Anti-PrP antibodies detected at terminal stage of prion-affected mouse

The causative agent of prion diseases is the pathological isoform (PrP^Sc) of the host-encoded cellular prion protein (PrP^C). PrP^Sc has an identical amino acid sequence to PrP^C; thus, it has been assumed that an immune response against PrP^Sc could not be found in prion-affected animals. In this study, we found the anti-prion protein (PrP) antibody at the terminal stage of mouse scrapie. Several sera from mice in the terminal stage of scrapie reacted to the recombinant mouse PrP (rMPrP) molecules and brain homogenates of mouse prion diseases. These results indicate that mouse could recognize PrP^C or PrP^Sc as antigens by the host immune system. Furthermore, immunization with rMPrP generates high titers of anti-PrP antibodies in wild-type mice. Some anti-PrP antibodies immunized with rMPrP prevent PrP^Sc replication in vitro. The mouse sera from terminal prion disease have several wide epitopes, although mouse sera immunized with rMPrP possess narrow epitopes.     

The POM Monoclonals: A Comprehensive Set of Antibodies to Non-Overlapping Prion Protein Epitopes

PrP^Sc, a misfolded and aggregated form of the cellular prion protein PrP^C, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP^C in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP^C and PrP^Sc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP^C. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP^C. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP^Sc. Other antibodies immunoprecipitate PrP^C, but not PrP^Sc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP^C and PrP^Sc. Amino-proximal antibodies were found to react with repetitive PrP^C epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.     

Different antigenicities of the N‐terminal region of cellular and scrapie prion proteins

Limited information is available about conformational differences between the abnormal isoform of prion protein (PrP^Sc) and cellular prion protein (PrP^C) under native conditions. To clarify conformational differences between these two isoforms, PrP‐deficient mice were immunized with brain homogenates of normal and scrapie‐infected animals. All mice generated anti‐PrP antibodies. Peptide array analysis of these serum samples revealed a distinctive epitope of PrP^Sc consisting of QGSPGGN (PrP41–47) at the N‐terminus. This study demonstrated a conformational dissimilarity at the N‐terminus between PrP^Sc and PrP^C, a finding that may provide novel information about conformational features of PrP^Sc.     

Prion Protein—Antibody Complexes Characterized by Chromatography-Coupled Small-Angle X-Ray Scattering

Aberrant self-assembly, induced by structural misfolding of the prion proteins, leads to a number of neurodegenerative disorders. In particular, misfolding of the mostly α-helical cellular prion protein (PrP^C) into a β-sheet-rich disease-causing isoform (PrP^Sc) is the key molecular event in the formation of PrP^Sc aggregates. The molecular mechanisms underlying the PrP^C-to-PrP^Sc conversion and subsequent aggregation remain to be elucidated. However, in persistently prion-infected cell-culture models, it was shown that treatment with monoclonal antibodies against defined regions of the prion protein (PrP) led to the clearing of PrP^Sc in cultured cells. To gain more insight into this process, we characterized PrP-antibody complexes in solution using a fast protein liquid chromatography coupled with small-angle x-ray scattering (FPLC-SAXS) procedure. High-quality SAXS data were collected for full-length recombinant mouse PrP [denoted recPrP(23–230)] and N-terminally truncated recPrP(89–230), as well as their complexes with each of two Fab fragments (HuM-P and HuM-R1), which recognize N- and C-terminal epitopes of PrP, respectively. In-line measurements by fast protein liquid chromatography coupled with SAXS minimized data artifacts caused by a non-monodispersed sample, allowing structural analysis of PrP alone and in complex with Fab antibodies. The resulting structural models suggest two mechanisms for how these Fabs may prevent the conversion of PrP^C into PrP^Sc.     

Trans-Dominant Inhibition of Prion Propagation In Vitro Is Not Mediated by an Accessory Cofactor

Previous studies identified prion protein (PrP) mutants which act as dominant negative inhibitors of prion formation through a mechanism hypothesized to require an unidentified species-specific cofactor termed protein X. To study the mechanism of dominant negative inhibition in vitro, we used recombinant PrP^C molecules expressed in Chinese hamster ovary cells as substrates in serial protein misfolding cyclic amplification (sPMCA) reactions. Bioassays confirmed that the products of these reactions are infectious. Using this system, we find that: (1) trans-dominant inhibition can be dissociated from conversion activity, (2) dominant-negative inhibition of prion formation can be reconstituted in vitro using only purified substrates, even when wild type (WT) PrP^C is pre-incubated with poly(A) RNA and PrP^Sc template, and (3) Q172R is the only hamster PrP mutant tested that fails to convert into PrP^Sc and that can dominantly inhibit conversion of WT PrP at sub-stoichiometric levels. These results refute the hypothesis that protein X is required to mediate dominant inhibition of prion propagation, and suggest that PrP molecules compete for binding to a nascent seeding site on newly formed PrP^Sc molecules, most likely through an epitope containing residue 172.     

Pathologic Prion Protein Infects Cells by Lipid-Raft Dependent Macropinocytosis

Transmissible spongiform encephalopathies, including variant-Creutzfeldt-Jakob disease (vCJD) in humans and bovine spongiform encephalopathies in cattle, are fatal neurodegenerative disorders characterized by protein misfolding of the host cellular prion protein (PrP^C) to the infectious scrapie form (PrP^Sc). However, the mechanism that exogenous PrP^Sc infects cells and where pathologic conversion of PrP^C to the PrP^Sc form occurs remains uncertain. Here we report that similar to the mechanism of HIV-1 TAT-mediated peptide transduction, processed mature, full length PrP contains a conserved N-terminal cationic domain that stimulates cellular uptake by lipid raft-dependent, macropinocytosis. Inhibition of macropinocytosis by three independent means prevented cellular uptake of recombinant PrP; however, it did not affect recombinant PrP cell surface association. In addition, fusion of the cationic N-terminal PrP domain to a Cre recombinase reporter protein was sufficient to promote both cellular uptake and escape from the macropinosomes into the cytoplasm. Inhibition of macropinocytosis was sufficient to prevent conversion of PrP^C to the pathologic PrP^Sc form in N2a cells exposed to strain RML PrP^Sc infected brain homogenates, suggesting that a critical determinant of PrP^C conversion occurs following macropinocytotic internalization and not through mere membrane association. Taken together, these observations provide a cellular mechanism that exogenous pathological PrP^Sc infects cells by lipid raft dependent, macropinocytosis.     

Antibodies to a Nonconjugated Prion Protein Peptide 95-123 Interfere with PrP<Superscript><Emphasis Type="Bold">Sc</Emphasis></Superscript> Propagation in Prion-Infected Cells

Summary 1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP^Sc propagation in prion-infected cells. 2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP^Sc) and normal prion protein (PrP^c), respectively. Sera positive in all tests were chosen for PrP^Sc inhibition studies in cell culture. 3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95–123 led to a transient reduction of PrP^Sc levels in persistently prion-infected cells. 4. Thus, anti-PrP antibodies interfering with PrP^Sc propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95–123 for the treatment of prion diseases.     

Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence

Prion diseases are associated with the misfolding of the host-encoded cellular prion protein (PrP^C) into a disease associated form (PrP^Sc). Recombinant PrP can be refolded into either an α-helical rich conformation (α-PrP) resembling PrP^C or a β-sheet rich, protease resistant form similar to PrP^Sc. Here, we generated tetracysteine tagged recombinant PrP, folded this into α- or β-PrP and determined the levels of FlAsH fluorescence. Insertion of the tetracysteine tag at three different sites within the 91-111 epitope readily distinguished β-PrP from α-PrP upon FlAsH labeling. Labelling of tetracysteine tagged PrP in the α-helical form showed minimal fluorescence, whereas labeling of tagged PrP in the β-sheet form showed high fluorescence indicating that this region is exposed upon conversion. This highlights a region of PrP that can be implicated in the development of diagnostics and is a novel, protease free mechanism for distinguishing PrP^Sc from PrP^C. This technique may also be applied to any protein that undergoes conformational change and/or misfolding such as those involved in other neurodegenerative disorders including Alzheimer’s, Huntington’s and Parkinson’s diseases.     

Recombinant Prion Protein Refolded with Lipid and RNA Has the Biochemical Hallmarks of a Prion but Lacks In Vivo Infectivity

During prion infection, the normal, protease-sensitive conformation of prion protein (PrP^C) is converted via seeded polymerization to an abnormal, infectious conformation with greatly increased protease-resistance (PrP^Sc). In vitro, protein misfolding cyclic amplification (PMCA) uses PrP^Sc in prion-infected brain homogenates as an initiating seed to convert PrP^C and trigger the self-propagation of PrP^Sc over many cycles of amplification. While PMCA reactions produce high levels of protease-resistant PrP, the infectious titer is often lower than that of brain-derived PrP^Sc. More recently, PMCA techniques using bacterially derived recombinant PrP (rPrP) in the presence of lipid and RNA but in the absence of any starting PrP^Sc seed have been used to generate infectious prions that cause disease in wild-type mice with relatively short incubation times. These data suggest that lipid and/or RNA act as cofactors to facilitate the de novo formation of high levels of prion infectivity. Using rPrP purified by two different techniques, we generated a self-propagating protease-resistant rPrP molecule that, regardless of the amount of RNA and lipid used, had a molecular mass, protease resistance and insolubility similar to that of PrP^Sc. However, we were unable to detect prion infectivity in any of our reactions using either cell-culture or animal bioassays. These results demonstrate that the ability to self-propagate into a protease-resistant insoluble conformer is not unique to infectious PrP molecules. They suggest that the presence of RNA and lipid cofactors may facilitate the spontaneous refolding of PrP into an infectious form while also allowing the de novo formation of self-propagating, but non-infectious, rPrP-res.     

Binding of bovine T194A PrPC by PrPSc-specific antibodies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP^C) into an infectious, pathological conformation (PrP^Sc). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP^Sc-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP^C associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP^C that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP^C misfolding.     

Characterization of Conformation-dependent Prion Protein Epitopes

Whereas prion replication involves structural rearrangement of cellular prion protein (PrP^C), the existence of conformational epitopes remains speculative and controversial, and PrP transformation is monitored by immunoblot detection of PrP(27–30), a protease-resistant counterpart of the pathogenic scrapie form (PrP^Sc) of PrP. We now describe the involvement of specific amino acids in conformational determinants of novel monoclonal antibodies (mAbs) raised against randomly chimeric PrP. Epitope recognition of two mAbs depended on polymorphisms controlling disease susceptibility. Detection by one, referred to as PRC5, required alanine and asparagine at discontinuous mouse PrP residues 132 and 158, which acquire proximity when residues 126–218 form a structured globular domain. The discontinuous epitope of glycosylation-dependent mAb PRC7 also mapped within this domain at residues 154 and 185. In accordance with their conformational dependence, tertiary structure perturbations compromised recognition by PRC5, PRC7, as well as previously characterized mAbs whose epitopes also reside in the globular domain, whereas conformation-independent epitopes proximal or distal to this region were refractory to such destabilizing treatments. Our studies also address the paradox of how conformational epitopes remain functional following denaturing treatments and indicate that cellular PrP and PrP(27–30) both renature to a common structure that reconstitutes the globular domain.     

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 μg/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.     

In Vivo Generation of Neurotoxic Prion Protein: Role for Hsp70 in Accumulation of Misfolded Isoforms

Prion diseases are incurable neurodegenerative disorders in which the normal cellular prion protein (PrP^C) converts into a misfolded isoform (PrP^Sc) with unique biochemical and structural properties that correlate with disease. In humans, prion disorders, such as Creutzfeldt-Jakob disease, present typically with a sporadic origin, where unknown mechanisms lead to the spontaneous misfolding and deposition of wild type PrP. To shed light on how wild-type PrP undergoes conformational changes and which are the cellular components involved in this process, we analyzed the dynamics of wild-type PrP from hamster in transgenic flies. In young flies, PrP demonstrates properties of the benign PrP^C; in older flies, PrP misfolds, acquires biochemical and structural properties of PrP^Sc, and induces spongiform degeneration of brain neurons. Aged flies accumulate insoluble PrP that resists high concentrations of denaturing agents and contains PrP^Sc-specific conformational epitopes. In contrast to PrP^Sc from mammals, PrP is proteinase-sensitive in flies. Thus, wild-type PrP rapidly converts in vivo into a neurotoxic, protease-sensitive isoform distinct from prototypical PrP^Sc. Next, we investigated the role of molecular chaperones in PrP misfolding in vivo. Remarkably, Hsp70 prevents the accumulation of PrP^Sc-like conformers and protects against PrP-dependent neurodegeneration. This protective activity involves the direct interaction between Hsp70 and PrP, which may occur in active membrane microdomains such as lipid rafts, where we detected Hsp70. These results highlight the ability of wild-type PrP to spontaneously convert in vivo into a protease-sensitive isoform that is neurotoxic, supporting the idea that protease-resistant PrP^Sc is not required for pathology. Moreover, we identify a new role for Hsp70 in the accumulation of misfolded PrP. Overall, we provide new insight into the mechanisms of spontaneous accumulation of neurotoxic PrP and uncover the potential therapeutic role of Hsp70 in treating these devastating disorders.     

Oxidation of Helix-3 Methionines Precedes the Formation of PK Resistant PrPSc

While elucidating the peculiar epitope of the α-PrP mAb IPC2, we found that PrP^Sc exhibits the sulfoxidation of residue M213 as a covalent signature. Subsequent computational analysis predicted that the presence of sulfoxide groups at both Met residues 206 and 213 destabilize the α-fold, suggesting oxidation may facilitate the conversion of PrP^C into PrP^Sc. To further study the effect of oxidation on prion formation, we generated pAbs to linear PrP peptides encompassing the Helix-3 region, as opposed to the non-linear complexed epitope of IPC2. We now show that pAbs, whose epitopes comprise Met residues, readily detected PrP^C, but could not recognize most PrP^Sc bands unless they were vigorously reduced. Next, we showed that the α-Met pAbs did not recognize newly formed PrP^Sc, as is the case for the PK resistant PrP present in lines of prion infected cells. In addition, these reagents did not detect intermediate forms such as PK sensitive and partially aggregated PrPs present in infected brains. Finally, we show that PrP molecules harboring the pathogenic mutation E200K, which is linked to the most common form of familial CJD, may be spontaneously oxidized. We conclude that the oxidation of methionine residues in Helix-3 represents an early and important event in the conversion of PrP^C to PrP^Sc. We believe that further investigation into the mechanism and role of PrP oxidation will be central in finally elucidating the mechanism by which a normal cell protein converts into a pathogenic entity that causes fatal brain degeneration.     

The N-Terminal Sequence of Prion Protein Consists an Epitope Specific to the Abnormal Isoform of Prion Protein (PrPSc)

The conformation of abnormal prion protein (PrP^Sc) differs from that of cellular prion protein (PrP^C), but the precise characteristics of PrP^Sc remain to be elucidated. To clarify the properties of native PrP^Sc, we attempted to generate novel PrP^Sc-specific monoclonal antibodies (mAbs) by immunizing PrP-deficient mice with intact PrP^Sc purified from bovine spongiform encephalopathy (BSE)-affected mice. The generated mAbs 6A12 and 8D5 selectivity precipitated PrP^Sc from the brains of prion-affected mice, sheep, and cattle, but did not precipitate PrP^C from the brains of healthy animals. In histopathological analysis, mAbs 6A12 and 8D5 strongly reacted with prion-affected mouse brains but not with unaffected mouse brains without antigen retrieval. Epitope analysis revealed that mAbs 8D5 and 6A12 recognized the PrP subregions between amino acids 31–39 and 41–47, respectively. This indicates that a PrP^Sc-specific epitope exists in the N-terminal region of PrP^Sc, and mAbs 6A12 and 8D5 are powerful tools with which to detect native and intact PrP^Sc. We found that the ratio of proteinase K (PK)-sensitive PrP^Sc to PK-resistant PrP^Sc was constant throughout the disease time course.     

Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells without cell lysis and proteinase K treatment

Prion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrP^Sc). PrP^Sc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrP^C) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrP^Sc (PrP^Sc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrP^Sc (PrP^Sc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrP^Sc can be directly detected from cells persistently infected with prions using anti-PrP monoclonal antibody (mAb) 132 that recognizes epitope consisting of mouse PrP amino acids 119–127. The novel cell-based ELISA could distinguish prion-infected cells from prion-uninfected cells without cell lysis and PK treatment. MAb 132 could detect both PrP^Sc-sen and PrP^Sc-res even if all PrP^Sc molecules were not detected. The analytical dynamic range for PrP^Sc detection was approximately 1 log. The coefficient of variation and signal-to-background ratio were 7%–11% and 2.5–3.3, respectively, demonstrating the reproducibility of this assay. The addition of a cytotoxicity assay immediately before PrP^Sc detection did not affect the following PrP^Sc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrP^Sc detection were completed in the same plate. The simplicity and non-requirement for cell lysis or PK treatment are advantages for the high throughput screening of anti-prion compounds.     

Molecular model of an alpha-helical prion protein dimer and its monomeric subunits as derived from chemical cross-linking and molecular modeling calculations

Prions are the agents of a series of lethal neurodegenerative diseases. They are composed largely, if not entirely, of the host-encoded prion protein (PrP), which can exist in the cellular isoform PrP^C and the pathological isoform PrP^Sc. The conformational change of the α-helical PrP^C into β-sheet-rich PrP^Sc is the fundamental event of prion disease. The transition of recombinant PrP from a PrP^C-like into a PrP^Sc-like conformation can be induced in vitro by submicellar concentrations of SDS. An α-helical dimer was identified that might represent either the native state of PrP^C or the first step from the monomeric PrP^C to highly aggregated PrP^Sc. In the present study, the molecular structure of these dimers was analyzed by introducing covalent cross-links using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Inter- and intramolecular bonds between directly neighboured amino groups and carboxy groups were generated. The bonds formed in PrP dimers of recombinant PrP (90-231) were identified by tryptic digestion and subsequent mass spectrometric analysis. Intra- and intermolecular cross-links between N-terminal glycine and three acidic amino acid side chains in the globular part of PrP were identified, showing the N-terminal amino acids (90-124) are not as flexible as known from NMR analysis. When the cross-linked sites were used as structural constraint, molecular modeling calculations yielded a structural model for PrP dimer and its monomeric subunit, including the folding of amino acids 90-124 in addition to the known structure. Molecular dynamics of the structure after release of the constraint indicated an intrinsic stability of the domain of amino acids 90-124.     

The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt–Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.     

PrPSc-specific antibodies do not induce prion disease or misfolding of PrPC in highly susceptible Tga20 mice

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders caused by misfolding of a cellular protein PrP^C into an infectious conformation PrP^Sc. Previously our group demonstrated induction of PrP^Sc-specific antibodies with a SN6b vaccine that targets regions of the protein that are exposed upon misfolding. There are concerns that these antibodies could function as templates to promote misfolding and cause disease. To evaluate the consequences of prolonged exposure to PrP^Sc-specific antibodies in a prion sensitized animal, tga20 mice were vaccinated with the SN6b vaccine. No clinical signs of disease were detected up to 255 d post-vaccination, and postmortem assay of brains and spleens revealed no proteinase-K resistant PrP. These results suggest that vaccinating against TSEs with the SN6b antigen is safe from the standpoint of prion disease induction.     

PrPST,a Soluble,Protease Resistant and Truncated PrP Form Features in the Pathogenesis of a Genetic Prion Disease

While the conversion of PrP^C into PrP^Sc in the transmissible form of prion disease requires a preexisting PrP^Sc seed, in genetic prion disease accumulation of disease related PrP could be associated with biochemical and metabolic modifications resulting from the designated PrP mutation. To investigate this possibility, we looked into the time related changes of PrP proteins in the brains of TgMHu2ME199K/wt mice, a line modeling for heterozygous genetic prion disease linked to the E200K PrP mutation. We found that while oligomeric entities of mutant E199KPrP exist at all ages, aggregates of wt PrP in the same brains presented only in advanced disease, indicating a late onset conversion process. We also show that most PK resistant PrP in TgMHu2ME199K mice is soluble and truncated (PrP^ST), a pathogenic form never before associated with prion disease. We next looked into brain samples from E200K patients and found that both PK resistant PrPs, PrP^ST as in TgMHu2ME199K mice, and “classical” PrP^Sc as in infectious prion diseases, coincide in the patient''s post mortem brains. We hypothesize that aberrant metabolism of mutant PrPs may result in the formation of previously unknown forms of the prion protein and that these may be central for the fatal outcome of the genetic prion condition.