Biomedical subjects

Stephen J DeArmond

Publications and source records attributed to Stephen J DeArmond.

At least 19 recordsLinked to original sources

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

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

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

Human growth hormone-related iatrogenic Creutzfeldt-Jakob disease with abnormal imaging.

BACKGROUND: Although more than 160 cases of iatrogenic Creutzfeldt-Jakob disease (iCJD) from human growth hormone (hGH) treatment have been documented, to our knowledge abnormal cerebellar findings on magnetic resonance imaging (MRI) have not been reported. OBJECTIVE: To report a case of hGH-related iCJD with abnormal cerebellar MRI findings on fluid-attenuated inversion recovery (FLAIR) and diffusion-weighted MRI (DWI). DESIGN: Case report. SETTING: Outpatient neurology clinic at a university medical center. PATIENT: A 33-year old man who had subacute gait ataxia and blurred vision. RESULTS: Beginning 19 years prior, this patient had received cadaveric pituitary-derived hGH treatment for at least 5 years. Magnetic resonance imaging revealed FLAIR and DWI abnormalities, particularly in the cerebellum. He died 7 months after disease onset of autopsy-confirmed iCJD. Pathological changes corresponded largely to MRI findings. CONCLUSIONS: To our knowledge, this is the first case of hGH-related iCJD with FLAIR and DWI abnormalities within the cerebellum. As symptoms referable to the cerebellum occur early in iCJD, it suggests that these MRI sequences may allow earlier diagnosis of this form of prion disease.

Adult↗

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↗

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↗

PubMed2004

Neurotoxicity of intra-arterial papaverine preserved with chlorobutanol used for the treatment of cerebral vasospasm after aneurysmal subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Papaverine is used to vasodilate cerebral arteries undergoing vasospasm from subarachnoid hemorrhage. However, papaverine inhibits cellular respiration in vitro and could cause neurotoxicity in humans. METHODS: We studied 5 consecutive patients with cerebral vasospasm who were treated with intra-arterial papaverine preserved with chlorobutanol and imaged with MRI fluid-attenuated inversion recovery and diffusion-weighted imaging after treatment. One patient had histological analysis of the brain at autopsy. RESULTS: All 5 patients exhibited marked neurological decline immediately after treatment, and this was sustained through hospital discharge. In all cases, MRI images showed selective gray matter-only signal changes within the vascular territory treated with papaverine. Histological analysis of 1 case brought to autopsy showed selective injury to islands of neurons with relative sparing of white matter. CONCLUSIONS: Intra-arterial delivery of papaverine preserved with chlorobutanol into vasospastic anterior cerebral arteries may result in marked neurological deterioration with selective gray matter changes on MRI imaging. This effect is consistent with a permanent toxic effect to human brain. It is unclear whether this toxicity is caused by papaverine or chlorobutanol, and its use in the treatment of cerebral vasospasm should be reserved for cases without alternatives.

Aged↗

PubMed2004

Synthetic mammalian prions.

Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of beta sheet-rich structures. Fibrils consisting of recMoPrP(89-230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89-231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

Amyloid↗

PubMed2004

Time-controlled transcardiac perfusion cross-linking for the study of protein interactions in complex tissues.

Because of their sensitivity to solubilizing detergents, membrane protein assemblies are difficult to study. We describe a protocol that covalently conserves protein interactions through time-controlled transcardiac perfusion cross-linking (tcTPC) before disruption of tissue integrity. To validate tcTPC for identifying protein-protein interactions, we established that tcTPC allowed stringent immunoaffinity purification of the gamma-secretase complex in high salt concentrations and detergents and was compatible with mass spectrometric identification of cross-linked aph-1, presenilin-1 and nicastrin. We then applied tcTPC to identify more than 20 proteins residing in the vicinity of the cellular prion protein (PrPC), suggesting that PrP is embedded in specialized membrane regions with a subset of molecules that, like PrP, use a glycosylphosphatidylinositol anchor for membrane attachment. Many of these proteins have been implicated in cell adhesion/neuritic outgrowth, and harbor immunoglobulin C2 and fibronectin type III-like motifs.

Amino Acid Sequence↗

PubMed2004

Discovering the mechanisms of neurodegeneration in prion diseases.

The purpose of my chapter in this issue of Neuroscience Reviews dedicated to Dr. Lawrence Eng is to summarize my contributions to understanding the mechanisms of neurodegeneration in prion diseases. I explain that I was able to advance the field of prion disease neuropathology largely because of the foundation of neurochemistry and immunohistochemistry that I learned while working 5 years in Dr. Eng's laboratory. In my review, I relate how my Neuropathology Research Laboratory began as a collaboration with Dr. Stanley Prusiner 20 years ago that led from immunohistochemical staining of amyloid plaques in rodent and human brains using prion protein-specific antibodies to molecular evidence that the abnormal prion protein, PrP(Sc), is the cause of the clinically relevant neuropathological changes in animal and human prion diseases.

Animals↗

PubMed2004

The neurodegeneration sequence in prion diseases: evidence from functional, morphological and ultrastructural studies of the GABAergic system.

Loss of the GABAergic system of neurons has been reported to be the first detectable neuropathological change in prion diseases, which features the accumulation of an aberrant isoform of the prion protein (PrP(Sc)). To determine the timing of GABAergic system dysfunction and degeneration and its relationship to PrP(Sc) accumulation during the course of prion disease in Syrian hamsters, we applied 3 approaches: i) quantifying GABA-immunopositive neurons and their processes by light and electron microscopy to test for selective loss; ii) measuring evoked [3H]-GABA release from synaptosomes to test for functional abnormalities; and iii) determining the kinetics of PrP(Sc) accumulation in subcellular fractions to correlate it with GABAergic dysfunction. At the terminal stages of disease, we found a significant increase in the number of GABA-positive and -negative presynaptic boutons with abnormally aggregated synaptic vesicles. At the same stage, we also found an equal degree of GABA-immunopositive and -immunonegative presynaptic bouton loss. In contrast, GABA-positive neocortical cell bodies increased, based on stereologic estimates in the terminal stage of scrapie. In the context of these abnormalities, evoked release of [3H]-GABA from cortical and thalamic synaptosomes was significantly decreased, which correlated well with the accumulation of PrP(Sc) in synaptosomes and cell membrane fractions.

Animals↗

PubMed2004

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains.

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP(Sc)(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4 degrees C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP(Sc)(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a beta-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP(Sc)(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP(Sc) in the brains of Tg196 mice. Our results argue that MoPrP(Sc)(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP(C)(P101L) as well as in Tg196 mice expressing low levels of MoPrP(C)(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a beta-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.

Animals↗

PubMed2003

Trafficking of prion proteins through a caveolae-mediated endosomal pathway.

To understand the posttranslational conversion of the cellular prion protein (PrPC) to its pathologic conformation, it is important to define the intracellular trafficking pathway of PrPC within the endomembrane system. We studied the localization and internalization of PrPC in CHO cells using cryoimmunogold electron microscopy. At steady state, PrPC was enriched in caveolae both at the TGN and plasma membrane and in interconnecting chains of endocytic caveolae. Protein A-gold particles bound specifically to PrPC on live cells. These complexes were delivered via caveolae to the pericentriolar region and via nonclassical, caveolae-containing early endocytic structures to late endosomes/lysosomes, thereby bypassing the internalization pathway mediated by clathrin-coated vesicles. Endocytosed PrPC-containing caveolae were not directed to the ER and Golgi complex. Uptake of caveolae and degradation of PrPC was slow and sensitive to filipin. This caveolae-dependent endocytic pathway was not observed for several other glycosylphosphatidyl inositol (GPI)-anchored proteins. We propose that this nonclassical endocytic pathway is likely to determine the subcellular location of PrPC conversion.

Animals↗

PubMed2003

Cytosolic prion protein in neurons.

Localizing the cellular prion protein (PrPC) in the brain is necessary for understanding the pathogenesis of prion diseases. However, the precise ultrastructural localization of PrPC still remains enigmatic. We performed the first quantitative study of the ultrastructural localization of PrPC in the mouse hippocampus using high-resolution cryoimmunogold electron microscopy. PrPC follows the standard biosynthetic trafficking pathway with a preferential localization in late endosomal compartments and on the plasma membrane of neurons and neuronal processes. PrPC is found with the same frequency within the synaptic specialization and perisynaptically, but is almost completely excluded from synaptic vesicles. Unexpectedly, PrP is also found in the cytosol in subpopulations of neurons in the hippocampus, neocortex, and thalamus but not the cerebellum. Cytosolic PrP may have altered susceptibility to aggregation, suggesting that these neurons might play a significant role in the pathogenesis of prion diseases, in particular those mammals harboring mutant PrP genes.

Animals↗

PubMed2003

Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.

Transgenic (Tg) mouse lines that express chimeric mouse-human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt-Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening the incubation time to approximately 110 days for prions from sCJD patients. The construct with a murine residue at position 96 resulted in lengthening the incubation time to more than 280 days for sCJD prions. When murine residues 96, 165, and 167 were expressed, the abbreviated incubation times for sCJD prions were abolished. Variant CJD prions showed prolonged incubation times between 300 and 700 days in Tg(MHu2M) mice on first passage and incubation times of approximately 350 days in Tg(MHu2M,M165V,E167Q) mice. On second and third passages of variant CJD prions in Tg(MHu2M) mice, multiple strains of prions were detected based on incubation times and the sizes of the protease-resistant, deglycosylated PrP(Sc) fragments. Our discovery of a previously undescribed chimeric transgene with abbreviated incubation times for sCJD prions should facilitate studies on the prion species barrier and human prion diversity.

Amino Acid Substitution↗

PubMed2003

Challenging the clinical utility of the 14-3-3 protein for the diagnosis of sporadic Creutzfeldt-Jakob disease.

BACKGROUND: Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal neurodegenerative disorder for which there is no noninvasive and disease-specific test for premortem diagnosis. Previous studies have suggested that, in the proper clinical context, the 14-3-3 protein in cerebrospinal fluid is a reliable marker for sporadic CJD. OBJECTIVE: To assess the sensitivity of the cerebrospinal fluid 14-3-3 protein test among patients with definite sporadic CJD. DESIGN AND SETTING: We reviewed cases of sporadic CJD referred to our institution that were ultimately proved by pathological examination and on which cerebrospinal fluid 14-3-3 testing had been performed. PARTICIPANTS: Patients with CJD referred to our institution for clinical and/or pathological evaluation (biopsy- or autopsy-confirmed diagnosis) from January 1, 1998, through July 15, 2002, and on whom 14-3-3 testing had been performed. Thirty-two such patients with definite sporadic CJD were identified. Main Outcome Measure The 14-3-3 test results, from various laboratories, in these 32 patients. RESULTS: Seventeen of the 32 patients had a positive result for the 14-3-3 test, yielding a sensitivity of only 53%. A positive 14-3-3 result was significantly correlated with a shorter time between disease onset and the lumbar puncture for the 14-3-3 test. CONCLUSIONS: Testing for the 14-3-3 protein is only modestly sensitive to sporadic CJD, and we caution against ruling out a diagnosis of the disease on the basis of a negative 14-3-3 result.

14-3-3 Proteins↗

PubMed2003

Perspectives on prion biology, prion disease pathogenesis, and pharmacologic approaches to treatment.

The main goals of this article have been to summarize our current understanding of the biology of PrP, the propagation of prions, and the etiology and pathogenesis of each form of prion disease (familial, sporadic, and infectious); and to review current rational pharmacologic strategies for treatment of prion diseases. Each of these subjects is presented primarily from the perspective of investigations performed by the prion disease research laboratories at the University of California in San Francisco and by its many collaborators in the United States and abroad. This review focuses on key results from the hundreds of transgenic mouse lines expressing different PrP constructs that have been used to determine the roles played by different PrPSc and PrPC domains in prion propagation and the prion disease phenotype.

Animals↗