PrP Sc Molecules Research Articles

Conformational diversity in purified prions produced in vitro.

Prion diseases are caused by misfolding of either wild-type or mutant forms of the prion protein (PrP) into self-propagating, pathogenic conformers, collectively termed PrPSc. Both wild-type and mutant PrPSc molecules exhibit conformational diversity in vivo, but purified prions generated by the serial protein misfolding cyclic amplification (sPMCA) technique do not display this same diversity in vitro. This discrepancy has left a gap in our understanding of how conformational diversity arises at the molecular level in both types of prions. Here, we use continuous shaking instead of sPMCA to generate conformationally diverse purified prions in vitro. Using this approach, we show for the first time that wild type prions initially seeded by different native strains can propagate as metastable PrPSc conformers with distinguishable strain properties in purified reactions containing a single active cofactor. Propagation of these metastable PrPSc conformers requires appropriate shaking conditions, and changes in these conditions cause all the different PrPSc conformers to converge irreversibly into the same single conformer as that produced in sPMCA reactions. We also use continuous shaking to show that two mutant PrP molecules with different pathogenic point mutations (D177N and E199K) adopt distinguishable PrPSc conformations in reactions containing pure protein substrate without cofactors. Unlike wild-type prions, the conformations of mutant prions appear to be dictated by substrate sequence rather than seed conformation. Overall, our studies using purified substrates in shaking reactions show that wild-type and mutant prions use fundamentally different mechanisms to generate conformational diversity at the molecular level.

Prion receptors, prion internalization, intra- and inter-cellular transport.

Cellular prion protein (PrP C ) is a membrane bound protein that undergoes several post translational modifications. It can exert pleiotropic functions either in physiological and pathological conditions. The disease-causing isoform of the prion protein, called PrP Sc , acts as corruptive seed able to establish a process of misfolding and aggregation of further PrP Sc molecules. Therefore, PrP C acts as substrate for PrP Sc formation and as receptor to convey neurotoxic functions typical of other prion-like proteins involved in several neurodegenerative conditions. PrP C is transported from the outer leaflet of the cellular membrane to the intracellular compartments through several endocytic pathways, including clathrin-dependent and independent mechanisms and interactions with different adaptor proteins. During prion infection, the pathological counterpart PrP Sc , can be uptaken by the cells with mechanisms partially overlapping the ones of the physiological form, PrP C . The intercellular routing of both prion protein isoforms involves the release through vesicles and tunneling nanotubes. From the extracellular milieu, PrP C and PrP Sc can be uptaken by adjacent cells, thus exerting physiological and pathological functions.

Distinct conformers of amyloid beta accumulate in the neocortex of patients with rapidly progressive Alzheimer's disease

Amyloid beta (Aβ) deposition in the neocortex is a major hallmark of Alzheimer's disease (AD), but the extent of deposition does not readily explain phenotypic diversity and rate of disease progression. The prion strain–like model of disease heterogeneity suggests the existence of different conformers of Aβ. We explored this paradigm using conformation-dependent immunoassay (CDI) for Aβ and conformation-sensitive luminescent conjugated oligothiophenes (LCOs) in AD cases with variable progression rates. Mapping the Aβ conformations in the frontal, occipital, and temporal regions in 20 AD patients with CDI revealed extensive interindividual and anatomical diversity in the structural organization of Aβ with the most significant differences in the temporal cortex of rapidly progressive AD. The fluorescence emission spectra collected in situ from Aβ plaques in the same regions demonstrated considerable diversity of spectral characteristics of two LCOs—quatroformylthiophene acetic acid and heptaformylthiophene acetic acid. Heptaformylthiophene acetic acid detected a wider range of Aβ deposits, and both LCOs revealed distinct spectral attributes of diffuse and cored plaques in the temporal cortex of rapidly and slowly progressive AD and less frequent and discernible differences in the frontal and occipital cortex. These and CDI findings indicate a major conformational diversity of Aβ accumulating in the neocortex, with the most notable differences in temporal cortex of cases with shorter disease duration, and implicate distinct Aβ conformers (strains) in the rapid progression of AD.

In Vitro Seeding Activity of Glycoform-Deficient Prions from Variably Protease-Sensitive Prionopathy and Familial CJD Associated with PrPV180I Mutation

Both sporadic variably protease-sensitive prionopathy (VPSPr) and familial Creutzfeldt-Jakob disease linked to the prion protein (PrP) V180I mutation (fCJDV180I) have been found to share a unique pathological prion protein (PrPSc) that lacks the protease-resistant PrPSc glycosylated at residue 181 because two of four PrP glycoforms are apparently not converted into the PrPSc from their cellular PrP (PrPC). To investigate the seeding activity of these unique PrPSc molecules, we conducted in vitro prion conversion experiments using serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays with different PrPC substrates. We observed that the seeding of PrPSc from VPSPr or fCJDV180I in the sPMCA reaction containing normal human or humanized transgenic (Tg) mouse brain homogenates generated PrPSc molecules that unexpectedly exhibited a dominant diglycosylated PrP isoform along with PrP monoglycosylated at residue 181. The efficiency of PrPSc amplification was significantly higher in non-CJDMM than in non-CJDVV human brain homogenate, whereas it was higher in normal TgVV than in TgMM mouse brain homogenate. PrPC from the mixture of normal TgMM and Tg mouse brain expressing PrPV180I mutation (Tg180) but not TgV180I alone was converted into PrPSc by seeding with the VPSPr or fCJDV180I. The RT-QuIC seeding activity of PrPSc from VPSPr and fCJDV180I was significantly lower than that of sCJD. Our results suggest that the formation of glycoform-selective prions may be associated with an unidentified factor in the affected brain and the glycoform-deficiency of PrPSc does not affect the glycoforms of in vitro newly amplified PrPSc.

Pharmacological Agents Targeting the Cellular Prion Protein.

Prion diseases are associated with the conversion of the cellular prion protein (PrPC), a glycoprotein expressed at the surface of a wide variety of cell types, into a misfolded conformer (the scrapie form of PrP, or PrPSc) that accumulates in brain tissues of affected individuals. PrPSc is a self-catalytic protein assembly capable of recruiting native conformers of PrPC, and causing their rearrangement into new PrPSc molecules. Several previous attempts to identify therapeutic agents against prion diseases have targeted PrPSc, and a number of compounds have shown potent anti-prion effects in experimental models. Unfortunately, so far, none of these molecules has successfully been translated into effective therapies for prion diseases. Moreover, mounting evidence suggests that PrPSc might be a difficult pharmacological target because of its poorly defined structure, heterogeneous composition, and ability to generate different structural conformers (known as prion strains) that can elude pharmacological intervention. In the last decade, a less intuitive strategy to overcome all these problems has emerged: targeting PrPC, the common substrate of any prion strain replication. This alternative approach possesses several technical and theoretical advantages, including the possibility of providing therapeutic effects also for other neurodegenerative disorders, based on recent observations indicating a role for PrPC in delivering neurotoxic signals of different misfolded proteins. Here, we provide an overview of compounds claimed to exert anti-prion effects by directly binding to PrPC, discussing pharmacological properties and therapeutic potentials of each chemical class.

Quick and sensitive SPR detection of prion disease-associated isoform (PrPSc) based on its self-assembling behavior on bare gold film and specific interactions with aptamer-graphene oxide (AGO)

Herein, we constructed a novel sandwich surface plasmon resonance (SPR) detection assay for sensitive prion disease-associated isoform (PrPSc) detection, utilizing bare gold film and apatamer-graphene oxide (AGO). Due to the self-assembling behavior of PrPSc on gold surface, the non-modified gold surface can be directly used as sensing surface for the quick detection, for the purpose to avoid the interference from the traditional, complex and changeable probe-modified sensing surface. And due to the highly specific affinity of AGO towards PrPSc, the sandwich type SPR sensor exhibits excellent analytical performance towards the discrimination and quantitation of PrPSc. A good linear relationship was obtained between SPR responses and the logarithm of PrPSc concentrations over a range of 0.001–1ng/mL. The detection sensitivity for PrPSc was improved by ∼156 orders of AGO compared with SPR direct detection format. Besides, morphological changes of the sensing film surfaces were investigated by high resolution AFM imaging, confirming the capture of PrPSc molecules and their further specific recognition by AGO. The specificity of the present biosensor was also investigated by PrPC and other regents as controls. By compared with other reported methods, the AGO enhanced sandwich SPR assay was confirmed to be efficient, sensitive, and with wide working range.

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

ABSTRACTPrion-infected cells have been used for analyzing the effect of compounds on the formation of abnormal isoform of prion protein (PrPSc). PrPSc is usually detected using anti-prion protein (PrP) antibodies after the removal of the cellular isoform of prion protein (PrPC) by proteinase K (PK) treatment. However, it is expected that the PK-sensitive PrPSc (PrPSc-sen), which possesses higher infectivity and conversion activity than the PK-resistant PrPSc (PrPSc-res), is also digested through PK treatment. To overcome this problem, we established a novel cell-based ELISA in which PrPSc 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 PrPSc-sen and PrPSc-res even if all PrPSc molecules were not detected. The analytical dynamic range for PrPSc 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 PrPSc detection did not affect the following PrPSc detection. Thus, all the procedures including cell culture, cytotoxicity assay, and PrPSc 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.

Re-infection of the prion from the scrapie‑infected cell line SMB-S15 in three strains of mice, CD1, C57BL/6 and Balb/c.

It is well known that the SMB-S15 cell line was originally established by cultures from the brains of mice affected by the Chandler scrapie strain, and this cell line may express PrPSc permanently. However, the infectivity of the S15-derived prions on experimental animals has not yet been well documented. In the present study, the cell lysates of SMB-S15 were intracerebrally inoculated into three different strains of mice, namely C57BL/6, Balb/c and CD1. Prion protein (PRNP) gene sequencing revealed the same encoded PrP proteins in the sequences of amino acids in the three strains of mice, in addition to a synonymous single nucleotide polymorphism (SNP) in CD1 mice. All infected mice developed typical experimental transmissible spongiform encephalopathies (TSEs) approximately six months post-infection. The clinical features of three infected mice were comparable. The pathogenic characteristics, such as the electrophoretic and glycosylation profiles and proteinase K (PK) resistance of PrPSc molecules, as well as the neuropathological characteristics, such as spongiform vacuolation, PrPSc deposits in cortex regions, astrogliosis and activated microglia, were also similar in all three strains of infected mice. However, PrPSc deposits in the cerebellums of CD1 mice were significantly fewer, which was linked with the observation that lower numbers of CD1 mice presented cerebellum-associated symptoms. Successive inoculation of the individual strains of mice with brain homogenates from the infected mice also induced typical experimental scrapie. The data in the present study thus confirm that the prion agent in SMB-S15 cells causes stable infectivity in different types of mice with distinct phenotypes after long-term propagation in vitro. The present study also provides further scrapie rodent models, which may be used in further studies.

Peripherally administrated prions reach the brain at sub-infectious quantities in experimental hamsters

The mechanisms implicated in prion infection and tissue distribution are not completely understood. In this study we investigated the levels of 263K prions in brain and spleen of Syrian hamsters few days after intra-peritoneal challenge. For this purpose we utilized the PMCA technology which permits to detect as little as few PrPSc molecules. Our results show that peripherally administered prions directly reach the brain, although at levels below the minimum necessary to produce disease. PrPSc remains in the brain several days after administration suggesting inefficient clearance or early replication. Understanding the fate of the infectious agent after administration and its uptake in different organs and fluids may provide useful information to develop strategies to minimize further spreading of prion diseases.

Anti-prion activity of a panel of aromatic chemical compounds: in vitro and in silico approaches.

The prion protein (PrP) is implicated in the Transmissible Spongiform Encephalopathies (TSEs), which comprise a group of fatal neurodegenerative diseases affecting humans and other mammals. Conversion of cellular PrP (PrPC) into the scrapie form (PrPSc) is the hallmark of TSEs. Once formed, PrPSc aggregates and catalyzes PrPC misfolding into new PrPSc molecules. Although many compounds have been shown to inhibit the conversion process, so far there is no effective therapy for TSEs. Besides, most of the previously evaluated compounds failed in vivo due to poor pharmacokinetic profiles. In this work we propose a combined in vitro/in silico approach to screen for active anti-prion compounds presenting acceptable drugability and pharmacokinetic parameters. A diverse panel of aromatic compounds was screened in neuroblastoma cells persistently infected with PrPSc (ScN2a) for their ability to inhibit PK-resistant PrP (PrPRes) accumulation. From ∼200 compounds, 47 were effective in decreasing the accumulation of PrPRes in ScN2a cells. Pharmacokinetic and physicochemical properties were predicted in silico, allowing us to obtain estimates of relative blood brain barrier permeation and mutagenicity. MTT reduction assays showed that most of the active compounds were non cytotoxic. Compounds that cleared PrPRes from ScN2a cells, were non-toxic in the MTT assay, and presented a good pharmacokinetic profile were investigated for their ability to inhibit aggregation of an amyloidogenic PrP peptide fragment (PrP109–149). Molecular docking results provided structural models and binding affinities for the interaction between PrP and the most promising compounds. In summary, using this combined in vitro/in silico approach we have identified new small organic anti-scrapie compounds that decrease the accumulation of PrPRes in ScN2a cells, inhibit the aggregation of a PrP peptide, and possess pharmacokinetic characteristics that support their drugability. These compounds are attractive candidates for prion disease therapy.

Fate of pathological prion (PrPsc92–138) in soil and water: prion-clay nanoparticle molecular dynamics

Pathogenic prion protein scrapie (PrPsc) may contaminate soils for decades and remain in water in colloidal suspension, providing infection pathways for animals through the inhalation of ingested dust and soil particles, and drinking water. We used molecular dynamics simulations to understand the strong binding mechanism of this pathogenic peptide with clay mineral surfaces and compared our results to experimental works. We restricted our model to the moiety PrP(92–138), which is a portion of the whole PrPsc molecule responsible for infectivity and modeled it using explicit solvating water molecules in contact with a pyrophyllite cleavage plane. Pyrophyllite is taken as a model for common soil clay, but it has no permanent structural charge. However, partial residual negative charges occur on the cleavage plane slab surface due to a slab charge unbalance. The charge is isotropic in 2D and it was balanced with K+ ions. After partially removing potassium ions, the peptide anchors to the clay surface via up to 10 hydrogen bonds, between protonated lysine or histidine residues and the oxygen atoms of the siloxane cavities. Our results provide insight to the mechanism responsible for the strong association between the PrPsc peptide and clay nanoparticles and the associations present in contaminated soil and water which may lead to the infection of animals.

Neurodegeneration Induced by Clustering of Sialylated Glycosylphosphatidylinositols of Prion Proteins

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrP(Sc)). The mechanisms by which PrP(Sc) accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons, the addition of PrP(Sc) alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A(2) (cPLA(2)), and triggering synapse damage. These effects of PrP(Sc) are dependent upon its glycosylphosphatidylinositol (GPI) anchor, suggesting that it is the increased density of GPIs that occurs following the aggregation of PrP(Sc) molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrP(C)) also increased membrane cholesterol, activated cPLA(2), and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1, another GPI-anchored protein, and were dependent on the GPI anchor attached to PrP(C) containing two acyl chains and sialic acid. We propose that the aggregation of PrP(Sc), or the cross-linkage of PrP(C), causes the clustering of sialic acid-containing GPI anchors at high densities, resulting in altered membrane composition, the pathological activation of cPLA(2), and synapse damage.

Allelic origin of protease-sensitive and protease-resistant prion protein isoforms in Gerstmann-Sträussler-Scheinker disease with the P102L mutation.

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited prion disease associated with point mutations in the Prion Protein gene. The most frequent mutation associated with GSS involves a proline-to-leucine substitution at residue 102 of the prion protein, and is characterized by marked variability at clinical, pathological and molecular levels. Previous investigations of GSS P102L have shown that disease-associated pathological prion protein, or PrPSc, consists of two main conformers, which under exogenous proteolysis generates a core fragment of 21 kDa and an internal fragment of 8 kDa. Both conformers are detected in subjects with spongiform degeneration, whereas only the 8 kDa fragment is recovered in cases lacking spongiosis. Several studies have reported an exclusive derivation of protease-resistant PrPSc isoforms from the mutated allele; however, more recently, the propagation of protease-resistant wild-type PrPSc has been described. Here we analyze the molecular and pathological phenotype of six GSS P102L cases characterized by the presence of 21 and 8 kDa PrP fragments and two subjects with only the 8 kDa PrP fragment. Using sensitive protein separation techniques and Western blots with antibodies differentially recognizing wild-type and mutant PrP we observed a range of PrPSc allelic conformers, either resistant or sensitive to protease treatment in all investigated subjects. Additionally, tissue deposition of protease-sensitive wild-type PrPSc molecules was seen by conventional PrP immunohistochemistry and paraffin-embedded tissue blot. Our findings enlarge the spectrum of conformational allelic PrPSc quasispecies propagating in GSS P102L thus providing a molecular support to the spectrum of disease phenotypes, and, in addition, impact the diagnostic role of PrP immunohistochemistry in prion diseases.

Seeding Specificity and Ultrastructural Characteristics of Infectious Recombinant Prions

Infectious mouse prions can be produced from a mixture of bacterially expressed recombinant prion protein (recPrP), palmitoyloleoylphosphatidylglycerol (POPG), and RNA [Wang, F.; et al. (2010) Science 327, 1132]. In contrast, amyloid fibers produced from pure recPrP without POPG or RNA (recPrP fibers) fail to infect wild type mice [Colby, D.W.; et al. (2010) PLoS Pathog. 387, e1000736]. We compared the seeding specificity and ultrastructural features of infectious recombinant prions (recPrP(Sc)) with those of recPrP fibers. Our results indicate that PrP fibers are not able to induce the formation of PrP(Sc) molecules from wild type mouse brain homogenate substrate in serial protein misfolding cyclic amplification (sPMCA) reactions. Conversely, recPrP(Sc) molecules did not accelerate the formation of amyloid in vitro, under conditions that produce recPrP fibers spontaneously. Ultrastructurally, recombinant prions appear to be small spherical aggregates rather than elongated fibers, as determined by atomic force and electron microscopy. Taken together, our results show that recPrP(Sc) molecules and PrP fibers have different ultrastructural features and seeding specificities, suggesting that prion infectivity may be propagated by a specific and unique assembly pathway facilitated by cofactors.

In Situ Photodegradation of Incorporated Polyanion Does Not Alter Prion Infectivity

Single-stranded polyanions ≥40 bases in length facilitate the formation of hamster scrapie prions in vitro, and polyanions co-localize with PrPSc aggregates in vivo [1], [2]. To test the hypothesis that intact polyanionic molecules might serve as a structural backbone essential for maintaining the infectious conformation(s) of PrPSc, we produced synthetic prions using a photocleavable, 100-base oligonucleotide (PC-oligo). In serial Protein Misfolding Cyclic Amplification (sPMCA) reactions using purified PrPC substrate, PC-oligo was incorporated into physical complexes with PrPSc molecules that were resistant to benzonase digestion. Exposure of these nuclease-resistant prion complexes to long wave ultraviolet light (315 nm) induced degradation of PC-oligo into 5 base fragments. Light-induced photolysis of incorporated PC-oligo did not alter the infectivity of in vitro-generated prions, as determined by bioassay in hamsters and brain homogenate sPMCA assays. Neuropathological analysis also revealed no significant differences in the neurotropism of prions containing intact versus degraded PC-oligo. These results show that polyanions >5 bases in length are not required for maintaining the infectious properties of in vitro-generated scrapie prions, and indicate that such properties are maintained either by short polyanion remnants, other co-purified cofactors, or by PrPSc molecules alone.

Correlation of Polydispersed Prion Protein and Characteristic Pathology in the Thalamus in Variant Creutzfeldt‐Jakob Disease: Implication of Small Oligomeric Species

The vacuolation, neuronal loss and gliosis that characterize human prion disease pathology are accompanied by the accumulation of an aggregated, insoluble and protease-resistant form (termed PrP(Sc)) of the host-encoded normal cellular prion protein (PrP(C)). In variant Creutzfeldt-Jakob disease the frontal cortex and cerebellum exhibit intense vacuolation and the accumulation of PrP(Sc) in the form of amyloid plaques and plaque-like structures. In contrast the posterior thalamus is characterized by intense gliosis and neuronal loss, but PrP(Sc) plaques are rare and vacuolation is patchy. We have used sucrose density gradient centrifugation coupled with conformation dependent immunoassay to examine the biochemical properties of the PrP(Sc) that accumulates in these different brain regions. The results show a greater degree of PrP(Sc) polydisperal in thalamus compared with frontal cortex or cerebellum, including a subpopulation PrP(Sc) molecules in the thalamus that have sedimentation properties resembling those of PrP(C). Much effort has focused on identifying aspects of PrP(Sc) biochemistry that distinguish between different forms of human prion disease and contribute to differential diagnosis. Here we show that PrP(Sc) sedimentation properties, which can depend on aggregation state, correlate with, and may underlie the distinct neurodegenerative processes occurring in different regions of the variant Creutzfeldt-Jakob disease brain.

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.

Endogenous Proteolytic Cleavage of Disease-associated Prion Protein to Produce C2 Fragments Is Strongly Cell- and Tissue-dependent

The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions. We found the following: (i) the full-length to C2 ratio varies considerably depending on the infected cell or tissue. Thus, in primary neurons and brain tissue, PrP(Sc) accumulated predominantly as untrimmed species, whereas efficient trimming occurred in Rov and MovS cells, and in spleen tissue. (ii) Although C2 is generally considered to be the counterpart of the PrP(Sc) proteinase K-resistant core, the N termini of the fragments cleaved in vivo and in vitro can actually differ, as evidenced by a different reactivity toward the Pc248 anti-octarepeat antibody. (iii) In lysosome-impaired cells, the ratio of full-length versus C2 species dramatically increased, yet efficient prion propagation could occur. Moreover, cathepsin but not calpain inhibitors markedly inhibited C2 formation, and in vitro cleavage by cathepsins B and L produced PrP(Sc) fragments lacking the Pc248 epitope, strongly arguing for the primary involvement of acidic hydrolases of the endolysosomal compartment. These findings have implications on the molecular analysis of PrP(Sc) and cell pathogenesis of prion infection.

Complex Polyamines: Unique Prion Disaggregating Compounds

Among the candidate anti-prion chemotherapeutic agents identified to date, complex polyamines constitute the only class of compounds that possess the ability to remove pre-existing PrP(Sc) molecules from infected cells. The potency of branched polyamines such as cationic dendrimers increases with the density of positive charges on their surface. Cationic dendrimers appear to accumulate together with PrP(Sc) molecules in lysosomes, where the acidic environment facilitates dendrimer-mediated PrP(Sc) disaggregation. Dendrimers can disaggregate a range of different amyloid proteins by interacting with specific epitopes on each protein. Studies with model peptides suggest that dendrimers may cause fiber breakage and capping of elongating fibers. Potential limitations to the development of dendrimers as therapeutic compounds for neurodegenerative disorders of protein misfolding such as prion diseases include poor bioavailability, limited spectrum of activity, and detrimental neurological side effects. A related group of compounds, lipopolyamines, are smaller molecules containing a lipophilic tail that may assist membrane targeting. Developing strategies to enable the safe delivery of potent complex polyamines to the central nervous system represents a critical avenue for future research.

Human Prion Protein (PrP) 219K Is Converted to PrPSc but Shows Heterozygous Inhibition in Variant Creutzfeldt-Jakob Disease Infection

Prion protein gene (PRNP) E219K is a human polymorphism commonly occurring in Asian populations but is rarely found in patients with sporadic Creutzfeldt-Jakob disease (CJD). Thus the polymorphism E219K has been considered protective against sporadic CJD. The corresponding mouse prion protein (PrP) polymorphism variant (mouse PrP 218K) is not converted to the abnormal isoform (PrP(Sc)) and shows a dominant negative effect on wild-type PrP conversion. To define the conversion activity of this human molecule, we herein established knock-in mice with human PrP 219K and performed a series of transmission experiments with human prions. Surprisingly, the human PrP 219K molecule was converted to PrP(Sc) in variant CJD infection, and the conversion occurred more efficiently than PrP 219E molecule. Notably the knock-in mice with PRNP codon 219E/K showed the least efficient conversion compared with their hemizygotes with PRNP codon 219E/0 or codon 219K/0, or homozygotes with PRNP codon 219E/E or codon 219K/K. This phenomenon indicated heterozygous inhibition. This heterozygous inhibition was observed also in knock-in mice with PRNP codon 129M/V genotype. In addition to variant CJD infection, the human PrP 219K molecule is conversion-competent in transmission experiments with sporadic CJD prions. Therefore, the protective effect of PRNP E219K against sporadic CJD might be due to heterozygous inhibition.