The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections
2007; Springer Nature; Volume: 26; Issue: 17 Linguagem: Inglês
10.1038/sj.emboj.7601830
ISSN1460-2075
AutoresJoel C. Watts, Bettina Drisaldi, Vivian Ng, Jing Yang, Bob Strome, Patrick Horne, Man‐Sun Sy, Larry K.K. Yoong, Rebecca Young, Peter Mastrangelo, Catherine Bergeron, Paul E. Fraser, George A. Carlson, Howard T.J. Mount, Gerold Schmitt‐Ulms, David Westaway,
Tópico(s)Food Allergy and Anaphylaxis Research
ResumoArticle16 August 2007Open Access The CNS glycoprotein Shadoo has PrPC-like protective properties and displays reduced levels in prion infections Joel C Watts Joel C Watts Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Search for more papers by this author Bettina Drisaldi Bettina Drisaldi Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Vivian Ng Vivian Ng Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Jing Yang Jing Yang Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Bob Strome Bob Strome Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Patrick Horne Patrick Horne Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Man-Sun Sy Man-Sun Sy Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Larry Yoong Larry Yoong Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Rebecca Young Rebecca Young McLaughlin Research Institute, Great Falls, MT, USA Search for more papers by this author Peter Mastrangelo Peter Mastrangelo Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Catherine Bergeron Catherine Bergeron Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Search for more papers by this author Paul E Fraser Paul E Fraser Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Medical Biophysics, University of Toronto, Toronto, Canada Search for more papers by this author George A Carlson George A Carlson McLaughlin Research Institute, Great Falls, MT, USA Search for more papers by this author Howard TJ Mount Howard TJ Mount Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Medicine, University of Toronto, Toronto, Canada Search for more papers by this author Gerold Schmitt-Ulms Gerold Schmitt-Ulms Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Search for more papers by this author David Westaway Corresponding Author David Westaway Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Centre for Prions and Protein Folding Diseases, University of Alberta, Alberta, Canada Search for more papers by this author Joel C Watts Joel C Watts Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Search for more papers by this author Bettina Drisaldi Bettina Drisaldi Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Vivian Ng Vivian Ng Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Jing Yang Jing Yang Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Bob Strome Bob Strome Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Patrick Horne Patrick Horne Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Man-Sun Sy Man-Sun Sy Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Larry Yoong Larry Yoong Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Rebecca Young Rebecca Young McLaughlin Research Institute, Great Falls, MT, USA Search for more papers by this author Peter Mastrangelo Peter Mastrangelo Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Search for more papers by this author Catherine Bergeron Catherine Bergeron Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Search for more papers by this author Paul E Fraser Paul E Fraser Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Medical Biophysics, University of Toronto, Toronto, Canada Search for more papers by this author George A Carlson George A Carlson McLaughlin Research Institute, Great Falls, MT, USA Search for more papers by this author Howard TJ Mount Howard TJ Mount Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Medicine, University of Toronto, Toronto, Canada Search for more papers by this author Gerold Schmitt-Ulms Gerold Schmitt-Ulms Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Search for more papers by this author David Westaway Corresponding Author David Westaway Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada Centre for Prions and Protein Folding Diseases, University of Alberta, Alberta, Canada Search for more papers by this author Author Information Joel C Watts1,2, Bettina Drisaldi1, Vivian Ng1, Jing Yang1, Bob Strome1, Patrick Horne1, Man-Sun Sy3, Larry Yoong1, Rebecca Young4, Peter Mastrangelo1, Catherine Bergeron1,2, Paul E Fraser1,5, George A Carlson4, Howard TJ Mount1,6, Gerold Schmitt-Ulms1,2 and David Westaway 1,2,7 1Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada 2Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada 3Department of Pathology, School of Medicine, Case Western Reserve University, Cleveland, OH, USA 4McLaughlin Research Institute, Great Falls, MT, USA 5Department of Medical Biophysics, University of Toronto, Toronto, Canada 6Department of Medicine, University of Toronto, Toronto, Canada 7Centre for Prions and Protein Folding Diseases, University of Alberta, Alberta, Canada *Corresponding author. Centre for Prions and Protein Folding Diseases, University of Alberta, Room 116, Environmental Engineering Building, Edmonton, Alberta, Canada T6G 2M8. Tel.: +780 492 9377; Fax: +780 492 9352; E-mail: [email protected] The EMBO Journal (2007)26:4038-4050https://doi.org/10.1038/sj.emboj.7601830 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cellular prion protein, PrPC, is neuroprotective in a number of settings and in particular prevents cerebellar degeneration mediated by CNS-expressed Doppel or internally deleted PrP ('ΔPrP'). This paradigm has facilitated mapping of activity determinants in PrPC and implicated a cryptic PrPC-like protein, 'π'. Shadoo (Sho) is a hypothetical GPI-anchored protein encoded by the Sprn gene, exhibiting homology and domain organization similar to the N-terminus of PrP. Here we demonstrate Sprn expression and Sho protein in the adult CNS. Sho expression overlaps PrPC, but is low in cerebellar granular neurons (CGNs) containing PrPC and high in PrPC-deficient dendritic processes. In Prnp0/0 CGNs, Sho transgenes were PrPC-like in their ability to counteract neurotoxic effects of either Doppel or ΔPrP. Additionally, prion-infected mice exhibit a dramatic reduction in endogenous Sho protein. Sho is a candidate for π, and since it engenders a PrPC-like neuroprotective activity, compromised neuroprotective activity resulting from reduced levels may exacerbate damage in prion infections. Sho may prove useful in deciphering several unresolved facets of prion biology. Introduction Prions are the causative agents of neurodegenerative diseases, which include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, chronic wasting disease in mule deer and elk, and Creutzfeldt–Jakob Disease (CJD) in humans. The infectious agent is believed to consist of improperly folded forms of a host-encoded protein, the cellular prion protein (PrPC). Conversion of PrPC into the disease-associated isoform, PrPSc, is thought to be the primary pathogenic event, although the mechanisms by which PrPSc causes disease are poorly understood. PrPC is absolutely required for disease progression, as PrP knockout (Prnp0/0) mice do not succumb to disease and do not propagate infectivity following intracerebral challenge with infectious prions (Büeler et al, 1992, 1993). The mammalian prion protein family currently consists of two proteins: PrPC which is expressed at high levels in the central nervous system (CNS), and Doppel (Dpl), a molecule with a similar three-dimensional structure, whose postnatal expression is normally confined to the testis (Silverman et al, 2000; Mo et al, 2001). Whereas a role for Dpl in the proper functioning of the male reproductive system has been confirmed in two lines of Dpl knockout mice (Behrens et al, 2002; Paisley et al, 2004), the function of PrPC, a well-conserved neuronal glycoprotein, comprises a conundrum, in part because phenotypic alterations in Prnp0/0 mice have been subtle or disputed. One emerging area of consensus concerns a protective effect of PrPC against neuronal insults. In particular, PrPC is upregulated following ischemic brain damage, in both humans and mice (McLennan et al, 2004; Weise et al, 2004). PrPC deficiency in mice increases infarct size following cerebral artery occlusion and increases caspase 3 activation (Spudich et al, 2005), and PrPC overexpression improves neurological behavior and reduces infarct volume in a rat stroke model (Shyu et al, 2005). Another widely observed and perhaps related phenomenon involves the ability of PrPC to protect against a variety of proapoptotic stimuli (Kuwahara et al, 1999; Bounhar et al, 2001; Chiarini et al, 2002; Cui et al, 2003; Sakudo et al, 2005; Lee et al, 2006; Novitskaya et al, 2006). Strong evidence for a neuroprotective activity for PrPC against apoptosis in vivo has come from studies of transgenic (Tg) mice expressing internally deleted forms of PrP or wild-type (wt)Dpl within the CNS. The presence of Dpl in the brain of Prnp0/0 mice leads to a neurodegenerative syndrome characterized by a profound apoptotic loss of cerebellar cells (Nishida et al, 1999; Moore et al, 2001; Rossi et al, 2001). A similar phenotype is observed when N-terminally truncated versions of PrPC (ΔPrP) are expressed in the brain (Shmerling et al, 1998). Remarkably, both syndromes are abrogated by the coexpression of wt PrPC. Recently, it has been shown that a smaller deletion restricted to the well-conserved central domain of PrP is sufficient to elicit a highly toxic phenotype in Prnp0/0 mice (Baumann et al, 2007; Li et al, 2007). The above studies have led to a model in which Dpl and ΔPrP initiate aberrant signaling through a hypothetical prion ligand termed LPrP, a process which is blocked by PrPC binding (Flechsig et al, 2004). Assuming that the interaction between PrPC and LPrP represents an essential physiological event, the authors also proposed the existence of a PrPC-like protein termed π, which binds to LPrP and is capable of compensating for the absence of PrPC in Prnp0/0 mice. To this date, no candidates for π (or LPrP) have been put forward. However, an open reading frame was discovered which, when translated, exhibits homology to the central hydrophobic domain in PrPC. This gene, denoted Sprn ('shadow of the prion protein'), is present from zebrafish to humans and is predicted to encode a short protein, Shadoo (Sho) (Premzl et al, 2003). Sprn is located on chromosome 7 in mice, away from the Prn gene complex on chromosome 2. Building on the genetic interaction between PrPC and Dpl or ΔPrP, we have established an assay for PrPC activity in primary cultures of cerebellar granule cells (Drisaldi et al, 2004, and references therein). Here, cerebellar granule neurons (CGNs) cultured from Prnp0/0 mice are transfected with plasmids encoding Dpl or PrP alleles of interest and individual apoptotic events scored. This assay recapitulates the phenotypes produced by multiple PrP alleles in Tg mice, including neurotoxicity of both Dpl and ΔPrP, and neuroprotective activity of PrPC against the toxicity elicited by either Dpl or ΔPrP. In conjunction with biochemical and histological analyses, we have used the CGN assay to explore the properties of Sho. We now demonstrate that Sho is a GPI-anchored neuronal glycoprotein present in the CNS from early postnatal life. Not only is Sho PrPC-like in its ability to protect against both Dpl and ΔPrP toxicity in the CGN assay, but it is also strikingly reduced in prion infections. Results Sho protein expression in N2a cells and brain Motivated by the absence of obvious phenotypic defects in adult Prnp0/0 mice, we considered proteins that might overlap functionally with PrPC. One criterion was evolutionary conservation. In this regard, bioinformatic analyses by Premzl and co-workers have yielded an interesting candidate in the shape of the Sprn open reading frame present in genomic DNA of species from mammals to fish (Premzl et al, 2003, 2004; Miesbauer et al, 2006) and which, unlike the hypothetical Prnt gene (Makrinou et al, 2002; Premzl et al, 2004), is present in the mouse genome. The architecture of the predicted Sho protein loosely resembles the flexibly disordered PrP N-terminus (Figure 1). Analyses of Sprn expression by RT–PCR (Premzl et al, 2003) and interrogation of expressed sequence tag databases (UniGene) imply expression in embryonic and adult neuronal tissue as well as the retina and visual cortex, but have yet to document spliced mRNAs. Consequently, we evaluated Sho as a putative third member of the prion gene family. Figure 1.Domain structure of Dpl, PrPΔ32–121, PrP and Sho. α-Helices (A, B and C) are boxed. A basic repeat region in Sho is stippled and the hydrophobic domain in PrP and Sho is striped. Neuroprotective 'determinants', sequences required for PrP's neuroprotective action and mapped genetically, are shown in boxed numbers, and an expanded view shows the central hydrophobic regions of PrP and Sho aligned with the T-COFFEE algorithm. PrP and Sho deletions used in this study are bracketed alongside the alignment. Two residues that correspond to the N-termini of human PrP 'C1' fragment are underlined. 'Neurotoxic' and 'neuroprotective' refer to assays in cerebellar granule cell cultures or Tg mice (see main text). Download figure Download PowerPoint To address expression at the protein level, we raised antibodies against Sho. Two antisera ('04SH-1' and '06SH-3') were against a mouse Sho peptide consisting of residues 86–100, whereas a third ('06rSH-1') was against full-length recombinant mouse Sho(25–122) expressed in Escherichia coli (Figure 2A) and recognizes an N-terminal epitope contained within residues 30–61 (Supplementary Figure S1). Assessed by Western blot of tissue lysates, 06rSH-1 was virtually devoid of cross-reactive species (Supplementary Figure S1). Cross-reactive species of molecular weights incompatible with authentic Sho were present in analyses with antisera 04SH-1 and with 06SH-3, but these had varying intensities and/or different molecular weights for the two antisera. Consequently, the following comments are restricted to signals detected by two or more varieties of α-Sho antibodies. Figure 2.Analysis of recombinant Sho in E. coli and expression of murine Sho in cultured cells. (A) Schematic representation of the Sho protein. The location of the mapped epitopes for α-Sho peptide antisera (04SH-1 and 06SH-3) and α-recombinant Sho (06rSH-1) are shown. (B) Circular dichroism spectrum of recombinant mouse Sho, rSho(25–122). The spectral trace is consistent with a random coil configuration. (C) Cell surface expression of wt Sho and a mutant Sho allele lacking the hydrophobic tract in non-permeabilized transfected N2a cells as demonstrated by immunocytochemistry. Scale bar, 50 microns. (D) Diminution of Sho signal in the cell lysates of Sho-transfected N2a cells following pretreatment with increasing concentrations of PI-PLC. (E) Western blot showing expression of a wt Sho transgene in N2a cells with or without PNGaseF treatment. A lysate from cells transfected with empty vector is included to show antibody specificity. Download figure Download PowerPoint Similar to PrPC, murine Sho is revealed as being expressed at the cell surface, N-glycosylated and sensitive to the GPI anchor cleaving enzyme phosphatidylinositol-specific phospholipase C (PI-PLC) in transfected N2a neuroblastoma cells (Figure 2C–E). Following PNGaseF treatment, full-length Sho has a molecular weight of 9.1 kDa as assessed by SDS–PAGE (predicted 9.5 kDa). In addition to the full-length protein, a fraction of the protein in transfected N2a cells has a faster electrophoretic mobility (Figure 2E). In PrP, a well-documented physiological 'C1' cleavage occurs just before the hydrophobic tract at residues His111 and Met112 (human PrP numbering scheme, underlined; Figure 1) both in cultured cells and in the adult brain (Pan et al, 1992; Chen et al, 1995; Vincent et al, 2000). Since cell lysates for these analyses were prepared in the presence of protease inhibitors, it is possible that an analogous endoproteolytic processing could figure in the biogenesis of Sho. A detailed description of truncated forms of Sho will be presented elsewhere (Coomaraswamy et al, in preparation). Analysis of mouse tissue by Western blotting defined a predominant glycosylated protein species of similar molecular weight to transfected full-length Sho (approximately 18 kDa), and one that is developmentally regulated, appearing at embryonic day 16 and persisting in early postnatal life and in the brains of adults (Figure 3A and B). PNGaseF-sensitive bands of identical molecular weights were observed with two independent Sho antibodies, confirming the authenticity of the bands and the presence of Sho in the mouse CNS. Sho signal was emphasized in membrane-enriched preparations from adult mouse brains, corroborating its membrane anchorage (Figure 3C). In Western blots performed with α-Sho86–100 antisera, Sho signal increased following treatment with PNGaseF, suggesting that N-glycosylation at Asn107 partially occludes antibody binding at the adjacent epitope for the peptide-directed antiserum. Figure 3.Analysis of mouse Sho in tissue preparations. (A) Expression of Sho in whole mouse embryos assessed by Western blotting using a 12% Tris–glycine gel. Sho is expressed beginning at embryonic day 16. (B) Postnatal expression of Sho in neonatal and adult mouse brains with or without PNGaseF treatment assessed by Western blotting using 14% Tris–glycine (α-rSho) or 4–12% NuPAGE (α-Sho(86–100) peptide and α-PrP) gels. Full-length species before and after enzymatic treatment are bracketed. (C) Sho is present in a membrane-enriched fraction prepared from mouse brains. Membrane preparations with or without PNGaseF treatment were analyzed on 4–12% NuPAGE gels by Western blotting. Sho was detected using either α-Sho peptide polyclonal 04SH-1 (A–C) or α-recombinant Sho polyclonal 06rSH-1 (B), whereas PrP was probed with single-chain antibodies D13 (A–B) or D18 (C). Download figure Download PowerPoint Overlapping and complementary PrPC/Sho expression In situ hybridization using antisense-strand riboprobes prepared against the mouse Sprn open reading frame (but not sense-strand controls) yielded signals in the adult mouse CNS. Analyses of the hippocampus and cerebellum revealed prominent signals in the cell bodies of pyramidal cells and Purkinje cells, respectively (Figure 4B and J). By way of comparison, Prnp has a broader pattern of neuronal expression (Kretzschmar et al, 1986; Taraboulos et al, 1992). Immunohistochemistry for Sho protein yielded prominent signals in the same cell types defined by in situ hybridization (Figure 4D and L), that is, hippocampal neurons and cerebellar Purkinje cells. In the case of antisera 04SH-1, these signals were absent when antibodies were preincubated in a solution containing the Sho86–100 peptide immunogen (Figure 4C and K). Besides defining Sho as the 'second' cellular prion protein present in neurons of the adult CNS, these data define intracellular transport phenomena, as immunohistochemical signals were present in cell processes in addition to the cell bodies detected by antisense riboprobes (i.e., predicted to contain Sho mRNA). In the case of Purkinje cells, immunostaining was present not only in cell bodies but also prominent in their processes, specifically in the dendritic arborizations present within the molecular layer of the cerebellum (Figures 4L and 5F–H, signals detected with all three antisera). A related phenomenon was observed in the hippocampus, notably in CA1 pyramidal neurons. Here, Sho immunoreactivity was absent from axonal projections (with all three α-Sho antibodies), present in cell bodies (seen by all three α-Sho antibodies), and notable in the apical dendritic processes located in the stratum radiatum of the hippocampus (strong signals with 04SH-1 and 06SH-3, and a less intense signal with 06rSH-1) (Figures 4D and 5A–C). Figure 4.Localization of Sprn mRNA and Sho protein in the adult mouse brain. (A–H) The hippocampus, and (I–P) the cerebellum. wt C57/B6 mice are presented in all sections, with the exception of B6 congenic Prnp0/0 (E, M) and Tg(SHaPrP)7 mice (H, P). Panels A, C, E, G, I, K, M and O in the left-hand columns comprise negative controls (i.e., sense-strand riboprobes, peptide-directed antisera preincubated with a soluble form of the antigenic peptide, Prnp0/0 mice for PrP-directed antibodies) for analyses presented in the right-hand columns. In situ hybridization: panels A, B, and I, J represent hybridizations with Sho sense-strand (A, I) or antisense (B, J) cRNA probe. Sections are not counterstained and blue staining from NBT/BCIP substrate represents hybridization to Sprn mRNA. Immunohistochemistry: all other panels of mouse brains with genotypes as noted above. Anti-Sho antibody 04SH-1 (α-Sho) antibody was used with (C, K) or without (D, L) preincubation with Sho(86–100) peptide. Antibodies 7A12 and 3F4 were used for the detection of mouse PrP (E, F, M, N) and hamster PrP (G, H, O, P), respectively. Note the Sho staining of CA1 apical dendritic processes (D, black bracket) and Purkinje cell layer (L, white arrow), and absence of Purkinje cell-body staining with α-moPrP (N) and relative paucity of staining with α-HaPrP antibody (P, black arrow). Scale bar in panels A–H, and I–P, 100 μm. Download figure Download PowerPoint Figure 5.Continued. Download figure Download PowerPoint Figure 6.Reciprocal and overlapping expression of Sho and PrPC in the CNS. (A–E) CA1 hippocampal neurons of adult mice probed as indicated. The signal for Sho immunohistochemistry in apical dendrites of wt mice (A–C) has an equivalent in a 'negative image' (white brackets) in the molecular layer of the neuropil imaged for either mouse PrP (D) or hamster PrP in Tg(SHaPrP)7 mice (E). Apical dendrites were less intensely stained with the α-Sho 06rSH-1 antibody (C, black arrowhead) Analogous analyses are presented for Purkinje cells (F–J). Somatodendritic localization of Sho in Purkinje cell bodies and dendritic arborizations (F–H) is contrasted by a reciprocal 'negative image' in molecular layer of the neuropil imaged for either mouse PrP (I) or hamster PrP in Tg(SHaPrP)7 mice (J). Note the complete absence of MoPrP staining in cell bodies (I, black arrowheads). Somatodendritic localization of calbindin in Purkinje cells of wt mice (K) is shown for comparative purposes. (L–N) The cerebral cortex probed simultaneously with α-Sho (06rSH-1, green) and α-PrP (8H4, red) antibodies. Overlapping PrPC and Sho expression is observed in neurons of the cerebral cortex, including colocalization in cell bodies (N, white arrowheads). Scale bar, 25 μm (A–E), 10 μm (F–K) or 20 μm (L–N). Download figure Download PowerPoint Since PrP has been reported to possess an unusual property for a GPI-linked protein, the ability to undergo basolateral (dendritic) sorting in polarized cells in distinction to the more typical apical (axonal) sorting, parallel analyses were undertaken for PrPC and Sho. PrPC was examined at basal levels from the (endogenous) Prnp gene, or expressed from a cosmid transgene of the hamster PrP gene including 25 kb of sequences 5′ to the transcriptional start-site (Scott et al, 1989; Prusiner et al, 1990). In the case of wt mouse PrP, analyses were performed with 7A12 antibody (Li et al, 2000) using Prnp0/0 mice as negative controls (Figure 4E and M), whereas non-Tg wt mice served as negative controls for the use of the hamster PrP transgene-specific 3F4 antibody (Figure 4G and O). Besides the anticipated differences in signal levels between wt and Tg(SHaPrP)7 mice, the PrPC-directed antibodies yielded similar expression patterns, with widespread staining throughout the brain. PrPC was underrepresented in the cell bodies of the pyramidal neurons of the hippocampus but abundantly present in the stratum oriens containing the axonal projections, and also in the radiatum, lacunosum moleculare and molecular layers (Figure 4F and H). In the cerebellum, mouse PrPC was absent in wt Purkinje cells (as described previously (Liu et al, 2001; Baumann et al, 2007)) and hamster PrPC was represented in some but not all Purkinje cells of Tg(SHaPrP)7 mice, and at a level lower than that of the surrounding neuropil (Figure 4N and P). On the other hand, PrPC was present in the granule cell layer and abundant in the molecular layer. Strikingly, the abundant signal present in the radiatum layer was not totally uniform, and—by virtue of a negative staining effect—revealed the outlines of Purkinje cell dendritic arborizations in the cerebellum and the apical dendritic of CA1 neurons (Figure 5D–E, and I–J): these are structures with marked immunostaining with α-Sho86–100 (04SH-1 and 06SH-3) and α-rSho (06-rSH1); Figure 5A–C and F–H). These data therefore define a complementary 'interlocking' aspect to PrPC/Sho protein expression in the cerebellum and suggest a similar effect in the hippocampus. Although the most prominent Sho signals were obtained in the hippocampus and cerebellum (Figure 4), signals for both Sprn mRNA and Sho protein were also present in other areas of the brain including the cerebral cortex, the thalamus, and the medulla (Figure 5L–N; Supplementary Figure S2). Coincidence with neurofilament staining indicates that neurons are the prominent site of Sho expression (Supplementary Figure S2). PrPC signal was abundant in these regions (Figure 5, and data not shown), confirming that Sho and PrPC expression profiles overlap in certain areas of the brain. A further example of this phenomenon is apparent in the adult retina (Premzl et al, 2003; Chishti et al, in preparation). In overview, Sho expression within the adult mouse brain is either less widespread than PrPC, or basal Sho levels in certain areas fall below the detection threshold of our current procedures. Lastly, to assess whether there is a physiological cross-regulation effect between PrPC and Sho, we performed a number of analyses on Prnp0/0 mice (Supplementary Figure S4). These failed to reveal a clear upregulation of Sho in response to PrPC deficiency. Neuroprotective activity and the central region of PrP Two N-terminal PrP modules, the charged region and the copper-binding octarepeats, contribute to neuroprotective activity in a neuronal assay (Drisaldi et al, 2004). However, while others analyzed the same N-terminal modules, they highlighted a trafficking effect (Sunyach et al, 2003), so we sought determinants beyond these potential delivery signals. The C-terminus of PrPC comprises a three-helix bundle (Riek et al, 1996) similar to Dpl (Mo et al, 2001), and in the form of alleles such as PrPΔ32–121 (Shmerling et al, 1998), exhibits Dpl-like toxicity. We therefore focused on the 'remaining' area of PrP, residues 91–121 between the octarepeats and the structured domain (Figure 1) containing the hydrophobic domain (HD) with Sho homology. The deletion alleles were tested for their ability to engender stable forms of PrP. As anticipated from prior analyses of in-frame deletions (Holscher et al, 1998; Shmerling et al, 1998; Hegde et al, 1999), these forms of PrP were synthesized at levels similar to wt PrP, and also underwent similar maturation (Baumann et al, 2007; Li et al, 2007) (Supplementary Figure S3). Residues 100–105 are poorly
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