Nathan R. Deleault

Formation of native prions from minimal components in vitro

The conformational change of a host protein, PrPC, into a disease-associated isoform, PrPSc, appears to play a critical role in the pathogenesis of prion diseases such as Creutzfeldt–Jakob disease and scrapie. However, the fundamental mechanism by which infectious prions are produced in neurons remains unknown. To investigate the mechanism of prion formation biochemically, we conducted a series of experiments using the protein misfolding cyclic amplification (PMCA) technique with a preparation containing only native PrPC and copurified lipid molecules. These experiments showed that successful PMCA propagation of PrPSc molecules in a purified system requires accessory polyanion molecules. In addition, we found that PrPSc molecules could be formed de novo from these defined components in the absence of preexisting prions. Inoculation of samples containing either prion-seeded or spontaneously generated PrPSc molecules into hamsters caused scrapie, which was transmissible on second passage. These results show that prions able to infect wild-type hamsters can be formed from a minimal set of components including native PrPC molecules, copurified lipid molecules, and a synthetic polyanion.

RNA molecules stimulate prion protein conversion

Much evidence supports the hypothesis that the infectious agents of prion diseases are devoid of nucleic acid, and instead are composed of a specific infectious protein. This protein, PrPSc, seems to be generated by template-induced conformational change of a normally expressed glycoprotein, PrPC (ref. 2). Although numerous studies have established the conversion of PrPC to PrPSc as the central pathogenic event of prion disease, it is unknown whether cellular factors other than PrPC might be required to stimulate efficient PrPSc production. We investigated the biochemical amplification of protease-resistant PrPSc-like protein (PrPres) using a modified version of the protein-misfolding cyclic amplification method. Here we report that stoichiometric transformation of PrPC to PrPres in vitro requires specific RNA molecules. Notably, whereas mammalian RNA preparations stimulate in vitro amplification of PrPres, RNA preparations from invertebrate species do not. Our findings suggest that host-encoded stimulatory RNA molecules may have a role in the pathogenesis of prion disease. They also provide a practical approach to improve the sensitivity of diagnostic techniques based on PrPres amplification.

Isolation of phosphatidylethanolamine as a solitary cofactor for prion formation in the absence of nucleic acids.

Infectious prions containing the pathogenic conformer of the mammalian prion protein (PrPSc) can be produced de novo from a mixture of the normal conformer (PrPC) with RNA and lipid molecules. Recent reconstitution studies indicate that nucleic acids are not required for the propagation of mouse prions in vitro, suggesting the existence of an alternative prion propagation cofactor in brain tissue. However, the identity and functional properties of this unique cofactor are unknown. Here, we show by purification and reconstitution that the molecule responsible for the nuclease-resistant cofactor activity in brain is endogenous phosphatidylethanolamine (PE). Synthetic PE alone facilitates conversion of purified recombinant (rec)PrP substrate into infectious recPrPSc molecules. Other phospholipids, including phosphatidylcholine, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol, were unable to facilitate recPrPSc formation in the absence of RNA. PE facilitated the propagation of PrPSc molecules derived from all four different animal species tested including mouse, suggesting that unlike RNA, PE is a promiscuous cofactor for PrPSc formation in vitro. Phospholipase treatment abolished the ability of brain homogenate to reconstitute the propagation of both mouse and hamster PrPSc molecules. Our results identify a single endogenous cofactor able to facilitate the formation of prions from multiple species in the absence of nucleic acids or other polyanions.

Cofactor molecules maintain infectious conformation and restrict strain properties in purified prions.

Prions containing misfolded prion protein (PrPSc) can be formed with cofactor molecules using the technique of serial protein misfolding cyclic amplification. However, it remains unknown whether cofactors materially participate in maintaining prion conformation and infectious properties. Here we show that withdrawal of cofactor molecules during serial propagation of purified recombinant prions caused adaptation of PrPSc structure accompanied by a reduction in specific infectivity of >105-fold, to undetectable levels, despite the ability of adapted “protein-only” PrPSc molecules to self-propagate in vitro. We also report that changing only the cofactor component of a minimal reaction substrate mixture during serial propagation induced major changes in the strain properties of an infectious recombinant prion. Moreover, propagation with only one functional cofactor (phosphatidylethanolamine) induced the conversion of three distinct strains into a single strain with unique infectious properties and PrPSc structure. Taken together, these results indicate that cofactor molecules can regulate the defining features of mammalian prions: PrPSc conformation, infectivity, and strain properties. These findings suggest that cofactor molecules likely are integral components of infectious prions.

Selective incorporation of polyanionic molecules into hamster prions

The central pathogenic event of prion disease is the conformational conversion of a host protein, PrPC, into a pathogenic isoform, PrPSc. We previously showed that the protein misfolding cyclic amplification (PMCA) technique can be used to form infectious prion molecules de novo from purified native PrPC molecules in an autocatalytic process requiring accessory polyanions (Deleault, N. R., Harris, B. T., Rees, J. R., and Supattapone, S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 9741-9746). Here we investigated the molecular mechanism by which polyanionic molecules facilitate infectious prion formation in vitro.Ina PMCA reaction lacking PrPSc template seed, synthetic poly(A) RNA molecules induce hamster (Ha)PrPC to adopt a protease-sensitive, detergent-insoluble conformation reactive against antibodies specific for PrPSc. During PMCA, labeled nucleic acids form nuclease-resistant complexes with HaPrP molecules. Strikingly, purified HaPrPC molecules subjected to PMCA selectively incorporate an ∼1-2.5-kb subset of [32P]poly(A) RNA molecules from a heterogeneous mixture ranging in size from ∼0.1 to >6 kb. Neuropathological analysis of scrapie-infected hamsters using the fluorescent dye acridine orange revealed that RNA molecules co-localize with large extracellular HaPrP aggregates. These findings suggest that polyanionic molecules such as RNA may become selectively incorporated into stable complexes with PrP molecules during the formation of native hamster prions.

Species-Dependent Differences in Cofactor Utilization for Formation of the Protease-Resistant Prion Protein in Vitro

The cofactor preferences for in vitro propagation of the protease-resistant isoforms of the prion protein (PrP(Sc)) from various rodent species were investigated using the serial protein misfolding cyclic amplification (sPMCA) technique. Whereas RNA molecules facilitate hamster PrP(Sc) propagation, RNA and several other polyanions do not promote the propagation of mouse and vole PrP(Sc) molecules. Pretreatment of crude Prnp(0/0) (PrP knockout) brain homogenate with RNase A or micrococcal nuclease inhibited hamster but not mouse PrP(Sc) propagation in a reconstituted system. Mouse PrP(Sc) propagation could be reconstituted by mixing PrP(C) substrate with homogenates prepared from either brain or liver, but not from several other tissues that were tested. These results reveal species-specific differences in cofactor utilization for PrP(Sc) propagation in vitro and also demonstrate the existence of an endogenous cofactor present in brain tissue not composed of nucleic acids.

Accelerated Accumulation of Misfolded Prion Protein and Spongiform Degeneration in a Drosophila Model of Gerstmann–Sträussler–Scheinker Syndrome

Prion diseases are CNS disorders that can occur in sporadic, infectious, and inherited forms. Although all forms of prion disease are associated with the accumulation of pathogenic conformers of the prion protein, collectively termed PrPSc, the mechanisms by which PrPSc molecules form and cause neuronal degeneration are unknown. Using the bipartite galactosidase-4–upstream activating sequence expression system, we generated transgenic Drosophila melanogaster heterologously expressing either wild-type (WT) or mutant, disease-associated (P101L) mouse PrP molecules in cholinergic neurons. Transgenic flies expressing neuronal P101L PrP molecules exhibited severe locomotor dysfunction and premature death as larvae and adults. These striking clinical abnormalities were accompanied by age-dependent accumulation of misfolded PrP molecules, intracellular PrP aggregates, and neuronal vacuoles. In contrast, transgenic flies expressing comparable levels of WT PrP displayed no clinical, pathological, or biochemical abnormalities. These results indicate that transgenic Drosophila expressing neuronal P101L PrP specifically exhibit several hallmark features of human Gerstmann–Sträussler–Scheinker (GSS) syndrome. Because the rates of abnormal PrP accumulation and clinical progression are highly accelerated in Drosophila compared with the rates of these processes in rodents or humans, the P101L mutant may be used for future genetic and pharmacologic studies as a novel invertebrate model of GSS.

Copper (II) ions potently inhibit purified PrPres amplification.

The structural conversion of a host protein, PrPC, into a protease‐resistant isoform, PrPres, is the central event in the pathogenesis of infectious prion diseases. Purification of native PrPC molecules from hamster brain by either cation exchange or immobilized chelator chromatographic resins yielded preparations that supported efficient amplification of scrapie‐induced PrPres in vitro. Using these purified preparations, we determined that in vitro PrPres amplification was inhibited by CuCl2 and ZnCl2 at IC50 concentrations of ∼400u2003nm and 10u2003μm, respectively. In contrast, 100u2003μm MnCl2 did not directly inhibit PrPres amplification or block Cu2+‐mediated inhibition. Additionally, the inhibition of PrPres amplification by Cu2+ ions could be reversed by addition of either neocuproine or imidazole. Cu2+ inhibited PrPres amplification in both the presence and absence of stimulatory polyanion molecules. These biochemical findings support the hypothesis that Cu2+ ions might regulate the pathogenesis of prion diseases in vivo.

The effects of prion protein proteolysis and disaggregation on the strain properties of hamster scrapie

Native mammalian prions exist in self-propagating strains that exhibit distinctive clinical, pathological and biochemical characteristics. Prion strain diversity is associated with variations in PrP(Sc) conformation, but it remains unknown precisely which physical properties of the PrP(Sc) molecules are required to encipher mammalian prion strain phenotypes. In this study, we subjected prion-infected brain homogenates derived from three different hamster scrapie strains to either (i) proteinase K digestion or (ii) sonication, and inoculated the modified samples into normal hamsters. The results show that the strain-specific clinical features and neuropathological profiles of inoculated animals were not affected by either treatment. Similarly, the strain-dependent biochemical characteristics of the PrP(Sc) molecules (including electrophoretic mobility, glycoform composition, conformational stability and susceptibility to protease digestion) in infected animals were unaffected by either proteolysis or sonication of the original inocula. These results indicate that the infectious strain properties of native prions do not appear to be altered by PrP(Sc) disaggregation, and that maintenance of such properties does not require the N-domain (approximately residues 23-90) of the protease-resistant PrP(Sc) molecules or protease-sensitive PrP(Sc) molecules.

Post-transcriptional suppression of pathogenic prion protein expression in Drosophila neurons.

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue‐specific expression system to express wild‐type and disease‐associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease‐associated insertional PrP mutant. We also found that wild‐type PrP molecules are maintained in a detergent‐soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent‐insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.