Scrapie-infected Neuroblastoma Cells Research Articles

Validation of Poly(Propylene Imine) Glycodendrimers Towards Their Anti-prion Conversion Efficiency.

Prion diseases, such as the sporadic Creutzfeldt-Jakob disease (sCJD), are a class of fatal neurodegenerative disorders. Currently, there is no efficient treatment or therapy available. Hence, the search for molecules that may inhibit the conversion of the cellular prion protein (PrPC) into its pathological counterpart PrPScrapie (PrPSc) is of great urgency. Here, we report the generation- and dose-dependent biological action of dense-shell poly(propylene imine) (PPI) glycodendrimers by using scrapie-infected neuroblastoma (ScN2a) cells and the real-time quaking-induced conversion assay (RT-QuIC) for validation of anti-prion efficiencies. Whereas the 2nd and 3rd generation of PPI glycodendrimers exhibited anti-prion conversion efficiency in ScN2a cells validated by RT-QuIC analysis, we observed that the 4th generation of glycodendrimers had shown no significant effect. Translational RT-QuIC studies conducted with human prions derived from sCJD patients indicated an anti-prion conversion effect (not on PrPRes degradation) of PPI glycodendrimers against human prions with the highest inhibitory activity of the 4th generation of PPI glycodendrimers towards prion aggregation compared to the 2nd and 3rd generation. In conclusion, our study highlights the potential of PPI glycodendrimers as therapeutic compounds due to their anti-conversion activity on human prions in a PrPSc strain depending manner.

Quiescin-sulfhydryl oxidase inhibits prion formation in vitro.

Prions are infectious proteins that cause a group of fatal transmissible diseases in animals and humans. The scrapie isoform (PrPSc) of the cellular prion protein (PrPC) is the only known component of the prion. Several lines of evidence have suggested that the formation and molecular features of PrPSc are associated with an abnormal unfolding/refolding process. Quiescin-sulfhydryl oxidase (QSOX) plays a role in protein folding by introducing disulfides into unfolded reduced proteins. Here we report that QSOX inhibits human prion propagation in protein misfolding cyclic amplification reactions and murine prion propagation in scrapie-infected neuroblastoma cells. Moreover, QSOX preferentially binds PrPSc from prion-infected human or animal brains, but not PrPC from uninfected brains. Surface plasmon resonance of the recombinant mouse PrP (moPrP) demonstrates that the affinity of QSOX for monomer is significantly lower than that for octamer (312 nM vs 1.7 nM). QSOX exhibits much lower affinity for N-terminally truncated moPrP (PrP89-230) than for the full-length moPrP (PrP23-231) (312 nM vs 2 nM), suggesting that the N-terminal region of PrP is critical for the interaction of PrP with QSOX. Our study indicates that QSOX may play a role in prion formation, which may open new therapeutic avenues for treating prion diseases.

Direct Evidence of Generation and Accumulation of β-Sheet-rich Prion Protein in Scrapie-infected Neuroblastoma Cells with Human IgG1 Antibody Specific for β-Form Prion Protein

We prepared β-sheet-rich recombinant full-length prion protein (β-form PrP) (Jackson, G. S., Hosszu, L. L., Power, A., Hill, A. F., Kenney, J., Saibil, H., Craven, C. J., Waltho, J. P., Clarke, A. R., and Collinge, J. (1999) Science 283, 1935-1937). Using this β-form PrP and a human single chain Fv-displaying phage library, we have established a human IgG1 antibody specific to β-form but not α-form PrP, PRB7 IgG. When prion-infected ScN2a cells were cultured with PRB7 IgG, they generated and accumulated PRB7-binding granules in the cytoplasm with time, consequently becoming apoptotic cells bearing very large PRB7-bound aggregates. The SAF32 antibody recognizing the N-terminal octarepeat region of full-length PrP stained distinct granules in these cells as determined by confocal laser microscopy observation. When the accumulation of proteinase K-resistant PrP was examined in prion-infected ScN2a cells cultured in the presence of PRB7 IgG or SAF32, it was strongly inhibited by SAF32 but not at all by PRB7 IgG. Thus, we demonstrated direct evidence of the generation and accumulation of β-sheet-rich PrP in ScN2a cells de novo. These results suggest first that PRB7-bound PrP is not responsible for the accumulation of β-form PrP aggregates, which are rather an end product resulting in the triggering of apoptotic cell death, and second that SAF32-bound PrP lacking the PRB7-recognizing β-form may represent so-called PrP(Sc) with prion propagation activity. PRB7 is the first human antibody specific to β-form PrP and has become a powerful tool for the characterization of the biochemical nature of prion and its pathology.

A novel anti-prion protein monoclonal antibody and its single-chain fragment variable derivative with ability to inhibit abnormal prion protein accumulation in cultured cells

mAbs T1 and T2 were established by immunizing PrP gene ablated mice with recombinant MoPrP of residues 121-231. Both mAbs were cross-reactive with PrP from hamster, sheep, cattle and deer. A linear epitope of mAb T1 was identified at residues 137-143 of MoPrP and buried in PrP(C) expressed on the cell surface. mAb T1 showed no inhibitory effect on accumulation of PrP(Sc) in cultured scrapie-infected neuroblastoma (ScN2a) cells. In contrast, mAb T2 recognized a discontinuous epitope ranged on, or structured by, residues 132-217 and this epitope was exposed on the cell surface PrP(C). mAb T2 showed an excellent inhibitory effect on PrP(Sc) accumulation in vitro at a 50% inhibitory concentration of 0.02 microg/ml (0.14 nM). The scFv form of mAb T2 (scFv T2) was secreted in neuroblastoma (N2a58) cell cultures by transfection through eukaryotic secretion vector. Coculturing of ScN2a cells with scFv T2-producing N2a58 cells induced a clear inhibitory effect on PrP(Sc) accumulation, suggesting that scFv T2 could potentially be an immunotherapeutic tool for prion diseases by inhibition of PrP(Sc) accumulation.

Human Prion Protein (PrP) 219K Is Converted to PrPSc but Shows Heterozygous Inhibition in Variant Creutzfeldt-Jakob Disease Infection

Prion protein gene (PRNP) E219K is a human polymorphism commonly occurring in Asian populations but is rarely found in patients with sporadic Creutzfeldt-Jakob disease (CJD). Thus the polymorphism E219K has been considered protective against sporadic CJD. The corresponding mouse prion protein (PrP) polymorphism variant (mouse PrP 218K) is not converted to the abnormal isoform (PrP(Sc)) and shows a dominant negative effect on wild-type PrP conversion. To define the conversion activity of this human molecule, we herein established knock-in mice with human PrP 219K and performed a series of transmission experiments with human prions. Surprisingly, the human PrP 219K molecule was converted to PrP(Sc) in variant CJD infection, and the conversion occurred more efficiently than PrP 219E molecule. Notably the knock-in mice with PRNP codon 219E/K showed the least efficient conversion compared with their hemizygotes with PRNP codon 219E/0 or codon 219K/0, or homozygotes with PRNP codon 219E/E or codon 219K/K. This phenomenon indicated heterozygous inhibition. This heterozygous inhibition was observed also in knock-in mice with PRNP codon 129M/V genotype. In addition to variant CJD infection, the human PrP 219K molecule is conversion-competent in transmission experiments with sporadic CJD prions. Therefore, the protective effect of PRNP E219K against sporadic CJD might be due to heterozygous inhibition.

Immunolocalisation of PrP Sc in scrapie-infected N2a mouse neuroblastoma cells by light and electron microscopy

The causative agent of transmissible spongiform encephalopathies (TSE) is PrP Sc, an infectious, misfolded isoform of the cellular prion protein (PrP C). The localisation and trafficking of PrP Sc and sites of conversion from PrP C to PrP Sc are under debate, particularly since most published work did not discriminate between PrP C and PrP Sc. Here we describe the localisation of PrP C and PrP Sc in a scrapie-infected neuroblastoma cell line, ScN2a, by light and electron microscopic immunolocalisation. After eliminating PrP C with proteinase K, PrP Sc was detected at the plasma membrane, endocytosed via clathrin-coated pits and delivered to early endosomes. Finally, PrP Sc was detected in late endosomes/lysosomes. As we detected PrP Sc at the cell surface, in early endosomes and in late endosomes/lysosomes, i.e. locations where PrP C is also present, our data imply that the conversion process could take place at the plasma membrane and/or along the endocytic pathway. Finally, we observed the release of PrP C/PrP Sc via exocytotic pathways, i.e. via exosomes and as an opaque electron-dense mass which may represent a mechanism of intercellular spreading of infectious prions.

Stress-protective signalling of prion protein is corrupted by scrapie prions

Studies in transgenic mice revealed that neurodegeneration induced by scrapie prion (PrP(Sc)) propagation is dependent on neuronal expression of the cellular prion protein PrP(C). On the other hand, there is evidence that PrP(C) itself has a stress-protective activity. Here, we show that the toxic activity of PrP(Sc) and the protective activity of PrP(C) are interconnected. With a novel co-cultivation assay, we demonstrate that PrP(Sc) can induce apoptotic signalling in PrP(C)-expressing cells. However, cells expressing PrP mutants with an impaired stress-protective activity were resistant to PrP(Sc)-induced toxicity. We also show that the internal hydrophobic domain promotes dimer formation of PrP and that dimerization of PrP is linked to its stress-protective activity. PrP mutants defective in dimer formation did not confer enhanced stress tolerance. Moreover, in chronically scrapie-infected neuroblastoma cells the amount of PrP(C) dimers inversely correlated with the amount of PrP(Sc) and the resistance of the cells to various stress conditions. Our results provide new insight into the mechanism of PrP(C)-mediated neuroprotection and indicate that pathological PrP conformers abuse PrP(C)-dependent pathways for apoptotic signalling.

Vaccine Approaches to Prevent and Treat Prion Infection

Prion diseases are transmissible neurodegenerative diseases of humans and animals. The prion agent consists of a misfolded protein, PrPSc (prion protein, scrapie form), of a glycosylphosphatidylinositol-anchored host protein, PrPC (PrP cellular form) of unknown function. During prion replication, PrPSc induces host PrPC to adopt its pathogenic conformation. Some PrPSc may aggregate to microscopically visible, extracellular prion plaques that stain for amyloid. The development of antiprion vaccines presents some challenges. While there is strong self-tolerance to an endogenous antibody response to PrPC and PrPSc, highly potent monoclonal antibodies (mAbs) have been raised in mice in which the prion protein gene has been deleted by gene targeting. These mAbs have been demonstrated to be antiprion-active in permanently scrapie-infected neuroblastoma (ScN2a) cells, primarily when bound to one of four epitopes (the octarepeat region, the region around codons 90-110, helix 1 region codons 145-160, and the extreme C-terminal codons 210-220). The mAbs directed against codon regions 90-110 or 145-160 are also antiprion-active in vivo, but only after intraperitoneal infection with prions, not intracerebral infection, suggesting their blood-brain barrier (BBB) impermeability. The challenge will be to make antibodies, or recombinant derivatives thereof, BBB permeable; this is preferably achieved by monovalent antibody fragments since divalent ones were found to be neurotoxic. Self-tolerance of wild-type animals to PrP immunizations was found to be of extrathymic origin. Even though antibodies raised in wild-type mice were found to display antiprion activity in ScN2a cells, these mice did not have significant extensions of incubation times when challenged intraperitoneally with prions. A general low affinity of these antibody responses to native surface-bound PrPC may account for this. Since wild-type mice were found to develop sufficient T-cell responses to codon regions 145-160 and 210-220, we believe that there is a theoretical chance of a successful vaccination therapy. The influence of the way the immunogen is presented has already been shown to be of major importance for the ensuing immune response, in that presentation of PrP with CpG oligodeoxynucleotides as adjuvant or viral packaging improved antibody responses. Major progress for active immunizations may therefore be expected in this field. Eradication programs will be one of the most important uses of active immunization protocols. For this purpose, vaccines will have to be inexpensive, easy to handle, and effective. In the short term, passive immunizations will likely be most promising for therapy of prion disease, including for human medical interventions. Active immunization protocols are less likely to succeed quickly, and will take years if not decades to be validated for domestic or free-ranging animals.

Sodium valproate does not augment Prpsc in murine neuroblastoma cells

Sodium valproate (VPA) has been reported to increase the accumulation of the pathologic isoform of prion protein (PrPsc) in scrapie-infected murine neuroblastoma cells. In this study, the effect of VPA on PrPsc accumulation was investigated in murine N2a neuroblastoma cells chronically infected with scrapie strain 22L (N2a-22L). No accumulation of PrPsc was detected after short-term (3 days) or long-term (21 days) treatment of N2a-22L cells with 4.8, 12, 18 or 24 microM VPA. Higher VPA concentrations (240 and 600 microM) also failed to augment PrPsc expression. In conclusion, in our experimental conditions, no deleterious effect was induced by VPA on prions replication.

Cyclodextrins Inhibit Replication of Scrapie Prion Protein in Cell Culture

Prion diseases are fatal neurodegenerative disorders that are caused by the conversion of a normal host-encoded protein, PrP(C), to an abnormal, disease-causing form, PrP(Sc). This paper reports that cyclodextrins have the ability to reduce the pathogenic isoform of the prion protein PrP(Sc) to undetectable levels in scrapie-infected neuroblastoma cells. Beta-cyclodextrin removed PrP(Sc) from the cells at a concentration of 500 microM following 2 weeks of treatment. Structure activity studies revealed that antiprion activity was dependent on the size of the cyclodextrin. The half-maximal inhibitory concentration (IC(50)) for beta-cyclodextrin was 75 microM, whereas alpha-cyclodextrin, which possessed less antiprion activity, had an IC(50) of 750 microM. This report presents cyclodextrins as a new class of antiprion compound. For decades, the pharmaceutical industry has successfully used cyclodextrins for their complex-forming ability; this ability is due to the structural orientation of the glucopyranose units, which generate a hydrophobic cavity that can facilitate the encapsulation of hydrophobic moieties. Consequently, cyclodextrins could be ideal candidates for the treatment of prion diseases.

The tyrosine kinase inhibitor imatinib mesylate delays prion neuroinvasion by inhibiting prion propagation in the periphery

Prion diseases are fatal neurodegenerative disorders with no effective therapy. A hallmark of prion disease is the conversion of the normal cellular form of prion protein PrP(C) into a disease-associated isoform PrP(Sc). The authors recently have shown that a tyrosine kinase inhibitor, imatinib mesylate, induces clearance of PrP(Sc) via specific inhibition of c-Abl in prion-infected cell culture models. In this study, the authors assessed the in vivo effects of imatinib mesylate on prion disease using a scrapie-infected mouse model and further investigated prion infectivity of the drug-treated scrapie-infected neuroblastoma (ScN2a) cells. The authors found that imatinib mesylate abolished prion infectivity to almost undetectable level in ScN2a cells and the level of PrP(Sc) was significantly decreased by the drug in scrapie-infected mouse spleens as well as in ScN2a cells. Moreover, the drug treatment at an early phase of peripheral scrapie infection delayed the appearance of PrP(Sc) in the central nervous system (CNS) and onset of clinical disease in mice. However, neither intraperitoneal nor intracerebroventricular delivery of the drug exerted any PrP(Sc) clearance effect in the CNS.

Structure–activity relationship study of 9-aminoacridine compounds in scrapie-infected neuroblastoma cells

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.

A Trans-dominant Negative 37 kDa/67 kDa Laminin Receptor Mutant Impairs PrPSc Propagation in Scrapie-infected Neuronal Cells

The 37kDa/67kDa laminin receptor (LRP/LR) has been identified as a cell surface receptor for cellular and infectious prion proteins. Here, we show that an N-terminally truncated LRP mutant encompassing the extracellular domain of the LRP/LR (LRP102-295::FLAG) reduces the binding of recombinant cellular huPrP to mouse neuroblastoma cells, and infectious moPrP27-30 to BHK cells, and interferes with the PrP(Sc) propagation in scrapie-infected neuroblastoma cells (N2aSc(+)). A cell-free binding assay demonstrated the direct binding of the LRP102-295::FLAG mutant to both PrP(c) and PrP(Sc). These results, together with the finding that endogenous LRP levels remain unaffected by the expression of the mutant, indicate that the secreted LRP102-295::FLAG mutant may act in a trans-dominant negative manner as a decoy by trapping PrP molecules. The LRP mutant might represent a potential therapeutic tool for the treatment of TSEs.

Increased susceptibility to oxidative stress in scrapie-infected neuroblastoma cells is associated with intracellular iron status

The molecular mechanism of neurodegeneration in prion diseases remains largely uncertain, but one of the features of infected cells is higher sensitivity to induced oxidative stress. In this study, we have investigated the role of iron in hydrogen peroxide (H 2O 2)-induced toxicity in scrapie-infected mouse neuroblastoma N2a (ScN2a) cells. ScN2a cells were significantly more susceptible to H 2O 2 toxicity than N2a cells as revealed by cell viability (MTT) assay. After 2 h exposure, significant decrease in cell viability in ScN2a cells was observed at low concentrations of extracellular H 2O 2 (5–10 μM), whereas N2a cells were not affected. The increased H 2O 2 toxicity in ScN2a cells may be related to intracellular iron status since ferrous iron (Fe 2+) chelator 2,2′-bipyridyl (BIP) prevented H 2O 2-induced decrease in cell viability. Further, the level of calcein-sensitive labile iron pool (LIP) was significantly increased in ScN2a cells after H 2O 2 treatment. Finally, the production of reactive oxygen species (ROS) was inhibited by 30% by iron chelators desferrioxamine (DFO) and BIP in ScN2a cells, whereas no significant effect of iron chelators on basal ROS production was observed in N2a cells. This study indicates that cellular resistance to oxidative stress in ScN2a cells is associated with intracellular status of reactive iron.

Increased iron-induced oxidative stress and toxicity in scrapie-infected neuroblastoma cells

The mechanisms behind the pathology of prion diseases are still unknown, but accumulating evidence suggests oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we have investigated iron-induced oxidative stress in scrapie-infected mouse neuroblastoma N2a (ScN2a) cells. Uninfected N2a and ScN2a cells were treated with ferric ammonium citrate (FAC) for 1–16 h, and the levels of labile iron pool (LIP), the formation of reactive oxygen species (ROS), cell viability and ferritin protein levels were measured. The increase in LIP in N2a cells was transient with a quick recovery to normal levels within 4 h accompanied by a moderate increase of formation of ROS after 3 h followed by the decrease to the basal level. In ScN2a cells, the increase in LIP was lower, but the process of recovery was prolonged and accompanied by high ROS formation and decreased cell viability. Ferritin protein levels were significantly lower in ScN2a cells than in wild-type cells in all iron treatments. These results suggest that ScN2a cells are more sensitive to iron treatment as compared to wild-type cells with respect to ROS formation and cell viability, and that ferritin deficiency in infected cells may contribute to iron-induced oxidative stress in scrapie-infected cells.

Notch-1 activation and dendritic atrophy in prion disease

In addition to neuronal vacuolation and astrocytic hypertrophy, dendritic atrophy is a prominent feature of prion disease. Because increased Notch-1 expression and cleavage releasing its intracellular domain (NICD) inhibit both dendrite growth and maturation, we measured their levels in brains from mice inoculated with Rocky Mountain Laboratory (RML) prions. The level of NICD was elevated in the neocortex, whereas the level of beta-catenin, which stimulates dendritic growth, was unchanged. During the incubation period, levels of the disease-causing prion protein isoform, PrPSc, and NICD increased concomitantly in the neocortex. Additionally, increased levels of Notch-1 mRNA and translocation of NICD to the nucleus correlated well with regressive dendritic changes. In scrapie-infected neuroblastoma (ScN2a) cells, the level of NICD was elevated compared with uninfected control (N2a) cells. Long neurofilament protein-containing processes extended from the surface of N2a cells, whereas ScN2a cells had substantially shorter processes. Transfection of ScN2a cells with a Notch-1 small interfering RNA decreased Notch-1 mRNA levels, diminished NICD concentrations, and rescued the long process phenotype. These results suggest that PrPSc in neurons and in ScN2a cells activates Notch-1 cleavage, resulting in atrophy of dendrites in the CNS and shrinkage of processes on the surface of cultured cells. Whether diminishing Notch-1 activation in vivo can prevent or even reverse neurodegeneration in prion disease remains to be established.

Changed iron regulation in scrapie-infected neuroblastoma cells

Prion diseases are characterized by the conversion of the normal cellular prion protein PrP C into a pathogenic isoform, PrP Sc. The mechanisms involved in neuronal cell death in prion diseases are largely unknown, but accumulating evidence has demonstrated oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we report changes in cellular iron metabolism in scrapie-infected mouse neuroblastoma N2a cells (ScN2a). We detected twofold lower total cellular iron and calcein-chelatable cytosolic labile iron pool (LIP) in ScN2a cells as compared to the N2a cells. We also measured in ScN2a cells significantly lower activities of iron regulatory proteins 1 and 2 (IRP1 and IRP2, respectively), regulators of cellular iron by sensing cytosolic free iron levels and controlling posttranscriptionally the expression of the major iron transport protein transferrin receptor 1 (TfR1) and the iron sequestration protein ferritin. IRP1 and IRP2 protein levels were decreased by 40% and 50%, respectively, in ScN2a cells. TfR1 protein levels were fourfold reduced and ferritin levels were threefold reduced in ScN2a cells. TfR1 and ferritin mRNA levels were significantly reduced in ScN2a cells. ScN2a cells responded normally to iron and iron chelator treatment with respect to the activities of IRP1 and IRP2, and biosynthesis of TfR1 and ferritin. However, the activities of IRP1 and IRP2, and protein levels of TfR1 and ferritin, were still significantly lower in iron-depleted ScN2a cells as compared to the N2a cells, suggesting lower need for iron in ScN2a cells. Our results demonstrate that scrapie infection leads to changes in cellular iron metabolism, affecting both total cellular and cytosolic free iron, and the activities and expression of major regulators of cellular iron homeostasis.

Manipulation of PrP res production in scrapie-infected neuroblastoma cells

In the present study the accumulation of protease resistant prion protein (PrP res) in scrapie-infected neuroblastoma cells (ScN2a cells) was shown to be dependent on culture conditions. The highest levels of PrP res were found in slow growing cells. Further increases in PrP res accumulation were observed in ScN2a cells treated with retinoic acid, a compound that is associated with neuronal differentiation. The effects of retinoic acid were dose-dependent with a maximal effect at 200 ng/ml. A similar increase in PrP res was observed in another prion-infected cell line, scrapie-mouse brain (SMB) cells, treated with retinoic acid while retinoic acid increased the amount of PrP C in non-infected cells. Other drugs reported to cause neuronal differentiation, such as phorbol esters, did not increase the PrP res content of ScN2a cells. The survival of retinoic acid-treated ScN2a cells co-cultured with microglia was significantly reduced when compared to untreated ScN2a cells and an inverse correlation was demonstrated between the PrP res content of cells and their survival when co-cultured with microglia. The production of interleukin-6 by microglia cultured with retinoic acid-treated ScN2a cells was significantly higher than that of microglia cultured with untreated ScN2a cells.

Manipulation of PrPres production in scrapie-infected neuroblastoma cells

In the present study the accumulation of protease resistant prion protein (PrP res ) in scrapie-infected neuroblastoma cells (ScN2a cells) was shown to be dependent on culture conditions. The highest levels of PrP res were found in slow growing cells. Further increases in PrP res accumulation were observed in ScN2a cells treated with retinoic acid, a compound that is associated with neuronal differentiation. The effects of retinoic acid were dose-dependent with a maximal effect at 200 ng/ml. A similar increase in PrP res was observed in another prion-infected cell line, scrapie-mouse brain (SMB) cells, treated with retinoic acid while retinoic acid increased the amount of PrP C in non-infected cells. Other drugs reported to cause neuronal differentiation, such as phorbol esters, did not increase the PrP res content of ScN2a cells. The survival of retinoic acid-treated ScN2a cells co-cultured with microglia was significantly reduced when compared to untreated ScN2a cells and an inverse correlation was demonstrated between the PrP res content of cells and their survival when co-cultured with microglia. The production of interleukin-6 by microglia cultured with retinoic acid-treated ScN2a cells was significantly higher than that of microglia cultured with untreated ScN2a cells.

Quinoline derivatives are therapeutic candidates for transmissible spongiform encephalopathies.

We previously reported that quinacrine inhibited the formation of an abnormal prion protein (PrPres), a key molecule in the pathogenesis of transmissible spongiform encephalopathy, or prion disease, in scrapie-infected neuroblastoma cells. To elucidate the structural aspects of its inhibiting action, various chemicals with a quinoline ring were screened in the present study. Assays of the scrapie-infected neuroblastoma cells revealed that chemicals with a side chain containing a quinuclidine ring at the 4 position of a quinoline ring (represented by quinine) inhibited the PrPres formation at a 50% inhibitory dose ranging from 10(-1) to 10(1) micro M. On the other hand, chemicals with a side chain at the 2 position of a quinoline ring (represented by 2,2'-biquinoline) more effectively inhibited the PrPres formation at a 50% inhibitory dose ranging from 10(-3) to 10(-1) micro M. A metabolic labeling study revealed that the action of quinine or biquinoline was not due to any alteration in the biosynthesis or turnover of normal prion protein, whereas surface plasmon resonance analysis showed a strong binding affinity of biquinoline with a recombinant prion protein. In vivo studies revealed that 4-week intraventricular infusion of quinine or biquinoline was effective in prolonging the incubation period in experimental mouse models of intracerebral infection. The findings suggest that quinoline derivatives with a nitrogen-containing side chain have the potential of both inhibiting PrPres formation in vitro and prolonging the incubation period of infected animals. These chemicals are new candidates for therapeutic drugs for use in the treatment of transmissible spongiform encephalopathies.