Anti-prion activity found in beetle grub hemolymph of Trypoxylus dichotomus septentrionalis

No remedies for prion disease have been established, and the conversion of normal to abnormal prion protein, a key event in prion disease, is still unclear. Here we found that substances in beetle grub hemolymph, after they were browned by aging for a month or heating for hours, reduced abnormal prion protein (PrP) levels in RML prion-infected cells. Active anti-prion components in the hemolymph were resistant to protease treatment and had molecular weights larger than 100 kDa. Aminoguanidine treatment of the hemolymph abolished its anti-prion activity, suggesting that Maillard reaction products are enrolled in the activity against the RML prion. However, levels of abnormal PrP in RML prion-infected cells were not decreased by incubation with the Maillard reaction products formed by amino acids or bovine serum albumin. The anti-prion components in the hemolymph modified neither cellular or cell-surface PrP levels nor lipid raft or autophagosome levels. The anti-prion activity was not observed in cells infected with 22 L prion or Fukuoka-1 prion, suggesting the anti-prion action is prion strain-dependent. Although the active components of the hemolymph need to be further evaluated, the present findings imply that certain specific chemical structures in the hemolymph, but not chemical structures common to all Maillard reaction products, are involved in RML prion formation or turnover, without modifying normal PrP expression. The anti-prion components in the hemolymph are a new tool for elucidating strain-dependent prion biology.     

Treatment of transmissible spongiform encephalopathy by intraventricular drug infusion in animal models

The therapeutic efficacy of direct drug infusion into the brain, the target organ of transmissible spongiform encephalopathies, was assessed in transgenic mice intracerebrally infected with 263K scrapie agent. Pentosan polysulfate (PPS) gave the most dramatic prolongation of the incubation period, and amphotericin B had intermediate effects, but antimalarial drugs such as quinacrine gave no significant prolongation. Treatment with the highest dose of PPS at an early or late stage of the infection prolonged the incubation time by 2.4 or 1.7 times that of the control mice, respectively. PPS infusion decreased not only abnormal prion protein deposition but also neurodegenerative changes and infectivity. These alterations were observed within the brain hemisphere fitted with an intraventricular infusion cannula but not within the contralateral hemisphere, even at the terminal disease stage long after the infusion had ended. Therapeutic effects of PPS were also demonstrated in mice infected with either RML agent or Fukuoka-1 agent. However, at doses higher than that providing the maximal effects, intraventricular PPS infusion caused adverse effects such as hematoma formation in the experimental animals. These findings indicate that intraventricular PPS infusion might be useful for the treatment of transmissible spongiform encephalopathies in humans, providing that the therapeutic dosage is carefully evaluated.     

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.     

Striatal pathology underlies prion infection-mediated hyperactivity in mice

Although prion diseases are most commonly modeled using the laboratory mouse, the diversity of prion strains, behavioral testing and neuropathological assessments hamper our collective understanding of mouse models of prion disease. Here we compared several commonly used murine strains of prions in C57BL/6J female mice in a detailed home cage behavior detection system and a systematic study of pathological markers and neurotransmitter systems. We observed that mice inoculated with RML or 139A prions develop a severe hyperactivity phenotype in the home cage. A detailed assessment of pathology markers, such as microglial marker IBA1, astroglial marker GFAP and degeneration staining indicate early striatal pathology in mice inoculated with RML or 139A but not in those inoculated with 22L prions. An assessment of neuromodulatory systems including serotonin, dopamine, noradrenalin and acetylcholine showed surprisingly little decline in neuronal cell bodies or their innervations of regions controlling locomotor behavior, except for a small decrease in dopaminergic innervations of the dorsal striatum. These results implicate the dorsal striatum in mediating the major behavioral phenotype of 139A and RML prions. Further, they suggest that measurements of activity may be a sensitive manner in which to diagnose murine prion disease. With respect to neuropathology, our results indicate that pathological stains as opposed to neurotransmitter markers are much more informative and sensitive as markers of prion disease in mouse models.Key words: PrP, neurodegeneration, protein misfolding, home-cage, transmissible spongiform encephalopathy     

Effect of glycans and the glycophosphatidylinositol anchor on strain dependent conformations of scrapie prion protein: improved purifications and infrared spectra

Mammalian prion diseases involve conversion of normal prion protein, PrP(C), to a pathological aggregated state (PrP(res)). The three-dimensional structure of PrP(res) is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent β-sheet content. PrP(res) molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild-type PrP(res) with GPI- and glycan-deficient PrP(res) produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP(res) revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to β-sheets. These PrP(res) spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrP(res) were nearly indistinguishable. With ME7 PrP(res), modest differences between the wild-type and anchorless spectra were detected, notably an ～2 cm(-1) shift in an apparent β-sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP(res), at least relative to the differences observed between strains, but can subtly affect turns and certain β-sheet components. Recently reported H-D exchange analyses of anchorless PrP(res) preparations strongly suggested the presence of strain-dependent, solvent-inaccessible β-core structures throughout most of the C-terminal half of PrP(res) molecules, with no remaining α-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrP(res).     

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNP^Sc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNP^Sc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNP^Sc in addition to attenuation of microgliosis and neuroprotection.     

The aminoglycoside G418 hinders de novo prion infection in cultured cells

The study of prions and the discovery of candidate therapeutics for prion disease have been facilitated by the ability of prions to replicate in cultured cells. Paradigms in which prion proteins from different species are expressed in cells with low or no expression of endogenous prion protein (PrP) have expanded the range of prion strains that can be propagated. In these systems, cells stably expressing a PrP of interest are typically generated via coexpression of a selectable marker and treatment with an antibiotic. Here, we report the unexpected discovery that the aminoglycoside G418 (Geneticin) interferes with the ability of stably transfected cultured cells to become infected with prions. In G418-resistant lines of N2a or CAD5 cells, the presence of G418 reduced levels of protease-resistant PrP following challenge with the RML or 22L strains of mouse prions. G418 also interfered with the infection of cells expressing hamster PrP with the 263K strain of hamster prions. Interestingly, G418 had minimal to no effect on protease-resistant PrP levels in cells with established prion infection, arguing that G418 selectively interferes with de novo prion infection. As G418 treatment had no discernible effect on cellular PrP levels or its localization, this suggests that G418 may specifically target prion assemblies or processes involved in the earliest stages of prion infection.     

The stoichiometry of host PrPC glycoforms modulates the efficiency of PrPSc formation in vitro

A central event in the formation of infectious prions is the conformational change of a host-encoded glycoprotein, PrPC, into a pathogenic isoform, PrPSc. However, the molecular requirements for efficient PrP conversion remain unknown. In this study, we employed the recently developed protein misfolding cyclic amplification (PMCA) and scrapie cell assay (SCA) techniques to study the role of N-linked glycosylation on prion formation in vitro. The results show that unglycosylated PrPC molecules are required to propagate mouse RML prions, whereas diglycosylated PrPC molecules are required to propagate hamster Sc237 prions. Furthermore, the formation of Sc237 prions is inhibited by substoichiometric levels of hamster unglycosylated PrPC molecules. Thus, interactions between different PrPC glycoforms appear to control the efficiency of prion formation in a species-specific manner.     

Mefloquine, an antimalaria drug with antiprion activity in vitro, lacks activity in vivo

In view of the effectiveness of antimalaria drugs inhibiting abnormal protease-resistant prion protein (PrP-res) formation in scrapie agent-infected cells, we tested other antimalarial compounds for similar activity. Mefloquine (MF), a quinoline antimalaria drug, was the most active compound tested against RML and 22L mouse scrapie agent-infected cells, with 50% inhibitory concentrations of approximately 0.5 and approximately 1.2 microM, respectively. However, MF administered to mice did not delay the onset of intraperitoneally inoculated scrapie agent, the result previously observed with quinacrine. While most anti-scrapie agent compounds inhibit PrP-res formation in vitro, many PrP-res inhibitors have no activity in vivo. This underscores the importance of testing promising candidates in vivo.     

Biological and biochemical characteristics of prion strains conserved in persistently infected cell cultures

Abnormal prion protein (PrP(Sc)) plays a central role in the transmission of prion diseases, but the molecular basis of prion strains with distinct biological characteristics remains to be elucidated. We analyzed the characteristics of prion disease by using mice inoculated with the Chandler and Fukuoka-1 strains propagated in a cultured mouse neuronal cell line, GT1-7, which is highly permissive to replication of the infectious agents. Strain-specific biological characteristics, including clinical manifestations, incubation period as related to the infectious unit, and pathological profiles, remained unchanged after passages in the cell cultures. We noted some differences in the biochemical aspects of PrP(Sc) between brain tissues and GT1-7 cells which were unlikely to affect the strain phenotypes. On the other hand, the proteinase K-resistant PrP core fragments derived from Fukuoka-1-infected tissues and cells were slightly larger than those from Chandler-infected versions. Moreover, Fukuoka-1 infection, but not Chandler infection, gave an extra fragment with a low molecular weight, approximately 13 kDa, in both brain tissues and GT1-7 cells. This cell culture model persistently infected with different strains will provide a new insight into the understanding of the molecular basis of prion diversity.     

Chronic subclinical prion disease induced by low-dose inoculum

We have compared the transmission characteristics of the two mouse-adapted scrapie isolates, ME7 and Rocky Mountain Laboratory (RML), in tga20 mice. These mice express elevated levels of PrP protein compared to wild-type mice and display a relatively short disease incubation period following intracerebral prion inoculation. Terminal prion disease in tga20 mice induced by ME7 or RML was characterized by a distinct pattern of clinical signs and different incubation times. High-dose RML inoculated intracerebrally into tga20 mice induced the most rapid onset of clinical signs, with mice succumbing to terminal disease after only 58 +/- 3 days. In contrast, high-dose ME7 gave a mean time to terminal disease of 74 +/- 0 days. Histological examination of brain sections from prion-inoculated tga20 mice at terminal disease showed that ME7 gave rise to a more general and extensive pattern of vacuolation than RML. Low-dose inoculum failed to induce terminal disease but did cause preclinical symptoms, including the appearance of reversible clinical signs. Some mice oscillated between showing no clinical signs and early clinical signs for many months but never progressed to terminal disease. Brain tissue from these mice with chronic subclinical prion disease, sacrificed at >200 days postinoculation, contained high levels of infectivity and showed the presence of PrP(Sc). Parallel analysis of brain tissue from mice with terminal disease showed similar levels of infectivity and detectable PrP(Sc). These results show that high levels of infectivity and the presence of the abnormal isomer of PrP can be detected in mice with subclinical disease following low-dose prion inoculation.     

Accelerated high fidelity prion amplification within and across prion species barriers

Experimental obstacles have impeded our ability to study prion transmission within and, more particularly, between species. Here, we used cervid prion protein expressed in brain extracts of transgenic mice, referred to as Tg(CerPrP), as a substrate for in vitro generation of chronic wasting disease (CWD) prions by protein misfolding cyclic amplification (PMCA). Characterization of this infectivity in Tg(CerPrP) mice demonstrated that serial PMCA resulted in the high fidelity amplification of CWD prions with apparently unaltered properties. Using similar methods to amplify mouse RML prions and characterize the resulting novel cervid prions, we show that serial PMCA abrogated a transmission barrier that required several hundred days of adaptation and subsequent stabilization in Tg(CerPrP) mice. While both approaches produced cervid prions with characteristics distinct from CWD, the subtly different properties of the resulting individual prion isolates indicated that adaptation of mouse RML prions generated multiple strains following inter-species transmission. Our studies demonstrate that combined transgenic mouse and PMCA approaches not only expedite intra- and inter-species prion transmission, but also provide a facile means of generating and characterizing novel prion strains.     

Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171

Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA), did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.     

Styrylbenzoazole derivatives for imaging of prion plaques and treatment of transmissible spongiform encephalopathies

Recent prevalence of acquired forms of transmissible spongiform encephalopathies (TSEs) has urged the development of early diagnostic measures as well as therapeutic interventions. To extend our previous findings on the value of amyloid imaging probes for these purposes, styrylbenzoazole derivatives with better permeability of blood-brain barrier (BBB) were developed and analyzed in this study. The new styrylbenzoazole compounds clearly labeled prion protein (PrP) plaques in brain specimens from human TSE in a manner irrespective of pathogen strain, and a representative compound BF-168 detected abnormal PrP aggregates in the brain of TSE-infected mice when the probe was injected intravenously. On the other hand, most of the compounds inhibited abnormal PrP formation in TSE-infected cells with IC50 values in the nanomolar range, indicating that they represent one of the most potent classes of inhibitor ever reported. BF-168 prolonged the lives of mice infected intracerebrally with TSE when the compound was given intravenously at the preclinical stage. The new compounds, however, failed to detect synaptic PrP deposition and to show pathogen-independent therapeutic efficacy, similar to the amyloid imaging probes we previously reported. The compounds were BBB permeable and non-toxic at doses for imaging and treatment; therefore, they are expected to be of practical use in human TSE.     

The efficacy of tetracyclines in peripheral and intracerebral prion infection

We have previously shown that tetracyclines interact with and reverse the protease resistance of pathological prion protein extracted from scrapie-infected animals and patients with all forms of Creutzfeldt-Jakob disease, lowering the prion titre and prolonging survival of cerebrally infected animals. To investigate the effectiveness of these drugs as anti-prion agents Syrian hamsters were inoculated intramuscularly or subcutaneously with 263K scrapie strain at a 10(-4) dilution. Tetracyclines were injected intramuscularly or intraperitoneally at the dose of 10 mg/kg. A single intramuscular dose of doxycycline one hour after infection in the same site of inoculation prolonged median survival by 64%. Intraperitoneal doses of tetracyclines every two days for 40 or 44 days increased survival time by 25% (doxycycline), 32% (tetracycline); and 81% (minocycline) after intramuscular infection, and 35% (doxycycline) after subcutaneous infection. To extend the therapeutic potential of tetracyclines, we investigated the efficacy of direct infusion of tetracyclines in advanced infection. Since intracerebroventricular infusion of tetracycline solutions can cause overt acute toxicity in animals, we entrapped the drugs in liposomes. Animals were inoculated intracerebrally with a 10(-4) dilution of the 263K scrapie strain. A single intracerebroventricular infusion of 25 microg/20 microl of doxycycline or minocycline entrapped in liposomes was administered 60 days after inoculation, when 50% of animals showed initial symptoms of the disease. Median survival increased of 8.1% with doxycycline and 10% with minocycline. These data suggest that tetracyclines might have therapeutic potential for humans.     

Strain-Dependent Effect of Macroautophagy on Abnormally Folded Prion Protein Degradation in Infected Neuronal Cells

Prion diseases are neurodegenerative disorders caused by the accumulation of abnormal prion protein (PrP^Sc) in the central nervous system. With the aim of elucidating the mechanism underlying the accumulation and degradation of PrP^Sc, we investigated the role of autophagy in its degradation, using cultured cells stably infected with distinct prion strains. The effects of pharmacological compounds that inhibit or stimulate the cellular signal transduction pathways that mediate autophagy during PrP^Sc degradation were evaluated. The accumulation of PrP^Sc in cells persistently infected with the prion strain Fukuoka-1 (FK), derived from a patient with Gerstmann–Sträussler–Scheinker syndrome, was significantly increased in cultures treated with the macroautophagy inhibitor 3-methyladenine (3MA) but substantially reduced in those treated with the macroautophagy inducer rapamycin. The decrease in FK-derived PrP^Sc levels was mediated, at least in part, by the phosphatidylinositol 3-kinase/MEK signalling pathway. By contrast, neither rapamycin nor 3MA had any apparently effect on PrP^Sc from either the 22L or the Chandler strain, indicating that the degradation of PrP^Sc in host cells might be strain-dependent.     

Structural biology of ex vivo mammalian prions

The structures of prion protein (PrP)–based mammalian prions have long been elusive. However, cryo-EM has begun to reveal the near-atomic resolution structures of fully infectious ex vivo mammalian prion fibrils as well as relatively innocuous synthetic PrP amyloids. Comparisons of these various types of PrP fibrils are now providing initial clues to structural features that correlate with pathogenicity. As first indicated by electron paramagnetic resonance and solid-state NMR studies of synthetic amyloids, all sufficiently resolved PrP fibrils of any sort (n > 10) have parallel in-register intermolecular β-stack architectures. Cryo-EM has shown that infectious brain-derived prion fibrils of the rodent-adapted 263K and RML scrapie strains have much larger ordered cores than the synthetic fibrils. These bona fide prion strains share major structural motifs, but the conformational details and the overall shape of the fibril cross sections differ markedly. Such motif variations, as well as differences in sequence within the ordered polypeptide cores, likely contribute to strain-dependent templating. When present, N-linked glycans and glycophosphatidylinositol (GPI) anchors project outward from the fibril surface. For the mouse RML strain, these posttranslational modifications have little effect on the core structure. In the GPI-anchored prion structures, a linear array of GPI anchors along the twisting fibril axis appears likely to bind membranes in vivo, and as such, may account for pathognomonic membrane distortions seen in prion diseases. In this review, we focus on these infectious prion structures and their implications regarding prion replication mechanisms, strains, transmission barriers, and molecular pathogenesis.     

Subclinical prion disease induced by oral inoculation

Natural transmission of prion disease is believed to occur by peripheral infection such as oral inoculation. Following this route of inoculation, both the peripheral nervous system and the lymphoreticular system may be involved in the subsequent neuroinvasion of the central nervous system by prions, which may not necessarily result in clinical signs of terminal disease. Subclinical prion disease, characterized by the presence of infectivity and PrP(Sc) in the absence of overt clinical signs, may occur. It is not known which host factors contribute to whether infection with prions culminates in a terminal or subclinical disease state. We have investigated whether the level of host PrP(c) protein expression is a factor in the development of subclinical prion disease. When RML prion inoculum was inoculated by either the i.c. or intraperitoneal route, wild-type and tga20 mice both succumbed to terminal prion disease. In contrast, orally inoculated tga20 mice succumbed to terminal prion disease, whereas wild-type mice showed no clinical signs. However, wild-type mice sacrificed 375 or 525 days after oral inoculation harbored significant levels of brain PrP(Sc) and infectivity. These data show that same-species transmission of prions by the oral route in animals that express normal levels of PrP(c) can result in subclinical prion disease. This indicates that the level of host PrP(c) protein expression is a contributing factor to the regulation of development of terminal prion disease. Events that increase PrP(c) expression may predispose a prion-infected animal to the more deleterious effects of prion pathology.     

Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication

Neuroblastoma-derived N2a-PK1 cells, fibroblastic LD9 cells, and CNS-derived CAD5 cells can be infected efficiently and persistently by various prion strains, as measured by the standard scrapie cell assay. Swainsonine, an inhibitor of Golgi α-mannosidase II that causes abnormal N-glycosylation, strongly inhibits infection of PK1 cells by RML, 79A and 22F, less so by 139A, and not at all by 22L prions, and it does not diminish propagation of any of these strains in LD9 or CAD5 cells. Misglycosylated PrP(C) formed in the presence of swainsonine is a good substrate for conversion to PrP(Sc), and misglycosylated PrP(Sc) is fully able to trigger infection and seed the protein misfolding cyclic amplification reaction. Distinct subclones of PK1 cells mediate swainsonine inhibition to very different degrees, implicating misglycosylation of one or more host proteins in the inhibitory process. The use of swainsonine and other glycosylation inhibitors described herein enhances the ability of the cell panel assay to differentiate between prion strains. Moreover, as shown elsewhere, the susceptibility of prions to inhibition by swainsonine in PK1 cells is a mutable trait.