Scrapie-infected Neuroblastoma Cells Research Articles

Establishment of a chicken monoclonal antibody panel against mammalian prion protein.

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.

Copper binding to PrP C may inhibit prion disease propagation

Although it has been well established that PrP C, the normal isoform of PrP Sc, is a copper-binding protein, the role of this metal in the function of PrP C 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 PrP Sc 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 PrP Sc. 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 PrP C 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 PrP C was intracellular. We hypothesized that PrP C internalization by copper may hinder PrP Sc interaction with this molecule, and thereby affect prion disease propagation.

Evaluation of quinacrine treatment for prion diseases.

Based on in vitro observations in scrapie-infected neuroblastoma cells, quinacrine has recently been proposed as a treatment for Creutzfeldt-Jakob disease (CJD), including a new variant CJD which is linked to contamination of food by the bovine spongiform encephalopathy (BSE) agent. The present study investigated possible mechanisms of action of quinacrine on prions. The ability of quinacrine to interact with and to reduce the protease resistance of PrP peptide aggregates and PrPres of human and animal origin were analyzed, together with its ability to inhibit the in vitro conversion of the normal prion protein (PrPc) to the abnormal form (PrPres). Furthermore, the efficiencies of quinacrine and chlorpromazine, another tricyclic compound, were examined in different in vitro models and in an experimental murine model of BSE. Quinacrine efficiently hampered de novo generation of fibrillogenic prion protein and PrPres accumulation in ScN2a cells. However, it was unable to affect the protease resistance of preexisting PrP fibrils and PrPres from brain homogenates, and a "curing" effect was obtained in ScGT1 cells only after lengthy treatment. In vivo, no detectable effect was observed in the animal model used, consistent with other recent studies and preliminary observations in humans. Despite its ability to cross the blood-brain barrier, the use of quinacrine for the treatment of CJD is questionable, at least as a monotherapy. The multistep experimental approach employed here could be used to test new therapeutic regimes before their use in human trials.

Prion Diseases: Time for a Therapy ?

Prion diseases are transmissible neurodegenerative disorders of humans and animals for which no effective treatment is available. Conformationally altered, proteaseresistant forms of the prion protein (PrP), termed PrPsc, are critical for disease transmissibility and pathogenesis, thus representing a primary target for therapeutic strategies. Different approaches affecting the clearance of PrPsc, inhibiting or preventing its conformation change are currently under investigation. Some compounds antagonize prion propagation in cellular and / or animal models of the disease. These molecules include polyanions, polyene antibiotics, tetrapyrroles, branched polyamines and compounds termed beta-sheet breakers whose direct target its the abnormal conformational change for PrPsc. The suitability of these compounds for therapy is limited, primarily because they cannot cross the blood-brain barrier (BBB) and / or show severe toxicity. A variety of drugs already used for treatment of unrelated human diseases and known to penetrate the BBB have now been screened. The tricyclic derivatives of acridine and phenothiazine inhibit PrPsc formation in scrapie-infected neuroblastoma cells, confirming earlier reports on anti-prion activity of these compounds. In addition, activation of the immune system has been proposed to increase the clearance of PrPsc and reduce the prion infectivity and antibodies can exert antiaggregation activity. We showed that tetracyclines reverse abnormal physicochemical properties and abolish the neurotoxicity of PrP peptides in vitro. These compounds interact with PrPsc from brain tissue of BSE-affected cattle and patients with new-variant Creutzfeldt-Jakob disease (vCJD). Syrian hamsters injected with tetracycline-treated inoculum showed a significant delay in the onset of clinical signs of disease and prolonged survival time. When tetracycline was pre-incubated with a high dilution of the scrapie-infected inoculum, one third of the hamsters did not develop disease. These conformation-based approaches appear to hold the most promise for prion diseases and for the neurodegenerative disorders associated with protein misfolding. Although several classes of compounds offer anti-prion action their use in clinical practice is limited by the fact they have little or no efficacy at the onset of the disease. Thus, the discovery of more efficient drugs must run in parallel with the development of diagnostic tests to identify TSE earlier.

Differential Inhibition of Prion Propagation by Enantiomers of Quinacrine

Prion diseases are fatal neurologic disorders caused by accumulation of a pathogenic isoform (PrPSc) of the prion protein (PrP). The recent discovery of the inhibitory action of quinacrine on PrPSc formation in scrapie-infected neuroblastoma (ScN2a) cells raised the possibility of a treatment for patients with prion disease. To investigate the efficacy of quinacrine enantiomers, we measured the inhibitory effect of these isomers on PrPSc formation in ScN2a cells. (S)-quinacrine exhibited superior antiprion activity compared with (R)-quinacrine and two generic quinacrines that appear to be racemates. Treatment with these various forms of quinacrine did not induce adverse changes affecting cell survival and the expression of marker proteins over a range of potentially therapeutic concentrations. Thus, quinacrine enantiomers demonstrated stereoselectivity on prion elimination but not cytotoxicity in ScN2a cells. Our results raise the possibility that in vivo treatment using one enantiomer of quinacrine may be superior to a racemic mixture, which is the form that is generally used when quinacrine is employed to treat parasitic diseases.

Specific inhibition of pathological prion protein accumulation by small interfering RNAs.

Development of transmissible spongiform encephalopathies (TSEs) pathogenesis requires the presence of both the normal host prion protein (PrP-sen) and the abnormal pathological proteinase-K resistant isoform (PrP-res). PrP-res forms highly insoluble aggregates, with self-perpetuating properties, by binding and converting PrP-sen molecules into a likeness of themselves. In the present report, we show that small interfering RNA (siRNA) duplexes trigger specific Prnp gene silencing in scrapie-infected neuroblastoma cells. A non-passaged, scrapie-infected culture transfected with siRNA duplexes is depleted of PrP-sen and rapidly loses its PrP-res content. The use of different murine-adapted scrapie strains and host cells did not influence the siRNA-induced gene silencing efficiency. More than 80% of transfected cells were positive for the presence of fluorescein-labeled siRNA duplexes. No cytotoxicity associated with the use of siRNA was observed during the time course of these experiments. Despite a transient abrogation of PrP-res accumulation, our results suggest that the use of siRNA may provide a new and promising therapeutic approach against prion diseases.

Inhibition of complex glycosylation increases the formation of PrPsc.

N-linked glycans with complex structure have a major role in the biological activity of a wide variety of cell surface and secreted glycoproteins. Here, we show that geldanamycin, an inhibitor of Hsp90, interferes with the formation of complex glycosylated mammalian prion protein (PrPC). Similarly to inhibitors of alpha-mannosidases, geldanamycin stabilized a high mannose PrPC glycoform and prevented the subsequent processing into complex structures. Moreover, a PrP/Grp94 complex could be isolated from geldanamycin-treated cells, suggesting that Grp94 might play a role in the processing of PrPC in the endoplasmic reticulum. Inhibition of complex glycosylation did not interfere with the glycosylphosphatidylinositol (GPI) anchor attachment and cellular trafficking of high mannose PrPC to the outer leaflet of the plasma membrane. In scrapie-infected neuroblastoma cells, however, high mannose PrPC glycoforms were preferred substrates for the formation of PrP-scrapie (PrPSc). Our study reveals that complex glycosylation is dispensable for the cellular trafficking of PrPC, but modulates the formation of PrPSc.

Potent inhibition of scrapie prion replication in cultured cells by bis-acridines.

Prion diseases are characterized by an accumulation of PrP(Sc), a misfolded isoform of the normal cellular prion protein, PrP(C). We previously reported the bioactivity of acridine-based compounds against PrP(Sc) replication in scrapie-infected neuroblastoma cells and now report the improved potency of bis-acridine compounds. Bis-acridines are characterized by a dimeric motif, comprising two acridine heterocycles tethered by a linker. A library of bis-(6-chloro-2-methoxy-acridin-9-yl) and bis-(7-chloro-2-methoxy-benzo[b][1,5]naphthyridin-10-yl) analogs was synthesized to explore the effect of structurally diverse linkers on PrP(Sc) replication in scrapie-infected neuroblastoma cells. Structure-activity analysis revealed that linker length and structure are important determinants for inhibition of prion replication in cultured scrapied cells. Three bis-acridine analogs, (6-chloro-2-methoxy-acridin-9-yl)-(3-[4-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-piperazin-1-yl]-propyl)-amine, N,N'-bis-(6-chloro-2-methoxy-acridin-9-yl)-1,8-diamino-3,6-dioxaoctane, and (1-[[4-(6-chloro-2-methoxy-acridin-9-ylamino)-butyl]-[3-(6-chloro-2-methoxy-acridin-9-ylamino)-propyl]-carbamoyl]-ethyl)-carbamic acid tert-butyl ester, showed half-maximal inhibition of PrP(Sc) formation at 40, 25, and 30 nM, respectively, and were not cytotoxic to uninfected neuroblastoma cells at concentrations of 500 nM. Our data suggest that bis-acridine analogs may provide a potent alternative to the acridine-based compound quinacrine, which is currently under clinical evaluation for the treatment of prion disease.

Increased expression of normal prion protein (PRPC) in schizophrenia and bipolar disorder

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.

Temporal and spatial relationship between the death of PrP-damaged neurones and microglial activation.

Previous studies have demonstrated a role for microglia in the neuronal loss that occurs in the transmissible spongiform encephalopathies or prion diseases. In the present studies, the processes that lead to the death of neurones treated with synthetic peptides derived from the prion protein (PrP) were fully activated within 1 h, although neuronal cell death was not seen until 24 h later. Similarly, neurones exposed to PrP peptides for only 1 h activated microglia and a temporal relationship between the production of interleukin-6, an indicator of microglial activation, and microglial killing of PrP-treated neurones was also demonstrated. Activation of microglia and microglia-mediated killing of PrP-treated neurones or scrapie-infected neuroblastoma cells were maximal only when microglia were in direct contact with neurones.

Synthetic Miniprion PrP106

Elucidation of structure and biological properties of the prion protein scrapie (PrP(Sc)) is fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease-specific isoforms and the pathogenesis of prion diseases. Unfortunately, the insolubility and heterogeneity of PrP(Sc) have limited these studies. The observation that a construct of 106 amino acids (termed PrP106 or miniprion), derived from mouse PrP and containing two deletions (Delta 23-88, Delta 141-176), becomes protease-resistant when expressed in scrapie-infected neuroblastoma cells and sustains prion replication when expressed in PrP(0/0) mice prompted us to generate a corresponding synthetic peptide (sPrP106) to be used for biochemical and cell culture studies. sPrP106 was obtained successfully with a straightforward procedure, which combines classical stepwise solid phase synthesis with a purification strategy based on transient labeling with a lipophilic chromatographic probe. sPrP106 readily adopted a beta-sheet structure, aggregated into branched filamentous structures without ultrastructural and tinctorial properties of amyloid, exhibited a proteinase K-resistant domain spanning residues 134-217, was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. These features are central properties of PrP(Sc) and make sPrP106 an excellent tool for investigating the molecular basis of the conformational conversion of PrP(C) into PrP(Sc) and prion disease pathogenesis.

Valproic acid treatment results in increased accumulation of prion proteins.

PrP(Sc), the only identified component of the prion, is an aberrant isoform of PrP(C), a glycoprotein of unknown function. In this study, it was shown that valproic acid, a widely used antiepileptic drug, can cause an increase of several orders of magnitude in the accumulation of PrP(C) in normal neuroblastoma cells (N2a), and of both PrP isoforms in scrapie infected neuroblastoma cells (ScN2a). Although preliminary results indicate that valproic acid administration to hamsters inoculated with prions had no significant effect on disease incubation time, it is suggested that administration of valproic acid to humans at risk of developing Creutzfeldt-Jakob disease should be evaluated with caution.

Altered insulin receptor processing and function in scrapie-infected neuroblastoma cell lines

The underlying neurochemical changes contributing to prion-induced neurodegeneration remain largely unknown. This study shows that scrapie infection induced a 2-fold increase of insulin receptor (IR) protein and aberrantly processed IR β-chain in scrapie-infected N2a neuroblastoma cells (ScN2a) as measured by Western blot of immunoprecipitated IR, in the absence of increased IR mRNA. Elevated IR protein level was further confirmed in an independently scrapie-infected neuroblastoma cell line N1E-115 (ScN1E-115). Proliferation studies showed that the increased IR level in ScN2a did not result in an increased insulin-mediated cell growth compared to normal N2a cells. Binding studies indicated that this apparent paradox was due to a 65% decrease in specific [ 125I]insulin binding sites in ScN2a when compared to the amount of immunoreactive IR, although the IR binding affinity was unchanged. Analysis of insulin stimulated IR tyrosine phosphorylation showed a slight but not significant reduction in ScN2a, when related to the increased level of immunoreactive IR. However, comparing the IR tyrosine phosphorylation to the loss of binding sites in ScN2a, we demonstrated an increased IR tyrosine phosphorylation of the remaining functional IR. In addition to these differences in IR properties, the basal extracellular signal regulated kinase-2 (ERK2) phosphorylation detected by Western blot, was significantly elevated and the insulin stimulated ERK2 phosphorylation was subsequently decreased in ScN2a. Together, these data show that scrapie infection affects the level and processing of the IR and signal transduction mediated by the IR in neuroblastoma cells, as well as induces an elevated basal ERK2 phosphorylation. Aberrant regulation of neuroprotective receptors may contribute to neurodegeneration in prion diseases.

Deletion of beta-strand and alpha-helix secondary structure in normal prion protein inhibits formation of its protease-resistant isoform.

A fundamental event in the pathogenesis of transmissible spongiform encephalopathies (TSE) is the conversion of a normal, proteinase K-sensitive, host-encoded protein, PrP-sen, into its protease-resistant isoform, PrP-res. During the formation of PrP-res, PrP-sen undergoes conformational changes that involve an increase of beta-sheet secondary structure. While previous studies in which PrP-sen deletion mutants were expressed in transgenic mice or scrapie-infected cell cultures have identified regions in PrP-sen that are important in the formation of PrP-res, the exact role of PrP-sen secondary structures in the conformational transition of PrP-sen to PrP-res has not yet been defined. We constructed PrP-sen mutants with deletions of the first beta-strand, the second beta-strand, or the first alpha-helix and tested whether these mutants could be converted to PrP-res in both scrapie-infected neuroblastoma cells (Sc(+)-MNB cells) and a cell-free conversion assay. Removal of the second beta-strand or the first alpha-helix significantly altered both processing and the cellular localization of PrP-sen, while deletion of the first beta-strand had no effect on these events. However, all of the mutants significantly inhibited the formation of PrP-res in Sc(+)-MNB cells and had a greatly reduced ability to form protease-resistant PrP in a cell-free assay system. Thus, our results demonstrate that deletion of the beta-strands and the first alpha-helix of PrP-sen can fundamentally affect PrP-res formation and/or PrP-sen processing.

Up-regulation of Functionally Impaired Insulin-like Growth Factor-1 Receptor in Scrapie-infected Neuroblastoma Cells

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie-infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in the level of IGF-1R in two independently scrapie-infected neuroblastomas, ScN2a and ScN1E-115 cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80% decrease in specific (125)I-IGF-1-binding sites compared with N2a cells. This decrease in IGF-1R-binding sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1 stimulated IGF-1Rbeta tyrosine phosphorylation showed no major change in the dose-response between the cell types, possibly due to altered tyrosine kinase signaling in scrapie-infected neuroblastoma cells. Altogether these data indicate that scrapie infection affects the expression, binding affinity, and signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and function may weaken the trophic support in scrapie-infected neurons and thereby contribute to neurodegeneration in prion diseases.

Killing of prion-damaged neurones by microglia.

The loss of neurones that occurs in the transmissible spongiform encephalopathies, or prion diseases, can be reproduced in vitro by incubating neuronal cultures with either peptides derived from the prion protein or with partially purified prion preparations. In the present studies, the extent of neuronal loss on exposure to these prions or prion peptides was increased by the addition of microglia, a process that was dependent upon the number of microglia added, the concentration of prions/peptides present and the degree of fibrillarity of the prion peptides. Microglia also killed scrapie-infected neuroblastoma cells expressing infectious PrP(SC). Microglia secreted low amounts of interleukin (IL)-6 when incubated with peptides alone but up to 10 times as much IL-6 when incubated with peptide-treated neurones, suggesting that microglia recognise peptide-induced changes in neurones.

Scrapie prion protein accumulation by scrapie-infected neuroblastoma cells abrogated by exposure to a prion protein antibody.

Exposure of susceptible neuroblastoma N2a cells to mouse scrapie prions leads to infection, as evidenced by the continued presence of the scrapie form of the prion protein (PrP(Sc)) and infectivity after 300 or more cell doublings. We find that exposure to phosphatidylinositol-specific phospholipase C (PIPLC) or to the monoclonal anti-prion protein (PrP) antibody 6H4 not only prevents infection of susceptible N2a cells but also cures chronically scrapie-infected cultures, as judged by the long-term abrogation of PrP(Sc) accumulation after cessation of treatment. A nonpassaged, stationary infected culture rapidly loses PrP(Sc) when exposed to the antibody or PIPLC, indicating that the PrP(Sc) level is determined by steady state equilibrium between formation and degradation, and that depletion of the cellular form of PrP can interrupt the propagation of PrP(Sc). These findings encourage the belief that passive immunization may provide a therapeutic approach to prion disease.

Branched polyamines cure prion-infected neuroblastoma cells.

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP(Sc), the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529-14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 microg of PPI generation 4.0/ml for 4 weeks not only reduced PrP(Sc) to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the beta-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP(Sc) to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP(Sc) from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP(Sc) from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP(Sc) clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.

Lysosomotropic agents and cysteine protease inhibitors inhibit scrapie-associated prion protein accumulation.

We report that lysosomotropic agents and cysteine protease inhibitors inhibited protease-resistant prion protein accumulation in scrapie-infected neuroblastoma cells. The inhibition occurred without either apparent effects on normal prion protein biosynthesis or turnover or direct interactions with prion protein molecules. The findings introduce two new classes of inhibitors of the formation of protease-resistant prion protein.

Cationic Lipopolyamines Induce Degradation of PrPSc in Scrapie-Infected Mouse Neuroblastoma Cells

In prion diseases the endogenous prion protein (PrPC) is converted into an abnormally folded isoform, denoted PrPSc, which represents the major component of infectious scrapie prions. The mechanism of the conversion is largely unknown, but the conversion is thought to occur after PrPC has reached the plasma membrane. Here we show that exogenous administration of the cationic lipopolyamine DOSPA interfered with the accumulation of PrPSc in scrapie-infected neuroblastoma cells. Structural analysis of the compounds tested revealed that inhibition of PrPSc was specific for lipids with a headgroup composed of the polyamine spermine and a quarternary ammonium ion between the headgroup and the lipophilic tail. The cationic lipopolyamine DOSPA induced the cellular degradation of preexisting PrPSc aggregates within 12 hours and interfered with the de novo synthesis of PrPSc. Biosynthesis of PrPC, or the assembly of sphingolipid-cholesterol microdomains (rafts) on the plasma membrane, were not affected by this inhibitor. After removal of DOSPA and replating into normal medium propagation of PrPSc commenced, although initially at a reduced rate. Incubation of ScN2a cells in free spermidine had no inhibitory effect on the accumulation of PrPSc. Our results indicate that membrane targeting of a small polyamine molecule creates a potent inhibitor of PrPSc propagation and offers the possibility to degrade preexisting PrPSc aggregates in living cells.