Immunohistochemical localization of ADP-ribosylarginine hydrolase in rodent CNS

Brain Research 746 Ž1997. 1–9

Research report

Immunohistochemical localization of ADP-ribosylarginine hydrolase in rodent CNS Tsuyoshi Miyaoka a,b , Mikako Tsuchiya a , Kazuo Yamada a , Muhammad Badruzzaman a , Chikako Yamamori a,b , Hiroshi Ishino b , Makoto Shimoyama a, ) a

Department of Biochemistry, Shimane Medical UniÕersity, Izumo 693, Japan Department of Psychiatry, Shimane Medical UniÕersity, Izumo 693, Japan

b

Accepted 10 September 1996

Abstract Polyclonal antibodies were generated against ADP-ribosylarginine hydrolase ŽAAH., using recombinant fusion protein of rat AAH and glutathione-S-transferase as a immunogen, and affinity-purified. Western blotting showed that the antibodies recognized in mouse brain homogenate a single protein with a molecular mass of 38 kDa, the expected size for mouse AAH. An analysis using the antibodies revealed that heavy labelings were apparent in various brain regions. In the cerebral cortex, pyramidal cells in layers III and V were the most heavily labeled. In the hippocampal formation, labeling was present on the pyramidal neurons and granule cells. The most heavily immunostained cell type was the pyramidal neuron of CA3. In the cerebellum, Purkinje cells were the most heavily labeled. Less intense staining was present over the granule cells. In the basal ganglia, neurons in the caudate nucleus and large multipolar cells in the amygdaloid complex were immunoreactive. Heavy labeling was seen in many midbrain and brainstem nuclei. Neurons in the habenula and ependymal cells were stained heavily. On Western blot analysis of rat cerebrospinal fluid ŽCSF., the anti-AAH antibodies recognized a protein with a molecular mass of 38 kDa. This is apparently the first evidence of a widespread but distinctive distribution of AAH in neurons of mouse brain and the presence of extracellular AAH in rat CSF. Keywords: ADP-ribosylarginine hydrolase; Immunohistochemistry; Mouse brain; Rat CSF; Western blotting; Arginine specific ADP-ribosylation

1. Introduction Arginine-specific ADP-ribosylation is a post-translational modification of proteins, catalyzed by argininespecific ADP-ribosyltransferases such as cholera toxin, transferring the ADP-ribose moiety from NAD to arginine residues in cellular proteins w30x. In nitrogen fixing bacteria, Rhodospirillum rubrum, ADP-ribosylation-de-ADPribosylation of a specific arginine-residue of dinitrogenase reductase has been shown to regulate the enzyme activity w10x. ADP-ribosyltransferase activities have also been detected in turkey erythrocytes w13x, rabbit skeletal muscle

Abbreviations: BSA, bovine serum albumin; DTT, dithiothreitol; GST, glutathione-S-transferase; HPLC, high-performance liquid chromatography; IgG, immunoglobulin G; MSH, 2-mercaptoethanol; PMSF, phenylmethylsulfonyl fluoride; SDSrPAGE, sodium dodecyl sulfaterpolyacrylamide gel electrophoresis ) Corresponding author. Fax: q81 Ž853. 23-6420.

w23x, and chicken leukocytes w12x and some were purified and cloned w28,31x. On the other hand, an ADP-ribosylarginine hydrolase ŽAAH. activity, which catalyzes de-ADP-ribosylation, cleaves the bond between ADP-ribose and arginine, was also found in various animal tissues w5,8,14–16,22x. It is now considered that arginine-specific ADP-ribosyltransferase and AAH could catalyze the opposing arms of an ADP-ribosylation cycle in which arginine is modified and regenerated w24,25x. The endogenous ADP-ribose acceptor protein substrates in this potential regulatory pathway and the role of the modification in metabolism remain unclear. The presence of ADP-ribosyltransferases was noted in the mammalian brain and its regulatory functions in the nervous system have been proposed. Coggins et al. w7x reported that neuronal phosphoprotein B-50rGAP-43, which is associated with growth and regeneration within the nervous system, was a substrate for endogenous ADPribosyltransferases and a regulatory mechanism at neuronal membranes was suggested. Schuman et al. w21x detected an arginine-specific ADP-ribosyltransferase in rat hippocam-

0006-8993r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 6 . 0 1 1 2 6 - 2

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Fig. 1. Specificity of the anti-AAH antibodies and detection of AAH protein in the mouse forebrain. Recombinant GST-AAH Ž2 m g, lane 1., thrombin-digested recombinant GST-AAH Ž2 m g, lane 2., mouse brain homogenate Ž80 m g, lane 3. and purified GST from mouse brain Ž10 m g, lane 4. were separated on SDSrPAGE followed by protein staining ŽA. and immunoblotting with anti-AAH antibodies ŽB..

pus and reported that nicotinamide and phylloquinone, specific inhibitors of the transferase, prevented long-term potentiation ŽLTP. in the hippocampus. From these findings, it seems that ADP-ribosylation may play important roles in neural functions in the brain. The brain shows the highest activity of AAH among tissues examined in the rat w8,17x. AAH, purified from rat brain, is a soluble protein with a molecular mass of 38 kDa. In the presence of Mg 2q and DTT, it cleaves the a-anomer of ADP-ribosylarginine, the stereospecific product of the transferase reaction. Though the tissue distribution of AAH has noted in assays of activity and Western blot analysis w8,17x, less is known of the distribution of AAH in the brain. Factors such as neurotransmitters distribute in specific brain regions and have specific functions at each region. Therefore, the distinct distribution of AAH in the brain may aid

in elucidating physiological functions of the enzyme as well as ADP-ribosylation-de-ADP-ribosylation cycle in the brain. Affinity-purified polyclonal antibodies specific to AAH were used to obtain evidence of distinct distributions of AAH in the mouse brain, which has higher activity of the hydrolase than the rat brain w17x. 2. Materials and methods 2.1. Preparation of recombinant GST-AAH fusion protein Recombinant GST and GST-AAH fusion protein Ž10 mg. were prepared as described elsewhere w19x. To prepare affinity matrices for purification of antibodies against AAH, recombinant GST or GST-AAH Ž10 mg each. was coupled to 1 ml of CNBr-activated Sepharose 4B ŽPharmacia..

Fig. 2. Immunohistochemical distribution of AAH in the mouse sensory-motor cortex. AAH-immunoreactive neurons were heterogeneously distributed among cellular layers: neurons in layer II and IV were not immunostained, but neurons in layers III and V were strongly immunostained. wm, white matter. Bar s 200 m m. Fig. 3. Immunohistochemical distribution of AAH in the mouse hippocampus. The hippocampus was characterized by the intensely immunostained pyramidal cells of CA1 ; 3. The stratum lucidum Žsl. Žarrow heads. was intensely stained. The dentate gyrus was characterized by moderate immunostaining of granule cells within the stratum granulosum Žsg. Žarrows.. Dendrites from all pyramidal cells along Ammon’s Horn can be traced in the stratum radiatum Žsr.. Bar s 200 m m. Fig. 4. Immunohistochemical distribution of AAH in the mouse amygdala. In the amygdala complex, large multipolar cells were immunostained Žarrows.. Bar s 200 m m.

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2.2. Preparation of affinity-purified antibodies against AAH New Zealand white rabbits were immunized initially with 100 m g of the recombinant GST-AAH fusion protein

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in complete Freund’s adjuvant ŽDIFCO. and every 2 weeks with 50 m g of the protein in incomplete Freund’s adjuvant ŽDIFCO.. One week after the seventh immunization, the rabbit was bled for the serum. Ammonium sulfate was

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Table 1 Distribution of AAH in mouse brain Brain region

0.2 M Tris-glycine buffer, pH 2.5, then the pH of the eluate was adjusted to the neutral range. Staining intensity

Regions which showed heavy ; very heavy immunoreactivity Cerebral cortex Pyramidal cells Žlayers III, V, VI. 4 Hippocampal formation Pyramidal cells ŽCA3. 3 Hypothalamus 3; 4 Amygdala 3; 4 Basal ganglia Caudate-putamen 3 Claustrum 3 Habenula Medial habenular nucleus 4 Lateral habenular nucleus 3 Dorsal thalamus 3; 4 Lower brainstem Red nucleus 4 Darkschewitsch nucleus 3 Medial parabrachial nucleus 3 Cerebellum Purkinje cells 4 Deep nuclei 3 Ependymal cells 4 Regions which showed no; less immunoreactivity Cerebral cortex Granule cells Žlayers II, IV. 0 Basal ganglia Globus pallidus 0r1 Corpus callosum 0 Habenula Habenular commissure 0 Lower brainstem Superior colliculus 0r1 Cerebellum Granule cells 0r1 Regional labeling is given: 0, immunonegative, 0r1, less labeling, 3, heavy labeling, 4, very heavy labeling.

added to the serum to 33% saturation, then the precipitate was dissolved in 10 mM phosphate buffer ŽPB, pH 7.5. containing 0.15 M NaCl Žphosphate-buffered saline, PBS.. After passing through a GST-Sepharose column to remove antibodies against GST, AAH-specific antibodies were purified further from the serum on a GST-AAH-Sepharose column. After the serum had been applied to a GST-AAH column, the column was washed with 20 mM Tris-HCl buffer, pH 7.5, containing 0.15 M NaCl ŽTris-buffered saline, TBS.. Specific antibodies to AAH were eluted with

2.3. Immunohistochemistry Ten adult BALBrc mice age from 6 to 8 weeks were anesthetized with diethyl ether, then transcardially perfused with PBS, and fixed by perfusing with 0.1 M PB containing 4% paraformaldehyde ŽpH 7.5.. The brains were removed and postfixed for 72 h at 48C with the same fixative solution, embedded in paraffin, and cut into 6 m m-thick sections. After deparaffinization, sections were treated with alcohol containing hydrogen peroxide, exposed to 10% goat serum, and then incubated with the rabbit anti-AAH antibodies Ž1:1000 dilution. for 24 h at 48C in a humid chamber. All subsequent incubations were performed at room temperature for 30 min. After a rinse in PBS, the sections were incubated with goat biotinylated anti-rabbit IgG for 3 h, washed again with PBS, and incubated with peroxidase-conjugated streptoavidin for 1 h, then the color was developed with dimethylaminoazobenzene Ž40 m grml. and 0.002% hydrogen peroxide. Finally, nuclei in cells were stained by hematoxylin. Nissle staining of adjacent sections was done to observe the neural shape. Controls included both the replacement of primary antibodies with normal rabbit serum and liquid phase preabsorption of the primary antibodies with recombinant AAH protein. 2.4. Gel electrophoresis and immunoblotting Proteins were electrophoresed by SDSrPAGE according to Laemmli w9x and then electrophoretically transferred onto a nitrocellulose membrane w27x. The membrane was incubated for 1 h at room temperature with 2.5% Žwrv. nonfat dry milk in TBS, then for 18 h with the polyclonal antibodies diluted 1:1000 in TBS containing 2.5% BSA, and for 1 h with peroxidase-conjugated anti-rabbit IgG Žgoat.. Immunoreactive proteins were then detected using ECL kits ŽAmersham.. 2.5. Affinity-purification of mouse brain GST Mouse brain cytosol was applied to glutathione-Sepharose affinity column, and GST was eluted by 10 mM glutathione. The purified preparation contained two pro-

Fig. 5. Immunohistchemical distribution of AAH in the mouse habenula and ependymal cells. Upper panel: anti-AAH antibodies stained neurons in the medial and lateral habenular nuclei, and ependymal cells around the ventricle. LHb, lateral habenular nucleus; MHb, medial habenular nucleus; sm, stria medullaris thalami. Bar s 200 m m. Lower panel: ependymal cells in the choroid plexus of the lateral ventricle showed very strong immunoreactivity. Bar s 100 m m. Fig. 6. Immunohistochemical distribution of AAH in the mouse cerebellar cortex. The cerebellar cortex was characterized by an intensely immunostained Purkinje cell layer ŽPc., which separates a moderate immunoreactive molecular layer Žmol. from the slightly immunoreactive granule cell layer Žgcl.. wm, white matter. Bar s 200 m m.

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teins of 26 and 27 kDa corresponding to the sizes of endogenous GST in mouse brain w26x. 2.6. Miscellaneous Brains were homogenized with 5 volumes of 0.25 M sucrose containing 20 mM TrisrHCl buffer, pH 7.5, 2 mM EDTA, 1 mM MSH and 1 mM PMSF. The homogenate was centrifuged for 1 h at 100 000 = g, and the supernatant was used as brain cytosol. CSF was aspirated from the cisterna magna of Jcl:Wistar rats anesthetized with diethyl ether, using a syringe with a 21-gauge needle. The CSF was centrifuged for 10 min at 4000 = g and the supernatant was used for analysis. Protein concentration was determined according to Bradford w3x using bovine serum albumin as a standard. AAH assay was carried out according to the methods described elsewhere w19x.

3. Results 3.1. Polyclonal antibodies and immunoblotting Polyclonal antibodies against AAH were obtained by immunization of rabbits with recombinant GST-rat AAH fusion protein, and purified on a GST-AAH column after the immune serum had been absorbed by recombinant GST protein. On Western blots, the anti-AAH antiserum reacted with GST-AAH and AAH portion Ž38 kDa. of thrombin-digested GST-AAH but not with GST portion of the recombinant protein or affinity-purified mouse brain GST ŽFig. 1.. In the mouse brain homogenate, the antibodies detected a single band of 38 kDa ŽFig. 1, lane 3., corresponding to mouse AAH w17,24x. Thus, the antibodies specifically recognized AAH in mouse brain. 3.2. AAH immunohistochemistry Specific distribution patterns of AAH-immunoreactive products were observed in some regions of mouse brain, at light microscopic levels ŽTable 1.. The characteristic regions are described below, with photographs. No staining was observed with antibodies that had been incubated in the presence of the AAH antigen prior to the experiment Ždata not shown.. 3.2.1. Cerebral neocortex In the cerebral cortex, layers III and V were immunostained strongly by the anti-AAH antibodies ŽFig. 2.. Especially in layer III, there was a heavy labeling of pyramidal cells. Moderate labeling was seen in small neurons of layer I and spindle cells in layer VI, while granule cells in layers II and IV were not stained. 3.2.2. Hippocampal formation Though all portion of the hippocampus contained immunoreactive neurons, most prominent were the pyramidal

cells of CA1 ; 3, especially CA3 ŽFig. 3.. The stratum lucidum in this region was more intensely stained than the stratum radiatum. In addition to the prominent immunostaining within the hippocampus, the granule cells in the dentate gyrus were moderately stained. Immunostained neurons were also present in the hilus. 3.2.3. Basal ganglia The cell bodies and processes of neurons in the caudate-putamen were strongly immunostained with the anti-AAH antibodies Ždata not shown.. In the globus pallidus, neurons and passing fiber bundles were not immunostained Ždata not shown.. In the amygdaloid complex, intensely immunoreactive large multipolar cells were present ŽFig. 4.. 3.2.4. Habenula and ependymal cells Neurons in the habenula were immunostained heavily with the anti-AAH antibodies. Closely packed, round neurons and the neuropil in the medial habenular nucleus were densely stained, while loosely arranged neurons and the neuropil in the lateral habenular nucleus were moderately stained ŽFig. 5, upper panel.. Ependymal cells around the ventricles and in the choroid plexus showed the strongest immunoreactivity of all the mouse brain cells examined ŽFig. 5.. 3.2.5. Cerebellar cortex, midbrain nuclei Purkinje cells were densely immunostained in all folia of the cerebellar cortex ŽFig. 6.. Compared to the Purkinje cells, granule cells were less intensely immunostained. The deep cerebellar nuclei contained numerous labeled neurons Ždata not shown.. In the midbrain, heavy immunoreactivity was evident in large cells in the red nucleus and pontine nuclei, and medium-sized neurons of the substantia nigra Ždata not shown.. 3.2.6. Detection of AAH in rat CSF Since ependymal cells which secrete various factors into the CSF were heavily stained with the anti-AAH antibodies, we examined the possibility that AAH also is secreted by ependymal cells into the CSF. As shown in Fig. 7, on Western blotting, the antibodies detected a protein with a molecular mass of 38 kDa in the CSF. Significant AAH activity was detected in rat CSF and the specific activity was 4.5-fold higher than that of rat brain cytosol AAH ŽTable 2.. These results provide evidence for the presence of AAH in CSF.

4. Discussion ADP-ribosylation is one post-translational modification of proteins and is considered to function as a mechanism regulating protein activity. Other workers reported findings of ADP-ribosyltransferase activity in the mammalian brain

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Fig. 7. Detection of AAH in CSF by Western blotting. Rat CSF Ž1 m l, lanes 1 and 3. and rat brain cytosol Ž50 m g, lanes 2 and 4. were fractionated by SDSrPAGE followed by protein staining Žlanes 1 and 2. and immunoblot analysis with anti-AAH antibodies Žlanes 3 and 4..

and proposed regulatory roles for this activity in CNS w7,21x, little information is available concerning the precise localization of ADP-ribosyltransferase protein in the brain. The brain has an abundance of AAH w8,17x, which catalyzes de-ADP-ribosylation of protein, and the AAH was easily obtained, as a recombinant protein. To elucidate the physiological significance of ADP-ribosylation in the brain, we considered it important to examine the localization of AAH, using affinity-purified antibodies raised against the recombinant AAH protein. Neurons stained with anti-AAH antibodies were present throughout the brain, with high levels in the cerebral cortex, hippocampus, basal ganglia, cerebellum and habenula. This prominent staining of neural cell bodies is indicative of a large intracellular pool of AAH in neurons. Table 2 AAH activity in rat CSF and brain cytosol Source

Activity Žnmolrmin per mg.

CSF Brain cytosol

8.15 1.83

Rat CSF Ž0.4 m g. or brain cytosol Ž10 m g. were incubated with 200 m M ADP-ribosylw 14 Cxarginine at 308C for 30 min in 50 mM TrisrHCl buffer ŽpH 7.5. containing 10 mM MgCl 2 and 5 mM DTT, and amount of w 14 Cxarginine released during the reaction was determined w19x.

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In the cerebral cortex, differences in staining were detected between cortical layers, the most prominently labeled neurons being pyramidal neurons localized in layers III and V. Pyramidal neurons in the hippocampus and Purkinje cells in the cerebellum were the most heavily immunostained cells in each region. These findings of a widespread and distinctive distribution of AAH in mouse brain are taken to mean that AAH may contribute in basic functions of some types of neurons. Cerebral cortex, amygdala, hippocampus and Purkinje cells are regions relating to memory and long-term potentiation ŽLTP. or long-term depression ŽLTD. w1,2,6,11x can be considered. The relation of ADP-ribosylation to LTP in the hippocampus have been reported w4,18,21x. The latter authors reported that CA1 tissue from the hippocampus possessed both arginine- or cysteine-specific ADP-ribosyltransferase activities that were dramatically stimulated by nitric oxide and attenuated by the inhibitors of monoADP-ribosyltransferase activity, nicotinamide and phylloquinone and that extracellular application of these inhibitors prevented LTP. Based on these results, they proposed that ADP-ribosyltransferase plays a role in LTP as a potential target for nitric oxide. Our observation of a high AAH content in hippocampal neurons supports the notion that ADP-ribosylation regulates LTP in a reversible manner and may have a role in memory. Basal ganglia, midbrain and brainstem nuclei, habenula are important regions for interneuronal signal transduction and neural signals are actively concentrated into these regions. Intracellular signal transduction is also active in these regions. ADP-ribosylation may have a role of intracellular signal transduction. Coggins et al. w7x demonstrated that the neuronal growth-associated phosphoprotein B-50rGAP-43, which interacts with Go protein in neuronal cells, can serve as a substrate for endogenous ADPribosyltransferases, thereby suggesting the possibility that ADP-ribosylation involves intracellular signal transduction in neurons. Thus, the abundance of AAH in these regions parallels the modulator function of ADP-ribosylation, as a reversible reaction in cellular signaling in neuronal system. In our immunohistochemical study with anti-AAH antibodies, ependymal cells around the ventricles and in the choroid plexus showed the strongest staining among various cells of the mouse brain. In rat CSF, a protein immunoreactive to the antibodies and the catalytic activity of AAH also were detected. Since ependymal cells are thought to secrete various substances into the CSF, AAH present in the CSF may be synthesized and secreted by ependymal cells. Though we have no direct evidence that the extracellular AAH is actively secreted rather than released from dead cells, recent studies of arginine-specific ADP-ribosyltransferase clearly indicate the presence of the transferase on the extracellular surface of lymphocytes or skeletal muscle cells w20,29x. Thus, extracellular AAH can

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attack ADP-ribosylated arginine residues of cell surface proteins modified by the ectoADP-ribosyltransferase and make the extracellular ADP-ribosylation reversible. To our knowledge, this is the first report of extracellular AAH. In this study, we demonstrated the presence of AAH in various brain regions, which may reflect the presence of enzymes catalyzing formation of the AAH substrate, arginine-specific ADP-ribosyltransferases. The regional localization of the transferase is the subject of ongoing study. The role of reversible ADP-ribosylation of arginine residues of proteins in CNS can be given further attention, based on the new evidence.

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Acknowledgements w17x

Part of this work was supported by a Grant-in-Aid for Scientific Research on Priority Areas, 05271103, from the Ministry of Education, Science, Sports and Culture of Japan, and a grant from the Kato Memorial Bioscience Foundation. We thank S. Yamashita and H. Osago for the technical assistance. This work was carried out in the Department of Biochemistry, Shimane Medical University.

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