-positive cells (red) were also positive for GFAP (green), but some GFAP-positive cells were not DSD-positive, indicating that a group of astrocytes express DSD. In contrast, the vicinity of the cell body of the Purkinje cells did not show immunopositivity to GFAP. Chicken Bergmann glial cells in the cerebellum are almost negative to GFAP, unlike their mammalian counterparts (Kálmán et al., 1993). A paired box transcription factor, Pax-7, is known to be localized within the nuclei of the Bergmann glial cells in the adult chicken cerebellum (Shin et al., 2003). Therefore, by double-labeling immunofluorescence microscopy, we examined whether the nucleus of these DSD-positive cells was Pax-7-positive. As shown in Fig. 4c, in the Purkinje cell layer, the cells with a Pax-7-positive nucleus (red in Fig. 4c) also showed intense staining for DSD in the cytoplasm of the cell body (green in Fig. 4c). These results indicate that the DSD-positive cells in the proximity of the Purkinje cell bodies were Bergmann glial cells.
Cerebrum We examined the distribution of DSD in the cerebrum (Fig. 5). DSD immunoreactivity was found in the cytoplasm of a group of cells with many processes. The fine processes of these cells were stained as intensely as the cytoplasm of the cell body (Fig. 5b). As shown in Fig. 5c, by double-labeling immunofluorescence microscopy, the nucleus of the DSD-positive cells (the cells with red cytoplasm in Fig. 5c) showed no staining for the neuronal
marker NeuN (Mullen et al., 1992). These results indicated that the DSD-positive cells are astrocytes, not neurons with NeuN-positive nucleus.
Discussion In this study, we examined biochemically and immunohistochemically the tissue and cellular distribution of DSD. In chicken kidney cortex, only epithelial cells of the proximal tubules were strongly stained for DSD. In chicken liver, only hepatocytes exhibited intense staining for DSD, with the intensity levels evenly distributed throughout the liver lobule. The cellular localization of DSD in these tissues was identical to that reported for DAO in mammals (Horiike et al., 1985), suggesting that DSD plays a role in removing d-serine from the circulating blood. In chicken cerebellar cortex, DSD was found to localize exclusively in Bergmann glial cells and astrocytes. This DSD localization was identical to that found for DAO in the mammalian cerebellum (Horiike et al., 1987). In chicken cerebrum, astrocytes showed intense staining for DSD in the cytoplasm of the cell body and processes. This result is consistent with the fact that the d-serine contents in chicken cerebrum are very low (Nagata et al., 1994). In mammals, DAO is solely responsible for the degradation of dserine in the brain. In the mammalian cerebrum DAO activity is not detected (Horiike et al., 1994), and d-serine concentration is unusually high (Nagata et al., 1994; Hashimoto and Oka, 1997). It is believed that the high levels of d-serine are important for NMDAR-dependent functions in the mammalian cerebrum (Hunt
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and Castillo, 2012; Zorumski and Izumi, 2012). The present results suggest that in chicken cerebrum NMDAR does not need high concentrations of d-serine. In conclusion, we identified DSD-positive cells in the chicken kidney, liver, cerebrum, and cerebellum. Except for the cerebrum, the cellular localization of DSD is identical to that of DAO in mammals. In chicken cerebrum, the astrocytes showed intense staining for DSD, explaining the low d-serine level in this region. Acknowledgements We thank Mr. Takefumi Yamamoto, Central Research Laboratory of our university, for technical advice regarding immunohistochemistry. The present study was supported by a Grant-in-aid for Scientific Research (C) (no. 24590351) (to H.T.) and a Grant-in-aid for Scientific Research (B) (no. 22370026) (to K.K., M.M., and H.T.) from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, as well as a Grant-in-aid (Heisei era24) from Shiga University of Medical Science. References Díaz RC, Montaner B, Pérez TR. Immunochemical study of transforming growth factor- in the kidney of the rat and chicken. Histochem Cell Biol 1996;105:475–8. Dixon M, Kleppe K. d-Amino acid oxidase. II. Specificity, competitive inhibition and reaction sequence. Biochim Biophys Acta 1965;96:357–67. Hashimoto A, Oka T. Free d-Aspartate and d-serine in the mammalian brain and periphery. Prog Neurobiol 1997;52: 325–53. Horiike K, Arai R, Tojo H, Yamano T, Nozaki M, Maeda T. Histochemical staining of cells containing flavoenzyme d-amino acid oxidase based on its enzymatic activity: application of a coupled peroxidation method. Acta Histochem Cytochem 1985;18:539–50. Horiike K, Tojo H, Arai R, Yamano T, Nozaki M, Maeda T. Localization of d-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum. Brain Res Bull 1987;19:587–96. Horiike K, Tojo H, Arai R, Nozaki M, Maeda T. d-Amino-acid oxidase is confined to the lower brain stem and cerebellum in rat brain: regional differentiation of astrocytes. Brain Res 1994;652:297–303. Horiike K, Ishida T, Tanaka H, Arai R. Distribution of d-amino acid oxidase and d-serine in vertebrate brains. J Mol Catal B 2001;12:37–41. Hunt DL, Castillo PE. Synaptic plasticity of NMDA receptors: mechanisms and functional implications. Curr Opin Neurobiol 2012;22:496–508.
Ito T, Hemmi H, Kataoka K, Mukai Y, Yoshimura T. A novel zincdependent d-serine dehydratase from Saccharomyces cerevisiae. Biochem J 2008;409:399–406. Ito T, Koga K, Hemmi H, Yoshimura T. Role of zinc ion for catalytic activity in d-serine dehydratase from Saccharomyces cerevisiae. FEBS J 2012;279:612–24. Kálmán M, Székely AD, Csillag A. Distribution of glial fibrillary acidic protein-immunopositive structures in the brain of the domestic chicken (Gallus domesticus). J Comp Neurol 1993;330: 221–37. Marchi WJ, Johnston GAR. The oxidation of glycine by d-amino acid oxidase in extracts of mammalian central nervous tissue. J Neurochem 1969;16:355–61. Mullen RJ, Buck CR, Smith AM. NeuN, a neuronal specific nuclear protein in vertebrates. Development 1992;116:201–11. Nagata Y, Horiike K, Maeda T. Distribution of free d-serine in vertebrate. Brain Res 1994;634:291–5. Papouin T, Ladépêche L, Ruel J, Sacchi S, Labasque M, Hanini M, et al. Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists. Cell 2012;150: 633–46. Pollegioni L, Sacchi S. Metabolism of the neuromodulator d-serine. Cell Mol Life Sci 2010;67:2387–404. Schell MJ. The N-methyl d-aspartate receptor glycine site and dserine metabolism: an evolutionary perspective. Philos Trans R Soc Lond B 2004;359:943–64. Shin DH, Lee KS, Lee Y, Chang YP, Kim JW, Choi YS, et al. Pax7 immunoreactivity in the post-natal chicken central nervous system. Anat Histol Embryol 2003;32:378–83. Tanaka H, Yamamoto A, Ishida T, Horiike K. Simultaneous measurement of d-serine dehydratase and d-amino acid oxidase activities by the detection of 2-oxo-acid formation with reversephase high-performance liquid chromatography. Anal Biochem 2007;362:83–8. Tanaka H, Yamamoto A, Ishida T, Horiike K. d-Serine dehydratase from chicken kidney: a vertebral homologue of the cryptic enzyme from Burkholderia cepacia. J Biochem 2008;143:49–57. Tanaka H, Yamamoto A, Ishida T, Horiike K. Discovery of d-serine dehydratase in vertebrate and its deficiency in mammals. Tanpakushitsu Kakusan Koso 2009;54:1190–6. Tanaka H, Senda M, Venugopalan N, Yamamoto A, Senda T, Ishida T, et al. Crystal structure of a zinc-dependent d-serine dehydratase from chicken kidney. J Biol Chem 2011;286:27548–58. Wolosker H, Dumin E, Balan L, Foltyn VN. d-Amino acids in the brain: d-serine in neurotransmission and neurodegeneration. FEBS J 2008;275:3514–26. Zorumski CF, Izumi Y. NMDA receptors and metaplasticity: mechanisms and possible roles in neuropsychiatric disorders. Neurosci Biobehav Rev 2012;36:989–1000.
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Immunohistochemical localization of d-serine dehydratase in chicken tissues Yoshihiro Nishimura a , Hiroyuki Tanaka b,∗ , Tetsuo Ishida b , Shinji Imai a , Yoshitaka Matsusue a , Yasutoshi Agata b , Kihachiro Horiike b a b
Department of Orthopaedic Surgery, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Seta, Ohtsu, Shiga 520-2192, Japan
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Article history: Received 13 November 2013 Received in revised form 21 December 2013 Accepted 22 December 2013 Keywords: d-Serine dehydratase d-Serine Immunohistochemistry Chicken brain Astrocyte
a b s t r a c t Chicken d-serine dehydratase (DSD) degrades d-serine to pyruvate and ammonia. The enzyme requires both pyridoxal 5 -phosphate and Zn2+ for its activity. d-Serine is a physiological coagonist that regulates the activity of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate. We have recently found in chickens that d-serine is degraded only by DSD in the brain, whereas it is also degraded to 3hydroxypyruvate by d-amino acid oxidase (DAO) in the kidney and liver. In mammalian brains, d-serine is degraded only by DAO. It has not been clarified why chickens selectively use DSD for the control of d-serine concentrations in the brain. In the present study, we measured DSD activity in chicken tissues, and examined the cellular localization of DSD using a specific anti-chicken DSD antibody. The highest activity was found in kidney. Skeletal muscles and heart showed no activity. In chicken brain, cerebellum showed about 6-fold-higher activity (1.1 ± 0.3 U/g protein) than cerebrum (0.19 ± 0.03 U/g protein). At the cellular level DSD was demonstrated in proximal tubule cells of the kidney, in hepatocytes, in Bergmann-glia cells of the cerebellum and in astrocytes. The finding of DSD in glial cells seems to be important because d-serine is involved in NMDAR-dependent brain functions. © 2014 Elsevier GmbH. All rights reserved.
Introduction Chicken d-serine dehydratase (DSD) is one of the founding members of the recently discovered pyridoxal 5 -phosphate (PLP)dependent enzyme family that requires a catalytic Zn2+ ion in the active site (Ito et al., 2008; Tanaka et al., 2011). DSD catalyzes the dehydration of d-serine to produce pyruvate and ammonia. When O2 molecules are available, d-serine can also be degraded by damino acid oxidase (DAO), a flavin-dependent enzyme, through oxidative deamination. In the case of DAO, 3-hydroxypyruvate, ammonia, and hydrogen peroxide are produced. We have developed a method to measure DSD- and DAO-dependent degradation of d-serine simultaneously by quantifying the respective products of pyruvate and 3-hydroxypyruvate (Tanaka et al., 2007). By using this method, we have found that in chicken brain d-serine is
Abbreviations: DAB, 3,3 -diaminobenzidine; DAO, d-amino acid oxidase; DSD, d-serine dehydratase; GFAP, glial fibrillary acidic protein; HRP, horseradish peroxidase; NMDAR, N-methyl-d-aspartate receptor; PBS, phosphate-buffered saline; PLP, pyridoxal 5 -phosphate. ∗ Corresponding author. E-mail address: [email protected] (H. Tanaka).
degraded only by DSD, whereas in chicken kidney and liver, both DSD and DAO are responsible for d-serine degradation. d-Serine and glycine are physiological allosteric activators of the N-methyl-d-aspartate receptor (NMDAR) for l-glutamate (Schell, 2004; Wolosker et al., 2008; Papouin et al., 2012). Therefore, the control of regional concentrations of d-serine in the central nervous system is important. In mammalian brains, lower levels of d-serine are found where DAO activity is higher (Nagata et al., 1994; Horiike et al., 2001) suggesting the importance of DAO in the regulation of NMDAR activities (Pollegioni and Sacchi, 2010). Because mammals lack DSD, DAO is the only enzyme to degrade d-serine in mammals. However, avian and many other non-mammalian vertebrate species have DSD in addition to DAO (Tanaka et al., 2009). DAO can degrade various d-amino acids and glycine (Dixon and Kleppe, 1965; Marchi and Johnston, 1969). In fact, d-serine is not the best substrate for DAO. However, it is the best substrate for DSD, which shows very narrow substrate specificity (Tanaka et al., 2008). These different properties between DAO and DSD seem to have implications for the loss of DSD in mammals and the exclusive expression of DSD in chicken brains. However, it has not been clarified why mammals have lost DSD and why avian species utilize only DSD in the brain. To elucidate the physiological role of DSD further, it is important not only to understand its enzymatic properties (Tanaka et al., 2008,
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2011; Ito et al., 2012) but also to reveal its cellular localization in various tissues. We have developed a specific polyclonal antibody against chicken DSD (Tanaka et al., 2008). In the present study, using this antibody, we identified DSD-expressing cells in various chicken tissues. Materials and methods Tissue collection Small organ parts (1–5 g) were removed from four adult male chickens (White Leghorn) killed at a local slaughterhouse. Blocks 5 mm3 were made from the individual parts and immediately fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde for 4 h at room temperature. The remaining portions were immediately frozen in liquid nitrogen, and then stored at −80 ◦ C until use. Assay of DSD activity Each chicken tissue (1–2 g) was homogenized on ice with 3 volumes of 10 mM potassium phosphate, pH 7.2, containing 50 M pyridoxal 5 -phosphate (PLP), using a glass homogenizer. The standard assay mixture (100 L) contained 50 mM potassium phosphate, pH 7.8, 50 mM d-serine, and 10 M PLP. The reaction was initiated by the addition of a 5-L aliquot of each homogenate, run at 37 ◦ C for 30 min, and then stopped with the addition of 40 L of 12.5% trichloroacetic acid. After the addition of 5 nmol of 2-oxoglutaric acid (5 L of 1 mM stock solution in H2 O) as an internal standard, the mixture was centrifuged at 10,000 × g for 10 min at 4 ◦ C. As we described previously (Tanaka et al., 2007), an aliquot of the supernatant (100 L) was treated with 3-methyl-2benzothiazoline hydrazone hydrochloride to convert 2-oxo-acids into the corresponding azine derivatives, which were then quantified by HPLC on a Cosmosil 3C18 column (Nacalai Tesque, Japan). One unit of DSD activity was defined as the amount of enzyme producing 1 mol of pyruvate per min at 37 ◦ C under the standard assay conditions. Western blot analysis Aliquots of the total tissue homogenates prepared for the DSD assay were incubated in the presence of 2% SDS and 5% 2mercaptoethanol at about 80 ◦ C for 3 min, and then separated by SDS-PAGE using a 12.5% gel (ATTO, Tokyo, Japan). The protein contents of the tissue homogenates were determined using a BioRad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA). The proteins were transferred to Clear Blot Membrane-P using a semidry blotter (ATTO). All subsequent procedures were carried out at room temperature. The membrane was rinsed twice by incubation with 100 mL of PBS containing 0.1% Tween 20 (Buffer A) for 5 min, and then incubated with PBS containing 5% (w/v) skim milk and 0.1% Tween 20 for 1 h. The anti-chicken DSD antibody (3 mg/mL stock) (Tanaka et al., 2008) was diluted 10,000-fold with Buffer A containing 1% bovine serum albumin. The blocked membrane was incubated with the diluted antibody for 1 h. The membrane was washed three times with Buffer A, and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibodies (1:5000, diluted with Buffer A) (Amersham ECL Plus Western Blotting Reagent Pack; GE Healthcare, UK) for 1 h at room temperature. After five washes with Buffer A, the antibodies were detected using Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Amersham, UK). The specificity of the anti-chicken DSD antibody was confirmed using negative controls with the diluted antibody (1:10,000) pre-incubated overnight with 2 mg/mL of the DSD purified from chicken kidney (Tanaka et al., 2008) at 4 ◦ C.
Table 1 Distribution of DSD activity in chicken tissues. Tissues
DSD activity (U/g protein)
Kidney Liver Whole brain Cerebrum Cerebellum Brain stem Midbrain Heart Skeletal muscle
4.4 ± 1.4 0.40 ± 0.10 0.40 ± 0.20 0.19 ± 0.03 1.1 ± 0.3 0.40 ± 0.10 0.25 ± 0.10 ND ND
Values are the mean ± SD determined for 4–5 male chickens. No DSD activity was detected for heart and skeletal muscles (ND, not detected).
Immunohistochemistry The fixed tissues were embedded in paraffin wax. Three-mthick paraffin sections were cut and placed on silane-coated glass slides, and then deparaffinized by incubation for 10 min in xylene. The sections were rehydrated through consecutive incubations in 100%, 90%, 80%, and 70% ethanol, and finally washed with PBS. The sections were first incubated in 3% H2 O2 in methanol for 20 min at room temperature to inhibit endogenous peroxidase. Then, the sections were washed with PBS and incubated overnight at 4 ◦ C with the anti-DSD antibody diluted in PBS containing 1% BSA (1:500). After removing unbound antibodies by washing with PBS, the sections were incubated with an HRP-labeled goat antibody against rabbit IgG (Histofine Simple Stain Max PO (Multi); Nichirei Bioscience, Tokyo, Japan) twofold diluted with PBS containing 1% BSA. The sections were washed with PBS, and then peroxidase activity was developed by incubation with 3,3 -diaminobenzidine (DAB) using a Simple Stain DAB Solution (Nichirei Bioscience). Counterstaining was performed with hematoxylin. Then, the sections were dehydrated in ethanol, cleared with xylene, coverslipped with Crystal/Mount (Biomeda, Pittsburgh, PA, USA), and observed with a Nikon Microphoto-FXA (Nikon Co., Tokyo, Japan). The specificity of the immunohistochemical procedures was evaluated by omitting the primary anti-DSD antibody or the secondary antibody in the above procedures. Immunofluorescence microscopy Three-m-thick deparaffinized sections were incubated overnight at 4 ◦ C with the anti-DSD antibody (1:1000). The sections were washed three times with PBS, incubated with
Fig. 1. Western blot detection of DSD in whole tissue homogenates with the antichicken DSD antibody. Aliquots of whole tissue homogenates (containing 20 g of protein) were loaded on a 12.5% SDS-PAGE gel and consequently transferred to a PVDF membrane. The immunoblotting was done using the antibody (1:10,000, final concentration of 0.3 g/mL). The lane of purified DSD is the DSD (2 g) purified from chicken kidney.
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Fig. 2. Immunohistochemical detection of DSD in chicken kidney cortex. (a) The collecting tubules were visualized by Alcian blue staining (Díaz et al., 1996). Strong staining was found only in the renal proximal tubules. (b) Higher magnification shows intense staining in the cytoplasm of the epithelial cells. G, glomerulus; PT, proximal tubule; DT, distal tubule; CT, collecting tubule. Scale bar = 50 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Alexa-Fluor® 594-labeled donkey anti-rabbit antibodies (1:500, Invitrogen, Eugene, OR, USA) for 1 h at room temperature, and rinsed in PBS three times. The sections were coverslipped using Gel/Mount (Biomeda) and examined with an LSM 510 META (Carl Zeiss Corporation, Oberkochen, Germany). For double staining of DSD and glial fibrillary acidic protein (GFAP) or NeuN (Mullen et al., 1992) or Pax-7 (Shin et al., 2003), the sections were incubated overnight at 4 ◦ C with the anti-DSD antibody (1:1000) and mouse monoclonal anti-GFAP antibody (1:1000; Chemicon, Temecula, CA, USA) or mouse monoclonal anti-NeuN antibody (1:1000; Chemicon) or mouse monoclonal anti-Pax-7 antibody (1:1000; R&D Systems, Minneapolis, MN, USA), respectively, rinsed three
times in PBS, and then incubated with Alexa-Fluor 594-labeled donkey anti-rabbit antibodies (1:500) and Alexa-Fluor® 488labeled donkey anti-mouse IgG antibodies (1:500) for 1 h at room temperature. Results Distribution of DSD in chicken tissues Table 1 summarizes DSD activity in chicken tissues. The highest DSD activity was found in the kidney (4.4 ± 1.4 U/g protein). The liver and brain showed DSD activity of 0.4 U/g protein, about 10%
Fig. 3. Immunohistochemical detection of DSD in chicken liver. (a) Strong cytoplasmic staining was found only in hepatocytes. Higher magnification shows the interlobular bile duct (panel b) and artery (panel c). PV, portal vein; BD, interlobular bile duct; A, interlobular artery. Scale bar = 50 m.
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Fig. 4. DSD immunolocalization in chicken cerebellum. (a) The cytoplasm of the cell body and long processes of Bergmann glial cells was stained for DSD. The astrocytes in the granular layer were stained in the cytoplasm. (b) Confocal microscopy analysis. Merged image of DSD staining (red) and GFAP (green). DSD-positive cells (red) were also positive for GFAP (green) in the granular layer, located immediately beneath the Purkinje cell layer. (c) Merged image of DSD (green) and Pax-7 (red). The nuclei of the DSD-positive cells were stained for Pax-7. PL, Purkinje cell layer; ML, molecular layer; GL, granular layer; P, the soma of the Purkinje cell. Scale bar = 25 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
of that in the kidney. In the chicken brain regions, the cerebellum showed about 6-fold-higher DSD activity than the cerebrum. We could not detect DSD activity in the skeletal muscles and heart. Specificity of DSD antibody The specificity and sensitivity of the anti-DSD antibody were examined by Western blot analysis of the total homogenate of chicken tissues. Except for the skeletal muscles and heart, a distinct signal was observed for all the tissues examined only at a molecular weight corresponding to that of purified DSD (40 kDa) (Fig. 1). The strength of the signal correlated well with the tissue levels of DSD activity (Table 1 and Fig. 1). These results demonstrated that the antibody is highly specific for DSD. Immunohistochemical localization of DSD The cellular distribution of DSD in the following tissues was revealed by using DAB as a substrate for the final HRP reaction, which resulted in a brown stain. Tissues were counterstained with hematoxylin (blue stain). No DSD immunoreactivity was detected in parallel incubations of control sections when the anti-DSD antibody was omitted or pre-absorbed with 2 mg/mL of DSD purified from chicken kidney.
Kidney cortex Strong staining for DSD was found only in the renal proximal tubules (Fig. 2a). No significant staining was found in the glomeruli, renal distal tubules, and collecting tubes. Higher magnification revealed non-granular staining in the cytoplasm of the epithelial cells of the proximal tubules (Fig. 2b), consistent with the cytoplasmic localization of DSD (Tanaka et al., 2008). Liver All of the hepatocytes exhibited intense and homogeneous staining in the cytoplasm (Fig. 3a). The intensity was evenly distributed between the different liver lobules. In the interlobular regions, no DSD staining was found for arterioles, bile ducts, and veins (Fig. 3b and c). Cerebellar cortex In the granular layer, granule cells were unstained for DSD (Fig. 4a). A group of cells with many short processes exhibited intense cytosolic staining for DSD. The cytoplasm of the processes of these cells was also stained for DSD, and the DSD-positive processes wrapped the granule cells. Purkinje cells were unstained for DSD (Fig. 4). In the vicinity of the cell body of the Purkinje cells, a small group of cells showed intense staining for DSD in their cell body. These cells had long
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Fig. 5. DSD immunolocalization in chicken cerebrum. (a) Strong cytoplasmic staining was found in the cells with short processes. Higher magnification shows the staining of the fine processes (panel b). (c) Double labeling of DSD (red) and NeuN (green). The nuclei of the DSD-positive cells (red cytoplasm) showed no staining for NeuN. Scale bar = 25 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
fiber-like processes running across the molecular layer, and the cytoplasm of the processes was stained for DSD. As shown in Fig. 4b, in the granular layer DSD-positive cells (red) were also positive for GFAP (green), but some GFAP-positive cells were not DSD-positive, indicating that a group of astrocytes express DSD. In contrast, the vicinity of the cell body of the Purkinje cells did not show immunopositivity to GFAP. Chicken Bergmann glial cells in the cerebellum are almost negative to GFAP, unlike their mammalian counterparts (Kálmán et al., 1993). A paired box transcription factor, Pax-7, is known to be localized within the nuclei of the Bergmann glial cells in the adult chicken cerebellum (Shin et al., 2003). Therefore, by double-labeling immunofluorescence microscopy, we examined whether the nucleus of these DSD-positive cells was Pax-7-positive. As shown in Fig. 4c, in the Purkinje cell layer, the cells with a Pax-7-positive nucleus (red in Fig. 4c) also showed intense staining for DSD in the cytoplasm of the cell body (green in Fig. 4c). These results indicate that the DSD-positive cells in the proximity of the Purkinje cell bodies were Bergmann glial cells.
Cerebrum We examined the distribution of DSD in the cerebrum (Fig. 5). DSD immunoreactivity was found in the cytoplasm of a group of cells with many processes. The fine processes of these cells were stained as intensely as the cytoplasm of the cell body (Fig. 5b). As shown in Fig. 5c, by double-labeling immunofluorescence microscopy, the nucleus of the DSD-positive cells (the cells with red cytoplasm in Fig. 5c) showed no staining for the neuronal
marker NeuN (Mullen et al., 1992). These results indicated that the DSD-positive cells are astrocytes, not neurons with NeuN-positive nucleus.
Discussion In this study, we examined biochemically and immunohistochemically the tissue and cellular distribution of DSD. In chicken kidney cortex, only epithelial cells of the proximal tubules were strongly stained for DSD. In chicken liver, only hepatocytes exhibited intense staining for DSD, with the intensity levels evenly distributed throughout the liver lobule. The cellular localization of DSD in these tissues was identical to that reported for DAO in mammals (Horiike et al., 1985), suggesting that DSD plays a role in removing d-serine from the circulating blood. In chicken cerebellar cortex, DSD was found to localize exclusively in Bergmann glial cells and astrocytes. This DSD localization was identical to that found for DAO in the mammalian cerebellum (Horiike et al., 1987). In chicken cerebrum, astrocytes showed intense staining for DSD in the cytoplasm of the cell body and processes. This result is consistent with the fact that the d-serine contents in chicken cerebrum are very low (Nagata et al., 1994). In mammals, DAO is solely responsible for the degradation of dserine in the brain. In the mammalian cerebrum DAO activity is not detected (Horiike et al., 1994), and d-serine concentration is unusually high (Nagata et al., 1994; Hashimoto and Oka, 1997). It is believed that the high levels of d-serine are important for NMDAR-dependent functions in the mammalian cerebrum (Hunt
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and Castillo, 2012; Zorumski and Izumi, 2012). The present results suggest that in chicken cerebrum NMDAR does not need high concentrations of d-serine. In conclusion, we identified DSD-positive cells in the chicken kidney, liver, cerebrum, and cerebellum. Except for the cerebrum, the cellular localization of DSD is identical to that of DAO in mammals. In chicken cerebrum, the astrocytes showed intense staining for DSD, explaining the low d-serine level in this region. Acknowledgements We thank Mr. Takefumi Yamamoto, Central Research Laboratory of our university, for technical advice regarding immunohistochemistry. The present study was supported by a Grant-in-aid for Scientific Research (C) (no. 24590351) (to H.T.) and a Grant-in-aid for Scientific Research (B) (no. 22370026) (to K.K., M.M., and H.T.) from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, as well as a Grant-in-aid (Heisei era24) from Shiga University of Medical Science. References Díaz RC, Montaner B, Pérez TR. Immunochemical study of transforming growth factor- in the kidney of the rat and chicken. Histochem Cell Biol 1996;105:475–8. Dixon M, Kleppe K. d-Amino acid oxidase. II. Specificity, competitive inhibition and reaction sequence. Biochim Biophys Acta 1965;96:357–67. Hashimoto A, Oka T. Free d-Aspartate and d-serine in the mammalian brain and periphery. Prog Neurobiol 1997;52: 325–53. Horiike K, Arai R, Tojo H, Yamano T, Nozaki M, Maeda T. Histochemical staining of cells containing flavoenzyme d-amino acid oxidase based on its enzymatic activity: application of a coupled peroxidation method. Acta Histochem Cytochem 1985;18:539–50. Horiike K, Tojo H, Arai R, Yamano T, Nozaki M, Maeda T. Localization of d-amino acid oxidase in Bergmann glial cells and astrocytes of rat cerebellum. Brain Res Bull 1987;19:587–96. Horiike K, Tojo H, Arai R, Nozaki M, Maeda T. d-Amino-acid oxidase is confined to the lower brain stem and cerebellum in rat brain: regional differentiation of astrocytes. Brain Res 1994;652:297–303. Horiike K, Ishida T, Tanaka H, Arai R. Distribution of d-amino acid oxidase and d-serine in vertebrate brains. J Mol Catal B 2001;12:37–41. Hunt DL, Castillo PE. Synaptic plasticity of NMDA receptors: mechanisms and functional implications. Curr Opin Neurobiol 2012;22:496–508.
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