Inhibition of PrPSc formation in scrapie infected N2a cells by 5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine derivatives

Prion diseases are fatal, neurodegenerative diseases characterized by the structural conversion of the normal, cellular prion protein, PrP^C into an abnormally structured, aggregated and partially protease-resistant isoform, termed PrP^Sc. Although substantial research has been directed toward development of therapeutics targeting prions, there is still no curative treatment for the disease. Benzoxazines are bicyclic heterocyclic compounds possessing several pharmaceutically important properties, including neuroprotection and reactive oxygen species scavenging. In an effort to identify novel inhibitors of prion formation, several 5,7,8-trimethyl-1,4-benzoxazine derivatives were evaluated in vitro for their effectiveness on the expression levels of normal PrP^C and its conversion to the abnormal isoforms of PrP^Sc in a scrapie-infected cell culture model. The most potent compound was 2-(4-methoxyphenyl)-5,7,8-trimethyl-3,4-dihydro-2H-1,4-benzoxazine, with a diminishing effect on the formation of PrP^Sc, thus establishing a class of compounds with a promising therapeutic use against prion diseases.     

Piperazine derivatives inhibit PrP/PrP propagation in vitro and in vivo

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc^C into a disease-associated isoform PrP^Sc. PrP^Sc accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP^Sc species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP^Sc formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge).     

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.     

The cellular prion protein (PrPC): Its physiological function and role in disease

Prion diseases are caused by conversion of a normal cell-surface glycoprotein (PrP^C) into a conformationally altered isoform (PrP^Sc) that is infectious in the absence of nucleic acid. Although a great deal has been learned about PrP^Sc and its role in prion propagation, much less is known about the physiological function of PrP^C. In this review, we will summarize some of the major proposed functions for PrP^C, including protection against apoptotic and oxidative stress, cellular uptake or binding of copper ions, transmembrane signaling, formation and maintenance of synapses, and adhesion to the extracellular matrix. We will also outline how loss or subversion of the cytoprotective or neuronal survival activities of PrP^C might contribute to the pathogenesis of prion diseases, and how similar mechanisms are probably operative in other neurodegenerative disorders.     

Pressure-assisted dissociation and degradation of “proteinase K-resistant” fibrils prepared by seeding with scrapie-infected hamster prion protein

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP^C, into a fibrous pathogenic form, PrP^Sc, which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP^Sc into PrP^C is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP^Sc from PrP^C eventually in vivo. Here we show that, by applying pressures at kbar range, the “proteinase K-resistant” fibrils (rHaPrP^res) prepared from hamster prion protein (rHaPrP [23–231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP^res fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP^Sc from PrP^C is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP^Sc from various systems of pathogenic concern.     

Sequestration of free cholesterol in cell membranes by prions correlates with cytoplasmic phospholipase A<Subscript>2</Subscript>activation

Background  

The transmissible spongiform encephalopathies (TSEs), otherwise known as the prion diseases, occur following the conversion of the normal cellular prion protein (PrP^C) to an alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain leads to neurodegeneration through an unidentified mechanism. Since many neurodegenerative disorders including prion, Parkinson's and Alzheimer's diseases may be modified by cholesterol synthesis inhibitors, the effects of prion infection on the cholesterol balance within neuronal cells were examined.     

Identification of an Intracellular Site of Prion Conversion

Prion diseases are fatal, neurodegenerative disorders in humans and animals and are characterized by the accumulation of an abnormally folded isoform of the cellular prion protein (PrP^C), denoted PrP^Sc, which represents the major component of infectious scrapie prions. Characterization of the mechanism of conversion of PrP^C into PrP^Sc and identification of the intracellular site where it occurs are among the most important questions in prion biology. Despite numerous efforts, both of these questions remain unsolved. We have quantitatively analyzed the distribution of PrP^C and PrP^Sc and measured PrP^Sc levels in different infected neuronal cell lines in which protein trafficking has been selectively impaired. Our data exclude roles for both early and late endosomes and identify the endosomal recycling compartment as the likely site of prion conversion. These findings represent a fundamental step towards understanding the cellular mechanism of prion conversion and will allow the development of new therapeutic approaches for prion diseases.     

The Octarepeat Region of the Prion Protein Is Conformationally Altered in PrPSc

Background

Prion diseases are fatal neurodegenerative disorders characterized by misfolding and aggregation of the normal prion protein PrP^C. Little is known about the details of the structural rearrangement of physiological PrP^C into a still-elusive disease-associated conformation termed PrP^Sc. Increasing evidence suggests that the amino-terminal octapeptide sequences of PrP (huPrP, residues 59–89), though not essential, play a role in modulating prion replication and disease presentation.

Methodology/Principal Findings

Here, we report that trypsin digestion of PrP^Sc from variant and sporadic human CJD results in a disease-specific trypsin-resistant PrP^Sc fragment including amino acids ∼49–231, thus preserving important epitopes such as the octapeptide domain for biochemical examination. Our immunodetection analyses reveal that several epitopes buried in this region of PrP^Sc are exposed in PrP^C.

Conclusions/Significance

We conclude that the octapeptide region undergoes a previously unrecognized conformational transition in the formation of PrP^Sc. This phenomenon may be relevant to the mechanism by which the amino terminus of PrP^C participates in PrP^Sc conversion, and may also be exploited for diagnostic purposes.     

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrP^Sc), an abnormal conformer of the normal cellular prion protein PrP^C. Depletion of PrP^C in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrP^C expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrP^C levels were reduced by ∼70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrP^Sc formation in the thalamic infusion site by ∼75%, it did not suppress PrP^Sc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrP^C in the brain is required for successful therapy of prion diseases.     

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP^C) into disease-specific forms, termed PrP^Sc, that have the ability to interact with PrP^C promoting its conversion to PrP^Sc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP^C region involved in the interaction with PrP^Sc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP^Sc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.     

Glimepiride Reduces the Expression of PrPC,Prevents PrPSc Formation and Protects against Prion Mediated Neurotoxicity

Background

A hallmark of the prion diseases is the conversion of the host-encoded cellular prion protein (PrP^C) into a disease related, alternatively folded isoform (PrP^Sc). The accumulation of PrP^Sc within the brain is associated with synapse loss and ultimately neuronal death. Novel therapeutics are desperately required to treat neurodegenerative diseases including the prion diseases.

Principal Findings

Treatment with glimepiride, a sulphonylurea approved for the treatment of diabetes mellitus, induced the release of PrP^C from the surface of prion-infected neuronal cells. The cell surface is a site where PrP^C molecules may be converted to PrP^Sc and glimepiride treatment reduced PrP^Sc formation in three prion infected neuronal cell lines (ScN2a, SMB and ScGT1 cells). Glimepiride also protected cortical and hippocampal neurones against the toxic effects of the prion-derived peptide PrP82–146. Glimepiride treatment significantly reduce both the amount of PrP82–146 that bound to neurones and PrP82–146 induced activation of cytoplasmic phospholipase A₂ (cPLA₂) and the production of prostaglandin E₂ that is associated with neuronal injury in prion diseases. Our results are consistent with reports that glimepiride activates an endogenous glycosylphosphatidylinositol (GPI)-phospholipase C which reduced PrP^C expression at the surface of neuronal cells. The effects of glimepiride were reproduced by treatment of cells with phosphatidylinositol-phospholipase C (PI-PLC) and were reversed by co-incubation with p-chloromercuriphenylsulphonate, an inhibitor of endogenous GPI-PLC.

Conclusions

Collectively, these results indicate that glimepiride may be a novel treatment to reduce PrP^Sc formation and neuronal damage in prion diseases.     

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrP<Superscript>C</Superscript>

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrP^Sc of the cellular prion protein PrP^C. Expression of PrP^C is a prerequisite for prion infection, and conformational conversion of PrP^C is induced upon its direct interaction with PrP^Sc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrP^C as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrP^Sc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrP^C α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrP^C at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aβ oligomers characteristic for Alzheimer’s disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrP^C and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.     

Brain delivery of AAV9 expressing an anti-PrP monovalent antibody delays prion disease in mice

Prion diseases are caused by a conformational modification of the cellular prion protein (PrP^C) into disease-specific forms, termed PrP^Sc, that have the ability to interact with PrP^C promoting its conversion to PrP^Sc. In vitro studies demonstrated that anti-PrP antibodies inhibit this process. In particular, the single chain variable fragment D18 antibody (scFvD18) showed high efficiency in curing chronically prion-infected cells. This molecule binds the PrP^C region involved in the interaction with PrP^Sc thus halting further prion formation. These findings prompted us to test the efficiency of scFvD18 in vivo. A recombinant Adeno-Associated Viral vector serotype 9 was used to deliver scFvD18 to the brain of mice that were subsequently infected by intraperitoneal route with the mouse-adapted scrapie strain RML. We found that the treatment was safe, prolonged the incubation time of scrapie-infected animals and decreased the burden of total proteinase-resistant PrP^Sc in the brain, suggesting that scFvD18 interferes with prion replication in vivo. This approach is relevant for designing new therapeutic strategies for prion diseases and other disorders characterized by protein misfolding.     

Glypican-1 Mediates Both Prion Protein Lipid Raft Association and Disease Isoform Formation

In prion diseases, the cellular form of the prion protein, PrP^C, undergoes a conformational conversion to the infectious isoform, PrP^Sc. PrP^C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI) anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs). We show that heparin displaces PrP^C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP^C. We then utilised a transmembrane-anchored form of PrP (PrP-TM), which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP^C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP^C from rafts, promoting its endocytosis. Glypican-1 and PrP^C colocalised on the cell surface and both PrP^C and PrP^Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP^Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP^C on the β-secretase cleavage of the Alzheimer''s amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP^C and PrP^Sc in lipid rafts.     

Lipopolysaccharide induced conversion of recombinant prion protein

The conformational conversion of the cellular prion protein (PrP^C) to the β-rich infectious isoform PrP^Sc is considered a critical and central feature in prion pathology. Although PrP^Sc is the critical component of the infectious agent, as proposed in the “protein-only” prion hypothesis, cellular components have been identified as important cofactors in triggering and enhancing the conversion of PrP^C to proteinase K resistant PrP^Sc. A number of in vitro systems using various chemical and/or physical agents such as guanidine hydrochloride, urea, SDS, high temperature, and low pH, have been developed that cause PrP^C conversion, their amplification, and amyloid fibril formation often under non-physiological conditions. In our ongoing efforts to look for endogenous and exogenous chemical mediators that might initiate, influence, or result in the natural conversion of PrP^C to PrP^Sc, we discovered that lipopolysaccharide (LPS), a component of gram-negative bacterial membranes interacts with recombinant prion proteins and induces conversion to an isoform richer in β sheet at near physiological conditions as long as the LPS concentration remains above the critical micelle concentration (CMC). More significant was the LPS mediated conversion that was observed even at sub-molar ratios of LPS to recombinant ShPrP (90–232).     

Anti-prion activity of an RNA aptamer and its structural basis

Prion proteins (PrPs) cause prion diseases, such as bovine spongiform encephalopathy. The conversion of a normal cellular form (PrP^C) of PrP into an abnormal form (PrP^Sc) is thought to be associated with the pathogenesis. An RNA aptamer that tightly binds to and stabilizes PrP^C is expected to block this conversion and to thereby prevent prion diseases. Here, we show that an RNA aptamer comprising only 12 residues, r(GGAGGAGGAGGA) (R12), reduces the PrP^Sc level in mouse neuronal cells persistently infected with the transmissible spongiform encephalopathy agent. Nuclear magnetic resonance analysis revealed that R12, folded into a unique quadruplex structure, forms a dimer and that each monomer simultaneously binds to two portions of the N-terminal half of PrP^C, resulting in tight binding. Electrostatic and stacking interactions contribute to the affinity of each portion. Our results demonstrate the therapeutic potential of an RNA aptamer as to prion diseases.     

Sialylation of Prion Protein Controls the Rate of Prion Amplification,the Cross-Species Barrier,the Ratio of PrPSc Glycoform and Prion Infectivity

The central event underlying prion diseases involves conformational change of the cellular form of the prion protein (PrP^C) into the disease-associated, transmissible form (PrP^Sc). PrP^C is a sialoglycoprotein that contains two conserved N-glycosylation sites. Among the key parameters that control prion replication identified over the years are amino acid sequence of host PrP^C and the strain-specific structure of PrP^Sc. The current work highlights the previously unappreciated role of sialylation of PrP^C glycans in prion pathogenesis, including its role in controlling prion replication rate, infectivity, cross-species barrier and PrP^Sc glycoform ratio. The current study demonstrates that undersialylated PrP^C is selected during prion amplification in Protein Misfolding Cyclic Amplification (PMCAb) at the expense of oversialylated PrP^C. As a result, PMCAb-derived PrP^Sc was less sialylated than brain-derived PrP^Sc. A decrease in PrP^Sc sialylation correlated with a drop in infectivity of PMCAb-derived material. Nevertheless, enzymatic de-sialylation of PrP^C using sialidase was found to increase the rate of PrP^Sc amplification in PMCAb from 10- to 10,000-fold in a strain-dependent manner. Moreover, de-sialylation of PrP^C reduced or eliminated a species barrier of for prion amplification in PMCAb. These results suggest that the negative charge of sialic acid controls the energy barrier of homologous and heterologous prion replication. Surprisingly, the sialylation status of PrP^C was also found to control PrP^Sc glycoform ratio. A decrease in PrP^C sialylation levels resulted in a higher percentage of the diglycosylated glycoform in PrP^Sc. 2D analysis of charge distribution revealed that the sialylation status of brain-derived PrP^C differed from that of spleen-derived PrP^C. Knocking out lysosomal sialidase Neu1 did not change the sialylation status of brain-derived PrP^C, suggesting that Neu1 is not responsible for desialylation of PrP^C. The current work highlights previously unappreciated role of PrP^C sialylation in prion diseases and opens multiple new research directions, including development of new therapeutic approaches.     

RNA and CuCl2 induced conformational changes of the recombinant ovine prion protein

Prion diseases are a group of neurodegenerative illnesses caused by conformational conversion of benign, α-helix rich cellular prion protein (PrP^C) into the highly stable, β-sheet rich scrapie prion protein (PrP^Sc) isoform. To date, the role of RNA on the conformational conversion of ovine prion protein in vitro remains unknown. To examine the effect of the interaction between RNA and PrP^C, conformations of recombinant ovine prion protein PrP^23–256 (OvPrP^23–256) binding various concentrations of RNA were analyzed by circular dichroism (CD) spectrum. The results indicated that the conformational conversion of OvPrP^23–256 was triggered by RNA with a decrease in α-helix content and increase in β-sheet. Moreover, the conformation of OvPrP^23–256 interacting with both RNA and CuCl₂ was also examined by CD spectrum, which showed that α-helix content decreased while β-sheet increased dramatically. Proteinase K digestion assay disclosed that the recombinant ovine PrP^C acquired PK resistance after RNA and/or Cu²⁺ treatment. It confirmed that the RNA/Cu²⁺ treatment in vitro altered the biochemical properties of ovine PrP^C. The implication of this finding, with respect to PrP^Sc, is that a dysfunctional state of a normal physiological process possibly facilitates diseases. The information gained from this study may provide useful approaches to study the pathogenesis of prion diseases.     

Glycosylphosphatidylinositol Anchor Analogues Sequester Cholesterol and Reduce Prion Formation

A hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP^C where C is cellular) into an alternatively folded, disease-related isoform (PrP^Sc, where Sc is scrapie), the accumulation of which is associated with synapse degeneration and ultimately neuronal death. The formation of PrP^Sc is dependent upon the presence of PrP^C in specific, cholesterol-sensitive membrane microdomains, commonly called lipid rafts. PrP^C is targeted to these lipid rafts because it is attached to membranes via a glycosylphosphatidylinositol anchor. Here, we show that treatment of prion-infected neuronal cell lines (ScN2a, ScGT1, or SMB cells) with synthetic glycosylphosphatidylinositol analogues, glucosamine-phosphatidylinositol (glucosamine-PI) or glucosamine 2-O-methyl inositol octadecyl phosphate, reduced the PrP^Sc content of these cells in a dose-dependent manner. In addition, ScGT1 cells treated with glucosamine-PI did not transmit infection following intracerebral injection to mice. Treatment with glucosamine-PI increased the cholesterol content of ScGT1 cell membranes and reduced activation of cytoplasmic phospholipase A₂ (PLA₂), consistent with the hypothesis that the composition of cell membranes affects key PLA₂-dependent signaling pathways involved in PrP^Sc formation. The effect of glucosamine-PI on PrP^Sc formation was also reversed by the addition of platelet-activating factor. Glucosamine-PI caused the displacement of PrP^C from lipid rafts and reduced expression of PrP^C at the cell surface, putative sites for PrP^Sc formation. We propose that treatment with glucosamine-PI modifies local micro-environments that control PrP^C expression and activation of PLA₂ and subsequently inhibits PrP^Sc formation.