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We previously reported that treatment with P-glycoprotein (P-gp) substrates promotes in vitro invasion in multidrug-resistant (MDR) breast cancer cells. This effect is initiated by the P-gp pump function and mediated by interaction of P-gp with some unknown component(s). However, the underlying mechanism(s) remains poorly understood. Here we confirm a novel physical interaction between P-gp and cellular prion protein (PrPc). Blocking P-gp activity or depletion of PrPc inhibited paclitaxel (P-gp substrate)- induced invasion. Paclitaxel further facilitated the formation of P-gp/PrPc clusters residing in caveolar domains and promoted the association of P-gp with caveolin-1. Both caveolin-1 and the integrity of caveolae were required for the drug-induced invasion. In addition, the P-gp/PrPc complex also played an important role in anti-apoptotic activity of MCF7/Adr cells.These data provide new insights into the mode by which MDR breast cancers evade cytotoxic attacks from P-gp substrates and also suggest a role for P-gp/ PrPc interaction in this process. [ABSTRACT FROM AUTHOR]

Abstract: While the prion protein (PrP) is clearly involved in neuropathology, its physiological roles remain elusive. Here, we demonstrate PrP functions in cell–substrate interaction in Drosophila S2, N2a and HeLa cells. PrP promotes cell spreading and/or filopodia formation when overexpressed, and lamellipodia when downregulated. Moreover, PrP normally accumulates in focal adhesions (FAs), and its downregulation leads to reduced FA numbers, increased FA length, along with Src and focal adhesion kinase (FAK) activation. Furthermore, its overexpression elicits the formation of novel FA-like structures, which require intact reggie/flotillin microdomains. Altogether, PrP modulates process formation and FA dynamics, possibly via signal transduction involving FAK and Src. [Copyright &y& Elsevier]

Abstract: Spongiform degeneration is characterized by vacuolation in nervous tissue accompanied by neuronal death and gliosis. Although spongiform degeneration is a hallmark of prion diseases, this pathology is also present in the brains of patients suffering from Alzheimer’s disease, diffuse Lewy body disease, human immunodeficiency virus (HIV) infection, and Canavan’s spongiform leukodystrophy. The shared outcome of spongiform degeneration in these diverse diseases suggests that common cellular mechanisms must underlie the processes of spongiform change and neurodegeneration in the central nervous system. Immunohistochemical analysis of brain tissues reveals increased ubiquitin immunoreactivity in and around areas of spongiform change, suggesting the involvement of ubiquitin–proteasome system dysfunction in the pathogenesis of spongiform neurodegeneration. The link between aberrant ubiquitination and spongiform neurodegeneration has been strengthened by the discovery that a null mutation in the E3 ubiquitin–protein ligase mahogunin ring finger-1 (Mgrn1) causes an autosomal recessively inherited form of spongiform neurodegeneration in animals. Recent studies have begun to suggest that abnormal ubiquitination may alter intracellular signaling and cell functions via proteasome-dependent and proteasome-independent mechanisms, leading to spongiform degeneration and neuronal cell death. Further elucidation of the pathogenic pathways involved in spongiform neurodegeneration should facilitate the development of novel rational therapies for treating prion diseases, HIV infection, and other spongiform degenerative disorders. [Copyright &y& Elsevier]

Abstract: Cell based models used for the study of prion diseases have traditionally employed mouse-adapted strains of sheep scrapie prions. To date, attempts to generate human prion propagation in cell culture have been unsuccessful. Rabbit kidney epithelial cells (RK13) are permissive to infection with prions from a variety of species upon expression of cognate PrP transgenes. We explored RK13 cells expressing human PrP for their utility as a cell line capable of sustaining infection with human prions. RK13 cells processed exogenously expressed human PrP similarly to exogenously expressed mouse PrP but were not permissive to infection when exposed to sporadic Creutzfeldt–Jakob disease prions. Transmission of the same sporadic Creutzfeldt Jakob disease prions to wild-type mice generated a strain of mouse-adapted human prions, which efficiently propagated in RK13 cells expressing mouse PrP, demonstrating these cells are permissive to infection by mouse-adapted human prions. Our observations underscore the likelihood that, in contrast to prions derived from non-human mammals, additional unidentified cofactors or subcellular environment are critical for the generation of human prions. [Copyright &y& Elsevier]

Tauopathies, including Alzheimer's disease (AD), are manifested by the deposition of well-characterized amyloid aggregates of Tau protein in the brain. However, it is rather unlikely that these aggregates constitute the major form of Tau responsible for neurodegenerative changes. Currently, it is postulated that the intermediates termed as soluble oligomers, assembled on the amyloidogenic pathway, are the most neurotoxic form of Tau. However, Tau oligomers reported so far represent a population of poorly characterized, heterogeneous and unstable assemblies. In this study, to obtain the oligomers, we employed the aggregation-prone K18 fragment of Tau protein with deletion of Lys280 (K18Δ280) linked to a hereditary tauopathy. We have described a new procedure of inducing aggregation of mutated K18 which leads either to the formation of nontoxic amyloid fibrils or neurotoxic globular oligomers, depending on its phosphorylation status. We demonstrate that PKA-phosphorylated K18Δ280 oligomers are toxic to hippocampal neurons, which is manifested by loss of dendritic spines and neurites, and impairment of cell-membrane integrity leading to cell death. We also show that N1, the soluble N-terminal fragment of prion protein (PrP), protects neurons from the oligomers-induced cytotoxicity. Our findings support the hypothesis on the neurotoxicity of Tau oligomers and neuroprotective role of PrP-derived fragments in AD and other tauopathies. These observations could be useful in the development of therapeutic strategies for these diseases. • Oligomers of phosphorylated K18 fragment of Tau with Lys280 deletion are neurotoxic. • N1 fragment of prion protein inhibits neurotoxicity of the oligomers of K18Δ280. • Protective action of N1 can result from its interaction with K18Δ280 oligomers. • N1 binds stronger to phosphorylated Tau and K18Δ280 than to their unmodified forms. • N1 induces association of the oligomers of K18Δ280 into larger assemblies. [ABSTRACT FROM AUTHOR]

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrPC) into its pathogenic, amyloidogenic isoform (PrPSc) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer’s disease and Pick’s disease. In the present study, we have examined the effects of LF on PrPSc formation by using cell culture models. Bovine LF inhibited PrPSc accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrPC by diminishing its internalization and was capable of interacting with PrPC in addition to PrPSc. Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities. [ABSTRACT FROM AUTHOR]

Abstract: The cellular prion protein (PrPc) undergoes a physiological cleavage between amino acids 111 and 112, thereby leading to the secretion of an amino-terminal fragment referred to as N1. This proteolytic event is either constitutive or regulated by protein kinase C (PKC) and is operated by the disintegrins ADAM9/ADAM10 or ADAM17 respectively. We recently showed that the stimulation of the M1/M3 muscarinic receptors potentiates this cleavage via the phosphorylation and activation of ADAM17. We have examined the contribution of various PKC isoforms in the regulated processing of PrPc. First we show that the PDBu- and carbachol-stimulated N1 secretions are blocked by the general PKC inhibitor GF109203X. We establish that HEK293 and human-derived rhabdhomyosarcoma cells over-expressing constitutively active PKCα, PKCδ or PKCε, but not PKCζ, produce increased amounts of N1 and harbor enhanced ability to hydrolyze the fluorimetric substrate of ADAM17, JMV2770. Conversely, over-expression of the corresponding dominant negative proteins abolishes PDBU-stimulated N1 secretion and restores N1 to levels comparable to constitutive production. Moreover, deletion of PKCα lowers N1 recovery in primary cultured fibroblasts. Importantly, mutation of threonine 735 of ADAM17 significantly lowers the PDBu-induced N1 formation while transient over-expression of constitutively active PKCα, PKCδ or PKCε, but not PKCζ, induced both the phosphorylation of ADAM17 on its threonine residues and N1 secretion. As a corollary, T735A mutation concomitantly reversed PKCα-, PKCδ- and PKCε-induced ADAM17 phosphorylation and N1 recovery. Finally, we established that PKCε-dependent N1 production is fully prevented by ADAM17 deficiency. Altogether, the present results provide strong evidence that the activation of PKCα, δ and ε, but not ζ, isoforms leads to increased N1 secretion via the phosphorylation and activation of ADAM17, a process that likely accounts for M1/M3 muscarinic receptors-mediated control of N1 production. [Copyright &y& Elsevier]

Transmissible spongiform encephalopathies (TSEs) are a group of diseases of infectious, sporadic and genetic origin, found in higher organisms and caused by the pathological form of the prion protein. The inheritable subgroup of TSEs is linked to insertional or point mutations in the prion gene prnp, which favour its misfolding and are passed on to offspring in an autosomal-dominant fashion. The large majority of patients with these diseases are heterozygous for the prnp gene, leading to the coexpression of the wild-type (wt) (PrPC) and the mutant forms (PrPmut) in the carriers of these mutations. To mimic this situation in vitro, we produced Fischer rat thyroid cells coexpressing PrPwt alongside mutant versions of mouse PrP including A117V, E200K and T182A relevant to the human TSE diseases Gestmann–Sträussler–Scheinker (GSS) disease and familial Creutzfeldt–Jakob disease (fCJD). We found that coexpression of mutant PrP with wt proteins does not affect the glycosylation pattern or the biochemical characteristics of either protein. However, FRET and co-immunoprecipitation experiments suggest an interaction occurring between the wt and mutant proteins. Furthermore, by comparing the intracellular localization and detergent-resistant membrane (DRM) association in single- and double-expressing clones, we found changes in the intracellular/surface ratio and an increased sequestration of both proteins in DRMs, a site believed to be involved in the pathological conversion (or protection thereof) of the prion protein. We, therefore, propose that the mutant forms alter the subcellular localization and the membrane environment of the wt protein in co-transfected cells. These effects may play a role in the development of these diseases. [ABSTRACT FROM AUTHOR]

The protein-only hypothesis posits that the infectious agent causing transmissible spongiform encephalopathies consists of protein and lacks any informational nucleic acids. This agent, termed prion by Stanley Prusiner, is thought to consist partly of PrPSc, a conformational isoform of a normal cellular protein termed PrPC. Scientists and lay persons have been fascinated by the prion concept, and it has been subjected to passionate critique and intense experimental scrutiny. As a result, PrPC and its isoforms rank among the most intensively studied proteins encoded by the mammalian genome. Despite all this research, both the physiological function of PrPC and the molecular pathways leading to neurodegeneration in prion disease remain unknown. Here we review the salient traits of those diseases ascribed to improper behavior of the prion protein and highlight how the physiological functions of PrPC may help explain the toxic phenotypes observed in prion disease. [ABSTRACT FROM AUTHOR]

Prion diseases are caused by an infectious agent constituted by a rogue protein called prion (PrPSc) of neuronal origin (PrPc) and are exemplified by Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy in cattle. Considerable efforts have been made to understand the cerebral damage caused by these diseases but a clear comprehensive view cannot be achieved without defining the neurophysiological function of PrPc. This lack of information is in part attributable to our ignorance of the precise localization of PrPc in the brain neuronal cell. One relevant option to explore this aspect is to undertake PrP immunohistochemistry at the electron-microscopy level, knowing that this challenge raises major technical constraints. In describing the attempts and restrictions of the various approaches used, we review here the efforts that have been invested in this particular field of prionology. The common result emerging from these contributions is that the synapse could be the site at which PrPc exerts its critical activity. This location suggests, in the perspective of synaptic regulation, that PrPc can be assigned multiple biological functions and supports the novel concept that prion-like changes are involved in long-term memory formation. The synaptic trait of PrPc and PrPSc suggests that synapse loss is the key event in neuronal death. Interestingly, synaptic alterations are also considered to be predominant in the pathophysiological mechanism in Alzheimer, Parkinson and Huntington diseases. All these brain disorders, characterized by the formation of a specific amyloid protein of synaptic origin, can be classified under the heading of amyloidogenic synaptopathies. [ABSTRACT FROM AUTHOR]

Abstract: Prion diseases are associated with conformational change in the copper-binding protein PrP. The copper-binding sites in PrP are located in the N-terminal region of the molecule and comprise a series of tandem repeats of the sequence PHGGGWGQ together with two histidines at residues 96 and 111 (human PrP numbering). The co-ordination of copper ions within the non-octapeptide repeat metal ion-binding site involves Met109 (human numbering, which corresponds with Met112 in ovine PrP) and the binding of copper to this site leads to an increase in β-sheet formation in PrP. Here we have investigated the influence of the M112T polymorphism on copper-induced structural changes in ovine recombinant PrP. M112ARQ and T112ARQ ovine PrP show similar secondary structure although M112ARQ appears more thermostable than T112ARQ. Following treatment with copper, M112ARQ showed a greater increase in β-sheet content than did T112ARQ when measured by CD spectroscopy and by ELISA using anti-PrP monoclonal antibodies. These biochemical and biophysical differences between M112ARQ and T112ARQ correlate with similar differences seen between allelic variants of ovine PrP associated with susceptibility and resistance to classical scrapie. These observations suggest that T112ARQ may provide a measure of resistance to classical scrapie pathogenesis compared to M112ARQ. [Copyright &y& Elsevier]

Exosomes are small membranous vesicles secreted by a number of cell types and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of multivesicular bodies (MVB) to form intraluminal vesicles (ILV). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell–cell signalling, removal of unwanted proteins, and the transfer of pathogens between cells, such as HIV-1. Another such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Interestingly, this work is mirrored by studies on another protein involved in neurodegenerative disease, the amyloid precursor protein (APP) which is associated with Alzheimer’s disease (AD). Recent work has found APP proteolytic fragments in association with exosomes, suggesting a common pathway previously unknown for proteins associated with neurodegenerative diseases. This review will be discussing the current literature regarding the role of exosomes in secretion of the proteins, PrP and APP, and the subsequent implications for neurodegenerative disease. [ABSTRACT FROM AUTHOR]

Abstract: The cellular prion protein (PrPC) is highly conserved in the evolution of mammals, and therefore, thought to have important cellular functions. Despite decades of intensive research, the physiological function of PrPC remains enigmatic. We carried out a yeast two-hybrid screen on a bovine brain cDNA expression library and identified the transmembrane protein tetraspanin-7 (CD231), as a PrPC interacting protein. We confirmed the interaction between PrPC and tetraspanin-7 by yeast two-hybrid assay, immunofluorescent co-localization, and immunocoprecipitation. Our mutational studies further demonstrated that PrPC specifically binds tetraspanin-7 through the region corresponding to bovine PrP154–182 containing alpha-helix 1. [Copyright &y& Elsevier]

Abstract: Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrPCWD of elk can readily induce the conversion of normal cervid PrP (PrPC) molecules to a protease-resistant form, but is less efficient in converting the PrPC of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrPCWD-induced conversion can be greatly enhanced in all species. Our results demonstrate that PrPC from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrPC can apparently overcome the structural barriers between more distant species. [Copyright &y& Elsevier]

The biological role of the scrapie isoform of prion protein (PrPSc) as an infectious agent in numerous human and non-human disorders of the central nervous system is well established. In contrast, and despite decades of intensive research, the physiological function of the endogenous cellular form of the prion protein (PrPC) remains elusive. In mammals, the ubiquitous expression of PrPC suggests biological functions other than its pathological role in propagating the accumulation of its misfolded isotype. Other functions that have been attributed to PrPC include signal transduction, synaptic transmission and protection against cell death through the apoptotic pathway. More recently, immunoregulatory properties of PrPC have been reported. We review accumulating in vitro and in vivo evidence regarding physiological functions of PrPC. [ABSTRACT FROM AUTHOR]

Abstract: Copper is an essential trace element for successful pregnancy. However, the mechanisms by which copper is transported from maternal circulation to the fetus have not been clearly elucidated. Two proteins, cellular prion (PrPC) and COMMD1, are known to be responsible for prion diseases and canine copper toxicosis, respectively, and are thought to play a role in copper homeostasis. However, their placental expression and localization throughout human gestation are still unknown. In this study, we used quantitative RT-PCR, western blotting and immunohistochemistry to investigate in detail the expression and localization of PrPC and COMMD1 proteins in human placenta throughout pregnancy. Our results show that both proteins are expressed in human placenta. PrPC showed the highest mRNA and protein expression levels during the first trimester of pregnancy. PrPC and COMMD1 proteins are similarly localized within the placental villi. Both proteins are present in the syncytiotrophoblast, the cytotrophoblast, vascular endothelial cells and Hofbauer cells. These data offer some insights into possible roles for PrPC and COMMD1 within the placenta. [Copyright &y& Elsevier]

The deposition of proteins in the form of amyloid fibrils and plaques is the characteristic feature of a number of neurodegenarative conditions affecting the nervous system. These disorders include prion and Alzhelmer's disease and are of enormous importance for public health. It has become apparent over the last 20 years that specificity and application in prion disease's diagnostic and therapeutic situations are the most important considerations in designing strategies for the generation of antiprion antibodies. Specific antiprion therapeutics have been suggested and the establishment of the 'proof-of-principle' that the use of epitope-specific antiprion antibodies leads to indefinite delay of disease onset, has increased momentum for its use, althoug caution should be exerted prior to the application of new therapeutic strategies in a clinical set up. Furthermore, in vivo stimulation of immune-competent cells to specifically recognize and neutralize the abnormally folded isoform should also be pursued. [ABSTRACT FROM AUTHOR]

The prion protein (PrPC) has a primary role in the pathogenesis of transmissible spongiform encephalopathies, which causes prion disorders partially due to Ca2+ dysregulation. In our previous work, we found that overexpressed PrPC in gastric cancer was involved in apoptosis, cell proliferation, and metastasis of gastric cancer. To better understand how PrPC acts in gastric cancer, a human microarray was performed to select differentially regulated genes that correlate with the biological function of PrPC. The microarray data were analyzed and revealed 3798 genes whose expression increased at least 2-fold in gastric cancer cells transfected with PrPC. These genes encode proteins involved in several aspects of cell biology, among which, we specially detected molecules related to calcium, especially the S100 calcium-binding proteins, and found that PrPC upregulates S100A1, S100A6, S100B, and S100P but downregulates CacyBP in gastric cancer cells. We also found that intracellular Ca2+ levels in cells transfected with PrPC increased, whereas these levels decreased in knockdowns of these cells. Taken together, PrPC might increase intracellular Ca2+, partially through calcium-binding proteins, or PrPC might upregulate the expression of S100 proteins, partially through stimulating the intracellular calcium level in gastric cancer. Though the underlying mechanisms need further exploration, this study provides a new insight into the role of PrPC in gastric cancer and enriches our knowledge of prion protein. [ABSTRACT FROM AUTHOR]

Knowledge of the physiological function of cellular prion protein has been acquired from prion diseases such as Creutzfeldt–Jakob disease, as well as PRNP knock out and transgenic mice. Recent progress in neurobiology has further delineated the neuroprotective role played by cellular prion protein. In this paper, we review the role of cellular prion protein in cell survival including its antiapoptotic effect on Bax-mediated cell death and its responses to various environmental stresses including oxidative stress, and ischemia. Finally, we discuss the significance of cellular prion protein in different neurodegenerative diseases and the possible development of future therapies. [ABSTRACT FROM AUTHOR]

Transmissible spongiform encephalopathies (TSEs) are neurological disorders that include genetic, infectious and sporadic forms of human Creutzfeldt-Jakob disease (CJD). The pathogenic agent is the prion protein that is composed of an abnormal isoform (PrPSc) of a host-encoded protein (PrPC). Analysis of the relative amounts of PrPSc glycoforms has been used to discriminate between various agents involved in TSE. The distribution and efficiency of conversion to PrPSc can be influenced by differences in the expression of PrPC. However, little attention has been given so far to the banding patterns of PrPC. Using four different antibodies recognizing amino- and carboxyl-terminal PrP sequences we analysed the glycoforms of PrPC in seven regions of the human brain using brains obtained from six subjects. For determination of the staining intensities, signals were quantified by densitometry and reproducible patterns were accomplished by many repeated immunoblot analyses. When amino-terminal binding antibodies were used for detection, PrPC in the frontal neocortex, nucleus lentiformis, thalamus, hippocampus and cerebellum displayed a glycotype with high staining of the diglycosylated isoforms. This was different from patterns in the pons and medulla oblongata, which showed a high intensity of the nonglycosylated isoform, and PrPC proteins, approximately 27 kDa in size, exhibited high staining using the carboxyl-terminal binding antibodies. This intense staining followed from an overlay of full-length and truncated PrPC isoforms. Furthermore, we found marked differences in the expression of PrPC. Variations in the processing of PrPC may lead to interregional differences in the glycoform composition of PrPSc in human brains. [ABSTRACT FROM AUTHOR]

Prion diseases are neurodegenerative disorders which cause Creutzfeldt–Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. The infectious agent is a protease resistant isoform (PrPSc) of a host encoded prion protein (PrPC). PrPSc proteins are characterized according to size and glycoform pattern. We analyzed the glycoform patterns of PrPC obtained from humans, sheep, cattle and mice to find interspecies variability for distinct differentiation among species. To obtain reliable results, the imaging technique was used for measurement of the staining band intensities and reproducible profiles were achieved by many repeated immunoblot analysis. With a set of antibodies, we discovered two distinct patterns which were not species-dependent. One pattern is characterized by high signal intensity for the di-glycosylated isoform using antibodies that bind to the N-terminal region, whereas the other exhibits high intensity for protein bands at the size of the nonglycosylated isoform using antibodies recognizing the C-terminal region. This pattern is the result of an overlap of the nonglycosylated full-length and the glycosylated N-terminal truncated PrPC isoforms. Our data demonstrate the importance of antibody selection in characterization of PrPC. [ABSTRACT FROM AUTHOR]

Cellular prion protein changes conformation during transformation to an infectious scrapie isoform. One measure of transformation is the development of partial resistance to protease treatment. A fraction of human and bovine plasma was identified containing activity that facilitates transformation of cellular prion protein to a protease resistant isoform in the presence of RNA in the absence of seeded scrapie prion protein. Purification of proteins from this fraction led to the identification of α2-macroglobulin as an active component suggesting that it may facilitate conformational changes in prion protein in spontaneous forms of prion disease. [ABSTRACT FROM AUTHOR]

Abstract: Cellular prion protein, PrPC, is essential for the development of prion diseases where it is considered to be a substrate for the formation of the disease-associated conformer, PrPSc. In sheep, PrPC is abundant in neuronal tissue and is also found at lower concentrations in a range of non-neuronal tissues, including mammary gland. Here, we demonstrate the presence of soluble PrPC in the non-cellular, non-lipid fraction of clarified ovine milk. Compared with brain-derived PrPC, ovine milk PrPC displays an increased electrophoretic mobility. Ovine milk PrPC is mainly present as three species that differ in the extent of their N-linked glycosylation, with glycoform profiles varying among animals. Similar PrPC species are also present in fresh and commercial homogenised/pasteurised bovine milk, with additional N-terminal PrPC fragments detectable in ruminant milk and commercial milk products. [Copyright &y& Elsevier]

Conversion of the cellular isoform of the prion protein (PrPC) into the disease-associated isoform (PrPSc) plays a key role in the development of prion diseases. Within its cellular pathway, PrPC undergoes several posttranslational modifications, i.e., the attachment of two N-linked glycans and a glycosyl phosphatidyl inositol (GPI) anchor, by which it is linked to the plasma membrane on the exterior cell surface. To study the interaction of PrPC with model membranes, we purified posttranslationally modified PrPC from transgenic Chinese hamster ovary (CHO) cells. The mono-, di- and oligomeric states of PrPC free in solution were analyzed by analytical ultracentrifugation. The interaction of PrPC with model membranes was studied using both lipid vesicles in solution and lipid bilayers bound to a chip surface. The equilibrium and mechanism of PrPC association with the model membranes were analyzed by surface plasmon resonance. Depending on the degree of saturation of binding sites, the concentration of PrPC released from the membrane into aqueous solution was estimated at between 10-9 and 10-7 M. This corresponds to a free energy of the insertion reaction of -48 kJ/mol. Consequences for the conversion of PrPC to PrPSc are discussed. [ABSTRACT FROM AUTHOR]

Abstract: A focused library of variously substituted 9-aminoacridine compounds was screened for bioactivity against accumulation of the infectious prion protein isoform, denoted PrPSc, in a cell model of prion replication. The efficacy of compounds against PrPSc accumulation was influenced by both substituents of the distal tertiary amine and acridine heterocycle, while cellular cytotoxicity was encoded in the acridine heterocycle substituents. [Copyright &y& Elsevier]

Abstract: PrPC is a glycosylphosphatidylinositol (GPI) anchored glycoprotein of unknown function. Misfolding of normal cellular PrPC to the pathogenic PrPSc is the hallmark of prion diseases (transmissible spongiform encephalopathies). Prion diseases are characterized by extensive neurodegeneration and early death. Understanding how PrPC maintains its correct conformation is a major endeavor of current inquiry. Here we demonstrate a novel interaction between PrPC and the J protein family member, Rdj2 (DjA2; Dj3, Dnj3, Cpr3, and Hirip4). The importance of the J protein family in the cellular folding machinery has been recognized for many years. The PrPC/Rdj2 association was direct and concentration-dependent. Other J proteins such as CSPα and auxilin did not associate with PrPC in the absence of ATP, demonstrating the specificity of the PrPC/J protein interaction. These findings suggest that the J protein family serves as a ‘folding catalyst’ for PrPC and implicates Rdj2 as a factor in the protection against prion diseases. [Copyright &y& Elsevier]

Abstract: RK13 cell lines generated to express bovine PrPC with a four extra octarepeat insertional mutation (Bo-10ORPrPC) show partially insoluble PrPC and lower rates of cell growth when compared to either the same cells expressing wild type Bo-6ORPrPC or the original RK13 cell line. The expression of Bo-10ORPrPC in cell cultures was also associated with changes in cell size and reorganization of the actin cytoskeleton. This last process was reversed by Clostridium difficile toxin-B, a specific inhibitor of small GTPase proteins. Further, in clones expressing Bo-10ORPrPC, increased proportions of cells at cell cycle stage G2/M were observed. Proteasome inhibitors caused a further expansion of G2/M-stage cells that was more marked in cell lines expressing Bo-10ORPrPC than those expressing Bo-6ORPrPC, while this effect was minimal or null in the original RK13 cell line. Hence, the presence of Bo-10ORPrPC in RK13 cells promotes cell cycle arrest at G2/M, and the effect is amplified by proteasome inhibition. These findings suggest a role for PrPC in cell morphology and cell cycle regulation, and open new avenues for understanding the mechanisms underlying PrP mutation-associated diseases. [Copyright &y& Elsevier]

Abstract: The family of transmissible spongiform encephalopathies (TSE), also termed prion diseases, is a group of fatal, neurodegenerative diseases characterized by the accumulation of a misfolded protein, the disease-associated prion protein PrPSc. This glycoprotein differs in secondary structure from its normal, cellular isoform PrPC, which is physiologically expressed mostly by neurons. Scrapie is a prion disease first described in the 18th century in sheep and goats, and has been established as a model in rodents to study the pathogenesis and pathology of prion diseases. Assuming a multitude of molecular parameters change in the tissue in the course of the disease, FTIR microspectroscopy has been proposed as a valuable new method to study and identify prion-affected tissues due to its ability to detect a variety of changes in molecular structure and composition simultaneously. This paper reviews and discusses results from previous FTIR microspectroscopic studies on nervous tissue of scrapie-infected hamsters in the context of histological and molecular alterations known from conventional pathogenesis studies. In particular, data from studies reporting on disease-specific changes of protein structure characteristics, and also results of a recent study on hamster dorsal root ganglia (DRG) are discussed. These data include an illustration on how the application of a brilliant IR synchrotron light source enables the in situ investigation of localized changes in protein structure and composition in nervous cells or tissue due to PrPSc deposition, and a demonstration on how the IR spectral information can be correlated with results of complementary studies using immunohistochemistry and x-ray fluorescence techniques. Using IR microspectroscopy, some neurons exhibited a high accumulation of disease-associated prion protein evidenced by an increased amount of β-sheet at narrow regions in or around the infected nervous cells. However, not all neurons from terminally diseased hamsters showed PrPSc deposition. Generally, the average spectral differences between all control and diseased DRG spectra are small but consistent as demonstrated by independent experiments. Along with studies on the purified misfolded prion protein, these data suggest that synchrotron FTIR microspectroscopy is capable of detecting the misfolded prion protein in situ without the necessity of immunostaining or purification procedures. [Copyright &y& Elsevier]

Transmissible spongiform encephalopathies (TSEs) in humans and animals are attributed to protein-only infectious agents, called prions. Prions have been proposed to arise from the conformational conversion of the cellular protein PrPC into a misfolded form (e.g., PrPSc for scrapie), which precipitates into aggregates and fibrils. It has been proposed that the conversion process is triggered by the interaction of the infectious form (PrPSc) with the cellular form (PrPC) or might result from a mutation in the gene for PrPC. However, until recently, all efforts to reproduce this process in vitro had failed, suggesting that host factors are necessary for prion replication. In this review we discuss recent findings such as the cellular factors that might be involved in the conformational conversion of prion proteins and the potential mechanisms by which they could operate. [ABSTRACT FROM AUTHOR]

Abstract: Cellular prion protein (PrPc) has been associated with some physiological functions in the last few years. In a previous paper, we have demonstrated an increased hippocampal synaptic transmission in adult mice lacking this protein. In the present study, we investigate the impact of aging on the generation and maintenance of hippocampal long-term Potentiation (LTP) in 9-month-old mice devoid of PrPc protein (Prnp0/0). We observed a lower threshold for inducing LTP in 9-month-old Prnp0/0 mice compared to wild-type ones at the same age. The maintenance of dentate gyrus LTP was more persistent in hippocampal slices from Prnp0/0 mice. Furthermore, the expression of mRNA for NR2A and NR2B subunits of the NMDA glutamatergic receptor in hippocampus of aged Prnp0/0 animals showed an increase compared to the wild type. We propose that increased hippocampal glutamatergic transmission in Prnp0/0 mice is related to the enhanced plasticity and persistence of the dentate LTP. [Copyright &y& Elsevier]

Abstract: Neuronal cell death is considered to be a hallmark in prion diseases. These disorders are believed to result from the post-translational conversion of a normal cell membrane sialoglycoprotein PrPC, composed primarily of α-helical structure, into a disease specific isoform, PrPSc that is rich in β-sheet and partially proteinase-resistant. Recent in vivo studies indicate that prion replication can be inhibited by anti-PrP monoclonal antibodies that led to the indefinite delay in the development of prion disease. The recent report by Solforosi and colleagues has increased the need to understand pathway(s) leading to prion-associated apoptosis and neuronal death thought to be the cause of death in transmissible spongiform encephalopathy (TSE) individuals. Furthermore, these reports increased momentum about the use of antibody-based therapy in prion diseases, although great caution should be exerted when using anti-prion antibodies directly into the central nervous system (CNS) with special emphasis on refined strategies such as specific targeting of regions of the prion protein thought not to be involved in signalling pathways. [Copyright &y& Elsevier]

We previously found elevated levels of prion protein (PrPC) in the blood plasma of 16 patients with renal failure. We studied a further 20 patients with renal failure, and all had a significantly higher PrPC concentration than healthy normal subjects ( P < 0·0001). Renal dialysis did not remove plasma PrPC in these patients. Because dialysis patients receive heparin during dialysis, which could potentially bind to PrPC, the concentration of PrPC was measured in patients receiving heparin for cardiopulmonary bypass and was found to be similar to normal controls. We also studied several other groups with chronic illnesses and found that patients with thrombotic thrombocytopenic purpura and sickle cell anaemia had normal plasma PrPC levels, but that those with β-thalassaemia had slightly elevated levels of plasma PrPC. This suggests that the observations in renal failure were not just part of a generalized response to chronic illness or acute phase reaction. The mechanism of elevated plasma PrPC levels in renal disease is unknown, but this shows that plasma PrPC is not a specific marker of neurological disease or Creutzfeldt–Jakob disease. [ABSTRACT FROM AUTHOR]

Cellular prion protein (PrPC), a glycosylphosphatidylinositol-anchored membrane protein, was found in our lab to be widely expressed in gastric cancer cell lines. In order to evaluate its biological significance in human gastric cancer, we investigated its expression in a large series of gastric tissue samples (n = 124) by immuno histochemical staining with the monoclonal antibody 3F4. Compared with normal tissues, gastric adenocarcinoma showed increased PrPC expression, correlated with the histopathological differentiation (according to the WHO and Lauren classifications) and tumor progression (as documented by pTNM staging). To better understand the underlying mechanism, we introduced the PrPC and two pairs of RNAi into the poorly differentiated gastric cancer cell line AGS and found that PrPC suppressed ROS and slowed down apoptosis in transfected cells. Further study proved that the apoptosis-related protein Bcl-2 was upregulated whereas p53 and Bax were downregulated in the PrPC-transfected cells. A reverse effect was observed in PrPC siRNA-transfected cells. These results strongly suggested that PrPC might play a role as an effective antiapoptotic protein through Bcl-2-dependent apoptotic pathways in gastric cancer cells. Further study into the mechanism of these relationships might enrich the knowledge of PrP, better our understanding of the nature of gastric carcinoma, and further develop possible strategies to block or reverse the development of gastric carcinoma. Copyright © 2006 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Recent reports have shown that prions, the causative agent of transmissible spongiform encephalopathies, accumulate in the skeletal muscle of diseased animals and man. In an attempt to characterise in this tissue the prion protein (PrPC), whose conformational rearrangement governs the generation of prions, we have analysed the protein in primary cultured murine myocytes and in different skeletal muscle types. Our results indicate that the expression and cellular processing of PrPC change during myogenesis, and in muscle fibres with different contractile properties. These findings imply a potential role for PrPC in the skeletal muscle physiology, but may also explain the different capability of muscles to sustain prion replication. [Copyright &y& Elsevier]

Abstract: We used a bovine brain cDNA library to perform a yeast two-hybrid assay with bovine mature PrPC as bait. The screening result showed that αB-crystalline interacted with PrPC. The interaction was further evaluated both in vivo and in vitro with different methods, such as immunofluorescent colocalization, native polyacrylamide-gel electrophoresis, and IAsys biosensor assays. The results suggested that αB-crystalline may have the ability to refold denatured prion proteins, and provided first evidence that αB-crystalline is directly associated with prion protein. [Copyright &y& Elsevier]

Abstract: Pathologic prion protein (PrPSc), implicated in transmissible spongiform encephalopathies, is detected by antibody-based tests or bioassays to confirm the diagnosis of prion diseases. Presently, the Western blot or an ELISA is officially used to screen the brain stem in cattle for the presence of PrPSc. The immuno-polymerase chain reaction (IPCR), a technique whereby the exponential amplification ability of PCR is coupled to the detection of proteins by antibodies in an ELISA format, was applied in a modified real-time IPCR method to detect ultra-low levels of prion protein. Using IPCR, recombinant hamster PrPC was consistently detected at 1fg/mL and proteinase K (PK)-digested scrapie infected hamster brain homogenates diluted to 10−8 (approximately 10–100 infectious units) was detected with a semi-quantitative dose response. This level of detection is 1million-fold more sensitive than the levels detected by Western blot or ELISA and poises IPCR as a method capable of detecting PrPSc in the pre-clinical phase of infection. Further, the data indicate that unless complete PK digestion of PrPC in biological materials is verified, ultrasensitive assays such as IPCR may inaccurately classify a sample as positive. [Copyright &y& Elsevier]

Abstract: Recent studies suggest that the disease isoform of prion protein (PrPSc) is non-neurotoxic in the absence of cellular isoform of prion protein (PrPC), indicating that PrPC may participate directly in the neurodegenerative damage by itself. Meanwhile, transgenic mice harboring a high-copy-number of wild-type mouse (Mo) PrPC develop a spontaneous neurological dysfunction in an age-dependent manner, even without inoculation of PrPSc and thus, investigations of these aged transgenic mice may lead to the understanding how PrPC participate in the neurotoxic property of PrP. Here we demonstrate mitochondria-mediated neuronal apoptosis in aged transgenic mice overexpressing wild-type MoPrPC (Tg(MoPrP)4053/FVB). The aged mice exhibited an aberrant mitochondrial localization of PrPC concomitant with decreased proteasomal activity, while younger littermates did not. Such aberrant mitochondrial localization was accompanied by decreased mitochondrial manganese superoxide dismutase (Mn-SOD) activity, cytochrome c release into the cytosol, caspase-3 activation, and DNA fragmentation, most predominantly in hippocampal neuronal cells. Following cell culture studies confirmed that decrease in the proteasomal activity is fundamental for the PrPC-related, mitochondria-mediated apoptosis. Hence, the neurotoxic property of PrPC could be explained by the mitochondria-mediated neuronal apoptosis, at least in part. [Copyright &y& Elsevier]

Human plasminogen has been shown to interact with the abnormal disease-specific prion protein. Till now, no data are available for patients with Creutzfeldt–Jakob disease (CJD). Therefore, we compared plasminogen concentrations and plasminogen activities in patients with sporadic CJD and controls with other dementia, which were collected in the framework of the German CJD Surveillance study. Patients with CJD had significantly higher plasminogen concentrations than patients with other forms of dementia and plasminogen specific activities were lower in CJD patients. The reasons for these abnormalities are not clear at the moment. The results may reflect a disease-specific prion protein and plasminogen interaction in patients with CJD. Other possible explanations are plasminogen polymorphisms and genotypes with distinct plasminogen activity levels in CJD than in controls, which should be a subject for further studies. [Copyright &y& Elsevier]

Abstract: For the development of specific immunological assays, the binding of a specific antibody (Ab) to the target antigen (Ag) has to be relatively strong. In this study, we have utilized affinity capillary electrophoresis (ACE), a form of capillary zone electrophoresis (CZE) to determine the binding constant (Kb) of specific Abs against bovine serum albumin (BSA) and the healthy prion protein (PrPc), in buffer solutions at fixed pHs, approximating in vivo conditions. We have also utilized capillary isoelectric focusing (cIEF) to determine the complexity and recognition of the various isoforms of PrPc Abs towards their Ag, PrPc. Only ACE and CZE have been used to derive Kb values. The selected Abs for the prion protein can recognize both healthy and diseased states of the protein and are commercially available. The Kb values of PrPc Abs appear to be as strong as the anti-BSA (Ab to BSA) and other reported Kb values for proteins of similar size to PrPc. This appears to be one of the few reports on Kb values for any PrPc Abs, and their applications for in vitro immunoassays (e.g., enzyme-linked immunosorbent assays (ELISAs)). Such assays are being used to detect the infectious agent, PrPres, in brain and related matter/tissues. [Copyright &y& Elsevier]

The cellular prion protein (PrPc) is highly conserved in mammals and expressed widely in different tissues but its physiological role remains elusive. Recently, the human PrPc was shown to possess nucleic acid binding and chaperoning properties similar to human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, a key viral factor in virus structure and replication. These findings prompted us to determine if PrPc could influence HIV-1 replication. We used the human 293T cell line as a model system, since only a very low level of PrPc accumulates in these cells. Expression of PrP at a high level resulted in a specific decrease of HIV-1 Env and Vpr expression. Despite similar levels of intracellular Gag, virus production was reduced by eightfold and infectivity by three- to fourfold in the presence of PrPc. A PrPc mutant lacking the glycosylphosphatidylinositol (GPI) anchor peptide did not impair HIV-1 production, suggesting that PrPc trafficking is critical for this inhibitory effect. Coexpressing HIV-1 and PrPc in these cells also caused a fraction of PrPc to become partially proteinase K-resistant (PrPres), further illustrating the interactions between HIV-1 and PrPc. [Copyright &y& Elsevier]

W. M. Williams, E. R. Stadtman and J. Moskovitz (2004) Neuropathology and Applied Neurobiology, doi: 10.1111/j.1365-2990.2004.00523.x Ageing and exposure to oxidative stress in vivo differentially affect cellular levels of PrPc in mouse cerebral microvessels and brain parenchyma The biological function of cellular prion protein PrPc has not been established, despite in vitro studies suggesting antioxidant activity or link to signal transduction pathways. In this study, mice were exposed to hyperoxia to establish whether oxidative stress affected prion expression in vivo. C57Bl/6J mice aged 6, 18, and 24 months, maintained under normoxic conditions, exhibited age-related increases in PrPc in both cerebral microvessels and in microvessel-depleted brain homogenate. We demonstrate that PrPc is differentially affected by exposure to hyperoxia in vivo for 1 (24 h) or 2 (48 h) days, or for 1 day hyperoxia, followed by 1 day normoxia. Brain parenchymal cells from 6-month-old mice exposed to 1 day hyperoxia showed elevation of a glycosylated ∼36 kDa form, whereas in 24-month-old mice cellular prion level was substantially reduced. Extending hyperoxia from 1 to 2 days resulted in significantly reduced PrPc level, regardless of age. Parenchymal PrPc is substantially elevated in 6-month-old mice, but declines in 18- and 24-month-old animals following 1 day hyperoxia. By contrast, PrPc content in cerebral microvessels from 6-month-old mice declined after a 2 day exposure to hyperoxia, while microvessels from 24-month-old brains showed elevated prion levels 24 h after hyperoxia. Moreover, unglycosylated 25–30 kDa PrPc, and a previously undescribed 50–64 kDa band containing at least some glycosylated protein, predominated in microvessels with lesser content of the glycosylated ∼36 kDa form. Cellular content of these unglycosylated forms was correlated with age, while the response to hyperoxia was evident in both unglycosylated and glycosylated forms of the protein following 1 and 2 day exposures. The observed elevation of the 25–30 and 50–64 kDa bands of microvessel PrPc is not sustainable following 1 day hyperoxia, but returns to near normoxic levels within 24 h after hyperoxia. We also show in a knockout mouse for methionine sulfoxide reductase (MsrA), the enzyme responsible for reducing methionine sulfoxide back to methionine, and a regulator of cellular antioxidant defence, that following hyperoxia brain PrPc in the null mutant is elevated relative to PrPc content in the parent strain. Our results show up-regulated PrPc expression or reduced turnover in response to age-related, and hyperoxia-induced oxidative stress. [ABSTRACT FROM AUTHOR]

Although it has been well established that PrPC, the normal isoform of PrPSc, is a copper-binding protein, the role of this metal in the function of PrPC as well as in prion disease pathology remains unclear. Here, we show that when scrapie-infected neuroblastoma cells were cultured in the presence of copper, the accumulation of PrPSc in these cells was markedly reduced. In addition, our results indicate that when normal neuroblastoma cells were cultured in the presence of copper ions, they could no longer bind and internalize PrPSc. In another set of experiments, copper was added to the drinking water of normal and scrapie-infected hamsters. Our results show that administration of copper to normal hamsters induced cerebellar PrPC accumulation. Most important, a significant delay in prion disease onset was observed when scrapie-infected hamsters were treated with copper. As shown before for neuroblastoma cells, also in vivo most of the copper-induced accumulation of PrPC was intracellular. We hypothesized that PrPC internalization by copper may hinder PrPSc interaction with this molecule, and thereby affect prion disease propagation. [Copyright &y& Elsevier]

Prions are composed of an isoform of a normal sialoglycoprotein called PrPc, whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrPc-binding prot [ABSTRACT FROM AUTHOR]