Prion-infected Cell Lines Research Articles

Utility of RNAi-mediated prnp gene silencing in neuroblastoma cells permanently infected by prions: Potentials and limitations

Prion diseases are incurable, transmissible neurodegenerative disorders in humans and animals. Because the disease-associated isoform of prion protein, PrPSc, is conformationally converted from cellular prion protein, PrPC, knockdown of PrPC expression by RNA interference (RNAi) implicates therapy for prion diseases. In this study, introduction of small interfering (si) and small hairpin (sh) RNAs targeting the prion protein gene (prnp) transcripts triggered specific gene silencing and reduced the PrPC level in both prion-free and -infected neuroblastoma cell lines. Furthermore, this approach suppressed PrPSc formation and ultimately eliminated PrPSc from prion-infected cell lines. However, prolonged culture of cured cells resulted in reappearance of PrPSc in the cell population, presumably by de novo PrPSc formation from residual PrPC uncontrolled by RNAi and PrPSc remained under the detection limit. Protein misfolding cyclic amplification assays further confirmed that lysate of cured cells was sufficient to support PrPSc propagation. Our data not only suggest a potential treatment option but also implicate a caveat for using an RNAi approach for prion diseases. These findings provide critical information required to advance RNAi-based prevention and therapy for prion diseases of humans and animals.

Identifying Key Components of the PrPC-PrPSc Replicative Interface

In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.

Docosahexaenoic and eicosapentaenoic acids increase prion formation in neuronal cells

BackgroundThe transmissible spongiform encephalopathies, otherwise known as prion diseases, occur following the conversion of the cellular prion protein (PrPC) to an alternatively folded, disease-associated isoform (PrPSc). Recent studies suggest that this conversion occurs via a cholesterol-sensitive process, as cholesterol synthesis inhibitors reduced the formation of PrPSc and delayed the clinical phase of scrapie infection. Since polyunsaturated fatty acids also reduced cellular cholesterol levels we tested their effects on PrPSc formation in three prion-infected neuronal cell lines (ScGT1, ScN2a and SMB cells).ResultsWe report that treatment with docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA) or the cholesterol synthesis inhibitor simvastatin reduced the amounts of free cholesterol in membrane extracts from prion-infected neuronal cells. Simvastatin reduced cholesterol production while DHA and EPA promoted the conversion of free cholesterol to cholesterol esters. Crucially, while simvastatin reduced PrPSc formation, both DHA and EPA significantly increased the amounts of PrPSc in these cells. Unlike simvastatin, the effects of DHA and EPA on PrPSc content were not reversed by stimulation of cholesterol synthesis with mevalonate. Treatment of ScGT1 cells with DHA and EPA also increased activation of cytoplasmic phospholipase A2 and prostaglandin E2 production. Finally, treatment of neuronal cells with DHA and EPA increased the amounts of PrPC expressed at the cell surface and significantly increased the half-life of biotinylated PrPC.ConclusionWe report that although treatment with DHA or EPA significantly reduced the free cholesterol content of prion-infected cells they significantly increased PrPSc formation in three neuronal cell lines. DHA or EPA treatment of infected cells increased activation of phospholipase A2, a key enzyme in PrPSc formation, and altered the trafficking of PrPC. PrPC expression at the cell surface, a putative site for the PrPSc formation, was significantly increased, and the rate at which PrPC was degraded was reduced. Cholesterol depletion is seen as a potential therapeutic strategy for prion diseases. However, these results indicate that a greater understanding of the precise relationship between membrane cholesterol distribution, PrPC trafficking, cell activation and PrPSc formation is required before cholesterol manipulation can be considered as a prion therapeutic.

Cholesterol esterification reduces the neurotoxicity of prions

The transmissible spongiform encephalopathies develop following the conversion of a host-encoded protein (PrP C) into abnormally folded, disease-related isoforms (PrP Sc). Here we report that three acyl-coenzyme A:cholesterol acyltransferase (ACAT) inhibitors, TMP-153, FR179254 or YIC-C8-434, were more toxic to prion-infected neuronal cell lines (ScGT1 and ScN2a cells) than to their uninfected equivalents (GT1 and N2a cells). The toxicity of ACAT inhibitors for ScGT1 cells was not reversed by the addition of cholesterol esters, rather it was increased by the addition of free cholesterol indicating that the toxicity of ACAT inhibitors was related to the increased free cholesterol content of cells rather than reduced amounts of cholesterol esters. This hypothesis was strengthened by the observation that the addition of free cholesterol killed ScGT1, but not GT1 cells. Treatment with ACAT inhibitors increased caspase-3 activity and prostaglandin E 2 production in ScGT1 cells but not in GT1 cells. The addition of the phospholipase A 2 (PLA 2) inhibitors (AACOCF 3 or MAFP) reduced prostaglandin E 2 production and protected ScGT1 cells against the toxicity of ACAT inhibitors. These results indicate that cholesterol esterification is an important cellular response that reduces PrP Sc-induced activation of PLA 2 and protects against cell death in ScGT1 cells.

Gene expression profile of quinacrine‐cured prion‐infected mouse neuronal cells

Prion diseases are transmissible fatal neurodegenerative diseases of humans and animals, characterised by the presence of an abnormal isoform (scrapie prion protein; PrP(Sc)) of the endogenous cellular prion protein (PrP(C)). The pathological mechanisms at the basis of prion diseases remain elusive, although the accumulation of PrP(Sc) has been linked to neurodegeneration. Different genomic approaches have been applied to carry out large-scale expression analysis in prion-infected brains and cell lines, in order to define factors potentially involved in pathogenesis. However, the general lack of overlap between the genes found in these studies prompted us to carry an analysis of gene expression using an alternative approach. Specifically, in order to avoid the complexities of shifting gene expression in a heterogeneous cell population, we used a single clone of GT1 cells that was de novo infected with mouse prion-infected brain homogenate and then treated with quinacrine to clear PrP(Sc). By comparing the gene expression profiles of about 15 000 genes in quinacrine-cured and not cured prion-infected GT1 cells, we investigated the influence of the presence or the absence of PrP(Sc). By real-time PCR, we confirmed that the gene encoding for laminin was down-regulated as a consequence of the elimination of PrP(Sc) by the quinacrine treatment. Thus, we speculate that this protein could be a specific candidate for further analysis of its role in prion infection and pathogenesis.

Prion Strain- and Species-Dependent Effects of Antiprion Molecules in Primary Neuronal Cultures

Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents.

Packaging of prions into exosomes is associated with a novel pathway of PrP processing

Prion diseases are fatal, transmissible neurodegenerative disorders associated with conversion of the host-encoded prion protein (PrP(C)) into an abnormal pathogenic isoform (PrP(Sc)). Following exposure to the infectious agent (PrP(Sc)) in acquired disease, infection is propagated in lymphoid tissues prior to neuroinvasion and spread within the central nervous system. The mechanism of prion dissemination is perplexing due to the lack of plausible PrP(Sc)-containing mobile cells that could account for prion spread between infected and uninfected tissues. Evidence exists to demonstrate that the culture media of prion-infected neuronal cells contain PrP(Sc) and infectivity but the nature of the infectivity remains unknown. In this study we have identified PrP(C) and PrP(Sc) in association with endogenously expressing PrP neuronal cell-derived exosomes. The exosomes from our prion-infected neuronal cell line were efficient initiators of prion propagation in uninfected recipient cells and to non-neuronal cells. Moreover, our neuronal cell line was susceptible to infection by non-neuronal cell-derived exosome PrP(Sc). Importantly, these exosomes produced prion disease when inoculated into mice. Exosome-associated PrP is packaged via a novel processing pathway that involves the N-terminal modification of PrP and selection of distinct PrP glycoforms for incorporation into these vesicles. These data extend our understanding of the relationship between PrP and exosomes by showing that exosomes can establish infection in both neighbouring and distant cell types and highlight the potential contribution of differentially processed forms of PrP in disease distribution. These data suggest that exosomes represent a potent pool of prion infectivity and provide a mechanism for studying prion spread and PrP processing in cells endogenously expressing PrP.

Similar Structure−Activity Relationships of Quinoline Derivatives for Antiprion and Antimalarial Effects

Prion diseases are invariably fatal neurodegenerative diseases, in which the infectious agent consists of PrP(Sc), a pathogenic misfolded isoform of the normal cellular prion protein (PrP(C)). Until now, no pharmacological options exist for these novel pathogens. Here we describe the screening of a series of polyquinolines and quinolines linked to a large variety of terminal groups for their ability to cure a persistently prion infected cell line (ScN2a). Several compounds showed antiprion activity in the nanomolar range. The most active molecule, named 42, had a half-effective concentration (EC50) for antiprion activity of 50 nM. In a library of quinoline derivatives we were able to identify several structure-activity relationships (SAR). Remarkably, antiprion SAR in ScN2a cells were similar to antimalarial SAR in a cell model of malaria, particularly for the sulfonamide quinoline derivatives, suggesting that some molecular targets of antiprion and antimalarial substances overlap.

Prion infection influences murine endogenous retrovirus expression in neuronal cells

Prions as causative agents of transmissible spongiform encephalopathies have been well investigated in experimental and modelling work. However, little is known about the molecular pathogenesis of prion-induced encephalopathies, the role of co-factors, and the interaction of prions with cellular components. We investigated the influence of prion infection on expression of murine endogenous retroviruses (ERVs), which compose approximately 10% of the mouse genome. Hypothalamic neuronal cells (GT1) and neuroblastoma cells (N2a) were examined. Both cell lines can be persistently infected with mouse adapted prion strains, i.e., RML. Using a mammalian retrovirus-specific DNA microarray and quantitative PCR methods, we compared the expression profiles of ERVs in prion-infected, uninfected, and anti-prion compound-treated murine neuronal cell lines, including clonal cell populations. The results suggest that prion infection influences ERV expression in neuronal cell lines, that this influence is cell line-specific, ERV-specific, and responsive to anti-prion compound treatment.

The AGAAAAGA Palindrome in PrP Is Required to Generate a Productive PrPSc-PrPC Complex That Leads to Prion Propagation

The molecular hallmark of prion disease is the conversion of normal prion protein (PrPC) to an insoluble, proteinase K-resistant, pathogenic isoform (PrPSc). Once generated, PrPSc propagates by complexing with, and transferring its pathogenic conformation onto, PrPC. Defining the specific nature of this PrPSc-PrPC interaction is critical to understanding prion genesis. To begin to approach this question, we employed a prion-infected neuroblastoma cell line (ScN2a) combined with a heterologous yeast expression system to independently model PrPSc generation and propagation. We additionally applied fluorescence resonance energy transfer analysis to the latter to specifically study PrP-PrP interactions. In this report we focus on an N-terminal hydrophobic palindrome of PrP (112-AGAAAAGA-119) thought to feature intimately in prion generation via an unclear mechanism. We found that, in contrast to wild type (wt) PrP, PrP lacking the palindrome (PrPDelta112-119) neither converted to PrPSc when expressed in ScN2a cells nor generated proteinase K-resistant PrP when expressed in yeast. Furthermore, PrPDelta112-119 was a dominant-negative inhibitor of wtPrP in ScN2a cells. Both wtPrP and PrPDelta112-119 were highly insoluble when expressed in yeast and produced distinct cytosolic aggregates when expressed as fluorescent fusion proteins (PrP::YFP). Although self-aggregation was evident, fluorescence resonance energy transfer studies in live yeast co-expressing PrPSc-like protein and PrPDelta112-119 indicated altered interaction properties. These results suggest that the palindrome is required, not only for the attainment of the PrPSc conformation but also to facilitate the proper association of PrPSc with PrPC to effect prion propagation.

Phospholipase A2 Inhibitors or Platelet-activating Factor Antagonists Prevent Prion Replication

A key feature of prion diseases is the conversion of the cellular prion protein (PrP(C)) into disease-related isoforms (PrP(Sc)), the deposition of which is thought to lead to neurodegeneration. In this study a pharmacological approach was used to determine the metabolic pathways involved in the formation of protease-resistant PrP (PrP(res)) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). Daily treatment of these cells with phospholipase A(2) (PLA(2)) inhibitors for 7 days prevented the accumulation of PrP(res). Glucocorticoids with anti-PLA(2) activity also prevented the formation of PrP(res) and reduced the infectivity of SMB cells. Treatment with platelet-activating factor (PAF) antagonists also reduced the PrP(res) content of cells, while the addition of PAF reversed the inhibitory effect of PLA(2) inhibitors on PrP(res) formation. ScGT1 cells treated with PLA(2) inhibitors or PAF antagonists for 7 days remained clear of detectable (PrPres) when grown in control medium for a further 12 weeks. Treatment of non-infected cells with PLA(2) inhibitors or PAF antagonists reduced PrP(C) levels suggesting that limiting cellular PrP(C) may restrict prion formation in infected cells. These data indicate a pivotal role for PLA(2) and PAF in controlling PrP(res) formation and identify them as potential therapeutic agents.

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.

Squalestatin Cures Prion-infected Neurons and Protects Against Prion Neurotoxicity

A key feature of prion diseases is the conversion of the normal, cellular prion protein (PrP(C)) into beta-sheet-rich disease-related isoforms (PrP(Sc)), the deposition of which is thought to lead to neurodegeneration. In the present study, the squalene synthase inhibitor squalestatin reduced the cholesterol content of cells and prevented the accumulation of PrP(Sc) in three prion-infected cell lines (ScN2a, SMB, and ScGT1 cells). ScN2a cells treated with squalestatin were also protected against microglia-mediated killing. Treatment of neurons with squalestatin resulted in a redistribution of PrP(C) away from Triton X-100 insoluble lipid rafts. These effects of squalestatin were dose-dependent, were evident at nanomolar concentrations, and were partially reversed by cholesterol. In addition, uninfected neurons treated with squalestatin became resistant to the otherwise toxic effect of PrP peptides, a synthetic miniprion (sPrP106) or partially purified prion preparations. The protective effect of squalestatin, which was reversed by the addition of water-soluble cholesterol, correlated with a reduction in prostaglandin E(2) production that is associated with neuronal injury in prion disease. These studies indicate a pivotal role for cholesterol-sensitive processes in controlling PrP(Sc) formation, and in the activation of signaling pathways associated with PrP-induced neuronal death.

Cultured cell sublines highly susceptible to prion infection.

Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP(Sc)). In order to derive cell lines producing sufficient quantities of PrP(Sc) for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrP(Sc). Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrP(Sc) levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.

Abnormal plasma membrane properties and functions in prion-infected cell lines.

A long trail of evidence indicates that the formation of PrPSc or its accumulation causes the neuronal dysfunction and clinical features of prion diseases. The results of our current line of studies argue that the main neuropathological and clinical features of prion diseases are explained by altered ion channel function secondary to decreased plasma membrane fluidity. This kind of mechanism has the potential to functionally disconnect neuronal networks and cause neuronal vacuolation. Our laboratory is currently focusing its investigations on pathogenic mechanisms that have the potential to link the formation of PrPSc with plasma membrane abnormalities in prion diseases. In summary, the first hypothesis suggests that the conversion of PrPC to PrPSc affects plasma membrane fluidity directly, which secondarily alters the properties and functions of its components. In contrast, the second hypothesis argues that PrPSc accumulation alters the ability of chaperones to correctly fold plasma-membrane proteins during their synthesis, which directly affects the properties of nascent proteins and secondarily affects membrane fluidity. Our current investigations are attempting to determine which of these mechanisms are plausible and, then, which is primary.