Biomedical subjects

Byron Caughey

Publications and source records attributed to Byron Caughey.

At least 19 recordsLinked to original sources

PubMed2006

Structural properties of prion protein protofibrils and fibrils: an experimental assessment of atomic models.

Decades after the prion protein was implicated in transmissible spongiform encephalopathies, the structure of its toxic isoform and its mechanism of toxicity remain unknown. By gathering available experimental data, albeit low resolution, a few pieces of the prion puzzle can be put in place. Currently, there are two fundamentally different models of a prion protofibril. One has its building blocks derived from a molecular dynamics simulation of the prion protein under amyloidogenic conditions, termed the spiral model. The other model was constructed by threading a portion of the prion sequence through a beta-helical structure from the Protein Data Bank. Here we compare and contrast these models with respect to all of the available experimental information, including electron micrographs, symmetries, secondary structure, oligomerization interfaces, enzymatic digestion, epitope exposure, and disaggregation profiles. Much of this information was not available when the two models were introduced. Overall, we find that the spiral model is consistent with all of the experimental results. In contrast, it is difficult to reconcile several of the experimental observables with the beta-helix model. While the experimental constraints are of low resolution, in bringing together the previously disconnected experiments, we have developed a clearer picture of prion aggregates. Both the improved characterization of prion aggregates and the existing atomic models can be used to devise further experiments to better elucidate the misfolding pathway and the structure of prion protofibrils.

Amyloid↗

PubMed2006

Prions and their partners in crime.

Prions, the infectious agents of transmissible spongiform encephalopathies (TSEs), have defied full characterization for decades. The dogma has been that prions lack nucleic acids and are composed of a pathological, self-inducing form of the host's prion protein (PrP). Recent progress in propagating TSE infectivity in cell-free systems has effectively ruled out the involvement of foreign nucleic acids. However, host-derived nucleic acids or other non-PrP molecules seem to be crucial. Interactions between TSE-associated PrP and its normal counterpart are also pathologically important, so the physiological functions of normal PrP and how they might be corrupted by TSE infections have been the subject of recent research.

Animals↗

PubMed2006

Molecular heterosis of prion protein beta-oligomers. A potential mechanism of human resistance to disease.

The gene encoding prion protein is polymorphic in human populations, with over 40% of native Europeans, for example, being heterozygous for the Met-129 and Val-129 alleles. The polymorphism affects both the incidence and the clinical presentation of a range of prion diseases, with heterozygotes generally showing the highest levels of resistance. It has been suggested that an earlier epidemic of prion diseases exerted balancing selection on the two alleles, and we have previously demonstrated that the two encoded proteins have potentially compensating tendencies to form amyloid and soluble beta-oligomers, respectively, in vitro. More strikingly, here we demonstrate that mixed oligomers, composed of both allelic forms, show an extreme sluggishness in converting to amyloid in comparison with oligomers homogenous for either allele. It may be that this example of molecular heterosis in vitro provides the basis for maintenance of the polymorphism in the population and that beta-oligomers represent a form of PrP sequestered from pathogenic amyloid formation in vivo.

Alleles↗

PubMed2006

Structural insights into the interaction between prion protein and nucleic acid.

The infectious agent of transmissible spongiform encephalopathies (TSE) is believed to comprise, at least in part, the prion protein (PrP). Other molecules can modulate the conversion of the normal PrP(C) into the pathological conformer (PrP(Sc)), but the identity and mechanisms of action of the key physiological factors remain unclear. PrP can bind to nucleic acids with relatively high affinity. Here, we report small-angle X-ray scattering (SAXS) and nuclear magnetic resonance spectroscopy measurements of the tight complex of PrP with an 18 bp DNA sequence. This double-stranded DNA sequence (E2DBS) binds with nanomolar affinity to the full-length recombinant mouse PrP. The SAXS data show that formation of the rPrP-DNA complex leads to larger values of the maximum dimension and radius of gyration. In addition, the SAXS studies reveal that the globular domain of PrP participates importantly in the formation of the complex. The changes in NMR HSQC spectra were clustered in two major regions: one in the disordered portion of the PrP and the other in the globular domain. Although interaction is mediated mainly through the PrP globular domain, the unstructured region is also recruited to the complex. This visualization of the complex provides insight into how oligonucleotides bind to PrP and opens new avenues to the design of compounds against prion diseases.

Animals↗

PubMed2006

Fractionation of prion protein aggregates by asymmetrical flow field-flow fractionation.

Achieving the successful separation and analysis of amyloid and other large protein aggregates can be a difficult proposition. Field-flow fractionation (FFF) is a flow-based separation method like chromatography; however, FFF is capable of high-resolution separations in the absence of a stationary matrix. Thus, FFF is a relatively gentle technique and is well suited to the task of separating large macromolecules and macromolecular complexes. Flow field-flow fractionation (FlFFF), one of the techniques in the FFF family, has been used to successfully fractionate a wide size range of prion protein aggregates, allowing their subsequent characterization by several biophysical and biochemical methods. The ability to easily adjust the strength of the field used during separation means that FlFFF could be applied to particles ranging from 1 nm to nearly 100 mum in size. This flexibility, coupled with the ability to produce fast, high-resolution separations, makes FFF a potentially valuable tool in the field of amyloid research.

Chemical Fractionation↗

PubMed2006

Searching for anti-prion compounds: cell-based high-throughput in vitro assays and animal testing strategies.

The transmissible spongiform encephalopathies (TSEs) or prion diseases are infectious neurodegenerative diseases of mammals. Protease-resistant prion protein (PrP-res) is only associated with TSEs and thus has been a target for therapeutic intervention. The most effective compounds known against scrapie in vivo are inhibitors of PrP-res in infected cells. Mouse neuroblastoma (N2a) cells have been chronically infected with several strains of mouse scrapie including RML and 22L. Also, rabbit epithelial cells that produce sheep prion protein in the presence of doxycycline (Rov9) have been infected with sheep scrapie. Here a high-throughput 96-well plate PrP-res inhibition assay is described for each of these scrapie-infected cell lines. With this dot-blot assay, thousands of compounds can easily be screened for inhibition of PrP-res formation. This assay is designed to find new PrP-res inhibitors, which may make good candidates for in vivo anti-scrapie testing. However, an in vitro assay can only suggest that a given compound might have in vivo anti-scrapie activity, which is typically measured as increased survival times. Methods for in vivo testing of compounds for anti-scrapie activity in transgenic mice, a much more lengthy and expensive process, are also discussed.

Animals↗

PubMed2006

Enhanced antiscrapie effect using combination drug treatment.

Combination treatment with pentosan polysulfate and Fe(III)meso-tetra(4-sulfonatophenyl)porphine in mice beginning 14 or 28 days after scrapie inoculation significantly increased survival times. This increase may be synergistic, implying that the compounds act cooperatively in vivo. Combination therapy may therefore be more effective for treatment of transmissible spongiform encephalopathies and other protein-misfolding diseases.

Animals↗

PubMed2006

A porphyrin increases survival time of mice after intracerebral prion infection.

Prion diseases, including scrapie, are incurable neurodegenerative disorders. Some compounds can delay disease after a peripheral scrapie inoculation, but few are effective against advanced disease. Here, we tested multiple related porphyrins, but only Fe(III)meso-tetra(4-sulfonatophenyl)porphine injected into mouse brains after intracerebral scrapie inoculation substantially increased survival times.

Animals↗

PubMed2006

Potent antiscrapie activities of degenerate phosphorothioate oligonucleotides.

Although transmissible spongiform encephalopathies (TSEs) are incurable, a key therapeutic approach is prevention of conversion of the normal, protease-sensitive form of prion protein (PrP-sen) to the disease-specific protease-resistant form of prion protein (PrP-res). Here degenerate phosphorothioate oligonucleotides (PS-ONs) are introduced as low-nM PrP-res conversion inhibitors with strong antiscrapie activities in vivo. Comparisons of various PS-ON analogs indicated that hydrophobicity and size were important, while base composition was only minimally influential. PS-ONs bound avidly to PrP-sen but could be displaced by sulfated glycan PrP-res inhibitors, indicating the presence of overlapping binding sites. Labeled PS-ONs also bound to PrP-sen on live cells and were internalized. This binding likely accounts for the antiscrapie activity. Prophylactic PS-ON treatments more than tripled scrapie survival periods in mice. Survival times also increased when PS-ONs were mixed with scrapie brain inoculum. With these antiscrapie activities and their much lower anticoagulant activities than that of pentosan polysulfate, degenerate PS-ONs are attractive new compounds for the treatment of TSEs.

Animals↗

PubMed2006

Prion strain-dependent differences in conversion of mutant prion proteins in cell culture.

Although the protein-only hypothesis proposes that it is the conformation of abnormal prion protein (PrP(Sc)) that determines strain diversity, the molecular basis of strains remains to be elucidated. In the present study, we generated a series of mutations in the normal prion protein (PrP(C)) in which a single glutamine residue was replaced with a basic amino acid and compared their abilities to convert to PrP(Sc) in cultured neuronal N2a58 cells infected with either the Chandler or 22L mouse-adapted scrapie strain. In mice, these strains generate PrP(Sc) of the same sequence but different conformations, as judged by infrared spectroscopy. Substitutions at codons 97, 167, 171, and 216 generated PrP(C) that resisted conversion and inhibited the conversion of coexpressed wild-type PrP in both Chandler-infected and 22L-infected cells. Interestingly, substitutions at codons 185 and 218 gave strain-dependent effects. The Q185R and Q185K PrP were efficiently converted to PrP(Sc) in Chandler-infected but not 22L-infected cells. Conversely, Q218R and Q218H PrP were converted only in 22L-infected cells. Moreover, the Q218K PrP exerted a potent inhibitory effect on the conversion of coexpressed wild-type PrP in Chandler-infected cells but had little effect on 22L-infected cells. These results show that two strains with the same PrP sequence but different conformations have differing abilities to convert the same mutated PrP(C).

Amino Acid Sequence↗

PubMed2006

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.

Animals↗

PubMed2006

Inhibition of protease-resistant prion protein formation in a transformed deer cell line infected with chronic wasting disease.

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

Animals↗

PubMed2006

Mouse-adapted scrapie infection of SN56 cells: greater efficiency with microsome-associated versus purified PrP-res.

The process by which transmissible spongiform encephalopathy agents, or prions, infect cells is unknown. We employed a new differentiable cell line (SN56) susceptible to infection with three mouse-adapted scrapie strains to gain insight into the cellular infection process. The effect of disease-associated PrP (PrP-res) association with microsomal membranes on infection efficiency was examined by comparing sustained PrP-res production in cells treated with either scrapie brain microsomes or purified, detergent-extracted PrP-res. When normalized for quantity of input PrP-res, scrapie brain microsomes induced dramatically enhanced persistent PrP-res formation compared to purified PrP-res. Infected SN56 cells released low levels of PrP-res into the culture supernatant, which also efficiently initiated infection in recipient cells. Interestingly, microsomes labeled with a fluorescent marker were internalized by SN56 cells in small vesicles, which were subsequently found in neuritic processes. When bound to culture wells to reduce internalization during the infection process, scrapie microsomes induced less long-term PrP-res production than suspended microsomes. Long-term differentiation of infected SN56 cells was accompanied by a decrease in PrP-res formation. Our observations provide evidence that infection of cells is aided by the association of PrP-res with membranes and/or other microsomal constituents.

Animals↗

PubMed2005

Comparison of protease-resistant prion protein inhibitors in cell cultures infected with two strains of mouse and sheep scrapie.

The transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases. A primary therapeutic target for TSE intervention has been a protease-resistant form of prion protein known as PrP(Sc) or PrP-res. In vitro testing of mouse scrapie-infected cell cultures has identified many PrP-res inhibitors that also have activity in vivo. Here we identify 32 new inhibitors of two strains of mouse scrapie PrP-res. Furthermore, to investigate the species-specificity of these and other PrP-res inhibitors, we have developed a high-throughput cell culture assay based on Rov9 cells chronically-infected with sheep scrapie. Of 32 inhibitors of murine PrP-res that were also tested in the Rov9 cells, only six showed inhibitory activity against sheep PrP-res. The three most potent inhibitors of both murine and ovine PrP-res formation (with 50% inhibition at < or =5 microM) were tannic acid, pentosan polysulfate and Fe(III) deuteroporphyrin 2,4-bisethyleneglycol. The latter two have anti-mouse scrapie activity in vivo. These results identify new inhibitors of murine and ovine PrP-res formation and reinforce the idea that compounds effective against PrP-res from one species or strain cannot be assumed to be active against others.

Animals↗

PubMed2005

The most infectious prion protein particles.

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.

Animals↗

PubMed2005

Anchorless prion protein results in infectious amyloid disease without clinical scrapie.

In prion and Alzheimer's diseases, the roles played by amyloid versus nonamyloid deposits in brain damage remain unresolved. In scrapie-infected transgenic mice expressing prion protein (PrP) lacking the glycosylphosphatidylinositol (GPI) membrane anchor, abnormal protease-resistant PrPres was deposited as amyloid plaques, rather than the usual nonamyloid form of PrPres. Although PrPres amyloid plaques induced brain damage reminiscent of Alzheimer's disease, clinical manifestations were minimal. In contrast, combined expression of anchorless and wild-type PrP produced accelerated clinical scrapie. Thus, the PrP GPI anchor may play a role in the pathogenesis of prion diseases.

Animals↗

PubMed2005

Uptake and neuritic transport of scrapie prion protein coincident with infection of neuronal cells.

Invasion of the nervous system and neuronal spread of infection are critical, but poorly understood, steps in the pathogenesis of transmissible spongiform encephalopathies or prion diseases. To characterize pathways for the uptake and intraneuronal trafficking of infectious, protease-resistant prion protein (PrP-res), fluorescent-labeled PrP-res was used to infect a neuronally derived murine cell line (SN56) and adult hamster cortical neurons in primary culture. Concurrent with the establishment of persistent scrapie infection, SN56 cells internalized PrP-res aggregates into vesicles positive for markers for late endosomes and/or lysosomes but not synaptic, early endocytic, or raft-derived vesicles. Internalized PrP-res was then transported along neurites to points of contact with other cells. Similar trafficking was observed with dextran, Alzheimer's Abeta1-42 fibrils and noninfectious recombinant PrP fibrils, suggesting that PrP-res is internalized by a relatively nonspecific pinocytosis or transcytosis mechanism. Hamster cortical neurons were also capable of internalizing and disseminating exogenous PrP-res. Similar trafficking of exogenous PrP-res by cortical neurons cultured from the brains of PrP knock-out mice showed that uptake and neuritic transport did not require the presence of endogenous cellular PrP. These experiments visualize and characterize the initial steps associated with prion infection and transport within neuronal cells.

Animals↗

PubMed2004

Prion diseases--close to effective therapy?

The transmissible spongiform encephalopathies could represent a new mode of transmission for infectious diseases--a process more akin to crystallization than to microbial replication. The prion hypothesis proposes that the normal isoform of the prion protein is converted to a disease-specific species by template-directed misfolding. Therapeutic and prophylactic strategies to combat these diseases have emerged from immunological and chemotherapeutic approaches. The lessons learned in treating prion disease will almost certainly have an impact on other diseases that are characterized by the pathological accumulation of misfolded proteins.

Animals↗