Trafficking of PrPc to mitochondrial raft-like microdomains during cell apoptosis

The cellular form of prion protein (PrP^c) is a highly conserved cell surface GPI-anchored glycoprotein that was identified in cholesterol-enriched, detergent-resistant microdomains, named “rafts.” The association with these specialized portions of the cell plasma membrane is required for conversion of PrP^c to the transmissible spongiform encephalopathy-associated protease-resistant isoform. Usually, PrP^c is reported to be a plasma membrane protein, however several studies have revealed PrP^c as an interacting protein mainly with the membrane/organelles, as well as with cytoskeleton network. Recent lines of evidence indicated its association with ER lipid raft-like microdomains for a correct folding of PrP^c, as well as for the export of the protein to the Golgi and proper glycosylation. During cell apoptosis, PrP^c can undergo intracellular re-localization, via ER-mitochondria associated membranes (MAM) and microtubular network, to mitochondrial raft-like microdomains, where it induced the loss of mitochondrial membrane potential and citochrome c release, after a contained raise of calcium concentration. We suggest that PrP^c may play a role in the multimolecular signaling complex associated with cell apoptosis Lipid rafts and their components may, thus, be investigated as pharmacological targets of interest, introducing a novel and innovative task in modern pharmacology, i.e., the development of glycosphingolipid targeted drugs.     

Trafficking of PrPc to mitochondrial raft-like microdomains during cell apoptosis

The cellular form of prion protein (PrP^c) is a highly conserved cell surface GPI-anchored glycoprotein that was identified in cholesterol-enriched, detergent-resistant microdomains, named “rafts.” The association with these specialized portions of the cell plasma membrane is required for conversion of PrP^c to the transmissible spongiform encephalopathy-associated protease-resistant isoform. Usually, PrP^c is reported to be a plasma membrane protein, however several studies have revealed PrP^c as an interacting protein mainly with the membrane/organelles, as well as with cytoskeleton network. Recent lines of evidence indicated its association with ER lipid raft-like microdomains for a correct folding of PrP^c, as well as for the export of the protein to the Golgi and proper glycosylation. During cell apoptosis, PrP^c can undergo intracellular re-localization, via ER-mitochondria associated membranes (MAM) and microtubular network, to mitochondrial raft-like microdomains, where it induced the loss of mitochondrial membrane potential and citochrome c release, after a contained raise of calcium concentration. We suggest that PrP^c may play a role in the multimolecular signaling complex associated with cell apoptosis

Lipid rafts and their components may, thus, be investigated as pharmacological targets of interest, introducing a novel and innovative task in modern pharmacology, i.e., the development of glycosphingolipid targeted drugs.     

C18:3-GM1a induces apoptosis in Neuro2a cells: enzymatic remodeling of fatty acyl chains of glycosphingolipids

GM1a [Gal beta1-3GalNAc beta1-4(NeuAc alpha2-3)Gal beta1-4Glc beta1-1Cer] is known to support and protect neuronal functions. However, we report that alpha-linolenic acid-containing GM1a (C18:3-GM1a), which was prepared using the reverse hydrolysis reaction of sphingolipid ceramide N-deacylase, induced apoptosis in neuronal cells. Intranucleosomal DNA fragmentation, chromatin condensation, and caspase activation, all typical features of apoptosis, were observed when mouse neuroblastoma Neuro2a cells were cultured with C18:3-GM1a but not GM1a containing stearic acid (C18:0) or oleic acid (C18:1). The phenotype of Neuro2a cells induced by C18:3-GM1a was similar to that evoked by lyso-GM1a. However, lyso-GM1a caused a complete disruption of lipid microdomains of Neuro2a cells and hemolysis of sheep erythrocytes, whereas C18:3-GM1a did neither. C18:3-GM1a, but not lyso-GM1a, was found to be abundant in lipid microdomains after the removal of loosely bound GM1a by BSA. The activation of stress-activated protein kinase/c-Jun N-terminal kinase in Neuro2a cells was observed with lyso-GM1a but not C18:3-GM1a. These results indicate that the mechanism of apoptosis induced by C18:3-GM1a is distinct from that caused by lyso-GM1a. This study also clearly shows that fatty acid composition of gangliosides significantly affected their pharmacological activities when added to the cell cultures and suggests why naturally occurring gangliosides do not possess polyunsaturated fatty acids as a major constituent.     

Small stress molecules inhibit aggregation and neurotoxicity of prion peptide 106-126

In prion diseases, the posttranslational modification of host-encoded prion protein PrP^c yields a high β-sheet content modified protein PrP^sc, which further polymerizes into amyloid fibrils. PrP106-126 initiates the conformational changes leading to the conversion of PrP^c to PrP^sc. Molecules that can defunctionalize such peptides can serve as a potential tool in combating prion diseases. In microorganisms during stressed conditions, small stress molecules (SSMs) are formed to prevent protein denaturation and maintain protein stability and function. The effect of such SSMs on PrP106-126 amyloid formation is explored in the present study using turbidity, atomic force microscopy (AFM), and cellular toxicity assay. Turbidity and AFM studies clearly depict that the SSMs—ectoine and mannosylglyceramide (MGA) inhibit the PrP106-126 aggregation. Our study also connotes that ectoine and MGA offer strong resistance to prion peptide-induced toxicity in human neuroblastoma cells, concluding that such molecules can be potential inhibitors of prion aggregation and toxicity.     

Prion protein is a component of the multimolecular signaling complex involved in T cell activation

In this study we analyzed the interaction of prion protein PrP^C with components of glycosphingolipid-enriched microdomains in lymphoblastoid T cells. PrP^C was distributed in small clusters on the plasma membrane, as revealed by immunoelectron microscopy. PrP^C is present in microdomains, since it coimmunoprecipitates with GM3 and the raft marker GM1. A strict association between PrP^C and Fyn was revealed by scanning confocal microscopy and coimmunoprecipitation experiments. The phosphorylation protein ZAP-70 was immunoprecipitated by anti-PrP after T cell activation. These results demonstrate that PrP^C interacts with ZAP-70, suggesting that PrP^C is a component of the multimolecular signaling complex within microdomains involved in T cell activation.     

Sialic Acid on the Glycosylphosphatidylinositol Anchor Regulates PrP-mediated Cell Signaling and Prion Formation

The prion diseases occur following the conversion of the cellular prion protein (PrP^C) into disease-related isoforms (PrP^Sc). In this study, the role of the glycosylphosphatidylinositol (GPI) anchor attached to PrP^C in prion formation was examined using a cell painting technique. PrP^Sc formation in two prion-infected neuronal cell lines (ScGT1 and ScN2a cells) and in scrapie-infected primary cortical neurons was increased following the introduction of PrP^C. In contrast, PrP^C containing a GPI anchor from which the sialic acid had been removed (desialylated PrP^C) was not converted to PrP^Sc. Furthermore, the presence of desialylated PrP^C inhibited the production of PrP^Sc within prion-infected cortical neurons and ScGT1 and ScN2a cells. The membrane rafts surrounding desialylated PrP^C contained greater amounts of sialylated gangliosides and cholesterol than membrane rafts surrounding PrP^C. Desialylated PrP^C was less sensitive to cholesterol depletion than PrP^C and was not released from cells by treatment with glimepiride. The presence of desialylated PrP^C in neurons caused the dissociation of cytoplasmic phospholipase A₂ from PrP-containing membrane rafts and reduced the activation of cytoplasmic phospholipase A₂. These findings show that the sialic acid moiety of the GPI attached to PrP^C modifies local membrane microenvironments that are important in PrP-mediated cell signaling and PrP^Sc formation. These results suggest that pharmacological modification of GPI glycosylation might constitute a novel therapeutic approach to prion diseases.     

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP^C) from a mainly α-helical to a β-sheet rich PrP-scrapie (PrP^Sc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP^Sc, nor the normal function of PrP^C is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP^C mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP^C increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP^C on ferritin iron content is enhanced by stimulating PrP^C endocytosis, and reversed by cross-linking PrP^C on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP^102L decreases ferritin iron content significantly relative to PrP^C expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP^C nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP^C in cellular iron uptake and transport to ferritin, and dysfunction of PrP^C as a significant contributing factor of brain iron imbalance in prion disorders.     

Overexpression of Nonconvertible PrPcΔ114–121 in Scrapie-Infected Mouse Neuroblastoma Cells Leads to trans-Dominant Inhibition of Wild-Type PrPSc Accumulation

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP^Sc of a normal cellular glycoprotein, PrP^c, which is characterized by a high content of β-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109–122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrP^c molecule, should play a role in promoting the conversion into PrP^Sc. By using persistently scrapie-infected mouse neuroblastoma (Sc⁺-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrP^c that are capable of blocking the conversion of endogenous, wild-type PrP^c into PrP^Sc. We constructed a deletion mutant (PrP^cΔ114–121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109–122). Transient transfections of mammalian expression vectors encoding either wild-type PrP^c or PrP^cΔ114–121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrP^c versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc⁺-MNB cells revealed that PrP^cΔ114–121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP^Sc derived from endogenous PrP^c. Thus, we showed that the presence of amino acids 114 to 121 of mouse PrP^c plays an important role in the conversion process of PrP^c into PrP^Sc and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP^Sc accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.     

Identifying critical sites of PrPc-PrPSc interaction in prion-infected cells by dominant-negative inhibition

A direct physical interaction of the prion protein isoforms is a key element in prion conversion. Which sites interact first and which parts of PrP^c are converted subsequently is presently not known in detail. We hypothesized that structural changes induced by PrP^Sc interaction occur in more than one interface and subsequently propagate within the PrP^C substrate, like epicenters of structural changes. To identify potential interfaces we created a series of systematically-designed mutant PrPs and tested them in prion-infected cells for dominant-negative inhibition (DNI) effects. This showed that mutant PrPs with deletions in the region between first and second α-helix are involved in PrP-PrP interaction and conversion of PrP^C into PrP^Sc. Although some PrPs did not reach the plasma membrane, they had access to the locales of prion conversion and PrP^Sc recycling using autophagy pathways. Using other series of mutant PrPs we already have identified additional sites which constitute potential interaction interfaces. Our approach has the potential to characterize PrP-PrP interaction sites in the context of prion-infected cells. Besides providing further insights into the molecular mechanisms of prion conversion, this data may help to further elucidate how prion strain diversity is maintained.     

Immunohistochemical characterization of cell types expressing the cellular prion protein in the small intestine of cattle and mice

The gastrointestinal tract is thought to be the main site of entry for the pathological isoform of the prion protein (PrP^Sc). Prion diseases are believed to result from a conformational change of the cellular prion protein (PrP^c) to PrP^Sc. Therefore, PrP^c expression is a prerequisite for the infection and spread of the disease to the central nervous system. However, the distribution of PrP^c in the gut is still a matter of controversy. We therefore investigated the localization of PrP^c in the bovine and murine small intestine. In cattle, most PrP^c positive epithelial cells were detected in the duodenum, while a few positive cells were found in the jejunum. PrP^c was expressed in serotonin producing cells. In bovine Peyer’s patches, PrP^c was distributed in extrafollicular areas, but not in the germinal centre of the jejunum and ileum. PrP^c was expressed in myeloid lineage cells such as myeloid dendritic cells and macrophages. In mice, PrP^c was expressed in some epithelial cells throughout the small intestine as well as in cells such as follicular dendritic cell in the germinal centre of Peyer’s patches. In this study, we demonstrate that there are a number of differences in the localization of PrP^c between the murine and bovine small intestines.     

The octarepeat region of prion protein,but not the TM1 domain,is important for the antioxidant effect of prion protein

The cellular prion protein (PrP^c) plays a crucial role in the pathogenesis of prion diseases, but its physiological function is far from understood. Several candidate functions have been proposed including binding and internalization of metal ions, a superoxide dismutase-like activity, regulation of cellular antioxidant activities, and signal transduction. The transmembrane (TM1) region of PrP^c (residues 110–135) is particularly interesting because of its very high evolutionary conservation. We investigated a possible role of TM1 in the antioxidant defense, by assessing the impact of overexpressing wt-PrP or deletion mutants in N₂A mouse neuroblastoma cells on intracellular reactive oxygen species (ROS) levels. Under conditions of oxidative stress, intracellular ROS levels were significantly lowered in cells overexpressing either wild-type PrP^c (wt-PrP) or a deletion mutant affecting TM1 (Δ8TM1-PrP), but, as expected, not in cultures overexpressing a deletion mutant lacking the octapeptide region (Δocta-PrP). Overexpression of wt-PrP, Δ8TM1-PrP, or Δocta-PrP did not affect basal ROS levels. Interestingly, the mitochondrial membrane potential was significantly lowered in Δocta-PrP-transfected cultures in the absence of oxidative stress. We conclude that the protective effect of PrP^c against oxidative stress involves the octarepeat region but not the TM1 domain nor the high-affinity copper binding site described for human residues His96/His111.     

Interactome Analyses Identify Ties of PrPC and Its Mammalian Paralogs to Oligomannosidic N-Glycans and Endoplasmic Reticulum-Derived Chaperones

The physiological environment which hosts the conformational conversion of the cellular prion protein (PrP^C) to disease-associated isoforms has remained enigmatic. A quantitative investigation of the PrP^C interactome was conducted in a cell culture model permissive to prion replication. To facilitate recognition of relevant interactors, the study was extended to Doppel (Prnd) and Shadoo (Sprn), two mammalian PrP^C paralogs. Interestingly, this work not only established a similar physiological environment for the three prion protein family members in neuroblastoma cells, but also suggested direct interactions amongst them. Furthermore, multiple interactions between PrP^C and the neural cell adhesion molecule, the laminin receptor precursor, Na/K ATPases and protein disulfide isomerases (PDI) were confirmed, thereby reconciling previously separate findings. Subsequent validation experiments established that interactions of PrP^C with PDIs may extend beyond the endoplasmic reticulum and may play a hitherto unrecognized role in the accumulation of PrP^Sc. A simple hypothesis is presented which accounts for the majority of interactions observed in uninfected cells and suggests that PrP^C organizes its molecular environment on account of its ability to bind to adhesion molecules harboring immunoglobulin-like domains, which in turn recognize oligomannose-bearing membrane proteins.     

Role of Lipid Rafts and GM1 in the Segregation and Processing of Prion Protein

The prion protein (PrP^C) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrP^C is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrP^C raft association, together with raft lipid composition, appears essential for the conversion of PrP^C into the scrapie isoform PrP^Sc, and the development of prion disease. Controversial findings were reported on the nature of PrP^C-containing rafts, as well as on the distribution of PrP^C between rafts and non-raft membranes. We investigated PrP^C/ganglioside relationships and their influence on PrP^C localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrP^C conformations coexist: in lipid rafts PrP^C is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrP^Sc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrP^C conformation and interaction with lipid bilayers, without modifying PrP^C distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

In-situ analysis of cellular poly(ADP-ribose) production in scrapie-infected mouse neuroblastoma cells

Transmissible spongiform encephalopathies (TSEs), also called prion diseases, are characterized by formation of the disease-associated isoform of prion protein (PrP^Sc), which arises from a normal isoform termed PrP^c by a post-translational conversion process occurring in an autocatalytic fashion. Oxidative stress has been proposed as a pathogenetic mechanism in TSEs and increased lipid peroxidation has recently been described in prion-infected cell cultures, suggesting an intrinsic link between the presence of prions and oxidative stress. We investigated if poly(ADP-ribose) formation can be detected in cultured cells upon prion infection, as this NAD⁺-consuming and DNA strand break-activated nuclear enzymatic reaction has the potential to cause rapid and lethal NAD⁺ depletion in cells under severe oxidative stress. Poly(ADP-ribose) production was analysed by immunofluorescence in freshly scrapie-infected Neuro2a–D11 mouse neuroblastoma cells, which had been confirmed by immunocytochemistry to produce PrP^Sc, and in uninfected controls. No spontaneous poly(ADP-ribose) specific signals were observed in infected or in uninfected cells, while both cell types readily reacted to H₂O₂ treatment with poly(ADP-ribose) synthesis in a dose-dependent manner, with no obvious difference in staining intensity at any dose tested. In summary, our data reveal that replication of scrapie agent in neuroblastoma cells can proceed without detectable stimulation of the cellular poly(ADP-ribosyl)ation system.     

Heparin-like molecules bind differentially to prion-proteins and change their intracellular metabolic fate

PrP^Sc is the only known component of the scrapie prion. The difference between PrP^Sc and its normal isoform PrP^c is probably conformational, since no difference has been found in the amino acid sequence or postranslational modifications between both proteins. Heparan sulfate (HS) has been shown to be a component of amyloid plaques in a number of diseases including the prion diseases. We now present evidence that PrP can specifically bind to heparin-like compounds and that this interaction might have a physiological significance. HS can increase the concentration of PrP in normal neuroblastoma cells, whereas low molecular weight heparin (LMWH) does not. In contrast, LMWH and other heparin-like molecules, excluding HS, can inhibit the synthesis of PrP^Sc in scrapie infected cells and reverse their phenotype back to normal as judged by measurement of PrP^Sc by immunoblotting and by infectivity experiments. Whether an interaction between PrP and glycosaminoglycans plays a direct role in the conversion of PrP^c into PrP^Sc remains to be established. © 1993 Wiley-Liss, Inc.     

Comparison of the Anti-Prion Mechanism of Four Different Anti-Prion Compounds,Anti-PrP Monoclonal Antibody 44B1, Pentosan Polysulfate,Chlorpromazine, and U18666A,in Prion-Infected Mouse Neuroblastoma Cells

Molecules that inhibit the formation of an abnormal isoform of prion protein (PrP^Sc) in prion-infected cells are candidate therapeutic agents for prion diseases. Understanding how these molecules inhibit PrP^Sc formation provides logical basis for proper evaluation of their therapeutic potential. In this study, we extensively analyzed the effects of the anti-PrP monoclonal antibody (mAb) 44B1, pentosan polysulfate (PPS), chlorpromazine (CPZ) and U18666A on the intracellular dynamics of a cellular isoform of prion protein (PrP^C) and PrP^Sc in prion-infected mouse neuroblastoma cells to re-evaluate the effects of those agents. MAb 44B1 and PPS rapidly reduced PrP^Sc levels without altering intracellular distribution of PrP^Sc. PPS did not change the distribution and levels of PrP^C, whereas mAb 44B1 appeared to inhibit the trafficking of cell surface PrP^C to organelles in the endocytic-recycling pathway that are thought to be one of the sites for PrP^Sc formation. In contrast, CPZ and U18666A initiated the redistribution of PrP^Sc from organelles in the endocytic-recycling pathway to late endosomes/lysosomes without apparent changes in the distribution of PrP^C. The inhibition of lysosomal function by monensin or bafilomycin A1 after the occurrence of PrP^Sc redistribution by CPZ or U18666A partly antagonized PrP^Sc degradation, suggesting that the transfer of PrP^Sc to late endosomes/lysosomes, possibly via alteration of the membrane trafficking machinery of cells, leads to PrP^Sc degradation. This study revealed that precise analysis of the intracellular dynamics of PrP^C and PrP^Sc provides important information for understanding the mechanism of anti-prion agents.     

Fluorimetric Analysis of Copper Transport Mechanisms in the B104 Neuroblastoma Cell Model: A Contribution from Cellular Prion Protein to Copper Supplying

Dysregulated body copper homeostasis can negatively impact neuronal functions, but full knowledge of the mechanisms underlying the cell metal distribution has not been achieved yet. The high-affinity copper transporter 1 (Ctr1) is considered the main route for cell copper entry, while the cellular prion protein (PrP^C) is presumed to be involved in the same process. Anchored to the outer side of the plasma membrane, this protein has the ability to bind copper ions and undergo internalization. To provide indications about the contribution of Ctr1 and PrP^C proteins in cell copper transport, we used a fluorimetric method to characterize the kinetic properties of ion internalization in a neuroblastoma cell model, overexpressing prion protein (B104). Biochemical characteristics of intake delineated in the presence of other metal ions and an excess of extracellular potassium were compatible with PrP^C-mediated endocytotic transport. Accordingly, inhibition of clathrin-dependent endocytosis by hypertonic shock and enzymatic removal of surface prion protein reduced copper influx by the same extent. On the whole, experimental evidence collected in a neuron-like cell model sustains a role for PrP^C in mediating copper uptake by clathrin-dependent endocytosis.     

Antibodies to a Nonconjugated Prion Protein Peptide 95-123 Interfere with PrP<Superscript><Emphasis Type="Bold">Sc</Emphasis></Superscript> Propagation in Prion-Infected Cells

Summary 1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP^Sc propagation in prion-infected cells. 2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP^Sc) and normal prion protein (PrP^c), respectively. Sera positive in all tests were chosen for PrP^Sc inhibition studies in cell culture. 3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95–123 led to a transient reduction of PrP^Sc levels in persistently prion-infected cells. 4. Thus, anti-PrP antibodies interfering with PrP^Sc propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95–123 for the treatment of prion diseases.     

Proteomic consequences of expression and pathological conversion of the prion protein in inducible neuroblastoma N2a cells

Neurodegenerative diseases are often associated with misfolding and deposition of specific proteins in the nervous system. The prion protein, which is associated with transmissible spongiform encephalopathies (TSEs), is one of them. The normal function of the cellular form of the prion protein (PrP^C) is mediated through specific signal transduction pathways and is linked to resistance to oxidative stress, neuronal outgrowth and cell survival. In TSEs, PrP^C is converted into an abnormally folded isoform, called PrP^Sc, that may impair the normal function of the protein and/or generate toxic aggregates. To investigate these molecular events we performed a two-dimensional gel electrophoresis comparison of neuroblastoma N2a cells expressing different amounts of PrP^C and eventually infected with the 22L prion strain. Mass spectrometry and peptide mass fingerprint analysis identified a series of proteins with modified expression. They included the chaperones Grp78/BiP, protein disulfide-isomerase A6, Grp75 and Hsp60 which had an opposite expression upon PrP^C expression and PrP^Sc production. The detection of these proteins was coherent with the idea that protein misfolding plays an important role in TSEs. Other proteins, such as calreticulin, tubulin, vimentin or the laminin receptor had their expression modified in infected cells, which was reminiscent of previous results. Altogether our data provide molecular information linking PrP expression and misfolding, which could be the basis of further therapeutic and pathophysiological research in this field.Key words: chaperones, neuroblastoma, prion, proteomics