The cellular mechanisms by which prions cause neurological dysfunction are poorly understood. To address this issue, we have been using cultured cells to analyze the localization, biosynthesis, and ...metabolism of PrP molecules carrying mutations associated with familial prion diseases. We report here that mutant PrP molecules are delayed in their maturation to an endoglycosidase H-resistant form after biosynthetic labeling, suggesting that they are impaired in their exit from the endoplasmic reticulum (ER). However, we find that proteasome inhibitors have no effect on the maturation or turnover of either mutant or wild-type PrP molecules. Thus, in contrast to recent studies from other laboratories, our work indicates that PrP is not subject to retrotranslocation from the ER into the cytoplasm prior to degradation by the proteasome. We find that in transfected cells, but not in cultured neurons, proteasome inhibitors cause accumulation of an unglycosylated, signal peptide-bearing form of PrP on the cytoplasmic face of the ER membrane. Thus, under conditions of elevated expression, a small fraction of PrP chains is not translocated into the ER lumen during synthesis, and is rapidly degraded in the cytoplasm by the proteasome. Finally, we report a previously unappreciated artifact caused by treatment of cells with proteasome inhibitors: an increase in PrP mRNA level and synthetic rate when the protein is expressed from a vector containing a viral promoter. We suggest that this phenomenon may explain some of the dramatic effects of proteasome inhibitors observed in other studies. Our results clarify the role of the proteasome in the cell biology of PrP, and suggest reasonable hypotheses for the molecular pathology of inherited prion diseases.
Approx. 15% of human prion diseases have a pattern of autosomal dominant inheritance, and are linked to mutations in the gene encoding PrP (prion protein), a GPI ...(glycosylphosphatidylinositol)-anchored protein whose function is not clear. The cellular mechanisms by which PrP mutations cause disease are also not known. Soon after synthesis in the ER (endoplasmic reticulum), several mutant PrPs misfold and become resistant to phospholipase cleavage of their GPI anchor. The biosynthetic maturation of the misfolded molecules in the ER is delayed and, during transit in the secretory pathway, they form detergent-insoluble and protease-resistant aggregates, suggesting that intracellular PrP aggregation may play a pathogenic role. We have investigated the consequence of deleting residues 114-121 within the hydrophobic core of PrP on the aggregation and cellular localization of two pathogenic mutants that accumulate in the ER and Golgi apparatus. Compared with their full-length counterparts, the deleted molecules formed smaller protease-sensitive aggregates and were more efficiently transported to the cell surface and released by phospholipase cleavage. These results indicate that mutant PrP aggregation and intracellular retention are closely related and depend critically on the integrity of the hydrophobic core. The discovery that Delta114-121 counteracts misfolding and improves the cellular trafficking of mutant PrP provides an unprecedented model for assessing the role of intracellular aggregation in the pathogenesis of prion diseases.
Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, whose primary mechanisms or causes are still not defined and for which no effective treatment is available. We have recently ...reported that before disease onset the level of tyrosine nitrated proteins is increased in the G93A SOD1 transgenic mouse model of ALS. In the present investigation, we carried out a proteomic analysis of spinal cord extracts from G93A SOD1 mice at the presymptomatic stage of the disease to further unravel primary events in the pathogenesis and tentatively screen for potential pharmacological targets. Using a robust two-dimensional gel electrophoresis-based proteomic approach, we detected a number of proteins differentially represented in presymptomatic mice in comparison with controls. Alterations of these proteins correlate with mitochondrial dysfunction, aggregation, and stress response. Moreover, we found a variation in the isoform pattern of cyclophilin A, a molecular chaperone that protects cells from the oxidative stress.
In prion diseases, the infectious isoform of the prion protein (PrPSc) may subvert a normal, physiological activity of the cellular isoform (PrPC). A deletion mutant of the prion protein (Δ105–125) ...that produces a neonatal lethal phenotype when expressed in transgenic mice provides a window into the normal function of PrPC and how it can be corrupted to produce neurotoxic effects. We report here the surprising and unexpected observation that cells expressing Δ105–125 PrP and related mutants are hypersensitive to the toxic effects of two classes of antibiotics (aminoglycosides and bleomycin analogues) that are commonly used for selection of stably transfected cell lines. This unusual phenomenon mimics several essential features of Δ105–125 PrP toxicity seen in transgenic mice, including rescue by co-expression of wild type PrP. Cells expressing Δ105–125 PrP are susceptible to drug toxicity within minutes, suggesting that the mutant protein enhances cellular accumulation of these cationic compounds. Our results establish a screenable cellular phenotype for the activity of neurotoxic forms of PrP, and they suggest possible mechanisms by which these molecules could produce their pathological effects in vivo.
Into the fold: Prion diseases are neurodegenerative disorders characterized by the accumulation in the brain of a self‐replicating, misfolded isoform (PrPSc) of the cellular prion protein (PrPC). No ...therapies are available for these pathologies. We capitalized on previously described cell‐based assays to screen a library of small molecules, and identified 55, a compound capable of counteracting both prion replication and toxicity. Compound 55 may represent the starting point for the development of a completely new class of therapeutics for prion diseases.
Prion diseases comprise a group of rapidly progressive and invariably fatal neurodegenerative disorders for which there are no effective treatments. While conversion of the cellular prion protein ...(PrP
C
) to a β-sheet rich isoform (PrP
Sc
) is known to be a critical event in propagation of infectious prions, the identity of the neurotoxic form of PrP and its mechanism of action remain unclear. Insights into this mechanism have been provided by studying PrP molecules harboring deletions and point mutations in the conserved central region, encompassing residues 105-125. When expressed in transgenic mice, PrP deleted for these residues (Δ105-125) causes a spontaneous neurodegenerative illness that is reversed by co-expression of wild-type PrP. In cultured cells, Δ105-125 PrP confers hypersensitivity to certain cationic antibiotics and induces spontaneous ion channel activity that can be recorded by electrophysiological techniques. We have utilized these drug-hypersensitization and current-inducing activities to identify which PrP domains and subcellular locations are required for toxicity. We present an ion channel model for the toxicity of Δ105-125 PrP and related mutants and speculate how a similar mechanism could mediate PrP
Sc
-associated toxicity. Therapeutic regimens designed to inhibit prion-induced toxicity, as well as formation of PrP
Sc
, may prove to be the most clinically beneficial.
Mutation in the prion gene PRNP accounts for 10-15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated ...the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrPC). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrPC. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases.
Insight into the normal function of PrP(C), and how it can be subverted to produce neurotoxic effects, is provided by PrP molecules carrying deletions encompassing the conserved central region. The ...most neurotoxic of these mutants, Δ105-125 (called ΔCR), produces a spontaneous neurodegenerative illness when expressed in transgenic mice, and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous, or can be exerted on neighboring cells, is unknown. To investigate this question, we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice, which are marked by the presence or absence of GFP, are differentiated together to yield neurons, astrocytes, and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity, we assayed sensitivity of the cells to the cationic antibiotic, Zeocin. In a previous study, we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics, an effect that is suppressed by co-expression of wild type PrP, similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system, we find that while ΔCR-dependent toxicity is cell-autonomous, the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C), and the cellular mechanisms underlying the rescuing process.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Prion diseases are fatal neurodegenerative disorders that can arise sporadically, be genetically inherited or acquired through infection. The key event in these diseases is misfolding of the cellular ...prion protein (PrP(C)) into a pathogenic isoform that is rich in beta-sheet structure. This conformational change may result in the formation of PrP(Sc), the prion isoform of PrP, which propagates itself by imprinting its aberrant conformation onto PrP(C) molecules. A great deal of effort has been devoted to developing protocols for purifying PrP(Sc) for structural studies, and testing its biological properties. Most procedures rely on protease digestion, allowing efficient purification of PrP27-30, the protease-resistant core of PrP(Sc). However, protease treatment cannot be used to isolate abnormal forms of PrP lacking conventional protease resistance, such as those found in several genetic and atypical sporadic cases.
We developed a method for purifying pathological PrP molecules based on sequential centrifugation and immunoprecipitation with a monoclonal antibody selective for aggregated PrP. With this procedure we purified full-length PrP(Sc) and mutant PrP aggregates at electrophoretic homogeneity. PrP(Sc) purified from prion-infected mice was able to seed misfolding of PrP(C) in a protein misfolding cyclic amplification reaction, and mutant PrP aggregates from transgenic mice were toxic to cultured neurons.
The immunopurification protocol described here isolates biologically active forms of aggregated PrP. These preparations may be useful for investigating the structural and chemico-physical properties of infectious and neurotoxic PrP aggregates.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract A nine-octapeptide insertional mutation in the prion protein (PrP) causes a fatal neurodegenerative disorder in both humans and transgenic mice. To determine the precise cellular ...localization of this mutant PrP (designated PG14), we have generated transgenic mice expressing PG14-EGFP, a fluorescent fusion protein that can be directly visualized in vivo . Tg(PG14-EGFP) mice develop an ataxic neurological illness characterized by astrogliosis, PrP aggregation, and accumulation of a partially protease-resistant form of the mutant PrP. Strikingly, PG14-EGFP forms numerous fluorescent aggregates in the neuropil and white matter of multiple brain regions. These aggregates are particularly prominent along axonal tracts in both brain and peripheral nerve, and similar intracellular deposits are visible along the processes of cultured neurons. Our results reveal intra-axonal aggregates of a mutant PrP, which could contribute to the pathogenesis of familial prion disease by disrupting axonal transport.
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