ARTICLE |
Correspondence to: Göran Andersson, Div. of Pathology F 46, Huddinge University Hospital, S-141 86 Huddinge, Sweden. E-mail: Goran.Andersson@impi.ki.se
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Summary |
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Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocytemacrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 1020 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC50 for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate > tungstate > phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in -motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system. (J Histochem Cytochem 49:379396, 2001)
Key Words: bone, brain, ganglion, macrophage, osteoclasts, osteopontin, protein phosphatase, sensory, sympathetic
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Introduction |
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PURPLE ACID PHOSPHATASES (PAPs) are acid metallohydrolases that contain a binuclear Fe3+M2+ center in their active site, where M = Fe or Zn (
Mammalian PAPs exist in two interconvertible states: pink, reduced and enzymatically active, with a mixed-valent Fe(II)-Fe(III) cluster, and purple, oxidized and catalytically inactive, with the dinuclear pair as Fe(III)-Fe(III). The mammalian serine/threonine protein phosphatases calcineurin (Type 2B) (
In humans and rats, TRAP has been detected in a wide variety of tissues as a minor acid phosphatase isoenzyme. However, it is highly expressed in certain activated macrophages and bone-resorbing osteoclasts (
Calcineurin, protein phosphatase-1 (PP-1) (
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Materials and Methods |
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Experimental Animals
Five 3-week-old SpragueDawley rats of unspecified gender were used in each experiment. Rats were kept under controlled light/dark conditions with food and water available ad libitum. The rats were decapitated and tissues were dissected out for partial purification of TRAP and for RNA preparation, as described in the following sections.
Tissues for immunohistochemistry and in situ hybridization histochemistry were dissected out after perfusion via the ascending aorta with 4% paraformaldehyde in 0.1 M Sörensen's phosphate buffer, pH 7.4, immersed in the same fixative for 24 hr, and then embedded in paraffin. Sections (4.5 µm thick) were adhered to slides (SuperFrost; Menzel-Gläser, Braunschweig, Germany) and processed for immunohistochemistry or ISH histochemistry (see below). Before perfusion the animals were anesthetized with a lethal dose of sodium pentobarbital (60 mg/ml).
The use of animals in this study was approved by the local ethical committee of the Karolinska Institute (S 170/98).
Partial Purification of TRAP from Rat Neural Tissues and Bone
TRAP was purified from the rat brain, spinal cord, trigeminal ganglion, and bone essentially as described previously (
Assay Procedures
The TRAP enzyme activity in each purified sample was assayed in 96-well plates using p-nitrophenylphosphate (pNPP) as substrate in an incubation medium (200 µl) containing (final concentrations): 2.5 mM pNPP (ditris salt; Sigma), 0.1 M sodium acetate buffer, pH 5.8, 0.2 M KCl, 0.1% Triton X-100, 10 mM sodium tartrate, and the reducing agents ascorbic acid (1 mM) and FeCl3 (100 µM). The p-nitrophenol liberated after 1-hr incubation at 37C was converted into p-nitrophenolate by the addition of 50 µl of 0.9 M NaOH, and the absorbance was measured at 405 nm using a Spectramax250 spectrophotometer (Molecular Devices; Sunnyvale, CA). One unit (U) of TRAP activity corresponds to 1 µmol of p-nitrophenol liberated per minute at 37C. To oxidize the reduced enzyme, 10 mM hydrogen peroxide (H2O2) was added, and after incubation for 10 min at room temperature (RT), the substrate (pNPP) was added. The concentration of inhibitor required to reduce enzyme activity to 50% of the activity in the absence of inhibitor (IC50) for the oxyanions molybdate, tungstate, and phosphate was calculated using linear regression analysis. Km for pNPP was calculated using the method of LineweaverBurke.
Electrophoresis and Immunoblotting Analysis
Partially purified TRAP (30 mU) from each tissue was subjected to SDS-PAGE in 12% slab minigels according to the procedure described by
Total RNA Preparation and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
Total RNA was extracted according to the method of
The RT reaction was performed on 1 µg of mRNA or 5 µg of total RNA (for nested PCR) with 200 U Superscript II RNase H- Reverse Transcriptase (Gibco; Paisley, UK) at 37C, using oligo d(T)1218 (Gibco) as primer, according to the manufacturer's protocol. Negative controls included reactions in which the reverse transcriptase was excluded from the reaction mixture.
PCR amplification was performed on aliquots of cDNA corresponding to 200 ng mRNA or 800 ng total RNA (for nested PCR) using 2.5 U Taq polymerase in the presence of 1.5 mM MgCl2 and 1 x Q-Solution according to the manufacturer's protocol (QIAGEN). The 5'- TRAP primer (positions 3247; 5'-TTCTGTTCCAGGAGCTT-3') and 3'- TRAP primer (positions 600616; 3'-CAGTCGGTCGTCGGACT-5') were added (150 ng/reaction) (positions calculated from the rat TRAP cDNA sequence available at GenBank/EMBL Data Bank accession no. M76110). The expected size of the TRAP cDNA fragment is 586 bp.
For nested PCR, the 5'-TRAP primer 5'-CGCCAGAACCGTGCAGA-3' (positions 160176) was included in the second amplification, generating a band with a predicted size of 356 bp. The cDNA was amplified in a PerkinElmer Cetus Instruments DNA ThermalCycler, running 25, 30, 35, or 40 cycles under the following conditions: 94C, 1 min; 56C, 1 min; 72C, 1 min. In the nested PCR, cDNA was amplified in a PerkinElmer Cetus Instruments DNA GeneAmp PCR system 9700 running 40 cycles twice under the following conditions: 94C, 30 sec; 56C, 30 sec; 72C, 30 sec. The PCR products were electrophoresed on a 1.5% agarose gel containing ethidium bromide, 0.15 µg/ml.
PCR analysis for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was performed on all of the samples as an internal control for amplification of equal amounts of RNA. The primers 5'-CTGAACGGGAAGCTCACTGG-3' and 3'-TGAGGTCCACCACCCTGTTG-5'corresponding to positions 664683 and 976957 in the rat GAPDH cDNA sequence (
Sequencing of TRAP cDNA PCR Product
Twenty ng of PCR product from the trigeminal ganglion RT-PCR reaction using 5'-TRAP primer (positions 3449; 5'-TTCTGTTCCAGGAGCTT-3') and 3'-TRAP primer (positions 602618; 3'-CAGTCGGTCGTCGGACT-5') was ligated into a pCR 2.1 vector and then transformed into TOP 10 F' cells using a TA Cloning Kit (Invitrogen; Leek, The Netherlands) according to the manufacturer's protocol. Cells were plated onto Luria-Bertani-41 (LB) plates containing ampicillin (50 g/ml) and 5-bromo-4-chloro-3-indolyl · -D-galactopyranoside (X-gal) (40 µl; 40 mg/ml) and screened for colonies containing vector with insert using blue/white selection.
Plasmids from selected colonies were miniprepared according to the protocol of Maniatis (
Immunohistochemistry
Paraffin sections of brain, spinal cord, spinal ganglia, trigeminal ganglion, celiacsuperior mesenteric ganglion complex, superior cervical ganglion, and long bones were deparaffinized and rehydrated, and then incubated in a microwave oven in citrate buffer, pH 6.0 (1.8 mM citric acid, 9.8 mM sodium citrate) at 1100 W for 3 min and then at 160 W for another 12 min. After cooling to RT, immunostaining was achieved using 200 µl polyclonal antiserum, diluted 1:500, against TRAP as previously described (
Incubation of parallel sections with primary antibodies preabsorbed with 1 µg purified PAP antigen or with normal swine serum (X0901; DAKO) diluted 1:500 served as controls.
Some of the sections were counterstained with Ehrlich hematoxylin before dehydration and mounting.
The localization of TRAP immunoreactivity and the anatomic structures were identified by using a stereotaxic atlas (
DIG Labeling of RNA Probe
The plasmid pT7T3 19U (Pharmacia) containing rat TRAP cDNA fragment of 832 bp (
In Situ Hybridization Histochemistry
Paraffin sections prepared as described above were heated overnight at 55C. After deparaffinization and rehydration, the sections were washed in PBS (0.145 M NaCl, 0.01 M Na2PO4). After treatment with pepsin (1.5 mg/ml) (Boehringer Mannheim) in 0.02 M HCl at 37C for 20 min and washing in PBS, the sections were postfixed in 2% paraformaldehyde in PBS for 5 min at RT and then washed in PBS. The sections were prehybridized in 2 x SSC (1 x SSC = 150 mM NaCl, 15 mM sodium citrate)/50% formamide at 42C for 2 hr. Hybridization was performed in 30 µl of a solution containing 2 x SSC, 2 x Denhardt's, 1 mM EDTA, 0.25 mg/ml tRNA, 100 mg/ml dextran sulfate, 50% formamide, and 2.5 ng/ml probe at 42C overnight. The sections were washed twice in 2 x SSC for 10 min and then three times in 0.1 x SSC for 15 min at RT. The detection was performed using the DIG Nucleic Acid Detection Kit (Boehringer Mannheim) according to the manufacturer's protocol. After counterstaining with Ehrlich hematoxylin, the sections were dehydrated and mounted. Sections exposed to DIG-labeled sense probes served as negative controls.
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Results |
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Partial Purification and Characterization of TRAP Activity from Rat Neural Tissue
TRAP was partially purified from trigeminal ganglion, spinal cord, brain and, as a comparison, from rat bone, according to an established procedure (
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To examine the sensitivity to different oxyanion inhibitors of TRAP from the trigeminal ganglion, spinal cord, brain, and bone, the IC50 values for the known PAP/TRAP inhibitors molybdate, tungstate, and phosphate were determined (Table 1). The IC50 values were derived from linear regression analysis. The sensitivity of TRAP enzyme activity for these inhibitors was similar in the different tissues, and the rank order of potency was molybdate>tungstate>phosphate.
Identification of TRAP Protein and mRNA in Neural Tissue
Immunoblotting analysis of partially purified TRAP (30 mU) from the trigeminal ganglion, spinal cord, and brain showed the appearance of a protein band with the expected size of 3537 kD in the absence of -mercaptoethanol as the disulfide reductant (Fig 1). Recombinant rat TRAP (100 ng) expressed from a Baculovirus vector was used as a positive control (
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Nested RT-PCR of cDNA from bone, trigeminal ganglion, spinal cord, and brain was performed with 40 cycles, resulting in a PCR product of 356 bp corresponding to the theoretical size (Fig 2C).
Immunohistochemical Localization of TRAP
TRAP-like immunoreactivity was encountered in neuronal cell bodies in both the central (Fig 3 Fig 4 Fig 5 Fig 6 Fig 7) and peripheral (Fig 8 and Fig 9) nervous systems of untreated rats. The signal was seen in the cytoplasm of the cells but was not uniformly distributed. Instead, the signal appeared in aggregated structures (see Fig 5 and Fig 7 Fig 8 Fig 9). Immunoreactive material could also be seen in neuronal processes close to the cell soma (Fig 5A). A very weak immunoreactive signal was observed in nerve fibers in the dorsal horn of the spinal cord. TRAP-immunoreactive material was also seen in glial cells, e.g., in the white matter of corpus callosum (Fig 3A3C).
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Central Nervous System. Wide distribution of TRAP-like immunoreactivity was observed in neurons in the rat brain and spinal cord. A large number of neuron populations were labeled but with a signal of variable intensity. The overall distribution is shown in the set of color micrographs (Fig 3A3C) representing different levels of the brain. A strong signal was noted in the neurons in the septum and diagonal bands (Fig 3A), ventral pallidum, cerebral cortex, and piriform cortex (Fig 3A3C). Intensely stained cells were also observed in the triangular septal nucleus. In the cerebral cortex, the most intense staining was seen in neurons in Layers V and VIb, somewhat less intense in Layer VIa, less in Layers II and III, and very weak in Layer I (Fig 3A3C). All of the pyramidal cells in hippocampal fields CA3 and CA4 (Fig 3B3C, Fig 6, and Fig 7B) were intensely immunoreactive, whereas in the CA1 (Fig 6 and Fig 7A) and CA2 (Fig 6) fields a strong signal was limited to scattered neurons. A strong signal could be demonstrated in neurons in the hilar region of the hippocampus, whereas the majority of the granule cells of the dentate gyrus exhibited a weaker signal (Fig 6 and Fig 7B). A few scattered granule cells in the dentate gyrus had a stronger TRAP immunoreactivity (Fig 7B). Strong TRAP-like immunoreactivity could be seen in neurons in the exterior globus pallidus (Fig 3B and Fig 8D) and the caudate nucleus (Fig 3B and Fig 8B), particularly in larger cells (Fig 8B), and in the interior globus pallidus (Fig 3B and Fig 8A) and medial habenula (Fig 3B). Neurons in the thalamus and hypothalamus were intensely stained, with particularly strong labeling in the reticular, rhomboid, anterodorsal, and ventral posterolateral thalamic nuclei (Fig 3B). Moderate staining was observed in the dorsal nucleus of the hippocampal commissure, ventromedial hypothalamic nucleus, and basolateral amygdaloid nucleus. Weak labeling was observed in the dorsomedial and ventrolateral laterodorsal thalamic nuclei. There was intense TRAP-like immunoreactivity in neurons of the substantia nigra pars compacta and reticulata (Fig 3C and Fig 8C), neurons of the Darkschewitsch nucleus, the lateral mammillary nucleus, the magnocellular red nucleus, and the postcommissurial magnocellular nucleus (Fig 3C).
In the pons and medulla oblongata, intensely stained neurons were present, e.g., in the dorsal part of the central gray and the deep gray layer of the superior colliculus (Fig 5A), the spinal trigeminal nucleus, the inferior olive, and the parvocellular reticular nucleus alpha (Fig 4B). A strong signal was seen in the neurons in the spinal cord (Fig 4C), particularly within -motor neurons in the ventral spinal cord (Fig 4C and Fig 5B). A very weak signal was noted in nerve fibers in the dorsal horn of the spinal cord. In the olfactory bulb, intense staining of nerve cells was observed in the mitral cell layer and external plexiform layer (Fig 4A). Scattered cells were also detected in the internal plexiform and granular layers and in the glomerular layer. Medium immunoreactivity could be seen in the mammillary nucleus. In the cerebellum, intense labeling was observed in the Purkinje cells, and in scattered neurons in the molecular and granular layer (Fig 4B, Fig 9A, and Fig 9B).
Peripheral Nervous System. TRAP-like immunoreactivity was observed in all neuronal cell bodies in both sensory ganglia, and paravertebral and prevertebral sympathetic ganglia (Fig 10 and Fig 11). Medium to large nerve cells in sensory ganglia, including the trigeminal ganglion (Fig 10A and Fig 10C) and dorsal root ganglia (Fig 11C and Fig 11F), were intensely labeled, whereas smaller neurons exhibited a weaker staining intensity. Similarly, in the superior cervical ganglion (Fig 11A and Fig 11D) and the celiac ganglion (Fig 11B and Fig 11E), larger neurons were more intensely stained than smaller nerve cells, although the difference in cell size was not as marked as in the sensory ganglia.
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Controls. No signal could be seen in sections incubated with primary antiserum preabsorbed with the antigen (see Fig 3D, Fig 9C, and Fig 10D) or after omission of the primary antiserum (not shown).
Histochemical Localization of TRAP mRNA by In Situ Hybridization
Central Nervous System.
The distribution of TRAP mRNA expression closely resembled that of the TRAP-like immunoreactivity in the spinal cord (Fig 4D and Fig 5C). In the cerebellum, a strong signal was seen in Purkinje cells (Fig 9D and Fig 9E), and intense labeling was also observed in some cells in the molecular layer (Fig 9D and Fig 9E).
Peripheral Nervous System. In the trigeminal ganglion, TRAP mRNA expression was most marked in small to medium-sized neurons, whereas the larger neurons were weakly labeled (Fig 10B and Fig 10E).
Controls. No signal could be seen after hybridization with labeled sense probe (Fig 9F) or after treatment with RNase before the hybridization (not shown).
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Discussion |
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This article describes the characterization and distribution of TRAP (or PAP) in the central and peripheral nervous systems of the rat. The TRAP enzyme is commonly isolated as the purple and catalytically inactive Fe(III)Fe(III) variant (
After the demonstration of TRAP enzymatic activity, it was of interest to investigate whether the TRAP enzyme could be demonstrated at the protein and mRNA levels, to ascertain that the TRAP enzyme activity indeed reflects the presence of TRAP protein. Furthermore, the levels of TRAP enzyme activity and TRAP mRNA, respectively, were compared among different tissues using a TRAP assay and RT-PCR. The results from these experiments show that TRAP enzyme activity and mRNA levels correlate well. In the morphological studies there was some discrepancy with regard to the granular layer of the cerebellar cortex. The granule cells showed TRAP-like immunoreactivity but no or very weak signal for TRAP mRNA. This could represent low sensitivity of the ISH histochemical method used or a fast turnover of the TRAP mRNA in these cells.
The PAP enzymes expressed in bone and spleen are monomeric proteins ranging in size between 34 and 37 kD, depending largely on differences in the post-translational modifications (
Immunohistochemical analysis showed a widespread distribution of TRAP-like immunoreactivity in both the central and peripheral nervous systems. In view of the suggested close evolutionary relationship between the PAP/TRAPs and the protein phosphatases PP1, PP2A, and PP2B (or calcineurin) (
The isoforms of PP-1 are also extensively distributed in the brain ( and PP-1
1 and contains small amounts of PP-1
mRNA (
and PP-1
1 immunoreactivity to dendritic spines has been observed in the caudate nucleus and hippocampus (
The distribution of PP-2A in the rat brain (
FRAP enzyme activity has been shown to occur in the nervous system, and the localization of TRAP to neurons of sensory ganglia coincides with that of FRAP. However, FRAP is restricted to small sensory neurons (see
The TRAP-immunoreactive material had a very distinct pattern, particularly in the large motor neurons in the spinal cord and in sensory ganglia, where the immunoreactive material was seen as aggregated round structures all over the cytoplasm. Electron microscopic analysis will be necessary to identify these structures, but it is possible that, similarly to the subcellular localization in osteoclasts, the TRAP-immunoreactive material may reside in lysosome-like structures (
A role for TRAP/PAP enzymes in the formation of oxygen-derived free radicals has been indicated by several studies (
Copperzinc superoxide dismutase (CuZn SOD), an enzyme that may function both as a free radical scavenger and as a generator of ROS, has a widespread distribution in the rat brain (
In bone, TRAP has been ascribed a role in bone resorption on the basis of its localization to osteoclasts (
Interestingly, OPN mRNA expression was recently demonstrated in neurons in the rat brain (
Notably, there was an induction of OPN mRNA in the rat striatum and hippocampus after global forebrain ischemia (
In conclusion, the present data demonstrate that an enzymatically active TRAP protein occurs in the rat nervous system. Whether or not there are specific molecular forms of TRAP in the nervous system awaits further analysis. The distribution of TRAP within the nervous system appears to indicate that a large number of neuronal cell types have the capacity to synthesize this enzyme but, depending on the levels, that it may contribute differentially to neuronal survival and response to oxidative stress. Co-localization studies to identify, by neurotransmitter content or glial markers, which populations of cells express different levels of TRAP will be the starting point for understanding whether TRAP may be involved in pathological processes such as Alzheimer's disease.
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Acknowledgments |
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Supported by grants from the Swedish Medical Research Council (10363), the Research Funds of Karolinska Institutet, and EC Biotechnology Program 6.1 (contract B104-CT-98-0385).
We are grateful for the expert technical assistance of Maria Norgård.
Received for publication May 31, 2000; accepted October 12, 2000.
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