In vitro prion protein conversion in detergent-solubilized membranes

A fundamental event in the pathogenesis of prion disease is the conversion of PrP(C), a normal glycophosphatidyl-anchored glycoprotein, into an infectious isoform designated PrP(Sc). In a modified version of the protein misfolding cyclic amplification (PMCA) technique [Saborio et al. (2001) Nature 411, 810-813], protease-resistant PrP(Sc)-like molecules (PrPres) can be amplified in vitro in a species- and strain-specific manner from crude brain homogenates, providing a biochemical model of the prion conversion reaction [Lucassen et al. (2003) Biochemistry 42, 4127-4135]. In this study, we investigated the ability of enriched membrane subsets and detergent-solubilized membrane preparations to support PrPres amplification. Membrane fractionation experiments showed that purified synaptic plasma membrane preparations enriched in PrP(C) but largely depleted of late endosomal and lysosomal markers were sufficient to support PrPres amplification. Detergent solubilization experiments showed that a small group of select detergents could be used to produce soluble preparations that contain PrP(C) and fully support PrPres amplification. The stability of PrPres amplification ability in detergent-solubilized supernatants was dependent on detergent concentration. These results lead to the surprising conclusion that membrane attachment is not required for PrP(C) to convert efficiently into PrPres in vitro and also indicate that biochemical purification of PrPres amplification factors from brain homogenates is a feasible approach.     

Strain-specific kinetics of prion protein formation in vitro and in vivo

The molecular basis of prion strain diversity is proposed to be encoded by distinct conformations of the abnormal scrapie isoform of the prion protein (PrP(Sc)). PrP(Sc) formation for the hyper (HY) and drowsy (DY) strains of the transmissible mink encephalopathy (TME) agent was investigated using the cell-free PrP conversion reaction to determine the role of distinct PrP(Sc) conformations in the rate of in vitro conversion of cellular PrP into protease-resistant PrP. PrP conversion increased at an exponential rate for both TME strains until peak levels were reached at 72-96 h of reaction time. The amount and rate of PrP conversion for HY TME was greater than those for DY TME between 48 h and the peak level of PrP conversion. Between 96 and 120 h, there was a negative rate of PrP conversion; and between 120 and 168 h, the net rate of HY and DY PrP conversion approached zero. These findings suggest that PrP conversion can occur in three distinct stages: an elongation phase, a depolymerization phase, and a steady-state phase. Strain-specific properties between the TME strains were identified only during the elongation phase. The steady-state phase could be disrupted by the addition of PrP(Sc) to, or by sonication of, the cell-free PrP conversion reaction. These treatments resulted in an increase in the amount of PrP conversion that was equal to or greater than that found during the peak level of PrP conversion for both TME strains, indicating that the steady-state phase was in dynamic equilibrium. In a related study, the rate of accumulation of HY and DY PrP(Sc) in hamster brain exhibited a strain-specific pattern that had similarities to the strain-specific PrP conversion reaction during the elongation phase. These results suggest that strain-specific conformations of PrP(Sc) have the ability to influence the rate of additional PrP(Sc) formation from cellular PrP both in vitro and in vivo.     

Cytoplasmic prion protein induces forebrain neurotoxicity

The prion protein (PrP) is essential for the pathogenesis of prion disease. PrP has been detected in the cytosol of neurons and transgenic mice expressing PrP in the cytosol (cyPrP) under a pan-neuronal promoter developed rapid cerebellar granule neuron degeneration. Yet, it remains unclear whether cyPrP is capable to cause toxicity in other neuronal populations. Here, we report that transgenic mice expressing cyPrP in the forebrain neurons developed behavioral abnormalities including clasping and hyperactivity. These mice had reduced thickness in cortex and developed astrogliosis in hippocampal and cortical regions. Moreover, cyPrP in these mice was recognized by the A11 anti-oligomer antibody and was associated with the hydrophobic lipid core of membranes, indicating that cyPrP oligomer caused membrane perturbation contributes to cyPrP neurotoxicity. Together, our results clearly revealed that cyPrP is able to cause toxicity in different neuronal populations, supporting a role of cyPrP in PrP-mediated neurodegenerative disorders.     

Membrane interaction of off-pathway prion oligomers and lipid-induced on-pathway intermediates during prion conversion: A clue for neurotoxicity

Soluble oligomers of prion proteins (PrP), produced during amyloid aggregation, have emerged as the primary neurotoxic species, instead of the fibrillar end-products, in transmissible spongiform encephalopathies. However, whether the membrane is among their direct targets, that mediate the downstream adverse effects, remains a question of debate. Recently, questions arise from the formation of membrane-active oligomeric species generated during the β-aggregation pathway, either in solution, or in lipid environment. In the present study, we characterized membrane interaction of off-pathway oligomers from recombinant prion protein generated along the amyloid aggregation and compared to lipid-induced intermediates produced during lipid-accelerated fibrillation. Using calcein-leakage assay, we show that the soluble prion oligomers are the most potent in producing leakage with negatively charged vesicles. Binding affinities, conformational states, mode of action of the different PrP assemblies were determined by thioflavin T binding-static light scattering experiments on DOPC/DOPS vesicles, as well as by FTIR-ATR spectroscopy and specular neutron reflectivity onto the corresponding supported lipid bilayers. Our results indicate that the off-pathway PrP oligomers interact with lipid membrane via a distinct mechanism, compared to the inserted lipid-induced intermediates. Thus, separate neurotoxic mechanisms could exist following the puzzling intermediates generated in the different cell compartments. These results not only reveal an important regulation of lipid membrane on PrP behavior but may also provide clues for designing stage-specific and prion-targeted therapy.     

Molecular mechanisms of neurotoxicity of pathological prion protein

Transmissible Spongiform Encephalopathies or prion related disorders are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and accumulation of a misfolded form of the cellular prion protein (PrP), denoted PrP(Sc). Although the mechanism of neurodegeneration and the involvement of PrP(Sc) is far from clear, data indicates that neuronal apoptosis might be related to activation of several signaling pathways, including proteasome dysfunction, alterations in prion maturation pathway and endoplasmic reticulum (ER) stress. In this article we describe recent studies investigating the molecular mechanism of PrP(Sc) neurotoxicity. We propose a model in which the key step in the pathogenesis of prion disorders, independent on their etiology, is the alteration of ER-homeostasis due to drastic modifications of the physicochemical properties of PrP, leading to the activation of ER-dependent signaling pathways that controls cellular survival.     

In vitro conversion and seeded fibrillization of posttranslationally modified prion protein

The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrP(C) into an aggregated, β-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.     

Structural characterization of beta-sheeted oligomers formed on the pathway of oxidative prion protein aggregation in vitro

The pathology of transmissible spongiform encephalopathies (TSEs) is strongly associated with the structural conversion of the cellular prion protein (PrPC) into a misfolded isoform (PrPSc) that assembles into amyloid fibrils. Since increased levels of oxidative stress have been linked to prion diseases, we investigated the metal-induced oxidation of human PrP (90-231). A novel in vitro conversion assay based on aerobic incubation of PrP in the presence of elemental copper pellets at pH 5 was established, resulting in aggregation of highly beta-sheeted prion proteins. We show for the first time that two discrete oligomeric species of elongated shape, approx. 25 mers and 100 mers, are formed on the pathway of oxidative PrP aggregation in vitro, which are well characterized regarding shape and size using small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and electron microscopy (EM). Considering that small oligomers of highly similar size have recently been reported to show the highest specific infectivity within TSE-infected brain tissues of hamsters, the novel oligomers observed in this study are interesting candidates as agent causing neurodegenerative and/or self-propagating effects. Moreover, our results significantly strengthen the theory that oxidative stress might be an influence that leads to substantial structural conversions of PrP in vivo.     

In vivo cytotoxicity of the prion protein fragment 106-126

Transmissible spongiform encephalopathies are fatal neurological diseases characterized by astroglyosis, neuronal loss, and by the accumulation of the abnormal isoform of the prion protein. The amyloid prion protein fragment 106-126 (P106-126) has been shown to be toxic in cultured hippocampal neurons (). Here, we show that P106-126 is also cytotoxic in vivo. Taking advantage of the fact that retina is an integral part of the central nervous system, the toxic effect of the peptide was investigated by direct intravitreous injection. Aged solutions of P106-126 induced apoptotic-mediated retinal cell death and irreversibly altered the electrical activity of the retina. Neither apoptosis nor electroretinogram damages were observed with freshly diluted P106-126, suggesting that the toxicity is linked to the aggregation state of the peptide. The retina provides a convenient in vivo system to look for potential inhibitors of cytotoxicity associated with spongiform encephalopathies.     

Amidation and structure relaxation abolish the neurotoxicity of the prion peptide PrP106-126 in vivo and in vitro

One of the major pathological hallmarks of transmissible spongiform encephalopathies (TSEs) is the accumulation of a pathogenic (scrapie) isoform (PrP(Sc)) of the cellular prion protein (PrP(C)) primarily in the central nervous system. The synthetic prion peptide PrP106-126 shares many characteristics with PrP(Sc) in that it shows PrP(C)-dependent neurotoxicity both in vivo and in vitro. Moreover, PrP106-126 in vitro neurotoxicity has been closely associated with the ability to form fibrils. Here, we studied the in vivo neurotoxicity of molecular variants of PrP106-126 toward retinal neurons using electroretinographic recordings in mice after intraocular injections of the peptides. We found that amidation and structure relaxation of PrP106-126 significantly reduced the neurotoxicity in vivo. This was also found in vitro in primary neuronal cultures from mouse and rat brain. Thioflavin T binding studies showed that amidation and structure relaxation significantly reduced the ability of PrP106-126 to attain fibrillar structures in physiological salt solutions. This study hence supports the assumption that the neurotoxic potential of PrP106-126 is closely related to its ability to attain secondary structure.     

Disease-associated prion protein oligomers inhibit the 26S proteasome

The mechanism of cell death in prion disease is unknown but is associated with the production of a misfolded conformer of the prion protein. We report that disease-associated prion protein specifically inhibits the proteolytic beta subunits of the 26S proteasome. Using reporter substrates, fluorogenic peptides, and an activity probe for the beta subunits, this inhibitory effect was demonstrated in pure 26S proteasome and three different cell lines. By challenge with recombinant prion and other amyloidogenic proteins, we demonstrate that only the prion protein in a nonnative beta sheet conformation inhibits the 26S proteasome at stoichiometric concentrations. Preincubation with an antibody specific for aggregation intermediates abrogates this inhibition, consistent with an oligomeric species mediating this effect. We also present evidence for a direct relationship between prion neuropathology and impairment of the ubiquitin-proteasome system (UPS) in prion-infected UPS-reporter mice. Together, these data suggest a mechanism for intracellular neurotoxicity mediated by oligomers of misfolded prion protein.     

In vitro amplification of protease-resistant prion protein requires free sulfhydryl groups

Prions, the infectious agents of transmissible spongiform encephalopathies, are composed primarily of a misfolded protein designated PrP(Sc). Prion-infected neurons generate PrP(Sc) from a host glycoprotein designated PrP(C) through a process of induced conformational change, but the molecular mechanism by which PrP(C) undergoes conformational change into PrP(Sc) remains unknown. We employed an in vitro PrP(Sc) amplification technique adapted from protein misfolding cyclic amplification (PMCA) to investigate the mechanism of prion-induced protein conformational change. Using this technique, PrP(Sc) from diluted scrapie-infected brain homogenate can be amplified >10-fold without sonication when mixed with normal brain homogenate under nondenaturing conditions. PrP(Sc) amplification in vitro exhibits species and strain specificity, depends on both time and temperature, only requires membrane-bound components, and does not require divalent cations. In vitro amplification of Syrian hamster Sc237 PrP(Sc) displays an optimum pH of approximately 7, whereas amplification of CD-1 mouse RML PrP(Sc) is optimized at pH approximately 6. The thiolate-specific alkylating agent N-ethylmaleimide (NEM) as well as the reversible thiol-specific blockers p-hydroxymercuribenzoic acid (PHMB) and mersalyl acid inhibited PrP(Sc) amplification in vitro, indicating that the conformational change from PrP(C) to PrP(Sc) requires a thiol-containing factor. Our data provide the first evidence that a reactive chemical group plays an essential role in the conformational change from PrP(C) to PrP(Sc).     

Mutant prion protein-mediated aggregation of normal prion protein in the endoplasmic reticulum: implications for prion propagation and neurotoxicity

Familial prion disorders are believed to result from spontaneous conversion of mutant prion protein (PrPM) to the pathogenic isoform (PrPSc). While most familial cases are heterozygous and thus express the normal (PrPC) and mutant alleles of PrP, the role of PrPC in the pathogenic process is unclear. Plaques from affected cases reveal a heterogeneous picture; in some cases only PrPM is detected, whereas in others both PrPC and PrPM are transformed to PrPSc. To understand if the coaggregation of PrPC is governed by PrP mutations or is a consequence of the cellular compartment of PrPM aggregation, we coexpressed PrPM and PrPC in neuroblastoma cells, the latter tagged with green fluorescent protein (PrPC-GFP) for differentiation. Two PrPM forms (PrP231T, PrP217R/231T) that aggregate spontaneously in the endoplasmic reticulum (ER) were generated for this analysis. We report that PrPC-GFP aggregates when coexpressed with PrP231T or PrP217R/231T, regardless of sequence homology between the interacting forms. Furthermore, intracellular aggregates of PrP231T induce the accumulation of a C-terminal fragment of PrP, most likely derived from a potentially neurotoxic transmembrane form of PrP (CtmPrP) in the ER. These findings have implications for prion pathogenesis in familial prion disorders, especially in cases where transport of PrPM from the ER is blocked by the cellular quality control.     

Flexible N-terminal region of prion protein influences conformation of protease-resistant prion protein isoforms associated with cross-species scrapie infection in vivo and in vitro

Transmissible spongiform encephalopathy (TSE) diseases are characterized by the accumulation in brain of an abnormal protease-resistant form of the host-encoded prion protein (PrP), PrP-res. PrP-res conformation differs among TSE agents derived from various sources, and these conformational differences are thought to influence the biological characteristics of these agents. In this study, we introduced deletions into the flexible N-terminal region of PrP (residues 34-124) and investigated the effect of this region on the conformation of PrP-res generated in an in vitro cell-free conversion assay. PrP deleted from residues 34 to 99 generated 12-16-kDa protease-resistant bands with intact C termini but variable N termini. The variable N termini were the result of exposure of new protease cleavage sites in PrP-res between residues 130 and 157, suggesting that these new cleavage sites were caused by alterations in the conformation of the PrP-res generated. Similarly truncated 12-16-kDa PrP bands were also identified in brain homogenates from mice infected with mouse-passaged hamster scrapie as well as in the cell-free conversion assay using conditions that mimicked the hamster/mouse species barrier to infection. Thus, by its effects on PrP-res conformation, the flexible N-terminal region of PrP seemed to influence TSE pathogenesis and cross-species TSE transmission.     

In vitro assay for fragmentation of amyloid fibers of yeast prion protein

In prion propagation, fragmentation of amyloid fibers, as well as conformational conversion of prion protein, is critical: the latter increases the net amount of abnormal prion proteins and the former multiplies number of seeds. We present here a method for in vitro measurement of fragmentation of amyloid fibers of yeast Sup35 prion protein. In this method, amyloid fibers are tethered to the surface of magnetic beads. Fragmentation of the fibers results in release of fiber fragments into the medium, which are then quantified by immunoblotting. This method is versatile for other amyloid fibers.     

Dominant-negative effects of the N-terminal half of prion protein on neurotoxicity of prion protein-like protein/doppel in mice

Prion protein-like protein/doppel is neurotoxic, causing ataxia and Purkinje cell degeneration in mice, whereas prion protein antagonizes doppel-induced neurodegeneration. Doppel is homologous to the C-terminal half of prion protein but lacks the amino acid sequences corresponding to the N-terminal half of prion protein. We show here that transgenic mice expressing a fusion protein consisting of the N-terminal half, corresponding to residues 1-124, of prion protein and doppel in neurons failed to develop any neurological signs for up to 730 days in a background devoid of prion protein. In addition, the fusion protein prolonged the onset of ataxia in mice expressing exogenous doppel. These results suggested that the N-terminal part of prion protein has a neuroprotective potential acting both cis and trans on doppel. We also show that prion protein lacking the pre-octapeptide repeat (Delta25-50) or octapeptide repeat (Delta51-90) region alone could not impair the antagonistic function against doppel.     

In vitro and in vivo antagonists against parathyroid hormone-related protein

Four analogues of parathyroid hormone-related protein (PTHrP), PTHrP(7-34)NH2, (10-34)NH2, (15-34)NH2 and (20-34)NH2, were synthesized and their antagonistic activity against PTHrP(1-34) was examined in vitro and in vivo. In vitro studies revealed that all four analogues antagonized PTHrP-stimulated cyclic AMP production in rat osteosarcoma cells (ROS 17/2.8), and that PTHrP(7-34)NH2 and PTHrP(10-34)NH2 had potent antagonistic activity. In vivo experiments in nude mice also revealed that PTHrP(7-34)NH2 completely inhibited hypercalcemia induced by PTHrP(1-34), indicating that these analogues antagonize the effects of PTHrP(1-34) in vitro and in vivo.     

In vitro amplification of misfolded prion protein using lysate of cultured cells

Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrP(C)). PrP(Sc) was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrP(C) in cell lysate was a critical factor to drive efficient PrP(Sc) amplification, our results demonstrate that cell lysate in which PrP(C) is present abundantly serves as an excellent substrate source for PMCA.     

In vivo aggregation of the HET-s prion protein of the fungus Podospora anserina

We have proposed that the [Het-s] infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. The HET-s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility. Under the prion form, the HET-s protein causes a cell death reaction when co-expressed with the HET-S protein, from which it differs by only 13 amino acid residues. We show here that the HET-s protein can exist as two alternative states, a soluble and an aggregated form in vivo. As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET-s--green fluorescent protein (GFP) fusion protein. The HET-s protein is aggregated in vivo when highly expressed. However, we could not demonstrate HET-s aggregation at wild-type expression levels, which could indicate that only a small fraction of the HET-s protein is in its aggregated form in vivo in wild-type [Het-s] strains. The antagonistic HET-S form is soluble even at high expression level. A double amino acid substitution in HET-s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion. Together, these results further support the model that the [Het-s] element corresponds to an abnormal self-perpetuating aggregated form of the HET-s protein.