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Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrPSc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrPSc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-009-0633-x) contains supplementary material, which is available to authorized users.

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time-and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small ␤-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or ␤-oligomers was substantially reduced when expression of endogenous PrP C was down-regulated by small interfering RNAs. In opposition to the ␤-oligomer and amyloid fibrils of rPrP, the monomeric ␣-helical form of rPrP stimulated neurite outgrowth and survival of neurons. These studies illustrated that both soluble ␤-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP C is required for mediating the toxicity of abnormally folded external PrP aggregates.Several neurodegenerative maladies including Alzheimer, Parkinson, Huntington, and prion diseases have been related to the age-dependent accumulation of amyloid deposits in the brain (1, 2). A common feature among these and other "conformational" diseases is the conversion of specific proteins or peptides into polymeric forms that are characterized by cross-␤-sheet structures and referred to as amyloid (3). Even though the amyloidogenic proteins have no obvious sequence similarity, they share similar conformational features within the amyloid form (4, 5).In addition to the amyloid fibrils/deposits, the formation of nonfibrillar soluble oligomers has been observed for a number of proteins associated with conformational diseases including ␣-synuclein (6, 7), A␤ peptides (8, 9), transthyretin (10), lysozyme (11), and prion protein (12, 13). Soluble oligomers were found either as a prefibrillar intermediate that formed on the kinetic pathway to the mature amyloid fibrils (14 -17) or as off-pathway products produced via alternative aggregation mechanisms (13,18,19). For the past several years, a substantial body of evidence has accumulated indicating that soluble oligomers are toxic species that are actively involved in the impairment of cellular functions in neurodegenerative diseases. Furthermore, non-fibrillar oligomers have been shown to be intrinsically toxic to cells even when formed from proteins that are not related to any known conformational disease (20,21). This implies that oligomeric species share a common mechanism of cytotoxicity regardless of the specific protein from which they are generated (22,23). On the other hand, there is considerable debate as to whether mature amyloid fibrils also exhibit toxici...

In recent studies, the amyloid fibrils produced in vitro from recombinant prion protein encompassing residues 89-230 (rPrP 89-230) were shown to produce transmissible form of prion disease in transgenic mice (Legname et al., (2004) Science 305, 673-676). Long incubation time observed upon inoculation of the amyloid fibrils, however, suggests that the fibrils generated in vitro have low infectivity titers. These results emphasize the need to define optimal conditions for prion conversion in vitro, under which high levels of infectivity can be generated in a cell-free system. Because copper(II) has been implicated in normal and pathological functions of the prion protein, here we investigated the effect of Cu(2+) on cell-free conversion of recombinant PrP. Our results show that at pH 7.2 and at micromolar concentrations, Cu(2+) inhibited conversion of full-length recombinant PrP (rPrP 23-230) into amyloid fibrils. This effect was most pronounced for Cu(2+), and less so for Zn(2+), while Mn(2+) had no effect on the conversion. Cu(2+)-dependent inhibition of the amyloid formation was less effective at pH 6.0, at which rPrP 23-230 displays lower Cu(2+)-binding capacity. Using rPrP 89-230, we found that Cu(2+)-dependent inhibition occurred even in the absence of octarepeat region; however, it was less effective. Our further studies indicated that Cu(2+) inhibited conversion by stabilizing a nonamyloidogenic PK-resistant form of alpha-rPrP. Remarkably, Cu(2+) also had a profound effect on preformed amyloid fibrils. When added to the fibrils, Cu(2+) induced long-range coiling of individual fibrils and enhanced their PK-resistance. It, however, produced only minor changes in their secondary structures. In addition, Cu(2+) induced further aggregation of the amyloid fibrils into large clumps, presumably, through interfibrillar coordination of copper ions by octarepeats. Taken together, our studies suggest that the role of Cu(2+) in the pathogenesis of prion diseases is complex. Because Cu(2+) may inhibit prion replication, while at the same time stabilize disease-specific isoform against proteolytic clearance, the final outcome of copper-induced effect on progression of prion disease may not be straightforward.

a b s t r a c tSome pathogenic mutations associated with Alzheimer's disease are thought to affect structuraldynamic properties and the lateral dimerization of amyloid precursor protein (APP) in neuron membrane. Dimeric structure of APP transmembrane fragment Gln 686 -Lys 726 was determined in membrane-mimicking dodecylphosphocholine micelles using high-resolution NMR spectroscopy.The APP membrane-spanning a-helix Lys 699 -Lys 724 self-associates in a left-handed parallel dimer through extended heptad repeat motif I 702 X 3 M 706 X 2 G 709 X 3 A 713 X 2 I 716 X 3 I 720 X 2 I 723 , whereas the juxtamembrane region Gln 686 -Val 695 constitutes the nascent helix, also sensing the dimerization. The dimerization mechanism of APP transmembrane domain has been described at atomic resolution for the first time and is important for understanding molecular events of APP sequential proteolytical cleavage resulting in amyloid-b peptide. Structured summary of protein interactions:APPjmtm and APPjmtm bind by comigration in gel electrophoresis (View interaction) APPjmtm and APPjmtm bind by nuclear magnetic resonance (View interaction).

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In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP Sc produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP Sc recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a ␤-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP Sc , and the other to classical PrP Sc . Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP Sc with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.Keywords: prion protein; amyloid fibrils; conformational transition; proteinase K; Creutzfeldt-Jakob disease Several severe neurodegenerative diseases including Creutzfeldt-Jakob disease (CJD), Gerstmann-StrausslerSheinker disease, and fatal familial insomnia are associated with misfolding and aggregation of the prion protein (Prusiner 2001). Prion maladies manifest themselves in infectious, familial, and sporadic forms. To explain the infectious form of the prion diseases, the "protein-only hypothesis" postulates that an abnormal isoform of the prion protein, PrP Sc , acts as an infectious agent and propagates its pathological conformation in an autocatalytic manner using the normal isoform of the same protein, PrP C , as a substrate (Prusiner 1982). PrP C and PrP Sc differ substantially in their conformations. Unlike PrP C , PrP Sc is a multimeric assembly characterized by enhanced resistance to proteinase K (PK) digestion and by an increase in the amount of ␤-sheet structure Reprint requests to: Ilia V. Baskakov, 725 W. Lombard Street, Baltimore, MD 21201, USA; e-mail: Baskakov@umbi.umd.edu; fax: (410) 706-8184.Abbreviations: CJD, Creutzfeldt-Jakob disease; spCJD, sporadic CJD; PrP, prion protein; PrP C , the normal, cellular isoform of PrP; PrP Sc , the abnormal, infections isoform of PrP; rPrP, recombinant PrP; rPrP 89-230, recombinant PrP encompassing residues 89-230; rPrP 23-230, full-length recombinant PrP; ␣-rPrP 89-230, ␣-helical isoform of rPrP 89-231; ␣-rPrP 23-230, ␣-helical isoform of rPrP 23-231; PK, proteinase K; PrP 27-30, PK-resistant core of classical PrP ...

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