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The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64'752 unique siRNAs targeting 21'584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51'128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies., Competing Interests: The authors have declared that no competing interests exist.

In solid cancers, high expression of the cellular prion protein (PrPC) is associated with stemness, invasiveness, and resistance to chemotherapy, but the role of PrPC in tumor response to radiotherapy is unknown. Here, we show that, in neuroblastoma, breast, and colorectal cancer cell lines, PrPC expression is increased after ionizing radiation (IR) and that PrPC deficiency increases radiation sensitivity and decreases radiation-induced radioresistance in tumor cells. In neuroblastoma cells, IR activates ATM that triggers TAK1-dependent phosphorylation of JNK and subsequent activation of the AP-1 transcription factor that ultimately increases PRNP promoter transcriptional activity through an AP-1 binding site in the PRNP promoter. Importantly, we show that this ATM-TAK1-PrPC pathway mediated radioresistance is activated in all tumor cell lines studied and that pharmacological inhibition of TAK1 activity recapitulates the effects of PrPC deficiency. Altogether, these results unveil how tumor cells activate PRNP to acquire resistance to radiotherapy and might have implications for therapeutic targeting of solid tumors radioresistance.

The phenotypes of calbindin-D9k - ( CaBP-9k -) knockout (KO), calbindin - D28k - ( CaBP-28k -) KO, and CaBP-9k/28k -KO mice are similar to those of wild-type (WT) mice due to the compensatory action of other calcium transport proteins. In this study, we investigated the expression of cellular prion protein (PrP C ) in the brains of CaBP-9k -, CaBP-28k -, and CaBP-9k/28k -KO mice. PrP C expression was significantly upregulated in the brain of all three strains. Levels of phospho-Akt (Ser473) and phospho-Bad (Ser136) were significantly elevated, but those of phospho-ERK and phospho-Bad (Ser155 and 112) were significantly reduced in the brains of CaBP-9k -, CaBP-28k -, and CaBP-9k/28k -KO mice. The expressions of the Bcl-2, p53, Bax, Cu/Zn-SOD, and Mn-SOD proteins were decreased in the brains of all KO mice. Expression of the endoplasmic reticulum marker protein BiP/GRP78 was decreased, and that of the CHOP protein was increased in the brains of those KO mice. To identify the roles of CaBP-28k , we transfected PC12 cells with siRNA for CaBP-28k and found increased expression of the PrP C protein compared to the levels in control cells. These results suggest that CaBP-28k expression may regulate PrP C protein expression and these mice may be vulnerable to the influence of prion disease.

Accumulation of conformationally altered cellular proteins (i.e., prion protein) is the common feature of prions and other neurodegenerative diseases. Previous studies demonstrated that the lack of terminal sequence of cellular prion protein (PrPC), necessary for the addition of glycosylphosphatidylinositol lipid anchor, leads to a protease-resistant conformation that resembles scrapie-associated isoform of prion protein. Moreover, mice overexpressing the truncated form of PrPC showed late-onset, amyloid deposition, and the presence of a short protease-resistant PrP fragment in the brain similar to those found in Gerstmann-Sträussler-Scheinker disease patients. Therefore, the physiopathological function of truncated_/anchorless 23-230 PrPC (Δ23-230 PrPC) has come into focus of attention. The present study aims at revealing the physiopathological function of the anchorless PrPC form by identifying its interacting proteins. The truncated_/anchorless Δ23-230 PrPC along with its interacting proteins was affinity purified using STrEP-Tactin chromatography, in-gel digested, and identified by quadrupole time-of-flight tandem mass spectrometry analysis in prion protein-deficient murine hippocampus (HpL3-4) neuronal cell line. Twenty-three proteins appeared to interact with anchorless Δ23-230 PrPC in HpL3-4 cells. Out of the 23 proteins, one novel protein, pyruvate kinase isozymes M1/M2 (PKM2), exhibited a potential interaction with the anchorless Δ23-230 form of PrPC. Both reverse co-immunoprecipitation and confocal laser-scanning microscopic analysis confirmed an interaction of PKM2 with the anchorless Δ23-230 form of PrPC. Furthermore, we provide the first evidence for co-localization of PKM2 and PrPC as well as PrPC-dependent PKM2 expression regulation. In addition, given the involvement of PrPC in the regulation of apoptosis, we exposed HpL3-4 cells to staurosporine (STS)-mediated apoptotic stress. In response to STS-mediated apoptotic stress, HpL3-4 cells transiently expressing 23-230-truncated PrPC were markedly less viable, were more prone to apoptosis and exhibited significantly higher PKM2 expressional regulation as compared with HpL3-4 cells transiently expressing full-length PrPC (1-253 PrPC). The enhanced STS-induced apoptosis was shown by increased caspase-3 cleavage. Together, our data suggest that the misbalance or over expression of anchorless Δ23-230 form of PrPC in association with the expressional regulation of interacting proteins could render cells more prone to cellular insults-stress response, formation of aggregates and may ultimately be linked to the cell death.

The cellular prion protein (PrP(C)) plays a key role in prion diseases when it converts to the pathogenic form scrapie prion protein. Increasing knowledge of its participation in prion infection contrasts with the elusive and controversial data regarding its physiological role probably related to its pleiotropy, cell-specific functions, and cellular-specific milieu. Multiple approaches have been made to the increasing understanding of the molecular mechanisms and cellular functions modulated by PrP(C) at the transcriptomic and proteomic levels. Gene expression analyses have been made in several mouse and cellular models with regulated expression of PrP(C) resulting in PrP(C) ablation or PrP(C) overexpression. These analyses support previous functional data and have yielded clues about new potential functions. However, experiments on animal models have shown moderate and varied results which are difficult to interpret. Moreover, studies in cell cultures correlate little with in vivo counterparts. Yet, both animal and cell models have provided some insights on how to proceed in the future by using more refined methods and selected functional experiments.

Although structurally and biochemically similar to the cellular prion (PrP(C)), doppel (Dpl) is unique in its biological functions. There are no reports about any neurodegenerative diseases induced by Dpl. However the artificial expression of Dpl in the PrP-deficient mouse brain causes ataxia with Purkinje cell death. Abundant Dpl proteins have been found in testis and depletion of the Dpl gene (Prnd) causes male infertility. Therefore, we hypothesize different regulations of Prnd in the nerve and male productive systems. In this study, by electrophoretic mobility shift assays we have determined that two different sets of transcription factors are involved in regulation of the Prnd promoter in mouse neuronal N2a and GC-1 spermatogenic (spg) cells, i.e., upstream stimulatory factors (USF) in both cells, Brn-3 and Sp1 in GC-1 spg cells, and Sp3 in N2a cells, leading to the expression of Dpl in GC-1 spg but not in N2a cells. We have further defined that, in N2a cells, Dpl induces oxidative stress and apoptosis, which stimulate ataxia-telangiectasia mutated (ATM)-modulating bindings of transcription factors, p53 and p21, to Prnp promoter, resulting the PrP(C) elevation for counteraction of the Dpl cytotoxicity; in contrast, in GC-1 spg cells, phosphorylation of p21 and N-terminal truncated PrP may play roles in the control of Dpl-induced apoptosis, which may benefit the physiological function of Dpl in the male reproduction system.

We employed a sensitive mass spectrometry-based method to deconstruct, confirm, and quantitate the prions present in elk naturally infected with chronic wasting disease and sheep naturally infected with scrapie. We used this approach to study the oxidation of a methionine at position 216 (Met216), because this oxidation (MetSO216) has been implicated in prion formation. Three polymorphisms (Ile218, Val218, and Thr218) of sheep recombinant prion protein were prepared. Our analysis showed the novel result that the proportion of MetSO216 was highly dependent upon the amino acid residue at position 218 (I > V > T), indicating that Ile218 in sheep and elk prion protein (PrP) renders the Met216 intrinsically more susceptible to oxidation than the Val218 or Thr218 analogue. We were able to quantitate the prions in the attomole range. The presence of prions was verified by the detection of two confirmatory peptides: GENFTETDIK (sheep and elk) and ESQAYYQR (sheep) or ESEAYYQR (elk). This approach required much smaller amounts of tissue (600 μg) than traditional methods of detection (enzyme-linked immunosorbent assay, Western blot, and immunohistochemical analysis) (60 mg). In sheep and elk, a normal cellular prion protein containing MetSO216 is not actively recruited and converted to prions, although we observed that this Met216 is intrinsically more susceptible to oxidation.

Growing evidence suggests that a physiological activity of the cellular prion protein (PrP(C)) plays a crucial role in several neurodegenerative disorders, including prion and Alzheimer's diseases. However, how the functional activity of PrP(C) is subverted to deliver neurotoxic signals remains uncertain. Transgenic (Tg) mice expressing PrP with a deletion of residues 105-125 in the central region (referred to as ΔCR PrP) provide important insights into this problem. Tg(ΔCR) mice exhibit neonatal lethality and massive degeneration of cerebellar granule neurons, a phenotype that is dose dependently suppressed by the presence of wild-type PrP. When expressed in cultured cells, ΔCR PrP induces large, ionic currents that can be detected by patch-clamping techniques. Here, we tested the hypothesis that abnormal ion channel activity underlies the neuronal death seen in Tg(ΔCR) mice. We find that ΔCR PrP induces abnormal ionic currents in neurons in culture and in cerebellar slices and that this activity sensitizes the neurons to glutamate-induced, calcium-mediated death. In combination with ultrastructural and biochemical analyses, these results demonstrate a role for glutamate-induced excitotoxicity in PrP-mediated neurodegeneration. A similar mechanism may operate in other neurodegenerative disorders attributable to toxic, β-rich oligomers that bind to PrP(C).

Prion protein (PrP(C) ), is a glycoprotein that is expressed on the cell surface. The current study examines the role of PrP(C) in early human embryogenesis using human embryonic stem cells (hESCs) and tetracycline-regulated lentiviral vectors that up-regulate or suppresses PrP(C) expression. Here, we show that expression of PrP(C) in pluripotent hESCs cultured under self-renewal conditions induced cell differentiation toward lineages of three germ layers. Silencing of PrP(C) in hESCs undergoing spontaneous differentiation altered the dynamics of the cell cycle and changed the balance between the lineages of the three germ layers, where differentiation toward ectodermal lineages was suppressed. Moreover, over-expression of PrP(C) in hESCs undergoing spontaneous differentiation inhibited differentiation toward lineages of all three germ layers and helped to preserve high proliferation activity. These results illustrate that PrP(C) is involved in key activities that dictate the status of hESCs including regulation of cell cycle dynamics, controlling the switch between self-renewal and differentiation, and determining the fate of hESCs differentiation. This study suggests that PrP(C) is at the crossroads of several signaling pathways that regulate the switch between preservation of or departure from the self-renewal state, control cell proliferation activity, and define stem cell fate., (© 2012 The Authors Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Programmed cell death is regulated by prototypes of a large family of Bcl-2-like proteins such as Bax and Bcl-2. A neuroprotective role for cellular prion protein (PrPc) on programmed cell death has been reported, although the cytosolic accumulation of PrPc correlates with toxicity and death of some neurons by apoptosis. In order to understand the signalling function of PrPc in promoting survival or suppressing cell death, we analyzed the expression and co-localization of PrPc, Bax and Bcl-2 proteins in the developing cerebellum of rats treated at PD10 (postnatal day 10) with the chemotherapeutic drug cisplatin (cisPt) or the new platinum (Pt) compound [Pt(O,O'-acac)(γ-acac)(DMS)] (PtAcacDMS). Differences in the expression of PrPc, Bax and Bcl-2 were found in proliferating cells and immature Purkinje neurons. One day after administration (PD11), cisPt markedly increased the apoptosis of the proliferating cells of the EGL (external granular layer); at the same time, several apoptotic bodies with strong Bax immunoreactivity were noticed. After PtAcacDMS, changes in PrPc and apoptotic proteins, with respect to the controls, were found but Bax immunopositive apoptotic bodies were not detectable, which could mean that apoptotic cell death of proliferating cells is preserved. Co-localization was clearly detected in the Purkinje cell population and may explain better the mechanisms by which PrPc and the apoptotic proteins function, and particularly the role of PrPc. Considering the reactivity of Purkinje neurons to these proteins at PD11 and Pd17, at least PrPc expression increased after cisPt and PtAcacDMS treatments or, if PrPc decreased, balanced itself with Bcl-2. The noteworthiness of this finding is that it emphasizes that most of the post-mitotic Purkinje cells need to be rescued, otherwise they undergo degeneration and are not replaced. Based on the effects of both Pt compounds on Purkinje cell differentiation, it should be emphasized that PrPc, together with the synergistic action of the co-localized anti-apoptotic protein, acts as a neuroprotective protein countering cytotoxicity in the postnatal critical phases of cerebellum development., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The prion protein (PrP(C)) is a cell surface protein expressed mainly in the nervous system. In addition to the role of its abnormal conformer in transmissible spongiform encephalopathies, normal PrP(C) may be implicated in other degenerative conditions often associated with inflammation. PrP(C) is also present in cells of hematopoietic origin, including T cells, dendritic cells, and macrophages, and it has been shown to modulate their functions. Here, we investigated the impact of inflammation and stress on the expression and function of PrP(C) in neutrophils, a cell type critically involved in both acute and chronic inflammation. We found that systemic injection of LPS induced transcription and translation of PrP(C) in mouse neutrophils. Up-regulation of PrP(C) was dependent on the serum content of TGF-β and glucocorticoids (GC), which, in turn, are contingent on the activation of the hypothalamic-pituitary-adrenal axis in response to systemic inflammation. GC and TGF-β, either alone or in combination, directly up-regulated PrP(C) in neutrophils, and accordingly, the blockade of GC receptors in vivo curtailed the LPS-induced increase in the content of PrP(C). Moreover, GC also mediated up-regulation of PrP(C) in neutrophils following noninflammatory restraint stress. Finally, neutrophils with up-regulated PrP(C) presented enhanced peroxide-dependent cytotoxicity to endothelial cells. The data demonstrate a novel interplay of the nervous, endocrine, and immune systems upon both the expression and function of PrP(C) in neutrophils, which may have a broad impact upon the physiology and pathology of various organs and systems.

The mammalian cellular prion protein (PrP(C) ) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc) ), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about PrP(Sc) conversion and its role in TSEs, the normal function of PrP(C) has not been elucidated. In adult mammals, PrP(C) is most abundant in the central nervous tissue, with intermediate levels in the intestine and heart, and lower levels in the pancreas and liver. PrP(C) is expressed during neurogenesis throughout development, and it has recently been proposed that PrP(C) participates in neural cell differentiation during embryogenesis. In order to establish the developmental timing and to address the cell-specific expression of PrP(C) during mammalian development, we examined PrP(C) expression in bovine gametes and embryos through gestation Day 39. Our data revealed differential levels of Prnp mRNA at Days 4 and 18 in pre-attachment embryos. PrP(C) was detected in the developing central and peripheral nervous systems in Day-27, 32-, and -39 embryos. PrP(C) was particularly expressed in differentiated neural cells located in the marginal regions of the central nervous system, but was absent from mitotically active, periventricular areas. Moreover, a PrP(C) cell-specific pattern of expression was detected in non-nervous tissues, including liver and mesonephros, during these stages. The potential participation of PrP(C) in neural cell differentiation is supported by its specific expression in differentiated states of neurogenesis., (Copyright © 2012 Wiley Periodicals, Inc.)

The human prion protein fragment, PrP (106-126), may contain a majority of the pathological features associated with the infectious scrapie isoform of PrP, known as PrP(Sc). Based on our previous findings that hypoxia protects neuronal cells from PrP (106-126)-induced apoptosis and increases cellular prion protein (PrP(C)) expression, we hypothesized that hypoxia-related genes, including hypoxia-inducible factor-1 alpha (HIF-1α), may regulate PrP(C) expression and that these genes may be involved in prion-related neurodegenerative diseases. Hypoxic conditions are known to elicit cellular responses designed to improve cell survival through adaptive processes. Under normoxic conditions, a deferoxamine-mediated elevation of HIF-1α produced the same effect as hypoxia-inhibited neuron cell death. However, under hypoxic conditions, doxorubicin-suppressed HIF-1α attenuated the inhibitory effect on neuron cell death mediated by PrP (106-126). Knock-down of HIF-1α using lentiviral short hairpin (sh) RNA-induced downregulation of PrP(C) mRNA and protein expression under hypoxic conditions, and sensitized neuron cells to prion peptide-mediated cell death even in hypoxic conditions. In PrP(C) knockout hippocampal neuron cells, hypoxia increased the HIF-1α protein but the cells did not display the inhibitory effect of prion peptide-induced neuron cell death. Adenoviruses expressing the full length Prnp gene (Ad-Prnp) were utilized for overexpression of the Prnp gene in PrP(C) knockout hippocampal neuron cells. Adenoviral transfection of PrP(C) knockout cells with Prnp resulted in the inhibition of prion peptide-mediated cell death in these cells. This is the first report demonstrating that expression of normal PrP(C) is regulated by HIF-1α, and PrP(C) overexpression induced by hypoxia plays a pivotal role in hypoxic inhibition of prion peptide-induced neuron cell death. These results suggest that hypoxia-related genes, including HIF-1α, may be involved in the pathogenesis of prion-related diseases and as such may be a therapeutic target for prion-related neurodegenerative diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Malignant gliomas recur even after extensive surgery and chemo-radiotherapy. Although a relatively novel chemotherapeutic agent, temozolomide (TMZ), has demonstrated promising activity against gliomas, the effects last only a few months and drug resistance develops thereafter in many cases. It has been acknowledged that glioma cells respond to TMZ treatment by undergoing G2/M arrest, but not apoptosis. Here we demonstrate a phase-specific chemotherapy resistance due to cellular prion protein (PrPc) in human glioma cells upon TMZ treatment. TMZ-induced G2/M-arrested cultures show an upregulation of PrPc expression and are more resistant, whereas G1/S-phase cells that show decreased levels of PrPc are more sensitive to apoptosis. Furthermore, an investigation into the biological significance of PrPc association with par-4 provided the first evidence of a relationship between the endogenous levels of PrPc and the resistance of glioma cells to the apoptotic effects of TMZ. Upon TMZ treatment, PrPc exerts its antiapoptotic activity by inhibiting PKA-mediated par-4 phosphorylation that are important for par-4 activation, nuclear entry and initiation of apoptosis. In context with cell cycle-dependent responses to chemotherapy, the data from this study suggest the possibility of exploiting the PrPc-dependent pathway to improve the efficacy of TMZ-based regimen for patients with gliomas., (Copyright © 2011 UICC.)

Prion diseases are characterised by the accumulation of PrP(Sc), an abnormally folded isoform of the cellular prion protein (PrP(C)), in affected tissues. Following peripheral exposure high levels of prion-specific PrP(Sc) accumulate first upon follicular dendritic cells (FDC) in lymphoid tissues before spreading to the CNS. Expression of PrP(C) is mandatory for cells to sustain prion infection and FDC appear to express high levels. However, whether FDC actively replicate prions or simply acquire them from other infected cells is uncertain. In the attempts to-date to establish the role of FDC in prion pathogenesis it was not possible to dissociate the Prnp expression of FDC from that of the nervous system and all other non-haematopoietic lineages. This is important as FDC may simply acquire prions after synthesis by other infected cells. To establish the role of FDC in prion pathogenesis transgenic mice were created in which PrP(C) expression was specifically "switched on" or "off" only on FDC. We show that PrP(C)-expression only on FDC is sufficient to sustain prion replication in the spleen. Furthermore, prion replication is blocked in the spleen when PrP(C)-expression is specifically ablated only on FDC. These data definitively demonstrate that FDC are the essential sites of prion replication in lymphoid tissues. The demonstration that Prnp-ablation only on FDC blocked splenic prion accumulation without apparent consequences for FDC status represents a novel opportunity to prevent neuroinvasion by modulation of PrP(C) expression on FDC.

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases attributed to misfolding of the cellular prion protein, PrP(C), into a β-sheet-rich, aggregated isoform, PrP(Sc). We previously found that expression of mouse PrP with the two amino acid substitutions S170N and N174T, which result in high structural order of the β2-α2 loop in the NMR structure at pH 4.5 and 20°C, caused transmissible de novo prion disease in transgenic mice. Here we report that expression of mouse PrP with the single-residue substitution D167S, which also results in a structurally well ordered β2-α2 loop at 20°C, elicits spontaneous PrP aggregation in vivo. Transgenic mice expressing PrP(D167S) developed a progressive encephalopathy characterized by abundant PrP plaque formation, spongiform change, and gliosis. These results add to the evidence that the β2-α2 loop has an important role in intermolecular interactions, including that it may be a key determinant of prion protein aggregation.

Unlabelled: The PrP(C) is expressed in several cell types but its physiological function is unknown. Some studies associate the PrP(C) with copper metabolism and the antioxidant activity of SOD. Our hypothesis was that changes in PrP(C) expression lead to abnormal copper regulation and induce SOD downregulation in the vascular wall., Objectives: to study whether the PrP(C) expression undergoes induction by agents that trigger endoplasmic reticulum stress (ERS) and, in this context, to evaluate the SOD activity., Methods: To trigger ERS, in vitro, rabbit aortic smooth muscle cells were challenged for 4, 8 and 18 hours, with angiotensin-II, tunicamycin and 7-ketocholesterol. For in vivo studies rabbit aortic arteries were subjected to injury by balloon catheter., Results: In vitro baseline SOD activity, determined through inhibition of cytochrome-c reduction, was 13.9±1.2 U/mg protein, angiotensin-II exposed for 8 hours produced an increase in SOD activity, and cellular copper concentration was about 9 times greater only under these conditions. Western blotting analysis for SOD isoenzymes showed an expression profile that was not correlated with the enzymatic activity. PrP(C) expression decreased after exposure to all agents after different incubation periods. RT-PCR assay showed increased mRNA expression for PrP(C) only in cells stimulated for 8 hours with the different stressors. The PrP(C) mRNA expression in rabbit aortic artery fragments, subjected to balloon catheter injury, showed a pronounced increase immediately after overdistension. The results obtained indicated a PrP(C) protection factor during the early part of the ERS exposure period, but did not demonstrate a SOD-like profile for the PrP(C)., (Copyright © 2009 Elsevier GmbH. All rights reserved.)

The infectivity associated with prion disease sets it apart from a large group of late-onset neurodegenerative disorders that shares the characteristics of protein aggregation and neurodegeneration. The unconventional infectious agent, PrP(Sc), is an aberrantly folded form of the normal prion protein (PrP(C)) and the PrP(C)-to-PrP(Sc) conversion is a critical pathogenic step in prion disease. Using the Protein Misfolding Cyclic Amplification technique, we converted folded bacterially expressed recombinant PrP into a proteinase K-resistant and aggregated conformation (rPrP-res) in the presence of anionic lipid and RNA molecules. Moreover, high prion infectivity was demonstrated by intracerebral inoculation of rPrP-res into wild-type mice, which caused prion disease with a short incubation period. The establishment of the in vitro recombinant PrP conversion assay makes it feasible for us to explore the molecular basis behind the intriguing properties associated with prion infectivity., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Mammalian prions cause fatal neurodegenerative conditions including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. Prion infections are typically associated with remarkably prolonged but highly consistent incubation periods followed by a rapid clinical phase. The relationship between prion propagation, generation of neurotoxic species and clinical onset has remained obscure. Prion incubation periods in experimental animals are known to vary inversely with expression level of cellular prion protein. Here we demonstrate that prion propagation in brain proceeds via two distinct phases: a clinically silent exponential phase not rate-limited by prion protein concentration which rapidly reaches a maximal prion titre, followed by a distinct switch to a plateau phase. The latter determines time to clinical onset in a manner inversely proportional to prion protein concentration. These findings demonstrate an uncoupling of infectivity and toxicity. We suggest that prions themselves are not neurotoxic but catalyse the formation of such species from PrP(C). Production of neurotoxic species is triggered when prion propagation saturates, leading to a switch from autocatalytic production of infectivity (phase 1) to a toxic (phase 2) pathway.

The mammalian cellular prion protein (PrP(C)) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc)), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrP(C) is poorly understood. Given the abundant expression of PrP(C) in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrP(C) participates in neural cell differentiation. In the present study, we investigated the role of PrP(C) in neural development during early embryogenesis. In bovine fetuses, PrP(C) was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrP(C) contributed to the process of neural differentiation during early embryogenesis. PrP(C) showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrP(C) expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells differentiation by retinoic acid (RA) resulted in up-regulation of PrP(C) at Day 20 and nestin at Day 12. PrP(C) expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrP(C) knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis of bovine fetuses suggests the participation of PrP(C) in neural cell differentiation during early embryogenesis. The positive association between PrP(C) and nestin expression provide evidence for the contribution of PrP(C) to ES cell differentiation into neural progenitor cells., (Copyright © 2010 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)

The microtubule associated protein tau plays a crucial role in Alzheimer's disease and in many neurodegenerative disorders collectively known as tauopathies. Recently, tau pathology has been also documented in prion diseases although the possible molecular events linking these two proteins are still unknown. We have investigated the fate of normal cellular prion protein (PrP(C)) in primary cortical neurons overexpressing tau protein. We found that overexpression of tau reduces PrP(C) expression at the cell surface and causes its accumulation and aggregation in the cell body but does not affect its maturation and glycosylation. Trapped PrP(C) forms detergent-insoluble aggregates, mainly composed of un-glycosylated and mono-glycosylated forms of prion protein. Interestingly, co-transfection of tau gene in cortical neurons with a proteasome activity reporter, consisting of a short peptide degron fused to the carboxyl-terminus of green fluorescent protein (GFP-CL1), results in down-regulation of the proteasome system, suggesting a possible mechanism that contributes to intracellular PrP(C) accumulation. These findings open a new perspective for the possible crosstalk between tau and prion proteins in the pathogenesis of tau induced-neurodegeneration.

The SPRN gene encodes the Shadoo glycoprotein (Sho), a central nervous system-expressed member of the prion protein superfamily. Sho has similarity to two features within PrPC's natively unstructured N-terminus, a hydrophobic domain and tandem repeats with positively charged residues. Indeed, scrutiny of Sho's biochemical properties in uninfected cells has revealed overlaps with the properties of PrPC, these including shared protein binding partners. SPRN is conserved in mammals, as is the prion gene PRNP, but in sheep SPRN and PRNP are both marked by polymorphic variation, suggestive of a shared selection pressure within these scrapie disease-prone livestock animals. In rodent models of prion disease there are reduced levels of Sho in infected tissues, defining a form of cross-regulation between full-length Sho holoprotein and PrPSc. In human prion disease an SPRN signal peptide polymorphism is associated with risk for sporadic Creutzfeldt-Jakob Disease (CJD), while two patients with early-onset variant CJD carried putatively inactive SPRN alleles. Further investigation of Sho as a novel tracer or modifier for the accumulation of pathologic forms of PrP may prove advantageous.

The definite physiological role of the cellular prion protein (PrP(c)) remains elusive. There is ample in vitro and in vivo evidence suggesting a neuroprotective role for PrP(c). On the other hand, several in vitro and in vivo studies demonstrated detrimental effects of PrP(c) overexpression through activation of a p53 pathway. Recently, we reported that transient overexpression of PrP(c) in human embryonic kidney 293 cells elicits proteome expression changes which point to deregulation of proteins involved in energy metabolism and cellular homeostasis. Here we report proteome expression changes following stable PrP(c) overexpression in human neuronal SH-SY5Y cells. In total 18 proteins that are involved in diverse biological processes were identified as differentially regulated. The majority of these proteins is involved in cell signaling, cytoskeletal organization and protein folding. Annexin V exhibited a several fold up-regulation following stable PrP(c) overexpression in SH-SY5Y cells. This finding has been reproduced in alternative, mouse N2a and human SK-N-LO neuroblastoma cell lines transiently overexpressing PrP(c). Annexin V plays an important role in maintenance of calcium homeostasis which when disturbed can activate a p53-dependent cell death. Although we did not detect changes in p53 expression between PrP(c) overexpressing SH-SY5Y and control cells, deregulation of several proteins including annexin V, polyglutamine tract-binding protein-1, spermine synthase and transgelin 2 indicates disrupted cellular equilibrium. We conclude that stable PrP(c) overexpression in SH-SY5Y cells is sufficient to perturb cellular balance but insufficient to affect p53 expression., ((c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Prions are infectious proteins that are able to recruit a normal cellular prion protein and convert it into a prion. The mechanism of this conversion is unknown. Detailed analysis of the normal cellular prion protein and a corresponding prion has shown they possess identical post-translational modifications and differ solely in conformation. Recent work has suggested that the oxidized form of the methionine at position 213 (Met213) plays a role in the conversion of the normal cellular prion protein to the prion conformation and is a prion-specific covalent signature. We developed a sensitive method of quantitating the methionine sulfoxide present at position 213 (MetSO213) and used this method to measure the changes in MetSO213 over the time course of an intracranial challenge, using the 263K strain of hamster-adapted scrapie. These results indicate that the proportion of Met213 that is oxidized decreases over the course of the disease. We examined the quantity of MetSO213 in PrP(C) and compared it to the amount found in animals terminally afflicted with the 263K, 139H, and drowsy strains of hamster-adapted scrapie. These strains show only low levels of MetSO213 that is comparable to that of PrP(C). These data suggest that MetSO213 does not appear to be a prion-specific covalent signature.

Protein misfolding cyclic amplification (PMCA) is a cell-free assay mimicking the prion replication process. However, constraints affecting PMCA have not been well-defined. Although cellular prion protein (PrP(C)) is required for prion replication, the influence of PrP(C) abundance on PMCA has not been assessed. Here, we show that PMCA was enhanced by using mouse brain material in which PrP(C) was overexpressed. Tg(MoPrP)4112 mice overexpressing PrP(C) supported more sensitive and efficient PMCA than wild type mice. As brain homogenate of Tg(MoPrP)4112 mice was diluted with PrP(C)-deficient brain material, PMCA became less robust. Our studies suggest that abundance of PrP(C) is a determinant that directs enhancement of PMCA. PMCA established here will contribute to optimizing conditions to enhance PrP(Sc) amplification by using concentrated PrP(C) source and expands the use of this methodology.

The host encoded cellular prion protein (PrP(C)) is an N-linked glycoprotein tethered to the cell membrane by a glycophosphatidylinositol (GPI) anchor. Under certain conditions, PrP(C) can undergo conversion into a conformationally-altered isoform (PrP(Sc)) widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Understanding the tissue-specific expression of PrP(C) is crucial considering that cells expressing high levels of PrP(C) bear a risk for conversion and accumulation of PrP(Sc). In the present study, fifteen bovine somatic tissues were analyzed for PrP(C) expression by quantitative western blot and immunohistochemistry. Quantitative western blot analysis revealed highest expression of PrP(C) in cerebellum, obex and spinal cord. Intermediate levels were detected in thymus, intestine, nerve, heart and spleen, and lower levels in lung, muscle, kidney, lymph node, skin, pancreas and liver. Immunohistochemical analysis detected intense cellular-specific PrP(C) staining in neurons, thymocytes and lymphocytes. PrP(C) was also detected in the enteric wall, pancreatic islets of langerhans, myocardium, pulmonary alveolar sacs, renal glomeruli and dermal epithelial cells. This study demonstrated the quantitatively varied, wide-spread, tissue- and cell-specific expression pattern of PrP(C) in bovine somatic tissues. The importance of this study is to lay the foundation for understanding the tissue-specific expression of PrP(C) and to consider the potential participation of more bovine tissues in the transmission of BSE infection.

Prion leads to fatal transmissible spongiform encephalopathies. Cellular prion protein (PrPc) is necessary in prion disease. At present, it is demonstrated that PrPc plays a protective role in several carcinomas, such as gastric and breast cancer. We designed four 19-nt siRNAs according to cDNA sequence of human PrPc and constructed retrovirus-based RNAi vectors. We evaluated the inhibitive effect of these sequences on HuPrPc (human PrPc) and selected out three sequences with stable and efficient inhibition. And the efficiency of si626 reached more than 85%, which effect was significant. Next, we performed cell invasion assays of PC3M-si292 and PC3M-si626 in which PrPc was inhibited. And it showed that the cell invasive ability decreased in PrPc knock-down cell lines. This will make preparations for the further research on gene therapy of prion diseases and PrPc related carcinoma treatment and PrPc could be considered as a potential therapeutic target molecule in prostate cancer treatment.

Prion mRNA translation inhibition by antisense oligodeoxynucleotides (asODN) incorporated into immunoliposomes was investigated. It was shown that asODN complementary to cap region, start-codon region and a part of open reading frame can decrease the prion expression by 80% in L1210 cell line and by 60% in prion-replicating organs of laboratory rats. These results give grounds for further research asODN to be used as a means of prevention and treatment of prion infections.

The PrP gene encodes the cellular isoform of the prion protein (PrP(c)) which has been shown to be crucial to the development of transmissible spongiform encephalopathies (TSEs). PrP knock-out mice, which do not express endogenous PrP(c), exhibit resistance to TSE disease. The regulation of PrP gene expression represents, therefore, a crucial factor in the development of TSEs. Two sequence motifs in the PrP promoter (positions -287 to -263 from transcriptional start) were previously reported as being highly conserved, and it was suggested that they represent binding sites for as yet unidentified transcription factors. To test this hypothesis, binding of nuclear proteins was analyzed by electrophoretic mobility shift assays using ovine or murine cells and tissues with radiolabeled DNA probes containing the conserved motif sequences. Specific binding was observed to both motifs, and polymorphic variants of these motifs exhibited differential binding. Two proteins bound to these motifs were identified as the Yin Yang 1 (YY1) (motif 1) and E4BP4 (motif 2) transcription factors. Functional promoter analysis of four different promoter variants revealed that motif 1 (YY1) was associated with inhibitory activity in the context of the PrP promoter, whereas motif 2 (E4BP4) was linked to a slight enhancing activity. This represents the first demonstration of binding of nuclear factors to two highly conserved DNA sequence motifs within mammalian PrP promoters. The action of these factors on the PrP promoter is haplotype-specific, leading us to propose that the prion protein expression pattern and, with it, the distribution of TSE infectivity may be associated with PrP promoter genotype.

Increasing evidence suggests that the cellular prion protein (PrP(C)) plays a protective role in response to oxidative stress, but the molecular mechanism is unclear. Here, we demonstrate that murine neuro-2a and human HeLa cells rapidly respond to an increase of intracellular copper concentration by up-regulating ataxia-telangiectasia mutated (ATM)-mediated transcription of PrP(C). Copper stimulation activates ATM by phosphorylation at Ser-1981, which leads to phosphorylation of p53 at Ser-15 and the initiation of the mitogen-activated protein kinase kinase/extracellular-related kinases/extracellular-related kinases (MEK/ERK)/Sp1 pathway. As results, Sp1 and p53 bind to the PrP promoter, leading to increase PrP(C) expression. Elevated PrP(C) correlates with reduction of intracellular copper concentration and suppression of Cu(II)-induced accumulation of reactive oxygen species and cell death. Depletion of PrP(C), ATM, p53, and/or Sp1 further demonstrates that ATM is a key regulatory protein to promote activation of p53 and Sp1 leading to PrP(C) elevation, which is required to reduce Cu(II) toxic effects and may play an important role in modulation of intracellular copper concentration.

The physiological role of the cellular prion protein (PrP (c)) is still not fully understood. Current evidence strongly suggests that PrP (c) overexpression in different cell lines sensitizes cells to apoptotic stimuli through a p53 dependent pathway. On the other hand, an expression of PrP (c) in PrP (c)-deficient cells undergoing apoptosis exhibited repeatedly antiapoptotic effects. Therefore, the presence/absence and/or the level of PrP (c) expression seem to be critical for the fluctuation between PrP (c)'s pro- and antiapoptotic properties. The present study examined whether an overexpression of PrP (c) itself, without addition of any apoptotic agent, can lead to proteome changes that might account for the higher responsiveness to apoptotic stimuli. Beyond this, we examined whether the sole introduction of PrP (c) into PrP (c)-deficient cells could be sufficient to up-regulate antiapoptotic proteins capable of mitigating apoptosis. For this purpose, we used two cell lines, one expressing [human embryonic kidney (HEK) 293 cells] and the other lacking (mouse neuronal PrP (c)-deficient cells) endogenous PrP (c). Protein profiling following transient PrP (c) overexpression in HEK 293 cells revealed a major PrP (c) involvement in regulation of proteins participating in energy metabolism and cellular homeostasis, whereas transient introduction of PrP (c) into mouse neuronal PrP (c)-deficient cells resulted mainly in the regulation of proteins involved in protection against oxidative stress and apoptosis. In addition, we report for the first time that PrP (c) overexpression influenced the regulation of several proteins known to have contributory roles in the pathogenesis of Alzheimer disease (AD). Revealing the correlation between presence/absence and/or different levels of PrP (c) expression with the regulation of certain cellular proteins might further contribute to our understanding of the complex role of PrP (c) in cell physiology.

Cell models for prion diseases are mainly of neuronal origin. However, the pathological isoform PrP(Sc) of cellular prion protein (PrP(c)) and prion infectivity are found in a variety of extraneural tissues in prion diseases. Although many cell types are not able to propagate PrP(Sc), little is known about cellular mechanism counteracting prion infection. It is desirable to identify neuronal or non-neuronal cell models that restrict PrP(Sc) generation or propagate PrP(Sc) only transiently. Neuroendocrine cells are derived from tumours forming the interface between endocrine and nervous system. We investigated the susceptibility of such murine cell lines to prion infection, which were in principle able to transiently propagate PrP(Sc). Surprisingly and in contrast to neuronal cells prion infection was abrogated by rapid and PrP(Sc)-specific down-regulation of PrP(c) expression upon exposure to prion-infected material. Cell lines described here provide novel models for studying PrP(c) regulation and intrinsic cellular defence mechanisms upon prion exposure.

The trafficking of normal cellular prion protein (PrPC) is believed to control its conversion to the altered conformation (designated PrPSc) associated with prion disease. Although anchored to the membrane by means of glycosylphosphatidylinositol (GPI), PrPC on neurons is rapidly and constitutively endocytosed by means of coated pits, a property dependent upon basic amino acids at its N-terminus. Here, we show that low-density lipoprotein receptor-related protein 1 (LRP1), which binds to multiple ligands through basic motifs, associates with PrPC during its endocytosis and is functionally required for this process. Moreover, sustained inhibition of LRP1 levels by siRNA leads to the accumulation of PrPC in biosynthetic compartments, with a concomitant lowering of surface PrPC, suggesting that LRP1 expedites the trafficking of PrPC to the neuronal surface. PrPC and LRP1 can be co-immunoprecipitated from the endoplasmic reticulum in normal neurons. The N-terminal domain of PrPC binds to purified human LRP1 with nanomolar affinity, even in the presence of 1 muM of the LRP-specific chaperone, receptor-associated protein (RAP). Taken together, these data argue that LRP1 controls both the surface, and biosynthetic, trafficking of PrPC in neurons.

It is established that except for already known influence of pentosan polysulphate (SP-54) on the expression of pathological prion, this preparation has an inhibiting effect in respect of physiological prion. Moreover, the reduction of concentration of physiologycal prion is registered in the central and peripheral organs of the prion-replicating system. It was also shown that inhibition of the studied protein leads to the growth in copper and zinc concentration in the proper organs and tissues, but at the same time activity of Cu/Zn-dependent superoxide dismutase does not change.

The prion protein (PrP) is the key protein implicated in diseases known as transmissible spongiform encephalopathies. PrP has been shown to be a metallo-protein that binds copper (Cu), and copper might have a role in the normal function of the protein. Conversely, PrP expression in yeast led us to suggest that the protein might be involved in the regulation of Cu homeostasis. In the presence of excess Cu in the growth medium, PrP expression limited the increase of the total number of Cu atoms per cell to a maximum of 14-fold compared with mock control cells, which showed a 52-fold increased intracellular Cu level. Conclusively, we suggest that PrP expression itself has a regulatory or buffering function for the cellular Cu level in yeast cells, most likely due to binding of Cu to the multiple Cu binding sites.

Certain applications in the prion field require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, which are normally generated as a result of spontaneous oxidation or degradation.

Prion protein (PrP(C)) via its isoform PrP(SC) is involved in the pathogenesis of transmissible spongiform encephalopathies (TSEs). We observed that murine erythroleukemia (MEL) cells arrested in phase G(1) undergo transcriptional activation of Prn-p gene. Here, we explored the potential role of activation of Prn-p gene and cytosolic accumulation of PrP(C) in growth arrest, differentiation, and apoptotic DNA fragmentation by stably transfecting MEL and N2a cells with Prn-p cDNA. Stably transfected MEL cells (clones # 6, 12, 20, 38, and 42) were assessed for growth and differentiation, while clones N2a13 and N2a8 of N2a cells for growth and apoptosis by flow cytometry using Annexin V and propidium iodide (PI). Our results indicate that (a) Induction of terminal differentiation of stably transfected MEL cells led to growth arrest, activation of Prn-p gene, concomitant expression of transfected Prn-p cDNA, suppression of bax gene, cytosolic accumulation of PrP(C), and DNA fragmentation. The latter was also induced in non-differentiated MEL cells growing under serum-free conditions; (b) similarly, serum deprivation promoted growth arrest, apoptosis/necrosis associated with DNA fragmentation in parental N2a and N2a13 cells that produced relative high level of PrP(C) and not PrP(SC). These data indicate that activation of Prn-p gene and expression of transfected Prn-p cDNA in cells of both hematopoietic and neuronal origin occurred concomitantly, and led to cytosolic accumulation of PrP(C) and DNA damage induced by serum deprivation. PrP(C) production failed to protect DNA fragmentation induced by serum deprivation. The question how does PrP(C) contribute to growth arrest and DNA fragmentation is discussed., ((c) 2007 Wiley-Liss, Inc.)

The prion protein (PrP) level in muscle has been reported to be elevated in patients with inclusion-body myositis, polymyositis, dermatomyositis, and neurogenic muscle atrophy, but it is not clear whether the elevated PrP accumulation in the muscles is sufficient to cause muscle diseases. We have generated transgenic mice with muscle-specific expression of PrP under extremely tight regulation by doxycycline, and we have demonstrated that doxycycline-induced overexpression of PrP strictly limited to muscles leads to a myopathy characterized by increased variation of myofiber size, centrally located nuclei, and endomysial fibrosis, in the absence of intracytoplasmic inclusions, rimmed vacuoles, or any evidence of a neurogenic disorder. The PrP-induced myopathy correlates with accumulation of an N-terminal truncated PrP fragment in the muscle, and the muscular PrP displayed consistent mild resistance to protease digestion. Our findings indicate that overexpression of wild-type PrP in skeletal muscles is sufficient to cause a primary myopathy with no signs of peripheral neuropathy, possibly due to accumulation of a cytotoxic truncated form of PrP and/or PrP aggregation.

Propagation of the scrapie isoform of the prion protein (PrP(Sc)) depends on the expression of endogenous cellular prion (PrP(C)). During oral infection, PrP(Sc) propagates, by conversion of the PrP(C) to PrP(Sc), from the gastrointestinal tract to the nervous system. Intestinal epithelium could serve as the primary site for PrP(C) conversion. To investigate PrP(C) sorting in epithelia cells, we have generated both a green fluorescent protein (EGFP) or hemagglutinin (HA) tagged human PrP(C) (hPrP(C)). Combined molecular, biochemical, and single living polarized cell imaging characterizations suggest that hPrP(C) is selectively targeted to the apical side of Madin-Darby canine kidney (MDCKII) and of intestinal epithelia (Caco2) cells.

Ovine PBMCs (peripheral blood mononuclear cells) express PrP(C) [cellular PrP (prion-related protein)] and have the potential to harbour and release disease-associated forms of PrP during scrapie in sheep. Cell-surface PrP(C) expression by PBMCs, together with plasma PrP(C) levels, may contribute to the regulatory mechanisms that determine susceptibility and resistance to natural scrapie in sheep. Here, we have correlated cell-surface PrP(C) expression on normal ovine PBMCs by FACS with the presence of PrP(C) in plasma measured by capture-detector immunoassay. FACS showed similar levels of cell-surface PrP(C) on homozygous ARR (Ala136-Arg154-Arg171), ARQ (Ala136-Arg154-Gln171) and VRQ (Val136-Arg154-Gln171) PBMCs. Cell-surface ovine PrP(C) showed modulation of N-terminal epitopes, which was more evident on homozygous ARR cells. Ovine plasma PrP(C) levels showed genotypic variation and the protein displayed C-terminal epitopes not available in cell-surface PrP(C). Homozygous VRQ sheep showed the highest plasma PrP(C) level and homozygous ARR animals the lowest. For comparison, similar analyses were performed on normal bovine PBMCs and plasma. PrP(C) levels in bovine plasma were approx. 4-fold higher than ovine homozygous ARQ plasma despite similar levels of PBMC cell-surface PrP(C) expression. Immunoassays using C-terminal-specific anti-PrP monoclonal antibodies as capture and detector reagents revealed the highest level of PrP(C) in both ovine and bovine plasma, whilst lower levels were detected using N-terminal-specific monoclonal antibody FH11 as the capture reagent. This suggested that a proportion of plasma PrP(C) was N-terminally truncated. Our results indicate that the increased susceptibility to natural scrapie displayed by homozygous VRQ sheep correlates with a higher level of plasma PrP(C).

The cellular prion protein (PrP(c)) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrP(c) is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrP(c) expression and other parameters such as age of the animals under study or stage of lactation.

Background: The amino terminal half of the cellular prion protein PrPc is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein., Results: Fusion proteins of PrPc and GFP were expressed at high level in E.coli and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrPc the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in E.coli, mutations introduced randomly in the PrPc protein sequence that affected folding could be selected directly by recovery of GFP fluorescence., Conclusion: Use of GFP as a measure of refolding of PrPc fusion proteins in vitro and in vivo proved informative. Refolding in vitro suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrPc structure and PrPc-ligand interaction.

The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.

PrPC is a ubiquitously expressed glycophos-phatidylinositol-linked cell-surface glycoprotein found primarily in neural tissue. Although its normal function has not been established, there is evidence suggesting that PrPC is involved in cell signalling and cellular homeostasis. This suggests that variation in neuronal expression levels of this protein contributes towards pathogenicity induced by neurotropic agents. We have investigated the pathological response to infection with herpes simplex virus type-1 (HSV-1) in strains of mice that express different levels of PrPC. Prnp-/- mice fail to express PrPC due to an interruption in the open reading frame of the Prnp gene, whilst tg19 and tga20 mice express approximately 5 and 10 times more PrPC protein, respectively, than wild-type animals. Mice that express normal or increased levels of PrPC protein were more susceptible to acute HSV-1 infection than Prnp-/- mice. Following ear pinna inoculation with HSV-1 SC16, the order of susceptibility was tga20>tg19>wild-type>Prnp-/-. This trend was reversed when latent virus was assessed. Prnp-/- mice expressed significantly higher levels of latency-associated transcript-positive neurons in various tissues when compared with wild-type, tg19 and tga20 mice. Collectively, our data show that acute HSV-1 replication proceeds more efficiently in neuronal tissue that expresses PrPC protein and lends support to the view that this protein is involved in regulation of neurotropic viral pathogenesis. This suggests that interference of PrPC expression, or possible biochemical pathways associated with its function, may serve as an effective means of limiting the pathogenesis of acute HSV-1 infection.

Full-length Bos taurus PrPC protein was obtained in the eu- and prokaryotic expression systems. Immunoblotting and indirect enzyme immunoassay demonstrated high specificity and antigenic activity of full-length proteins in the reactions with monoclonal antibodies (anti-SAF-32 and VRQ-84). Membrane location of recombinant PrPC protein in insect cells was shown by immunofluorescent analysis.

Neuronal degeneration and astrogliosis are hallmarks of prion disease. Synthetic prion protein (PrP) peptide 106-126 (PrP106-126) can induce death of neurons and proliferation of astrocytes in vitro and this neurotoxic effect depends on the expression of cellular PrP (PrPC) and is hence believed to be PrP(C) -mediated. To further elucidate the involvement of PrPC in PrP106-126-induced neurotoxicity, we determined the expression of PrP mRNA in primary culture of rat cortical neuron cells, cerebellar granule cells, and astrocytes following treatment with 50 microM of PrP106-126 scrambled PrP106-126 by quantitative real-time RT-PCR. As shown by MTT test, PrP106-126 induced significant death of neuron cells and marked proliferation of astrocytes after 10 days of treatment. Under the same treatment regimens, the level of PrP gene expression was significantly down-regulated in cortical neuron cell cultures and cerebellar granule cell cultures and was up-regulated in astrocyte cultures. The altered PrP gene expression occurred as early as 3 days after the treatment. After 10 days of treatment, while the cultured cortical neurons underwent further apoptosis, their expression of PrP gene started to recover gradually. These findings indicate that PrP 106-126 regulates transcription of the PrP gene and this activity is associated with its neurotoxicity in primary rat neuronal cultures.

The conversion of the cellular prion protein (PrPc) into a pathogenic isoform (PrP(Sc)) is one of the underlying events in the pathogenesis of the fatal transmissible spongiform encephalopathies (TSEs). Numerous compounds have been described to inhibit prion replication and PrP(Sc) accumulation in cell culture. Among these, the drug suramin induces aggregation and re-targeting of PrPc to endocytic compartments. Plasma membrane and sites of conversion into PrP(Sc) are thereby bypassed. In the present study, a library of suramin analogues was tested as a potential class of new anti-prion compounds and the molecular mechanisms underlying these effects were analysed. Treatment of prion-infected neuroblastoma cells with compounds containing symmetrical aromatic sulfonic acid substitutions inhibited de novo synthesis of PrP(Sc) and induced aggregation and reduction of the half-life of PrPc without downregulating PrPc cell surface expression. Half-molecule compounds lacking the symmetrical bipolar structure or the anionic groups had no effect on PrP(Sc) synthesis or PrPc solubility. Cell surface expression of PrPc was necessary for the activity of effective compounds. Suramin derivatives did not induce aggregation of PrPc when transport along the secretory pathway was compromised, suggesting that their effects occur at a post trans-Golgi network (TGN) site, possibly close to the compartment of conversion into PrP(Sc). In vitro studies with recombinant PrP demonstrated that the inhibitory effect correlated with direct binding to PrP and induction of insoluble PrP aggregates. Our data reveal an anti-prion effect that differs from those characterising other sulphated polyanions and is dependent on the presence of the symmetrical anionic structure of these molecules.