Biomedical subjects

Stanley B Prusiner

Publications and source records attributed to Stanley B Prusiner.

At least 19 recordsLinked to original sources

PubMed2007

Structure-activity relationship study of prion inhibition by 2-aminopyridine-3,5-dicarbonitrile-based compounds: parallel synthesis, bioactivity, and in vitro pharmacokinetics.

2-Aminopyridine-3,5-dicarbonitrile compounds were previously identified as mimetics of dominant-negative prion protein mutants and inhibit prion replication in cultured cells. Here, we report findings from a comprehensive structure-activity relationship study of the 6-aminopyridine-3,5-dicarbonitrile scaffold. We identify compounds with significantly improved bioactivity (approximately 40-fold) against replication of the infectious prion isoform (PrPSc) and suitable pharmacokinetic profiles to warrant evaluation in animal models of prion disease.

Aminopyridines↗

PubMed2006

Continuum of prion protein structures enciphers a multitude of prion isolate-specified phenotypes.

On passaging synthetic prions, two isolates emerged with incubation times differing by nearly 100 days. Using conformational-stability assays, we determined the guanidine hydrochloride (Gdn.HCl) concentration required to denature 50% of disease-causing prion protein (PrP(Sc)) molecules, denoted as the [Gdn.HCl](1/2) value. For the two prion isolates enciphering shorter and longer incubation times, [Gdn.HCl](1/2) values of 2.9 and 3.7 M, respectively, were found. Intrigued by this result, we measured the conformational stabilities of 30 prion isolates from synthetic and naturally occurring sources that had been passaged in mice. When the incubation times were plotted as a function of the [Gdn.HCl](1/2) values, a linear relationship was found with a correlation coefficient of 0.93. These findings demonstrate that (i) less stable prions replicate more rapidly than do stable prions, and (ii) a continuum of PrP(Sc) structural states enciphers a multitude of incubation-time phenotypes. Our data argue that cellular machinery must exist for propagating a large number of different PrP(Sc) conformers, each of which enciphers a distinct biological phenotype as reflected by a specific incubation time. The biophysical explanation for the unprecedented plasticity of PrP(Sc) remains to be determined.

Animals↗

PubMed2006

Expression mapping of tetracycline-responsive prion protein promoter: digital atlasing for generating cell-specific disease models.

We present a digital atlas system that allows mapping of molecular expression patterns at cellular resolution through large series of histological sections. Using this system, we have mapped the distribution of a distinct marker, encoded by the LacZ reporter gene driven by the tetracycline-responsive prion protein promoter in double transgenic mice. The purpose is to evaluate the suitability of this promoter mouse line for targeting genes of interest to specific brain regions, essential for construction of inducible transgenic disease models. Following processing to visualize the promoter expression, sections were counterstained to simultaneously display cytoarchitectonics. High-resolution mosaic images covering entire coronal sections were collected through the mouse brain at intervals of 200 microm. A web-based application provides access to a customized virtual microscopy tool for viewing and navigation within and across the section images. For each section image, the nearest section in a standard atlas is defined, and annotations of key structures and regions inserted. Putative categorization of labeled cells was performed with use of distribution patterns, followed by cell size and shape, as parameters that were compared to legacy data. Among the ensuing results were expression of this promoter in putative glial cells in the cerebellum (and not in Purkinje cells), in putative glial cells in the substantia nigra, in pallidal glial cells or interneurons, and in distinct cell layers and regions of the hippocampus. The study serves as a precursor for a database resource allowing evaluation of the suitability of different promoter mouse lines for generating disease models.

Animals↗

PubMed2006

Structure-activity relationship study of 9-aminoacridine compounds in scrapie-infected neuroblastoma cells.

A focused library of variously substituted 9-aminoacridine compounds was screened for bioactivity against accumulation of the infectious prion protein isoform, denoted PrP(Sc), in a cell model of prion replication. The efficacy of compounds against PrP(Sc) accumulation was influenced by both substituents of the distal tertiary amine and acridine heterocycle, while cellular cytotoxicity was encoded in the acridine heterocycle substituents.

Aminacrine↗

PubMed2006

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation.

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.

Amino Acid Sequence↗

PubMed2006

Human prions and plasma lipoproteins.

Prions are composed solely of an alternatively folded isoform of the prion protein (PrP), designated PrP(Sc). The polyoxometalate phosphotungstic acid has been used to separate PrP(Sc) from its precursor PrP(C) by selective precipitation; notably, native PrP(Sc) has not been solubilized by using nondenaturing detergents. Because of the similarities between PrP(Sc) and lipoproteins with respect to hydrophobicity and formation of phosphotungstic acid complexes, we asked whether these molecules are bound to each other in blood. Here we report that prions from the brains of patients with sporadic Creutzfeldt-Jakob disease (CJD) bind to very low-density (VLDL) and low-density (LDL) lipoproteins but not to high-density lipoproteins (HDL) or other plasma components, as demonstrated both by affinity assay and electron microscopy. Immunoassays demonstrated that apolipoprotein B (apoB), which is the major protein component of VLDL and LDL, bound PrP(Sc) through a highly cooperative process. Approximately 50% of the PrP(Sc) bound to LDL particles was released after exposure to 4 M guanidine hydrochloride at 80 degrees C for 20 min. The apparent binding constants of native human (Hu) PrP(Sc) or denatured recombinant HuPrP(90-231) for apoB and LDL ranged from 28 to 212 pM. Whether detection of PrP(Sc) in VLDL and LDL particles can be adapted into an antemortem diagnostic test for prions in the blood of humans, livestock, and free-ranging cervids remains to be determined.

Brain↗

PubMed2006

Changes in neuropeptide expression in mice infected with prions.

Prion diseases are neurodegenerative disorders characterized by accumulation of an aberrantly folded isoform (PrP(Sc)) of the normal prion protein (PrP(C)). Using in situ hybridization and immunohistochemistry, we have studied changes in the expression of neuropeptides, acetylcholinesterase and tyrosine hydroxylase in CD1 and FVB wild-type mouse strains, as well as in PrP(C) null mice and in mice overexpressing PrP(C) following intracerebral inoculation with RML or Me7 prions. In the immunohistochemical analysis, neuropeptide Y (NPY), enkephalin and dynorphin-like immunoreactivities increased in mossy fibers of CD1 and FVB mice inoculated with either RML- or Me7 prions, whereas cholecystokinin-like immunoreactivity was decreased. These changes in peptide levels were paralleled by an increase in the transcripts in granule cells for neuropeptide Y, enkephalin, and cholecystokinin. However, the dynorphin transcript was decreased in the granule cells. The changes occurred more rapidly in PrP(C)-overexpressing compared to wild-type mice, and could not be found at all in PrP(C)-knockout mice. These changes in peptide expression, which mostly occur before appearance of symptoms of disease, may reflect attempts to initiate protective and/or regenerative processes.

Animals↗

PubMed2006

Quinacrine is mainly metabolized to mono-desethyl quinacrine by CYP3A4/5 and its brain accumulation is limited by P-glycoprotein.

Quinacrine (QA), an antimalarial drug used for over seven decades, has been found to have potent antiprion activity in vitro. To determine whether QA can be used to treat prion diseases, we investigated its metabolism and ability to traverse the blood-brain barrier in mice. In vitro and in vivo, we identified by liquid chromatography-tandem mass spectrometry the major metabolic pathway of QA as N-desethylation and compared our results with an authentic reference compound. The major human cytochrome (P450) isoforms involved in QA mono-desethylation were identified as CYP3A4/5 by using specific chemical and antibody inhibition as well as cDNA-expressed P450 studies. QA transport from the basolateral to apical side in multidrug resistance protein 1 gene (MDR1)-transfected Madin-Darby canine kidney (MDCK) cells was markedly greater than in control MDCK cells and was inhibited by the potent P-glycoprotein (P-gp) inhibitor GG918 (N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-iso-1-quinolynyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamine). In MDR1-knockout (KO) mice, QA brain levels were 6 to 9 times higher after a single i.v. dose of 2 mg/kg QA and 49 times higher after multiple oral doses of 10 mg/kg/day QA for 7 days, compared with those in wild-type (WT) FVB mice. In contrast, the QA levels in plasma, liver, spleen, and kidney were similar after a single 2 mg/kg i.v. dose and <2 times greater after 10 mg/kg oral doses in MDR1-KO mice compared with WT mice. These results indicate that P-gp plays a critical role in transporting QA from the brain.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2006

Prion infection of mouse neurospheres.

Only a few cell lines have been infected with prions, offering limited genetic diversity and sensitivity to several strains. Here we report that cultured neurospheres expressing cellular prion protein (PrP(C)) can be infected with prions. Neurosphere lines isolated from the brains of mice at embryonic day 13-15 grow as aggregates and contain CNS stem cells. We produced neurosphere cultures from FVB/NCr (FVB) mice, from transgenic (Tg) FVB mice that overexpress mouse PrP-A (Tg4053), and from congenic FVB mice with a targeted null mutation in the PrP gene (Prnp(0/0)) and incubated them with the Rocky Mountain Laboratory prion strain. While monitoring the levels of disease-causing PrP (PrP(Sc)) at each passage, we observed a dramatic rise in PrP(Sc) levels with time in the Tg4053 neurosphere cells, whereas the level of PrP(Sc) decayed to undetectable levels in cell cultures lacking PrP. PrP(Sc) levels in cultures from FVB mice initially declined but then increased with passage. Prions produced in culture were transmissible to mice and produced disease pathology. Intracellular aggregates of PrP(Sc) were present in cells from infected cultures. The susceptibility of neurosphere cultures to prions mirrored that of the mice from which they were derived. Neurosphere lines from Tg4053 mice provide a sensitive in vitro bioassay for mouse prions; neurosphere lines from other Tg mice overexpressing PrP might be used to assay prions from other species, including humans.

Animals↗

PubMed2006

Transmission of elk and deer prions to transgenic mice.

Chronic wasting disease (CWD) is a fatal prion disease in deer and elk. Unique among the prion diseases, it is transmitted among captive and free-ranging animals. To facilitate studies of the biology of CWD prions, we generated five lines of transgenic (Tg) mice expressing prion protein (PrP) from Rocky Mountain elk (Cervus elaphus nelsoni), denoted Tg(ElkPrP), and two lines of Tg mice expressing PrP common to white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus), denoted Tg(DePrP). None of the Tg(ElkPrP) or Tg(DePrP) mice exhibited spontaneous neurologic dysfunction at more than 600 days of age. Brain samples from CWD-positive elk, white-tailed deer, and mule deer produced disease in Tg(ElkPrP) mice between 180 and 200 days after inoculation and in Tg(DePrP) mice between 300 and 400 days. One of eight cervid brain inocula transmitted disease to Tg(MoPrP)4053 mice overexpressing wild-type mouse PrP-A in approximately 540 days. Neuropathologic analysis revealed abundant PrP amyloid plaques in the brains of ill mice. Brain homogenates from symptomatic Tg(ElkPrP) mice produced disease in 120 to 190 days in Tg(ElkPrP) mice. In contrast to the Tg(ElkPrP) and Tg(DePrP) mice, Tg mice overexpressing human, bovine, or ovine PrP did not develop prion disease after inoculation with CWD prions from among nine different isolates after >500 days. These findings suggest that CWD prions from elk, mule deer, and white-tailed deer can be readily transmitted among these three cervid species.

Animals↗

PubMed2006

Inactivation of prions by acidic sodium dodecyl sulfate.

Prompted by the discovery that prions become protease-sensitive after exposure to branched polyamine dendrimers in acetic acid (AcOH) (S. Supattapone, H. Wille, L. Uyechi, J. Safar, P. Tremblay, F. C. Szoka, F. E. Cohen, S. B. Prusiner, and M. R. Scott, J. Virol. 75:3453-3461, 2001), we investigated the inactivation of prions by sodium dodecyl sulfate (SDS) in weak acid. As judged by sensitivity to proteolytic digestion, the disease-causing prion protein (PrPSc) was denatured at room temperature by SDS at pH values of < or =4.5 or > or =10. Exposure of Sc237 prions in Syrian hamster brain homogenates to 1% SDS and 0.5% AcOH at room temperature resulted in a reduction of prion titer by a factor of ca. 10(7); however, all of the bioassay hamsters eventually developed prion disease. When various concentrations of SDS and AcOH were tested, the duration and temperature of exposure acted synergistically to inactivate both hamster Sc237 prions and human sporadic Creutzfeldt-Jakob disease (sCJD) prions. The inactivation of prions in brain homogenates and those bound to stainless steel wires was evaluated by using bioassays in transgenic mice. sCJD prions were more than 100,000 times more resistant to inactivation than Sc237 prions, demonstrating that inactivation procedures validated on rodent prions cannot be extrapolated to inactivation of human prions. Some procedures that significantly reduced prion titers in brain homogenates had a limited effect on prions bound to the surface of stainless steel wires. Using acidic SDS combined with autoclaving for 15 min, human sCJD prions bound to stainless steel wires were eliminated. Our findings form the basis for a noncorrosive system that is suitable for inactivating prions on surgical instruments, as well as on other medical and dental equipment.

Animals↗

PubMed2006

Discovering DNA encodes heredity and prions are infectious proteins.

The resemblance between the discoveries that DNA is the basis of heredity and that prions are infectious proteins is remarkable. Though four decades separated these two discoveries, the biochemical methodologies and scientific philosophies that were employed are surprisingly similar. In both cases, bioassays available at the time that the projects were initiated proved to be inadequate to support purification studies. Improved bioassays allowed the transforming principle (TP) to be purified from pneumococci and prions from scrapie-infected hamster brains. Publications describing TP as composed of DNA prompted some scientists to contend that undetected proteins must contaminate TP enriched fractions. The simplicity of DNA was thought to prevent it from encoding genetic information. By the time prions were discovered, the genomes of all infectious pathogens including viruses, bacteria, fungi and parasites had been shown to be comprised of nucleic acids and so an antithetical refrain became widely echoed: DNA or RNA molecules must be hiding among the proteins of prions. Finding the unexpected and being asked to demonstrate unequivocally the absence of a possible contaminant represent uncanny parallels between the discoveries that DNA encodes the genotype and that prions are infectious proteins.

Animals↗

PubMed2005

Solid-state NMR structural studies of the fibril form of a mutant mouse prion peptide PrP89-143(P101L).

The peptide fragment 89-143 of the prion protein (carrying a P101L mutation) is biologically active in transgenic mice when in a fibrillar form. Injection of these fibrils into transgenic mice (expressing full length PrP with the P101L mutation) induces a neurodegenerative prion disease (Kaneko et al., J. Mol. Biol. 295 (2000) 997). Here we present solid-state NMR studies of PrP(89-143)(P101L) fibrils, probing the conformation of residues in the hydrophobic segment 112-124 with chemical shifts. The conformations of glycine residues were analyzed using doubly (13)C=O labeled peptides by two-dimensional (2D) double-quantum correlation, and double-quantum filtered dephasing distance measurements. MQ-NMR experiments were carried out to probe the relative alignment of the individual peptides fibrils. These NMR studies indicate that the 112-124 segment adopts an extended beta-sheet conformation, though not in a parallel, in register alignment. There is evidence for conformational variability at Gly 113. DQ correlation experiments provide useful information in regions with conformational heterogeneity.

Amyloid↗

PubMed2005

Selective precipitation of prions by polyoxometalate complexes.

The efficacy of a variety of polyoxometalate anions in selectively precipitating the infectious isoform of the prion protein (PrPSc) over the normal, cellular protein (PrPC) was tested as a means of improving the sensitivity of a conformation-dependent immunoassay (CDI). Experiments utilizing neutralized aqueous solutions containing MgCl2.6H2O and Na2H[PW12O40] (1) indicated that use of the former is detrimental to selectivity, while use of the latter gives optimal selectivity at a concentration of 2.48% w/v, much higher than the 0.31% w/v employed in the current CDI protocol. Similar experiments using Na4[SiW12O40] (2), K5[BW12O40] (3), and Na6[H2W12O40] (4) were performed to probe the role of charge density in polyoxometalate anions possessing the Keggin structure. These, coupled with assignment of solution structures via NMR and infrared spectroscopy, revealed the following trend in optimal concentrations: [HBW11O39]8- < [SiW11O39]8- < [PW11O39]7- = [H2W12O40]6-. Interestingly, use of the salts K10[Zn4(H2O)2(PW9O34)2] (5), Na16[Zn4(H2O)2(P2W15O56)2] (6), and Na27[NaAs4W40O140] (7), containing larger polyoxometalate anions, was found to suppress PrPSc precipitation in a concentration-dependent manner. Taken together, the results suggest a precipitation mechanism wherein Keggin-type anions link multiple PrPSc moieties by binding a somewhat hindered cleft featuring one or more positively charged residues.

Crystallography, X-Ray↗

PubMed2005

Diagnosis of human prion disease.

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrPSc) used limited proteolysis to digest the precursor cellular PrP (PrPC). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrPSc, we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrPSc in all 10-24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, approximately 90% of the total PrPSc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrPC. Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

Biopsy↗

PubMed2005

Strain-specified characteristics of mouse synthetic prions.

Synthetic prions were produced in our laboratory by using recombinant mouse prion protein (MoPrP) composed of residues 89-230. The first mouse synthetic prion strain (MoSP1) was inoculated into transgenic (Tg) 9949 mice expressing N-terminally truncated MoPrP(Delta23-88) and WT FVB mice expressing full-length MoPrP. On first and second passage in Tg9949 mice, MoSP1 prions caused disease in 516 +/- 27 and 258 +/- 25 days, respectively; numerous, large vacuoles were found in the brainstem and gray matter of the cerebellum. MoSP1 prions passaged in Tg9949 mice were inoculated into FVB mice; on first and second passage, the FVB mice exhibited incubation times of 154 +/- 4 and 130 +/- 3 days, respectively. In FVB mice, vacuolation was less intense but more widely distributed, with numerous lesions in the hippocampus and cerebellar white matter. This constellation of widespread neuropatho-logic changes was similar to that found in FVB mice inoculated with Rocky Mountain Laboratory (RML) prions, a strain derived from a sheep with scrapie. Conformational stability studies showed that the half-maximal GdnHCl (Gdn1/2) concentration for denaturation of MoSP1 prions passaged in Tg9949 mice was approximately 4.2 M; passage in FVB mice reduced the Gdn1/2 value to approximately 1.7 M. RML prions passaged in either Tg9949 or FVB mice exhibited Gdn1/2 values of approximately 1.8 M. The incubation times, neuropathological lesion profiles, and Gdn1/2 values indicate that MoSP1 prions differ from RML and many other prion strains derived from sheep with scrapie and cattle with bovine spongiform encephalopathy.

Animals↗

PubMed2005

Notch-1 activation and dendritic atrophy in prion disease.

In addition to neuronal vacuolation and astrocytic hypertrophy, dendritic atrophy is a prominent feature of prion disease. Because increased Notch-1 expression and cleavage releasing its intracellular domain (NICD) inhibit both dendrite growth and maturation, we measured their levels in brains from mice inoculated with Rocky Mountain Laboratory (RML) prions. The level of NICD was elevated in the neocortex, whereas the level of beta-catenin, which stimulates dendritic growth, was unchanged. During the incubation period, levels of the disease-causing prion protein isoform, PrPSc, and NICD increased concomitantly in the neocortex. Additionally, increased levels of Notch-1 mRNA and translocation of NICD to the nucleus correlated well with regressive dendritic changes. In scrapie-infected neuroblastoma (ScN2a) cells, the level of NICD was elevated compared with uninfected control (N2a) cells. Long neurofilament protein-containing processes extended from the surface of N2a cells, whereas ScN2a cells had substantially shorter processes. Transfection of ScN2a cells with a Notch-1 small interfering RNA decreased Notch-1 mRNA levels, diminished NICD concentrations, and rescued the long process phenotype. These results suggest that PrPSc in neurons and in ScN2a cells activates Notch-1 cleavage, resulting in atrophy of dendrites in the CNS and shrinkage of processes on the surface of cultured cells. Whether diminishing Notch-1 activation in vivo can prevent or even reverse neurodegeneration in prion disease remains to be established.

Active Transport, Cell Nucleus↗

PubMed2005

Prion clearance in bigenic mice.

The clearance of prions from the brain was investigated in bigenic mice designated Tg(tTA : PrP(+/0))3, in which expression of the cellular prion protein (PrP(C)) was regulated by oral doxycycline administration. With suppression of PrP(C) expression, the incubation time for RML prions was prolonged almost threefold from approximately 150 to approximately 430 days. To determine the clearance rate of disease-causing PrP(Sc), bigenic mice were given oral doxycycline beginning 98 days after inoculation with RML prions and sacrificed at various time points over the subsequent 56 days. The half-life (t1/2) for PrP(Sc) was approximately 1.5 days in mouse brain, in reasonable agreement with the apparent t1/2 of 30 h that was determined in a separate study for scrapie-infected mouse neuroblastoma (ScN2a) cells in culture. Both protease-sensitive and -resistant conformers of PrP(Sc) were cleared at the same rate. The t1/2 value for PrP(C) clearance from brain was approximately 18 h, which was considerably longer than the t1/2 of 5 h found in ScN2a cells. The capability of the brain to clear prions raises the possibility that PrP(Sc) is normally made at low levels and continually cleared, and that PrP(Sc) may have a function in cellular metabolism. Moreover, these bigenic mice make it possible to determine both components of PrP(Sc) accumulation, i.e. the rates of formation and clearance, for various strains of prions exhibiting different incubation times.

Animals↗