Aggregation of cellular prion protein is initiated by proximity-induced dimerization

Prion diseases or transmissible spongiform encephalopathies (TSEs) are infectious and fatal neurodegenerative disorders in humans and animals. Pathological features of TSEs include the conversion of cellular prion protein (PrP(C)) into an altered disease-associated conformation generally designated PrP(Sc), abnormal deposition of PrP(Sc) aggregates, and spongiform degeneration of the brain. The molecular steps leading to PrP(C) aggregation are unknown. Here, we have utilized an inducible oligomerization strategy to test if, in the absence of any infectious prion particles, the encounter between PrP(C) molecules may trigger its aggregation in neuronal cells. A chimeric PrP(C) composed of one (Fv1) or two (Fv2) modified FK506-binding protein (Fv) fused with PrP(C) were created, and transfected in N2a cells. Similar to PrP(C), Fv1-PrP and Fv2-PrP were glycosylated, displayed normal localization, and anti-apoptotic function. When cells were treated with the dimeric Fv ligand AP20187, to induce dimerization (Fv1) or oligomerization (Fv2) of PrP(C), both dimerization and oligomerization of PrP(C) resulted in the de novo production, release and deposition of extracellular PrP aggregates. Aggregates were insoluble in non-ionic detergents and partially resistant to proteinase K. These findings demonstrate that homologous interactions between PrP(C) molecules may constitute a minimal and sufficient molecular event leading to PrP(C) aggregation and extracellular deposition.     

Tethering the N-terminus of the prion protein compromises the cellular response to oxidative stress

The role of the N-terminal half of the prion protein (PrPC) in normal cellular function and pathology remains enigmatic. To investigate the biological role of the N-terminus of PrP, we examined the cellular properties of a construct of murine PrP, PrP-DA, in which the N-terminus is tethered to the membrane by an uncleaved signal peptide and which retains the glycosyl-phosphatidylinositol anchor. Human neuroblastoma SH-SY5Y cells expressing PrP-DA were more susceptible to hydrogen peroxide and copper induced toxicity than wtPrP expressing cells. The PrP-DA expressing cells had an increased level of intracellular free radicals and reduced levels of superoxide dismutase and glutathione peroxidase as compared to the wtPrP expressing cells. The membrane topology, cell surface location, lipid raft localisation, intracellular trafficking and copper-mediated endocytosis of PrP-DA were not significantly different from wtPrP. However, cells expressing PrP-DA accumulated an N-terminal fragment that was resistant to proteinase K. The data presented here are consistent with the N-terminal region of PrPC having a role in the cellular response to oxidative stress, and that tethering this region of the protein to the membrane compromises this function through the accumulation of a protease-resistant N-terminal fragment, similar to that seen in some forms of human prion disease.     

Molecular morphology and toxicity of cytoplasmic prion protein aggregates in neuronal and non-neuronal cells

Recent studies have revealed that accumulation of prion protein (PrP) in the cytoplasm results in the production of aggregates that are insoluble in non-ionic detergents and partially resistant to proteinase K. Transgenic mice expressing PrP in the cytoplasm develop severe ataxia with cerebellar degeneration and gliosis, suggesting that cytoplasmic PrP may play a role in the pathogenesis of prion diseases. The mechanism of cytoplasmic PrP neurotoxicity is not known. In this report, we determined the molecular morphology of cytoplasmic PrP aggregates by immunofluorescence and electron microscopy, in neuronal and non-neuronal cells. Transient expression of cytoplasmic PrP produced juxtanuclear aggregates reminiscent of aggresomes in human embryonic kidney 293 cells, human neuroblastoma BE2-M17 cells and mouse neuroblastoma N2a cells. Time course studies revealed that discrete aggregates form first throughout the cytoplasm, and then coalesce to form an aggresome. Aggresomes containing cytoplasmic PrP were 1-5-microm inclusion bodies and were filled with electron-dense particles. Cytoplasmic PrP aggregates induced mitochondrial clustering, reorganization of intermediate filaments, prevented the secretion of wild-type PrP molecules and diverted these molecules to the cytoplasm. Cytoplasmic PrP decreased the viability of neuronal and non-neuronal cells. We conclude that any event leading to accumulation of PrP in the cytoplasm is likely to result in cell death.     

Extracellular copper ions regulate cellular prion protein (PrPC) expression and metabolism in neuronal cells

The physiological functions of cellular prion protein (PrP(C)) remain unclear. It has been demonstrated that PrP(C) is a copper binding protein and proposed that its functions could be strictly linked to copper metabolism and neuroprotection. The aim of this study was to clarify how extracellular copper modifies PrP(C) expression and metabolism in cultured neurones. We reported here that copper delivered at physiological concentrations significantly decreases PrP(C) mRNA expression in GN11 neurones. Moreover, copper increases the release of PrP(C) into the culture medium. These results indicate that extracellular copper strongly affects the amount of cellular PrP and might represent an interesting strategy to decrease the expression of PrP(C) in neurones and its conversion in the pathological isoform PrP(Sc).     

Anterograde and retrograde intracellular trafficking of fluorescent cellular prion protein

In order to investigate the microtubule-associated intracellular trafficking of the NH2-terminal cellular prion protein (PrPC) fragment [Biochem. Biophys. Res. Commun. 313 (2004) 818], we performed a real-time imaging of fluorescent PrPC (GFP-PrPC) in living cells. Such GFP-PrPC exhibited an anterograde movement towards the direction of plasma membranes at a speed of 140-180 nm/s, and a retrograde movement inwardly at a speed of 1.0-1.2 microm/s. The anterograde and retrograde movements of GFP-PrPC were blocked by a kinesin family inhibitor (AMP-PNP) and a dynein family inhibitor (vanadate), respectively. Furthermore, anti-kinesin antibody (alpha-kinesin) blocked its anterograde motility, whereas anti-dynein antibody (alpha-dynein) blocked its retrograde motility. These data suggested the kinesin family-driven anterograde and the dynein-driven retrograde movements of GFP-PrPC. Mapping of the interacting domains of PrPC identified amino acid residues indispensable for interactions with kinesin family: NH2-terminal mouse (Mo) residues 53-91 and dynein: NH2-terminal Mo residues 23-33, respectively. Our findings argue that the discrete N-terminal amino acid residues are indispensable for the anterograde and retrograde intracellular movements of PrPC.     

Prion protein expression modulates neuronal copper content

The prion protein is a copper (Cu)-binding protein. The abnormal isoform of this protein is associated with the transmissible spongiform encephalopathies or prion diseases. In prion diseases, the prion protein loses its Cu binding capacity. The effect of prion protein expression on the Cu content of the brain was investigated. Transgenic mice, either overexpressing the prion protein or expressing a mutant form lacking the Cu-binding region of the protein, were compared with wild-type mice and mice in which expression of the protein was knocked out. Age-dependent differences in Cu content of the brain were detected. Also, synaptosomal fractions from the brains of the mice showed different Cu content depending on the expression of the prion protein. Mice expressing prion protein, but without the Cu-binding domain showed reduced Cu content. Mice overexpressing the prion protein showed little difference in Cu in the brain compared with wild type but also the prion protein expressed by the mice showed a reduction in the level of Cu bound. These results confirm that prion protein expression modulates the Cu level found at the synapse and this effect is dependent on its Cu binding capacity. Loss of normal Cu binding by the prion protein altered age-related increases in metals in the brain. This may explain why many forms of human prion disease do not develop until late in life.     

朊蛋白的细胞生物学研究

朊蛋白病是人和牛羊等哺乳动物所患的致命性的神经系统变性疾病,它是由机体内正常的朊蛋白改变构象后所引起的疾病。本综述对朊蛋白在细胞生物学领域的认知和理解进行了归纳总结,阐述了正常和异常朊蛋白的翻译、表达、定位、裂解、转化等一系列过程,是对疾病本质的有益探索。     

Post-transcriptional suppression of pathogenic prion protein expression in Drosophila neurons

A wealth of evidence supports the view that conformational change of the prion protein, PrPC, into a pathogenic isoform, PrPSc, is the hallmark of sporadic, infectious, and inherited forms of prion disease. Although the central role played by PrPSc in the pathogenesis of prion disease is appreciated, the cellular mechanisms that recognize PrPSc and modulate its production, clearance, and neural toxicity have not been elucidated. To address these questions, we used a tissue-specific expression system to express wild-type and disease-associated PrP molecules heterologously in Drosophila melanogaster. Our results indicate that Drosophila brain possesses a specific and saturable mechanism that suppresses the accumulation of PG14, a disease-associated insertional PrP mutant. We also found that wild-type PrP molecules are maintained in a detergent-soluble conformation throughout life in Drosophila brain neurons, whereas they become detergent-insoluble in retinal cells as flies age. PG14 protein expression in Drosophila eye did not cause retinal pathology. Our work reveals the presence of mechanisms in neurons that specifically counterbalance the production of misfolded PrP conformations, and provides an opportunity to study these processes in a model organism amenable to genetic analysis.     

Prion protein scrapie and the normal cellular prion protein

Prions are infectious proteins and over the past few decades, some prions have become renowned for their causative role in several neurodegenerative diseases in animals and humans. Since their discovery, the mechanisms and mode of transmission and molecular structure of prions have begun to be established. There is, however, still much to be elucidated about prion diseases, including the development of potential therapeutic strategies for treatment. The significance of prion disease is discussed here, including the categories of human and animal prion diseases, disease transmission, disease progression and the development of symptoms and potential future strategies for treatment. Furthermore, the structure and function of the normal cellular prion protein (PrP^C) and its importance in not only in prion disease development, but also in diseases such as cancer and Alzheimer's disease will also be discussed.     

Axonal transport of the cellular prion protein is increased during axon regeneration

The cellular prion protein, PrPc, is a glycosylphosphatidylinositol-anchored cell surface glycoprotein and a protease-resistant conformer of the protein may be the infectious agent in transmissible spongiform encephalopathies. PrPc is localized on growing axons in vitro and along fibre bundles that contain elongating axons in developing and adult brain. To determine whether the growth state of axons influenced the expression and axonal transport of PrPc, we examined changes in the protein following post-traumatic regeneration in the hamster sciatic nerve. Our results show (1) that PrPc in nerve is significantly increased during nerve regeneration; (2) that this increase involves an increase in axonally transported PrPc; and (3) that the PrPc preferentially targeted for the newly formed portions of the regenerating axons consists of higher molecular weight glycoforms. These results raise the possibility that PrPc may play a role in the growth of axons in vivo, perhaps as an adhesion molecule interacting with the extracellular environment through specialized glycosylation.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Loss of the cellular prion protein affects the Ca2+ homeostasis in hippocampal CA1 neurons

Previous neurophysiological studies on prion protein deficient (Prnp(-/-)) mice have revealed a significant reduction of slow afterhyperpolarization currents (sI(AHP)) in hippocampal CA1 pyramidal cells. Here we aim to determine whether loss of PrP(C.) directly affects the potassium channels underlying sI(AHP) or if sI(AHP) is indirectly disturbed by altered intracellular Ca(2+) fluxes. Patch-clamp measurements and confocal Ca(2+) imaging in acute hippocampal slice preparations of Prnp(-/-) mice compared to littermate control mice revealed a reduced Ca(2+) rise in CA1 neurons lacking PrP(C) following a depolarization protocol known to induce sI(AHP). Moreover, we observed a reduced Ca(2+) influx via l-type voltage gated calcium channels (VGCCs). No differences were observed in the protein expression of the pore forming alpha1 subunit of VGCCs Prnp(-/-) mice. Surprisingly, the beta2 subunit, critically involved in the transport of the alpha1 subunit to the plasma membrane, was found to be up-regulated in knock out hippocampal tissue. On mRNA level however, no differences could be detected for the alpha1C, D and beta2-4 subunits. In conclusion our data support the notion that lack of PrP(C.) does not directly affect the potassium channels underlying sI(AHP), but modulates these channels due to its effect on the intracellular free Ca(2+) concentration via a reduced Ca(2+) influx through l-type VGCCs.     

Reversible aggregation of mouse prion protein derivatives with PrPSC-like structural properties

Three carbamylated derivatives of reduced mouse prion protein (mPrP) were isolated during the aborted oxidative folding in the presence of urea. These three prion protein derivatives (mPrP-a, mPrP-b, and mPrP-c) exist as monomer in the acidic solution (pH < 2.0) and exhibit prevalent random coil structure. However, they undergo rapid aggregation and transformation to a predominant -sheet structure upon exposure to ionic buffer with pH greater than 3.0. The stability of aggregates of mPrP conformers is in part dependent upon the time that they were allowed to develop. The nascent aggregates comprise a significant fraction of loosely packed mPrP monomers that can be dissociated by treatment with strong acidic solution. Matured aggregates acquired through prolonged sample incubation contain more tightly packed mPrP monomers that cannot be dissociated by strong acid but can be disaggregated by denaturant. The properties of reversible aggregation of mPrP-a, mPrP-b, and mPrP-c bear a striking resemblance to that observed with aggregates of hamster PrP^SC.     

PrPc on the road: trafficking of the cellular prion protein

The glycosylphosphatidylinositol (GPI)-anchored cellular prion protein (PrPc) has a fundamental role in prion diseases. Intracellular trafficking of PrPc is important in the generation of protease resistant PrP species but little is known of how endocytosis affects PrPc function. Here, we discuss recent experiments that have illuminated how PrPc is internalized and what are the possible destinations taken by the protein. Contrary to what would be expected for a GPI-anchored protein there is increasing evidence that clathrin-mediated endocytosis and classical endocytic organelles participate in PrPc trafficking. Moreover, the N-terminal domain of PrPc may be involved in sorting events that can direct the protein during its intracellular journey. Indeed, the concept that the GPI-anchor determines PrPc trafficking has been challenged. Cellular signaling can be triggered or be regulated by PrPc and we suggest that endocytosis of PrPc may influence signaling in several ways. Definition of the processes that participate in PrPc endocytosis and intracellular trafficking can have a major impact on our understanding of the mechanisms involved in PrPc function and conversion to protease resistant conformations.     

Chimeric prion protein expression in cultured cells and transgenic mice.

The efficient expression of exogenous prion protein (PrP) molecules in mouse neuroblastoma cells that are chronically infected with murine scrapie prions (ScN2a cells; Butler, D.A., et al., 1988, J. Virol. 62, 1558-1564) and in transgenic mice is described. This technology allows investigation of the PrP molecule for structural regions involved in determining species specificity, as well as ablation experiments designed to address the functionality of particular regions of the PrP molecule. Previous reports demonstrated that the PrP gene specifies the host range for susceptibility of transgenic animals to prions (Scott, M., et al., 1989, Cell 59, 847-857; Prusiner, S.B., et al., 1990, Cell 63, 673-686). Consistent with these results, we showed that Syrian hamster (SHa) PrP is ineligible for efficient conversion to PrPSc in ScN2a cells. By constructing a series of chimeric mouse (Mo)/SHaPrP genes, we developed an epitopically tagged functional variant of the MoPrP gene, which can efficiently form protease-resistant PrP molecules upon expression in ScN2a cells. The presence of a defined epitope for an SHa-specific monoclonal antibody allows the products of this chimeric gene to be discriminated from endogenous MoPrP and creates a useful reagent for exploring structure/function relationships via targeted mutagenesis. In addition, we developed a transgenic mouse expression vector by manipulation of an SHaPrP cosmid clone. This vector permits the efficient expression of foreign PrP genes in the brains of transgenic animals, enabling pathological consequences of in vitro mutagenesis to be studied.     

Evaluation of non-immunoaffinity methods for isolation of cellular prion protein from bovine brain

Transmissible spongiform encephalopathies (TSEs) are progressive neurodegenerative diseases that affect the central nervous system of many animals, including humans. Research suggests that TSEs are caused by conversion of the cellular prion protein (PrP^C), which is encoded in many tissues, especially brain, to the pathological form (PrP^Sc). This conversion affects PrP^Sc structure, conferring different biochemical properties, such as the increased resistance to proteinase K, that have been widely used for its purification. By contrast, PrP^C is less resistant and its isolation is more challenging. Here, we propose a purification strategy to efficiently recover PrP^C from healthy bovine brain using conventional non-immunoaffinity methods. The applicability of extraction using detergents, size exclusion chromatography, diafiltration with molecular weight cutoff (MWCO) filters, and immobilized metal affinity chromatography (IMAC) using Western blot (WB) analysis to detect the presence of PrP^C is discussed in detail.     

A systematic investigation of production of synthetic prions from recombinant prion protein

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic'' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.