Cellular Prion Protein Is Expressed in a Subset of Neuroendocrine Cells of the Rat Gastrointestinal Tract
Department of Histology and Pathology (ZM,KP,MEB,MPS) and Morphology and Image Unit, School of Medicine, Foundation for Applied Medical Research (LG), University of Navarra, Pamplona, Spain
Correspondence to: María P. Sesma, Dept. of Histology and Pathology, University of Navarra, 31008 Pamplona, Spain. E-mail: mpsesma{at}unav.es
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: prion gastrointestinal tract neuroendocrine cells
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PrPc is a membrane sialoglycoprotein expressed at high concentrations in the CNS (Moudjou et al. 2001) and at lower concentrations in many other peripheral tissues (Fournier 2001
). In the case of acquired prion diseases, the infectious agent must first enter and then spread to the CNS. The lymphoreticular system plays an important role in the replication of the scrapie protein (Brown et al. 1999
). On the other hand, the pathways of neuroinvasion are less known, although some routes have been proposed (Beekes et al. 1998
; Aguzzi 2001
). Many authors look to the GI tract as the principal site of entry for the infectious agent (Prinz et al. 2003
), but how this passage through the GI mucosa occurs is one of the main questions. PrP is believed to be incorporated from the plasma membrane into endocytic organelles by constitutive transport, although most PrPc recycles back to the membrane without degradation. However, no agreement exists about the intracellular route. Reports on both transport in clathrin-coated vesicles (Shyng et al. 1995
) and caveolae-mediated uptake exist (Vey et al. 1996
; Peters et al. 2003
). Several works have suggested that PrPc is concentrated in caveolae-like domains (CLDs), cell structures involved in crucial physiological processes (Kurzchalia and Parton 1999
; Parton 2003
). This is why CLDs appear to have an important role in the PrPc-to-PrPsc conversion process.
To understand the process of how and where the infectious agent enters, it is important to know the cell types that express host PrPc along the GI wall. However, few studies have investigated PrPc in the GI tract, and those few have been carried out in mouse (Ford et al. 2002), hamster, and human (Fournier et al. 1998
,2000
), with a lack of consistency in results. We have studied the rat GI tract because the only PrPc expression analysis in this species has been done with brain, placenta, uterus, and testis (Tanji et al. 1995
).
The aims of the present work were to detect, by a combination of cellular and molecular techniques, the presence of PrPc in rat GI tract and to identify PrPc-producing cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Antisera
Two antisera against prion protein were used in this study: monoclonal 6H4 (Prionics; Zurich, Switzerland) and polyclonal IgG affinity-purified 91511 (Assay Designs; Ann Arbor, MI) antibodies. In addition, various antisera against some neuroendocrine cell markers (Table 1) were used for further identification of PrP-immunoreactive cells.
|
RNA Extraction and RT-PCR
Total RNA was obtained with the Ultraspec RNA Kit (Biotecx; Houston, TX), according to the manufacturer's instructions. The RNA concentration was spectrophotometrically determined. RNA was retrotranscribed with SuperScript II RNase-free Reverse Transcriptase (Invitrogen; Carlsbad, CA). PrP primers (Table 2) were designed based on the published rat sequence [GenBank accession numbers D50092 (exons 1, 2) and D50093 (exon 3)] and synthesized by Invitrogen. PCR was first performed to rule out genomic DNA contamination, employing a pair of primers including exons 2 and 3 of the PrP gene. Then PCR was carried out with two sets of primers that amplified the exon 3 region that encodes for PrP protein. PCR was performed in a total volume of 50 µl by using 2 µl of cDNA, 5 µl of 10 x buffer, 1.5 µl of MgCl2 (50 mM), 1 µl of dNTPs (10 mM), 1 µl of each primer (10 µM), 38 µl of ddH2O, 0.5 µl of BioTaq DNA polymerase (5 U/µl; Bioline, Canton, MA). After incubation at 94C for 10 min in a P-E Thermocycler-2400 (Perkin-Elmer; Foster City, CA), 30 cycles were performed for 30 sec at 94C, 30 sec at 59C, and 1 min at 72C, followed by 10 min at 72C. Water blank and non-RT samples were used as negative controls. To assess RNA integrity, rat GAPDH was amplified on the same samples. The primers used for GAPDH are shown in Table 2. PCR was performed in a total volume of 50 µl by using 1 µl of cDNA, 5 µl of 10 x buffer, 2.5 µl of MgCl2 (50 mM), 2.5 µl of dNTPs (10 mM), 2 µl of each primer (20 ng/µl), 34.5 µl of ddH2O, 0.5 µl of BioTaq DNA polymerase (5 U/µl; Bioline). After incubation at 95C for 5 min, 30 cycles were performed for 1 min at 95C, 1 min at 54C, and 1 min at 72C, followed by 10 min at 72C. Images were processed with Scion Image software (Scion; Frederick, MD) and captured in TIFF format.
|
Double Immunofluorescence
Tissue sections were deparaffinized, rehydrated, and microwave-preheated as described above. Slides were incubated with 2% bovine serum albumin (BSA; Sigma, St Louis, MO) in TBS for 30 min and incubated overnight at 4C with a mixture of the specific antisera (monoclonal and polyclonal) at their optimal dilutions. The slides were washed in TBS and incubated with a mixture of Alexa Fluor 568 TRITCgoat anti-mouse and Alexa Fluor 488 FITCgoat anti-rabbit antisera (Molecular Probes; Eugene, OR) for 45 min. Secondary antibody dilutions were 1:200 for PrPc and 1:400 for the rest of the primary antisera. Then slides were washed six times in 0.1% Tween-20 in TBS. Finally, slides were mounted in PBSglycerol. Each secondary reagent was confirmed to be species-specific by secondary antibody crossover (mouse or rabbit primary antibody followed by anti-rabbit or anti-mouse secondary, respectively). Sections were examined in a Nikon Eclipse E800M microscope equipped with filters giving excitation at a wavelength of 510560 nm for TRITC and 465495 nm for FITC, and images were captured in TIFF format with Analysis Soft Imaging System Gmbh software.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunoreactivity for PrPc in epithelial cells of the GI tract had already been described in human (Fournier et al. 1998,2000
; Pammer et al. 2000
), mouse (Ford et al. 2002
), and hamster (Fournier et al. 1998
,2000
). However, there is no agreement on which cell type expresses PrPc. In rat (present work), human (Pammer et al. 2000
), and mouse (Ford et al. 2002
) GI tract, PrPc immunoreactivity has been detected in endocrine cells. On the contrary, other authors have described PrPc immunolabeling in mucous and parietal cells in humans (Fournier et al. 1998
,2000
; Pammer et al. 2000
) and in mucous, parietal, and goblet cells in hamsters (Fournier et al. 1998
,2000
). Recently, PrPc has been localized in cellcell junction domains of human enterocytes (Morel et al. 2004
). These apparent discrepancies may be due to differences in the antisera, the fixation process, or the pretreatments used, or to changes in the pattern of expression of the protein among species. In fact, different subcellular locations for PrPc in the same cell type have been reported with the use of different antibodies (Fournier et al. 2000
). In transgenic mice expressing the green fluorescent protein reporter gene under the control of bovine PrP promoter, correlation between fluorescence and PrPc immunohistochemistry has been found but no fluorescence has been observed in gut epithelial cells (Lemaire-Vieille et al. 2000
). In the same way, other authors did not find PrPc in gut epithelium of neonatal mice (Okamoto et al. 2003
). These findings suggest that efficiency of the antisera and tissue processing are critical factors in detection of PrPc.
Our immunocytochemical findings are in agreement with the Western blot results because the more intense bands were found in GI regions (antrum and corpus) in which a high number of PrPc-positive cells were detected. In addition, demonstration of PrPc mRNA by RT-PCR further supports the expression of prion protein in the rat GI tract.
Few works have demonstrated the nature of PrPc positive cells as endocrine cells using phenotypic markers. The first report related to PrPc immunoreactivity in endocrine cells was in human antrum (Pammer et al. 2000). Using consecutive sections, PrPc-positive cells were identified as G-cells, although no images demonstrating this fact were shown. Similarly, in rat antrum we have also found co-localization between G and PrPc but a few 5HT- and Som-positive cells also showed PrPc labeling. Furthermore, human fundic glands were reported to be negative (Pammer et al. 2000
). On the contrary, in rat corpus we have found a high number of PrPc-positive cells in the basal region of the glands that co-localized with histamine, ghrelin, or Som. For small and large intestine, our results agree with those of Ford et al. (2002)
in relation to the co-localization between 5HT and PrPc. Nevertheless, the present study demonstrated that only a subset of 5HT cells were PrPc-immunoreactive and that staining for PrPc was also present in subpopulations of G, Som, and PYY cells. The presence of PrPc in subpopulations of particular neuroendocrine cell types suggests a tightly regulated expression of this protein.
The functional significance of the PrPc present in the neuroendocrine cells is not yet known. How the infectious agent crosses the gut wall and reaches germinal centers of lymphoid tissues or the peripheral nervous system also remains cryptic. The incorporation of PrPsc as a host PrPc-dependent process is a widely accepted idea because PrP knockout mice do not develop infection (Bueler et al. 1992). This is probably why some authors have pointed to enteroendocrine cells and other cell types, such as dendritic cells and intraepithelial lymphocytes, as the most probable PrPsc target cell populations (Ford et al. 2002
).
With regard to PrPsc interacting molecules, the infectious agent could interact with PrPc expressed by GI epithelial cells, forming a large molecular complex that would later be incorporated (Horiuchi et al. 1995; Fournier et al. 1998
). On the other hand, evidence has also pointed to the 37-kD laminin receptor precursor (LRP) as a PrPsc receptor (Rieger et al. 1997
; Shmakov et al. 2000
; Ghosh 2002
). LRP expression in human intestinal brush border (Shmakov et al. 2000
) and LRPPrPsc interaction (Rieger et al. 1997
) have been reported. PrPsc-receptor complexes would further reach the lymphoreticular system (van Keulen et al. 1996
; Hill et al. 1999
; Hilton et al. 2002
) and peripheral nerves (Groschup et al. 1996
; McBride and Beekes 1999
) before spreading to the CNS. As a possible infection transfer mechanism between cells, an intercellular traffic of glycosylphosphatidylinositol-anchored PrPc has been described (Liu et al. 2002
). This could explain PrPsc dissemination towards the nervous system once it crosses the epithelial barrier, because many nerve endings are present in tight contact with epithelial cells.
Only one theory exists about the role of enteroendocrine cells in the pathogenesis of spongiform encephalopathies. This theory postulates that changes in the properties of the GI epithelium might activate cells so that PrPsc could enter into them (Argenzio 1997). Here, PrPsc could replicate and then be secreted to reach the enteric nervous system or other non-enteric target cells throughout the bloodstream. In fact, a secretory form of PrPc has been reported (Hay et al. 1987
). In addition, PrPc expression in secretory cells, such as neuroendocrine cells, supports the possible involvement of this protein in the regulated secretory pathway, as has been suggested by other authors who also found PrPc in exocrine cells (Bendheim et al. 1992
; Fournier et al. 1998
). Nevertheless, PrPc expression in only specific neuroendocrine cell subpopulations is an intriguing point. The physiological role of PrPc might be connected to regulatory substances secreted by some neuroendocrine cells, but further research needs to be conducted.
In summary, PrPc is present in some but not all neuroendocrine cell types along the rat GI tract. This specific pattern indicates both a highly regulated expression and a particular role of this protein in these cells. Moreover, our results suggest that neuroendocrine cells may play an important role in the internalization of the PrPsc from gut lumen. Nevertheless, further studies are needed to verify this hypothesis.
![]() |
Acknowledgments |
---|
We thank Teresa Sabata, Paz Zamora, Silvia González, and Ainhoa Urbiola for technical assistance.
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aguzzi A (2001) Peripheral prion pursuit. J Clin Invest 108:661662
Argenzio RA (1997) Neuro-immune pathobiology of infectious enteric disease. Adv Exp Med Biol 412:2129[Medline]
Beekes M, McBride PA, Baldauf E (1998) Cerebral targeting indicates vagal spread of infection in hamsters fed with scrapie. J Gen Virol 79:601607[Abstract]
Bendheim PE, Brown HR, Rudelli RD, Scala LJ, Goller NL, Wen GY, Kascsak RJ, et al. (1992) Nearly ubiquitous tissue distribution of the scrapie agent precursor protein. Neurology 42:149156[Abstract]
Brown KL, Stewart K, Ritchie DL, Mabbott NA, Williams A, Fraser H, Morrison WI, et al. (1999) Scrapie replication in lymphoid tissues depends on prion protein-expressing follicular dendritic cells. Nat Med 5:13081312[CrossRef][Medline]
Bueler H, Fischer M, Lang Y, Bluethmann H, Lipp HP, DeArmond SJ, Prusiner SB, et al. (1992) Normal development and behaviour of mice lacking the neuronal cell-surface PrP protein. Nature 356:577582[CrossRef][Medline]
Ford MJ, Burton LJ, Morris RJ, Hall SM (2002) Selective expression of prion protein in peripheral tissues of the adult mouse. Neuroscience 113:177192[CrossRef][Medline]
Fournier JG (2001) Nonneuronal cellular prion protein. Int Rev Cytol 208:121160[Medline]
Fournier JG, Escaig-Haye F, Billette de Villemeur T, Robain O, Lasmezas CI, Deslys JP, Dormont D, et al. (1998) Distribution and submicroscopic immunogold localization of cellular prion protein (PrPC) in extracerebral tissues. Cell Tissue Res 292:7784[CrossRef][Medline]
Fournier JG, Escaig-Haye F, Grigoriev V (2000) Ultrastructural localization of prion proteins: physiological and pathological implications. Microsc Res Tech 50:7688[CrossRef][Medline]
Ghosh S (2002) Intestinal entry of prions. Z Gastroenterol 40:3740[CrossRef][Medline]
Groschup MH, Weiland F, Straub OC, Pfaff E (1996) Detection of scrapie agent in the peripheral nervous system of a diseased sheep. Neurobiol Dis 3:191195[CrossRef][Medline]
Hay B, Prusiner SB, Lingappa VR (1987) Evidence for a secretory form of the cellular prion protein. Biochemistry 26:81108115[Medline]
Hill AF, Butterworth RJ, Joiner S, Jackson G, Rossor MN, Thomas DJ, Frosh A, et al. (1999) Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet 353:183189[CrossRef][Medline]
Hilton DA, Ghani AC, Conyers L, Edwards P, McCardle L, Penney M, Ritchie D, et al. (2002) Accumulation of prion protein in tonsil and appendix: review of tissue samples. BMJ 325:633634
Horiuchi M, Yamazaki N, Ikeda T, Ishiguro N, Shinagawa M (1995) A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep. J Gen Virol 76:25832587[Abstract]
Kurzchalia TV, Parton RG (1999) Membrane microdomains and caveolae. Curr Opin Cell Biol 11:424431[CrossRef][Medline]
Lemaire-Vieille C, Schulze T, Podevin-Dimster V, Follet J, Bailly Y, Blanquet-Grossard F, Decavel JP, et al. (2000) Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice. Proc Natl Acad Sci USA 97:54225427
Liu T, Li R, Pan T, Liu D, Petersen RB, Wong BS, Gambetti P, et al. (2002) Intercellular transfer of the cellular prion protein. J Biol Chem 277:4767147678
McBride PA, Beekes M (1999) Pathological PrP is abundant in sympathetic and sensory ganglia of hamsters fed with scrapie. Neurosci Lett 265:135138[CrossRef][Medline]
Morel E, Fouquet S, Chateau D, Yvernault L, Frobert Y, Pincon-Raymond M, Chambaz J, et al. (2004) The cellular prion protein PrPC is expressed in human enterocytes in cell-cell junctional domains. J Biol Chem 279:14991505
Moudjou M, Frobert Y, Grassi J, La Bonnardiere C (2001) Cellular prion protein status in sheep: tissue-specific biochemical signatures. J Gen Virol 82:20172024
Okamoto M, Furuoka H, Horiuchi M, Noguchi T, Hagiwara K, Muramatsu Y, Tomonaga K, et al. (2003) Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice. Vet Pathol 40:723727
Pammer J, Cross HS, Frobert Y, Tschachler E, Oberhuber G (2000) The pattern of prion-related protein expression in the gastrointestinal tract. Virchows Arch 436:466472[CrossRef][Medline]
Parton RG (2003) Caveolaefrom ultrastructure to molecular mechanisms. Nat Rev Mol Cell Biol 4:162167[CrossRef][Medline]
Peters PJ, Mironov A Jr, Peretz D, van Donselaar E, Leclerc E, Erpel S, DeArmond SJ, et al. (2003) Trafficking of prion proteins through a caveolae-mediated endosomal pathway. J Cell Biol 162:703717
Prinz M, Huber G, Macpherson AJ, Heppner FL, Glatzel M, Eugster HP, Wagner N, et al. (2003) Oral prion infection requires normal numbers of Peyer's patches but not of enteric lymphocytes. Am J Pathol 162:11031111
Prusiner SB (1991) Molecular biology of prions causing infectious and genetic encephalopathies of humans as well as scrapie of sheep and BSE of cattle. Dev Biol Stand 75:5574[Medline]
Rieger R, Edenhofer F, Lasmezas CI, Weiss S (1997) The human 37-kDa laminin receptor precursor interacts with the prion protein in eukaryotic cells. Nat Med 3:13831388[Medline]
Sanchez MJ, Burrell MA (2002) Immunocytochemical detection of orexin A in endocrine cells of the developing mouse gut. J Histochem Cytochem 50:6369
Shmakov AN, Bode J, Kilshaw PJ, Ghosh S (2000) Diverse patterns of expression of the 67-kD laminin receptor in human small intestinal mucosa: potential binding sites for prion proteins? J Pathol 191:318322[CrossRef][Medline]
Shyng SL, Lehmann S, Moulder KL, Harris DA (1995) Sulfated glycans stimulate endocytosis of the cellular isoform of the prion protein, PrPC, in cultured cells. J Biol Chem 270:3022130229
Tanji K, Saeki K, Matsumoto Y, Takeda M, Hirasawa K, Doi K, Matsumoto Y, et al. (1995) Analysis of PrPC mRNA by in situ hybridization in brain, placenta, uterus and testis of rats. Intervirology 38:309315[Medline]
van Keulen LJ, Schreuder BE, Meloen RH, Mooij-Harkes G, Vromans ME, Langeveld JP (1996) Immunohistochemical detection of prion protein in lymphoid tissues of sheep with natural scrapie. J Clin Microbiol 34:12281231[Abstract]
Vey M, Pilkuhn S, Wille H, Nixon R, DeArmond SJ, Smart EJ, Anderson RG, et al. (1996) Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains. Proc Natl Acad Sci USA 93:1494514949