PrP mRNA Research Articles

Identification of the internal ribosome entry sites (IRES) of prion protein gene

Many studies demonstrated that there are several type bands of prion protein in cells. However, the formation of different prion protein bands is elusive. After several low molecular weight bands of prion protein appeared in SMB-S15 cells infected with scrapie agent Chandler, we think that IRES-dependent translation mechanism induced by prion is involved in the formation of prion protein bands. Then we designed a series of pPrP-GFP fusing plasmids and bicistronic plasmids to identify the IRES sites of prion protein gene and found 3 IRES sites inside of PrP mRNA. We also demonstrated that cap-independent translation of PrP was associated with the ER stress through Tunicamycin treatment. We still found that only IRE1 and PERK pathway regulated the IRES-dependent translation of PrP in this study. Our results indicated, we found that PrP gene had an IRES-dependent translation initiation mechanism and we successfully identified the IRESs inside of the prion protein gene.

CpG site DNA methylation patterns reveal a novel regulatory element in the mouse prion protein gene.

The cellular isoform of the prion protein (PrPC) plays critical roles in the development of prion disorders. Although PrP mRNA is ubiquitously present in a tissue-specific manner, the DNA methylation of PrP gene (Prnp) is still unknown. In this study, we demonstrated that the CpG island (CGI, positioned at −218 to +152 bp from the transcriptional start site) including the Prnp core promoter region was completely unmethylated in all tested tissues. On the other hand, CpG methylation in the CGI shore region (positioned at −599 to −238 bp) occurred in various tissue- and site-specific proportions. Interestingly, the correlation analysis between CpG methylation status and PrP mRNA levels showed that one CpG site methylation at −576 was negatively correlated with the PrP mRNA level (Pearson’s r = −0.374, P=0.035). Taken together, our results suggest that Prnp is a typical housekeeping gene and various methylation frequencies of the CGI shore region are likely to affect Prnp expression in a tissue-specific manner.

Hematological shift in goat kids naturally devoid of prion protein.

The physiological role of the cellular prion protein (PrPC) is incompletely understood. The expression of PrPC in hematopoietic stem cells and immune cells suggests a role in the development of these cells, and in PrPC knockout animals altered immune cell proliferation and phagocytic function have been observed. Recently, a spontaneous nonsense mutation at codon 32 in the PRNP gene in goats of the Norwegian Dairy breed was discovered, rendering homozygous animals devoid of PrPC. Here we report hematological and immunological analyses of homozygous goat kids lacking PrPC (PRNPTer/Ter) compared to heterozygous (PRNP+/Ter) and normal (PRNP+/+) kids. Levels of cell surface PrPC and PRNP mRNA in peripheral blood mononuclear cells (PBMCs) correlated well and were very low in PRNPTer/Ter, intermediate in PRNP+/Ter and high in PRNP+/+ kids. The PRNPTer/Ter animals had a shift in blood cell composition with an elevated number of red blood cells (RBCs) and a tendency toward a smaller mean RBC volume (P = 0.08) and an increased number of neutrophils (P = 0.068), all values within the reference ranges. Morphological investigations of blood smears and bone marrow imprints did not reveal irregularities. Studies of relative composition of PBMCs, phagocytic ability of monocytes and T-cell proliferation revealed no significant differences between the genotypes. Our data suggest that PrPC has a role in bone marrow physiology and warrant further studies of PrPC in erythroid and immune cell progenitors as well as differentiated effector cells also under stressful conditions. Altogether, this genetically unmanipulated PrPC-free animal model represents a unique opportunity to unveil the enigmatic physiology and function of PrPC.

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrPSc. Studies showed that recombinant cellular PrP, PrPC, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V136L141R154Q171 and A136F141R154Q171, and one genotype of white-tailed deer (Q95G96, X132,Y216) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrPC shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrPC and supports amplification of PrPSc and PrPCWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrPSc or PrPCWD production. Addition of 2 to 20 μM of manganese chloride (MnCl2) to unseeded PMCA resulted in conversion of recombinant PrPC to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.

G-quadruplexes within prion mRNA: the missing link in prion disease?

Cellular ribonucleic acid (RNA) plays a crucial role in the initial conversion of cellular prion protein PrPC to infectious PrPSc or scrapie. The nature of this RNA remains elusive. Previously, RNA aptamers against PrPC have been isolated and found to form G-quadruplexes (G4s). PrPC binding to G4 RNAs destabilizes its structure and is thought to trigger its conversion to PrPSc. Here it is shown that PrP messenger RNA (mRNA) itself contains several G4 motifs, located in the octarepeat region. Investigation of the RNA structure in one of these repeats by circular dichroism, nuclear magnetic resonance and ultraviolet melting studies shows evidence of G4 formation. In vitro translation of full-length PrP mRNA, naturally harboring five consecutive G4 motifs, was specifically affected by G4-binding ligands, lending support to G4 formation in PrP mRNA. A possible role of PrP binding to its own mRNA and the role of anti-prion drugs, many of which are G4-binding ligands, in prion disease are discussed.

PrPc is temporospatially expressed in molar development of rats

Odontogenesis, tooth development, is derived from two tissue components: ectoderm and neural crest-derived mesenchyme. Cyto-differentiation of odontogenic cells during development involves time-dependent and sequential regulation of genetic programs. This study was conducted to detect molecules implicated in cyto-differentiation of developing molar germs of rats. Differential display-PCR revealed that PrP(c) was differentially expressed between cap/early bell-staged germs (maxillary 3rd molar germs) and root formation-staged germs (maxillary 2nd molar germs) at postnatal day 9. Both levels of PrP(c) mRNA and protein expression were higher in the root formation stage than the cap/early bell stage and increased in a time-dependent manner. Immunofluorescence revealed for the first time that PrP(c) was not localized in the enamel organ, but localized in dental follicular cells for the development of the periodontal ligament and cementum as well as odontoblasts, both of which are of neural crest origin. These results suggest that the physiological functions of the PrP(c) in tooth development may be implicated in the differentiation of neural crest-derived mesenchyme including the periodontal tissues for root formation rather than epithelial tissue.

PrP mRNA and protein expression in brain and PrPc in CSF in Creutzfeldt-Jakob disease MM1 and VV2

Creutzfeldt-Jakob disease (CJD) is a heterogenic neurodegenerative disorder associated with abnormal post-translational processing of cellular prion protein (PrPc). CJD displays distinctive clinical and pathological features which correlate with the genotype at the codon 129 (methionine or valine: M or V respectively) in the prion protein gene and with size of the protease-resistant core of the abnormal prion protein PrPsc (type 1: 20/21 kDa and type 2: 19 kDa). MM1 and VV2 are the most common sporadic CJD (sCJD) subtypes. PrP mRNA expression levels in the frontal cortex and cerebellum are reduced in sCJD in a form subtype-dependent. Total PrP protein levels and PrPsc levels in the frontal cortex and cerebellum accumulate differentially in sCJD MM1 and sCJD VV2 with no relation between PrPsc deposition and spongiform degeneration and neuron loss, but with microgliosis, and IL6 and TNF-α response. In the CSF, reduced PrPc, the only form present in this compartment, occurs in sCJD MM1 and VV2. PrP mRNA expression is also reduced in the frontal cortex in advanced stages of Alzheimer disease, Lewy body disease, progressive supranuclear palsy, and frontotemporal lobe degeneration, but PrPc levels in brain varies from one disease to another. Reduced PrPc levels in CSF correlate with PrP mRNA expression in brain, which in turn reflects severity of degeneration in sCJD.

Expression of Prion Protein in Mouse Erythroid Progenitors and Differentiating Murine Erythroleukemia Cells

Prion diseases have been observed to deregulate the transcription of erythroid genes, and prion protein knockout mice have demonstrated a diminished response to experimental anemia. To investigate the role of the cellular prion protein (PrPC) in erythropoiesis, we studied the protein's expression on mouse erythroid precursors in vivo and utilized an in vitro model of the erythroid differentiation of murine erythroleukemia cells (MEL) to evaluate the effect of silencing PrPC through RNA interference.The expression of PrPC and selected differentiation markers was analyzed by quantitative multicolor flow cytometry, western blot analysis and quantitative RT-PCR. The silencing of PrPC expression in MEL cells was achieved by expression of shRNAmir from an integrated retroviral vector genome. The initial upregulation of PrPC expression in differentiating erythroid precursors was detected both in vivo and in vitro, suggesting PrPC's importance to the early stages of differentiation. The upregulation was highest on early erythroblasts (16200±3700 PrPC / cell) and was followed by the gradual decrease of PrPC level with the precursor's maturation reaching 470±230 PrPC / cell on most mature CD71−Ter119+ small precursors. Interestingly, the downregulation of PrPC protein with maturation of MEL cells was not accompanied by the decrease of PrP mRNA. The stable expression of anti-Prnp shRNAmir in MEL cells led to the efficient (>80%) silencing of PrPC levels. Cell growth, viability, hemoglobin production and the transcription of selected differentiation markers were not affected by the downregulation of PrPC.In conclusion, the regulation of PrPC expression in differentiating MEL cells mimics the pattern detected on mouse erythroid precursors in vivo. Decrease of PrPC protein expression during MEL cell maturation is not regulated on transcriptional level. The efficient silencing of PrPC levels, despite not affecting MEL cell differentiation, enables created MEL lines to be used for studies of PrPC cellular function.

The Effect of Temperature on Oogenesis and Brain Gene Expression of Hormones Involved in Reproduction and Growth in the Female Blue Gourami ( Trichogaster trichopterus )

Introduction: The blue gourami (Trichogaster trichopterus) female has an asynchronic ovary development and the FOM will occur only in the presence of the male. Thus, each stage of its gonadal development can be controlled and examined separately in the laboratory [4]. Previous studies in our laboratory have shown variations in hypothalamic and pituitary hormonal gene expression during reproduction stages. Gonadotropin releasing hormone 3 (GnRH3), and pituitary adenylate cyclase activating polypeptide (PACAP long and short form [PRP]) gene expression increased in reproductively active females [5,6], and beta-follicle stimulating hormone (beta FSH) and beta-luteinizing hormone (beta LH) gene expression were increased in females in the vitellogenic stage [4]. Accumulated data indicate an involvement of growth factors in the regulation of reproduction, e.g., growth hormone (GH) and prolactin (PRL) [2, 3]. The present study examined the effect of temperature on reproduction and growth-related factors in blue gourami (Trichogaster trichopterus) females under non-reproductive (NRC) and reproductive conditions. Methods: The females, maintained in three different containers at the temperatures as described in prevue study [1], gonads were sampled and mRNA levels of GnRH3, PACAP, PRP, IGF1, GH, beta-LH, beta-FSH and PRL were mustered[1]. Results: A higher percentage of oocytes in the advanced vitellogenic stage was found in FNRC kept at 27 °C than in those kept at 23 °C or 31 °C (P<0.05,Fig. 1a). In contrast, in FRM and FNRM, a higher percentage of oocytes at the FOM stage were observed at 23 °C, than at 27 °C (Fig. 1b). In FNRC kept at 23 oC and 27 oC, as compared to the group at 31 oC, significantly higher mRNA levels of brain GnRH3 were detected, whereas no significant differences in the mRNA levels of brain IGF-1, PACAP and PRP were observed in these females at 23 oC, 27 oC or 31 oC. In FRM, mRNA levels of brain GnRH3, IGF1, PACAP and PRP were higher when kept at 27 °C than at 23 °C (Figs. 2A-D). PRP mRNA levels were also higher at 27 °C than at 31 °C in these fish. On the other hand, mRNA levels of PACAP were greater in FNRM kept at 27 °C than in those fish maintained at 23 °C and 31°C (Fig. 2B); and mRNA levels of IGF-1 were higher in the brains of FNRM kept at 23 °C than in those kept at 31 °C (Fig. 2C).

PrP 106-126 Altered PrP mRNA gene expression in mouse microglia BV-2 Cells

Prion diseases are infectious and fatal neurodegenerative diseases. The pathogenic agent is an abnormal prion protein aggregate. Microglial activation in the centre nervous system is a characteristic feature of prion disease. In this study, we examined the effect of PrP 106-126 on PrP mRNA gene expression in Mouse microglia cells BV-2 by real-time quantitative PCR. PrP mRNA expression level was found to be significantly increased after 18 h exposure of BV-2 cells to PrP 106-126, with 3-fold increase after 18 h and 4.5-fold increase after 24 h and BV-2 cells proliferating occurred correspondingly. Our results provide the first in vitro evidence of the increase of PrP mRNA levels in microglial cells exposed to PrP 106-126, and indicate that microglial cells might play a critical role in prion pathogenesis.

Developmental expression of prion protein and its ligands stress‐inducible protein 1 and vitronectin

Prion protein (PrP(C)) is the normal isoform of PrP(Sc), a protein involved in neurodegenerative disorders. PrP(C) participates in neuritogenesis, neuroprotection, and memory consolidation through its interaction with the secreted protein stress-inducible protein 1 (STI1) and the extracellular matrix protein vitronectin (Vn). Although PrP(C) mRNA expression has been documented during embryogenesis, its protein expression patterns have not been evaluated. Furthermore, little is known about either Vn or STI protein expression. In this study, PrP(C), STI1, and Vn protein expression was explored throughout mouse embryonic life. We found that the distributions of the three proteins were spatiotemporally related. STI1 and Vn expression became evident at E8, earlier than PrP(C), in the nervous system and heart. At E10, we observed, in the spinal cord, a gradient of expression of the three proteins, more abundant in the notochord and floor plate, suggesting that they can have a role in axonal growth. As development proceeded, the three proteins were detected in other organs, suggesting that they may play a role in the development of nonneural tissues as well. Finally, although STI1 and Vn are PrP(C) ligands, their expression was not altered in PrP(C)-null mice.

PrP Expression, PrPSc Accumulation and Innervation of Splenic Compartments in Sheep Experimentally Infected with Scrapie

BackgroundIn prion disease, the peripheral expression of PrPC is necessary for the transfer of infectivity to the central nervous system. The spleen is involved in neuroinvasion and neural dissemination in prion diseases but the nature of this involvement is not known. The present study undertook the investigation of the spatial relationship between sites of PrPSc accumulation, localisation of nerve fibres and PrPC expression in the tissue compartments of the spleen of scrapie-inoculated and control sheep.Methodology/Principal FindingsLaser microdissection and quantitative PCR were used to determine PrP mRNA levels and results were compared with immunohistochemical protocols to distinguish PrPC and PrPSc in tissue compartments of the spleen. In sheep experimentally infected with scrapie, the major sites of accumulation of PrPSc in the spleen, namely the lymphoid nodules and the marginal zone, expressed low levels of PrP mRNA. Double immunohistochemical labelling for PrPSc and the pan-nerve fibre marker, PGP, was used to evaluate the density of innervation of splenic tissue compartments and the intimacy of association between PrPSc and nerves. Some nerve fibres were observed to accompany blood vessels into the PrPSc-laden germinal centres. However, the close association between nerves and PrPSc was most apparent in the marginal zone. Other sites of close association were adjacent to the wall of the central artery of PALS and the outer rim of germinal centres.Conclusions/SignificanceThe findings suggest that the degree of PrPSc accumulation does not depend on the expression level of PrPC. Though several splenic compartments may contribute to neuroinvasion, the marginal zone may play a central role in being the compartment with most apparent association between nerves and PrPSc.

Biosynthesis of Prion Protein Nucleocytoplasmic Isoforms by Alternative Initiation of Translation

The cellular prion protein PrP(C) is synthesized as a family of four distinct forms. Of these, (Cyt)PrP is a minor member that segregates outside of the secretory route and can generate cytotoxic forms. Using signal sequence mutants, we found that (Cyt)PrP is translated from a downstream AUG (coding for Met-8 in human PrP or Met-15 in Syrian hamster PrP). Shortening of the signal sequence dictated the spillage of this isoform into the cytosol, from where it accessed the nucleus or formed insoluble cytosolic aggregates if the proteasome is inhibited. The PrP isoform isolated from the nuclear fractions of cell and brain homogenates was partially SUMO-1-conjugated. Expression of HaPrP(M15) in cells caused an antiproliferative phenotype due to a cell cycle arrest at the G(0)/G(1) phase. The identification of this PrP isoform and its properties provides novel insight into PrP(C) physiological and pathological functions.

The role of calorie restriction and SIRT1 in prion-mediated neurodegeneration

A central focus of aging research is to determine how calorie restriction (CR) extends lifespan and delays diseases of aging. SIRT1, the mammalian ortholog of Sir2 in yeast, is a longevity factor which mediates dietary restriction in diverse species. In addition, SIRT1 plays a protective role in several models of neurodegenerative disease. We tested the role of SIRT1 in mediating the effects of CR in a mouse model of prion disease. Prion diseases are protein misfolding disorders of the central nervous system with many similarities to other neurodegenerative diseases, including deposition of aggregated protein, gliosis, and loss of synapses and neurons. We report that the onset of prion disease is delayed by CR and in the SIRT1 KO mice fed ad libitum. CR exerts no further effect on the SIRT1 KO strain, suggesting the effects of CR and SIRT1 deletion are mechanistically coupled. In conjunction, SIRT1 is downregulated in certain brain regions of CR mice. The expression of PrP mRNA and protein is reduced in the brains of CR mice and in SIRT1 knockout mice, suggesting a possible mechanism for the delayed onset of disease, as PrP levels are a critical determinant of how quickly mice succumb to prion disease. Surprisingly, CR greatly shortens the duration of clinical symptoms of prion disease and ultimately shortens lifespan of prion-inoculated mice in a manner that is independent of SIRT1. Taken together, our results suggest a more complex interplay between CR, SIRT1, and neurodegenerative diseases than previously appreciated.

Search for a scrapie-specific nucleic acid: a progress report.

Scrapie agent contains a proteinaceous component as well as an 'informational' molecule (suggested by the existence of distinct strains of scrapie). These operationally defined entities may be the same molecule, an infectious protein, or distinct, in which case a nucleic acid might encode the genetic information. Purification of scrapie agent enriched a protein, PrPSc, by virtue of its relative protease resistance. There is only a single PrP gene and the primary translation product of PrP mRNA is the same in normal and scrapie-infected brain; therefore the normal PrPC and the protease-resistant isoform, PrPSc, found in scrapie, probably result from different post-translational events. To search for scrapie-specific nucleic acid, globin RNA made in vitro was added to highly purified infectious preparations at a ratio of 10(3) molecules per infectious unit, nucleic acids were isolated and denatured, and cDNA synthesized using random oligonucleotide primers. Clones containing globin-related sequences were identified by in situ hybridization. 150 plaques not hybridizing to the globin probe were isolated. Inserts larger than 50 base pairs were analysed. By hybridization to a globin probe at reduced stringency all but four clones were found to contain small globin related inserts; two of these hybridized to hamster repetitive sequences as shown by Southern blot analysis. The other clones not related to hamster nucleic acids may be derived from unknown sources of contamination or from scrapie-specific nucleic acids.

Developmental regulation of prion protein mRNA in brain.

During development of the hamster brain, synthesis of the cellular isoform of the scrapie prion protein (PrPC) was found to be regulated. Low levels of PrP poly(A)+ mRNA were detectable one day after birth. PrP poly(A)+ mRNA reached maximal levels between 10 and 20 days post-partum; thereafter, no change in its level could be detected at ages up to 13 months. In contrast, myelin basic protein poly(A)+ mRNA was shown to reach maximal levels by 30 days of age and thereafter steadily declined in adult brain. Using monospecific PrP antisera, immunoprecipitable cell-free translation products were detected at low levels two days after birth and progressively increased up to 10 days of age. How the PrP mRNA participates in brain development and its function in scrapie prion infection are being investigated.

Increased PrP mRNA expression in lymphoid follicles of the ileal Peyer's patch of sheep experimentally exposed to the scrapie agent

To understand the functional role of cellular prion protein (PrP(C)) in the initiation and maintenance of prion disease within the host, it is important to obtain a more detailed understanding of PrP(C) transcription in tissues during the development of disease. Using an experimental model with oral infection, we examined the effect of scrapie and the accumulation of the scrapie related form of the prion protein (PrP(Sc)) on the expression level of PrP mRNA in the ileal Peyer's patch of sheep. In the early phase of infection, prior to PrP(Sc) accumulation, no effect on the PrP expression was detected. However, it was found that lambs with PrP genotypes associated with high susceptibility for scrapie generally had higher PrP mRNA levels than lambs with less susceptible genotypes. Further, in highly susceptible VRQ/VRQ sheep at a stage of disease with high accumulation of PrP(Sc), real-time RT-PCR and microdissection were used to investigate levels of PrP mRNA in four different tissue compartments. An increased level of PrP mRNA was found in lymphoid follicles of infected sheep compared with controls, indicating upregulation of PrP expression in the follicles to compensate for the loss of PrP(C) converted to PrP(Sc), or that PrP(Sc) accumulation directly or indirectly influences the PrP expression. Still, the PrP expression level in the follicles was low compared with the other compartments investigated, suggesting that although increased PrP expression could contribute to PrP(Sc) accumulation, other factors are also important in the processes leading to accumulation of PrP(Sc) in the follicles.

Characterization of Prion Susceptibility in Neuro2a Mouse Neuroblastoma Cell Subclones

In this study, we established Neuro2a (N2a) neuroblastoma subclones and characterized their susceptibility to prion infection. The N2a cells were treated with brain homogenates from mice infected with mouse prion strain Chandler. Of 31 N2a subclones, 19 were susceptible to prion as those cells became positive for abnormal isoform of prion protein (PrP(Sc)) for up to 9 serial passages, and the remaining 12 subclones were classified as unsusceptible. The susceptible N2a subclones expressed cellular prion protein (PrP(C)) at levels similar to the parental N2a cells. In contrast, there was a variation in PrP(C) expression in unsusceptible N2a subclones. For example, subclone N2a-1 expressed PrP(C) at the same level as the parental N2a cells and prion-susceptible subclones, whereas subclone N2a-24 expressed much lower levels of PrP mRNA and PrP(C) than the parental N2a cells. There was no difference in the binding of PrP(Sc) to prion-susceptible and unsusceptible N2a subclones regardless of their PrP(C) expression level, suggesting that the binding of PrP(Sc) to cells is not a major determinant for prion susceptibility. Stable expression of PrP(C) did not confer susceptibility to prion in unsusceptible subclones. Furthermore, the existence of prion-unsusceptible N2a subclones that expressed PrP(C) at levels similar to prion-susceptible subclones, indicated that a host factor(s) other than PrP(C) and/or specific cellular microenvironments are required for the propagation of prion in N2a cells. The prion-susceptible and -unsusceptible N2a subclones established in this study should be useful for identifying the host factor(s) involved in the prion propagation.

Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrPgene expression and genetic susceptibility to scrapie

BackgroundCellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs), and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes.ResultsPrevious to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes.ConclusionThe results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues.

The expression of prion protein (PrP(C)) in the megakaryocyte lineage.

Cellular prion protein (PrP(C)) is a naturally occurring protein in normal individuals which adopts an abnormal conformation, termed scrapie prion protein (PrP(Sc)) that is associated with disease. There is great concern that clinically asymptomatic variant Creutzfeldt-Jacob disease (vCJD) may transmit PrP(Sc) in blood transfusion products. PrP(C) is widely expressed and has been found in human blood. The majority of cellular borne PrP(C) is associated with platelets (84%). Although PrP(C) mRNA has been demonstrated in platelets, the quantity is unknown and may not reflect the total PrP(C) present. To investigate the expression of PrP(C) in the megakaryocyte lineage. The expression of PrP(C) was studied in CD34+ cells, cultured megakaryocytes and platelets using electron microscopy, flow cytometry, semi-quantitative RT-PCR and immunofluorescence confocal microscopy. The expression of PrP(C) appeared to increase with differentiation and polyploidization in the megakaryocyte lineage. PrP(C) was located within platelet alpha-granules and its source is likely to be from megakaryocyte precursors. If PrP(Sc) has a similar distribution, these results have implications for the selection of blood donors and preparation of cell-depleted blood products.