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Neuroscience Letters 192 (1995) 137-141

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Neurons of the peripheral nervous system express thymidine phosphorylase P.A. Eccleston, K. Funa, C.-H. H e l d i n * Ludwig Institute for Cancer Research, Biomedical Centre, S-751 24 Uppsala, Sweden

Received 17 January 1995; revised version received 5 May 1995; accepted 8 May 1995

Abstract

Platelet-derived endothelial cell growth factor (PD-ECGF) is an angiogenic factor which recently has been shown to be identical to thymidine phosphorylase. We describe here, high levels of expression of PD-ECGF/thymidine phosphorylase in neurons of the peripheral nervous system (PNS) but very little in the central nervous system (CNS). Monoclonal and polyclonal antibodies were used for the staining of sections of dorsal root ganglia, sympathetic cervical ganglia and the enteric plexus as well as the brain and spinal cord. In addition, in situ hybridisation confirmed the results of immunohistochemistry. The possible role of thymidine phosphorylase in the PNS is discussed.

Keywords: Sensory neurons; Peripheral nervous system; Central nervous system; Immunohistochemistry; Platelet-derived endothelial cell growth factor

Platelet-derived endothelial cell growth factor (PDECGF) is a protein of molecular weight 45 000, initially purified from human platelets, which stimulates chemotaxis of endothelial'cells in vitro and angiogenesis in vivo [1,2]. It was later found to be similar to the bacterial enzyme thymidine phosphorylase, and indeed to have thymidine phosphorylase activity [3-7]. Moreover, a molecule denoted gliostatin is almost identical to PDECGF and has been shown to have a survival effect on some central cortical neurons [8-10]. Thymidine phosphorylase (EC 2.4.2.4.) catalyses the reversible phosphorolysis of thymidine and most other pyrimidine-2-deoxyribosides as follows: thymidine+ orthophosphate ~ thymine + 2-deoxy-d-ribose-1phosphate. Thymine may be re-utilised for nucleoside synthesis, and pentose-l-phosphate converted into intermediates in the pentose phosphate shunt and glycolysis. The angiogenic effect of PD-ECGF/thymidine phosphorylase (PDECGF/TP) appears to be exerted by 2-deoxy-d-ribose, presumably derived from one of the products of the enzyme, i.e. 2-deoxy-d-ribose- 1-phosphate [ 11 ]. Thymidine phosphorylase does not appear to simply be a ubiquitous housekeeping enzyme since its expression * Corresponding author, Tel.: +46 18 174146; Fax: +46 18 506867.

shows a tissue specific pattern [12]. It is expressed at high levels in liver, lung, spleen, mature lymphocytes and lymph nodes and its expression may be elevated in some tumours [12,13]. In this study we describe high expression levels of this enzyme in neurons of the peripheral nervous system (PNS) but not the central nervous system (CNS) and we discuss its potential role in vivo. Dorsal root ganglia (DRG), sympathetic cervical ganglia, spinal cord, brain and colon were dissected from Sprague-Dawley rats of different postnatal ages under general anaesthesia by thiobarbital. Frozen tissue sections were fixed in acetone for immunohistochemistry and 4% paraformaldehyde for in situ hybridisation. Cells from rat sciatic nerve, from ld, 8d and adult nerves were dissociated and centrifuged onto microscope slides as described previously [14], in order to examine expression in Schwann cells at the level of the single cell. As controls, CHO cells (untransfected and transfected with PD-ECGF/TP cDNA) grown in serum containing medium were centrifuged onto microscope slides using a Shandon Cytospin centrifuge. ABC peroxidase immunohistochemistry was performed as described previously [14]. Two different antibodies were used for PD-ECGF/TP immunohistochemistry, KM-6 which is a rabbit polyclonal antiserum against PD-ECGF/TP purified from human platelets [15], and B4

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P.A. Eccleston et al. / Neuroscience Letters 192 (1995) 137-141

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Fig. 1. Western blot analysis of PD-ECGF/TP in 5-day-old rat dorsal root ganglia. An extract of dorsal root ganglia was electrophoresed under reducing conditions (2). Recombinant human PD-ECGF was used as a control (1). The blot was incubated with a rabbit antisera against PD-ECGF/TP (KM6), and immunocomplexes visualised with the ECL method. which is a monoclonal antibody against recombinant PDECGF/TP, and provided by A. Thomason, Amgen Inc., CA. The KM-6 antiserum was used at 1:1500 dilution and the monoclonal antibody at 10/*g/ml. As controls, the first antibodies were omitted or blocked with antigen, or irrelevant antibodies were used. To identify different cell types in the tissue sections, stainings were also performed using rabbit antisera against glial fibrillary acidic protein (GFAP) [16] and neurofilament (200 kDa subunit; Sigma Chemical Co.). In vitro transcription of cRNA probes and in situ hybridisation were performed as described previously [17]. A 1.5 kb E c o R I fragment of PD-ECGF/TP cDNA inserted in a Bluescript vector (Stratagene) was used as a template (a gift from K. Usuki). As a negative control, a probe labelled in the sense direction was used, and as a positive control, a platelet-derived growth factor (PDGF) A-chain probe was used. Western blot analyses on DRG from 5-day-old rats were carried out as described before [14]. A protein fraction obtained by the method of Wessel and Flugge [18], and recombinant PD-ECGF/TP as a control, were examined using KM6 polyclonal rabbit antisera (diluted

1:50). Fig. 1 shows that when an extract of DRG was analysed, the PD-ECGF/TP polyclonal antisera KM6 recog-

nised a single band of approximately 45 kDa which migrated with recombinant human PD-ECGF/TP. Using immunohistochemistry, we were able to detect PDECGF/TP in most of the neurons of the DRG (Figs. 2a,c). It was present in DRG of rats of all ages examined and no quantitative developmental changes could be observed (data not shown). Both the polyclonal antiserum (Fig. 2c) and a monoclonal (Fig. 2a) gave essentially the same resuits. Expression was very high in small dark neurons [19] corresponding to C-fibre sensory neurons. The level of expression was very variable in the neuronal population, and when the antibodies were used at high dilutions, some neurons were consistently unstained while others remained strongly labelled. Only low levels were detectable in the satellite cells and Schwann cells. When compared to neurofilament labelling (Fig. 2b), some axons appeared to have little or no labelling, suggesting that PDECGF/TP labelling was probably restricted to the axons of heavily labelled neurons. Freshly isolated and cultured Schwann cells only expressed low levels of PD-ECGF/TP (not shown). In situ hybridisation revealed that neurons of the DRG produced easily detectable levels of PD-ECGF/TP mRNA (Fig. 2f). This was similar in sections of DRG from ld, 8d and adult rats and all neurons appeared to be positive. Sense controls were negative (not shown). CHO cells transfected with PD-ECGF, used as a positive control also gave very heavy labelling, whereas untransfected cells were slightly positive, probably due to endogenous expression by these tumour cells (not shown). Schwann cells did not show expression of PD-ECGF/TP m R N A above background levels (Fig. 2f). To determine whether the striking expression of PDECGF/TP was restricted to sensory neurons we examined enteric neurons of the rat colon. Fig. 3a shows strong labelling of neurons by anti-PD-ECGF antibodies in the mesenteric plexus. These neurons were also neurofilament positive (Fig. 3b) but were not stained with antismooth muscle actin antibodies which, however, stained smooth muscle of the gut wall and blood vessels (not shown). In addition, expression of PD-ECGF/TP was examined in the sympathetic neurons of the superior cervical ganglia and was also found to be high in these neurons (Fig. 2e). However, the heterogeneous staining pattern which was found in the DRG was neither seen in these neurons nor in the enteric neurons. Sections of brain and spinal

Fig. 2. Expression of PD-ECGF/TP in the PNS. (a) 8-day-old rat dorsal root ganglia labelled with monoclonal anti-PD-ECGF/TP; (b) 8-day-old rat dorsal root ganglia labelled with rabbit anti-neurofilament; (c) 8-day-old rat dorsal root ganglia labelled with polyelonal rabbit anti-PD-ECGF/TP; (d) the control of (c) which was blocked with a 3-fold excess of human recombinant PD-ECGF/TP; (e) adult rat superior cervical ganglia labelled with monoclonal anti-PD-ECGF(bar = 20#m); (f) expression of PD-ECGF/TPmRNA in rat dorsal root ganglia. Sections of dorsal root ganglia from 8-dayold rats were hybridised with a 35S labelled cRNA probe. The neurons are clearly labelled and the controls did not show any specific labelling, viewed by dark field illumination (bar = 50/tm). Fig. 3. Expression of PD-ECGF/TP in the enteric nervous system. Sections of colon were labelled with monoclonal anti-PD-ECGF/TP(a), or with a anti-neurofilament monoclonal(b). Bar = 20~m.


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cord expressed very low levels of PD-ECGF/TP and the expression was only detected in the axons of neurons (not shown). We have demonstrated a high level of expression of PD-ECGF/TP in neurons of the DRG, superior cervical ganglia and enteric plexus. Indeed it was present in all the neuron types examined in the PNS but was low in the CNS. It could be seen in the axons of some PNS neurons and may be transported by axoplasmic flow to nerve endings. The reason for expression of such high levels by PNS neurons is currently not understood. The small dark sensory neurons express the highest levels of PD-ECGF protein. We did not examine metabolism/turnover but these cells were not highly labelled by in situ hybridisation, indicating that the protein may accumulate more in some neurons than others. Many different types of molecules act as putative neurotransmitters. Adenosine and ATP have long been studied as neurotransmitters and thus, it seems possible that thymine, thymidine or even ribose phosphate could act as neurotransmitters; if so, thymidine phosphorylase could regulate the synthesis of the neurotransmitter. Several studies indicate that PD-ECGF/TP is associated with more highly differentiated cells. Schwartz et al. [20] studied changes in thymidine salvage, as a population of human keratinocytes differentiated and showed that thymidine phosphorylase activity doubled as differentiation occurred, whilst thymidine kinase activity was rapidly lost. Moreover, neuroblastoma cells have been investigated and a more malignant, rapidly dividing cellline expressed 15 times less thymidine phosphorylase than a slowly dividing cell-line [21]. In addition, thymidine phosphorylase was expressed in quiescent astrocytes and not in the rapidly proliferating population [9]. It is possible that availability of thymidine phosphates plays a role in neuronal differentiation, as was suggested for keratinocytes. Asai et al. [10] showed that gliostatin/PD-ECGF/TP supported the survival of cortical neurons of the CNS. The mechanism for this is not understood and it is not known if the enzyme is a trophic substance in vivo. One possibility is that a precursor of thymidine metabolism, lacking in the culture medium, may be provided by the action of this enzyme. In a similar manner, thymidine phosphorylase could act as a survival factor on the neurons of the PNS, or alternatively on the satellite or Schwann cells surrounding them, or it could be transported to, and act in the CNS. PD-ECGF/TP has angiogenic activity in the chick chorioallantoic membrane [1]. The mechanism for this effect is likely to be indirect, involving products of its thymidine phosphorylase activity. Recent observations by Haraguchi et al. [11] suggest that 2-deoxy-d-ribose, formed from one of the products of the enzyme, is the angiogenic compound. Adenosine, a purine base, has

been demonstrated to stimulate angiogenesis [22], but thymidine and thymine were found to be inactive [11]. We are grateful to L. Jorge Gonez for helpful discussion, J. Cohen for comments on the manuscript, A. Thomason for his gift of B4 monoclonal antibody and recombinant PD-ECGF/TP, K. Usuki for the plasmid containing PD-ECGF/TP and CHO cells transfected with PDECGF/TP cDNA, A. Ahgren for technical assistance and I. Schiller for secretarial assistance. P.A. Eccleston was a Wellcome Traveling Research Fellow at the Ludwig Institute for Cancer Research. Partial support was provided by the Fredrik and Ingrid Thuring's foundation. [1] Ishikawa, F., Miyazono, K., Hellman, U., Drexler, H., Wernstedt, C., Hagiwara, K., Usuki, K., Takaku, F., Risau, W. and Heldin, C.-H., Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor, Nature, 338 (1989) 557-562. [2] Miyazono, K., Okabe, T., Urabe, A., Takaku, F. and Heldin, C.H., Purification and properties of an endothelial cell growth factor from human platelets, J. Biol. Chem., 262 (1987) 4098-4103. [3] Barton, G.J., Ponting, C.P., Spraggon, G., Finnis, C. and Sleep, D., Human PD-ECGFis homologous to Esherishia coli thymidine phosphorylase, Protein Sci., I (1992) 688-690. [4] Finnis, C., Dodsworth, N., Pollitt, C.E., Carr, G. and Sleep, D., Thymidine phosphorylase activity of platelet-derived endothelial cell growth factor is responsible for endothelial cell mitogenicity, Eur. J. Bioehem., 212 (1993) 201-210. [5] Furukawa, T., Yoshimura, A., Sumizawa, T., Haraguchi, M., Akiyama, S., Fukui, K., Ishizawa, M. and Yamada, Y., Angiogenic factor, Nature, 356 (1992) 668. [6] Moghaddam, A. and BrickneU, R., Expression of platelet-derived endothelial cell growth factor (PD-ECGF) in Escherichia coli and confirmation of its thymidine phosphorylase activity, Biochemistry, 31 (1992) 12141-12146. [7] Usuki, K., Saras, J., Waltenberger, J., Miyazono, K., Pierce, G., Thomason, A. and Heldin, C.-H., Platelet-derived endothelial cell growth factor has thymidine phosphorylase activity, Bioehem. Biophys. Res. Commun., 184 (1992) 1311-1316. [8] Asai, K., Hotta, T., Nakanishi, K., Ito, J., Tanaka, R., Otsuka, T., Matsui, N. and Kato, T., von Recklinghausen neurofibroma produces neuronal and glial growth-modulating factors, Brain Res., 556 (1991) 344-348. [9] Asai, K., Hirano, T., Kaneko, S., Moriyama, A., Nakanishi, K., Iosobe, I., Eksioglu, Y.Z. and Kato, T., A novel growth inhibitory factor, gliostatin, derived from neurofibroma, J. Neurochem., 59 (1992a) 307-317. [10] Asai, K., Nakanishi, K., Isobe, 1., Eksioglu, Y.Z., Hirano, A., Hama, K., Miyamoto, T. and Kato, T., Neurotrophic action of gliostatin on cortical neurons: identity of gliostatin and plateletderived endothelial cell growth factor, J. Biol. Chem., 267 (1992b) 20311-20316. [I1] Haraguchi, M., Miyadera, K., Uemura, K., Sumizawa, T., Furukawa, T., Yamada, K., Akiyama, S.-i. and Yamada, Y., Angiogenie activity of enzymes, Nature, 368 (1994) 198. [12] Yoshimura, A., Kuwazuru, Y., Fumkawa, T., Yoshida, H., Yamada, K. and Akiyama, S., Purification and tissue distribution of human thymidine phosphorylase; high expression in lymphocytes, reticuloeytes and tumonrs, Biochem. Biophys. Acta, 1034 (1990) 107-113. [13] Usuki, K., Heldin, N.-E., Miyazono, K., Ishikawa, F., Takaku, F., Westermark, B. and Heldin, C.-H., Production of platelet-derived endothelial cell growth factor by normal and transformed human cells in culture, Proe. Natl. Acad. Sci. USA, 86 (1989) 7427-


7431. [14] Eccleston, P.A, Funa, K. and Heldin, C.-H., Expression of platelet-derived growth factor (PDGF) and PDGF a- and fl-receptors in the peripheral nervous system: an analysis of sciatic nerve and dorsal root ganglia, Dev. Biol., 155 (1993) 459-470. [15] Miyazono, K. and Heldin, C.-H., High-yield purification of platelet-derived endothelial cell growth factor: structural characterization and establishment of a specific antiserum, Biochemistry, 28 (1989) 1704-1710. [16] Jessen, K.R., Thorpe, R. and Mirsky, R., Molecular identity, distribution and heterogeneity of glial fibrillary protein: an immunoblotting and immunohistochemical study of Schwann cells, satellite ceils, enteric glia and astrocytes, J. Neurocytol., 13 (1984) 187-200. [17] Hermanson, M., NistEr, M., Betsholtz, C., Heldin, C.-H., Westermark, B. and Funa, K., Endothelial cell hyperplasia in human glioblastoma: co-expression of mRNA for platelet-derived growth factor (PDGF) B chain and PDGF receptor suggests autocrine

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