PrP mRNA Research Articles

QUANTIFICATION OF PRION GENE EXPRESSION IN BRAIN AND PERIPHERAL ORGANS OF GOLDEN HAMSTER BY REAL-TIME RT-PCR

ABSTRACT Determination of tissue-specific expression of cellular prion protein (PrPc) is essential for understanding its poorly explained role in organisms. Herein we report on quantification of PrP mRNA in golden hamsters, a popular experimental model for studying mechanisms of transmissible spongiform encephalopathies (TSE), by real-time RT-PCR. Total RNA was isolated from four different regions of the brain and six peripheral organs of eight golden hamsters. PrP mRNA copy numbers were determined using absolute standard curve method with real-time quantitative PCR instrument. It was found that high mRNA levels were present in all four regions of the brain examined, including obex, neocortex, cerebellum, and thalamus. In peripheral organs examined, inguinal lymph node showed high level of the expression similar to that in overall brain; spleen, heart, liver, and lung showed moderate levels of the expression; and kidney showed the lowest expression. Our result is consistent with the potential involvement of different organs in prion diseases and offers essential data for further study of TSE mechanism in this animal model.

Cre-loxP mediated control of PrP to study transmissible spongiform encephalopathy diseases.

Expression of the PrP glycoprotein is essential for the development of the transmissible spongiform encephalopathy (TSE) or prion diseases. Although PrP is widely expressed in the mouse, the precise relevance of different PrP-expressing cell types to disease remains unclear. To address this, we generated two lines of floxed PrP gene-targeted transgenic mice using the Cre recombinase-loxP system. These floxed mice allow a functional PrP allele to be either switched "on" or "off." We demonstrate control of PrP expression for both alleles following Cre-mediated recombination, as determined by PrP mRNA and protein expression in the brain. Moreover, we show that Cre-mediated alteration of PrP expression in these mice has a major influence on the development of TSE disease. These floxed PrP mice will allow the involvement of PrP expression in specific cell types following TSE infection to be defined, which may identify potential sites for therapeutic intervention.

RNA mutagenesis and sporadic prion diseases

The extremely low incidence of sporadic prion diseases suggests that they may arise as a rare stochastic event in otherwise healthy animals or humans. Current hypotheses for sporadic prion disease include horizontal transmission, spontaneous conversion of Prp C into Prp Sc, and somatic mutation of the Prp gene. Here, we suggest RNA mutation as a possible initial event in the etiology of sporadic prion disease. The proposed model is based on (i) the fact that in Prp-expressing cells, mutations are statistically more likely to occur in the Prp mRNA population than in the corresponding two copies of the Prp gene, and (ii) the absence of RNA repair mechanisms analagous to those found for DNA mismatch correction resulting in a relatively higher rate of RNA mutations. Here, we suggest that translation of mutated Prp mRNA could lead to the synthesis of transient Prp Sc which results in the conversion of Prp C into Prp Sc and the propagation of a disease-associated isoform. This model points to RNA mutation as a possible mechanism for the generation of sporadic prion diseases and other pathological disorders in which infectious proteins other than Prp Sc might be implicated.

Prion Protein Accumulation and Neuroprotection in Hypoxic Brain Damage

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

Mutant PrP Is Delayed in Its Exit from the Endoplasmic Reticulum, but Neither Wild-type nor Mutant PrP Undergoes Retrotranslocation Prior to Proteasomal Degradation

The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.

Stimulation of cellular prion protein expression by TSH in human thyrocytes

The cellular isoform of prion protein (PrP C) is a cell-surface glycosyl-phosphatidylinositol-anchored protein which is ubiquitously expressed on the cell membrane. It may function as a cell receptor or as a cell adhesion molecule. Thyroid follicles, obtained from patients with Graves’ disease at thyroidectomy, were cultured in F-12/RPMI-1640 medium supplemented with 0.5% fetal bovine serum and bovine thyroid stimulating hormone (bTSH). Northern blot analyses revealed that bTSH increased the steady-state expression levels of PrP mRNA in a time- and dose-dependent manner. This increase was reproduced by dibutyryl-cAMP and 12-decanoylphorbol-13-acetate. The mRNA expression was greater in thyroid follicles in suspension culture than in thyrocytes cultured in a monolayer. These findings suggest that TSH stimulates PrP mRNA expression in thyrocytes through the protein kinase A and C pathways. The greater mRNA expression in thyroid follicles than in monolayer cells suggests that PrP C may be involved in structure formation or maintenance of thyroid follicles.

Tissue-specific expression pattern of bovine prion gene: quantification using real-time RT-PCR

In recent studies PrP mRNA was determined mostly by in situ hybridisation or Northern Blot analysis—methods not suitable for absolute quantification of mRNA copy numbers. Herein we report on bovine prion mRNA quantification using calibrated highly sensitive externally standardized real-time RT-PCR with LightCycler instrument. Total RNA was isolated from nine different regions of the CNS and seven peripheral organs. PrP c mRNA copy numbers could be determined in all tissues under study. In approval with prior studies high mRNA level was found in Neocortex and Cerebellum. Lymphatic organs showed at least as high expression levels of prion mRNA as overall brain. Lowest expression was detected in kidney. Results of our study provide insight into the involvement of different organs in pathogenesis with respect to prion mRNA expression. LightCycler technology is currently considered the most precise method for nucleic acid quantification and showed to be powerful tool for further studies on prion diseases pathogenesis.

Embryonic activation and developmental expression of the murine prion protein gene.

While it is well established that cellular prion protein (PrP(C)) expression is required for the development of transmissible spongiform encephalopathies (TSEs), the physiological function of PrP(C) has yet to be determined. A number of studies have examined PrP expression in different tissues and in the later stages of embryonic development. However, the relative levels of expression of PrP RNA and protein in tissues outside the central nervous system (CNS) is not well documented and the exact point of transcriptional activation of PrP during embryogenesis is unknown. We have studied PrP mRNA expression in murine embryos and both mRNA and protein expression in a variety of adult tissues. PrP RNA was detected at different levels in all tissues tested while PrP(C) protein was detectable in all adult tissues tested with the exception of kidney and liver. RNA and protein levels were also assessed at four points during postnatal brain development and levels of both were seen to increase with development. We also established that, during embryogenesis, induction of PrP RNA expression occurs between E8.5 and E9, during the period of transition from anaerobic to aerobic metabolism. Preliminary experiments investigating the effects of superoxide radicals on PrP expression in cultured neuroblastoma and astrocyte cells support the suggestion that PrP(C) forms part of a cellular antioxidant defense mechanism.

G1-dependent prion protein expression in human glioblastoma cell line T98G.

Human glioblastoma cell line T98G produced a cellular form of prion protein (PrP(C)), and we confirmed expression of PrP mRNA by RT-PCR. Immunoblot analysis of whole cell lysate revealed one major (35 kDa) and two faint bands (31, 25 kDa) that reacted with monoclonal anti-human PrP antibody 3F4. Cells treated with tunicamycin produced only a 25 kDa band, representing a deglycosylated form of PrP. Similarly, peptide: N-glycosidase F treatment of whole cell lysate altered the Asn-linked form to the deglycosylated form. When T98G cells were cultured for a longer period, the amount of PrP(C) per cell increased on Day 4 to 16 in a time-dependent manner. When the cells were cultured at high cell-density, the cells on Day 4 produced the same amount of PrP(C) as those on Day 16 of the usual culture. Moreover, in a serum-free medium, cells cultured at a low cell-density produced the same amount of PrP(C) as those cultured at the high cell-density. These results demonstrate that PrP(C) production in T98G cells was dependent on the phase of the cell cycle, probably the G1 phase.

In situ hybridization analysis of PrP mRNA in human CNS tissues.

Expression of the prion protein gene (Prnp) and production of the PrP protein are essential requirements for acquisition and spread of transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) in humans. Here we have developed an in situ hybridization method for use on human post-mortem central nervous system (CNS) tissues in order to determine those cell which are transcribing the Prnp gene and thus expressing PrP mRNA. Tissues from 11 adult individuals (age range 21-79 years) were analysed. Similar to previous studies in other animal systems, it was shown that PrP production occurs primarily in neuronal populations throughout the human brain. Neurones of the hippocampus, cortex, thalamus, cerebellum and medulla all synthesize PrP mRNA at readily detectable levels. No age-related differences were observed between the cases studied. It was also found that the ependymal cells produced PrP mRNA; these were the only non-neuronal cell type expressing the Prnp gene in the CNS. It is hoped that the information produced here will be helpful in understanding the pathology associated with CJD and other prion diseases in humans.

Onset of ataxia and Purkinje cell loss in PrP null mice inversely correlated with Dpl level in brain.

PrP knockout mice in which only the open reading frame was disrupted ('Zürich I') remained healthy. However, more extensive deletions resulted in ataxia, Purkinje cell loss and ectopic expression in brain of Doppel (Dpl), encoded by the downstream gene, PRND: A new PrP knockout line, 'Zürich II', with a 2.9 kb PRNP: deletion, developed this phenotype at approximately 10 months (50% morbidity). A single PRNP: allele abolished the syndrome. Compound Zürich I/Zürich II heterozygotes had half the Dpl of Zürich II mice and developed symptoms 6 months later. Zürich II mice transgenic for a PRND:-containing cosmid expressed Dpl at twice the level and became ataxic approximately 5 months earlier. Thus, Dpl levels in brain and onset of the ataxic syndrome are inversely correlated.

Sheep and goats: natural and experimental TSEs and factors influencing incidence of disease.

The major factor influencing incidence of disease following challenge with transmissible spongiform encephalopathies (TSEs) in sheep is the allotype at amino acid numbers 136, 154 and 171 of the PrP protein. There are at least two groups of TSEs, one which targets the amino acid encoded at position 136 and the other which is more influenced by the amino acid at codon 171. Within these groups of TSE types, there may additionally be sub-types, as resistance to some, but not all, "136-type" TSEs can also be affected by the amino acid at codon 154. In goats, there are also PrP polymorphisms which apparently influence incubation period of TSE disease, however, this has not found to be true for cattle and BSE incidence. Sheep PrP amino acid codons 136, 154 and 171 do not explain everything about, for example, natural scrapie occurrence in sheep flocks, and attention is now turning to the flanking regions of the PrP gene looking for sequence differences in gene expression control motifs which may also have an influence on disease development. The sheep PrP gene produces two mRNAs in peripheral tissues, the result of alternative polyadenylation in the 3' untranslated region of the gene. Results from transfection assays of murine neuroblastoma cells with constructs expressing different regions of ovine PrP mRNA have revealed the presence of sequences in the 3' untranslated region that modulate protein synthesis and have therefore the potential to affect disease progression.

Phosmet induces up-regulation of surface levels of the cellular prion protein.

Chronic (2 day) exposure of human neuroblastoma cells to the organophosphate pesticide phosmet induced a marked concentration-dependent increase in the levels of PrP present on the cell surface as assessed by biotin labelling and immunoprecipitation. Levels of both phospholipase C (PIPLC)-releasable and non-releasable forms of PrP were increased on the plasma membrane. These increases appear to be due to post-transcriptional mechanisms, since PrP mRNA levels as assessed by Northern blotting were unaffected by phosmet treatment. These data raise the possibility that phosmet exposure could increase the susceptibility to the prion agent by altering the levels of accessible PrP.

Differential allelic expression of PrP mRNA in carriers of the E200K mutation.

Creutzfeldt-Jakob disease (CJD) linked to the E200K mutation of the prion protein (PrP) gene presents with a wide range of age at disease onset. Since most patients are heterozygous for the mutation, we tested whether differential expression of mutant versus wild-type (wt) PrP may affect the age at disease onset in carriers of the mutation. We measured wt and mutant PrP protein and mRNA in Epstein-Barr virus (EBV)-transformed B cells of either E200K CJD patients or healthy E200K carriers. Our results suggests that while in most healthy carriers the expression of wt PrP was higher than that of E200K PrP, most of the E200K CJD patients express equal levels of both PrP proteins. Similar results were obtained for either PrP protein or PrP mRNA. These results suggest that preferential expression of PrP from the wt allele may modulate the outbreak of the disease in carriers of prion mutations. This notion is consistent with the results obtained in transgenic mice carrying a human PrP gene, which suggest that endogenous PrP protects mice from contracting scrapie after inoculation with human CJD brain. Similar mechanisms may prevail in other inherited diseases with variable phenotypes.

Expression of PrP mRNA Is Regulated by a Fragment of MRP8 in Human Fibroblasts

Prion protein (PrPc) is expressed in many tissues, both in human and animals. The scrapie isoform of PrPchas been shown to cause neurodegeneration. In other studies it has been demonstrated that overexpression of the PrP gene can result in nonneuronal tissue degradation. Little is known, however, about the normal function of PrPcand prion protein gene regulation. Using cultured periodontal ligament cells as an experimental model, we have demonstrated the stimulation of PrP mRNA expression by MRP8 (migration inhibitory factor-related protein). Additionally, we have shown that PDGF has an opposite effect acting as a suppresser. We propose that a correlation exists between PrPcmRNA expression and cell growth arrest and differentiation.

PRP27–30Is a Normal Soluble Prion Protein Fragment Released by Human Platelets

Prion diseases are neurodegenerative disorders characterized by the accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system. PrPScisoforms differ from their normal homologue (PrPC), in that they possess increased β-sheet conformation, are partially protease resistant and may be associated with amyloid deposition. Amyloid proteins are thought to derive from soluble precursors or fragments thereof, present in biological fluids, which in the disease state undergo conformational change leading to aggregation and deposition in target tissues. We report here that platelets carry PrP mRNA and release PrPC, a sialoglycoprotein bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Soluble PrPCand a N-terminal truncated PrPCisoform starting at position 90 are secreted by resting and agonist-stimulated platelets and are detectable after partial deglycosylation of releasates. N-terminal sequence analysis of the soluble 27–30 kDa isoform, GQGGGTHSQ(W)NKP, revealed homology to scrapie PrP27–30, the protease resistant core derived from PrPSc. These findings indicate that in addition to PrPC, platelets process a soluble PrP27–30isoform. Whether this isoform can be converted into scrapie PrP27–30remains to be determined.

A cellular form of prion protein (PrPC) exists in many non-neuronal tissues of sheep.

A cellular form of the prion protein (PrPC) is thought to be a substrate for an abnormal isoform of th eprion protein (PrPSc) in scrapie. PrPC is abundant in tissues of the central nervous system, but little is known about the distribution of PrPC in non-neuronal tissues of sheep, the natural host of scrapie. This study investigated the tissue distribution of PrPC in sheep. Although PrPC was abundant in neuronal tissues, it was detected in non-neuronal tissues such as spleen, lymph node, lung, heart, kidney, skeletal muscle, uterus, adrenal gland, parotid gland, intestine, proventriculus, abomasum and mammary gland. Neither PrPC nor PrP mRNA was detected in the liver. The tissue distribution of PrPC appears to be inconsistent with the tissues which possess scrapie infectivity, suggesting that factor(s) specific to certain cell types may be required to support multiplication of the scrapie agent.

Developmental expression of the prion protein gene in glial cells

Replication of prions is dependent on the presence of the host protein PrP c. During the course of disease, PrP c is converted into an abnormal isoform, PrP sc, which accumulates in the brain. Attempts to identify the cell type(s) in which prion replication and PrP conversion occur have reached conflicting results. Although PrP mRNA is present in high amounts in neurons throughout the life of the animal, PrP Sc initially accumulates in astrocytes and possibly other glial cells and, later in the course of the disease, spreads diffusely in the tissue, often in white matter. We report here that PrP mRNA is expressed not only in neurons but also in astrocytes and oligodendrocytes throughout the brain of postnatal hamsters and rats. The level of glial PrP mRNA expression in neonatal animals was comparable to that of neurons and increased two-fold during postnatal development. A substantial portion of brain PrP mRNA is therefore contributed by glial cells. Our results provide an explanation for the accumulation of PrP sc, in white matter tissue and in the cytoplasm of glial cells and argue for a direct involvement of glia in prion propagation.

Tight Hormonal Control of PRP Gene Expression in Endocrine Pancreatic Cells

In this work, we have studied whether PrP, a protein found predominantly on the surface of neurons, could also exist in pancreatic endocrine cells. For this purpose, we have compared the expression of PrP mRNAs in insulin- (beta) and glucacon- (alpha) producing cells to that of neuronal and nonneuronal cells. We show that PrP mRNAs are expressed in all the beta and alpha cells tested at levels similar to those observed in neuronal cells. We also show that the expression of PrP is tightly regulated by hormones. Indeed, recombinant human growth hormone and dexamethasone, 2 factors implicated in beta cell maturation, increase PrP mRNA steady state level. PrP can thus be added to the list of neuronal factors expressed in the endocrine pancreas. Moreover, beta cells could represent a new model to study the control of PrP expression.

129/Ola mice carrying a null mutation in PrP that abolishes mRNA production are developmentally normal.

The neural membrane glycoprotein PrP is implicated in the pathogenesis of the transmissible spongiform encephalopathies; however, the normal function of PrP and its precise role in disease are not understood. Recently, gene targeting has been used to produce mice with neo/PrP fusion transcripts, but no detectable PrP protein in the brain (1). Here we report the use of a different targeting strategy, to produce inbred mice with a complete absence of both PrP protein and mRNA sequences. At 7 mo of age, these mice show no overt phenotypic abnormalities despite the normal high levels of expression of PrP during mouse development. The mice are being used in experiments designed to address the role of PrP in the pathogenesis of scrapie and the replication of infectivity.